E. Gregory MacEwen

Spontaneously occurring tumors of companion animals as models for human cancer.

Spontaneous tumors in companion animals (dog and cat) offer a unique opportunity as models for human cancer biology and translational cancer therapeutics. The relatively high incidence of some cancers, similar biologic behavior, large body size, comparable responses to cytotoxic agents, and shorter overall lifespan are the factors that contribute to the advantages of the companion animal model. The tumor types that offer the best comparative interest include lymphoma/leukemia, osteosarcoma, STS, melanoma, and mammary tumors. With the increase in new therapeutic agents (traditional chemotherapy, gene therapy, biologic agents, etc.), the companion animal model can provide useful populations to test new agents where efficacy and toxicity can be examined.

IGF‐1 receptor contributes to the malignant phenotype in human and canine osteosarcoma

To further define the role of insulin‐like growth factor‐1 (IGF‐1) and its receptor (IGF‐1R) in osteosarcoma (OS), human OS cell lines with low (SAOS‐2) and high (SAOS‐LM2) metastatic potential and three canine OS‐derived cell lines were studied. Cell lines were evaluated for: IGF‐1R expression; expression of IGF binding proteins (IGFBPs); effect of IGF‐1 on tumor cell growth, invasion, expression of urokinase plasminogen activator (uPA), and soluble uPA receptor (suPAR), and; ectopic and orthotopic tumorigenicity of the canine OS cells in athymic mice. All cell lines exhibited steady‐state mRNA expression of IGF‐1R. The SAOS‐2 and SAOS‐LM2 cells expressed 9,138 and 10,234 cell‐associated binding sites, respectively. Canine OS cells expressed from 1,728 to 3,883 binding sites. Two IGF‐1‐treated cell lines displayed enhanced proliferation. Two cell lines formed colonies in semisolid media, and IGF‐1 increased colony number. Matrigel invasion was enhanced in one cell line following IGF‐1 treatment. uPA and suPAR were unchanged in SAOS‐2 and SAOS‐LM2 cells following IGF‐1 treatment, but the highly metastatic OS line SAOS‐LM2 expressed five times more suPAR and displayed enhanced invasion compared to the parental, low metastatic SAOS‐2. IGFBP‐5 was detected in four of five cell lines, and IGFBP‐3 was detected in two canine OS cell lines. Two canine OS lines were tumorigenic, and one metastasized spontaneously. In conclusion, OS cells express IGF‐1R, which can contribute to their growth and invasion. There is suggestive evidence that increasing receptor number may contribute to in vivo tumorigenesis. Additional studies are needed to determine how IGF‐1/IGF‐1R interactions contribute to the malignant phenotype of OS.

Canine Oral Melanoma: Comparison of Surgery Versus Surgery Plus Corynebacterium parvum

Eighty-nine dogs with malignant oral melanoma were selected for study. All dogs were clinically staged and treated with either surgical excision alone or surgery plus C. parvum immunotherapy. There was no difference in survival time between the two treatment groups. However, in dogs with advanced disease (Stages II, III) there was a statistical difference between surgery alone versus surgery plus C. parvum (p = 0.01). Dogs with Stage I disease (tumor less than 2 cm diameter) had a statistically improved survival (p = 0.02) regardless of the therapy given. These results suggest that C. parvum, when combined with surgery, may have antitumor activity in the canine melanoma model.

Systemic Administration of an Attenuated, Tumor-Targeting Salmonella typhimurium to Dogs with Spontaneous Neoplasia: Phase I Evaluation

Purpose: Genetically modified bacteria are a potentially powerful anticancer therapy due to their tumor targeting capacity, inherent antitumor activity, and ability to serve as efficient vectors for gene delivery. This study sought to characterize the acute and short-term toxicities and tumor colonization rates of a genetically modified Salmonella typhimurium (VNP20009) in dogs with spontaneous tumors, in the context of a phase I dose escalation trial. Experimental Design: Forty-one pet dogs with a variety of malignant tumors received weekly or biweekly i.v. infusions of VNP20009, at doses ranging from 1.5 × 105 to 1 × 108 cfu/kg. Vital signs and clinicopathologic variables were monitored regularly. Incisional biopsies were obtained before and 1 week following the first infusion for histopathology and bacterial culture. Results: The nominal maximum tolerated dose was 3 × 107 cfu/kg, with refractory fever and vomiting being the dose-limiting toxicities. One treatment-related acute death occurred. Bacteria were cultured from tumor tissue in 42% of cases. Thirty-five patients were evaluable for antitumor response. Major antitumor responses were seen in 15% (4 complete response and 2 partial response), and disease stabilization for at least 6 weeks in 10%. Conclusions: Administration of VNP20009 at doses with acceptable toxicity results in detectable bacterial colonization of tumor tissue and significant antitumor activity in tumor-bearing dogs.

Preclinical trial of doxorubicin entrapped in sterically stabilized liposomes in dogs with spontaneously arising malignant tumors

AbstractPurpose: To prospectively evaluate the short-term toxicoses associated with pegylated-liposomal doxorubicin (Doxil) administered to dogs with measurable tumors of various histologic types and sites. Preliminary information regarding efficacy was also generated. Methods: A group of 51 dogs with histologically confirmed malignancies received a total of 103 Doxil treatments given i.v. every 3 weeks at dosages ranging from 0.75 to 1.1 mg/kg in the context of a phase I dose-escalation trial. Acute and short-term toxicities as well as tumor response and duration of response were characterized. Results: The maximally tolerated dose in tumor-bearing dogs was established as 1.0 mg/kg i.v. every 3 weeks. The dose-limiting toxicity was a cutaneous toxicity clinically resembling palmar-plantar erythrodysesthesia (PPES). An overall response rate of 25.5% was observed with five complete responders and eight partial responders. Conclusions: Doxil appeared to be well tolerated at dosages similar to those tolerated for free doxorubicin in tumor-bearing dogs. PPES was the dose-limiting toxicity encountered, rather than myelosuppresion as is the case with free doxorubicin in dogs. Doxil as a single agent may have a broad spectrum of activity and deserves further evaluation.

Cisplatin and Doxorubicin Combination Chemotherapy for the Treatment of Canine Osteosarcoma: A Pilot Study

Sixteen dogs with histologically confirmed appendicular osteosarcoma were treated by amputation followed by cisplatin and doxorubicin chemotherapy. All dogs began chemotherapy within 24 hours of surgery. Cisplatin was administered at 50 mg/m2 intravenously (IV) concurrent with saline-induced diuresis. Doxorubicin was administered 24 hours later at 15 mg/m2 as a slow IV bolus. This protocol was given on a 21-day cycle for 4 cycles. No dose delays were required, but dose reduction of doxorubicin was required in 2 dogs because of neutropenia. Thoracic radiography was performed every 2 months after completion of therapy to monitor for metastatic disease. Two dogs were still alive and free from disease at the time of last contact (24 and 75 months, respectively). Postmortem examinations were performed on 13 of the 14 dogs that died. Eight of these dogs were euthanized because of metastatic osteosarcoma. Of the remaining 5 dogs, euthanasia was performed because of complications of idiopathic megaesophagus (n = 1), arthritis (n = 2), and hemangiosarcoma (n = 2). The median disease-free interval and survival times were 15.7 and 18 months, respectively. When compared to a historical group of 36 dogs with appendicular osteosarcoma treated with surgery and 4 doses of cisplatin. both disease-free interval and overall survival were significantly longer in the study population (P < .015 and P < .007, respectively).

C-Met tyrosine kinase receptor expression and function in human and canine osteosarcoma cells

To further characterize the role of hepatocyte growth factor-scatter factor (HGF-SF) and its receptor (c-Met) in osteosarcoma (OS), human OS cell lines with low (SAOS-2) and high (SAOS-LM2) metastatic potential, and cell lines derived from spontaneous canine OS were studied. All cell lines were evaluated for c-Met and HGF-SF expression and receptor activation using Northern, RT-PCR, and Western blot analyses, respectively. Functional activity of receptor-ligand interaction was measured using c-Met phosphorylation status, proliferation assays (anchorage-dependent and -independent), Matrigel invasion, modulation of urokinase plasminogen activator (uPA) expression, and cell dispersion (scattering). All cell lines exhibited steady-state mRNA expression of c-Met. The canine OS cell lines also expressed HGF-SF mRNA as determined by RT-PCR analysis. Western analysis showed c-Met protein expression and HGF-stimulated (human) or constitutive (canine) receptor autophosphorylation. Treatment with recombinant human HGF resulted in enhanced proliferation in 3 of 5 OS cell lines and enhanced colony formation in 2 of 5 OS cell lines. Matrigel invasion was significantly enhanced in 3 of the cell lines and uPA levels were significantly increased in the SAOS-2 cells following HGF treatment. Scattering was enhanced in both the SAOS-2 and SAOS-LM2 cells. These data support the involvement of c-Met and HGF-SF in the growth and progression of human and canine OS, and may offer new targets for the development of therapeutic strategies for OS.

Obesity in the Dog: Role of the Adrenal Steroid Dehydroepiandrosterone (DHEA)

It is estimated that 25-50% of pet dogs are overweight. In rodents and humans the adrenal steroid dehydroepiandrosterone (DHEA) has been associated with loss of body weight and reduction in total body fat content. We have shown that administration of DHEA results in weight loss in spontaneously obese dogs. DHEA also lowered serum cholesterol, in particular, the low density lipoprotein component of the plasma cholesterol. We hypothesize that treatment of obese dogs with DHEA in combination with a low energy, high fiber diet will result in greater weight loss when compared with dogs on the same diet, without DHEA. In the current study, spontaneously obese dogs were fed a uniform low energy, high fiber diet and then randomized to receive DHEA or placebo. Preliminary results show that the mean total body weight lost for dogs receiving DHEA was 3.59 kg compared with 2.38 kg for dogs receiving placebo (P greater than 0.05). The percent excess body weight (above ideal body weight) lost for the DHEA group was 65.7 versus 31.4 for the placebo group (P less than 0.02). The percent excess body weight lost per month on the study for the DHEA group was 15 versus 8.2 for the placebo group (P = 0.069). Although these results are preliminary, they indicate that DHEA combined with a low energy, high fiber diet enhances the loss of excess body weight compared with diet modification alone.

A preliminary study on the evaluation of asparaginase. Polyethylene glycol conjugate against canine malignant lymphoma

Thirty‐seven dogs with malignant lymphoma were treated with either polyethylene glycol conjugated (PEG) asparaginase alone (10–30 IU/kg intraperitoneally [IP] weekly—20 dogs) or PEG‐asparaginase combined with one cycle of chemotherapy (vincristine, cyclophosphamide, methotrexate, and prednisone), followed by maintenance PEG‐asparaginase (30 IU/kg, IP weekly—17 dogs). In the 20 dogs (eight were chemotherapy resistant) treated with PEG‐asparaginase alone, seven had a complete response (CR), seven had a partial response (PR), five had no response (NR), and one was not evaluable (NE). The duration of response (CR + PR) ranged from 14 to 102 days (median, 48 days). In the eight chemotherapy‐resistant dogs (seven were previously resistant to L‐asparaginase) four had responses (one CR and three PR). In the 17 dogs treated with combined PEG‐aspnraginase and chemotherapy, 13 had a CR, two had a PR, and two had NR. None of the dogs had had prior chemotherapy, and the duration of response (CR + PR) ranged from 7 to 840+ days, with a median of 126+ days. Four dogs are still on maintenance PEG‐asparaginase at 16+, 21+, 26+, and 28+ months. Toxicity consisted of death due to massive tumor breakdown (two dogs), disseminated intravascular coagulation (DIC—one dog), hypersensitivity reaction (one dog), vomiting (three dogs) and soft stools (three dogs). Four normal dogs were given very high doses of PEG‐asparaginase (200 IU/kg and 1200 IU/kg) once weekly for two treatments without any significant toxicity. These results indicate that PEG‐asparaginase has antitumor activity in dogs with spontaneously occurring malignant lymphoma.

In vitro and in vivo canine mononuclear cell production of tumor necrosis factor induced by muramyl peptides and lipopolysaccharide

Tumor necrosis factor (TNF) is a potent mediator of tumor cell killing by activated monocytes and macrophages. We measured TNF activity induced by muramyl peptides and lipopolysaccharide (LPS) in normal dogs. Canine adherent mononuclear cells were isolated and cultured in either medium alone or medium containing muramyl dipeptide (MDP) or MDP plus LPS. After 18 h, culture supernatants were collected and assayed for TNF activity. Sera from dogs injected with liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (L-MTP-PE) were also evaluated for TNF activity. TNF activity both in supernatants and in sera was detected in a 18 h WEHI-164 cell cytotoxicity assay and was confirmed by a monoclonal antibody directed against recombinant human TNF-alpha. Results showed a significant increase in TNF activity from mononuclear cells exposed to MDP or MDP plus LPS of 20% and 88%, respectively; P < 0.0005. Serum TNF activity rapidly increased within 2-3 h post L-MTP-PE injection and subsequently declined to pretreatment level at 4 h post administration. This study demonstrates that MDP +/- LPS can stimulate canine adherent mononuclear cells to release TNF and intravenous injection of L-MTP-PE is capable of rendering the in vivo release of TNF in normal dogs.