Pilot trial of perampanel on peritumoral hyperexcitability and clinical outcomes in newly diagnosed high-grade glioma.

Background: Glutamatergic neuron-glioma synaptogenesis and peritumoral hyperexcitability promote glioma growth in a positive feedback loop. The objective of this study was to evaluate the feasibility and estimated effect sizes of the AMPA-R antagonist, perampanel, on intraoperative electrophysiologic hyperexcitability and clinical outcomes. Methods: An open-label trial was performed comparing perampanel to standard of care (SOC) in patients undergoing resection of newly-diagnosed radiologic high-grade glioma. Perampanel was administered as a pre-operative loading dose followed by maintenance therapy until progressive disease or up to 12-months. SOC treatment involved levetiracetam for 7-days or as clinically indicated. The primary outcome of hyperexcitability was defined by intra-operative electrocorticography high frequency oscillation (HFO) rates. Seizure-freedom and overall survival (OS) were estimated by the Kaplan-Meier method. Tissue concentrations of perampanel, levetiracetam, and metabolites were measured by mass spectrometry. Results: HFO rates were similar between perampanel-treated and SOC cohorts. The trial was terminated early after interim analysis for futility, and outcomes assessed in 11 patients (7 perampanel-treated, 4 SOC). Over a median 281 days of post-enrollment follow-up, 27% of patients had seizures, including 14% treated with perampanel and 50% treated with SOC. OS in perampanel-treated patients was similar to a glioblastoma reference cohort (p=0.81). Glutamate concentrations in surface biopsies were positively correlated with HFO rates in adjacent electrode contacts and were not significantly associated with treatment assignment or drug concentrations. Conclusions: A peri-operative loading regimen of perampanel was safe and well-tolerated, with similar peritumoral hyperexcitability as in levetiracetam-treated patients. Maintenance anti-glutamatergic therapy was not observed to impact survival outcomes.

Investigative needle core biopsies for multi-omics in Glioblastoma.

Glioblastoma (GBM) is a primary brain cancer with an abysmal prognosis and few effective therapies. The ability to investigate the tumor microenvironment before and during treatment would greatly enhance both understanding of disease response and progression, as well as the delivery and impact of therapeutics. Stereotactic biopsies are a routine surgical procedure performed primarily for diagnostic histopathologic purposes. The role of investigative biopsies - tissue sampling for the purpose of understanding tumor microenvironmental responses to treatment using integrated multi-modal molecular analyses ('Multi-omics") has yet to be defined. Secondly, it is unknown whether comparatively small tissue samples from brain biopsies can yield sufficient information with such methods. Here we adapt stereotactic needle core biopsy tissue in two separate patients. In the first patient with recurrent GBM we performed highly resolved multi-omics analysis methods including single cell RNA sequencing, spatial-transcriptomics, metabolomics, proteomics, phosphoproteomics, T-cell clonotype analysis, and MHC Class I immunopeptidomics from biopsy tissue that was obtained from a single procedure. In a second patient we analyzed multi-regional core biopsies to decipher spatial and genomic variance. We also investigated the utility of stereotactic biopsies as a method for generating patient derived xenograft models in a separate patient cohort. Dataset integration across modalities showed good correspondence between spatial modalities, highlighted immune cell associated metabolic pathways and revealed poor correlation between RNA expression and the tumor MHC Class I immunopeptidome. In conclusion, stereotactic needle biopsy cores are of sufficient quality to generate multi-omics data, provide data rich insight into a patient's disease process and tumor immune microenvironment and can be of value in evaluating treatment responses. One sentence summary: Integrative multi-omics analysis of stereotactic needle core biopsies in glioblastoma.

An oncolytic virus expressing a full-length antibody enhances antitumor innate immune response to glioblastoma.

Oncolytic herpes simplex virus-1 is capable of lysing tumor cells while alerting the immune system. CD47, in collaboration with SIRPα, represents an important immune checkpoint to inhibit phagocytosis by innate immune cells. Here we show locoregional control of glioblastoma by an oncolytic herpes virus expressing a full-length anti(α)-human CD47 IgG1 or IgG4 antibody. The antibodies secreted by the virus-infected glioblastoma cells block the CD47 'don't eat me' signal irrespective of the subclass; however, αCD47-IgG1 has a stronger tumor killing effect than αCD47-IgG4 due to additional antibody-dependent cellular phagocytosis by macrophages and antibody-dependent cellular cytotoxicity by NK cells. Intracranially injected αCD47-IgG1-producing virus continuously releases the respective antibody in the tumor microenvironment but not into systemic circulation; additionally, αCD47-IgG1-producing virus also improves the survival of tumor-bearing mice better than control oncolytic herpes virus combined with topical αCD47-IgG1. Results from immunocompetent mouse tumor models further confirm that macrophages, and to a lesser extent NK cells, mediate the anti-tumor cytotoxicity of antibody-producing oncolytic herpesviruses. Collectively, oncolytic herpes simplex virus-1 encoding full-length antibodies could improve immune-virotherapy for glioblastoma.

Annexin A2 regulates angiogenesis and invasion phenotypes of malignant glioma.

We have established a pair of animal models (J3T-1 and J3T-2) with different invasive and angiogenic phenotypes, and demonstrated that annexin A2 is expressed at higher levels in J3T-1 than J3T-2 cells. The function of annexin A2 in relation to angiogenesis and invasion was investigated using these models. Stable silencing or overexpression of annexin A2 in J3T-1 and J3T-2 cells (J3T-1shA and J3T-2A cells) was established and used. Thirty human glioblastoma samples were evaluated for expression of annexin A2, vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). Immunohistochemical and quantitative reverse-transcription polymerase chain reaction analyses revealed higher expression of annexin A2, VEGF and PDGF in J3T-1 and J3T-2A cells. Cultured J3T-1 and J3T-2A cells exhibited higher adhesive ability to endothelial cells. Histopathological analysis of animal brain tumors revealed that J3T-1 and J3T-2A tumors displayed marked angiogenesis and invasion along the neovasculature, whereas J3T-2 and J3T-1shA tumors exhibited diffuse, infiltrative invasion without angiogenesis. Positive expression of annexin A2 was observed in tumor cells surrounding dilated vessels in 25/30 human glioblastoma specimens. Our results reveal that the phenotype of glioma invasion is closely related to angiogenesis. We identify annexin A2 as a factor regulating angiogenesis and invasion of malignant gliomas.

Developmental expression of GPR3 in rodent cerebellar granule neurons is associated with cell survival and protects neurons from various apoptotic stimuli.

G-protein coupled receptor 3 (GPR3), GPR6, and GPR12 belong to a family of constitutively active Gs-coupled receptors that activate 3'-5'-cyclic adenosine monophosphate (cAMP) and are highly expressed in the brain. Among these receptors, the endogenous expression of GPR3 in cerebellar granule neurons (CGNs) is increased following development. GPR3 is important for neurite outgrowth and neural maturation; however, the physiological functions of GPR3 remain to be fully elucidated. Here, we investigated the survival and antiapoptotic functions of GPR3 under normal and apoptosis-inducing culture conditions. Under normal culture conditions, CGNs from GPR3-knockout mice demonstrated lower survival than did CGNs from wild-type or GPR3-heterozygous mice. Cerebellar sections from GPR3-/- mice at P7, P14, and P21 revealed more caspase-3-positive neurons in the internal granular layer than in cerebellar sections from wild-type mice. Conversely, in a potassium-deprivation model of apoptosis, increased expression of these three receptors promoted neuronal survival. The antiapoptotic effect of GPR3 was also observed under hypoxic (1% O2/5% CO2) and reactive oxygen species (ROS)-induced apoptotic conditions. We further investigated the signaling pathways involved in the GPR3-mediated antiapoptotic effect. The addition of the PKA inhibitor KT5720, the MAP kinase inhibitor U0126, and the PI3 kinase inhibitor LY294002 abrogated the GPR3-mediated antiapoptotic effect in a potassium-deprivation model of apoptosis, whereas the PKC inhibitor Gö6976 did not affect the antiapoptotic function of GPR3. Furthermore, downregulation of endogenous GPR3 expression in CGNs resulted in a marked reduction in the basal levels of ERK and Akt phosphorylation under normal culture conditions. Finally, we used a transient middle cerebral artery occlusion (tMCAO) model in wild-type and GPR3-knockout mice to determine whether GPR3 expression modulates neuronal survival after brain ischemia. After tMCAO, GPR3-knockout mice exhibited a significantly larger infarct area than did wild-type mice. Collectively, these in vitro and in vivo results suggest that the developmental expression of constitutively active Gs-coupled GPR3 activates the ERK and Akt signaling pathways at the basal level, thereby protecting neurons from apoptosis that is induced by various stimuli.

Histone Deacetylase Inhibitors Augment Antitumor Efficacy of Herpes-based Oncolytic Viruses.

Replication-conditional (oncolytic) mutants of herpes simplex virus (HSV), are considered promising therapeutic alternatives for human malignancies, and chemotherapeutic adjuvants are increasingly sought to augment their efficacy. Histone deacetylase (HDAC) inhibitors are a new class of antineoplastic agents because of their potent activity in growth arrest, differentiation, and apoptotic death of cancer cells. The ability of the HDAC inhibitors to upregulate exogenous transgene expression and inhibit interferon (IFN) responses prompted our exploration of their use in improving the antitumor efficacy of oncolytic HSV. We discovered that the yield of viral progeny increased significantly when cultured glioma cells were treated with HDAC inhibitors before viral infection. Valproic acid (VPA), a commonly used antiepileptic agent with HDAC inhibitory activity, proved most effective when used to treat glioma cells before viral infection, but not concomitantly with viral infection. Pretreatment with VPA inhibited the induction of several IFN-responsive antiviral genes, augmented the transcriptional level of viral genes, and improved viral propagation, even in the presence of type I IFNs. Moreover, VPA pretreatment improved the propagation and therapeutic efficacy of oncolytic HSV in a human glioma xenograft model in vivo. These findings indicate that HDAC inhibitors can improve the efficacy of tumor virotherapies.