Hassan M. Fathallah-Shaykh

Gene Transfer of IFN-γ into Established Brain Tumors Represses Growth by Antiangiogenesis

The experiments in this paper were designed to examine the therapeutic effects of adenoviral-mediated gene transfer of IFN-γ into a mouse model of an established metastatic brain tumor. Temperature-sensitive replication-defective adenovirus was generated for gene transfer of IFN-γ (AdIFN) and β-galactosidase (AdBGAL) cDNAs in vivo. In this model, treatment with AdIFN elicits prolonged survival times and brain tumor rejection. Evidence against an immune-mediated response accounting for this result include: 1) absence of a memory immune response upon challenge, 2) lack of antitumor effects at sites distal to inoculation of AdIFN, and 3) preservation of the therapeutic effects of AdIFN in scid and beige mice and in inducible NO synthase (iNOS) knockouts. High concentrations of IFN-γ do not inhibit tumor growth in vitro making it unlikely that the antitumor effect of this treatment acts directly on the growth of the tumor cells. However, gene transfer of IFN-γ inhibits neovascularization of the tumor in a 3LL-Matrigel assay in vivo, and AdIFN induces apoptosis of endothelial cells in vivo, supporting the idea that AdIFN represses tumor growth by inhibiting angiogenesis. The substantial non-immune-mediated therapeutic benefits of AdIFN in animals paves the way for devising novel strategies for treating human brain tumors.

A Phase 1 Trial of ABT-510 Concurrent With Standard Chemoradiation for Patients With Newly Diagnosed Glioblastoma

OBJECTIVE To determine the maximum tolerated dose of ABT-510, a thrombospondin-1 mimetic drug with antiangiogenic properties, when used concurrently with temozolomide and radiotherapy in patients with newly diagnosed glioblastoma. DESIGN Phase 1 dose-escalation clinical trial. SETTING Comprehensive Cancer Center, University of Alabama at Birmingham. Patients A total of 23 patients with newly diagnosed, histologically verified glioblastoma enrolled between April 2005 and January 2007. INTERVENTIONS Four cohorts of 3 patients each received subcutaneous ABT-510 injection at doses of 20, 50, 100, or 200 mg/d. The maximum cohort was expanded to 14 patients to obtain additional safety and gene expression data. The treatment plan included 10 weeks of induction phase (temozolomide and radiotherapy with ABT-510 for 6 weeks plus ABT-510 monotherapy for 4 weeks) followed by a maintenance phase of ABT-510 and monthly temozolomide. MAIN OUTCOME MEASURES Patients were monitored with brain magnetic resonance imaging and laboratory testing for dose-limiting toxicities, defined as grades 3 or 4 nonhematological toxicities and grade 4 hematological toxicities. Therapy was discontinued if 14 maintenance cycles were completed, disease progression occurred, or if the patient requested withdrawal. Disease progression, survival statistics, and gene expression arrays were analyzed. RESULTS There were no grade 3 or 4 dose-limiting toxicity events that appeared related to ABT-510 for the dose range of 20 to 200 mg/d. A maximum tolerated dose was not defined. Most adverse events were mild, and injection-site reactions. The median time to tumor progression was 45.9 weeks, and the median overall survival time was 64.4 weeks. Gene expression analysis using TaqMan low-density arrays identified angiogenic genes that were differentially expressed in the brains of controls compared with patients with newly diagnosed glioblastoma, and identified FGF-1 and TIE-1 as being downregulated in patients who had better clinical outcomes. CONCLUSIONS ABT-510, at subcutaneous doses up to 200 mg/d, is tolerated well with concurrent temozolomide and radiotherapy in patients with newly diagnosed glioblastoma, and low-density arrays provide a useful method of exploring gene expression profiles.

OPTIC NEUROPATHY IN PATIENTS WITH GLIOBLASTOMA RECEIVING BEVACIZUMAB

Bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor, was recently approved for treatment of glioblastoma. Initial data indicate increased response rates and progression-free survival compared to historical controls. Despite these promising data, we have identified several cases of severe optic neuropathy in patients with glioblastoma treated with bevacizumab. ### Methods. We performed a retrospective record review from 2005 to 2008 to identify adult patients with glioblastoma receiving bevacizumab who developed severe optic neuropathy. Five institutions participated, including the University of Virginia, UCLA, Columbia University, Rush University, and the University of Nebraska. The UCLA patient has already been reported in a larger case series discussing patients with glioblastoma receiving bevacizumab.1 Age at diagnosis, gender, radiation therapy data, chemotherapeutic regimens including the bevacizumab dosing schedule, ophthalmologic records, CSF results, and MRI were assessed. ### Standard protocol approvals, registrations, and patient consents. Each institution provided institutional review board approval. Since data were collected retrospectively without identifiers, institutional review boards did not require patient or surrogate consent. ### Results. Six patients (5 women) were identified. Median age at diagnosis was 61 years (range 37 to 68). Following surgery, all patients received fractionated radiation therapy with concomitant temozolomide. One patient received bevacizumab at initial diagnosis; 5 received it at progression. Tumors received 60 Gy delivered in a mean of 30 fractions. Mean radiation dose to the optic chiasm, left optic nerve, and right optic nerve was 5,602.4 cGy, 3,673 cGy, and …

Expression of PRMT5 correlates with malignant grade in gliomas and plays a pivotal role in tumor growth in vitro

Abstract Protein arginine methyltransferase 5 (PRMT5) catalyzes the formation of ω-NG,N′G-symmetric dimethylarginine residues on histones as well as other proteins. These modifications play an important role in cell differentiation and tumor cell growth. However, the role of PRMT5 in human glioma cells has not been characterized. In this study, we assessed protein expression profiles of PRMT5 in control brain, WHO grade II astrocytomas, anaplastic astrocytomas, and glioblastoma multiforme (GBM) by immunohistochemistry. PRMT5 was low in glial cells in control brain tissues and low grade astrocytomas. Its expression increased in parallel with malignant progression, and was highly expressed in GBM. Knockdown of PRMT5 by small hairpin RNA caused alterations of p-ERK1/2 and significantly repressed the clonogenic potential and viability of glioma cells. These findings indicate that PRMT5 is a marker of malignant progression in glioma tumors and plays a pivotal role in tumor growth.

Priming in the brain, an immunologically privileged organ, elicits anti-tumor immunity

A crucial question in the study of tumor neuro‐immunology concerns the capacity of the central nervous system to initiate and execute an immune response. In a 100% fatal rat malignant glioma model, genetically modified tumors secreting INF‐γ intracerebrally generate an immune response resulting in a substantial increase in survival time, tumor rejection and specific systemic immunity. Tumors modified to secrete IL‐2 alone do not change the biologic behavior of transfected gliomas. INF‐γ induces elevated expression of major‐histocompatibility‐complex‐class‐I and ‐class‐II molecules in microglia throughout the brain and invokes enhanced tumor infiltration by CD4, CD8 and NK cells. These findings demonstrate successful immunization against a central‐nervous‐system tumor by direct priming in the brain with a live growth‐competent tumor vaccine. Int. J. Cancer 75:266–276, 1998.

Genomic Expression Discovery Predicts Pathways and Opposing Functions behind Phenotypes

Discovering states of genetic expression that are true to a high degree of certainty is likely to predict gene function behind biological phenotypes. The states of expression (up- or down-regulated) of 19,200 cDNAs in 10 meningiomas are compared with normal brain by an algorithm that detects only 1 false measurement per 192,000; 364 genes are discovered. The expression data accurately predict activation of signaling pathways and link gene function to specific phenotypes. Meningiomas appear to acquire aberrant phenotypes by disturbing the balanced expression of molecules that promote opposing functions. The findings expose interconnected genes and propose a role of genomic expression discovery in functional genomics of living systems.

Survival in a transgenic model of fals is independent of inos expression

Mutations in the gene encoding Cu, Zn superoxide dismutase (SOD1) have been shown to cause one form of familial amyotrophic lateral sclerosis (FALS). Experiments have indicated a toxic gain of function for the abnormal enzyme, although the precise mechanisms underlying mutant (m)SOD1 toxicity are still unclear. One possible hypothesis is based on the fact that reduced zinc binding to mSOD1 results in the enzymes enhanced ability to interact with nitric oxide (NO), generate peroxynitrite, and catalyze the nitration of tyrosine residues within critical cellular proteins. If the nitration\peroxynitrite hypothesis is correct and NO is critical in the pathophysiologic process induced by mSOD1, then alterations in NO synthesis might be expected to substantially affect the disease course. Because of its expression in motor neurons, neuronal NO synthase (nNOS) has been previously studied, but ablation of its activity either by pharmacologic means or by genetic manipulation using nNOS knockout mice had no effect on the course of disease in mSOD1 transgenic mice. However, these experiments did not inhibit inducible(i) NOS to any significant extent and thus could not address a potentially important contribution from iNOS to the disease process. Expression of iNOS is greatly increased within the spinal cords of mSOD1 transgenic mice as they age and develop weakness. Importantly, iNOS expression is localized to astrocytes and microglia. These cell populations undergo activation during preclinically symptomatic time periods in mSOD1 mice and express critical proteins that might serve as targets for peroxynitrite mediated injury. For these reasons, we elected to study the effect of iNOS in mSOD1induced disease. Mice with targeted deletions of both iNOS alleles (B6,129 background), as well as transgenic mice expressing human SOD1 with a glycine to alanine change at codon 93, (B6SJL strain; JR2300) were obtained from Jackson Labs. These two lines were crossed and F1 offspring positive for the G93A transgene were crossed with F1 siblings lacking the transgene. The F2 generation therefore afforded progeny carrying all iNOS genotypes (iNOS 1\1, iNOS 1\2, and iNOS 2\2) with or without the G93A SOD1 transgene and were followed longitudinally for survival (see Fig). The mean survival for G93A SOD1 mice homozygous for both deleted iNOS alleles was 254 6 3 days. This value does not significantly differ from the mean survival of G93A SOD1 littermates heterozygous for the targeted iNOS allele (257 6 6 days) or G93A SOD1 mice with both wild-type iNOS alleles (253 6 5 days). Pathologic features including motor neuron loss, astrogliosis, and microglial activation were similar in spinal cord sections taken from both G93A SOD1 mice and G93A 93A SOD1 mice lacking iNOS (data not shown). The results of this study indicate that survival and disease course in mice expressing a G93A SOD1 mutation is independent of iNOS expression. Taken together with studies finding little effect of nNOS on mutant SOD1-induced disease, these results would appear to offer an in vivo challenge to the importance of the nitration hypothesis for FALS pathogenesis.

Gene Expression Analysis in Radiotherapy Patients and C57BL/6 Mice as a Measure of Exposure to Ionizing Radiation

Abstract Dose assessment after radiological disasters is imperative to decrease mortality through rationally directed medical intervention. Our goal was to identify biomarkers capable of qualitative (nonirradiated/irradiated) and/or quantitative (dose) assessment of radiation exposure. Using real-time quantitative PCR, biodosimetry genes were identified in blood samples from cancer patients undergoing total-body irradiation. Time- (5, 12, 23, 48 h) and dose- (0–8 Gy) dependent changes in gene expression were examined in C57BL/6 mice. A training set was used to derive weighted voting classification algorithms (nonirradiated/irradiated) and continuous regression (dose assessment) models that were tested in a separate validation set of mice. Of eight biodosimetry genes identified in cancer patients (ACTA2, BBC3, CCNG1, CDKN1A, GADD45A, MDK, SERPINE1, Tnfrsf10b), expression of BBC3, CCNG1, CDKN1A, SERPINE1 and Tnfrsf10b was significantly (P < 0.05) increased in irradiated mice. CCNG1 and CDKN1A expression segregated irradiated mice from controls with an accuracy, specificity and sensitivity of 96.3, 100.0 and 94.4%, respectively, at 48 h. Multiple linear regression analysis predicted doses for the 0-, 1-, 2-, 4-, 6- and 8-Gy treatment groups as 0.0 ± 0.2, 1.6 ± 1.0, 2.9 ± 1.4, 5.1 ± 2.0, 5.3 ± 0.7 and 10.5 ± 5.6 Gy, respectively. These results suggest that gene expression analysis could be incorporated into biodosimetry protocols for qualitative and quantitative assessment of radiation exposure.

Mathematical modeling of noise and discovery of genetic expression classes in gliomas.

The microarray array experimental system generates noisy data that require validation by other experimental methods for measuring gene expression. Here we present an algebraic modeling of noise that extracts expression measurements true to a high degree of confidence. This work profiles the expression of 19 200 cDNAs in 35 human gliomas; the experiments are designed to generate four replicate spots/gene with switching of probes. The validity of the extracted measurements is confirmed by: (1) cluster analysis that generates a molecular classification differentiating glioblastoma from lower-grade tumors and radiation necrosis; (2) By what other investigators have reported in gliomas using paradigms for assaying molecular expression other than gene profiling; and (3) Real-time RT–PCR. The results yield a genetic analysis of gliomas and identify classes of genetic expression that link novel genes to the biology of gliomas.

Effects of anti-angiogenesis on glioblastoma growth and migration: model to clinical predictions.

Glioblastoma multiforme (GBM) causes significant neurological morbidity and short survival times. Brain invasion by GBM is associated with poor prognosis. Recent clinical trials of bevacizumab in newly-diagnosed GBM found no beneficial effects on overall survival times; however, the baseline health-related quality of life and performance status were maintained longer in the bevacizumab group and the glucocorticoid requirement was lower. Here, we construct a clinical-scale model of GBM whose predictions uncover a new pattern of recurrence in 11/70 bevacizumab-treated patients. The findings support an exception to the Folkman hypothesis: GBM grows in the absence of angiogenesis by a cycle of proliferation and brain invasion that expands necrosis. Furthermore, necrosis is positively correlated with brain invasion in 26 newly-diagnosed GBM. The unintuitive results explain the unusual clinical effects of bevacizumab and suggest new hypotheses on the dynamic clinical effects of migration by active transport, a mechanism of hypoxia-driven brain invasion.