The Sixteenth Annual Blood-Brain Barrier Consortium meeting, organized by the Oregon Health and Science University Blood-Brain Barrier Program, will focus on translational research in the blood-brain barrier and neuro-oncology. Partial funding comes from a National Institutes of Health R13 meeting grant, with support from the National Institute of Neurological Disorders and Stroke, and the National Institute on Deafness and Other Communication Disorders. For more information, visit www.ohsu.edu/bbb or contact Emily Hochhalter by phone at 011-503-494-0614 or e-mail at hochhalt@ohsu.edu.

One of our greatest friends and supporters of neuro-oncology, Mike Traynor, died September 12, 2009, in Asheville, NC, after a brief illness. Mike grew up outside of Chicago, where he developed a love of motorcycling, a love that grew during his military service in Japan (and later would have an important effect on the neuro-oncology community). After his military service, Mike's career took him to Atlanta, where he was vice president and general manager of newspaper publishing corporations and served in many related professional associations. The business and motivational skills he developed served him extremely well in his organization and management of the Pediatric Brain Tumor Foundation (PBTF) and its Ride for Kids program in 1991.

Nothing endures but change.

—Heraclitus (540–480 BCE)

For eleven years, Neuro-Oncology has led the charge to educate and report on the progress of the broad community of scientists and physicians working, directly or indirectly, to improve care for patients with brain tumors. The journal started, as journals do, as a dream, a hope, a sparkle in someone's eye—in this case, founding editor Darell Bigner's eye, and the eyes of those at the Society for Neuro-Oncology and the Pediatric Brain Tumor Foundation who saw a need for a journal that would fulfill a particular mission: to provide the international neuro-oncology community with high-quality, rapid information from all areas of the field.

Glioblastomas (GBs) are malignant central nervous system tumors often associated with devastating symptoms. Patients with GB have a very poor prognosis and despite treatment most of them die within 12 months from diagnosis. Several pathways such as the RAS, TP53 and PIK3 as well as the cell cycle control have been identified to be disrupted in this tumor. However, emerging data suggest that these aberrations only represent a fraction of the genetic changes involved in gliomagenesis. In this study, we have applied a 32K clone-based genomic array, covering 99% of the current assembly of the human genome to the detailed profiling of a set of 78 GBs. Complex patterns of aberrations including high and narrow copy number amplicons as well as a number of homozygously deleted loci were identified. Amplicons which varied both in number (3 in average) and size (average 1.4 Mb) were frequently detected (79% of the samples). The loci encompassed not only previously reported oncogenes (as EGFR, PDGFRA, MDM2, CDK4) but also numerous novel genes as GBR10, MKLN1, PPARGC1A, HGF, NAV3,CNTN1,SYT1 and ADAMTSL3. BNC2, PTPLAD2 and PTPRE represent candidate tumor suppressor genes encompassed within homozygously deleted loci. Many of these genes are already linked to several forms of cancer; others represent new candidate genes that may serve as prognostic markers or even as therapeutic targets in the future. The large individual variation observed between the samples demonstrates the underlying complexity of the disease and strengthens the demand for an individualized therapy based on the genetic profile of the patient.

Inhibitors targeting the integrin vβ3 are promising new agents currently tested in clinical trials for supplemental therapy of glioblastoma multiforme (GBM). The aim of our study was to evaluate [18F]Galacto-RGD positron emission tomography (PET) for non-invasive imaging of vβ3 expression in patients with GBM suggesting eligibility for this kind of additional treatment. Patients with suspected or recurrent GBM were examined with [18F]Galacto-RGD PET. Standardized uptake values (SUVs) of tumor hotspots, galea and blood pool were derived by region-of-interest analysis. [18F]Galacto-RGD PET images were fused with cranial MRI scans for image guided surgery. Tumor samples taken from areas with intense tracer accumulation in the [18F]Galacto-RGD PET were analyzed histologically and immunhistochemically for vβ3 integrin expression. While normal brain tissue did not show significant tracer accumulation (mean SUV 0.09 ± 0.04), GBMs demonstrated significant but heterogeneous tracer uptake with a maximum in the highly proliferating and infiltrating areas of tumors (mean SUV 1.6 ± 0.5). Immunohistochemical staining was prominent in tumor microvessels as well as glial tumor cells. In areas of highly proliferating glial tumor cells, tracer uptake (SUVs) in the [18F]Galacto-RGD PET correlated to the immunohistochemical vβ3 integrin expression of corresponding tumor samples. These data suggest that [18F]Galacto-RGD PET successfully identifies vβ3 expression in patients with GBM and might be a promising tool for planning and monitoring individualized cancer therapies targeting this integrin.

Acquisition of the insidious invasiveness by malignant glioma cells involves multiple genetic alterations in signaling pathways. Slit2, a chemorepulsive factor, controls cell migration of neuronal and glial cells during development and inhibits chemotaxic migration of various types of cells in vitro. However, the role of Slit2 in vitro remains controversial and the biological significance of Slit2 expression in cancer cell invasion in vivo has not yet been determined. In the present study, we characterized the effects of Slit2 expression on the migration and invasion of invasive glioma cells in vitro and in vivo. By RT-PCR analyses, Slit2 was found to express at lower levels in primary glioma specimens and invasive glioma cells when compared with normal human brains and astrocytes. Ectopic expression of Slit2 or treatment with recombinant Slit2 on glioma cells attenuate cell migration and invasion through inhibition of Cdc42 activity in vitro. Cellular depletion of Robo1, a cognate receptor for Slit2 prevented Slit2 inhibition of Cdc42 activity and glioma cell migration. In vivo, expression of Slit2 by invasive SNB19 cells markedly inhibited glioma cell infiltration into the brain of mice. Moreover, impediment of glioma cell invasion by Slit2 did not affect the expression of N-cadherin and β-catenin in glioma cells. These results provide the first evidence demonstrating that Slit2–Robo1 inhibits glioma invasion through attenuating Cdc42 activity in vitro and in the brain. Understanding the mechanisms of Slit2–Robo1 inhibition of glioma cell invasion will foster new treatments for malignant gliomas.

Background: Giant cell glioblastoma (GC) is an uncommon subtype of glioblastoma multiforme (GBM). Consequently, the epidemiology, natural history and factors associated with outcome are not well defined.

Methods: Patients diagnosed with GC from 1988 to 2004 were identified in the Surveillance, Epidemiology and End Results (SEER) database. Outcomes were examined with Kaplan-Meier survival analysis and Cox models. For comparison, similar analyses were conducted for patients diagnosed with GBM.

Results: GC was identified in 1% of 16,430 patients diagnosed with either GC or GBM. Compared to GBM, GC showed similar gender and racial distributions. Likewise, tumor size and location were not significantly different between the two histologies. GC tended to occur in younger patients with a median age at diagnosis of 51 years compared to 62 years for GBM. Additionally, patients with GC were more likely to undergo complete resection compared to patients with GBM. For both histologies, young age, tumor size, extent of resection and the use of adjuvant RT were associated with improved survival. Cox modeling suggests the prognosis for GC is significantly superior to GBM (HR = 0.76, 95% CI 0.59-0.97) even after adjustment for factors impacting survival.

Conclusions: GC is an uncommon GBM subtype that tends to occur in younger patients. Prospective data defining optimal treatment for GC are unavailable; however, these retrospective findings suggest resection, as opposed to biopsy only, and adjuvant RT may improve survival. The prognosis of GC is superior to GBM and long-term survival is possible suggesting aggressive therapy is warranted.

The cell surface receptor protein tyrosine phosphatase mu (PTPµ) is a homophilic cell adhesion molecule expressed in CNS neurons and glia. Glioblastomas (GBMs) are the highest grade primary brain tumors with astrocytic similarity and are characterized by marked dispersal of tumor cells. PTPµ expression was examined in human GBM, low-grade astrocytoma, and normal brain tissue. These studies revealed a striking loss of PTPµ protein expression in highly dispersive GBMs compared to less dispersive low-grade astrocytomas and normal brain. We hypothesized that PTPµ contributes to contact inhibition of glial cell migration by transducing signals in response to cell adhesion. Therefore, loss of PTPµ may contribute to the extensive dispersal of GBMs. The migration of brain tumor cells was assessed in vitro using a scratch wound assay. Parental U-87 MG cells express PTPµ and exhibited limited migration. However, shRNA-mediated knockdown of PTPµ induced a morphological change and increased migration. Next, a brain slice assay replicating the three-dimensional environment of the brain was used. To assess migration, labeled U-87 MG glioma cells were injected into adult rat brain slices, and their movement was followed over time. Parental U-87 MG cells demonstrated limited dispersal in this assay. However, PTPµ shRNA induced migration and dispersal of U-87 MG cells in the brain slice. Finally, a mouse xenograft model of intracranially injected U-87 MG cells was used. PTPµ shRNA induced morphological heterogeneity in these xenografts. Together, these data suggest that loss of PTPµ in human glioblastomas contributes to tumor cell migration and dispersal, implicating loss of PTPµ in glioma progression.

Platelet-derived growth factor (PDGF) and its receptors (PDGFR) are frequently co-expressed in meningiomas, potentially contributing to their pathogenesis. The North American Brain Tumor Consortium (NABTC) conducted a phase II study to evaluate the therapeutic potential of imatinib mesylate (Gleevec®), a PDGFR inhibitor, in patients with recurrent meningiomas. Patients were stratified into benign (WHO grade I) meningiomas or atypical (WHO grade II) and malignant (WHO grade III) meningiomas. The primary endpoint was 6-month progressionfree survival (6M-PFS). Patients requiring enzyme-inducing anti-epileptic drugs were ineligible. Patients received imatinib at a dose of 600 mg/day for the first 4-week cycle and then gradually increased to 800 mg/day for subsequent cycles, if there were no unacceptable toxicities. Plasma concentrations of imatinib and its active metabolite, CGP74588, were assessed. Twenty-three heavily pretreated patients were enrolled into the study (13 benign, 5 atypical, and 5 malignant meningiomas), of whom 22 were eligible. The study was closed prematurely due to slow accrual. Tissue was available only from a minority of patients but in these specimens there was uniform distribution of PDGFR, the drug target. Imatinib was generally well tolerated. Of 19 patients evaluable for response, 10 progressed at the first scan, and 9 were stable. There were no complete or partial responses (PR). Overall median PFS was 2 months (range 0.7–34 months); 6M-PFS was 29.4%. For benign meningiomas, median PFS was 3 months (range 1.1–34 months); 6M-PFS was 45%. For atypical and malignant meningiomas, median PFS was 2 months (range 0.7–3.7 months); 6M-PFS was 0%. Cycle 1 trough concentrations of imatinib and CGP74588 were 2129±1600 ng/ml and 517±326 ng/ml, respectively. Single-agent imatinib was well-tolerated but had no significant activity in recurrent meningiomas. Trough plasma concentrations of imatinib exceeded those associated with imatinib activity in CML.

The REV3L gene, encoding the catalytic subunit of human polymerase , plays a significant role in the cytotoxicity, mutagenicity and chemoresistance of certain tumors. However, the role of REV3L in regulating the sensitivity of glioma cells to chemotherapy remains unknown. In this study, we investigated the expression of REV3L gene in 10 normal brain specimens and 30 human glioma specimens, and examined the value of REV3L as a potential modulator of cellular response to various DNA damaging agents. RT-PCR/Real-Time PCR analysis revealed that REV3L was overexpressed in human gliomas compared with normal brain tissues. Glioma cell model with stable overexpression of REV3L was set up and used to probe the role of REV3L in cisplatin treatment, upregulation of REV3L markedly attenuated cisplatin-induced apoptosis of mitochondrial apoptotic pathway. We, therefore, assessed the REV3L-targeted treatment modality that combines suppression of REV3L expression using RNA interference (RNAi) with the cytotoxic effects of DNA damaging agents. We found that down-regulation of REV3L expression significantly enhanced the sensitivity of glioma cells to cisplatin, which was evidenced by the increased apoptosis rate as well as marked alterations in Bcl-2, Bcl-xl and Bax expression levels, and meanwhile reduced mutation frequencies in surviving glioma cells. These results suggest that REV3L may potentially contribute to gliomagenesis and play a crucial role in regulating cellular response to DNA cross-linking agent cisplatin. Our findings provide a novel strategy that RNAi targeting REV3L combined with chemotherapy has synergistic therapeutic effects on glioma cells, which warrants further investigation as an effective therapeutic regimen for patients with this malignancy.

Human glioblastoma is notorious for its capacity to interfere with effective anti-tumor immune responses. B7–H1 is the third member of the B7 family that plays important roles in tumor immune evasion. Recent studies showed that brain tumor stem-like cells (TSCs) contributed to tumorigenesis and radioresistance. However, the relationship between B7–H1 and the clinical behavior of brain TSCs remain unclear. In the present study, we reported that B7–H1 was correlated with the malignancy grade of astrocytic tumors. There was significant up-regulation of B7–H1 at the growing edge of the tumors. Immunostaining and flow cytometry analysis indicated that B7–H1 was primarily expressed by Ki67 negative tumor cells. In vitro, tumors cultured under medium favoring the growth of neural stem cells were able to form spheres, along with expression of neural stem/progenitor cell markers. These cells were able to differentiate into different neural lineages when cultured in differentiation medium, indicating that these cells have TSCs characteristics. We also found that B7–H1 was expressed, but not exclusively on CD133⁺ stem cells. Interestingly, we found CD133^- tumor cells also had the capacity to form brain tumor. Our data establish a correlation between the expression of negative costimulatory molecule B7–H1 and the malignancy grade of human gliomas, suggesting B7–H1 can be a novel tumor marker & target for therapy although it is not the privilege of brain tumor stem-like cells.

We investigated the activation of PDGFRA, PDGFRB, EGFR and their downstream pathways in malignant peripheral nerve sheath tumours (MPNSTs). To this, PDGFRA, PDGFRB and EGFR were immunohistochemically, biochemically, cytogenetically and mutationally analysed along with the detection of their cognate ligands in 16 neurofibromatosis type 1 (NF1)-related and 11 sporadic MPNSTs. The activation of the downstream receptor pathways was also studied by means of AKT, ERK and mTOR western blotting experiments, as well as RAS, BRAF, PI3KCA and PTEN mutational analysis and fluorescent in situ hybridisation. PDGFRA, PDGFRB and EGFR were expressed/activated, with higher levels of EGFR expression/phosphorylation parallelling increasing EGFR gene copy numbers in the NF1-related cases (71%). Autocrine loop activation of these receptors along with their coactivation were suggested by the expression of the cognate ligands in the absence of mutations and the presence of RTK heterodimers, respectively. Both MPNST groups showed AKT, ERK and mTOR expression/phosphorylation. No BRAF, PI3KCA or PTEN mutations were found in either group of MPNSTs, but 18% of the sporadic MPNSTs showed RAS mutations. PTEN monosomy segregated with the NF1-related cases (50%, p=0.018), but PTEN protein was expressed in all but two cases. In conclusion, PDGFRA, PDGFRB and EGFR seem to be promising molecular targets for tailored treatments in MPNST. In particular, the ligand- and heterodimerizationdependent RTK activation/expression coupled with a downstream signalling phosphorylation, mediated by the upstream receptors or RAS activation, may provide a rationale to apply combined RTK and mTOR inhibitor treatments both to sporadic and NF1-related cases.

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Background. Recent studies have shown that the clinical outcome of anaplastic oligodendroglial tumors is variable, but also that the histological diagnosis is subject to interobserver variation. We investigated if the assessment of 1p/19q co-deletion, polysomy of chromosome 7, EGFR gene amplification (EGFR^amp) and loss of chromosome 10 or 10q offers additional prognostic information to the histological diagnosis and would allow molecular subtyping. Methods. For this study the clinical data and tumors samples of the patients included in the multicenter prospective phase III EORTC 26951 trial on the effects of adjuvant procarbazine, CCNU and vincristine chemotherapy in anaplastic oligodendroglial tumors were used. Fluorescent In Situ Hybridization (FISH) was used to assess copy number aberrations of chromosome 1p, 19q, 7, 10 and 10q, and the EGFR gene. Three different analyses were done: on all included patients based on local pathology diagnosis, on the patients with confirmed anaplastic oligodendroglial tumors on central pathology review, and on this latter group but with after exclusion of anaplastic oligoastrocytoma (AOA) with necrosis. As a reference set for glioblastoma, patients from the prospective randomized phase III on glioblastoma (EORTC 26981) were used as a benchmark. Results. In 257 of 368 patients central pathology review confirmed the presence of an anaplastic oligodendroglial tumor. Tumors with combined 1p and 19q loss (1p^loss19q^loss) were histopathologically diagnosed as anaplastic oligodendroglioma (AOD), were more frequently located in the frontal lobe and had a better outcome. Anaplastic oligodendroglial tumors with EGFR^amp were more frequently AOA, were more often localized outside the frontal lobe and have a survival similar to glioblastoma. Survival of patients with AOA harboring necrosis was in a similar range as glioblastoma while patients with AOA with only endothelial proliferation had better overall survival. In univariate analysis all molecular factors except loss of 10q were of prognostic significance, however on multivariate analysis a histopathological diagnosis of AOA, necrosis and 1p^loss19q^loss remained independent prognostic factors. Conclusion. AOA with necrosis are to be considered WHO grade IV tumors (glioblastoma). Of all molecular markers analyzed in this study especially loss of 1p/19q carried prognostic significance, while the others contributed little prognostic value to classical histology.

Cilengitide is a cyclic peptide antagonist of integrins vβ3 and vβ5 which is currently evaluated as a novel therapeutic agent for recurrent and newly diagnosed glioblastoma. Its mode of action is thought to be mainly antiangiogenic, but may include direct effects on tumor cells, notably on attachment, migration, invasion and viability. Here we show that, at clinically relevant concentrations, cilengitide (1–100 µM) induces detachment in some, albeit not all glioma cell lines, while the effect on cell viability is modest. Detachment induced by 2 cilengitide could not be predicted by the level of expression of the cilengitide target molecules, vβ3 and vβ5, at the cell surface. Glioma cell death induced by cilengitide was associated with the generation of caspase activity, but caspase activity was dispensable for cell death since ectopic expression of cytokine response modifier (crm)-A or coexposure to the broad spectrum caspase inhibitor, zVAD-fmk, were not protective. Moreover, forced expression of Bcl-X_L or altering the p53 status did not modulate cilengitide-induced cell death. No consistent effects of cilengitide on glioma cell migration or invasiveness were observed in vitro. Preliminary clinical results indicate a preferential benefit from cilengitide added to temozolomide-based radiochemotherapy in patients with O⁶– methyltransferase (MGMT) gene promoter methylation. Accordingly, we also examined whether the MGMT status determines glioma cell responses to cilengitide alone or in combination with temozolomide. Neither ectopic expression of MGMT in MGMT-negative cells nor silencing the MGMT gene in MGMT-positive cells altered their response to cilengitide alone or cilengitide in combination with temozolomide. These data suggest that the beneficial clinical effects derived from cilengitide in vivo may arise from altered perfusion which promotes temozolomide delivery to glioma cells.

A benefit of Temozolomide (TMZ) is that myelotoxicity (TOX) is uncommon. Recently, several small series report significant TOX resulting in treatment delays or death. The ability to predict risk of TOX may influence patient care. A retrospective review of 680 malignant glioma patients was completed. A clinical risk formula for TOX for each gender was developed by logistic regression. The variables which remained are assigned a score of 1 and are added together for a final risk score. Women experienced more TOX then men (p=0.015). For males, risk factors include: BSA >=2 (OR 2.712, p=0.04); Not on steroids (OR 2.214, p=0.06); and on Bowel medication (meds) (OR 3.955, p=0.008). For females, final factors include: No prior chemotherapy (OR 3.727, p=0.001); Creatinine >=1 (OR 6.08 p=0.002); Platelet t < 270k (OR 2.438, p=0.03); BSA < 2 (OR 4.178, p=0.04); not on GERD meds (OR 2.942, p=0.01); and on analgesics (OR 2.169, p=0.05). Age was included because of observable trends. Risk of developing TOX ranged from 0% to 33% (male) and 100% (females). Polymorphisms in NQO1, MGMT, and GSTP1 were related to risk of developing TOX in a subset of patients. TOX with TMZ is a significant clinical issue for those at risk. Use of a clinical model to predict risk and evaluation of identified genetic polymorphisms related to TOX may allow for individualized dosing optimizing patient management.

While meningiomas are known as slow growing extracerebral neoplasm the subgroup of secretory meningioma with histological benign caracteristics tends to cause disproportional peritumoral edema frequently leading to severe medical and neurological complications in the postoperative management. Among 1484 meningiomas which were operated in our institution between 1990 and 2007, 44 (3%) patients were found to have the histological diagnosis of a secretory meningioma. The clinical course, radiological appearance and histopathological features were retrospectively analyzed to point out the specifics of these benign lesions. Meningiomas were located at the convexity (n = 14), the cranial base (18) and the sphenoid ridge (12). Disproportional to the tumor size a severe, nearly hemispheric perifocal edema was seen on preoperative MR-imaging in 18 (41%) patients. Following surgical resection the postoperative course was uneventful in 29 patients. In 15 patients severe peritumoral edema continued or even increased on postoperative CT-imaging. Six patients showed midline shift and clinical worsening necessitating respirator assisted ventilation and intracranial pressure monitoring. An association between the extent of brain edema and number of PAS positive pseudopasmmomas was found (p<0.02). Further, the size of the edema correlated with the number of immunohistochemically detected cells expressing carcinoembryonic antigen (CEA) and cytokeratin (CK) (p<0.01). Mean MIB-1 proliferation index was 3.0% (range 0–17%) and did not correlate with edema or tumor recurrence. Secretory meningiomas are frequently associated with a severe peritumoral edema. The extent of edema correlates with immunohistochemically detected expression of CEA and CK. Extended perifocal edema in meningiomas is an unusual finding and should alert the neurosurgeon that surgery may aggravate edema excessively leading to a life threatening postoperative situation.

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In the presented study we described the role of 9β1 integrin in glioblastoma progression following its interaction with NGF. The level of expression of 9β1 on astrocytomas is correlated with increased grade of this brain tumor, and is the highest on glioblastoma, whereas normal astrocytes do not express this integrin. Two glioblastoma cell lines, LN229 and LN18 that are 9β1 integrin positive or negative, respectively, were used for 9β1 integrin-dependent NGF-induced tumor progression. NGF was a significant promoter of pro-migratory and proproliferative activities of glioblastoma cells through direct interaction with 9β1 integrin and activation of MAPK Erk1/2 pathway. The level of NGF increases approximately 3 fold in the most malignant glioma tissue if compared with normal brain. This increase is related to secretion of NGF by tumoral cells. Specific inhibitors of 9β1 integrin or gene silencing inhibited NGF- induced proliferation of LN229 cell line to the level showed by LN18 cells. VLO5 promoted 9β1-dependent programmed cell death by induction of intrinsic apoptosis pathway in cancer cells. LN229 cells were rescued from pro-apoptotic effect of VLO5 by the presence of NGF. This disintegrin significantly inhibited tumor growth induced by implantation of LN229 cells to the CAM of quail embryonic model and this inhibitory effect was significantly abolished by the presence of NGF. 9β1 integrin appears to be an interesting target for blocking of progression malignant gliomas, especially in light of the stimulatory effect of NGF on development of this tumor and ability to transfer pro-apoptotic signal in cancer cells.

Abstracts from the Twelfth International Symposium on Pediatric Neuro-Oncology, June 6-9, 2006, Nara Japan