Angiogenesis and Invasion Ai-01. Case Report: Optic Neuropathy in a Patient with Glioblastoma Receiving Bevacizumab

ANGIOGENESIS AND INVASION AI-01. CASE REPORT: OPTIC NEUROPATHY IN A PATIENT WITH GLIOBLASTOMA RECEIVING BEVACIZUMAB Robert A. Fishman , Erik Happ , Tracey Stevens , and Lara Kunschner ; Allegheny General Hospital Bevacizumab, a humanized monoclonal antibody developed to target and subsequently block vascular endothelial growth factor, received FDA approval for single agent treatment of recurrent glioblastoma multiforme (GBM) in 2009. Initial response rates and progression-free survival are promising for patients treated with bevacizumab, leading to its widespread adoption in both salvage therapy and as experimental initial therapy for GBM. Established bevacizumab side effects include hypertension, delayed/ impaired wound healing, gastrointestinal perforation, and proteinuria. Six cases have recently been reported of optic neuropathy following bevacizumab treatment for GBM (Sherman J, Aregawi A, Fathallah S, Bierman P. J., Linsky K, Larner S, Newman D, Schiff D, Neurology 2009;73;1924– 1926). We report here the case of a 24-year-old woman with neurofibromatosis type I (NF-1) and right thalamic GBM (WHO grade IV) who developed a severe, subacute optic neuropathy during treatment with bevacizumab and irinotecan for recurrent GBM following standard initial chemoradiotherapy with temozolomide. After completing 4 cycles of salvage chemotherapy with bevacizumab and irinotecan, the patient developed diffuse blurring of vision, which progressed over 3–4 weeks, and eventually total blindness. Six weeks following cessation of bevacizumab, the patient’s vision improved, with return of light perception and eventual return of baseline vision. Our review of the literature has revealed few proposed mechanisms that may lead to bevacizumab-induced optic neuropathy; these mechanisms include arterial thrombosis, decreased optic nerve tolerance to radiation therapy, and upregulation of VEGF receptors with ensuing neovascularization and optic ischemia following radiation therapy. Our patient’s follow-up will include stringent eye exams by an ophthalmologist, to include confrontational visual field testing, external and anterior segment examination, and dilated fundus examination. Our completed poster will include a summary of our case, magnetic resonance images of the tumor, and results of ophthalmological examination during and following bevacizumab treatment. AI-02. DEGRADOME PROFILING FAILS TO IDENTIFY A UNIQUE PROTEASE SIGNATURE IN PRIMARY MALIGNANT BRAIN TUMORS Diane M. Jaworski 1, Holly M. Stradecki 1, Paul L. Penar 1, William W. Pendlebury 1, Caroline J. Pennington 2, Dylan R. Edwards 2, William C. Broaddus 3, and Helen L. Fillmore 3; Univ. of Vermont College of Medicine; Univ. of East Anglia; Virginia Commonwealth Univ. The primary treatment challenge presented by glioblastoma multiforme (GBM) is the insidious propensity of glioma cells to aggressively infiltrate normal brain, making surgical resection palliative rather than curative. Much attention has been focused on the role of proteases in tumor invasion. Although the human degradome, the repertoire of proteases produced by cells, consists of at least 569 proteases, most studies have restricted their attention to matrix metalloproteinases (MMPs). However, the poor outcomes of the MMP inhibitor clinical trials suggest a considerable contribution of other protease families to glioma invasion. TaqMan low-density array (TLDA) microfluidic cards containing primer/probe sets for 380 of the most widely expressed degradome genes were used to compare the protease signature of normal brain (i.e., from patients undergoing surgery for epilepsy) with those of metastatic and primary brain tumors (e.g., meningioma, oligodendroglioma, and astrocytoma). Supervised cluster analysis revealed distinct tumor-specific degradome signatures for all tumor types except astrocytoma. In every GBM surgical specimen analyzed, 22 genes were upregulated and 2 genes were downregulated to at least 4 times the level in normal brain; however, none of those genes was specific for GBM. Although TLDA profiling did not identify a GBM-specific protease signature, patient-specific protease signatures were apparent. As proof of principle that TLDA could better define resection boundaries, magnetic resonance imaging-defined stereotactic samples of tumor core, tumor-stromal interface, and normal adjacent brain were analyzed from one patient. While the tumor core (102 genes) and tumor-stromal interface (139 genes) had significantly increased protease expression, the normal adjacent brain still had 84 genes increased to at least 4 times the level of expression of control samples, indicating the need for greater surgical margins. These observations suggest that TLDA screening may prove a useful presurgical tool to establish surgical borders and tailor-made antiprotease cocktail therapies that may be more efficacious than previous single MMP inhibitor therapies. AI-03. ROLE OF NEUROPILIN1 AND ITS CO-FACTORS VEGF AND SEMA3A IN REGULATING HETEROGENEOUS MICROENVIRONMENT OF GLIOBLASTOMA MULTIFORME Joydeep Mukherjee 1, Cynthia Hawkins 2, and Abhijit Guha 3; The Hospital for Sick Children; Department of Pathology, The Hospital for Sick Children; Department of Neurosurgery, Western Hospital AND The Hospital For Sick Children INTRODUCTION: Glioblastoma multiforme (GBM) is characterized by pathological heterogeneity reflecting molecular heterogeneity in response to interactions with the tumor microenvironment and stromal cells. In this study, we focused on regional differences in the expression and interactions of Neuropilin1 (Nrp1) with its cofactors to activate the vascular endothelial growth factor (VEGF)165-VEGFR2 signaling axis or the Semaphorin3A (SEMA3A)-PlexinA1 signaling axis, thereby regulating neo-angiogenesis, migration and invasion. METHODS/RESULTS: Twenty-five paired flashfrozen and paraffin-embedded sections from the hypoxic center and normoxic periphery of GBMs were obtained on the basis of intra-operative magnetic resonance imaging–based stereotaxy and neuropathological verification. Five non-neoplastic human brain specimens were used as controls. Laser capture microdissection (LCM) on serial flash frozen sections was used to isolate tumor cells and endothelial cells. Real-time-PCR and immunohistochemistry (IHC) was used to evaluate expression. Nrp1, SEMA3A and PlexinA1 were elevated at both the RNA and protein levels in the peripheral tumor cells, while VEGF165 and VEGFR2 were increased in the central tumor cells and endothelial cells, respectively. This suggested that spatial gradients of Sema3A and VEGF may promote differential Nrp1 binding within the GBM center and periphery. Using genetic and pharmacological approaches to modulate Nrp1, VEGF165, SEMA3A, and PlexinA1, we found that Nrp1-facilitated activation of VEGF165-VEGFR2 or SEMA3A-PlexinA1 signaling results in the activation and deactivation of alphaVbeta3 integrin, respectively, with subsequent activation of focal adhesion kinase and metalloproteases. CONCLUSIONS: Our studies demonstrate how molecular heterogeneity related to the Nrp1-mediated activation of the VEGF and SEMA3A signaling pathways differs between regions of GBMs to contribute to pathological heterogeneity. AI-04. EFEMP1 ENHANCES INVASION OF U251HF HUMAN GLIOMA CELLS VIA MODULATION OF MMP2 Peter D. Pioli , Shadi Milani , Mark E. Linskey , and Yi-Hong Zhou ; University of California-Irvine Malignant gliomas are highly invasive primary brain tumors. The extracellular proteases and their inhibitors play a major role in defining a glioma cell’s ability to remodel its surroundings, thus allowing infiltration into surrounding normal brain tissue. One such family of proteases is the matrix metalloprotease (MMP) family, which has demonstrated the ability to cleave various extracellular proteins and function in cell-cell signaling and also motility. MMP2 and MMP9 are the two MMP members particularly found in gliomas and can be associated with an increase of glioma cell invasiveness. Our recent study revealed a novel tumor suppression function of EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1), which suppressed the tumorigenicity of the human glioma cell line U251HF, partially via suppression of angiogenesis, in s.c. xenograft models. However, we also found that EFEMP1 increased U251HF cell invasion through a Matrigel substrate. EFEMP1 have been shown to affect the tumorigenicity of multiple cancer types. Dependent upon cell type, EFEMP1 has displayed proand antitumorigenic capacities. Consistent with our report of MMP2 enhancement of U251HF invasiveness, we found that MMP2 was upregulated at the protein level, along with increased activity in zymography assays of U251HF-overexpressing EFEMP1. Since real-time qRT-PCR showed that MMP2 was not increased by EFEMP1 at the mRNA level, it appears that EFEMP1 activation of MMP2 occurs post-transcriptionally. The MMP inhibitor TIMP3 was reported to be a strong binding protein of EFEMP1, which likely underlies EFEMP1’s function in activating MMP2 by sequestering TIMP3 and therefore preventing its inhibition of MMP2. In conclusion, our data revealed that glioma invasiveness is also under positive regulation by EFEMP1 through modulation of MMP2 activity. The mechanism of EFEMP1 regulation of MMP2, with or without involvement of TIMP3, is under further investigation. Published by Oxford University Press on behalf of the Society for Neuro-Oncology 2010 by gest on Sptem er 4, 2016 http://neuro-oy.oxfordjournals.org/ D ow nladed from AI-05. USE OF HUMAN PROGENITOR CELLS FOR CELL-BASED DELIVERY OF ANGIOSTATIC GENES TO BRAIN TUMORS Valentina Marchetti , Faith Barnett , Matthew Wang , Lea Scheppke , Javier Sanchez-Cespedes , Charles De Rossi , Glen Nemerow, Bruce Torbett , and Martin Friedlander ; The Scripps Research Institute Cell-based delivery of antitumor genes, using neural and mesenchymal stem cells, has been proposed for the treatment of glioma. Previously, we have identified a rodent bone marrow population of endothelial progenitor cells (EPCs) that selectively target gliomas in vivo. These cells were transducible with lentivirus vectors (Barnett et al., CNS 2004). Previous studies have described the angiostatic molecule T2, a fragment of tryptophanyl-tRNA synthetase (TrpRS) (Tzima et al., PNAS 2003). We have also demonstrated that T2 has a potent antiglioma effect in vivo (Dorrell et al., PNAS 2007). In these studies, we defined a subpopulation of the EPCs, CD44 high expressing cells, which migrate to the tumor mass as well as microsatellites in vivo in a 9L tumor model. CD44 cells migrate to implanted tumor masses if injected into the contralateral hemisphere but do not if seeded peripherally. In this study, we set out to identify analogous populations of human cells that can be transduced by adenoand lentiviral vectors in order to induce an antitumor response. We have identified two candidate populations, bone marrow CD33+ cells and cord blood CD14+ cells. We developed transduction protocols using lentivirus and adenovirus vectors, infecting 65% of CD33+ cells and 75% of CD14+ cells, respectively. T2 gene was inserted in adenoviral vectors and secreted by CD14+ cells. Western blot analysis confirmed T2 expression. In vitro, CD14+ cells targeted 9L and U87 glioma cell lines. In vivo, the activation of CD14+ cells with LPS gave similar results to those observed with murine CD44 cells. In conclusion, we have identified two human cell populations useful as vehicles for delivering antitumor genes to brain tumors. Future studies to assess the efficacy of antiangiogenic molecules in this cell-based tumor model are underway. Supported by grants from the National Eye Institute to MF (EY11254 and