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PDF file - 55K, Table describing survival times and causes of death for surgery historical control dogs.

PDF file - 63K, Table that summarizes the immune monitoring carried out, by dog.

PDF file - 75K, Contains descriptions of two methods used to acquire clinical histology and T cell response data.

PDF file - 72K, Table that contains clinical information on the dogs enrolled in the vaccination cohort arm of the study.

Despite the growing number of preclinical and clinical trials focused on immunotherapy for the treatment of malignant gliomas, the prognosis for this disease remains grim. Although some promising advances have been made, the immune response stimulated as a result of immunotherapeutic protocols has been inefficient at complete tumor elimination, primarily due to our lack of understanding of the necessary effector functions of the immune system. We previously demonstrated that a tumor lysate vaccine/Fc-OX40L therapy is capable of inducing enhanced survival and tumor elimination in the GL261 mouse glioma model. The following experiments were performed to determine the mechanism(s) of action of this therapy that elicits a potent antitumor immune response. The evidence subsequently outlined indicates a CD8+ T cell–independent and CD4+ T cell–, NK cell–, and B cell–dependent means of prolonged survival. CD8+ T cell–independent tumor clearance is surprising considering the current focus of many cancer immunotherapy protocols. These results provide evidence for CD8+ T cell–independent means of antitumor response and should lead to additional examination of the potential manipulation of this mechanism for future treatment strategies.

Immunotherapy for malignant gliomas has striking efficacy in rodent models but very limited effects in clinical trials, which may reflect immunological differences between induced and spontaneous tumors. The monocytes that heavily infiltrate GBM develop myeloid-derived suppressor cell (MDSC) phenotype, and these immunosuppressive cells are increased in GBM patients, but not in murine models. Canine primary brain tumors may offer a more faithful representation of the human disease due to similarities in clinical and pathobiological characteristics. We were recently first to identify MDSC in dogs and found significantly increased in number in dogs with advanced cancers. To add further support to pet dogs with glioma as a translational model, we assessed whether canine glioma patients also have elevated numbers of MDSC relative to healthy dogs. MDSC secrete arginase that removes L-arginine from the microenvironment profoundly impairing T cell proliferation and function. Therefore we also measured serum levels of arginase 1 in all dogs. The mean percentage of monocytic MDSC in peripheral blood of dogs with glioma (n = 26) was 7.51 ± 1.31, which was significantly greater than healthy controls (n = 10) with 0.74 ± 0.34 (P< 0.0001). The median percentages of granulocytic MDSC did not differ between dogs with glioma versus controls, 15.95% and 12.5% respectively. There were also significantly increased levels of arginase in the serum of dogs with gliomas, 8.00 ± 3.32 U/L, relative to that of healthy controls, 2.40 ± 1.40 U/L (P < 0.0001). These data demonstrate that some of the immunosuppressive mechanisms present in human gliomas patients are also found in dogs with malignant gliomas. These findings lend further support that the pet dog with spontaneous brain cancer that shares our environment is an excellent translational model for the human disease. We are currently working to correlate changes in numbers of MDSC and arginase levels in serum with disease burden as possible biomarkers for response to therapy.

Malignant and atypical meningiomas are resistant to standard therapies and associated with poor prognosis. Despite progress in the treatment of other tumors with therapeutic vaccines, this approach has not been tested preclinically or clinically in these tumors. Spontaneous canine meningioma is a clinically meaningful but underutilized model for preclinical testing of novel strategies for aggressive human meningioma. We treated 11 meningioma-bearing dogs with surgery and vaccine immunotherapy consisting of autologous tumor cell lysate combined with toll-like receptor ligands. Therapy was well tolerated, and only one dog had tumor growth that required intervention, with a mean follow up of 585 days. IFN-γ–elaborating T cells were detected in the peripheral blood of 2 cases, but vaccine-induced tumor-reactive antibody responses developed in all dogs. Antibody responses were polyclonal, recognizing both intracellular and cell surface antigens, and HSP60 was identified as one common antigen. Tumor-reactive antibodies bound allogeneic canine and human meningiomas, showing common antigens across breed and species. Histologic analysis revealed robust infiltration of antibody-secreting plasma cells into the brain around the tumor in posttreatment compared with pretreatment samples. Tumor-reactive antibodies were capable of inducing antibody-dependent cell-mediated cytotoxicity to autologous and allogeneic tumor cells. These data show the feasibility and immunologic efficacy of vaccine immunotherapy for a large animal model of human meningioma and warrant further development toward human trials. Cancer Res; 73(10); 2987–97. ©2013 AACR.

As the reliable identification of proteins by tandem mass spectrometry becomes increasingly common, the full characterization of large data sets of proteins remains a difficult challenge. Our goal was to survey the proteome of a human T-cell lymphoma-derived cell line in a single set of experiments and present an automated method for the annotation of lists of proteins. A downstream application of these data includes the identification of novel pathogenetic and candidate diagnostic markers of T-cell lymphoma. Total protein isolated from cytoplasmic, membrane, and nuclear fractions of the SUDHL-1 T-cell lymphoma cell line was resolved by SDS-PAGE, and the entire gel lanes digested and analyzed by tandem mass spectrometry. Acquired data files were searched against the UniProt protein database using the SEQUEST algorithm. Search results for each subcellular fraction were analyzed using INTERACT and ProteinProphet. All protein identifications with an error rate of less than 10% were directly exported into excel and analyzed using GOMiner (NIH/NCI). The Gene ontology molecular function and cell location data were summarized for the identified proteins and results exported as user-interactive directed acyclic graphs. A total of 1105 unique proteins were identified and fully annotated, including numerous proteins that had not been previously characterized in lymphoma, in functional categories such as cell adhesion, migration, signaling, and stress response. This study demonstrates the utility of currently available bioinformatics tools for the robust identification and annotation of large numbers of proteins in a batchwise fashion.

Gliomas, the most invasive tumors of the brain, are a disparate group of oncological diseases for which there is currently no cure. Despite the use of combination therapy (surgery, radiation and chemotherapy) as the standard of care for patients, limited progress has been made in the control of the disease. Similar to that in humans, dogs develop spontaneous brain tumors that often carry a dismal prognosis regardless of therapeutic intervention. There is an obvious need for new therapies to treat dogs with brain tumors. Cancer immunotherapy, with the premise that tumor-reactive lymphocytes are able to infiltrate the brain to eradicate microscopic disease, is emerging as a promising strategy to treat glioma. To date, very little is known regarding canine glioma treatment outcome and there has been no reported randomized clinical trials examining the use of immunotherapy for treatment of glioma in dogs. A clinical trial using immunotherapy with autologous glioma cell lysate vaccines, combined with the TLR9 agonist CpG oligodeoxynucleotide to treat dogs with glioma, is underway in our laboratory. The objective of the treatment is to induce specific anti-tumor immunity in the brain and to prevent tumor recurrence. Our preliminary results have shown that vaccinated dogs exhibited extended survival when compared with historical controls. Based on western blot and flow cytometric analysis, this therapeutic approach induced tumor-reactive antibody responses with elevation of IgG antibody levels after vaccination. Thus, the examination of immune responses in therapeutic trials conducted in dogs with gliomas should greatly help predict outcomes of similar treatments in humans, and additionally provide an opportunity to accelerate the development of more efficient immunotherapeutic cancer vaccines for treatment of intracranial malignances. Citation Format: Michelle Goulart, Elizabeth Pluhar, Brian Andersen, Charles Seiler, Matthew Hunt, Gerry O'Sullivan, Zoe Zhang, John Ohlfest. Immunotherapy as treatment for dogs with glioma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B20.

Malignant and atypical meningiomas are resistant to standard therapies, but therapeutic vaccines have not been tested preclinically or clinically. Pet dogs with naturally occurring meningioma are an underutilized model for aggressive human meningioma and an outstanding opportunity for assessing experimental therapeutic approaches. We treated 11 meningioma-bearing dogs with surgery and vaccine immunotherapy consisting of autologous tumor cell lysate combined with CpG or imiquimod. Interferon gamma-elaborating T cells were detected in the peripheral blood of two of cases, but vaccine-induced tumor-reactive antibody responses were found in the sera of all dogs. Systemic antibody responses were polyclonal, recognizing intracellular and cell surface antigens, and heat shock protein 60 was identified as one common antigen. Tumor-reactive antibodies bound allogeneic canine and human meningiomas, demonstrating common antigens across breed and species. Histological analysis revealed robust infiltration of antibody-secreting plasma cells into the brain around the tumor in treated compared to pre-treatment samples. Tumor-reactive antibodies were capable of inducing antibody dependent cell-mediated cytotoxicity to autologous and allogeneic tumor cells. Moreover, median survival for lysate/CpG and lysate/imiquimod vaccination was 646 days versus 222 days in historic surgery controls (p Citation Format: Brian M. Andersen, G Elizabeth Pluhar, Charles E. Seiler, Zhengming Xiong, Michelle R. Goulart, Matthew Gerry O'Sullivan, Matthew A. Hunt, Charles E. Schiaffo, David M. Ferguson, John R. Ohlfest. Preclinical testing of three immune adjuvants in vaccination therapy for invasive canine meningioma. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B32.