Evaluation of liposomal clodronate for treatment of malignant histiocytosis in dogs.

Malignant histiocytosis (MH) is an aggressive cancer derived from myeloid lineage cells in both dogs and humans. In dogs, the tumor is characterized by the rapid development of metastatic tumors in multiple sites, including especially the lungs and lymph nodes. Humans develop an analogous disease known as Langerhans cell histiocytosis, which primarily affects children and young adults. Because these tumors are often resistant to conventional chemotherapy, there is a need for newer therapeutic approaches. Systemic administration of liposomal clodronate (LC) has been shown to effectively deplete phagocytic cells (e.g., macrophages and dendritic cells) in mice. We investigated therefore whether LC could also be used to treat naturally occurring MH in dogs. First, the susceptibility of canine MH cells to LC-mediated killing was assessed in vitro. Then the clinical safety and effectiveness of LC as a treatment for MH was assessed in a pilot study in five pet dogs with spontaneous MH. We found that canine MH cells were very susceptible to LC-induced apoptotic cell death, whereas other tumor cell lines were resistant to killing by LC. Studies using labeled liposomes demonstrated that susceptibility to LC killing was directly related to the efficiency of liposome uptake. In pet dogs with spontaneous MH, we found that a short course of LC treatment elicited significant tumor regression in two of five treated animals. These findings suggest that liposomal delivery of clodronate and possibly other bisphosphonates may offer an effective new approach to treatment of histiocytic neoplasms in dogs and humans.

Rabacfosadine for naïve canine intermediate to large cell lymphoma: Efficacy and adverse event profile across three prospective clinical trials.

While current lymphoma therapies induce remission in most dogs, drug-resistant relapse is common, creating a need for novel agents. Rabacfosadine (RAB), a double prodrug of the acyclic nucleotide phosphonate 9-(2-phosphonylmethoxyethel) guanine (PMEG), preferentially targets lymphoma cells with reduced systemic toxicity compared with PMEG. Previous studies evaluating RAB administered every 21 days have suggested efficacy in both naïve and relapsed subjects; however, no large studies of RAB as a single agent have been reported in previously untreated dogs with intermediate to large cell lymphoma. The purpose of this study was to evaluate the safety and efficacy of RAB in dogs with previously untreated (excluding corticosteroids) lymphoma. Sixty-three dogs received up to five RAB treatments every 21 days (16 at 0.82 mg/kg and 47 at 1.0 mg/kg) as a 30 minutes intravenous infusion, with (n = 23) or without (n = 40) concurrent corticosteroids. Response assessment and adverse event (Ae) evaluation were performed every 21 days via Veterinary Cooperative Oncology Group (VCOG) criteria. The overall response rate was 87% (52% CR, 35% PR). The overall median progression free interval was 122 days (199 for CR, 89 for PR and 153 days for all responders). T-cell immunophenotype and corticosteroid pre-treatment were predictive of inferior outcomes on multivariate analysis. AEs were most commonly of gastrointestinal origin (hyporexia/diarrhoea) and generally resolved with supportive treatment and/or dosage adjustment. Three dogs experienced VCOG-CTCAE grade 5 delayed pulmonary fibrosis. In conclusion, RAB administered every 3 weeks is generally well tolerated and demonstrates substantial antitumour activity in dogs with previously untreated intermediate to large cell lymphoma.

Prospective evaluation of flow cytometric characteristics, histopathologic diagnosis and clinical outcome in dogs with naïve B-cell lymphoma treated with a 19-week CHOP protocol.

Canine B-cell lymphoma is a clinically heterogenous disease; however, it is generally treated as a single disease entity. The purpose of this clinical trial was to prospectively evaluate naïve canine B-cell lymphoma patients using histopathology, flow cytometry (FC) and a standardized chemotherapy protocol to better define subsets of this disease that may respond differently to treatment. Sixty-four dogs with naïve multicentric B-cell lymphoma were treated with a standardized 19-week CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy protocol. Most of the dogs (84.3%) were diagnosed with diffuse large B-cell lymphoma (DLBCL), followed by nodal marginal zone (7.8%), small B-cell (4.7%), Burkitt-like (1.6%) and follicular lymphoma (1.6%). FC confirmed the diagnosis of B-cell lymphoma in all cases. There were no clear phenotyping differences between the subtypes of B-cell lymphoma detectable by our FC panel. The histologic subtypes in this study exhibited a range of forward scatter values on flow cytometry, but all of the DLBCL cases were higher than a value of 469, while the only cases with a lower forward scatter value were follicular lymphoma and diffuse small B-cell lymphoma. Dogs with DLBCL had a significantly better objective response rate to the CHOP protocol (96.3%) than the non-DLBCL subtypes (70%, P = .024). The median progression-free survival time for patients with DLBCL (233 days) was significantly longer than that of all other histopathologic subgroups combined (163 days, P = .0005).

Cancer stem cell populations in lymphoma in dogs and impact of cytotoxic chemotherapy.

Cancer relapse following chemotherapy has been attributed in part to the presence of cancer stem cells (CSC), which drive tumour growth and metastasis and are highly resistant to the effects of cytotoxic chemotherapy. As a result, treatment with cytotoxic chemotherapy selects for drug-resistant CSC populations that eventually drive tumour recurrence. Little is known currently regarding the role of CSC in dogs with lymphoma, nor the impact of chemotherapy on CSC populations. Therefore, we prospectively quantitated CSC populations in dogs with B-cell (BCL) and T-cell lymphoma (TCL), using tumour aspirates and flow cytometric analysis with a panel of CSC markers. In addition, in vitro studies were carried out to determine the impact of chemotherapy resistance on the stem cell phenotype and stem cell properties of lymphoma cells. We found that the percentages of tumour cells expressing CSC markers were significantly increased in dogs with BCL, compared with B cells from normal lymph nodes. Similar findings were observed in dogs with TCL. In vitro studies revealed that lymphoma cells selected for resistance to CHOP chemotherapy had significantly upregulated expression of CSC markers, formed spheroids in culture more readily, and expressed significantly greater aldehyde dehydrogenase activity compared with chemotherapy-sensitive tumour cells. Similar results were observed in tumour samples dogs with relapsed BCL. These findings suggest that cytotoxic chemotherapy can lead to a relative enrichment of tumour cells with CSC properties, which may be associated with lymphoma recurrence.

Continuous low-dose oral chemotherapy for adjuvant therapy of splenic hemangiosarcoma in dogs.

BACKGROUND: Hemangiosarcoma (HSA) is a highly metastatic and often rapidly fatal tumor in dogs. At present, conventional adjuvant chemotherapy provides only a modest survival benefit for treated dogs. Continuous oral administration of low-dose chemotherapy (LDC) has been suggested as an alternative to conventional chemotherapy protocols. Therefore, we evaluated the safety and effectiveness of LDC using a combination of cyclophosphamide, etoposide, and piroxicam as adjuvant therapy for dogs with stage II HSA. HYPOTHESIS: We hypothesized that oral adjuvant therapy with LDC could be safely administered to dogs with HSA and that survival times would be comparable to those attained with conventional doxorubicin (DOX) chemotherapy. ANIMALS: Nine dogs with stage II splenic HSA were enrolled in the LDC study. Treatment outcomes were also evaluated retrospectively for 24 dogs with stage II splenic HSA treated with DOX chemotherapy. METHODS: Nine dogs with stage II splenic HSA were treated with LDC over a 6-month period. Adverse effects and treatment outcomes were determined. The pharmacokinetics of orally administered etoposide were determined in 3 dogs. Overall survival times and disease-free intervals were compared between the 9 LDC-treated dogs and 24 DOX-treated dogs. RESULTS: Dogs treated with LDC did not develop severe adverse effects, and long-term treatment over 6 months was well-tolerated. Oral administration of etoposide resulted in detectable plasma concentrations that peaked between 30 and 60 minutes after dosing. Both the median overall survival time and the median disease-free interval in dogs treated with LDC were 178 days. By comparison, the overall survival time and disease-free interval in dogs treated with DOX were 133 and 126 days, respectively. CONCLUSIONS: Continuous orally administered LDC may be an effective alternative to conventional high-dose chemotherapy for adjuvant therapy of dogs with HSA.

Evaluation of a novel tumor vaccine in dogs with hemangiosarcoma.

BACKGROUND: Hemangiosarcoma (HSA) is a highly metastatic and often rapidly fatal tumor of dogs. At present, adjuvant chemotherapy is the only proven effective treatment for dogs with HSA, though the benefits from chemotherapy are modest. Administration of immunotherapy together with chemotherapy has also been reported to improve survival in dogs with HSA. Therefore, we evaluated safety and immunologic responses to a novel tumor vaccine administered together with doxorubicin chemotherapy in dogs with different stages of HSA. HYPOTHESIS: That tumor vaccination could be safely and effectively combined with doxorubicin chemotherapy for treatment of dogs with HSA. ANIMALS: Twenty-eight dogs with various stages of HSA were enrolled in the study. METHODS: The HSA vaccine was prepared with lysates of allogeneic canine HSA cell lines mixed with an adjuvant composed of liposome-DNA complexes. Dogs received a series of 8 immunizations administered over a 22-week period, and most also received chemotherapy. Clinical adverse effects were noted, immune responses were measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, and survival times were calculated. RESULTS: The most common adverse effects observed in vaccinated dogs also treated with doxorubicin chemotherapy were diarrhea and anorexia. Vaccinated dogs were found to mount strong humoral immune responses against a control antigen and, most dogs also mounted antibody responses against canine HSA cells. Thirteen dogs with stage II splenic HSA that received the tumor vaccine plus doxorubicin chemotherapy had an overall median survival time of 182 days. CONCLUSIONS: We conclude that an allogeneic tumor lysate vaccine is safe in dogs with HSA and can elicit humoral immune responses in dogs that are receiving concurrent doxorubicin chemotherapy.

Doxorubicin and BAY 12-9566 for the treatment of osteosarcoma in dogs: a randomized, double-blind, placebo-controlled study.

BACKGROUND: This study was designed to assess the efficacy of a matrix metalloproteinase inhibitor in prolonging posttreatment survival for dogs with appendicular osteosarcoma after treatment with amputation and doxorubicin chemotherapy. HYPOTHESIS: Survival will be prolonged in dogs receiving BAY 12-9566. ANIMALS: The study included 303 dogs with appendicular osteosarcoma. METHODS: Dogs were treated with doxorubicin (30 mg/m2) every 2 weeks for 5 treatments starting 2 weeks after amputation. Dogs were randomly allocated to receive a novel nonpeptidic biphenyl inhibitor of matrix metalloproteinases (MMPs, BAY 12-9566; 4-[4-4-(chlorophenyl)phenyl]-4-oxo-2S-(phenylthiomethyl) butanoic acid) or placebo after doxorubicin chemotherapy. RESULTS: Median survival for all 303 dogs was 8 months; and 1-year, 2-year, and 3-year survival rates were 35%, 17%, and 9%, respectively. Treatment with BAY 12-9566 did not influence survival. Multivariate analysis revealed that increasing age (P = .004), increasing weight (P = .006), high serum alkaline phosphatase (ALP) (P = .012) and high bone ALP (P < .001) were independently associated with shorter median survival times. Additional analyses on available data indicated that as the number of mitotic figures in the biopsy increased (P = .013), and as plasma active MMP-2 concentrations increased (P = .027), the risk of dying increased. CONCLUSIONS AND CLINICAL IMPORTANCE: Doxorubicin is an effective adjuvant to amputation in prolonging survival for dogs with appendicular osteosarcoma.

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.

Evaluation of a xenogeneic VEGF vaccine in dogs with soft tissue sarcoma.

Active immunization against pro-angiogenic growth factors or their receptors is an emerging strategy for controlling tumor growth and angiogenesis. Previous studies in rodent tumor models have indicated that immunization against xenogeneic growth factors is more likely to induce effective anti-tumor responses than immunization against the autologous growth factor. However, the effectiveness or safety of the xenogeneic vaccination approach has not been previously assessed in a clinically relevant outbred, spontaneous tumor model. Therefore, we investigated the safety and anti-tumor and anti-angiogenic effects of a xenogeneic vascular endothelial cell growth factor (VEGF) vaccine in pet dogs with spontaneous cancer. Nine dogs with soft tissue sarcoma were immunized with a recombinant human VEGF vaccine over a 16-week period. The effects of immunization on antibodies to human and canine VEGF, circulating VEGF concentrations, tumor microvessel density (MVD), and tumor growth were assessed. The xenogeneic VEGF vaccine was well-tolerated by all dogs and resulted in induction of humoral responses against both human and canine VEGF in animals that remained in the study long enough to receive multiple immunizations. Three of five multiply immunized dogs also experienced sustained decreases in circulating plasma VEGF concentrations and two dogs had a significant decrease in tumor MVD. The overall tumor response rate was 30% for all treated dogs in the study. We conclude therefore that a xenogeneic VEGF vaccine may be a safe and effective alternative means of controlling tumor growth and angiogenesis.