Canine Glioma Research Articles

EXTH-52. GLIOBLASTOMA-TARGETING AUTOLOGOUS INDUCED NEURAL STEM CELL THERAPY: EVALUATING SAFETY, TOXICITY, PERSISTENCE, AND TRANSPLANT METHODS IN A POST-SURGICAL CANINE MODEL

Abstract BACKGROUND Glioblastoma patient survival statistics have remained unchanged for more than three decades. Despite tumor resection and chemoradiotherapy, recurrence is inevitable. Moreover, the invasive behavior of glioblastoma confounds treatment. To improve patient survival statistics, a targeted therapy that can home to distant tumor foci is desperately needed. Induced neural stem cells (iNSCs) armed with cytotoxic payloads have proven efficacious against human xenograft models of glioblastoma. To further propel iNSCs to human clinical trials, we investigated the safety, toxicity, and persistence of iNSCs in a canine model. METHODS Autologous iNSCs generated from the skin of four non-tumor-bearing, purpose-bred, male beagles were engineered to express TRAIL and thymidine kinase (TK). iNSCs were loaded with ferumoxytol to facilitate MRI-tracking. Canines were divided into two cohorts to denote iNSC administration route: scaffold encapsulation or intracerebroventricular (ICV). Two dose levels were investigated: 1′106 iNSCs/kg or 3′106 iNSCs/kg. The scaffold cohort received a single dose of iNSCs while the ICV cohort received three doses of iNSCs via a Rickham reservoir. To activate TK, canines were administered valganciclovir. Canine health was assessed via neurological exams, MRI, and serial blood, urine, and CSF analyses. RESULTS No acute injection reactions were observed. Three of four canines exhibited surgery-induced blindness. Urine and CSF analyses were unremarkable. Unexpectedly, blood analyses showed transient neutropenia. Hypodense signal was observed on all MRI sequences through endpoint. Post-mortem histopathology of the spleen, liver, and lung were unremarkable. As expected, brain tissues exhibited gliosis, fibrous thickening, and inflammation. Spinal cords exhibited acute hemorrhaging, attributed to perimortem CSF draws. Surprisingly, significant testicular degeneration was observed; this was confirmed to be caused by valganciclovir. In conclusion, iNSCs exhibit limited toxicity and warrant further exploration. FUTURE DIRECTIONS Prospective studies will investigate the efficacy of autologous iNSCs in a spontaneous canine glioma model in preparation for human clinical trials.

On-Tissue Spatially-Resolved Glycoproteomics Guided by N-Glycan Imaging Reveal Global Dysregulation of Canine Glioma Glycoproteomic Landscape

We identify N-linked glycoproteins and deduce their spatial localization using spatially-resolved glycoproteomic guided by MALDI mass spectrometry N-glycan imaging on formalin-fixed paraffin embedded (FFPE) canine high-grade glioma tissues. The glycan HexNAc4-Hex5-NeuAc2 was found to be predominantly expressed in necrotic regions, whereas high mannose HexNAc2-Hex5 was predominantly found in benign regions. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were analysed. Haptoglobin is the protein associated with HexNAc4-Hex5-NeuAc2. Additionally, we identified several high-mannose (Hex2-HexNAc5) glycopeptides enriched in benign regions. This is the first report that directly links glycan imaging with intact glycopeptide identification. Over 400 N-glycosylated, O-GalNAcylated, O mannosylated, and S- and O- GlcNAcylated glycopeptides from over 30 proteins were identified. While N-glycosylation and O- mannosylation were similar between benign and tumor/necrotic sections, S- and O-GlcNAc glycopeptides were significantly decreased in tumor/necrotic sections, whereas sialylated O-GalNAc glycopeptides were significantly upregulated.

Abstract 4568: The One Health Consortium and combination immunotherapy: evaluating M032, a genetically engineered HSV-1 expressing Il-12, in combination with a checkpoint inhibitor in canine glioma patients

Abstract Introduction: Malignant gliomas are the most common primary brain tumors in humans, accounting for approximately 30% of all primary CNS tumors in adults (Levin et al., 2001). Interestingly, pet dogs spontaneously and sporadically develop malignant glial brain tumors that resemble high-grade gliomas in humans; with similar incidence, treatments, and outcome patterns. (Priester and McKay, 1980; Snyder et al., 2006; Ostrom et al., 2013; Ostrom et al., 2014; Hicks et al., 2017) The most malignant of these tumors have been refractory to limited treatment options. Despite aggressive treatment, outcomes are dismal with median survivals just over one year in humans and two months in dogs. Novel treatments are greatly needed and combination therapies appear to hold promise. Background: This pre-clinical protocol, a dose-escalating phase I study in dogs with sporadic malignant glioma, represents a first in comparative oncology and combination immunotherapy. The trial is evaluating M032, an Interleukin-12 expressing Herpes Simplex virus, alone and combined with a checkpoint inhibitor, Indoximod. Note, prior studies have demonstrated human IL-12 to generate effective anti-tumoral response in canine patients (Pavlin et al., 2012). M032 is currently being tested in humans with high-grade malignant gliomas. Thus, in a novel fashion, both canine and human trials are proceeding concurrently, allowing a direct “head-to-head” comparison of safety and efficacy. Methods: Stage 1 of the trial, which is presently ongoing, involves catheter-based administration of MO32 alone into the tumor resection cavity at escalating doses in subsequent cohorts of dogs to establish maximum tolerable dose (MTD), detect dose limiting toxicities (DLT), quantify immune response via serum assays, and determine survival benefit. Stage 2 will involve administration of a fixed dose of MO32 derived from Stage 1 data followed by 4 weeks of daily Indoximod administration at a pre-determined dose, with interval comparative immune assays, neurological monitoring, and imaging surveillance. Indoximod is expected to blunt suppressive cellular (Tregs, MDSCs) immune response components, allowing a longer time for effective anti-viral responses and via cross-epitope spreading, an anti-tumor response that will be more durable than that observed with administration of virus alone. In all cases, tumor is collected, processed, and archived for future studies, including whole genome and RNA sequencing. Results: Preliminary data from Stage 1 have demonstrated a median survival of 188 days among all canines following infusion of M032 HSV (95% confidence interval of 83.2 to 292.8 days). Nine canines have died and eight are still alive following treatment. Current dose is 1 x 109 plaque-forming units. No dose limiting toxicities have been observed with infusion of M032 alone. Conclusions: The ability to compare human and dog responses in real time affords the most stringent test of suitability of the dog as a valid and informative model of human brain tumors. The results of this and subsequent studies will allow canine trials to properly inform the design of human trials and further support bi-translational studies and the One Medicine approach to clinical research and application. Citation Format: M. R. Chambers, R. T. Bentley, David Crossman, Jeremy B. Foote, J. W. Koehler, James M. Markert, Simon R. Platt, Nidal B. Omar, D. M. Self, Andy Shores, Don Sorjonen, Alicia M. Waters, Amy B. Yanke, G Y. Gillespie. The One Health Consortium and combination immunotherapy: evaluating M032, a genetically engineered HSV-1 expressing Il-12, in combination with a checkpoint inhibitor in canine glioma patients [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4568.

Cannabidiol Induces Apoptosis and Perturbs Mitochondrial Function in both Human and Canine Glioma Cells

The use of cannabidiol (CBD), the major non‐psychoactive compound derived from cannabis, as a nutraceutical in both human and veterinary medicine has increased steadily within the past ten years despite a lack of scientific evidence for its effectiveness. Of the cornucopia of ailments that CBD supposedly treats, epilepsy, pain, and cancer remain prominent areas of interest to scientists and the general public. In the world of cancer research, several publications have demonstrated that CBD is cytotoxic against breast, endometrial, glial, and blood cancers, and in all cases it appears that CBD triggers apoptosis through a cannabinoid receptor independent process. A recent publication suggests CBD causes cell death in leukemia cells via calcium dependent mitochondrial membrane disruption, leading to activation of intrinsic apoptosis. Other receptors, such as TRPV1, and pathways, such as autophagy, have been implicated in CBD‐induced cell death but the molecular details remain elusive. Because of their remarkable similarities, from a comparative oncology perspective, canine and human gliomas are of particular interest. Regardless of species, gliomas are aggressive tumors that are notoriously challenging to treat, given their resistance to multimodal treatment, thus creating a need for more effective therapeutics. We sought to characterize the mechanism of CBD‐induced cell death in both human and canine glioma cells. We show that CBD by itself is cytotoxic, anti‐proliferative, and anti‐migratory but at concentrations which may not be pharmacologically attainable. Using resazurin and the IncuCyte live cell imaging platform, we show that metabolic‐based assays artificially increase cell line sensitivity to CBD compared to the IncuCyte, suggesting that non‐cytotoxic concentrations of CBD perturb mitochondrial function. Furthermore, we show that CBD treatment results in the formation of large cellular structures whose existence are RIPK3‐dependent. After treatment with a variety of cell death inhibitors, we demonstrate that CBD‐induced cell death occurs via apoptosis; a classically necroptotic protein, RIPK3, also appears to be involved. Lastly, by combining CBD with the autophagy inhibitor hydroxychloroquine (HCQ) we show that sensitivity to CBD increases with HCQ treatment in canine cells, which suggests involvement of the autophagy pathway for mediating CBD‐induced cell death. These data demonstrate that human and canine glioma cells respond similarly to CBD, indicate a non‐canonical function of RIPK3, and support the involvement of autophagy in CBD‐induced cell death.Support or Funding InformationSupport provided by Extract Labs in Boulder, Colorado.

Differential Expression of miRNAs in Hypoxia ("HypoxamiRs") in Three Canine High-Grade Glioma Cell Lines.

Dogs with spontaneous high-grade gliomas increasingly are being proposed as useful large animal pre-clinical models for the human disease. Hypoxia is a critical microenvironmental condition that is common in both canine and human high-grade gliomas and drives increased angiogenesis, chemo- and radioresistance, and acquisition of a stem-like phenotype. Some of this effect is mediated by the hypoxia-induced expression of microRNAs, small (~22 nucleotides long), non-coding RNAs that can modulate gene expression through interference with mRNA translation. Using an in vitro model with three canine high-grade glioma cell lines (J3T, SDT3G, and G06A) exposed to 72 h of 1.5% oxygen vs. standard 20% oxygen, we examined the global “hypoxamiR” profile using small RNA-Seq and performed pathway analysis for targeted genes using both Panther and NetworkAnalyst. Important pathways include many that are well-established as being important in glioma biology, general cancer biology, hypoxia, angiogenesis, immunology, and stem-ness, among others. This work provides the first examination of the effect of hypoxia on miRNA expression in the context of canine glioma, and highlights important similarities with the human disease.

Microtubule-Associated Protein 2 Expression in Canine Glioma.

Canine glioma is considered a potential model for human glioma, with recent studies of occurrence, therapy, and reclassification supporting the value of the canine model. The current diagnosis of canine glioma is based on morphologic criteria and immunohistochemistry (IHC), including oligodendrocyte transcription factor 2 (Olig2), glial fibrillary acidic protein (GFAP), and 2′, 3′ cyclic nucleotide phosphatase (CNPase). Microtubule-associated protein 2 (MAP2) is a proven marker of human glioma and is used to complement the diagnosis and its specific immunoreactivity pattern contributes to the differentiation of astrocytomas from other glial tumors. The objective of this study was to evaluate whether canine gliomas express MAP2 and to explore differences in the pattern of immunolabeling between different gliomas. Seventy-eight cases of canine glioma were evaluated for MAP2 expression by immunohistochemistry. A glial origin was supported by Olig2 IHC in all cases. MAP2 immunolabeling was evaluated on a semi-quantitative basis, including the percentage of immunolabeled neoplastic cells, as well as the signal intensity, distribution, and pattern of immunolabeling. MAP2 was expressed in all cases, with significant correlation between diagnosis and signal intensity (P = 0.04). MAP2 immunolabeling distribution was dominated by diffuse (34/78; 44%), followed by patchy (20/78; 26%), multifocal to coalescing (16/78; 21%), and scattered (8/78; 10%). All oligodendrogliomas (53/53; 100%) and undefined gliomas (12/12; 100%) revealed a combination of perinuclear and cytoplasmic immunolabeling, and all but 3 astrocytomas had a combination of perinuclear and cytoplasmic processes immunolabeling (10/13; 77%). Significant correlation between immunolabeling pattern and diagnosis was obtained (P = 0.001). The study demonstrates that MAP2 is expressed in canine gliomas and the pattern of expression can also be applied to help distinguish astrocytomas from oligodendrogliomas and undefined gliomas.

GENE-57. COMPARATIVE MOLECULAR LIFE HISTORY OF SPONTANEOUS CANINE AND HUMAN GLIOMA

Abstract Diffuse gliomas are the commonest of malignant brain tumors with high-grade tumors carrying dismal prognosis. Preclinical models have proven themselves as poor predictors of clinical efficacy, attributed to the lack of a comparable tumor microenvironment. Comparative genomics of canine and human gliomas provide an attractive alternative modality to identify conserved drivers and underlying mutational processes of glioma as well as evaluate life history tradeoffs related to tissue context and aging that can shape type and relative timing of drivers in gliomagenesis. We performed a comparative genomics analysis between whole genome-, exome-, transcriptome- and methylation-sequencing of 77 canine gliomas, and human pediatric (n=217) and adult gliomas (n=822). We found alterations in common with those in human pediatric and adult gliomas in the Tp53 and cell cycle pathways, receptor tyrosine kinase and Idh1 R132 mutations. Canine gliomas showed similarity to human pediatric gliomas in terms of lower mutational rates (0.2–0.29 per megabase), hotspot mutations and amplifications of Pdgfrα, and robust aneuploidy constrained within the syntenic regions of Pdgfrα and Myc, but also in the known pediatric drivers, Hist1 and Acvr1 genes. A mutational signature reflecting homologous repair defect was detected in canine and pediatric but not adult gliomas, potentially resulting in the observed higher rates of genomic instability. Canine gliomas in majority classified as pediatric tumors using a commonly used human brain methylation classifier (Capper et al. 2018) and cell-of-origin analysis by deconvoluting canine transcriptome using lineage-specific gene signatures. By providing a large canine glioma genomic-sequencing dataset and comparing it with human glioma, our study provides unique insights into glioma etiology and the chronology of glioma-causing somatic alterations. Further, our results effectively position preclinical models of spontaneous canine glioma for use in understanding glioma drivers, and evaluate novel therapies targeting aneuploidy, especially for pediatric brain tumors.

ACTR-63. PHASE I DOSE ESCALATION STUDY OF PROCASPASE ACTIVATING COMPOUND-1 (PAC-1) IN COMBINATION WITH TEMOZOLOMIDE IN PATIENTS WITH RECURRENT ANAPLASTIC ASTROCYTOMA OR GLIOBLASTOMA

Abstract BACKGROUND Procaspase activating compound -1 (PAC-1) is a small molecule that catalyzes conversion of procaspase-3 to caspase-3 which induces apoptosis in cancer cells. Glioblastoma (GBM) is among the tumors with high concentrations of procaspase-3 and low levels of caspase-3. PAC-1 crosses the blood brain barrier and has been shown to synergize with temozolomide (TMZ) in canine malignant glioma and meningioma that arise spontaneously. METHODS This is a multicenter phase 1 dose-escalation study to assess the maximum tolerated dose (MTD) of PAC-1 administered days 1–21 in combination with TMZ days 8–12 at a dose of 150 mg/m2 of each 28 day cycle in subjects with recurrent anaplastic astrocytoma (AA) or GBM. A modified Fibonacci 3 + 3 design is used with up to 4 dose levels of PAC-1 (375, 500, 625 and 750 mg/day). Neurologic toxicity, including cognitive function, is closely monitored throughout the trial. INTERIM DATA: A total of 14 subjects have been enrolled to-date. Of these, 7 at dose level 1, PAC-1 375 mg/day (6 GBM, 1 AA; median age 58y, range 25–75) and 7 at dose level 2, PAC-1 500 mg/day (5 GBM, 2 AA; median age 51y, range 35–60). Best responses to-date were 2 subjects with a partial response and 2 with stable disease. Grade 3 (hepatotoxicity) and 4 (cerebral edema) was reported as possibly related to PAC-1 in 1 patient at dose level 1. The median number of cycles received was 4 (range, 1–12+) at dose level 1 and 2 (range, 1–3) at dose level 2. Enrollment to dose level 2 has been completed and data analysis is ongoing. Updated response and toxicity as well as pharmacokinetic data will be presented.

Phase I study of procaspase activating compound -1 (PAC-1) in combination with temozolomide (TMZ) for the treatment of recurrent malignant glioma.

TPS3164 Background: The caspase family of cysteine proteases play key roles in the initiation and execution of apoptosis. The activation of procaspase-3 to caspase-3 is critical in both the intrinsic and extrinsic apoptotic cascades. Procaspase-3 levels are elevated in many cancers, including glioblastoma (GBM). As a result, caspase-3 levels are abnormally low in these tumors; thus they avoid apoptosis. PAC-1 is a small molecule that directly activates procaspase-3 and induces apoptosis of cancer cells. PAC-1 has activity against a wide range of cancer cell lines, and in animal models of cancer. PAC-1 crosses the blood brain barrier and has been shown to synergize with TMZ in both canine malignant glioma and meningioma that arise spontaneously. Methods: This Phase I dose escalation study uses a modified- Fibonacci 3+3 design to determine the MTD of PAC-1 when combined with TMZ in patients with recurrent malignant gliomas: anaplastic astrocytoma (AA) and GBM (open to enrollment). Here, we focus on component 2 of the study. Primary objectives: to establish MTD of PAC-1 when combined with a fixed dose of TMZ, tolerability, and toxicity using CTCAE v.4. Secondary and correlative objectives: pharmacokinetics, pharmacodynamics, preliminary anti-tumor activity correlation with procaspase-3 expression in tumor tissue, radiographic response using the Response Assessment in Neuro-Oncology (RANO) criteria, and neurocognitive function using a validated test battery. Inclusion criteria: diagnosis of recurrent high grade glioma (AA or GBM), ECOG PS 0-2, adequate organ function. Exclusion criteria: received prior cytotoxic therapy in the last 3-6 weeks (duration based on prior therapy) or uncontrolled chronic illness. Administration and design, Component 2: PAC-1, orally administered, is dosed at 375-650 mg daily (up to 3 dose levels) on days 1-21 of each 28-day cycle. A fixed dose of TMZ, (150 mg/m2), is administered orally, days 8 -12 of each cycle. The study is currently enrolling patients for Component 2. Clinical trial information: NCT02355535.

Diagnostic accuracy of stereotactic brain biopsy for intracranial neoplasia in dogs: Comparison of biopsy, surgical resection, and necropsy specimens.

BackgroundStereotactic brain biopsy (SBB) is a technique that allows for definitive diagnosis of brain lesions. Little information is available regarding the diagnostic utility of SBB in dogs with intracranial diseases.ObjectiveTo investigate the diagnostic accuracy (DA) of SBB in dogs with brain tumors.AnimalsThirty‐one client‐owned dogs that underwent SBB followed by surgical resection or necropsy examinations.MethodsRetrospective observational study. Two pathologists blinded to SBB and reference standard diagnoses reviewed histologic specimens and typed and graded tumors according to World Health Organization and revised canine glioma classification criteria. Agreement between tumor type and grade from SBB were compared to reference standards and assessed using kappa statistics. Patient and technical factors associated with agreement also were examined.ResultsStereotactic brain biopsy specimens were obtained from 24 dogs with gliomas and 7 with meningiomas. Tumor type agreement between SBB and the reference standard was observed in 30/31 cases (κ = 0.95). Diagnostic concordance was perfect for meningiomas. Grade agreement among gliomas was observed in 18/23 cases (κ = 0.47). Stereotactic brain biopsy underrepresented the reference standard glioma grade in cases with disagreement. The DA of SBB was 81%, with agreement noted in 56/69 biopsy samples. Smaller tumors and fewer SBB specimens obtained were significantly associated with diagnostic discordance.Conclusions and Clinical ImportanceThe DA of SBB readily allows for the diagnosis of common brain tumors in dogs. Although glioma grade discordance was frequent, diagnoses obtained from SBB are sufficient to currently inform therapeutic decisions. Multiple SBB specimens should be collected to maximize DA.

Magnetic resonance imaging and pathological features of a mixed glioma in a dog: case report

ABSTRACT The aim of this report was to describe the magnetic resonance imaging (MRI) and pathological features of a canine mixed glioma. A 12-year-old boxer male dog was presented for necropsy along with data from an MRI evaluation conducted ante-mortem. The images were examined and showed a poorly demarcated prosencephalic lesion, hyperintense on T2W images, hypointense on T1W images and heterogeneously hyperintense on T2W FLAIR images. There was mild nonuniform contrast enhancement, apparent midline shift, moderate perilesional edema and marked distortion of the adjacent lateral ventricle. The brain was evaluated macroscopically, microscopically and immunohistochemically. Grossly, there was a poorly demarcated soft mass, with areas of hemorrhage, within the left parietal and temporal lobes. Histologically, there was a densely cellular mass composed of two geographically distinct populations of neoplastic cells. The first population was composed of small and round cells organized in a honeycomb pattern. The second population constituted of intermingled streams and bundles of neoplastic cells that were strongly immunolabeled for glial fibrillary acidic protein (GFAP). The diagnosis of a mixed glioma was based on MRI findings, and mainly on histological and immunohistochemical findings.

Naturally Occurring Canine Glioma as a Model for Novel Therapeutics

Background: Current animal models of glioma are limited to small animal models, which are less predictive of treatment of human disease. Canines often develop gliomas de novo, but the natural history of the disease is not well described. Objective: We provide data for naturally occurring canine gliomas; evaluating medical and surgical therapies. Methods: We reviewed medical records of pet dogs with a presumptive diagnosis of glioma from MRI imaging that underwent surgery as part of the Canine Brain Tumor Clinical Trials Program. Breed, age, sex, median progression-free, and overall survival times and cause of death were recorded for multivariate analysis. Results: Ninety five dogs (56 male; mean age = 8.3 years) were included, but nine were excluded as final pathology was non-neoplastic. Gross total resection was reported in 81 cases based on postoperative MRI. Seventy had high-grade tumors (grade III or IV). Eighty three dogs presented with seizures, being the most common presenting clinical sign. Median survival after surgery was 723 days (95% CI 343–1103) for grade II tumors, 301 days (197–404) for grade III and 200 days (126–274) for grade IV (p = .009 Kaplan–Meier survival analysis; Log Rank test). Age (cox regression, p = .14) or sex (Kaplan–Meier test, p = .22) did not predict survival. Conclusions: This study establishes normative data for a model exploiting dogs with naturally occurring glioma, which can be used to test novel therapies prior to translation to human trials. Further work will focus on the effects of different therapies, including chemotherapy, radiation therapy, and immunotherapy.

Checkpoint Blockade Reverses Anergy in IL-13Rα2 Humanized scFv-Based CAR T Cells to Treat Murine and Canine Gliomas

We generated two humanized interleukin-13 receptor α2 (IL-13Rα2) chimeric antigen receptors (CARs), Hu07BBz and Hu08BBz, that recognized human IL-13Rα2, but not IL-13Rα1. Hu08BBz also recognized canine IL-13Rα2. Both of these CAR T cell constructs demonstrated superior tumor inhibitory effects in a subcutaneous xenograft model of human glioma compared with a humanized EGFRvIII CAR T construct used in a recent phase 1 clinical trial (ClinicalTrials.gov: NCT02209376). The Hu08BBz demonstrated a 75% reduction in orthotopic tumor growth using low-dose CAR T cell infusion. Using combination therapy with immune checkpoint blockade, humanized IL-13Rα2 CAR T cells performed significantly better when combined with CTLA-4 blockade, and humanized EGFRvIII CAR T cells’ efficacy was improved by PD-1 and TIM-3 blockade in the same mouse model, which was correlated with the levels of checkpoint molecule expression in co-cultures with the same tumor in vitro. Humanized IL-13Rα2 CAR T cells also demonstrated benefit from a self-secreted anti-CTLA-4 minibody in the same mouse model. In addition to a canine glioma cell line (J3T), canine osteosarcoma lung cancer and leukemia cell lines also express IL-13Rα2 and were recognized by Hu08BBz. Canine IL-13Rα2 CAR T cell was also generated and tested in vitro by co-culture with canine tumor cells and in vivo in an orthotopic model of canine glioma. Based on these results, we are designing a pre-clinical trial to evaluate the safety of canine IL-13Rα2 CAR T cells in dog with spontaneous IL-13Rα2-positive glioma, which will help to inform a human clinical trial design for glioblastoma using humanized scFv-based IL-13Rα2 targeting CAR T cells.

A Revised Diagnostic Classification of Canine Glioma: Towards Validation of the Canine Glioma Patient as a Naturally Occurring Preclinical Model for Human Glioma.

The National Cancer Institute-led multidisciplinary Comparative Brain Tumor Consortium (CBTC) convened a glioma pathology board, comprising both veterinarian and physician neuropathologists, and conducted a comprehensive review of 193 cases of canine glioma. The immediate goal was to improve existing glioma classification methods through creation of a histologic atlas of features, thus yielding greater harmonization of phenotypic characterization. The long-term goal was to support future incorporation of clinical outcomes and genomic data into proposed simplified diagnostic schema, so as to further bridge the worlds of veterinary and physician neuropathology and strengthen validity of the dog as a naturally occurring, translationally relevant animal model of human glioma. All cases were morphologically reclassified according to a new schema devised by the entire board, yielding a majority opinion diagnosis of astrocytoma (43, 22.3%), 19 of which were low-grade and 24 high-grade, and oligodendroglioma (134, 69.4%), 35 of which were low-grade and 99 were high-grade. Sixteen cases (8.3%) could not be classified as oligodendroglioma or astrocytoma based on morphology alone and were designated as undefined gliomas. The simplified classification scheme proposed herein provides a tractable means for future addition of molecular data, and also serves to highlight histologic similarities and differences between human and canine glioma.

Abstract 1176: Genomic profiling of canine glioma: Comparative analyses with respect to drivers of human glioma

Abstract Sporadic glioma occurs in companion dogs at frequencies comparable to humans, despite differences in environmental exposures and age at time of diagnosis. Despite advances in molecular and phenotypic characterization of adult gliomas, accurate prognostication and curative treatment modalities are often limited by lack of animal translational models that can faithfully recapitulate underlying oncogenic processes and treatment response to newer therapies. Studying canine glioma at molecular level has several merits: First, it has a distinct advantage over genetically modified animal models to study natural course of glioma in dogs. Second, breed-specific elevated cancer risk, e.g., short-nosed breeds have higher risk for gliomas and smaller effective population size compared to humans potentially allow better characterization driver elements in the evolving canine glioma. Finally, dogs with the natural mammalian immune system have spontaneous occurrence of glioma, making it an ideal model for preclinical immunotherapy testing. Based on these merits, we performed whole genome, exome, transcriptome and methylation (reduced bisulfite) sequencing on 178 canine tumor and germline samples. As in humans, we show frequently occurring mutations and copy number alterations in canine gliomas in p53 pathway, cell cycle pathway (CDK4, CDKN2A), and receptor tyrosine kinases (EGFR, PDGFRA). We identified R132 mutations in the IDH1 gene reflecting a remarkable and species-agnostic but cancer-specific driving effect. Frequent whole chromosome gains were observed of syntenic region of chromosome 13, harboring human glioma oncogenes PDGFRA and MYC, but human glioma specific changes such as 1p/19q co-deletion and whole chromosome 10 loss/whole chromosome 7 gain were absent. We calculate mutational processes and highlight ones related to DNA damage repair and transcriptional strand bias in driving glioma in both species. Based on recent study, we also estimate relative timing of mutations in driver genes and copy number gains in canine glioma and compare those to human glioma in mapping life history of glioma, i.e., are canine glioma more similar to adult or pediatric human glioma cohort? In addition, transcriptional profiling delineated tumor-associated cell fractions from 22 immune cell types. The monocyte gene signature scored highest among canine glioma transcriptomes, in contrast to macrophages/microglia in human disease. Our results suggest similarities between the canine and human glioma microenvironment and we are validating those using canine immunohistochemistry. In bringing together a large canine glioma genomic and transcriptomic sequencing dataset and comparing to human glioma, our study provides unique new insights into glioma etiology and the chronology of glioma-causing somatic alterations. Citation Format: Samirkumar B. Amin, Juan Emmanuel Martinez-Ledesma, Beth Boudreau, Hoon Kim, Kevin C. Johnson, Peter V. Dickinson, Rebecca Packer, Amanda R. Taylor, John H. Rossmeisl, Amy Heimberger, Jonathan Levine, Roel Verhaak. Genomic profiling of canine glioma: Comparative analyses with respect to drivers of human glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1176.

Canine (Pet Dog) Tumor Microsurgery and Intratumoral Concentration and Safety of Metronomic Chlorambucil for Spontaneous Glioma: A Phase I Clinical Trial

Metronomic (daily low-dose) chlorambucil requires further study before use in human patients with glioma. The aim of this study was to investigate distribution and safety of metronomic chlorambucil in naturally occurring canine glioma. Eight client-owned (pet) dogs with newly diagnosed spontaneous glioma were prospectively enrolled. Chlorambucil was administered preoperatively at 4 mg/m2 every 24 hours for ≥3 days and continued postoperatively until death or dose-limiting adverse events. Chlorambucil concentrations in the surgical glioma specimen, cerebrospinal fluid, and serum were analyzed. Dogs additionally received lomustine postoperatively. Dogs were monitored for seizures, myoclonus, cytopenias, and tumor recurrence. Complete microsurgical resection was achieved in 7 oligodendrogliomas and 1 astrocytoma (6 high grade, 2 low grade). Median surgical glioma specimen chlorambucil concentration was 0.52 ng/g (range, 0-2.62 ng/g), or 37% (range, 0%-178%) of serum concentration. Median cerebrospinal fluid concentration was 0.1 ng/mL (range, 0-0.3 ng/mL). Chlorambucil was not associated with increase in seizure activity. Six dogs displayed prolonged seizure-free intervals. There was no myoclonus. Three dogs developed asymptomatic thrombocytopenia after 8-12 months of chlorambucil. Median progression-free survival was 253 days (range, 63-860 days). Median overall survival was 257 days (range, 64-860 days). The presence of intratumoral chlorambucil indicated an altered blood-brain barrier that varied from case to case. Despite sporadic previous reports of neurotoxicity, prolonged seizure-free intervals supported a high safety margin at this dose in this species. Metronomic chlorambucil was well tolerated. Spontaneous canine glioma offers a robust preclinical model.

Immunologic and gene expression profiles of spontaneous canine oligodendrogliomas.

Malignant glioma (MG), the most common primary brain tumor in adults, is extremely aggressive and uniformly fatal. Several treatment strategies have shown significant preclinical promise in murine models of glioma; however, none have produced meaningful clinical responses in human patients. We hypothesize that introduction of an additional preclinical animal model better approximating the complexity of human MG, particularly in interactions with host immune responses, will bridge the existing gap between these two stages of testing. Here, we characterize the immunologic landscape and gene expression profiles of spontaneous canine glioma and evaluate its potential for serving as such a translational model. RNA in situ hybridization, flowcytometry, and RNA sequencing were used to evaluate immune cell presence and gene expression in healthy and glioma-bearing canines. Similar to human MGs, canine gliomas demonstrated increased intratumoral immune cell infiltration (CD4+, CD8+ and CD4+Foxp3+ T cells). The peripheral blood of glioma-bearing dogs also contained a relatively greater proportion of CD4+Foxp3+ regulatory T cells and plasmacytoid dendritic cells. Tumors were strongly positive for PD-L1 expression and glioma-bearing animals also possessed a greater proportion of immune cells expressing the immune checkpoint receptors CTLA-4 and PD-1. Analysis of differentially expressed genes in our canine populations revealed several genetic changes paralleling those known to occur in human disease. Naturally occurring canine glioma has many characteristics closely resembling human disease, particularly with respect to genetic dysregulation and host immune responses to tumors, supporting its use as a translational model in the preclinical testing of prospective anti-glioma therapies proven successful in murine studies.

Cover Image, Volume 15, Issue 4

The cover image, by S. R. Lai et al., is based on the Original Article In vitro anti‐tubulin effects of mebendazole and fenbendazole on canine glioma cells, DOI: 10.1111/vco.12288 image

Epidermal growth factor receptor activity is elevated in glioma cancer stem cells and is required to maintain chemotherapy and radiation resistance.

Glioblastoma remains among the most aggressive of all human and canine malignancies, displaying high mortality rates and limited treatment options. We propose that given the similarities between canine and human gliomas, such as incidence of occurrence, histopathology, molecular characteristics, and response to therapy, that canine gliomas are a natural model of the human disease. A range of human and canine tumours have been shown to harbor specific subpopulations of cells with stem cell-like properties that initiate and maintain neoplasticity while resisting conventional therapies. Here, we show that both canine and human glioma cell lines contain a small population of cancer stem cells (CSCs), and by molecular profiling highlight the important role of the epidermal growth factor receptor (EGFR) pathway in canine CSCs. EGFR signaling is crucial in the regulation of cancer cell proliferation, migration and survival. To date EGFR-targeted interventions alone have been largely ineffective. Our findings confirm that specifically inhibiting EGFR signaling alone has no significant effect on the viability of CSCs. However inhibition of EGFR did enhance the chemo- and radio-sensitivity of both canine and human glioma CSCs, enabling this resistant, tumourigenic population of cells to be effectively targeted by conventional therapies.

Synergistic and targeted therapy with a procaspase-3 activator and temozolomide extends survival in glioma rodent models and is feasible for the treatment of canine malignant glioma patients.

PurposeGlioblastoma is a deadly brain cancer with a median survival time of ∼15 months. Ionizing radiation plus the DNA alkylator temozolomide (TMZ) is the current standard therapy. PAC-1, a procaspase-3 activating small molecule, is blood-brain barrier penetrant and has previously demonstrated ability to synergize with diverse pro-apoptotic chemotherapeutics. We studied if PAC-1 could enhance the activity of TMZ, and whether addition of PAC-1 to standard treatment would be feasible in spontaneous canine malignant gliomas.Experimental DesignUsing cell lines and online gene expression data, we identified procaspase-3 as a potential molecular target for most glioblastomas. We investigated PAC-1 as a single agent and in combination with TMZ against glioma cells in culture and in orthotopic rodent models of glioma. Three dogs with spontaneous gliomas were treated with an analogous human glioblastoma treatment protocol, with concurrent PAC-1.ResultsProcaspase-3 is expressed in gliomas, with higher gene expression correlating with increased tumor grade and decreased prognosis. PAC-1 is cytotoxic to glioma cells in culture and active in orthotopic rodent glioma models. PAC-1 added to TMZ treatments in cell culture increases apoptotic death, and the combination significantly increases survival in orthotopic glioma models. Addition of PAC-1 to TMZ and radiation was well-tolerated in 3 out of 3 pet dogs with spontaneous glioma, and partial to complete tumor reductions were observed.ConclusionsProcaspase-3 is a clinically relevant target for treatment of glioblastoma. Synergistic activity of PAC-1/TMZ in rodent models and the demonstration of feasibility of the combined regime in canine patients suggest potential for PAC-1 in the treatment of glioblastoma.