Administration of intratumoral GD2-directed interleukin-2 immunocytokine and local radiation therapy to activate immune rejection of spontaneous canine melanoma.

Canine malignant melanoma provides a clinically relevant, large animal parallel patient population to study the GD2-reactive hu14.18-IL-2 immunocytokine as it is similar to human melanoma and expresses GD2. The objectives of this study were to evaluate safety, radiation fractionation, and identify informative biomarkers of an in-situ tumor vaccine involving local radiation therapy plus intratumoral-immunocytokine in melanoma tumor-bearing dogs. Twelve dogs (six dogs/arm) with locally advanced or metastatic melanoma were randomized to receive a single 8 Gy fraction (arm A) or three 8 Gy fractions over 1 week (arm B) to the primary site and regional lymph nodes (when clinically involved) with the single or last fraction 5 days before intratumoral-immunocytokine at 12 mg/m 2 on 3 consecutive days. Serial tumor biopsies were obtained. All 12 dogs completed protocol treatment, and none experienced significant or unexpected adverse events. Evidence of antitumor activity includes one dog with a complete response at day 60, one dog with a partial response at day 60, and four dogs with mixed responses. Histology of serial biopsies shows a variably timed increase in intratumoral lymphocytic inflammation in some dogs. Canine NanoString analyses of serial biopsies identified changes in gene signatures of innate and adaptive cell types versus baseline. There were no significant differences in NanoString results between arm A and arm B. We conclude that intratumoral-immunocytokine in combination with local radiation therapy in canine melanoma is well tolerated and has antitumor activity with the potential to inform clinical development in melanoma patients.

Design of a randomized, placebo-controlled study evaluating efficacy and safety of a cancer preventative vaccine in dogs.

Preventative anti-cancer vaccination strategies have long been hampered by the challenge of targeting the diverse array of potential tumor antigens, with successes to date limited to cancers with viral etiologies. Identification and vaccination against frameshift neoantigens conserved across multiple species and tumor histologies is a potential cancer preventative strategy currently being investigated. Companion dogs spontaneously develop cancers at a similar incidence to those in people and are a complementary comparative patient population for the development of novel anti-cancer therapeutics. In addition to an intact immune system with tumors that arise in an autochthonous tumor microenvironment, dogs also have a shorter lifespan and temporally compressed tumor natural history as compared to humans, which allows for more rapid evaluation of safety, immunogenicity, and efficacy of cancer vaccination strategies. Here we describe the study protocol for the Vaccination Against Canine Cancer Study (VACCS), the largest interventional cancer clinical trial conducted in companion dogs to date. In addition to safety and immunogenicity, the primary endpoint of VACCS is the cumulative incidence (CI) of dogs developing malignant neoplasia of any type at the end of the study period. Secondary endpoints include changes in incidence of specific tumor types, survival times following neoplasia diagnosis, and all-cause mortality.

Safety and feasibility of an in situ vaccination and immunomodulatory targeted radionuclide combination immuno-radiotherapy approach in a comparative (companion dog) setting.

RATIONALE: Murine syngeneic tumor models have revealed efficacious systemic antitumor responses following primary tumor in situ vaccination combined with targeted radionuclide therapy to secondary or metastatic tumors. Here we present studies on the safety and feasibility of this approach in a relevant translational companion dog model (n = 17 dogs) with advanced cancer. METHODS: The three component of the combination immuno-radiotherapy approach were employed either separately or in combination in companion dogs with advanced stage cancer. In situ vaccination was achieved through the administration of hypofractionated external beam radiotherapy and intratumoral hu14.18-IL2 fusion immunocytokine injections to the index tumor. In situ vaccination was subsequently combined with targeted radionuclide therapy using a theranostic pairing of IV 86Y-NM600 (for PET imaging and subject-specific dosimetry) and IV 90Y-NM600 (therapeutic radionuclide) prescribed to deliver an immunomodulatory 2 Gy dose to all metastatic sites in companion dogs with metastatic melanoma or osteosarcoma. In a subset of dogs, immunologic parameters preliminarily assessed. RESULTS: The components of the immuno-radiotherapy combination were well tolerated either alone or in combination, resulting in only transient low grade (1 or 2) adverse events with no dose-limiting events observed. In subject-specific dosimetry analyses, we observed 86Y-NM600 tumor:bone marrow absorbed-dose differential uptakes ≥2 in 4 of 5 dogs receiving the combination, which allowed subsequent safe delivery of at least 2 Gy 90Y-NM600 TRT to tumors. NanoString gene expression profiling and immunohistochemistry from pre- and post-treatment biopsy specimens provide evidence of tumor microenvironment immunomodulation by 90Y-NM600 TRT. CONCLUSIONS: The combination of external beam radiotherapy, intratumoral immunocytokine, and targeted radionuclide immuno-radiotherapy known to have activity against syngeneic melanoma in murine models is feasible and well tolerated in companion dogs with advanced stage, spontaneously arising melanoma or osteosarcoma and has immunomodulatory potential. Further studies evaluating the dose-dependent immunomodulatory effects of this immuno-radiotherapy combination are currently ongoing.

Pilot study of safety and feasibility of DNA microseeding for treatment of spontaneous canine melanoma.

Spontaneous canine malignant melanoma provides an excellent pre-clinical model to study DNA vaccines for melanoma immunotherapy. A USDA-approved xenogeneic human tyrosinase (huTYR) plasmid DNA vaccine delivered intramuscularly induces detectable immune responses and has clinical activity in some dogs with melanoma. The objective of this pilot study was to evaluate the feasibility, safety and immunogenicity of huTYR plasmid DNA administered to the skin via microseeding in dogs with spontaneous melanoma. DNA microseeding utilizes a modified tattooing device as an alternate and potentially more potent delivery method for DNA immunization. DNA was delivered to shaved inner thigh skin of six companion dogs with melanoma approximately every 14 days for a planned total of four vaccination time points. An anti-huTYR ELISA was used to test pre- and post-treatment sera. Biopsies of treated skin were obtained for detection of huTYR transgene expression. DNA microseeding was well tolerated with no significant toxicity detected beyond local site irritation, and there were no signs of autoimmunity. huTYR-expressing cells were observed in biopsies of huTYR DNA microseeding sites. Increased humoral anti-huTYR antibodies were seen in two of five evaluable dogs following microseeding compared to baseline. DNA microseeding is well tolerated in companion dogs with melanoma. Further investigation is needed to determine if combining DNA microseeding with other immunotherapy regimens potentiates this delivery platform for cancer immunotherapy.

GS-9219/VDC-1101--a prodrug of the acyclic nucleotide PMEG has antitumor activity in spontaneous canine multiple myeloma.

BACKGROUND: Multiple myeloma (MM) is an important human and canine cancer for which novel therapies remain necessary. VDC-1101 (formerly GS-9219), a novel double prodrug of the anti-proliferative nucleotide analog 9-(2-phosphonylmethoxyethyl) guanine (PMEG), possesses potent cytotoxic activity in vitro in human lymphoblasts and leukemia cell lines and in vivo in spontaneous canine lymphoma. Given the similarity in lineage between lymphoma and MM, we hypothesized that VDC-1101 would be active against MM. RESULTS: We evaluated the in vitro antiproliferative effects of VDC-1101 against 3 human MM cell lines, and we performed a phase-II clinical trial in 14 dogs with spontaneous MM. Each dog was treated with a maximum of 6 doses of VDC-1101 monotherapy over 10-15 weeks. Dose-dependent antiproliferative activity was observed in all evaluated cell lines. Major antitumor responses (reduction of serum paraprotein and resolution of hypercalcemia, peripheral cytopenias and bone marrow plasmacytosis) were observed in 9 of 11 evaluable dogs for a median of 172 days, including a durable stringent complete response (>1047 days) in a dog with melphalan-refractory disease. 2 dogs were euthanized due to presumed pulmonary fibrosis; there were no other dose-limiting toxicities encountered. CONCLUSIONS: In conclusion, VDC-1101 has significant anti-tumor activity at well-tolerated doses in spontaneous canine MM.

Comparative gene expression profiling identifies common molecular signatures of NF-κB activation in canine and human diffuse large B cell lymphoma (DLBCL).

We present the first comparison of global transcriptional changes in canine and human diffuse large B-cell lymphoma (DLBCL), with particular reference to the nuclear factor-kappa B (NF-κB) pathway. Microarray data generated from canine DLBCL and normal lymph nodes were used for differential expression, co-expression and pathway analyses, and compared with analysis of microarray data from human healthy and DLBCL lymph nodes. The comparisons at gene level were performed by mapping the probesets in canine microarrays to orthologous genes in humans and vice versa. A considerable number of differentially expressed genes between canine lymphoma and healthy lymph node samples were also found differentially expressed between human DLBCL and healthy lymph node samples. Principal component analysis using a literature-derived NF-κB target gene set mapped to orthologous canine array probesets and human array probesets clearly separated the healthy and cancer samples in both datasets. The analysis demonstrated that for both human and canine DLBCL there is activation of the NF-κB/p65 canonical pathway, indicating that canine lymphoma could be used as a model to study NF-κB-targeted therapeutics for human lymphoma. To validate this, tissue arrays were generated for canine and human NHL and immunohistochemistry was employed to assess NF-κB activation status. In addition, human and canine B-cell lymphoma lines were assessed for NF-κB activity and the effects of NF-κB inhibition.

Safety and efficacy of a xenogeneic DNA vaccine encoding for human tyrosinase as adjunctive treatment for oral malignant melanoma in dogs following surgical excision of the primary tumor.

OBJECTIVE: To evaluate the safety and efficacy of a vaccine containing plasmid DNA with an insert encoding human tyrosinase (ie, huTyr vaccine) as adjunctive treatment for oral malignant melanoma (MM) in dogs. ANIMALS: 111 dogs (58 prospectively enrolled in a multicenter clinical trial and 53 historical controls) with stage II or III oral MM (modified World Health Organization staging scale, I to IV) in which locoregional disease control was achieved. PROCEDURES: 58 dogs received an initial series of 4 injections of huTyr vaccine (102 µg of DNA/injection) administered transdermally by use of a needle-free IM vaccination device. Dogs were monitored for adverse reactions. Surviving dogs received booster injections at 6-month intervals thereafter. Survival time for vaccinates was compared with that of historical control dogs via Kaplan-Meier survival analysis for the outcome of death. RESULTS: Kaplan-Meier analysis of survival time until death attributable to MM was determined to be significantly improved for dogs that received the huTyr vaccine, compared with that of historical controls. However, median survival time could not be determined for vaccinates because < 50% died of MM before the end of the observation period. No systemic reactions requiring veterinary intervention were associated with vaccination. Local reactions were primarily limited to acute wheal or hematoma formation, mild signs of pain at the injection site, and postvaccination bruising. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the safety and efficacy of the huTyr DNA vaccine in dogs as adjunctive treatment for oral MM. IMPACT FOR HUMAN MEDICINE: Response to DNA vaccination in dogs with oral MM may be useful in development of plasmid DNA vaccination protocols for human patients with similar disease.

Predictors of outcome in dogs with subcutaneous or intramuscular hemangiosarcoma.

OBJECTIVE: To identify prognostic factors in a large group of dogs with subcutaneous or intramuscular hemangiosarcoma (HSA) or both. Design-Multi-institutional retrospective cohort study. Animals-71 dogs with subcutaneous or intramuscular HSA. PROCEDURES: Medical records of affected dogs were reviewed. The following factors were evaluated for an association with outcome: dog age and sex, clinical signs, anemia, thrombocytopenia, neutrophilia, tumor stage at diagnosis, achievement of complete excision, intramuscular involvement, presence of gross disease, tumor recurrence, and treatment. RESULTS: Of the 71 cases identified, 16 (29%) had intramuscular tumor involvement. For all dogs, median time to tumor progression and overall survival time (OST) were 116 and 172 days, respectively; 25% survived to 1 year. Univariate analysis identified presence of clinical signs or metastasis at diagnosis, dog age, tumor size, use of any surgery, and presence of gross disease as predictors of time to tumor progression and OST. There was no significant difference in survival time between dogs with respect to type of HSA. Multivariate analysis confirmed that adequate local tumor control, tumor diameter ≤ 4 cm, presence of metastasis at diagnosis, and presence of gross disease were significantly associated with OST. CONCLUSIONS AND CLINICAL RELEVANCE: Subcutaneous and intramuscular HSA remains a heterogeneous group of tumors that generally carries a poor prognosis. Adequate local control of smaller tumors with no associated clinical signs or metastasis may provide the best chance of long-term survival.

Preclinical investigation of PEGylated tumor necrosis factor alpha in dogs with spontaneous tumors: phase I evaluation.

PURPOSE: Tumor necrosis factor-alpha (TNF) is a cytokine with potent antitumor activity; however, toxicity and short half-life have limited its utility. Polyethylene glycol (PEG) conjugation of biotherapeutics can decrease immunogenicity while improving bioactivity and half-life. PEGylation of TNF (PEG-TNF) significantly improved half-life and toxicity in mice, resulting in enhanced antitumor activity. This study characterized toxicity, biological effect, and antitumor activity of PEG-TNF in pet dogs with spontaneous cancer. EXPERIMENTAL DESIGN: A phase I clinical trial enrolled dogs with measurable tumors in which standard therapy had failed or been declined. Physiologic, hematologic, and biochemical parameters were evaluated and tumor biopsies obtained serially. A subset of patients underwent serial dynamic contrast-enhanced magnetic resonance imaging. RESULTS: Fifteen dogs were enrolled at doses from 20.0 to 30.0 microg/kg. Dose-limiting toxicity at 30.0 microg/kg consisted of vascular leak in one and hypotension/coagulopathy in one, establishing 26.7 microg/kg as the maximum tolerated dose. Mean elimination half-life was 15.3 +/- 4.9 hours. Biological activity (transient fever and leukopenia, increased tumor inflammation, and necrosis) was observed at all dosages. A significant increase in tumor blood flow was observed with dynamic contrast-enhanced magnetic resonance imaging. Minor/transient antitumor responses were observed in dogs with melanoma, squamous cell carcinoma, and mammary carcinoma, and a partial response was observed in a dog with angiosarcoma. CONCLUSIONS: Using a clinically relevant, spontaneous large animal model of neoplasia, we have shown that biologically effective doses of PEG-TNF can be administered safely, and that PEG-TNF administration is associated with encouraging biological activity. These results justify the clinical evaluation of PEG-TNF in human cancer.

RT-PCR-based tyrosine kinase display profiling of canine melanoma: IGF-1 receptor as a potential therapeutic target.

Canine malignant melanoma (CMM) resembles human malignant melanoma in terms of metastatic behavior, refractoriness to standard therapy, and tumor antigen expression but it is largely unknown how CMM resembles human melanoma with regard to molecular pathogenesis and cellular signaling. No attempt has been made to systematically define the repertoire of tyrosine kinases (TKs) expressed in CMM. This study used a reverse transcription-PCR display technique to evaluate the expression of multiple TKs in the 17CM98 CMM cell line. RT-PCR was performed using degenerate primers coding for highly conserved regions flanking the kinase domains of many TKs and the repertoire of TKs expressed was determined using standard molecular cloning techniques. Sequencing 46 clones yielded canine homologs of insulin-like growth factor-1 receptor (IGF-1R) (50%), JAK1 (17%), PDGFR-a (11%), FGFR1 (9%), Axl (7%), Abl (4%), and PTK2 (2%). Interestingly, IGF-1R, JAK1, and Axl were detected in human melanoma using similar techniques, supporting the cross-species validity of this assay. Given the abundance of IGF-1R clones, we determined the biological effect of rhIGF-1 in 17CM98 cells. IGF-1 stimulated cell proliferation and vascular endothelial growth factor production in 17CM98, and addition of the IGF-1R inhibitor ADW742 abrogated IGF-1-induced phenotypic changes. Expression of IGF-1R mRNA was detected in five of five additional CMM cell cultures, and IGF-1R protein was detected in five of six primary tumors evaluated, suggesting that IGF-1R expression may be common in CMM and may provide a novel target for future therapy. In conclusion, this study suggests that similar TKs are expressed in human and canine melanoma, and shows potential antitumor effects of IGF-1R inhibition in CMM.

Assessment of GS-9219 in a pet dog model of non-Hodgkin's lymphoma.

PURPOSE: To assess, in dogs with naturally occurring non-Hodgkin's lymphoma, pharmacokinetics, safety, and activity of GS-9219, a prodrug of the nucleotide analogue 9-(2-phosphonylmethoxyethyl) guanine (PMEG), which delivers PMEG and its phosphorylated metabolites to lymphoid cells with preferential cytotoxicity in cells with a high proliferation index such as lymphoid malignancies. EXPERIMENTAL DESIGN: To generate proof-of-concept, a phase I/II trial was conducted in pet dogs (n = 38) with naturally occurring non-Hodgkin's lymphoma using different dose schedules of GS-9219. A subset of dogs was further evaluated with 3'-deoxy-3'-(18)F-fluorothymidine positron emission tomography/computed tomography imaging before and after treatment. RESULTS: The prodrug had a short plasma half-life but yielded high and prolonged intracellular levels of the cytotoxic metabolite PMEG diphosphate in peripheral blood mononuclear cells in the absence of detectable plasma PMEG. Dose-limiting toxicities were generally manageable and reversible and included dermatopathy, neutropenia, and gastrointestinal signs. Antitumor responses were observed in 79% of dogs and occurred in previously untreated dogs and dogs with chemotherapy-refractory non-Hodgkin's lymphoma. The median remission durations observed compare favorably with other monotherapies in dogs with non-Hodgkin's lymphoma. High 3'-deoxy-3'-(18)F-fluorothymidine uptake noted in lymphoid tissues before treatment decreased significantly after treatment (P = 0.016). CONCLUSIONS: GS-9219 was generally well tolerated and showed significant activity against spontaneous non-Hodgkin's lymphoma as modeled in pet dogs and, as such, supports clinical evaluation in humans.

Clinical signs, treatments, and outcome in cats with transitional cell carcinoma of the urinary bladder: 20 cases (1990-2004).

OBJECTIVE: To characterize demographics and clinical signs and evaluate outcomes of treatments in cats with transitional cell carcinoma (TCC) of the urinary bladder. DESIGN: Retrospective case series. ANIMALS: 20 cats with TCC. PROCEDURES: Medical records of 20 cats with a bladder mass identified as a TCC that were examined at 2 veterinary institutions between 1990 and 2004 were evaluated. Signalment, treatments, and outcome were assessed. RESULTS: Breeds included domestic short hair (n=14), long hair (2), and medium hair (2) cats, Siamese (1), and Abyssinian (1). All cats had been neutered at an early age (< 1 year old; 13 neutered males and 7 spayed females). The median age at diagnosis of TCC was 15.2 years. The trigone region was affected in 9 cats. Treatments included piroxicam administration, chemotherapy, or surgery as single interventions or in combination; 6 cats were not treated. At the time of diagnosis, 3 cats had pulmonary metastasis and 1 cat had metastasis to local lymph nodes. Median survival time for all 20 cats was 261 days. Nearly all deaths were attributable to progressive disease in the urinary tract. Five cats were lost to follow-up. CONCLUSIONS AND CLINICAL RELEVANCE: In cats, TCC of the urinary bladder appears to be a rare and aggressive disease that is more prevalent in male cats and frequently develops at sites distant from the trigone (unlike TCC in dogs). Nevertheless, initial clinical signs of TCC in cats in this study were similar to those reported for affected dogs.

Comparison of results of computed tomography and radiography with histopathologic findings in tracheobronchial lymph nodes in dogs with primary lung tumors: 14 cases (1999-2002).

OBJECTIVE: To compare results of computed tomography (CT) and radiography with histopathologic findings in tracheobronchial lymph nodes (TBLNs) in dogs with primary lung tumors. DESIGN: Retrospective case series. ANIMALS: 14 client-owned dogs. PROCEDURES: Criteria for inclusion were diagnosis of primary lung tumor, use of thoracic radiography and CT, and histologic confirmation of TBLN status. Medical records were reviewed for signalment; history; and physical examination, clinicopathologic, radiographic, CT, surgical, and histopathologic findings. RESULTS: Tracheobronchial lymphadenopathy was not identified via radiography in any dogs. Tracheobronchial lymphadenopathy was diagnosed in 5 dogs via CT. Six dogs had histologic confirmation of metastasis to TBLNs. Radiographic diagnosis yielded 6 false-negative and no false-positive results for tracheobronchial lymphadenopathy. Computed tomography yielded 1 false-negative and no false-positive results. Sensitivity of CT for correctly assessing TBLN status was 83%, and specificity was 100%. Positive predictive value was 100%, and negative predictive value was 89%. Dogs with lymphadenopathy via CT, histologic confirmation of TBLN metastasis, or primary tumors with a histologic grade > 1 had significantly shorter survival times than their counterparts. CONCLUSIONS AND CLINICAL RELEVANCE: Results of CT evaluation of TBLN status were in agreement with histopathologic findings and more accurate than use of thoracic radiography for evaluating TBLNs in dogs with primary lung tumors. Computed tomography imaging should be considered as part of the staging process to more accurately assess the TBLNs in dogs with primary lung tumors.

Does L-asparaginase influence efficacy or toxicity when added to a standard CHOP protocol for dogs with lymphoma?

The purpose of this study was to evaluate response rates, 1st remission duration (FRD), and toxicity in dogs with previously untreated lymphoma receiving an identical CHOP-based combination chemotherapy protocol with or without L-asparaginase (LASP). One hundred fifteen dogs with lymphoma were scheduled to receive an identical CHOP-based chemotherapy protocol that included L-ASP. However, because of manufacturer-imposed random rationing, 31 dogs did not receive L-ASP as scheduled. The 2 treatment groups were statistically similar with respect to signalment and presence of historical negative prognostic factors. No difference was observed in the median FRD whether dogs did or did not receive L-ASP (206 versus 217 days, respectively; P = .67). No difference was observed in the median overall survival times between dogs receiving or not receiving L-ASP (310 versus 308 days, respectively; P = .84). No statistical difference was observed with respect to overall response rate between dogs that did or did not receive L-ASP (89.3% versus 87.1%, respectively; P = .75). Complete response rates between the groups also were no different (83.3% and 77.4% for L-ASP and non-L-ASP groups, respectively; P = .59). Prevalence of toxicity (neutropenia, diarrhea, or vomiting) and treatment delays (P = .80) also were similar between groups. The results of this study suggest that exclusion of L-ASP in this multidrug protocol does not significantly impact outcome. Therefore, it may be more appropriate to reserve the use of L-ASP for treating relapse in dogs with lymphoma that have failed induction therapy.

Phase I study of liposome-DNA complexes encoding the interleukin-2 gene in dogs with osteosarcoma lung metastases.

Systemic gene delivery using cationic liposome-DNA complexes (LDCs) has been shown to elicit potent antitumor activity in mice with tumor metastases to the lungs. However, intravenous gene delivery for treatment of established cancer has not been evaluated previously in a spontaneous, large animal model. We therefore evaluated the safety, toxicity, and efficacy of intravenous gene delivery, using LDCs in dogs with established tumor metastases. Twenty dogs with chemotherapy-resistant osteosarcoma metastases to the lungs received a series of intravenous infusions of cationic liposomes and plasmid DNA encoding the canine interleukin-2 (IL-2) cDNA. Effects of intravenous gene delivery on immune activation, clinical and hematologic parameters, tumor responses, and survival times were assessed. We found that slow intravenous administration of IL-2 LDCs resulted in detectable IL-2 transgene expression in lung tissues of dogs. Repeated intravenous infusions of LDCs were well tolerated by dogs with lung tumor metastases and elicited systemic immune activation, as reflected by fever, leukogram changes, monocyte activation, and increased natural killer cell activity. Three of 20 dogs experienced partial or complete regression of lung metastases after infusion of IL-2 LDCs. Overall survival times were significantly increased in treated dogs compared with historical control animals with the same stage of disease. We conclude that repeated intravenous infusion of LDCs in cancerbearing dogs is safe and well tolerated at low doses and may be capable of eliciting antitumor activity in some animals with advanced tumor metastases.

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 x 10(5) to 1 x 10(8) 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 x 10(7) 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 evaluation of a liposome-encapsulated formulation of cisplatin in clinically normal dogs.

OBJECTIVE: To determine the acute and short-term adverse effects of a liposome-encapsulated form of cisplatin at increasing dosages of up to twice the known maximally tolerated dose (MTD) of unencapsulated cisplatin in clinically normal dogs. ANIMALS: 4 healthy 2.5-year-old sexually intact female hound-type dogs. PROCEDURE: 4 dosages (70, 100, 125, and 150 mg/m2) were evaluated, and the 4 dogs received a total of 9 infusions (1 to 3 infusions/dog). Dogs were monitored to detect changes in clinical and clinicopathologic status. Evaluations consisting of a physical examination, CBC, serum biochemical analysis, and urinalysis were performed before and 7 and 21 days after each infusion. RESULTS: Acute anaphylactic-like reactions to liposome-encapsulated cisplatin were common but clinically manageable. Nephrotoxicosis and substantial myelosuppression, toxic effects commonly associated with unencapsulated cisplatin, were not observed in dogs treated with liposome-encapsulated cisplatin at dosages equivalent to twice the known MTD of unencapsulated cisplatin. CONCLUSIONS AND CLINICAL RELEVANCE: Liposome-encapsulated cisplatin can be safely administered to clinically normal dogs at dosages of up to 150 mg/m2 without the need for concurrent hydration protocols. This was a necessary prerequisite to enable phase I clinical trials in dogs with naturally developing cancers that could theoretically benefit from escalation in the dosage of cisplatin. Determination of an MTD, cumulative and long-term toxic effects, and efficacy can now be conducted in the context of phase I trials in tumor-bearing dogs.

Plasma synthesis of carbon magnetic nanoparticles and immobilization of doxorubicin for targeted drug delivery.

Using dense medium plasma technology, carbon magnetic nanoparticles (CMNP) were synthesized at room temperature and atmospheric pressure. Based on results from X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy and scanning electron microscopy, we conclude that these nanoparticles are composed of spherical particles, 40-50 nm in diameter, with iron/iron oxide particles dispersed in a carbon-based host-structure. Thermal gravimetry/differential thermal gravimetry analysis shows these nanoparticles are stable to temperatures as high as 600 degrees C. The synthesized CMNP were treated by argon-plasma, aminated with ethylene diamine and subsequently activated by generating aldehyde groups on them. Free doxorubicin (DOX) molecules were then immobilized onto the surfaces of activated CMNP particles to form CMNP-DOX conjugates. The corresponding loading efficiency was determined. The in vitro antiproliferative activity of immobilized doxorubicin in the conjugates was demonstrated in tumor cell cytotoxicity assays. It is suggested that this CMNP-DOX system can be used for targeted drug-delivery systems.

Imatinib mesylate inhibits platelet-derived growth factor activity and increases chemosensitivity in feline vaccine-associated sarcoma.

Feline vaccine-associated sarcoma (VAS) is a biologically aggressive soft-tissue sarcoma that can develop at sites where inactivated feline vaccines have been administered. We showed that platelet-derived growth factor (PDGF) and its receptor (PDGFR) play a role in the growth of VAS cells. The presence of PDGFR-beta was confirmed in each of five VAS cell lines evaluated, one non-vaccine-associated feline fibrosarcoma (FSA) cell line and a feline fibroblast-derived cell line. The PDGF/PDGFR signaling pathway was inhibited in the VAS cell lines and the FSA cell line using the tyrosine kinase inhibitor imatinib mesylate (formerly called STI-571). Imatinib inhibited PDGF-BB-induced autophosphorylation of PDGFR in VAS cells and feline FSA cells in vitro in a dose-dependent manner. Imatinib also significantly inhibited growth of feline VAS tumors in a murine xenograft model. Imatinib reversed the protective effect of PDGF-BB on growth inhibition by doxorubicin and carboplatin. PDGF-BB protected VAS cells from serum starvation and doxorubicin-induced apoptosis but not carboplatin-induced apoptosis, and imatinib eliminated this protection. These observations suggest that imatinib inhibits PDGFR tyrosine kinase activity in feline soft tissue sarcomas in vitro and inhibits tumor growth in a xenograft model.

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.