Development of an allogeneic whole-cell tumor vaccine expressing xenogeneic gp100 and its implementation in a phase II clinical trial in canine patients with malignant melanoma.

A xenogeneic melanoma-antigen-enhanced allogeneic tumor cell vaccine (ATCV) is an appealing strategy for anti-cancer immunotherapy due to its relative ease of production, and the theoretical possibility that presentation of a multiplex of antigens along with a xenogeneic antigen would result in cross-reaction between the xenogeneic homologs and self-molecules, breaking tolerance and ultimately resulting in a clinically relevant immune response. In this study, we evaluated the efficacy of such a strategy using a xenogeneic melanoma differentiation antigen, human glycoprotein 100 (hgp100) in the context of a phase II clinical trial utilizing spontaneously arising melanoma in pet dogs. Our results demonstrate that the approach was well tolerated and resulted in an overall response rate (complete and partial response) of 17% and a tumor control rate (complete and partial response and stable disease of >6 weeks duration) of 35%. Dogs that had evidence of tumor control had significantly longer survival times than dogs that did not experience control. Delayed type hypersensitivity (DTH) to 17CM98 canine melanoma cells used in the whole cell vaccine was enhanced by ATCV and correlated with clinical response. In vitro cytotoxicity was enhanced by ATCV, but did not correlate with clinical response. Additionally, anti-hgp100 antibodies were elicited in response to ATCV in the majority of patients tested; however, this also did not correlate with clinical response. This approach, along with further elucidation of the mechanisms of tumor protection after xenogeneic immunization, may allow the development of more rational vaccines. This trial also further demonstrates the utility of spontaneous tumors in companion animals as a valid translational model for the evaluation of novel vaccine therapies.

In vitro effects of 2-methoxyestradiol on canine tumour cells.

The in vitro antiproliferative, apoptotic and cell-cycle effects of 2-methoxyestradiol (2ME(2)), an endogenous oestrogen metabolite, were investigated using a variety of canine tumour cell lines. The cells were cultured under standard conditions and incubated with varying concentrations of 2ME(2). Inhibition of tumour cell proliferation was evaluated using a tetrazolium-based colorimetric assay. DNA content analysis was performed using propidium iodide staining and flow cytometry. Cytologic analysis with Leukostat staining solution and Hoechst 33342 staining and Annexin V-fluorescein isothiocyanate (FITC) fluorescence were used to quantify cell-cycle distribution and apoptosis induction. Tumour cell proliferation was inhibited by 50% at concentrations of 2ME(2) ranging from 0.88 to 7.67 microM, depending on the cell line tested. Profound G(2)/M phase arrest, an increase in binucleate cells and induction of apoptosis were observed in all cell lines tested, in a dose-dependent manner. Based on these results, this compound has potential as an agent for the treatment of canine cancer and warrants further investigation. The canine lymphoma cell line, 1771, was inhibited at concentrations that may be achievable in vivo.

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.

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).

Use of cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum (II), a liposomal cisplatin analogue, in cats with oral squamous cell carcinoma.

OBJECTIVE: To determine clinical response and toxic effects of cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum (II) (L-NDDP) administered i.v. at escalating doses to cats with oral squamous cell carcinoma (SCC). ANIMALS: 18 cats with oral SCC. PROCEDURE: Cats that failed to respond to conventional treatment or had nonresectable tumors were included. Data included a CBC, serum biochemical analyses, urinalysis, cytologic examination of a fine-needle aspirate of enlarged lymph nodes, and thoracic and oral radiographs for clinical staging. A starting dose (75 to 100 mg/m2 of L-NDDP) was administered i.v.. At 21-day intervals, subsequent doses increased by the rate of 5 or 10 mg/m2. Response was evaluated every 21 days by tumor measurement and thoracic radiography. Quality of life was assessed by owners, using a performance status questionnaire. RESULTS: On average, cats received 2 treatments. Toxicoses included an intermittent, acute anaphylactoid-parasympathomimetic reaction, lethargy or sedation (< or = 24 hours), inappetence or signs of depression (< or = 72 hours), mild to moderate increase in hepatic enzyme activity, and melena. Pulmonary, renal, or hematopoietic abnormalities were not evident. Performance status surveys indicated normal behavior and grooming or decreased activity and self-care (19/20 assessments), ate well with or without assistance (15/20), and did not lose weight (15/20). Median survival time was 59.8 days (mean, 54.1 days). CONCLUSIONS AND CLINICAL RELEVANCE: L-NDDP was ineffective for treatment of cats with oral SCC. None of the cats had a complete or partial remission. Acute toxicoses and poor therapeutic response limit therapeutic usefulness of L-NDDP in cats, unless dosage, frequency, and administration procedures can be improved.

The immunotherapeutic potential of activated canine alveolar macrophages and antitumor monoclonal antibodies in metastatic canine melanoma.

A variety of immune cell activators can enhance the cytotoxic effects of monocytes/macrophages including interferon-gamma (IFN-gamma) and muramyl peptides, which are under investigation for cancer therapy in humans and dogs. Pulmonary alveolar macrophages (PAMs) in particular, are strategically located within the lung and provide a potential defense against cancer cells metastatic to the lung. For this reason, we examined the in vitro cytotoxic potential of fresh and IFN-gamma-activated PAMs from normal dogs targeted to canine malignant melanoma cells with antiganglioside monoclonal antibodies (mAbs). Antiganglioside mAbs 14.G2a (anti-GD2) and R24 (anti-GD3), both in clinical trials for human neuroectodermal tumors including melanoma, significantly enhanced the cytotoxicity of canine melanoma mediated by canine PAMs. Further, the cytotoxicity mediated by recombinant canine IFN-gamma-activated canine PAMs, in combination with anti-GD2 ganglioside mAb 14.G2a, enhanced melanoma cytotoxicity above that seen with mAb 14.G2a alone. This documentation of antibody-dependent cellular cytotoxicity mediated by activated PAMs suggests that activation and targeting of resident pulmonary immune cells be pursued as a means to control pulmonary metastases.

Preclinical development of human granulocyte-macrophage colony-stimulating factor-transfected melanoma cell vaccine using established canine cell lines and normal dogs.

Tumor vaccines and gene therapy have received significant attention as means of increasing cellular and humoral immune responses to cancer. We conducted a pilot study of seven research dogs to determine whether intradermal injection of canine tumor cells transfected via the Accell particle-mediated gene transfer device with the cDNA for human granulocyte-macrophage colony-stimulating factor (hGM-CSF) would generate biologically relevant levels of protein and result in demonstrable histological changes at sites of vaccination. Tumor cell vaccines of 10(7) irradiated canine melanoma cells were nontoxic, safe, and well tolerated. No significant alterations in blood chemistry values or hematological profiles were detected. A histological review of control vaccine sites revealed inflammatory responses predominated by eosinophils, whereas vaccine sites with hGM-CSF-transfected tumor cells had an influx of neutrophils and macrophages. Enzyme-linked immunosorbent assays of skin biopsies from vaccine sites had local hGM-CSF production (8.68-16.82 ng/site of injection) at 24 hours after injection and detectable levels (0.014-0.081 ng/site) for < or =2 weeks following vaccination. Flow cytometric analysis of hGM-CSF-transfected cells demonstrated < or =25% transfection efficiency, and hGM-CSF levels obtained during time-course assays demonstrated biologically relevant levels for both irradiated and nonirradiated samples. These data demonstrate the in vivo biological activity of irradiated hGM-CSF-transfected canine tumor cells and help provide evidence for a valid translational research model of spontaneous tumors.

Evaluation of toxicosis of liposome-encapsulated cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum (II) in clinically normal cats.

OBJECTIVE: To determine adverse effects of single and multiple doses of liposome-encapsulated cis-bis-neodecanoato-trans-R,R-1,2-diaminocyclohexane platinum (II) (L-NDDP) administered IV to healthy adult cats. ANIMALS: 10 healthy adult cats. PROCEDURE: 8 cats were given a single dose of L-NDDP (at rates of 75, 100, 150, or 200 mg/m2), and 2 cats were given liposomal lipid (1,500 mg/m2). Six of the 10 cats were given doses of L-NDDP at the maximum tolerated dosage (100 mg/m2) or a lower dosage (75 mg of L-NDDP/m2) at 21-day intervals, for a total of 4 treatments. Hematologic and serum biochemical analyses, urinalyses, and physical examinations were used to monitor effects of L-NDDP. RESULTS: All cats had transient pyrexia, lethargy, vomiting (1 to 3 times/24 h), inappetence, and an acute species-specific infusion reaction that was prevented by administration of atropine-diphenhydramine. Dose-limiting toxicosis was evident as a 10-day course of lethargy, intermittent vomiting, and diarrhea. In cats given multiple doses, dose-related thrombocytopenia, cumulative myelosuppression, transient increased hepatic transaminase activity, and mild to moderate hepatic hydropic degeneration and proximal renal tubular lipidosis in excess of lipidosis expected for this species were detected. Bone marrow hypoplasia was detected in some cats that received higher doses (cumulative dosages of 300 or 400 mg of L-NDDP/m2). CONCLUSION: Cats can safely be given L-NDDP at potentially therapeutic dosages without inducing renal or pulmonary toxicoses. CLINICAL RELEVANCE: Because L-NDDP has better tumoricidal activity than cisplatin (in vivo and in vitro) and is not cross resistant, it may be similarly or more efficacious than cisplatin in humans and dogs.

In vitro and in vivo enhancement of canine pulmonary alveolar macrophage cytotoxic activity against canine osteosarcoma cells.

The combination of chemotherapy with immunotherapy may offer an advantage over either therapy alone and provide a greater potential for total tumor eradication. Monocyte/macrophage-mediated tumor cell killing is a major mechanism of the host's defense against primary and/or metastatic neoplasia. We evaluated the tumoricidal activity against canine osteosarcoma cells of canine pulmonary alveolar macrophages (PAM) exposed in vitro to two recombinant canine (rc) cytokines (rcTNF alpha and rcIFN gamma). We also evaluated the in vivo tumoricidal activity of PAM from dogs treated with the macrophage activator, liposome-encapsulated muramyl tripeptide-phosphatidyl-ethanolamine (L-MTP-PE) alone or in combination with doxorubicin (DOX). This study demonstrated that rcTNF alpha and rcIFN gamma significantly enhance in vitro canine PAM cytotoxicity against canine osteosarcoma cells, and that PAM from dogs treated with DOX + L-MTP-PE have enhanced cytotoxic activity against osteosarcoma cells when compared to dogs treated with DOX or L-MTP-PE alone. These findings support the rationale for combining a chemotherapy agent with an immunotherapy agent for the treatment of metastatic disease, and suggest a role for TNF alpha and IFN gamma as agents for stimulating the antitumor activity of macrophages.

Adjuvant therapy for melanoma in dogs: results of randomized clinical trials using surgery, liposome-encapsulated muramyl tripeptide, and granulocyte macrophage colony-stimulating factor.

Spontaneous canine oral melanoma (COM) is a highly metastatic cancer, resistant to chemotherapy, and can serve as a model for cancer immunotherapy. Liposome-encapsulated muramyl tripeptide-phosphatidylethanolamine (L-MTP-PE) can activate the tumoricidal activity of the monocyte-macrophage system following i.v. injection. The objective of these studies was to evaluate the therapeutic effectiveness of L-MTP-PE administered alone and combined with recombinant canine granulocyte macrophage colony-stimulating factor (rcGM-CSF) in dogs undergoing surgery for oral melanoma. Ninety-eight dogs with histologically confirmed, clinically staged, oral melanoma were entered into two randomized, double-blind, surgical adjuvant trials. In trial 1, 50 dogs were stratified based on clinical stage and randomized to once a week L-MTP-PE or lipid equivalent (control). When all of the clinical stages were combined, no difference in disease-free survival or in survival time (ST) were detected. However, within stage I, dogs receiving L-MTP-PE had a significant increase in ST compared with control, with 80% of the dogs treated with L-MTP-PE still alive at >2 years. Within each stage II and stage III, there was no difference detected between the treatment groups. In trial 2, 48 dogs were stratified on the basis of clinical stage and extent of surgery (simple resection or radical excision), treated with L-MTP-PE two times a week, and randomized to rcGM-CSF or saline (placebo) given s.c. daily for 9 weeks. Within each stage and when all of the stages were combined, there was no difference between the treatment groups. In both studies, stage I COM is associated with a better prognosis. No effect on survival was observed with regard to tumor location in the oral cavity, sex, type/extent of surgery, or age. In a subset of dogs tested, pulmonary alveolar macrophage cytotoxicity was enhanced with combined rcGM-CSF and L-MTP-PE but not in dogs treated with L-MTP-PE alone. The present study indicates that after surgery, L-MTP-PE administered alone or combined with rcGM-CSF showed no significant antitumor activity in treating advanced stage COM. In early stage COM, L-MTP-PE was shown to result in a prolongation of ST. Furthermore, this study provides additional rationale for the use of the dog model for human malignant melanoma.

An accelerated technique for irradiation of malignant canine nasal and paranasal sinus tumors.

Tumor and normal tissue response was assessed in 21 dogs with malignant nasal tumors given 42 Gy cobalt radiation in 9 or 10 fractions over 11 to 13 days. Local tumor/clinical relapse recurred in 68% of dogs, with a median relapse free interval (RFI) of 270 days. Median survival was 428 days. One year survival for all dogs was 60%. RFI and survival times are better than, or similar to, previous reports of dogs treated with radiotherapy only. Acute radiation effects were severe in one dog. Late effects were severe in six of 15 dogs (40%) with durable tumor control. Late effects included bilateral blindness (3), osteoradionecrosis (3), and seizures (1). These six dogs had a median survival of 705 days. Loss of vision occurred in at least one eye in nine dogs (47%). Tumor staging based on CT findings was predictive for survival duration. Tumor histology was not predictive of outcome. Labrador Retrievers were significantly over-represented. Despite comparable or improved tumor control and survival times provided by this accelerated protocol, relative to other radiotherapy reports, local failure remains the major cause of death, and late radiation effects can be severe in dogs with durable tumor control.

Development of human granulocyte-macrophage colony-stimulating factor-transfected tumor cell vaccines for the treatment of spontaneous canine cancer.

Cytokine gene-engineered tumor vaccines are currently an area of intense investigation in both basic research and clinical medicine. Our efforts to utilize tumor vaccines in an immunotherapeutic manner involve canines with spontaneous tumors. We hypothesized that canine tumor cells, transfected with human granulocyte-macrophage colony-stimulating factor (hGM-CSF) cDNA in a plasmid vector, would prove nontoxic following intradermal administration, generate biologically relevant levels of protein, effect local histological changes at the sites of vaccination, and create a systemic antitumor response. Sixteen tumor-bearing dogs were admitted to a study of ex vivo gene therapy. Tumor tissue was surgically removed, enzymatically and mechanically dissociated, irradiated, transfected, and intradermally injected back into the patients. The dogs were vaccinated with primary autologous tumor cells transfected with hGM-CSF or a reporter control gene. hGM-CSF protein was detected (0.07 to 14.15 ng/vaccination site) at 24 hr postinjection and dramatic histological changes were observed, characterized by neutrophil and macrophage infiltration at the sites of injection of hGM-CSF-transfected tumor cells. This was in stark contrast to the lesser neutrophilic and eosinophilic infiltrates found at control vaccination sites. Objective evidence of an antitumor response was observed in three animals. These data, in a large animal translational model of spontaneous tumors, demonstrate in vivo biological activity of hGM-CSF-transfected autologous tumor cell vaccines.

Peripheral neuroblastoma in a dog.

Primitive neuroectodermal tumors are composed of primitive neuroepithelial cells and include tumors of the central and peripheral nervous system. Neuroblastoma, medulloblastoma and retinoblastoma are examples of these rare malignant tumors that usually occur in young patients. This report describes a peripheral neuroblastoma in a 2 year old Boxer that presented with signs of renal disease and a palpable abdominal mass. The purpose of this paper is to describe the clinical presentation, imaging and immunohistological studies of this abdominal tumor in a young dog and to review the literature.

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

PURPOSE: 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.

Assessment of potential doubling time (Tpot), argyrophilic nucleolar organizer regions (AgNOR), and proliferating cell nuclear antigen (PCNA) as predictors of therapy response in canine non-Hodgkin's lymphoma.

The predictive potential of several proliferation indices for therapeutic outcome was investigated in 55 dogs with spontaneously occurring non-Hodgkin's lymphoma (NHL). Indices included potential doubling time (Tpot), argyrophilic nucleolar organizer region (AgNOR) frequency, and proliferating cell nuclear antigen labeling index (PCNA-LI). All tumors were of intermediate- or high-grade histology as assessed by the Working Formulation, and all dogs presented with disease of advanced clinical stage. All tumors were treated with an identical chemotherapeutic protocol. Tpot determination by a bromodeoxyuridine (BrdU) delayed-biopsy technique was readily applied in the dog. AgNOR frequency and PCNA-LI were easily obtained from archival, formalin-fixed, paraffin-embedded canine tissues. When accounting for all other prognostic variables by employing multivariate analysis, Tpot (p=0.017), and AgNOR frequency (p=0.021), but not PCNA-LI, were predictive of first remission duration. AgNOR frequency (p=0.033) was also predictive of survival time, and the predictive potential of Tpot approached significance (p=0.076). We conclude that Tpot and AgNOR frequency can be used as predictors of outcome in dogs with NHL, and spontaneous NHL in the dog may have significant potential as a model for further characterization of the association between tumor cell kinetics and chemoresponsiveness.

In vivo particle-mediated cytokine gene transfer into canine oral mucosa and epidermis.

Cytokines can stimulate immune effector cells present within the oral mucosa and epidermis to respond to vaccination or to combat cancer. However, intravenous cytokine delivery is often inefficient and frequently accompanied by systemic toxicity. The goal of this study was to evaluate dogs as a large animal model for gene therapy of cancer because they develop spontaneous oral and epidermal tumors. In this report, we demonstrate that particle-mediated gene transfer of beta-galactosidase, luciferase, interleukin-2, interleukin-6, and granulocyte-macrophage colony stimulating factor (GM-CSF) complementary DNA (cDNA) into the oral mucosa and epidermis of healthy dogs resulted in effective, localized, transgenic protein expression. Additionally, the epidermal sites transfected with GM-CSF developed a profound inflammatory reaction characterized by neutrophilic infiltration. Clinical pathology analyses were unremarkable. These results demonstrate that in vivo particle-mediated gene transfer of canine oral mucosa and epidermis with cytokine cDNA can result in production of biologically active transgenic cytokines with minimal toxicity. These findings have applications to cancer immunotherapy using a gene gun approach.

Amputation and carboplatin for treatment of dogs with osteosarcoma: 48 cases (1991 to 1993).

Forty-eight dogs with histologically confirmed appendicular osteosarcoma (OSA) entered a prospective clinical trial evaluating treatment with amputation and up to 4 doses of carboplatin given every 21 days. The median disease-free interval (DFI) was 257 days, with 31.2% of the dogs disease-free at 1 year. The median survival time was 321 days, with 35.4% of the dogs alive at 1 year. Dogs with proximal humeral OSA had shorter DFI (P = .016) and survival (P = .037) times than dogs with OSA at other locations. Dogs with lower body weights ( < 40 kg) had longer DFI (P = .0056) and survival (P = .007) times than larger dogs. Survival times for dogs that received carboplatin were statistically longer than those previously reported for amputation alone (P < .001). DFI and survival times are similar to those previously reported for 2 to 4 doses of cisplatin. Carboplatin appears to be a well-tolerated chemotherapeutic drug that can be given safely every 21 days at a dose of 300 mg/m2. Neutropenia was the dose-limiting toxicity in this study.

Canine osteosarcoma: amputation and chemoimmunotherapy.

Canine osteosarcoma is a highly metastatic cancer commonly seen in large breed dogs. At the time of diagnosis, approximately 90% to 95% of the dogs have established micrometastases. Dogs undergoing amputation alone have a median survival time of 3 to 4 months. Amputation followed by cisplatin chemotherapy increases median survival times to 9 to 11 months. When dogs are treated with amputation and cisplatin, followed by immunotherapy (with liposome-encapsulated muramyl tripeptide phosphatidylethanolamine), median survival times increase to 14.4 months, the longest reported median survival time for dogs with osteosarcoma treated by amputation and any form of adjuvant therapy.

Adjuvant therapy for osteosarcoma in dogs: results of randomized clinical trials using combined liposome-encapsulated muramyl tripeptide and cisplatin.

Two randomized, double-blind clinical trials in dogs with spontaneous appendicular osteosarcoma treated with combination chemoimmunotherapy are reported. In both trials, dogs without overt metastasis underwent complete amputation of the affected limb. In trial 1, 40 dogs were treated with cisplatin chemotherapy [(CDDP), 70 mg/m2 i.v. every 28 days x 4]. Following CDDP, dogs without evidence of overt metastasis (n = 25) were randomized to receive liposome-encapsulated muramyl tripeptide phosphatidylethanolamine ](L-MTP-PE), 2 mg/m2 i.v.) or placebo liposomes (lipid equivalent) twice weekly for 8 weeks. Of 14 dogs in the placebo group, 13 (93%) died of metastasis; the median survival time was 9.8 months. Of 11 dogs in the L-MTP-PE group, 8 (73%) developed metastasis; the median survival time was 14.4 months, which was significantly longer than that of the placebo group (P < 0.01). In trial 2, 64 dogs received CDDP (70 mg/m2 i.v. every 21 days x 4) and were randomized to concurrently receive L-MTP-PE (2 mg/m2 i.v.) twice or once weekly, or placebo liposomes once weekly for 8 weeks. Median survival times were 10.3, 10.5, and 7.6 months, respectively. There were no significant differences among the three treatment groups in trial 2. Survival times for dogs receiving L-MTP-PE in trial 1 were significantly longer than those for dogs in trial 2 that received four doses of CDDP concurrently with twice weekly L-MTP-PE (P < 0. 04). The results of the first trial confirm our previous observation that L-MTP-PE has antimetastatic activity in dogs with osteosarcoma when given following amputation. The results of the second trial demonstrate that there is no survival advantage of administering L-MTP-PE concurrently with CDDP.