Lactate dehydrogenase type A (LDH-A) is a metabolic key enzyme catalyzing pyruvate into lactate and is excessively expressed by tumor cells. Transforming growth factor-beta2 (TGF-beta2) is a key regulator of invasion in high-grade gliomas partially by inducing a mesenchymal phenotype and by remodeling the extracellular matrix. In this study, we tested the hypothesis that lactate metabolism regulates TGF-beta2 mediated migration of glioma cells. Small interfering RNA directed against LDH-A (siLDH-A) suppresses, and lactate induces, TGF-beta2 expression, suggesting that lactate metabolism is strongly associated with TGF-beta2 in glioma cells. We demonstrate that TGF-beta2 enhances expression, secretion and activation of MMP-2 and induces the cell surface expression of integrin alpha_vbeta₃ receptors. In spheroid and Boyden chamber migration assays, inhibition of MMP-2 activity using a specific MMP-2 inhibitor and blocking of integrin alpha_vbeta₃ abrogated glioma cell-migration stimulated by TGF-beta2. Furthermore, siLDH-A inhibited MMP2 activity leading to inhibition of glioma migration. Taken together, we define a LDH-A induced and TGF-beta2 coordinated regulatory cascade of transcriptional regulation of MMP-2 and integrin alpha_vbeta₃. This novel interaction between lactate metabolism and TGF-beta2 might constitute a crucial mechanism for glioma migration.

Dear Editor, We read with interest the article "Tetramethylpyrazine inhibits activities of glioma cells and glutamate neuro-excitotoxicity: potential therapeutic application for treatment of gliomas" by Fu et al. (Neuro-Oncology 2008;10:139–153).¹ We were surprised that the authors did not examine or discuss the possible role of the N-methyl-D-aspartate (NMDA) type of ionotropic glutamate receptor in mediating glutamate-induced [Ca²⁺] increase in glioma cells and glioma cell-induced neurotoxicity. In fact, the NMDA receptor was not mentioned anywhere in the article, although it has been implicated in mediating both glutamate excitotoxicity and glioma growth. There are a number of reasons why the involvement of NMDA receptors should have been investigated, or at least discussed.

The effects of the epidermal growth factor receptor (EGFR) inhibitor gefitinib on cell growth and signaling were evaluated in 3 medulloblastoma (MB) cell lines (D283, D341, Daoy), one supratentorial PNET cell line (PFSK) and 4 MB primary cultures. Cell lines showed diverse expression of EGFR and HER2, with high levels of constitutively activated HER2 in the HER2-overexpressing D341 and D283 cells. Gefitinib sensitivity varied across lines, being related neither with the expression of HER receptors, nor with receptor baseline activation. Gefitinib determined a G₀/G₁ arrest in all lines, whereas apoptosis was dose-dependently induced only in D283 and D341 cells. The molecular response to gefitinib was investigated in Daoy and D341 lines, which showed a higher (IC₅₀ 3.8 µM) and lower (IC₅₀ 6.6 µM) sensitivity to the agent, respectively. Gefitinib inhibited constitutive and EGF-triggered EGFR phosphorylation in both lines, whereas was ineffective on constitutive activation of HER2 in D341 cells. pAkt inhibition paralleled that of pEGFR, suggesting the presence of an autocrine gefitinib-sensitive EGFR/Akt pathway. On the whole, the EGF-dependent signaling was less responsive to ligand stimulation and gefitinib inhibition in D341 cells, that correlated with the lower sensitivity to gefitinib antiproliferative effect of this line. In vivo, the growth of D341 and Daoy xenografts was inhibited at 150 mg/Kg/day gefitinib by approximately 50%. Ectopically overexpressed HER2 in Daoy cells significantly increased sensitivity to gefitinib antitumor effects in vivo (TVI = 78%). Our data indicate that gefitinib might be a molecularly targeted agent for the treatment of MB.

Around 25% of all tumors in those aged 0–14 years and 9% in those aged 15 to 24 years involve the central nervous system (CNS). They are the most common cause of cancer-related deaths in this age. In adults aged 25–84 years the proportion of CNS tumors is 2%. 5 year overall survival is 10–15% and there is considerable morbidity in survivors. Comprehensive up to date population-based incidence data on these tumors are lacking. We present incidence rates for primary CNS tumors based on data that have been derived from the high quality national cancer registration system in England. 54,336 CNS tumors of malignant, benign and uncertain behaviour were registered across the whole of England from 1995 to 2003. The age standardized rates for all ages (0 to 84 years) was 9.21 per 100,000 person years. This is higher than previously reported for England since it includes non-malignant CNS tumors and hence gives a more accurate picture of burden of disease. The age standardized rates for those aged 0 to 14 years, 15 to 24 years and 25 to 84 years were 3.56, 3.26 and 14.57 per 100,000 person years respectively. We describe the changing patterns in the epidemiology of primary CNS tumors in these three age groups with respect to sex, behaviour and histology using the current WHO classification. This will provide a reference for future studies nationally and internationally and make comparisons relevant and meaningful.

Patients with Muir-Torre syndrome, an autosomal-dominant familial tumor condition caused by germline mutation of the DNA mismatch repair genes, MSH2 or MLH1, present with tumors of the sebaceous gland and visceral malignancies characterized by microsatellite instability. Here we show development of glioblastoma multiforme in a patient with Muir-Torre syndrome. Immunohistochemical analysis of the brain tumor and colon cancer revealed loss of the DNA mismatch repair gene detected by the genetic test, suggesting a pathogenic link.

The brain is a specialized immune site representing a unique tumor microenvironment. The availability of fresh brain tumor material for ex-vivo analysis is often limited as large parts of many brain tumors are resected using ultrasonic aspiration. We now analyzed ultrasonic tumor aspirates as a bio-source to study immune suppressive mechanisms in 83 human brain tumors. Lymphocyte-infiltrates in brain tumor tissues and ultrasonic aspirates were comparable with respect to lymphocyte content and viability. Applying ultrasonic aspirates, we detected massive infiltration of CD4+FOXP3+CD25^highCD127^low regulatory T cells (Tregs) in glioblastomas (n=29) and metastatic brain tumors (n=20). No Treg accumulation was observed in benign tumors such as meningiomas (n=10) and pituitary adenomas (n=5). A significant Treg increase in blood was only seen in patients with metastatic brain tumors. Tregs in high-grade tumors exhibited an activated phenotype as indicated by decreased proliferation and elevated CTLA-4 and FoxP3 expression relative to blood Tregs. Functional analysis showed that the tumor derived Tregs efficiently suppressed cytokine secretion and proliferation of autologous intratumoral lymphocytes. Most tumor infiltrating Tregs localized in close proximity to effector T cells, as visualized by immunohistochemistry. Furthermore, 61% of the malignant brain tumors expressed programmed death ligand-1 (PD-L1), while the inhibitory PD-1 receptor was expressed on CD4+ effector cells present in 26% of tumors. In conclusion, using ultrasonic tumor aspirates as a bio-source we identify Tregs and the PD-L1/PD-1 pathway as immune suppressive mechanisms in malignant but not benign human brain tumors.

The purpose of this study was to evaluate long-term survival in patients with nonpilocytic low-grade gliomas (LGGs). Records of 314 adult patients with nonpilocytic LGGs diagnosed between 1960 and 1992 at Mayo Clinic, Rochester, Minnesota, were retrospectively reviewed. Kaplan-Meier method estimated progression-free survival (PFS) and overall survival (OS). Median age at diagnosis was 36 years. Median follow-up was 13.6 years. Operative pathology revealed pure astrocytoma in 181 patients (58%), oligoastrocytoma in 99 (31%), and oligodendroglioma in 34 (11%). Gross total resection was achieved in 41 patients (13%), radical subtotal resection (rSTR) in 33 (11%), subtotal resection in 130 (41%), and biopsy only in 110 (35%). Median OS was 6.9 years (range, 1 month-38.5 years). Adverse prognostic factors for OS identified by multivariate analysis were size of 5 cm or larger, pure astrocytoma histology, Kernohan grade 2, undergoing less than rSTR, and presentation with sensory motor symptoms. Statistically significant adverse prognostic factors for PFS by multivariate analysis were only size of 5 cm or larger and undergoing less than rSTR. In patients who underwent less than rSTR, RT was associated with improved OS and PFS. A substantial proportion of patients have a good long-term prognosis after GTR and rSTR, with nearly half of patients free of recurrence 15 years after diagnosis. Postoperative RT was associated with improved OS and PFS and is recommended for patients after subtotal resection or biopsy.

Glioblastoma multiforme (GBM) occurs rarely in children. Relatively few studies have been performed on molecular properties of pediatric GBMs. To evaluate the genetic alterations in pediatric GBM (age≤18 years) with special reference to p53, p16 and p27 protein expression; epidermal growth factor receptor (EGFR) alterations and phosphate and tensin homolog gene (PTEN) deletion. Thirty cases of childhood GBMs reported between January 2002 to June 2007 were selected and H&E stained slides reviewed. Immunohistochemical staining was performed for EGFR, p53, p16, p27 and MIB 1 labeling index. Fluorescence in situ hybridization (FISH) analysis was performed to evaluate for EGFR amplification and PTEN deletion. Histopathological features and MIB-1 labeling index were similar to adult GBMs. p53 protein expression was observed in 63%. Though EGFR protein over-expression was noted in 23% cases, corresponding amplification of the EGFR gene was rare (5.5%). Deletion of the PTEN gene was also equally rare (5.5%). One case showed polysomy (chromosomal gains) of chromosomes 7 & 10. Loss of p16 and p27 immunoexpression was observed in 68% and 54 % cases respectively. In pediatric de novo/primary GBMs, deletion of PTEN and EGFR amplification are rare, while p53 alterations are more frequent as compared to pimary adult GBMs. Frequency of loss of p16 and p27 immunoexpression is similar to their adult counterparts. This suggests that pediatric malignant gliomas are distinctly different from adult GBMs highlighting the need for identification of molecular targets that may be adopted for future novel therapeutic strategies.

Both genetic and epigenetic mechanisms contribute to meningioma development by altering gene expression and protein function. To determine the relative contribution of each mechanism to meningioma development, we used an integrative approach measuring copy number and DNA methylation changes genome-wide. We found that genetic alterations affected 1.9%, 7.4%, and 13.3% of the 691 loci studied, whereas epigenetic mechanisms affected 5.4%, 9.9%, and 10.3% of these loci in Grade I, II, and III meningiomas, respectively. Genetic and epigenetic mechanisms rarely involved the same locus in any given tumor. The predilection for epigenetic rather than genetic silencing was exemplified at the 5' CpG island of WNK2, a serine-threonine kinase gene on chromosome 9q22.31. WNK2 is known to negatively regulate EGFR signaling via inhibition of MEK1, and point mutations have been reported in WNK1, WNK2 as WNK3 and WNK4. In meningiomas, WNK2 was aberrantly methylated in 83% and 71% of Grade II and III meningiomas, but rarely in a total of 209 tumors from thirteen other tumor types. Aberrant methylation of the CpG island was associated with decreased expression in primary tumors. WNK2 could be reactivated with a methylation inhibitor in IOMM-Lee, a meningioma cell line with a densely methylated WNK2 CpG island and lack of WNK2 expression. Expression of exogenous WNK2 inhibited colony formation, implicating it as a potential cell growth suppressor. These findings indicate that epigenetic mechanisms are common across meningiomas of all grades and that for specific genes such as WNK2, epigenetic alteration may be the dominant, grade-specific mechanism of gene inactivation.

Quality of life is an important area of clinical neuro-oncology that is increasingly relevant as survivorship increases and as patients experience potential morbidities associated with new therapies. This review of quality-of-life studies done in the brain tumor population aims to summarize what is currently known about quality of life in patients with both low-grade and high-grade tumors and suggest how we may use this knowledge to direct future research. To date reports on quality of life have been primarily qualitative and focused on specific symptoms such as fatigue, sleep disorders, and cognitive dysfunction, as well as some symptom clusters. However, the increasing interest in exploring quality of life as a primary endpoint for cancer therapy has established a need for prospective, controlled studies to assess baseline and serial quality-of-life parameters in brain-tumor patients in order to plan and evaluate appropriate and timely intervention for their symptoms.

Temozolomide (TMZ)-based therapy is the standard of care for patients with glioblastoma multiforme (GBM), and resistance to this drug in GBM is modulated by the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT). Expression of MGMT is silenced by promoter methylation in approximately half of GBM and clinical studies have shown that elevated MGMT protein levels or lack of MGMT promoter methylation is associated with TMZ resistance in some, but not all GBM tumors. The relationship between MGMT protein expression and tumor response to TMZ was evaluated in 4 GBM xenograft lines that had been established from patient specimens and maintained by serial subcutaneous passaging in nude mice. Three MGMT unmethylated tumors all displayed elevated basal MGMT protein expression, but only 2 of these were resistant to TMZ therapy (GBM43 and 44), while the other (GBM14) displayed a level of TMZ sensitivity that was similar in extent to that seen in a single MGMT hypermethylated line (GBM12). In tissue culture and animal studies, TMZ treatment resulted in robust and prolonged induction of MGMT expression in the resistant GBM43 and GBM44 xenograft lines, while MGMT induction was blunted and abbreviated in GBM14. Consistent with a functional significance of MGMT induction, treatment of GBM43 with a protracted low dose TMZ regimen was significantly less effective than a shorter high-dose regimen, while survival for GBM14 was improved with the protracted dosing regimen. In conclusion, MGMT expression is dynamically regulated in some MGMT non-methylated tumors, and in these tumors, protracted dosing regimens may not be effective.

The clinical approach of using antibodies that sequester molecules in the bloodstream is an elegant solution to the drug-uptake problem in neuro-oncology.

Background. Glutathione S-transferases (GSTs) are polymorphic enzymes that catalyze the glutathione conjugation of alkylating agents, platinum compounds, and free radicals formed by radiation used to treat medulloblastoma. We hypothesized that GST polymorphisms may be responsible, in part, for individual differences in toxicity and response in pediatric medulloblastoma.

Methods. We investigated the relationship between GSTM1 and GSTT1 polymorphisms and survival and toxicity in 42 children with medulloblastoma diagnosed and treated at Texas Children's Cancer Center. We conducted Kaplan-Meier analyses to determine if the GST polymorphisms were related to progression-free survival (PFS) and performed logistic regression to explore associations between GST polymorphisms and occurrence of grade 3 or greater myelosuppression, ototoxicity, nephrotoxicity, neurotoxicity, and intellectual impairment.

Results. Patients with at least one null genotype had a 4.3 (1.1-16.8), 3.7 (1-13.6), and 6.4 (1.2-34) times increased risk for any ≥Gr 3 toxicity, any ≥Gr 3 toxicity excluding peripheral neuropathy, and any ≥Gr 3 toxicity requiring omission or cessation of chemotherapy, respectively. Compared to all others, patients with at least one null genotype had in average 27.2 (p=0.0002), 29 (p=0.0004), and 21.7 (p=0.002) lower full-scale, performance, and verbal IQ scores, respectively.

Conclusion. GSTM1 and T1 polymorphisms may predict adverse events including cognitive impairment after therapy in patients with medulloblastoma. A larger study to validate these findings is under way

Normalization of tumor vasculature by antiangiogenic agents may improve the delivery of cytotoxic drugs to the tumor, leading to more effective therapy. In this study, pharmacokinetic and pharmacodynamic approaches were utilized to investigate how sunitinib at different dose levels affects brain distribution of temozolomide (TMZ), and to ascertain the relationship between intratumoral TMZ concentrations and tumor vascularity in an orthotropic human glioma model. Three groups of intracerebral U87MG tumor-bearing mice were given either vehicle, 20 mg/kg/d or 60 mg/kg/d sunitinib, for 7 days before receiving a steady-state regimen of TMZ that consisted of an IV bolus and a 3-h intraarterial infusion. TMZ concentrations in plasma, normal brain and brain tumor were determined, and several biomarkers related to the antiangiogenic activity of sunitinib were examined. TMZ distribution in the normal brain as indicated by the brain-to-plasma steady-state TMZ concentration ratios was analogous between the three treatment groups. The brain tumor-to-plasma steady-state TMZ concentration (ss Ct/Cp) ratio was significantly increased in the 20 mg/kg sunitinib group (0.98 ± 0.17) as compared with the control (0.76 ± 0.17) and 60 mg/kg sunitinib (0.68 ± 0.09) groups. The ss Ct/Cp ratios were significantly correlated with the Vascular Normalization Index (VNI), derived from the expression of CD31, collagen IV, and -smooth muscle actin, which represents the fraction of functioning vessels out of the total tumor vessels. In conclusion, the effect of sunitinib on the brain tumor distribution of TMZ was dose-dependent, and indicated optimal tumor exposure was achieved at a lower dose and associated with the VNI.

The flavonoid quercetin has been reported to inhibit the proliferation of cancer cells whereas it has no effect on non-neoplastic cells. U87-MG, U251, A172, LN229 and U373 malignant gliomas cells were treated with quercetin (50-200 µM). 24 hours after treatment quercetin did not cause cytotoxicity. Combining quercetin with TNF-related apoptosis-inducing ligand (TRAIL) strongly augmented TRAIL-mediated apoptosis in U87-MG, U251, A172, LN229 glioma cells. U373 cells could not be sensitized by quercetin to TRAIL-mediated apoptosis. We demonstrated that TRAIL-induced apoptosis is enhanced by quercetin-induced reduction of survivin protein levels. Upon treatment with quercetin the protein level of survivin was strongly suppressed in U87-MG, U251, A172, but not in U373 glioma cells. Quercetin exposure resulted in proteasomal degradation of survivin. TRAIL-quercetin induced apoptosis was markedly reduced by over-expression of survivin. In addition, we demonstrated that upon treatment with quercetin down-regulation of survivin was also regulated by the Akt pathway. Taken together, the present study suggests that quercetin sensitizes glioma cells to death-receptor mediated apoptosis by suppression of inhibitor of apoptosis proteins survivin.

High-grade gliomas release excitotoxic concentrations of glutamate, which has been shown to enhance tumor proliferation and migration. Alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) glutamate receptors are abundantly expressed at the invading edge of glioblastoma specimens, suggesting they may play an important biologic role in tumor invasion. In this study, we examined potential mechanisms by which AMPA receptor expression and stimulation promote glioma cell migration and invasion. Overexpression of GluR1, the most abundant AMPA receptor subunit in gliomas, positively correlated with glioma cell adhesion to types I and IV collagen which was decreased in cells with knockdown of GluR1 and with blocking antibodies to β1 integrin. Furthermore, stimulation of the AMPA receptor led to detachment of cells from the ECM. Immunoprecipitation studies showed that GluR1 associated with the actin cytoskeleton-linked protein band 4.1B (brain type), which may serve as a link between GluR1 and integrins. Overexpression of GluR1 correlated with increased cell surface expression of β1 integrin, increased phosphorylation of focal adhesion kinase (FAK-Y397) and enhanced the number of focal adhesion (FA) complexes. Cells overexpressing GluR1 had increased co-localization of actin and paxillin at FAs and, in several glioma cell lines, significantly increased invasion in an in vitro Matrigel transwell assay. Likewise, in an intracranial xenograft model, overexpression of GluR1 led to perivascular and subependymal glioma cell invasion similar to patterns of tumor dissemination described in human glioblastoma. Together, these results suggest that AMPA receptors may link signals from the ECM to sites of FA, where signal integration promotes tumor invasion.

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We have addressed the search of novel genetic prognostic markers in a selected cohort of patients with localized resectable neuroblastoma (NB), stroma poor, who underwent relapse or progression (group 1) or complete remission (group 2) over a minimum follow-up of 32 months from diagnosis. Twenty three Italian patients with localized resectable NB (stages 1 and 2) diagnosed from 1994 to 2005 were studied. All patients received surgical treatment. Chemotherapy was administered only to the three stage 2 patients who had MYCN amplified tumors. High-resolution array-comparative genomic hybridization (CGH) DNA copy-number analysis technology was used in order to identify novel prognostic markers. Chromosome 1p36.22p36.32 loss and 1q22qter gain, detected almost exclusively in group 1 patients, were significantly associated with poor event-free survival (EFS) (P = 0.0024 and P = 0.024, respectively). In contrast, patients with 7p11.2p22 gain, who belonged predominantly to group 2, had a significantly better EFS (P = 0.015). The frequency of 17q gain or 3 and 11q losses did not differ significantly in group 1 vs group 2 NBs. The sensitive technique used allowed us to define the smallest region of 1p deletion. In conclusion, 1q22qter gain and 7p11.2p22 gain might represent new prognostic markers in localized resectable NB, but the small study size and the retrospective nature of the findings warrant further validation of the results obtained in larger studies.

Purpose. To investigate the utility of postoperative chemotherapy to delay radiotherapy and to identify prognostic factors in early childhood medulloblastoma. Patients. From 1987–1993, children below three years of age registered to the HIT-SKK87 trial received systemic interval chemotherapy until craniospinal radiotherapy was applied at three years of age or at relapse. Children with postoperative residual tumor or metastatic disease received systemic induction chemotherapy prior to interval chemotherapy. Results. 29 children were eligible for analyses (median age, 1.7 years; median follow-up, 12.6 years). In children without macroscopic metastases, rates for 10-year progression-free and overall survival were 52.9±12.1% and 58.8±11.9% (complete resection), and 55.6±16.6% and 66.7±15.7% (incomplete resection), compared to 0% and 0% in children with macroscopic metastases. Survival was superior in 9 children with desmoplastic or extensive nodular histology compared to 20 children with classic medulloblastoma (10-year PFS 88.9±10.5% and 30.0±10.3%; P=.003; OS 88.9±10.5 and 40.0±11.0%; P=.006). Eleven out of twelve children with tumor progression during chemotherapy had classic medulloblastoma. After treatment, IQ-scores were inferior compared to non-irradiated children from the subsequent study HIT-SKK92. Classic histology, metastatic disease, and male gender, were independent adverse risk factors for PFS and OS in 72 children from this study and from HIT-SKK92. Conclusions. In terms of survival, craniospinal radiotherapy was successfully delayed especially in young children with medulloblastoma of desmoplastic/extensive nodular histology, which was a strong independent favourable prognostic factor. In consequence to the neurocognitive deficits of survivors, the emerging concepts to avoid craniospinal radiotherapy should rely on the histologic medulloblastoma subtype.

First-line therapy for patients with glioblastoma multiforme (GBM) includes treatment with radiation and temozolomide (TMZ), an oral DNA alkylating chemotherapy. Sensitivity of glioma cells to TMZ is dependent on the level of cellular O6-methylguanine-DNA methyltransferase (MGMT) repair activity. Several common coding region polymorphisms in the MGMT gene (L84F and the linked pair I143V/K178R) modify functional characteristics of MGMT and cancer risk. To determine whether these polymorphic changes influence the ability of MGMT to protect glioma cells from TMZ, we stably overexpressed enhanced green fluorescent protein (eGFP)-tagged MGMT constructs in U87MG glioma cells. We confirmed that the WT eGFP-MGMT protein is properly localized within the nucleus and found that L84F, I143V/K178R and L84F/I143V/K178R eGFP-MGMT variants exhibited nuclear localization patterns indistinguishable from WT. Using MTT proliferation and clonogenic survival assays, we confirmed that WT eGFP-MGMT expressing cells are resistant to TMZ treatment compared to control U87MG cells, and found that each of the polymorphic eGFP-MGMT variants confer similar resistance to TMZ. However, upon exposure to O6-BG, a synthetic MGMT inhibitor, the L84F and L84F/I143V/K178R variants were degraded more rapidly than WT or I143V/K178R in a proteasome-dependent manner. Despite the increased O6-BG stimulated protein turnover caused by the L84F alteration, cells expressing L84F eGFP-MGMT did not exhibit altered sensitivity to the combination of O6-BG and TMZ compared to WT expressing cells. In conclusion, we demonstrate that the L84F polymorphic variant has altered protein turnover without modifying sensitivity of U87 cells to TMZ or combined TMZ and O6-BG. These findings may provide a clue to determining the clinical significance of coding region polymorphisms.

Glioblastomas often show activation of epidermal growth factor receptor (EGFR) and loss of PTEN tumor suppressor, but it is not known if these two genetic lesions act together to transform cells. To answer this question, we infected PTEN-/- neural precursor cells with a retrovirus encoding EGFRvIII, which is a constitutively activated receptor. EGFRvIII PTEN-/- cells formed highly mitotic tumors with nuclear pleomorphism, necrotic areas and glioblastoma markers. The transformed cells showed increased cell proliferation, centrosome amplification, colony formation in soft agar, self-renewal, expression of the stem cell marker, CD133, as well as resistance to oxidative stress and ionizing radiation. The RAS/ERK and PI3K/Akt pathways were activated, and Chk1, the DNA damage regulator, was phosphorylated at S280 by Akt, suppressing Chk1 phosphorylation at S345 in response to ionizing irradiation. The PTEN-/- cells showed low levels of DNA damage in the absence of irradiation, which was increased by EGFRvIII expression. Finally, secondary changes occurred during tumor growth in mice. Cells from these tumors showed decreased tumor latencies and additional chromosomal aberrations. Most of these tumor lines showed translocations of mouse chromosome 15. Intracranial injections of one of these lines led to invasive, GFAP+, nestin+ tumors. These results provide a molecular basis for the occurrence of these two genetic lesions in brain tumors and point to a role in induction of genomic instability.

Purpose: Promyelocytic leukemia (PML) protein plays an essential role in the induction of apoptosis; its expression is reduced in various cancers. As the functional roles of PML in glioblastoma multiforme (GBM) have not been clarified, we assessed the expression of PML protein in GBM tissues and explored the mechanisms of PML-regulated cell-death in GBM cells. Experimental Design: We examined the PML mRNA level and the expression of PML protein in surgical GBM specimens. PML-regulated apoptotic mechanisms in GBM cells transfected with plasmids expressing the PML gene were examined. Results: The protein expression of PML was significantly lower in GBM- than non-neoplastic tissues; approximately 10% of GBM tissues were PML-null. The PML mRNA levels were similar in both tissue types. The overexpression of PML activated caspase-8 and induced apoptosis in GBM cells. In these cells, PML decreased the expression of transactivated forms of NFB/p65 and c-FLIP gene expression was suppressed. Therefore, PML-induced apoptosis resulted from the suppression of the transcriptional activity of NFB/p65. PML overexpression decreased phosphorylated IB and nuclear NFB/p65 and increased the expression of the suppressor of cytokine signaling (SOCS)-1. A proteasome inhibitor blocked the reduction of activated p65 by PML. Conclusions: The reduction of PML is associated with the pathogenesis of GBM. PML induces caspase-8-dependent apoptosis via the repression of NFB activation by which PML facilitates the proteasomal degradation of activated p65 and the sequestration of p65 with IB in the cytoplasm. This novel mechanism of PML-regulated apoptosis may represent a therapeutic target for GBM.

Methotrexate (MTX) is an important anticancer drug and the most efficient chemotherapy component in primary central nervous system lymphoma (PCNSL). A typical side effect of intravenous high-dose MTX is the occurrence of confluent CNS white matter changes (WMC). Since MTX directly interferes with methionine metabolism, we analyzed the impact of genetic variants of methionine metabolism on the occurrence of WMC as a model of MTX toxicity. In a retrospective analysis of 68 PCNSL patients treated with MTX-based polychemotherapy with (n=42) or without (n=26) intraventricular treatment, ten genetic variants influencing methionine metabolism were analyzed. Pearson's Chi-square test and multinominal regression analysis were used to define the relevance of these genetic variants for the occurrence of WMC. In this patient sample, the occurrence of WMC was significantly predicted by the TT genotype of methylenetetrahydrofolate reductase c.677C>T (²=8.67; p=0.013; df=2), the AA genotype of methylenetetrahydrofolate reductase c.1298A>C (²=13.5; p=0.001; df=2) and the GG genotype of transcobalamin 2 c.776C>G (²=19.73; p<0.001) in addition to male gender (²=11.95; p=0.001). These data strengthen the hypothesis that MTX effects are influenced by methionine metabolism which may offer new strategies to improve MTX-based therapies.

Malignant glioma is the most commonly occurring primary malignant brain tumor. It is difficult to treat and usually associated with an inexorable, rapidly fatal clinical course. Chemotherapy, radiotherapy, and surgical excision are core components in the management of malignant glioma. However, chemotherapy, even with the most active regimens currently available, achieves only modest improvement in overall survival. Novel agents and new approaches to therapy are required to improve clinical outcomes. Irinotecan, a first-line treatment for metastatic colorectal cancer and an agent with high activity against solid tumors of the gastrointestinal tract, is an inhibitor of topoisomerase I, a critical enzyme needed for DNA transcription. Irinotecan crosses the blood-brain barrier and, in preclinical investigations, has demonstrated cytotoxic activity against central nervous system tumor xenografts. Its antitumor activity has also been demonstrated against glioblastoma cells with multidrug resistance. Studies in adult and pediatric patients with recurrent, intractable malignant glioma have evaluated irinotecan as monotherapy and in combination with other agents, including temozolomide, carmustine, thalidomide, and bevacizumab. Studies of irinotecan in combination with other medications, particularly temozolomide and bevacizumab, have yielded promising results. Irinotecan monotherapy has demonstrated efficacy; however, its efficacy appears to be enhanced when it is used in combination with other chemotherapeutic agents. When administered concurrently with enzyme-inducing antiepileptic drugs, the dosage must be increased to compensate for enhanced cytochrome CY3A4/5 enzyme activity. Toxicities associated with irinotecan have been manageable; the most important dose-limiting toxicities are neutropenia and diarrhea. Irinotecan-based chemotherapy of malignant glioma merits further study.

Background: The epidemiology and natural history of adult gliosarcomas (GSMs) as well as patient and treatment factors associated with outcome are ill-defined.

Methods: Patients greater than 20 years of age with GSM diagnosed from 1988 to 2004 were identified in the Surveillance, Epidemiology and End Results (SEER) database. Kaplan-Meier survival analysis and Cox models were used to examine outcomes. Similar analyses were conducted for patients diagnosed with glioblastoma (GBM) over the same time period.

Results: GSM represented 2.2% of the 16,388 patients identified with either GSM or GBM. No significant differences between GSM and GBM were identified with respect to age, gender, race, tumor size or use of adjuvant radiation therapy (RT). Patients with GSM were more likely to have temporal lobe involvement and undergo some form of tumor resection. The most important analyzed factors influencing GSM overall survival were age, extent of resection and the use of adjuvant RT. After adjusting for factors impacting overall survival, the prognosis for GSM appears slightly worse than for GBM (HR = 1.17, 95% CI 1.05–1.31).

Conclusions: GSM is a rare malignancy that presents very similarly to GBM with a slightly greater propensity for temporal lobe involvement. Optimal treatment remains to be defined. However, these retrospective findings suggest tumor excision, as opposed to biopsy only, and adjuvant RT may improve outcome. Despite therapy, prognosis remains dismal and outcomes may be inferior to those seen in GBM patients.

The aim of this study was to determine the efficacy of sagopilone (ZK-EPO), a novel epothilone, compared with other anticancer agents in human orthotopic models of primary and secondary brain tumors. Autoradiography and pharmacokinetic analyses were performed on rats and mice to determine passage across the blood-brain barrier and organ distribution of sagopilone. Mice bearing intracerebral human tumors (U373 or U87 glioblastoma, MDA-MB-435 melanoma, or patient-derived non-small-cell lung cancer [NSCLC]) were treated with sagopilone 5–10 mg/kg, paclitaxel 8–12.5 mg/kg (or temozolomide 100 mg/kg), or control (vehicle only). Tumor volume was measured to assess antitumor activity. Sagopilone crossed the blood-brain barrier in both rat and mouse models, leading to therapeutically relevant concentrations in the brain with a long half-life. Sagopilone exhibited significant antitumor activity in both the U373 and U87 human glioblastoma models, while paclitaxel showed a limited effect in the U373 model. Sagopilone significantly inhibited the growth of tumors from CNS metastases models (MDA-MB-435 melanoma and patient-derived Lu7187 and Lu7466 NSCLC) implanted in the brains of nude mice, in contrast to paclitaxel or temozolomide. Sagopilone has free access to the brain. Sagopilone demonstrated significant antitumor activity in orthotopic models of both glioblastoma and CNS metastases compared with paclitaxel or temozolomide, underlining the value of further research evaluating sagopilone in the treatment of brain tumors. Sagopilone is currently being investigated in a broad phase II clinical trial program, including patients with glioblastoma, NSCLC, breast cancer, and melanoma.

Purpose: The primary objectives of this phase II study were to evaluate the use of pre-irradiation temozolomide followed by concurrent temozolomide and RT in patients with newly diagnosed anaplastic oligodendroglioma (AO) and mixed anaplastic oligoastrocytoma (MOA). Methods: Pre-irradiation temozolomide (150 mg/m2/day) was given on a 7-day on/7-day off schedule for up to 6 cycles. The primary endpoint was the response rate during the 6-month pre-RT chemotherapy. Tumor tissue was analyzed for the presence of chromosomal deletions on 1p and 19q, and for MGMT promoter methylation. Results: Forty-two patients were enrolled; 39 were eligible. The objective response rate was 32% (6% CR, 26% PR) and the rate of progression during pre-RT chemotherapy was 10%. The worst non-hematological toxicity was grade 4 in 3 patients (8%). Twenty-two patients completed concurrent chemotherapy and RT. There were no grade 4 non-hematological toxicities during the concurrent chemotherapy and RT. Seventeen of 28 (60.7%) evaluable cases had codeletion of 1p/19q; all 17 were free from progression at 6 months. Sixteen of 20 (80%) evaluable cases had MGMT promoter methylation; all 16 were free from progression at 6 months. Conclusions: The rate of progression of 10% during pre-RT temozolomide chemotherapy for newly diagnosed AO and MAO compared favorably to prior experience with pre-RT PCV chemotherapy (20% in RTOG 9402). The toxicity of the dose-intense pre-RT regimen used in this study may warrant evaluation of other, less intense dosing strategies. Future studies will need to prospectively stratify patients according to the presence of deletions of chromosomes 1p and 19q.

Purpose: To evaluate efficacy and magnetic resonance imaging (MRI) after intravenous bevacizumab and/or carboplatin in a human glioma animal model.

Experimental Design: Male nude rats with intracerebral UW28 human glioma xenografts were randomized to four groups: (1) controls (n = 9) ; (2) bevacizumab 10 mg/kg (n = 6) ; (3) carboplatin 200 mg/m2 (n = 6); and (4) bevacizumab plus carboplatin (n = 6) . MRI was performed on the day of treatment (day 7-10) and one week later, and rats were followed for survival. Dynamic MRI was done in three controls and three rats treated with bevacizumab ± carboplatin before and 24 hours after treatment.

Results: Median overall survival (OS) was: (1) controls, 16 days; (2) bevacizumab, 23 days; (3) carboplatin, 22 days; (4) bevacizumab plus carboplatin, 36 days. OS of group 4 was significantly longer than group 1 (p = 0.0011) , group 2 (p = 0.0014) and group 3 (p = 0.0015) , and rats had significantly larger tumors. No objective tumor responses were observed on MRI at one week after treatment; however, after bevacizumab dynamic MRI showed reduced gadolinium enhancement intensity and increased time to peak, consistent with decreased vascular permeability.

Conclusions: Carboplatin plus bevacizumab is effective and superior over bevacizumab or carboplatin monotherapy in this animal model. Increased survival concomitant with increased asymptomatic tumor volume is suggestive that vascular targeting with reduced peritumoral edema and mass effect contributes to the efficacy of bevacizumab. The promising survival data warrant future clinical trials using bevacizumab plus carboplatin.

One barrier to successful treatment of malignant glioma is resistance to alkylating agents such as temozolomide. The cytotoxic activity of temozolomide and other alkylating agents is believed to be manifested largely by the formation of O-methylguanine DNA adducts. Consequently, the primary mechanism of resistance to temozolomide is a function of the activity of the DNA repair enzyme O-methylguanine DNA methyltransferase (MGMT). Fortuitously, MGMT is inactivated after each reaction (ie, suicide enzyme). Therefore, if the rate of DNA alkylation were to outpace the rate of MGMT protein synthesis, the enzyme could, in theory, be depleted. Several studies have shown that prolonged exposure to temozolomide can deplete MGMT activity in blood cells, a process that could potentially increase the antitumor activity of the drug. To date, however, there are limited data demonstrating the depletion of MGMT activity in tumor tissue exposed to temozolomide. A variety of dosing schedules that increase the duration of exposure and the cumulative dose of temozolomide are currently being investigated for the treatment of glioma, with the goal of improving antitumor activity and overcoming resistance. These alternative dosing regimens have been shown to deplete MGMT activity in peripheral blood mononuclear cells, but the regimen that provides the best balance between enhanced antitumor activity and acceptable hematologic toxicity has yet to be determined.

Anti-angiogenic drugs have emerged as effective treatment options for patients with recurrent malignant gliomas (MG). Though this class of drugs is generally well-tolerated, rare life-threatening complications including thromboembolism, hemorrhage, and gastrointestinal (GI) perforation are reported. We describe six cases of GI perforation among 244 glioma patients (2.5%) during treatment with anti-angiogenic agents in combination with chemotherapy and corticosteroids. Two patients succumbed to this complication, and the others recovered. Because GI perforation is a life-threatening yet treatable complication, neuro-oncologists must have a low threshold to consider it in patients on anti-angiogenic drug therapy who present with abdominal pain and other GI complaints

Purpose: The introduction of methotrexate-based chemotherapy has improved median survival for patients with primary central nervous system lymphoma (PCNSL). Older age is a negative prognostic marker in patients with PCNSL and may increase the likelihood of methotrexate toxicity. We studied the response and adverse effects of intravenous high-dose methotrexate in patients who were ≥ 70 at the time of diagnosis. Patients and Methods: We identified 31 patients at our institution diagnosed with PCNSL at age ≥ 70 who were treated with high-dose methotrexate (3.5 gm/m2-8 gm/m2) as initial therapy from 1992-2006. The best response to methotrexate was determined by contrast enhanced MRI. Toxicity was analyzed by chart review. Results: Thirty-one patients with a median age of 74 received a total of 303 cycles of methotrexate. Overall, 87.9% of the cycles required dose reduction because of impaired creatinine clearance. The median number of cycles per patient was 8. In 30 evaluable patients the overall radiographic response rate was 96.7%, with 18 complete responses (60%) and 11 partial responses (36.7%). Progression free survival and overall survival were 7.1 months and 37 months, respectively. Grades I-IV toxicities were observed in 27/31 patients and included gastrointestinal disturbances in 58% (3.2% grade III), hematological complications in 80.6% (6.5% grade III) and renal toxicity in 29% (0% grade III/IV). Conclusions: High-dose methotrexate is associated with a high proportion of radiographic responses and a low proportion of grades III/IV toxicity in patients ≥70. High-dose methotrexate should be considered as a feasible treatment option in elderly patients with PCNSL.

Purpose: Convection-enhanced delivery (CED) with various drug carrier systems has recently emerged as a novel chemotherapeutic method to overcome the problems of current chemotherapies against brain tumors. Polymeric micelle systems have exhibited dramatically higher in vivo antitumor activity in systemic administration. This study investigated the effectiveness of CED with polymeric micellar doxorubicin (micellar DOX) in a 9L syngeneic rat model.

Experimental Design: Distribution, toxicity, and efficacy of free, liposomal, and micellar DOX infused by CED were evaluated.

Results: Micellar DOX achieved much wider distribution in brain tumor tissue and surrounding normal brain tissue compared to free DOX. Tissue toxicity increased at higher doses, but rats treated with micellar DOX showed no abnormal neurological symptoms at any dose tested (0.1-1.0 mg/mL). Micellar DOX infused by CED resulted in prolonged median survival (36 days) compared to free DOX (19.6 days; P=0.0173) and liposomal DOX (16.6 days; P=0.0007) at the same dose (0.2 mg/mL).

Conclusions: This study indicates the potential of CED with the polymeric micelle drug carrier system for the treatment of brain tumors.

Lumbosacral radiculopathy is a rare complication of radiotherapy and may be challenging to differentiate from diagnosis of a tumor recurrence. We reviewed the records of three patients with a past history of cancer and radiotherapy who were referred for suspicion of carcinomatous meningitis on lumbar MRI, but whose final diagnosis was radiation-induced lumbosacral radiculopathy. The three patients developed a progressive lumbosacral radiculopathy at 20, 13 and 47 years following lumbar radiotherapy delivered for renal cancer, Hodgkin’s disease and a seminoma, respectively. MRI showed a diffuse, nodular enhancement of the cauda equina nerve roots on T1 sequences, suggestive of leptomeningeal metastasis. A slowly progressive clinical course over several years and negative CSF cytologic analysis ruled out the diagnosis of carcinomatous meningitis. Because of the radiological findings, a biopsy was performed in two patients. In the first, a biopsy limited to the arachnoid excluded a malignant infiltration. In the second, a biopsy of the enhancing lesions demonstrated spinal root cavernomas. These observations, together with three recent case reports in the literature, delineate a syndrome of "radiation-induced lumbosacral radiculopathy with multiple spinal root cavernomas" which mimics carcinomatous meningitis on MRI. Its diagnosis is important in order to avoid inappropriate treatment and useless or dangerous spinal root biopsies.

Background: Glioblastoma multiforme (GBM) is a lethal primary malignant brain tumor in adults. R115777 (Zarnestra) is an oral agent with antiproliferative effects being a potent and selective inhibitor of FTPase. This multi-center, open-label, phase II study was designed to evaluate the efficacy and safety of R115777 given after surgery and prior to radiation in patients with newly diagnosed and residual enhancing GBM.

Methods: Following surgery, an MRI confirmed the presence of residual enhancing tumor. Patients on EIASDs received 600mg twice per day and those not on EIASDs received 300mg twice per day. One to three monthly cycles of R115777 was administered and radiation was initiated with progression or after three cycles. A cycle consisted of 3 weeks of continuous R115777 followed by a one week of rest prior. MRI's were done monthly. The primary endpoint was overall survival; secondary endpoints were tumor response rate and toxicity.

Results: A total of twenty-eight confirmed GBM patients entered the study. Fifteen patients (54%) were on EIASDs. The overall median time of survival is 7.7 months. There were no tumor responses. Eight patients (29%) had stable disease as the best response. The study was stopped early due to progression of the disease in 12 patients (48%). A total of 24 patients (85%) were off study before the planned treatment schedule for radiation therapy.

Conclusions: R115777 administered prior to radiation therapy in patients with newly diagnosed GBM and residual enhancing disease did not result in any measurable responses or improvement in survival. R115777 administered prior to radiation therapy is not recommended for the patients with newly diagnosed GBM.

In Reference to Lamborn et al. (Neuro-Oncology 2008;10:162-170)

Purpose: To describe the quality of life (QOL) of low-grade glioma (LGG) patients at baseline prior to chemotherapy and through 12 cycles of Temozolomide (TMZ) chemotherapy.

Methods: Patients with histologically-confirmed LGG with only prior surgery were given TMZ for 12 cycles. QOL assessments by the Functional Assessment of Cancer Therapy-Brain (FACT-Br) were obtained at baseline prior to chemotherapy and at 2 month intervals while receiving TMZ.

Results: Patients with LGG at baseline prior to chemotherapy have higher reported social well-being scores (mean difference = 5.0; p < 0.01), but have lower reported emotional well-being scores (mean difference = 2.2; p < 0.01) compared to a normal population. Compared to left hemisphere tumors, patients with right hemisphere tumors reported higher physical (p = 0.01) well being scores. 44% of patients could not drive, 26% of patients did not feel independent, and 26% of patients were afraid of having a seizure. Difficulty with work was noted in 24% of patients. Mean change scores at each chemotherapy cycle compared to baseline for all QOL subscales showed either no significant change or were significantly positive (p < 0.01).

Conclusion: Patients with LGG on TMZ at baseline prior to chemotherapy have reported QOL comparable to a normal population with the exception of social and emotional well-being, and those with right hemisphere tumors report higher physical well-being scores compared to those with left hemisphere tumors. While remaining on therapy, LGG patients are able to maintain their QOL in all realms. LGG patients' QOL may be further improved by addressing their emotional well-being and their loss of independence in terms of driving or working.

Survival rates of pediatric brain tumor patients have significantly improved over the years due to developments in diagnostic techniques, neurosurgery, chemotherapy, radiotherapy and supportive care. However, brain tumors are still an important cause of cancer-related deaths in children. Prognosis is still highly dependent on clinical characteristics, such as the age of the patient, tumor type, stage and localization, but increased knowledge about the genetic and biological features of these tumors is being obtained and might be useful to further improve the outcome of these patients. It has become clear that the deregulation of signaling pathways essential in brain development, e.g. SHH, Wnt and Notch, plays an important role in the pathogenesis and biological behavior of especially medulloblastomas. More recently, data have become available about the cells of origin of brain tumors and about the possible existence of brain tumor stem cells. Newly developed array-based techniques for studying gene expression, protein expression, copy number aberrations and epigenetic events have led to the identification of other potentially important biological abnormalities in pediatric medulloblastomas and ependymomas.

Purpose: To describe a cohort of patients with leptomeningeal melanomatosis (LM) and to determine prognostic factors for outcomes in these patients. The primary hypothesis was that more extensive burden of CNS metastasis at the time of diagnosis of LM (as evidenced by imaging of the CNS parenchyma and meninges, and CSF cytology status (positive versus negative) correlates poorer outcomes. Patients and Methods: The records of all patients with LM treated at M.D. Anderson Cancer Center between 1944 and 2002 were reviewed. Information on clinical course and outcomes was gathered. Univariate and multivariate analyses were performed on 110 patients using Cox proportional hazards regression analysis to examine the effects of possible predictive factors on survival. Results: The overall median survival from LM diagnosis was 10 weeks with 95% confidence interval from 8 weeks to 14 weeks. Eighty-six (78.2%) patients had cutaneous primary lesions, and 23 (20.9%) had melanoma of unknown primary site. The primary hypothesis was not proven. Neither the presence of parenchymal CNS metastases nor greater imaging evidence of LM nor positive CSF cytology at diagnosis correlated with survival outcomes. Univariate analyses revealed possible predictors of longer survival, including: the presence of supratentorial or spinal LM on imaging at diagnosis versus not, or any treatment of LM; while an elevated serum LDH at the time of LM diagnosis predicted shorter survival. Multivariate analysis revealed that a history of a primary melanoma lesion originating on the trunk predicted shorter survival after LM diagnosis (HR=2.0(1.0, 3.8), p = 0.035) and treatment with intrathecal chemotherapy predicted longer survival (HR=0.5(0.4, 0.8), p = 0.0036). The positive result with respect to treatment is unreliable due to the inability to remove treatment selection bias from the analysis. Conclusions: This retrospective analysis confirmed the dismal prognosis associated with LM. The amount of CNS tumor burden at the time of diagnosis of LM did not inversely correlate with survival outcomes, as hypothesized.

Pathological angiogenesis is a hallmark of cancer, specifically of glioblastomas, the most malignant and common primary brain tumor. Vascular endothelial growth factor (VEGF) is the key protein in the regulation of the hypervascular phenotype of primary, malignant brain tumors. In this study, we tested VEGF Trap, a soluble decoy receptor for VEGF, in an intracranial glioma model. VEGF Trap was administered in short or prolonged schedules to human glioma-bearing animals at different stages of disease. Of importance, VEGF Trap treatment was efficacious in both initial and advanced phases of tumor development, by significantly increasing overall survival. Furthermore, this effect was enhanced in animals treated with more prolonged regimens. In addition, we observed the emergence of a VEGF Trap-resistant phenotype characterized by tumor growth and increased invasiveness. Our results suggest that VEGF Trap will be effective in treating both patients with recurrent or progressive resectable glioblastoma, and patients that have undergone extensive initial surgery. Finally, our results indicate that the clinical success of VEGF Trap may depend on a prolonged treatment in combined therapy aiming to simultaneously inhibit angiogenesis and tumor invasion.

Differential diagnosis of brain tumor types is mainly based on cell morphology, and could benefit from additional markers. The cAMP second messenger system is involved in regulating cell proliferation and differentiation and is conceivably modulated during cancer transformation. It mainly activates protein kinases, that are in part docked to cytoskeleton, membranes or organelles by anchoring proteins, forming protein aggregates that are detergent-insoluble and not freely diffusible, and are characteristics for each cell type.

The intracellular distribution of the detergent-insoluble regulatory subunits (R) of the cAMP dependent protein kinase has been examined in mouse and rat glioma cells both in vitro and in vivo by immunohistochemistry. In normal rodent brains, the RII regulatory subunit is detergent-insoluble only in ependymal cells, while in the rest of brain it is present in soluble form. Immunohistochemistry shows that in both mouse and rat glioma cell lines RII is mainly detergent-insoluble. It is localized close to the nucleus, associated to smooth vesicles in the trans-Golgi network area. Both paclitaxel and vinblastine cause a redistribution of RII within the cell. Under conditions that increased intracellular cAMP, apoptosis of glioma cells was observed, and was accompanied by RII redistribution. Also in vivo, detergent-insoluble RII can be observed in mouse and rat gliomas, where it delineates the border between normal brain tissue and glioma. Therefore, intracellular distribution of detergent-insoluble RII can assist in detecting tumor cells within the brain, thus making the histologic diagnosis of brain tumors more accurate, and may represent an additional target for therapy.

Abstracts for the Eighth Congress of the European Association for Neuro-Oncology (EANO), September 12-14, 2008, Barcelona, Spain

The purpose of the present study was to investigate the prognostic value of magnetic resonance imaging contrast enhancement (CE) at the time of histological diagnosis specifically in a selected population of World Health Organization (WHO) Grade II gliomas. A total of 927 histologically proven WHO grade II gliomas were reviewed, for which contrast-enhanced magnetic resonance imaging was available at the time of histological diagnosis. Contrast enhancement patterns were classified into three categories: "patchy and faint", "nodular-like" and "ring-like". Contrast enhancement progression over time was recorded before oncological treatment on successive MRIs, when available. Contrast enhancement was present in 143 cases (15.9%), with 93 "patchy and faint", 50 "nodular-like" and no "ring-like" patterns. Contrast enhancement areas were time progressive before oncological treatment in 35 of the 56 available cases (62.5%). Regardless of its pattern, the presence of contrast enhancement was not significantly associated with a worsened prognosis (p=0.415) by univariate analysis. Only the "nodular-like" pattern of contrast enhancement (p<0.01) and the time progressive contrast enhancements (p<0.001) in the available subgroup proved to be statistically associated with survival since first oncological treatment. The present results show the necessity, in cases of WHO grade II gliomas, to study the contrast enhancement at the time of histological diagnosis and, whenever possible, to follow its progression over time before oncological treatment. Indeed, "nodular-like" contrast enhancements and time progressive contrast enhancements are associated with a worsened prognosis, both suggesting malignant transformation, eventhough histopathological examination can not initially disclose signs of malignancy in those areas.

We have employed a laser capture microdissection technique and single nucleotide polymorphism arrays to characterize genomic alterations associated with the development of glioblastomas. Combined analysis of loss of heterozygosity (LOH) and copy number revealed that more than half (56.3%) of the identified 254 LOH loci showed no copy number alteration, indicating the presence of copy number neutral LOH. Furthermore, we found a glioblastoma case which showed copy number neutral LOH in 18 of the 22 autosomes. These results were confirmed by quantitative real-time PCR, microsatellite analysis, and fluorescence in situ hybridization (FISH). The high rate of copy number neutral LOH suggests that epigenetic abnormally of many genes are involved in the development and progression of glioblastomas.

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.

Glioblastoma multiforme (GBM) arises from genetic and signaling abnormalities in components of signal transduction pathways involved in proliferation, survival, and the cell cycle axis. Studies to date with single-agent targeted molecular therapy have revealed only modest effects in attenuating the growth of these tumors, suggesting that targeting multiple aberrant pathways may be more beneficial. Heat shock protein 90 (HSP90) is a molecular chaperone that is involved in the conformational maturation of a defined group of client proteins, many of which are deregulated in GBM. 17-allylamino-17-demethoxygeldanamycin (17-AAG) is a well-characterized HSP90 inhibitor that should be able to target many of the aberrant signal transduction pathways in GBM. We assessed the ability of 17-AAG to inhibit the growth of glioma cell lines and glioma stem cells both in vitro and in vivo, as well as assessing its ability to synergize with radiation and/or temozolomide, the standard therapies for GBM. Our results reveal that 17-AAG is able to inhibit the growth of both human glioma cell lines and glioma stem cells in vitro, and is able to target the appropriate proteins within these cells. In addition, 17-AAG can inhibit the growth of intracranial tumors, and can synergize with radiation both in tissue culture and in intracranial tumors. This compound was not found to synergize with temozolomide in any of our models of gliomas. Our results suggest that HSP90 inhibitors like 17-AAG may have therapeutic potential in GBM, either as a single agent or in combination with radiation.

At least 10% of glioblastoma relapses occur at distant and even contralateral locations. This disseminated growth limits surgical intervention and contributes to neurological morbidity. Preclinical data pointed towards a role for temozolomide in reducing radiotherapy-induced gliomas cell invasiveness. Our objective was to develop and validate a new analysis tool of magnetic resonance imaging (MRI) data to examine the clinical recurrence pattern of glioblastomas. MRIcro software was used to map location and extent of initial preoperative and recurrent tumours on MRI of 63 patients of the EORTC 26981/22981/NCIC CE.3 study into the same stereotaxic space. This allowed us to examine changes of site and distance between the initial and the recurrent tumour on the group level. Thirty of the 63 patients were treated using radiotherapy while the other patients completed a radiotherapy-plus-temozolomide treatment. Baseline characteristics (median age, Karnofsky performance score) and outcome data (progression-free survival, overall survival) of the patients included in this analysis resemble those of the general study cohort. The patient groups did not differ in the promotor methylation status of methyl-guanyl-methly-transferase (MGMT). Overall frequency of distant recurrences was 20%. Analysis of recurrence pattern revealed no difference in the size of the recurrent tumour nor a differential effect on the distance of the recurrences from the preoperative tumour location between the groups. The data show the feasibility of group-wise recurrence pattern analysis. An effect of temozolomide treatment on the recurrence pattern in the EORTC 26981/22981/NCIC CE.3 study could not be demonstrated.

The treatment of malignant gliomas with current therapies remains a challenge in neuro-oncology. Our recent work showed that embryonic stem cell (ESC)-derived astrocytes conditionally expressing genes can be used to induce apoptosis in malignant glioma cells in vitro. The tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) gene has been shown to induce apoptosis in a variety of tumor cells including gliomas. The aim of this study is to assess the pro-apoptotic effects of transgenic TRAIL delivered by ESC-derived astrocytes on malignant gliomas in vivo. Malignant glioma A172 cells were used to induce heterotopic xenografts in nude mice. ESC-derived astrocytes conditionally expressing TRAIL were injected into the xenografts. TRAIL expression was documented in the malignant glioma xenografts by RT-PCR and immunohistochemistry after external gene induction. A significant reduction in tumor volume occurred 48 hours after a single injection (14%) and double (31%) injections in the experimental groups. TUNEL revealed abundant apoptotic tumor cells in the experimental groups. Seven days after injection, the tumor had undergone severe necrosis with only scattered residual tumor cells at the periphery. Death receptor (DR4) expression increased significantly in the experimental groups compared to controls. Our data suggest that ESC-derived astrocytes conditionally expressing TRAIL should be considered as vectors to deliver gene therapy for malignant gliomas.

Health-related quality of life (HRQOL) has become an increasingly important endpoint in cancer studies; however, the research into the HRQOL of patients with high-grade glioma (HGG) is sparse, compared with that for patients with other neoplasms. Owing to the specific location and poor prognosis, it is more important and difficult to study HRQOL in patients with HGG than in those with other tumors; furthermore, the study of HRQOL in patients with HGG differs from that for patients with other tumors. In this review, we identified and compared the most frequently used instruments to assess HRQOL; analyzed specific facets and determinants of HRQOL (such as sex, tumor location and histological classification, depression, and cognitive function), as well as the association between HRQOL and survival; and appraised the effects of new treatments on HRQOL in patients with HGG from randomized controlled trials. Furthermore, we detected broadly existed problems and many contradictory outcomes, and gave some proper interpretation and suggestions regarding them.

Introduction: The benefit of cytoreductive surgery for glioblastoma multiforme (GBM) is unclear and selection bias in past series has been observed. The ALA study investigated the influence of fluorescence-guided resections on outcome and generated an extensive database of GBM patients with optimized resections. We evaluated whether the RTOG recursive partitioning analysis (RPA) would predict survival of these patients and whether there was any benefit from extensive resections depending on RPA class.

Patients and Methods: 243 per protocol patients with newly diagnosed GBM were operated with or without ALA and treated by radiotherapy. Postoperative MR-imaging was obtained in all patients. Patients were allocated into RTOG-RPA classes III to V based on age, Karnofsky Performance Status, neurological condition and mental status (as derived from the National Institute of Health stroke score).

Results: Median overall survival among RPA classes III, IV, and V was 17.8, 14.7 and 10.7 months, respectively, with 2-year survival rates of 26%, 12%, and 7% (p=0.0007). Stratified for degree of resection, survival of patients with complete resections was longer in RPA classes IV and V (17.7 vs. 12.9, p=0.0015, and 13.7 vs. 10.4, p=0.0398; 2-year rates: 21.0 vs. 4.4% and 11.1 vs. 2.6%, respectively), but not clearly in the small subgroup of RPA class III patients (19.3 vs. 16.3 months, p=0.14).

Conclusion: Survival of patients from the ALA study is correctly predicted by the RTOG-RPA classes. Differences in survival depending on resection status, especially in RPA classes IV and V, support a causal influence of resection on survival.

Candidate gene investigations have indicated a significant role for epigenetic events in the pathogenesis of medulloblastoma, the most common malignant brain tumour of childhood. To assess the medulloblastoma epigenome more comprehensively, we undertook a genome-wide investigation to identify genes which display evidence of methylation-dependent regulation. Expression microarray analysis of medulloblastoma cell lines following treatment with a DNA methyltransferase inhibitor revealed de-regulation of multiple transcripts (3-6% of probes per cell line). Eighteen independent genes demonstrated >3-fold reactivation in all cell lines tested. Bisulphite sequence analysis revealed dense CpG island methylation associated with transcriptional silencing for 12 of these genes. Extension of this analysis to primary tumours and the normal cerebellum revealed three major classes of epigenetically-regulated genes; (i) normally methylated genes (DAZL, ZNF157, ASN) whose methylation reflects somatic patterns observed in the cerebellum, (ii) X-linked genes (MSN, POU3F4, HTR2C) which show disruption of their sex-specific methylation patterns in tumours, and (iii) tumour-specific methylated genes (COL1A2, S100A10, S100A6, HTATIP2, CDH1, LXN) which display enhanced methylation levels in tumours compared to the cerebellum. Detailed analysis of COL1A2 supports a key role in medulloblastoma tumorigenesis; dense biallelic methylation associated with transcriptional silencing was observed in 46/60 cases (77%). Moreover, COL1A2 status distinguished infant medulloblastomas of the desmoplastic histopathological sub-type, indicating a distinct molecular pathogenesis may underlie these tumours and their more favourable prognosis. These data reveal a more diverse and expansive medulloblastoma epigenome than previously understood and provide strong evidence that the methylation status of specific genes may contribute to the biological sub-classification of medulloblastoma.

Malignant peripheral nerve sheath tumors MPNST are sarcomas with poor prognosis and limited treatment options. Evidence for a role of EGFR and erbB2 in MPNST led us to systematically study these potential therapeutic targets in a larger tumor panel n=37. Multiplex ligation-dependent probe amplification MLPA and FISH analysis revealed increased EGFR dosage in 28% of MPNST. ERBB2 and 3 tumor suppressor genes PTEN, CDKN2A, TP53 were frequently lost or reduced. Reduction of CDKN2A was linked to appearance of metastasis. Comparison of corresponding neurofibromas and MPNST revealed an increase in genetic lesions in MPNST. No somatic mutations were found within tyrosine kinase encoding exons of EGFR and ERBB2. However, on the protein level expression of EGFR and erbB2 was frequently detected in MPNST. EGFR expression was significantly associated with increased EGFR gene dosage. EGFR ligands TGFA and EGF were more strongly expressed in MPNST than in neurofibromas. The effect of the drugs erlotinib and trastuzumab, which target EGFR and erbB2, was determined on MPNST cell lines. In contrast to trastuzumab, erlotinib mediated dose-dependent inhibition of cell proliferation. EGF-induced EGFR phosphorylation was attenuated by erlotinib. Summarized, our data indicate that EGFR and erbB2 are potential targets in treatment of MPNST patients.