Kevin A. Walter

Vascular Gene Expression in Nonneoplastic and Malignant Brain

Malignant gliomas are uniformly lethal tumors whose morbidity is mediated in large part by the angiogenic response of the brain to the invading tumor. This profound angiogenic response leads to aggressive tumor invasion and destruction of surrounding brain tissue as well as blood-brain barrier breakdown and life-threatening cerebral edema. To investigate the molecular mechanisms governing the proliferation of abnormal microvasculature in malignant brain tumor patients, we have undertaken a cell-specific transcriptome analysis from surgically harvested nonneoplastic and tumor-associated endothelial cells. SAGE-derived endothelial cell gene expression patterns from glioma and nonneoplastic brain tissue reveal distinct gene expression patterns and consistent up-regulation of certain glioma endothelial marker genes across patient samples. We define the G-protein-coupled receptor RDC1 as a tumor endothelial marker whose expression is distinctly induced in tumor endothelial cells of both brain and peripheral vasculature. Further, we demonstrate that the glioma-induced gene, PV1, shows expression both restricted to endothelial cells and coincident with endothelial cell tube formation. As PV1 provides a framework for endothelial cell caveolar diaphragms, this protein may serve to enhance glioma-induced disruption of the blood-brain barrier and transendothelial exchange. Additional characterization of this extensive brain endothelial cell gene expression database will provide unique molecular insights into vascular gene expression.

A New Subarachnoid Hemorrhage Grading System Based on the Glasgow Coma Scale: A Comparison with the Hunt and Hess and World Federation of Neurological Surgeons Scales in a Clinical Series

OBJECTIVE Although the Hunt and Hess Scale (HHS) and World Federation of Neurological Surgeons Scale (WFNSS) are the most widely used subarachnoid hemorrhage (SAH) grading systems, neither system has achieved universal acceptance. We propose a simplified grading system based entirely on the Glasgow Coma Scale (GCS), which compresses the 15-point GCS into five grades that are comparable with those of the HHS and WFNSS. We refer to this system as the GCS grading system and present a direct comparison with the HHS and WFNSS for predictive value regarding patient outcome and interrater reliability. METHODS We reviewed 291 consecutive patients with aneurysms treated at our institution between January 1992 and January 1996 and compared the admission grades from the GCS, WFNSS, and HHS with outcome measures at discharge from hospitalization. The Glasgow Outcome score was used as the major outcome measure to evaluate the predictive value of the three scales. Mortality and length of stay (LOS) were also evaluated as outcome measures. The predictive value of each scale was tested with an ordinal logistic regression model for Glasgow Outcome score, a logistic regression model for mortality data, and a linear regression model for LOS. RESULTS Using the logistic regression model, the GCS was the best predictor of discharge Glasgow Outcome score, with an odds ratio of 2.585 (P = 0.0001), compared with 2.311 (P = 0.0001) for the WFNSS and 2.262 (P = 0.0001) for the HHS. Using mortality data in the logistic model, the HHS was the best predictor, with an odds ratio of 3.391 (P = 0.0001), compared with 2.859 (P = 0.0001) for the GCS and 2.560 (P = 0.0001) for the WFNSS. Each of the three scales had a high predictive value for LOS, using a linear model. We discuss, however, the problematic nature of LOS as an outcome measure for SAH. Interrater reliability for each scale was evaluated using kappa statistics, based on 15 additional patients evaluated prospectively, and showed that the GCS grade also had the greatest interrater reliability, with a kappa of 0.46 (P = 0.0002), compared with 0.41 (P = 0.0005) for the HHS and 0.27 (P = 0.027) for the WFNSS. CONCLUSION We conclude that the GCS grade has equal or greater predictive value regarding outcome after SAH than do the currently used grading systems and that it has greater reproducibility across observers. Broader familiarity with the GCS among medical and paramedical personnel may further enhance the usefulness of the GCS grade over the HHS and WFNSS in providing a standardized, universally accepted grading system for SAH.

Cytotoxicity of taxol in vitro against human and rat malignant brain tumors.

Taxol is a novel antitumor alkaloid that has shown clinical activity against several tumors, including ovarian and breast carcinoma and melanoma. To evaluate taxols potential as a therapy for malignant brain tumors, we measured the sensitivity of four human (U87, U373, H80, and D324) and two rat (9L, F98) brain-tumor cell lines to taxol. The cells were exposed to taxol in vitro using a clonogenic assay. Log cell kill (LD90) occurred at concentrations of 42 (9L), 25 (F98), 19 (H80), 7.2 (U373), 9.1 (U87), and 3.9 nM (D324) when cells were continuously exposed to taxol for 6–8 days. The human cell lines were uniformly more sensitive to taxol than were the rat lines. The duration of exposure had a significant effect on taxols cytotoxicity. When cells were exposed to taxol for 1 h the LD90 increased to 890 nM for the 9L rat line and 280 nM for the human U373 line. On the basis of these results, we conclude that taxol has significant potency in vitro against malignant brain tumors and that the activity occurs at concentrations of taxol that have previously been shown to be effective for several tumors against which the drug is currently being evaluated clinically.

Autologous glioma cell vaccine admixed with interleukin-4 gene transfected fibroblasts in the treatment of patients with malignant gliomas

BackgroundThe prognosis for malignant gliomas remains dismal. We addressed the safety, feasibility and preliminary clinical activity of the vaccinations using autologous glioma cells and interleukin (IL)-4 gene transfected fibroblasts.MethodsIn University of Pittsburgh Cancer Institute (UPCI) protocol 95-033, adult participants with recurrent glioblastoma multiforme (GBM) or anaplastic astrocytoma (AA) received gross total resection (GTR) of the recurrent tumors, followed by two vaccinations with autologous fibroblasts retrovirally transfected with TFG-IL4-Neo-TK vector admixed with irradiated autologous glioma cells. In UPCI 99-111, adult participants with newly diagnosed GBM or AA, following GTR and radiation therapy, received two intradermal vaccinations with the TFG-IL4-Neo-TK-transfected fibroblasts admixed with type-1 dendritic cells (DC) loaded with autologous tumor lysate. The participants were evaluated for occurrence of adverse events, immune response, and clinical response by radiological imaging.Results and DiscussionIn UPCI 95-033, only 2 of 6 participants received the vaccinations. Four other participants were withdrawn from the trial because of tumor progression prior to production of the cellular vaccine. However, both participants who received two vaccinations demonstrated encouraging immunological and clinical responses. Biopsies from the local vaccine sites from one participant displayed IL-4 dose-dependent infiltration of CD4+ as well as CD8+ T cells. Interferon (IFN)-γ Enzyme-Linked Immuno-SPOT (ELISPOT) assay in another human leukocyte antigen (HLA)-A2+ participant demonstrated systemic T-cell responses against an HLA-A2-restricted glioma-associated antigen (GAA) epitope EphA2883–891. Moreover, both participants demonstrated clinical and radiological improvement with no evidence of allergic encephalitis, although both participants eventually succumbed with the tumor recurrence. In 99-111, 5 of 6 enrolled participants received scheduled vaccinations with no incidence of major adverse events. Monocyte-derived DCs produced high levels of IL-12 p70. Treatment was well tolerated; however, we were unable to observe detectable IFN-γ post-vaccine responses or prolonged progression-free survival in these participants.ConclusionFeasibility challenges inherent in the generation of a patient-specific gene transfection-based vaccine strongly suggests the need for more practical formulations that would allow for the timely administration of vaccines. Nevertheless, successful generation of type-1 DCs and preliminary safety in the current study provide a strong rationale for further efforts to develop novel glioma vaccines.

PATTERNS AND TIMING OF RECURRENCE AFTER TEMOZOLOMIDE-BASED CHEMORADIATION FOR GLIOBLASTOMA

PURPOSE To determine recurrence patterns of glioblastoma treated with temozolomide-based chemoradiation. METHODS Pretreatment and serial posttreatment magnetic resonance imaging scans of 54 patients were retrospectively evaluated. Central recurrence (i.e., local progression) and the development of new (i.e., interval appearance of discrete enhancing lesion) in-field, marginal, and distant recurrences were assessed, with the pattern of recurrence of individual lesions defined relative to the 95% isodose line (D(95)). Distant recurrences were defined as lesions completely outside D(95), marginal recurrences crossed D(95), and in-field recurrences were completely inside D(95). RESULTS At a median follow-up of 17 months, 39 of 54 (72%) patients developed recurrent glioblastoma. Among these 39 patients, central recurrence occurred in 80% (at a median of 7 months from diagnosis); new in-field recurrence developed in 33% (at a median of 14 months); marginal recurrences developed in 15% (at a median of 18 months); and distant recurrences developed in 20% (at a median of 11 months). The actuarial rates of central, new in-field, marginal, distant, and any new recurrences at 1-year were 46%, 15%, 3%, 14%, and 25% respectively, whereas at 2 years, the rates were 68%, 60%, 32%, 28%, and 66%, reflecting an increasing probability of new lesions developing at later time points. Ten patients developed subependymal recurrences, of whom 7 developed multiple subependymal lesions. CONCLUSIONS Whereas central recurrence of glioblastoma treated with radiation and temozolomide predominates and persists over time, new in-field, marginal, and distant recurrences commonly develop, particularly at later time points in patients with longer survival.

Stereotactic radiosurgery for glioblastoma: retrospective analysis

PurposeThis retrospective study was done to better understand the conditions for which stereotactic radiosurgery (SRS) for glioblastoma may be efficacious.MethodsBetween 2000 and 2007, 33 patients with a pathological diagnosis of glioblastoma received SRS with the Novalis® Shaped Beam Radiosurgery system. Eighteen patients (54%) underwent salvage SRS for recurrence while 15 (45%) patients received upfront SRS following standard fractionated RT for newly diagnosed glioblastoma.ResultsThere were no RTOG grade >2 acute side effects. The median survival after SRS was 6.7 months (range 1.4 – 74.7). There was no significant difference in overall survival (from the time of initial diagnosis) with respect to the timing of SRS (p = 0.2). There was significantly better progression free survival in patients treated with SRS as consolidation versus at the time of recurrence (p = 0.04). The majority of patients failed within or at the margin of the SRS treatment volume (21/26 evaluable for recurrence).ConclusionSRS is well tolerated in the treatment of glioblastoma. As there was no difference in survival whether SRS is delivered upfront or at recurrence, the treatment for each patient should be individualized. Future studies are needed to identify patients most likely to respond to SRS.

Plasmalemmal Vesicle Associated Protein-1 (PV-1) is a marker of blood-brain barrier disruption in rodent models

BackgroundPlasmalemmal vesicle associated protein-1 (PV-1) is selectively expressed in human brain microvascular endothelial cells derived from clinical specimens of primary and secondary malignant brain tumors, cerebral ischemia, and other central nervous system (CNS) diseases associated with blood-brain barrier breakdown. In this study, we characterize the murine CNS expression pattern of PV-1 to determine whether localized PV-1 induction is conserved across species and disease state.ResultsWe demonstrate that PV-1 is selectively upregulated in mouse blood vessels recruited by brain tumor xenografts at the RNA and protein levels, but is not detected in non-neoplastic brain. Additionally, PV-1 is induced in a mouse model of acute ischemia. Expression is confined to the cerebovasculature within the region of infarct and is temporally regulated.ConclusionOur results confirm that PV-1 is preferentially induced in the endothelium of mouse brain tumors and acute ischemic brain tissue and corresponds to blood-brain barrier disruption in a fashion analogous to human patients. Characterization of PV-1 expression in mouse brain is the first step towards development of rodent models for testing anti-edema and anti-angiogenesis therapeutic strategies based on this molecule.

Plasmalemmal Vesicle Associated Protein-1 Is a Novel Marker Implicated in Brain Tumor Angiogenesis

Purpose:Plasmalemmal vesicle associated protein-1 (PV-1) is up-regulated in the endothelium of human glioblastoma. We sought to further characterize the expression pattern of PV-1 in human brain tumors and interrogate its role in brain tumor angiogenesis. Experimental Design: Quantitative reverse transcription-PCR and in situ hybridization were used to measure PV-1 expression in a panel of 46 human brain tumors and related pathologic states. Matrigel tubulogenesis assays and cell migration assays were used to show function of PV-1 in primary human endothelial cells (HMVEC) under gene knockdown conditions. Results:PV-1 is selectively up-regulated in a variety of high-grade human brain tumors, including glioblastoma and metastatic carcinoma, as well as other cerebral disorders associated with blood-brain barrier disruption, such as acute ischemia. Expression levels were reduced in low-grade neoplasia; however, tumors associated with the ependyma and choroid plexus, known sites of PV-1 expression, also exhibited robust expression. Cerebral expression of PV-1 mRNA was confined to endothelial cells in all cases. PV-1 expression was induced in HMVEC cells in vitro by exposure to medium conditioned by U87MG and U251MG human brain tumor cell lines and by medium supplemented with exogenous vascular endothelial growth factor or scatter factor/hepatocyte growth factor. RNA interference–mediated inhibition of PV-1 induction in HMVEC cells blocked Matrigel-induced tubulogenesis and inhibited cell migration induced by conditioned medium or angiogenic growth factors. Conclusions: Our results confirm that PV-1 is preferentially induced in the endothelium of high-grade human brain tumors. Inhibition of PV-1 expression is associated with failure of endothelial differentiation in vitro. PV-1 represents a novel marker of brain tumor angiogenesis and integrity of the blood-brain barrier and is a potential therapeutic target.

Vascular gene expression patterns are conserved in primary and metastatic brain tumors

Malignant primary glial and secondary metastatic brain tumors represent distinct pathological entities. Nevertheless, both tumor types induce profound angiogenic responses in the host brain microvasculature that promote tumor growth. We hypothesized that primary and metastatic tumors induce similar microvascular changes that could function as conserved angiogenesis based therapeutic targets. We previously isolated glioma endothelial marker genes (GEMs) that were selectively upregulated in the microvasculature of proliferating glioblastomas. We sought to determine whether these genes were similarly induced in the microvasculature of metastatic brain tumors. RT-PCR and quantitative RT-PCR were used to screen expression levels of 20 candidate GEMs in primary and metastatic clinical brain tumor specimens. Differentially regulated GEMs were further evaluated by immunohistochemistry or in situ hybridization to localize gene expression using clinical tissue microarrays. Thirteen GEMs were upregulated to a similar degree in both primary and metastatic brain tumors. Most of these genes localize to the cell surface (CXCR7, PV1) or extracellular matrix (COL1A1, COL3A1, COL4A1, COL6A2, MMP14, PXDN) and were selectively expressed by the microvasculature. The shared expression profile between primary and metastatic brain tumors suggests that the molecular pathways driving the angiogenic response are conserved, despite differences in the tumor cells themselves. Anti-angiogenic therapies currently in development for primary brain tumors may prove beneficial for brain metastases and vice versa.

Primary spinal cord glioma: A Surveillance, Epidemiology, and End Results database study

To characterize the overall survival (OS) and cause specific survival (CSS), and variables affecting outcome, in patients with primary spinal cord astrocytoma (SCA) and ependymoma (SCE). About 664 patients with SCA and 1,057 patients with SCE were analyzed using the Surveillance, Epidemiology, and End Results database. For grade 1, 2, 3 and 4 SCA, the 5-year OS was 82, 70, 28 and 14%; the 5-year CSS was 89, 77, 36 and 20%. For SCA, lower grade, younger age, and undergoing resection significantly improved OS and CSS; treatment without radiotherapy was favorable for CSS. Smaller tumor size also improved survival. For grade 1, 2, and 3 SCE, the 5-year OS was 92, 97 and 58%; the 5-year CSS was 100, 98 and 64%. For SCE, lower grade, younger age, and undergoing resection significantly improved OS and CSS; treatment without radiotherapy was favorable for OS. Smaller tumor size did not confer a survival benefit. Patients with resected grade 2 spinal cord glioma who did not receive radiotherapy fared well with respect to OS and CSS. For patients with spinal cord glioma, the variables of histology, grade, age and undergoing resection are significant predictors of outcome. Though treatment with radiotherapy was associated with worse outcomes, this may reflect a bias in that patients who underwent radiotherapy were perhaps more likely to have had adverse risk factors. Given the retrospective nature of this study, specific recommendations about which situations warrant radiotherapy cannot be determined.