Neuro-Oncology

Published by Oxford University Press (OUP)
Online ISSN: 1523-5866
Print ISSN: 1522-8517
Publications
(A) Workflow of FIGPA. FIGPA begins with protein detection by performing PLA. Antibodies recognizing the target of interest are added and that signal is amplified through PCR of linked, tethered nucleic acids. The tissue samples are additionally fixed in 4% paraformaldehyde and digested with pepsin. The genetic detection then takes place as the samples are dehydrated and hybridized overnight with the FISH probe. After washing, the PLA detection probe, complementary to the PCR amplification product, is added. (B) The output of FIGPA consists of the combined protein detection signals (blue dots) and genetic detection signals (green and red dots). These outputs are visualized with a fluorescent microscope. 
FIGPA in fixed human tumor cells. (A) FIGPA in U87 glioblastoma cells transfected with EGFRvIII. Proximate green (Spectrum Green) and red (Spectrum Orange) signals represent the centromere of chromosome 7 and the EGFR gene locus, respectively; the cytoplasmic red dots (563-nm dye) visualize the EGFRvIII protein. The nucleus is counterstained with Hoechst 33342. (B) Use of Pacific Blue protein detection dye (455 nm) in place of red 563-nm dye. All magnifications × 400. 
FIGPA in paraffin-embedded tumor tissue. (A) Hematoxylin-and-eosin stain of glioblastoma sample. (B) FISH probe targeted for EGFR gene locus at chromosome 7p12 (red dots) and centromere of chromosome 7 (green dots). Insert: single cell nucleus showing this tumor to be highly amplified for EGFR at the genetic level. (C) PLA reliably captures EGFR protein expression at the cytoplasmic cell membrane and the cytoplasm. Nucleus counterstained with Hoechst 33342. Top, right insert: single cell with EGFR protein overexpression. Bottom, right insert: same cell as above with demarcated cytoplasmic ( c ) and nuclear ( n ) compartments. (D) Same tumor analyzed via FIGPA (green and red signals represent the FISH probes denoting the centromere of chromosome 7 and the EGFR gene locus, and Pacific Blue dye denotes EGFR protein expression), demonstrating that the EGFR gene is amplified and that EGFR protein expression is concordantly elevated. The protein signal captured via Pacific Blue protein detection dye (455 nm) shows a similar pattern as the protein signal captured via red dye (563 nm) in Panel c . Top, right insert: single cell with amplified EGFR gene locus and EGFR protein overexpression. Bottom, right insert: same cell as above with demarcated cytoplasmic ( c ) and nuclear ( n ) compartments. All magnifications × 400. 
FIGPA captures tissue heterogeneity. (A) Tumor section near a tumor blood vessel highlights the specificity of FIGPA by revealing that EGFR amplification is confined to tumor cells and EGFR protein expression is high in tumor cells but virtually absent in cells of the tumor vessel wall. Asterisk (*) denotes the tumor vessel lumen, and the dotted line indicates the vessel wall. (B) Molecular heterogeneity within a glioblastoma tumor with one tumor cell population showing EGFR amplification and EGFR protein overexpression, and one population showing EGFR wild-type status. Green and red signals represent the FISH probes denoting the centromere of chromosome 7 and the EGFR gene locus, and Pacific Blue dye denotes EGFR protein expression. All magnifications × 400. 
We present a novel methodology combining traditional fluorescent in situ hybridization with an in situ protein detection technology called proximity ligation assay. This method has potential to perform a detailed analysis of the relationship between gene status and corresponding protein expression in cells and tissues. We demonstrate that the fluorescent in situ gene protein assay methodology is capable of resolving gene and protein patterns simultaneously on a cell-by-cell basis.
 
Suppression of primary cultured MB xenograft tumor cells with SVV-001. (A) Quantitative assessment of cell proliferation (mean + SD) with a CCK8 assay. After cells were exposed to SVV-001 for 72 hours, assessment indicates 4 anaplastic and 1 classic MB were successfully infected (* P , .05 and ** P , .01). (B) Representative images show the changes of cell viability in the resistant (ICb-1494MB) and permissive (ICb-984MB and ICb-1572MB) models as detected with an MTT assay (magnification × 10). Compared with the control and resistant models (ICb-1494MB) in which the formation of dark blue intracellular crystals was evident, there was a significant decrease and loss of the stained viable cells in the permissive models ICb-984MB and ICb-1572MB, respectively. 
Intracellular replication of SVV-GFP in FACS-purified CD133 + cells, and suppression of neurosphere formation from single CSCs. (A) Representative graph showing the high-level infection of CD133 + and CD133 2 cells by SVV-GFP (MOI of 2000 for 48 hours) in the 2 
In vivo infectivity and killing of MB xenograft cells induced by SVV-001. (A) Overview of the experimental design. SVV-001 (5 × 10 12 vp / kg) was administered through a single tail vein injection. Mice ( n 1⁄4 2–3 per test) were then euthanized at predetermined time points (24 hours, 48 hours, and 6 days). (B) Time-course infectivity of SVV-001 in ICb-1299MB at 24 hours (a–d), 48 hours (e–h), and 6 days (i–l). Infection of xenograft tumor cells by SVV-001 was determined with IHC staining using mouse antibodies against SVV-001 capsid protein (2A9) (a–d, f, h, j, and l) and compared with the corresponding hematoxylin and eosin staining (e, j, i, and k). Dotted lines in b and d encircle microvessels. Note the pink cytoplasmic inclusions in k. (C) Selective spatial infectivity of SVV-001 in ICb-1572MB 48 hours after virus injection. SVV-001 infected not only the tumor mass (C3), but also the cells spread through cerebrospinal fluid (C1 and C2), as well as an invasive nodule (C4a) and nonvascularized single tumor cells (C4b) while leaving normal mouse brain cells (C1–C3), even those in close proximity to tumor cells (C4b), unharmed. Magnifications: × 4 (a, i, j, C1, C3), × 10 (e, f, C4a), and × 40 (b–d, g, h, k, l, C4b). 
Prolongation of animal survival times and elimination of preformed xenograft tumors. SVV-001 (5 × 10 12 vp / kg) was administered through a single tail vein injection 2 and 4 weeks after tumor cell transplantation ( n 1⁄4 10 per group). (A) Log-rank analysis of animal survival times showing significant improvement of animal survival times in mice treated with SVV-001 ( P , .01). (B) Hematoxylin and eosin (H&E) staining of paraffin sections showing elimination of xenograft tumors in 8 of the 10 long-term survivors, compared with the huge ICb MB xenografts in the mock-treated control groups (arrow). (C) IHC staining with human-specific antibodies against mitochondria in the 2 ICb-1299MB (mice #1 and #2) and 6 ICb-1572MB (mice #5– 10) mice in which no residual tumors were observed with H&E staining. Compared with the intense positivity (arrow) detected in the positive control section, no MT-positive cells were detected in the 8 brains, though a disturbance of the granular layer and micronodules with empty / shallow cytoplasm and condensed nuclei (circled in red) were seen. Magnification: × 40. 
Induction of autophagy and apoptosis by SVV-001. (A) Overview of the steps activating autophagy and the specific activator. (B) Changes of autophagy (upper panel) and apoptosis (lower panel) genes induced by SVV-001 in vitro. Primary cultured cells from the permissive model ICb-1299MB and the resistant model ICb-1595MB were treated with SVV-001 (MOI 25) for 24 hours before being subjected to western hybridization. The autophagy inducer rapamycin (10 nM) and inhibitor 3MA (10 m M) were also included as controls. (C) IHC detection of LC3B expression in vivo in the 2 permissive xenograft mouse models 48 hours after SVV-001 single tail vein injection. (D) Impact of autophagy inhibitors (3MA, vinblastine, pepstatin, and bafilomycin A1) and activator (rapamycin) on the cell killing (upper panel) and extracellular viral production (lower panel). Cell viabilities in cells treated with combined drug (inhibitor or activator) and SVV-001 were normalized to those treated with drug only and presented as percentages. Treatment with autophagy inhibitors led to increased cell viability in the permissive models (upper panel) and decreased extracellular viral production in both the permissive and resistant models (* P , .05 and ** P , .01 when compared with SVV-001 only) (lower panel). 
Background Seneca Valley virus (SVV-001) is a nonpathogenic oncolytic virus that can be systemically administered and can pass through the blood-brain barrier. We examined its therapeutic efficacy and the mechanism of tumor cell infection in pediatric malignant gliomas.Methods In vitro antitumor activities were examined in primary cultures, preformed neurospheres, and self-renewing glioma cells derived from 6 patient tumor orthotopic xenograft mouse models (1 anaplastic astrocytoma and 5 GBM). In vivo therapeutic efficacy was examined by systemic treatment of preformed xenografts in 3 permissive and 2 resistant models. The functional role of sialic acid in mediating SVV-001 infection was investigated using neuraminidase and lectins that cleave or competitively bind to linkage-specific sialic acids.ResultsSVV-001 at a multiplicity of infection of 0.5 to 25 replicated in and effectively killed primary cultures, preformed neurospheres, and self-renewing stemlike single glioma cells derived from 4 of the 6 glioma models in vitro. A single i.v. injection of SVV-001 (5 × 10(12) viral particles/kg) led to the infection of orthotopic xenografts without harming normal mouse brain cells, resulting in significantly prolonged survival in all 3 permissive and 1 resistant mouse models (P < .05). Treatment with neuraminidase and competitive binding using lectins specific for α2,3-linked and/or α2,6-linked sialic acid significantly suppressed SVV-001 infectivity (P < .01).ConclusionSVV-001 possesses strong antitumor activity against pediatric malignant gliomas and utilizes α2,3-linked and α2,6-linked sialic acids as mediators of tumor cell infection. Our findings support the consideration of SVV-001 for clinical trials in children with malignant glioma.
 
Suppression of primary cultured MB xenograft tumor cells with SVV-001. (A) Quantitative assessment of cell proliferation (mean + SD) with a CCK8 assay. After cells were exposed to SVV-001 for 72 hours, assessment indicates 4 anaplastic and 1 classic MB were successfully infected (* P , .05 and ** P , .01). (B) Representative images show the changes of cell viability in the resistant (ICb-1494MB) and permissive (ICb-984MB and ICb-1572MB) models as detected with an MTT assay (magnification × 10). Compared with the control and resistant models (ICb-1494MB) in which the formation of dark blue intracellular crystals was evident, there was a significant decrease and loss of the stained viable cells in the permissive models ICb-984MB and ICb-1572MB, respectively. 
Intracellular replication of SVV-GFP in FACS-purified CD133 + cells, and suppression of neurosphere formation from single CSCs. (A) Representative graph showing the high-level infection of CD133 + and CD133 2 cells by SVV-GFP (MOI of 2000 for 48 hours) in the 2 
In vivo infectivity and killing of MB xenograft cells induced by SVV-001. (A) Overview of the experimental design. SVV-001 (5 × 10 12 vp / kg) was administered through a single tail vein injection. Mice ( n 1⁄4 2–3 per test) were then euthanized at predetermined time points (24 hours, 48 hours, and 6 days). (B) Time-course infectivity of SVV-001 in ICb-1299MB at 24 hours (a–d), 48 hours (e–h), and 6 days (i–l). Infection of xenograft tumor cells by SVV-001 was determined with IHC staining using mouse antibodies against SVV-001 capsid protein (2A9) (a–d, f, h, j, and l) and compared with the corresponding hematoxylin and eosin staining (e, j, i, and k). Dotted lines in b and d encircle microvessels. Note the pink cytoplasmic inclusions in k. (C) Selective spatial infectivity of SVV-001 in ICb-1572MB 48 hours after virus injection. SVV-001 infected not only the tumor mass (C3), but also the cells spread through cerebrospinal fluid (C1 and C2), as well as an invasive nodule (C4a) and nonvascularized single tumor cells (C4b) while leaving normal mouse brain cells (C1–C3), even those in close proximity to tumor cells (C4b), unharmed. Magnifications: × 4 (a, i, j, C1, C3), × 10 (e, f, C4a), and × 40 (b–d, g, h, k, l, C4b). 
Prolongation of animal survival times and elimination of preformed xenograft tumors. SVV-001 (5 × 10 12 vp / kg) was administered through a single tail vein injection 2 and 4 weeks after tumor cell transplantation ( n 1⁄4 10 per group). (A) Log-rank analysis of animal survival times showing significant improvement of animal survival times in mice treated with SVV-001 ( P , .01). (B) Hematoxylin and eosin (H&E) staining of paraffin sections showing elimination of xenograft tumors in 8 of the 10 long-term survivors, compared with the huge ICb MB xenografts in the mock-treated control groups (arrow). (C) IHC staining with human-specific antibodies against mitochondria in the 2 ICb-1299MB (mice #1 and #2) and 6 ICb-1572MB (mice #5– 10) mice in which no residual tumors were observed with H&E staining. Compared with the intense positivity (arrow) detected in the positive control section, no MT-positive cells were detected in the 8 brains, though a disturbance of the granular layer and micronodules with empty / shallow cytoplasm and condensed nuclei (circled in red) were seen. Magnification: × 40. 
Induction of autophagy and apoptosis by SVV-001. (A) Overview of the steps activating autophagy and the specific activator. (B) Changes of autophagy (upper panel) and apoptosis (lower panel) genes induced by SVV-001 in vitro. Primary cultured cells from the permissive model ICb-1299MB and the resistant model ICb-1595MB were treated with SVV-001 (MOI 25) for 24 hours before being subjected to western hybridization. The autophagy inducer rapamycin (10 nM) and inhibitor 3MA (10 m M) were also included as controls. (C) IHC detection of LC3B expression in vivo in the 2 permissive xenograft mouse models 48 hours after SVV-001 single tail vein injection. (D) Impact of autophagy inhibitors (3MA, vinblastine, pepstatin, and bafilomycin A1) and activator (rapamycin) on the cell killing (upper panel) and extracellular viral production (lower panel). Cell viabilities in cells treated with combined drug (inhibitor or activator) and SVV-001 were normalized to those treated with drug only and presented as percentages. Treatment with autophagy inhibitors led to increased cell viability in the permissive models (upper panel) and decreased extracellular viral production in both the permissive and resistant models (* P , .05 and ** P , .01 when compared with SVV-001 only) (lower panel). 
Difficulties of drug delivery across the blood-brain barrier (BBB) and failure to eliminate cancer stem cells (CSCs) are believed to be the major causes of tumor recurrences in children with medulloblastoma (MB). Seneca Valley virus-001 (SVV-001) is a naturally occurring oncolytic picornavirus that can be systemically administered. Here, we report its antitumor activities against MB cells in a panel of 10 primary tumor-based orthotopic xenograft mouse models. We found that SVV-001 killed the primary cultured xenograft cells, infected and replicated in tumor cells expressing CSC surface marker CD133, and eliminated tumor cells capable of forming neurospheres in vitro in 5 of the 10 xenograft models. We confirmed that SVV-001 could pass through BBB in vivo. A single i.v. injection of SVV-001 in 2 anaplastic MB models led to widespread infection of the preformed intracerebellar (ICb) xenografts, resulting in significant increase in survival (2.2-5.9-fold) in both models and complete elimination of ICb xenografts in 8 of the 10 long-term survivors. Mechanistically, we showed that the intracellular replication of SVV-001 is mediated through a subverted autophagy that is different from the bona fide autophagic process induced by rapamycin. Our data suggest that SVV-001 is well suited for MB treatment. This work expands the current views in the oncolytic therapy field regarding the utility of oncolytic viruses in simultaneous targeting of stem and nonstem tumor cells.
 
Pretreatment characteristics* 
Overall survival* 
Preclinical studies support the concept that inhibition of protein kinase C (PKC) by tamoxifen (TAM) should provide both antineoplastic effects and radiosensitization. High-dose TAM (80 mg/m2 p.o. daily in divided doses) was given with and after conventional radiotherapy (XRT) to inhibit PKC-mediated signaling, which is known to be enhanced in glioblastoma (GBM). Seventy-seven patients were accrued between December 2000 and December 2001; two were ineligible and not included in the efficacy results. Pretreatment characteristics of the patients included the following: 52% were less than 60 years of age, 39% had a Zubrod score of 0, 70% had minor or no neurological symptoms, and 65% were Radiation Therapy Oncology Group-recursive partition analysis (RPA) class III and IV. Eighty-six percent of patients achieved acceptable dosing of TAM. Notable toxicity included late radiation grade 3 in two patients and thromboembolic events in 16 patients (two grade 2, 10 grade 3, three grade 4, and one grade 5), for an incidence of 20.8% (which is lower than expected, based on the literature for deep vein thrombophlebitis in GBM patients not receiving TAM). Median survival time (MST) was 9.7 months as compared (by three different statistical methodologies) to the historical GBM control database of 1457 RPA class III, IV, and V drug/XRT-treated patients. After controlling for RPA class IV, the MST was 11.3 months, which compares to the historical RPA control of 11.3 months (P = 0.37). The results obtained do not exhibit a substantial advance over those of previous studies with various XRT/drug doublets, including BCNU. However, as TAM does not have significant overlapping toxicities with most other drugs, its testing in a combined modality approach with other medications may be justified in future clinical trials. Historically, the incidence of thromboembolic events in GBM patients is approximately 30%. The lower-than-expected incidence seen here has also been observed in other high-dose TAM GBM studies. We speculate that TAM inhibited the PKC-mediated phosphorylation of coagulation factors.
 
Background Glioblastoma multiforme, a World Health Organization grade IV glioma, has a poor prognosis in humans despite current treatment options. Here, we present magnetic resonance imaging (MRI) data regarding the regression of aggressive rat F98 gliomas and human U87 glioma xenografts after treatment with the nitrone compound OKN-007, a disulfonyl derivative of α-phenyl-tert-butyl nitrone.MethodsMRI was used to assess tumor volumes in F98 and U87 gliomas, and bioluminescence imaging was used to measure tumor volumes in F98 gliomas encoded with the luciferase gene (F98(luc)). Immunohistochemistry was used to assess angiogenesis (vascular endothelial growth factor [VEGF] and microvessel density [MVD]), cell differentiation (carbonic anhydrase IX [CA-IX]), hypoxia (hypoxia-inducible factor-1α [HIF-1α]), cell proliferation (glucose transporter 1 [Glut-1] and MIB-1), proliferation index, and apoptosis (cleaved caspase 3) markers in F98 gliomas. VEGF, CA-IX, Glut-1, HIF-1α, and cleaved caspase 3 were assessed in U87 gliomas.ResultsAnimal survival was found to be significantly increased (P < .001 for F98, P < .01 for U87) in the group that received OKN-007 treatment compared with the untreated groups. After MRI detection of F98 gliomas, OKN-007, administered orally, was found to decrease tumor growth (P < .05). U87 glioma volumes were found to significantly decrease (P < .05) after OKN-007 treatment, compared with untreated animals. OKN-007 administration resulted in significant decreases in tumor hypoxia (HIF-1α [P < .05] in both F98 and U87), angiogenesis (MVD [P < .05], but not VEGF, in F98 or U87), and cell proliferation (Glut-1 [P < .05 in F98, P < .01 in U87] and MIB-1 [P < .01] in F98) and caused a significant increase in apoptosis (cleaved caspase 3 [P < .001 in F98, P < .05 in U87]), compared with untreated animals.ConclusionsOKN-007 may be considered as a promising therapeutic addition or alternative for the treatment of aggressive human gliomas.
 
Grade 3 or 4 adverse events related to imatinib 
Platelet-derived growth factor (PDGF) and its receptors (PDGFR) are frequently coexpressed in meningiomas, potentially contributing to their pathogenesis. The North American Brain Tumor Consortium 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 end point was 6-month progression-free survival (6M-PFS). Patients requiring enzyme-inducing antiepileptic 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. 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 2,129 +/- 1,600 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 chronic myelogenous leukemia.
 
Background Due to the redundancy of molecular pathways simultaneously involved in glioblastoma growth and angiogenesis, therapeutic approaches intervening at multiple levels seem particularly appealing.Methods This prospective, multicenter, single-arm phase II trial was designed to evaluate the antitumor activity of sunitinib, an oral small-molecule inhibitor of several receptor tyrosine kinases, in patients with first recurrence of primary glioblastoma using a continuous once-daily dosing regimen. Patients received a starting dose of sunitinib 37.5 mg, followed by a maintenance dose between 12.5 mg and 50 mg depending on drug tolerability. The primary endpoint was a 6-month progression-free survival (PFS) rate. Secondary endpoints included median PFS, overall survival (OS), safety/toxicity, quality of life, and translational studies on the expression of sunitinib target molecules.ResultsForty participants were included in this study, and no objective responses were detected. PFS6 was 12.5%, median PFS 2.2 months, and median OS 9.2 months. Five participants (12.5%) showed prolonged stable disease ≥6 months with a median PFS of 16.0 months (range, 6.4-41.4 mo) and a median OS of 46.9 months (range, 21.2-49.2 mo) for this subgroup. c-KIT expression in vascular endothelial cells (n = 14 participants) was associated with improved PFS. The most common toxicities were fatigue/asthenia, mucositis/dermatitis, dysesthesias, gastrointestinal symptoms, cognitive impairment, leukoctopenia, and thrombocytopenia. Two participants (5%) terminated treatment due to toxicity.Conclusion Continuous daily sunitinib showed minimal antiglioblastoma activity and substantial toxicity when given at higher doses. High endothelial c-KIT expression may define a subgroup of patients who will benefit from sunitinib treatment by achieving prolonged PFS.ClinicalTrials.gov Identifier: NCT00535379.
 
radioactive profile of the individual alkylation products that were formed by incubation of 3 H-BCnU with calf thymus Dna and resolved by HPLC. The identities of the individual peaks labeled a to G are provided in Table 1. 
The alkylation products formed by in vitro treatment of DNA with tritium-labeled 1,3-bis(2-chloroethyl)-1-nitrosourea ((3)H-BCNU) were identified and quantified. Twelve adducts were resolved by high-performance liquid chromatography (HPLC). The principal DNA adducts formed by BCNU treatment corresponded to N-7-(2-hydroxyethyl)guanine (N7-HOEtG) (26%), N-7-(2-chloroethyl)guanine (15%), and phosphotriesters (19%). In addition, several minor products were identified as 1,2-(diguan-7-yl)ethane, N-1-(2-hydroxyethyl)-2-deoxyguanosine, 1-(N-1-2-deoxyguanosinyl), 2-(N-3-2-deoxycytidyl)ethane cross-link, and O-6-(2-hydroxyethyl)-2-deoxyguanosine, and individually they represented 1% to 5% of the total alkylation. An HPLC-electrochemical method was applied to quantify the levels of N7-HOEtG in samples treated with BCNU. Treatment of either purified DNA or U87MG cells with various amounts of BCNU produced a linear increase in the amount of N7-HOEtG. These results demonstrated that the levels of N7-HOEtG formed by BCNU treatment could be used as a molecular dosimeter of BCNU treatment dose. We measured the levels of N7-HOEtG in DNA isolated from tumor samples taken from four patients with GBM tumors following stereotactic intratumoral injection with DTI-015 (BCNU-ethanol). The level of N7-HOEtG in these samples ranged from 14.7 to 121.9 micromol N7-HOETG/mol DNA within 1 cm of the site of injection. As the distance from the site of injection increased, the levels of N7-HOEtG in tumor DNA decreased. In two of the samples, the levels of N7-HOEtG were 0.2 to 0.3 micromol N7-HOETG/mol DNA at 3.5 to 3.9 cm from the site of injection, demonstrating significant distribution of BCNU in the tumor. The levels of N7-HOEtG in these tumor samples corresponded to BCNU treatment concentrations of 0.02 to 43.0 mM. These studies demonstrate that stereotactic intratumoral injection of DTI-015 into human GBM tumors produces high concentrations of BCNU up to 2.5 cm from the site of injection in some of the tumors. These observations suggest that intratumoral injection of DTI-015 may be of benefit in the treatment of primary and recurrent GBM tumors.
 
Patient characteristics 
The activity of single-agent targeted molecular therapies in glioblastoma has been limited to date. The North American Brain Tumor Consortium examined the safety, pharmacokinetics, and efficacy of combination therapy with sorafenib, a small molecule inhibitor of Raf, vascular endothelial growth factor receptor 2, and platelet-derived growth factor receptor-β, and temsirolimus (CCI-779), an inhibitor of mammalian target of rapamycin. This was a phase I/II study. The phase I component used a standard 3 × 3 dose escalation scheme to determine the safety and tolerability of this combination therapy. The phase II component used a 2-stage design; the primary endpoint was 6-month progression-free survival (PFS6) rate. Thirteen patients enrolled in the phase I component. The maximum tolerated dosage (MTD) for combination therapy was sorafenib 800 mg daily and temsirolimus 25 mg once weekly. At the MTD, grade 3 thrombocytopenia was the dose-limiting toxicity. Eighteen patients were treated in the phase II component. At interim analysis, the study was terminated and did not proceed to the second stage. No patients remained progression free at 6 months. Median PFS was 8 weeks. The toxicity of this combination therapy resulted in a maximum tolerated dose of temsirolimus that was only one-tenth of the single-agent dose. Minimal activity in recurrent glioblastoma multiforme was seen at the MTD of the 2 combined agents.
 
Background Inhibition of epidermal growth factor receptor (EGFR) and the mechanistic target of rapamycin (mTOR) may have synergistic antitumor effects in high-grade glioma patients.Methods We conducted a phase I/II study of the EGFR inhibitor erlotinib (150 mg/day) and the mTOR inhibitor temsirolimus. Patients initially received temsirolimus 50 mg weekly, and the dose adjusted based on toxicities. In the phase II component, the primary endpoint was 6-month progression-free survival (PFS6) among glioblastoma patients.ResultsTwenty-two patients enrolled in phase I, 47 in phase II. Twelve phase I patients treated at the maximum tolerated dosage were included in the phase II cohort for analysis. The maximum tolerated dosage was 15 mg temsirolimus weekly with erlotinib 150 mg daily. Dose-limiting toxicities were rash and mucositis. Among 42 evaluable glioblastoma patients, 12 (29%) achieved stable disease, but there were no responses, and PFS6 was 13%. Among 16 anaplastic glioma patients, 1 (6%) achieved complete response, 1 (6%) partial response, and 2 (12.5%) stable disease, with PFS6 of 8%. Tumor levels of both drugs were low, and posttreatment tissue in 3 patients showed no reduction in the mTOR target phosphorylated (phospho-)S6(S235/236) but possible compensatory increase in phospho-Akt(S473). Presence of EGFR variant III, phospho-EGFR, and EGFR amplification did not correlate with survival, but patients with elevated phospho-extracellular signal-regulated kinase or reduced phosphatase and tensin homolog protein expression had decreased progression-free survival at 4 months.Conclusion Because of increased toxicity, the maximum tolerated dosage of temsirolimus in combination with erlotinib proved lower than expected. Insufficient tumor drug levels and redundant signaling pathways may partly explain the minimal antitumor activity noted.
 
Progression-free (solid line) and OS (dashed line) curves. 
Patient characteristics
The upper row shows a baseline scan of a patient with a gadolinium-enhancing left temporal glioblastoma with vasogenic edema on FLAIR and increased rCBV and Ktrans, a measurement of vessel permeability and total vessel surface. The lower row shows a partial response after 4 weeks of treatment according to the Levin’s criteria but , 50% reduction in the sum of products of perpendicular diameters (Macdonald criteria). The reduction in contrast enhancement is accompanied by decreased vasogenic edema on FLAIR, rCBV, and Ktrans. 
Adverse events
The objective of this phase II single-arm study was to evaluate the efficacy and safety of pazopanib, a multi-targeted tyrosine kinase inhibitor, against vascular endothelial growth factor receptor (VEGFR)-1, -2, and -3, platelet-derived growth factor receptor-alpha and -beta, and c-Kit, in recurrent glioblastoma. Patients with < or =2 relapses and no prior anti-VEGF/VEGFR therapy were treated with pazopanib 800 mg daily on 4-week cycles without planned interruptions. Brain magnetic resonance imaging and clinical reassessment were made every 8 weeks. The primary endpoint was efficacy as measured by 6-month progression-free survival (PFS6). Thirty-five GBM patients with a median age of 53 years and median Karnofsky performance scale of 90 were accrued. Grade 3/4 toxicities included leukopenia (n = 1), lymphopenia (n = 2), thrombocytopenia (n = 1), ALT elevation (n = 3), AST elevation (n = 1), CNS hemorrhage (n = 1), fatigue (n = 1), and thrombotic/embolic events (n = 3); 8 patients required dose reduction. Two patients had a partial radiographic response by standard bidimensional measurements, whereas 9 patients (6 at the 8-week point and 3 only within the first month of treatment) had decreased contrast enhancement, vasogenic edema, and mass effect but <50% reduction in tumor. The median PFS was 12 weeks (95% confidence interval [CI]: 8-14 weeks) and only 1 patient had a PFS time > or =6 months (PFS6 = 3%). Thirty patients (86%) had died and median survival was 35 weeks (95% CI: 24-47 weeks). Pazopanib was reasonably well tolerated with a spectrum of toxicities similar to other anti-VEGF/VEGFR agents. Single-agent pazopanib did not prolong PFS in this patient population but showed in situ biological activity as demonstrated by radiographic responses. ClinicalTrials.gov identifier: NCT00459381.
 
Progression-free ( solid line ) and overall survival ( dashed line ) curves of 35 patients with recurrent glioblastoma in the phase II trial of romidepsin. 
Romidepsin, a potent histone deacetylase inhibitor, has shown activity in preclinical glioma models. The primary objectives of this trial were to determine the pharmacokinetics of romidepsin in patients with recurrent glioma on enzyme-inducing antiepileptic drugs (EIAEDs) and to evaluate the antitumor efficacy of romidepsin in patients with recurrent glioblastoma who were not receiving EIAEDs. Two dose cohorts were studied in the phase I component of the trial (13.3 and 17.7 mg/m(2)/d). Patients in the phase II component were treated with intravenous romidepsin at a dosage of 13.3 mg/m(2)/day on days 1, 8, and 15 of each 28-day cycle. Eight patients were treated on the phase I component. A similar romidepsin pharmacokinetic profile was demonstrated between patients receiving EIAEDs to those not receving EIAEDs. Thirty-five patients with glioblastoma were accrued to the phase II component. There was no objective radiographic response. The median progression-free survival (PFS) was 8 weeks and only 1 patient had a PFS time ≥6 months (PFS6 = 3%). To date, 34 patients (97%) have died, with a median survival duration of 34 weeks. Despite in vitro studies showing that romidepsin is primarily metabolized by CYP3A4, no decrease in exposure to romidepsin was seen in patients receiving potent CYP3A4 inducers. Romidepsin, at its standard dose and schedule, was ineffective for patients with recurrent glioblastomas. ClinicalTrials.gov identifier: NCT00085540.
 
Background Expected survival is a major factor influencing extent of treatment for symptomatic spinal bone metastases (SBM). Predictive models have been developed, but their use can lead to over- or undertreatment.. The study objective was to identify prognostic factors associated with survival in patients with symptomatic SBM and to create a validated risk stratification model.Methods All patients who were treated for symptomatic SBM between 2001 and 2010 were included in this single center retrospective study. Medical records were reviewed for type of primary cancer, performance status, presence of visceral, brain and bone metastases, number and location of spinal metastases, and neurological functioning. Performance status was assessed with the Karnofsky performance score and neurological functioning with the Frankel scale. Analysis was performed using Kaplan-Meier curves, univariate log-rank tests, Cox regression models, and Harrell's C statistic.ResultsA total of 1 043 patients were studied. The most prevalent tumors were those of breast (n = 299), lung (n = 250), and prostate (n = 215). Median follow-up duration was 6.6 years, and 6 patients were lost to follow-up. Based on the results of the uni- and multivariate analyses, 4 categories were created. Median survival in category A was 31.2 months (95% CI, 25.2-37.3 months), 15.4 months (95% CI, 11.9-18.2 months) for category B, 4.8 months (95% CI, 4.1-5.4 months) for category C, and 1.6 months (95% CI, 1.4-1.9 months) for category D. Harrell's C statistic was calculated after the model was applied to an external dataset, yielding a result of 0.69.Conclusion Assessing patients according to the presented model results in 4 categories with significantly different survival times.
 
Steady-state pharmacokinetic parameters for sorafenib and erlotinib 
Background The signal transduction pathways of epidermal growth factor receptor and Ras are both important in the growth of glioblastoma multiforme (GBM). We hypothesized that inhibition of both pathways would improve the survival time of patients with recurrent GBM.Methods Patients with recurrent/progressive GBM with 0-2 prior chemotherapy regimens received erlotinib 150 mg once daily and sorafenib 400 mg twice daily until progression. The primary endpoint was overall survival. Pharmacokinetic sampling was performed during cycle 1.ResultsThe median overall survival was 5.7 months. Progression-free survival at 6 months was 14%. Toxicity was manageable. Clearance of erlotinib was markedly enhanced by sorafenib.Conclusion The study did not meet its objective of a 30% increase in overall survival time compared with historical controls. Erlotinib and sorafenib have significant pharmacokinetic interactions that may negatively impact the efficacy of the combination regimen.
 
OS and death hazard over time. 
Hazard ratios in different time intervals from randomization
PFS and failure hazard over time. 
Hazard by treatment for MGMT methylated and unmethylated subsets. 
Relationship between progression and survival. 
We assessed the longitudinal hazard characteristics for death and progression in patients with glioblastoma, evaluated the impact of prognostic factors and treatment on the hazard within different time intervals to determine if effects are time varying, and quantified the influence of progression on survival. Among patients randomized to Radiation Therapy Oncology Group trial 0525, which compared dose-dense with standard-dose temozolomide, we estimated the hazards of death and treatment failure (death or progression) over time and their interdependence. The peak hazard of death was reached at around 16 months with a slow decline after that; the hazard of progression/death reached a peak at around 6 months and decreased dramatically thereafter. The survival advantages for patients with MGMT gene promoter methylation and recursive partitioning analysis class III were substantial in the first 2 years, but lessened thereafter. The progression-free survival benefit of dose-dense over standard-dose temozolomide occurred in the first 6 months (hazard ratio: 0.70; 95% CI: 0.58-0.86; P < .001), although it diminished thereafter. After adjusting for recursive partitioning analysis class and MGMT methylation status, the hazard ratio of death for patients who had progressed over nonprogressors was 6.59 (95% CI: 5.15-8.43; P < .001). After the peak hazard of death, a consistently high hazard remains, but it is lower than in the peak period. The progression hazard peak is earlier, and then hazard consistently declines. The rate of dying after disease progression is about 6.59 times the rate for nonprogressors, suggesting that progression-free survival may be a relevant clinical endpoint. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
 
(A) The 3 regions of interest were defined on the corresponding T1-weighted postcontrast images: (grey voxels) enhancing tumor, (black voxels) peritumoral tissue, and (white voxels) normal tissue on the contralateral side of tumor. (B) Typical MRS spectra were obtained from (left) the enhancing tumor and from (right) contralateral normal tissue. Tumor tissue is characterized by elevated Cho and decreased NAA. 
Acquisition parameters
Changes in (A) NAA / Cho levels and (B) Cho / Cr levels in enhancing tumor relative to baseline levels. NAA / Cho levels significantly increase at 2 wk posttreatment ( P 1⁄4 .048), and Cho / Cr significantly decreases at 2 wk posttreatment ( P 1⁄4 .016) indicated by asterisks. 
(A) Changes in NAA / Cho, Cho / Cr, and NAA / Cr from baseline in tumor voxels grouped by PFS-6 survivors (PFS . 6 mo) and non –PFS-6 survivors (PFS ≤ 6 mo). (B) Changes in NAA / Cho, Cho / Cr, and NAA / Cr from baseline in peritumoral voxels grouped by PFS-6 survivors (PFS . 6 mo) and non–PFS-6 survivors (PFS ≤ 6 mo). Error bars represent SEs of the mean. Numbers of patients are noted on the bottom. 
Background The prognosis for patients with recurrent glioblastoma remains poor. The purpose of this study was to assess the potential role of MR spectroscopy as an early indicator of response to anti-angiogenic therapy.Methods Thirteen patients with recurrent glioblastoma were enrolled in RTOG 0625/ACRIN 6677, a prospective multicenter trial in which bevacizumab was used in combination with either temozolomide or irinotecan. Patients were scanned prior to treatment and at specific timepoints during the treatment regimen. Postcontrast T1-weighted MRI was used to assess 6-month progression-free survival. Spectra from the enhancing tumor and peritumoral regions were defined on the postcontrast T1-weighted images. Changes in the concentration ratios of n-acetylaspartate/creatine (NAA/Cr), choline-containing compounds (Cho)/Cr, and NAA/Cho were quantified in comparison with pretreatment values.ResultsNAA/Cho levels increased and Cho/Cr levels decreased within enhancing tumor at 2 weeks relative to pretreatment levels (P = .048 and P = .016, respectively), suggesting a possible antitumor effect of bevacizumab with cytotoxic chemotherapy. Nine of the 13 patients were alive and progression free at 6 months. Analysis of receiver operating characteristic curves for NAA/Cho changes in tumor at 8 weeks revealed higher levels in patients progression free at 6 months (area under the curve = 0.85), suggesting that NAA/Cho is associated with treatment response. Similar results were observed for receiver operating characteristic curve analyses against 1-year survival. In addition, decreased Cho/Cr and increased NAA/Cr and NAA/Cho in tumor periphery at 16 weeks posttreatment were associated with both 6-month progression-free survival and 1-year survival.Conclusion Changes in NAA and Cho by MR spectroscopy may potentially be useful as imaging biomarkers in assessing response to anti-angiogenic treatment.
 
Kaplan-Meier survival curves stratified by radiologic progression status on 2D-T1 (top row), 3D-T1 (middle row), and FLAIR (bottom row) imaging at 8 weeks (left column) and 16 weeks (right column) after initiation of anti-VEGF therapy, with associated P values. At both 8 and 16 weeks, there was statistically significant difference between the survival curves for progressors (dashed curves) and nonprogressors (solid curves) on 2D-T1 and 3D-T1, but not FLAIR. 
Comparison of median survival time by progression status on MRI performed 8 and 16 weeks after initiation of anti-VEGF therapy
Kaplan-Meier survival curves stratified by radiologic progression status on 2D-T1 (top row) and 3D-T1 (bottom row) imaging at 8 weeks (left column) and 16 weeks (right column) after initiation of anti-VEGF therapy, with substratification of patients who had not progressed (nonprogressors) into those showing partial or complete response (responders) and those without response (nonresponder, nonprogressors [NR-NPs]). Listed P values are between responders and NR-NPs. At both 8 and 16 weeks, there was no statistically significant difference between the survival curves for responders (dot-dash curves) and NR-NPs (solid curves) on 2D-T1 or 3D-T1. Although not statistically significant, there is better visual separation of Kaplan-Meier curves between the responders and NR-NPs for 3D-T1 than for 2D-T1. 
Kaplan-Meier survival curves stratified into patients not progressing on T1 or FLAIR (nonprogressors) and progressing on FLAIR but not T1 (isolated FLAIR progressors) for 2D-T1 (top row) and 3D-T1 (bottom row) at 8 weeks (left column) and 16 weeks (right column) after initiation of anti-VEGF therapy. Isolated FLAIR progressors (dashed curves) had no statistically significant reduction in survival time, compared with the nonprogressors (solid curves). 
Comparison of median survival time for T1 nonprogressors by progression status on FLAIR MRI performed 8 and 16 weeks after initiation of anti-VEGF therapy
Background: RTOG 0625/ACRIN 6677 is a multicenter, randomized, phase II trial of bevacizumab with irinotecan or temozolomide in recurrent glioblastoma (GBM). This study investigated whether early posttreatment progression on FLAIR or postcontrast MRI assessed by central reading predicts overall survival (OS). Methods: Of 123 enrolled patients, 107 had baseline and at least 1 posttreatment MRI. Two central neuroradiologists serially measured bidimensional (2D) and volumetric (3D) enhancement on postcontrast T1-weighted images and volume of FLAIR hyperintensity. Progression status on all posttreatment MRIs was determined using Macdonald and RANO imaging threshold criteria, with a third neuroradiologist adjudicating discrepancies of both progression occurrence and timing. For each MRI pulse sequence, Kaplan-Meier survival estimates and log-rank test were used to compare OS between cases with or without radiologic progression. Results: Radiologic progression occurred after 2 chemotherapy cycles (8 weeks) in 9 of 97 (9%), 9 of 73 (12%), and 11 of 98 (11%) 2D-T1, 3D-T1, and FLAIR cases, respectively, and 34 of 80 (43%), 21 of 58 (36%), and 37 of 79 (47%) corresponding cases after 4 cycles (16 weeks). Median OS among patients progressing at 8 or 16 weeks was significantly less than that among nonprogressors, as determined on 2D-T1 (114 vs 278 days and 214 vs 426 days, respectively; P <. 0001 for both) and 3D-T1 (117 vs 306 days [P <. 0001] and 223 vs 448 days [P =. 0003], respectively) but not on FLAIR (201 vs 276 days [P =. 38] and 303 vs 321 days [P =. 13], respectively).Conclusion Early progression on 2D-T1 and 3D-T1, but not FLAIR MRI, after 8 and 16 weeks of anti-vascular endothelial growth factor therapy has highly significant prognostic value for OS in recurrent GBM.
 
Kaplan-Meier survival curves of all eligible patients (stage 1 + 1B combined). OS, overall survival; PFS, progression-free survival. 
Pretreatment molecular analysis among eligible patients
Survival
We conducted a phase II trial to evaluate the efficacy of dasatinib, a multitargeted tyrosine kinase inhibitor, for adults with recurrent glioblastoma (GBM). Eligibility requirements were Karnofsky performance status ≥60%; no concurrent hepatic enzyme-inducing anticonvulsants; prior treatment with surgery, radiotherapy, and temozolomide exclusively; and activation or overexpression of ≥2 putative dasatinib targets in GBM (ie, SRC, c-KIT, EPHA2, and PDGFR). Using a 2-stage design, 77 eligible participants (27 in stage 1, if favorable, and then 50 in stage 2) were needed to detect an absolute improvement in the proportion of patients either alive and progression-free patients at 6 months (6mPFS) or responding (any duration) from a historical 11% to 25%. A high rate of ineligibility (27%) to stage 1 precluded a powered assessment of efficacy, but there was also infrequent treatment-related toxicity at 100 mg twice daily. Therefore, the study was redesigned to allow intrapatient escalation by 50 mg daily every cycle as tolerated (stage 1B) before determining whether to proceed to stage 2. Escalation was tolerable in 10 of 17 (59%) participants evaluable for that endpoint; however, among all eligible patients (stages 1 and 1B, n = 50), there were no radiographic responses, median overall survival was 7.9 months, median PFS was 1.7 months, and the 6mPFS rate was 6%. The clinical benefit was insufficient to correlate tested biomarkers with efficacy. The trial was closed without proceeding to stage 2. Intraparticipant dose escalation was feasible, but dasatinib was ineffective in recurrent GBM. Clinical trials.gov identified. NCT00423735 (available at http://clinicaltrials.gov/ct2/show/NCT00423735). © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
 
The EORTC (26981-22981)/NCIC CTG (CE.3) RCT in newly diagnosed GBM showed increased survival with concomitant and adjuvant temozolomide (TMZ) added to radiotherapy (RT). Study pts were 18-71 (median 56) years; however a sub-group analysis noted a trend of decreasing benefit from the addition of TMZ with increasing age, such that for age 65-71, the hazard ratio of 0.8 did not reach statistical significance (p = 0.340). This may reflect a lack of efficacy in elderly patients, or simply be due to a lack of statistical power in this subgroup. Recent RCTs in elderly GBM found improved survival with RT compared to supportive care alone and detected non-inferiority of 40 Gy/15 vs. a 60 Gy/30 RT regimen and superior survival was noted for MGMT-methylated patients treated with TMZ alone vs RT alone. Based upon these results short-course hypofractionated RT is often recommended for elderly pts. However, whether the addition of TMZ to RT confers a survival advantage in elderly pts, particularly for those with methylated MGMT, remains unanswered.
 
The standard of care for patients with newly diagnosed glioblastoma was redefined in 2005 when the EORTC NCIC trial showed the superiority of concomitant and maintenance temozolomide (TMZ) in addition to radiotherapy over radiotherapy alone. Since then numerous efforts to build on this new regimen have been explored, mostly testing the hypotheses that prolonged administration of TMZ or inhibition of angiogenesis will provide survival benefit. The most promising anti-angiogenesis agent was bevacizumab, a monoclonal antibody targeting vascular endothelial growth factor (VEGF). This was based on encouraging response rates and progression-free survival data upon landmark analyses at 6 months. Other VEGF-targeting agents such as cediranib or VEGF trap were less convincing, both regarding safety and tolerability as well as efficacy. The conditional approval by the FDA in 2009 for bevacizumab in patients with recurrent glioblastoma was linked to the future demonstration of its efficacy in prospective trials in patients with newly diagnosed disease. Two such trials were performed largely in parallel, one by the RTOG (RTOG-0825) in the US, and one by Roche (AVAGlio), largely in Europe (1). Rather mature results from both trials were presented at the 2013 ASCO Meeting in Chicago (2,3). The results from both trials provide a rather uniform picture: Progression-free survival is significantly prolonged, quality of life during progression-free survival is preserved in the AVAGlio trial, but not in RTOG-0825, overall survival is not improved, and safety and tolerability are acceptable. Subgroup analyses, as available so far, do not identify specific subgroups of patients with a particularly convincing benefit from bevacizumab, and the comparison of both trials suggests no major impact of cross-over at progression.
 
Patient characteristics at the time of study enrollment 
Nadir hematologic profile related to topotecan 
Our purpose was to establish the maximum tolerated dosage (MTD) of daily i.v. topotecan with conventionally fractionated radiotherapy (XRT) for patients with intrinsic pontine glioma of childhood. Topotecan was given as a 30-min i.v. infusion 30-60 min before each XRT treatment given daily for 33 days. Total XRT dose was 59.4 Gy. Dose escalation of topotecan was carried out using a standard phase I design. Dose limiting toxicity (DLT) was defined as an absolute neutrophil count (ANC) of < or =500/mm(3) for > or =7 days; platelets of < or =50,000/mm(3) for > or =7 days; >7 days platelet transfusions; fever and neutropenia (ANC < or =500/mm(3) for > or =7 days); and/or any > or=grade 3 non-hematologic toxicity. In this multi-institutional phase I study, 17 patients <21 years with intrinsic pontine glioma were enrolled. Sixteen patients completed treatment. An ANC < or =500/mm(3) for > or =7 days occurred in 2/5 patients at 0.50 mg/m(2) of topotecan, which was the DLT. The remaining 14 patients received topotecan without experiencing DLT. One patient at 0.40 mg/m(2) died of disease progression while on treatment. There were 6 other grade 4 hematologic events (5 ANCs <500/mm(3), 1 hemoglobin <6. 5 g/dl) not meeting DLT criteria. No significant non-hematologic toxicities were seen. The actuarial median survival time is 15 months (95% confidence interval, 9.6-19 months); 1-year survival is 53%. DLT of daily topotecan with cranial XRT is grade 4 neutropenia for > or =7 days at 0.50 mg/m(2) x 33 (total dosage = 16.5 mg/m(2)); the recommended safe MTD of daily topotecan for further phase II testing is 0.40 mg/m(2) x 33 (total dosage = 13.2 mg/m(2)).
 
The North American Brain Tumor Consortium conducted a phase I trial of the combination 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide. Eligibility included a patient with a cancer type that was considered refractory to standard therapy. Prior nitrosourea treatments were not permitted. There were parallel dose escalations in two treatment schedules. Forty-five patients were enrolled during an 18-month period. The maximum tolerated doses (MTDs) when temozolomide followed BCNU (Arm A) were temozolomide at 550 mg/m2/p.o. and BCNU at 150 mg/m2/i.v.), whereas the MTD when temozolomide preceded BCNU (Arm B) was temozolomide at 400 mg/m2/p.o. and BCNU at 100 mg/m2/i.v. Toxicity was predominantly hematologic, although there were three instances of pulmonary toxicity, which in one case could have represented potentiation of nitrosourea-induced pulmonary fibrosis. The half-life of temozolomide was 1.86 (+/-0.31) h. There was a moderate relationship between dose and peak concentration and a strong relationship between dose and plasma concentration time curve. Pharmacokinetic parameters of temozolomide were unaffected by the treatment schedule, so the difference in MTD between the schedules is likely due to a biologic rather than a pharmacokinetic sequence interaction. There were 9 partial responses among 43 patients evaluable for response, including 5 of 25 with a histologic diagnosis of glioblastoma. The recommended dose and schedule for phase II trials of this regimen are BCNU 150 mg/m2/i.v. followed in 2 h by temozolomide 550 mg/m2/p.o. repeated every 6 weeks. We are also recommending screening and periodic pulmonary function testing during treatment to assess the possible potentiation of nitrosourea-induced pulmonary fibrosis.
 
Patients with glioma at 1.5T (left column) and 3T (right column). A and B. Axial FLAIR sequence at 1.5T (A) and 3T (B) of a 51-year-old woman with a left frontal glioma (WHO grade II) treated with radiation therapy 9 years before. Time interval between examinations : 3 days . C, D, E and F. Post-contrast 3D T1-w (C, D), and perfusion images (E, F) of a 56-year- old patient with a progressing grade II oligoastrocytoma treated 10 years before with surgery and radiation therapy, showing nodular contrast enhancement (white arrows) at 1.5 (C) and 3-T (D). Focal rCBV increase visible as a “hot spot” (high values in red, low values in blue) on co-registered perfusion parametric map and post-contrast 3D T1-w images (black arrows) at 1.5 (E) and 3T (F). Time interval between examinations: 7 days . 
Comparison between 1.5 and 3T. A. Graph showing linear regression (black line) with 95% CI (dotted lines) of lesion volumes at 1.5-T and 3-T (rspearman 1⁄4 0.96 [95% CI:0.92 2 0.98]) Each dot represents a patient . B. Graph showing linear regression (black line) with 95% CI (dotted lines) of rCBV values on PWI at 1.5T and 3T (rspearman 1⁄4 0.90 [95% CI:0.79 2 0.96]). 
Morphological imaging analysis at 1.5 and 3Tesla in the 30 participants studied
Contrast-to-noise ratio. Scatter-plot comparing lesion CNR on FLAIR sequences (CNRFLAIR) and lesion enhancement on 3D T1-w images after injection of contrast medium (CNRCONTRAST), using Wilcoxon matched-pairs signed-rank test ( P *). 
Glioma follow-up is based on MRI parameters, which are correlated with survival. Although established criteria are used to evaluate tumor response, radiological markers may be confounded by differences in instrumentation including the magnetic field strength. We assessed whether MRIs obtained at 3 Tesla (T) and 1.5T provided similar information. We retrospectively compared imaging features of 30 consecutive patients with WHO grades II and III gliomas who underwent MRI at 1.5T and 3T within a month of each other, without any clinical changes during the same period. We compared lesion volumes on fluid attenuation inversion recovery (FLAIR), ratio of cerebral blood volume (rCBV) on perfusion-weighted imaging, contrast-to-noise ratio (CNR) on FLAIR, and on post-gadolinium 3D T1-weighted sequences between 1.5T and 3T using intraclass correlation coefficient (ICC). Concordance between observers within and between modalities was evaluated using weighted-kappa coefficient (wκ). The mean ± SD delay between modalities (1.5T and 3T MRI) was 8.6 ± 5.6 days. Interobserver/intraobserver concordance for lesion volume was almost perfect for 1.5T (ICC = 0.96/0.97) and 3T (ICC = 0.99/0.98). Agreement between observers for contrast enhancement was excellent at 1.5T (wκ = 0.92) and 3T (wκ = 0.92). The tumor CNR was significantly higher for FLAIR at 1.5T (P < .001), but it was higher at 3T (P = .012) for contrast enhancement. Correlations between modalities for lesion volume (ICC = 0.97) and for rCBV values (ICC = 0.92) were almost perfect. In the follow-up of WHO grades II and III gliomas, 1.5T and 3T provide similar MRI features, suggesting that monitoring could be performed on either a 1.5 or a 3T MR magnet. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
 
Treatment for newly diagnosed anaplastic oligodendroglial tumors is controversial. Radiotherapy (RT) alone and in combination with chemotherapy (CT) are the most well studied strategies. However, CT alone is often advocated, especially in cases with 1p19q codeletion. We retrospectively identified 1013 adults diagnosed from 1981-2007 treated initially with RT alone (n = 200), CT + RT (n = 528), CT alone (n = 201), or other strategies (n = 84). Median overall survival (OS) was 6.3 years and time to progression (TTP) was 3.1 years. 1p19q codeletion correlated with longer OS and TTP than no 1p or 19q deletion. In codeleted cases, median TTP was longer following CT + RT (7.2 y) than following CT (3.9 y, P = .003) or RT (2.5 y, P < .001) alone but without improved OS; median TTP was longer following treatment with PCV alone than temozolomide alone (7.6 vs. 3.3 y, P = .019). In cases with no deletion, median TTP was longer following CT + RT (3.1 y) than CT (0.9 y, P = .0124) or RT (1.1 y, P < .0001) alone; OS also favored CT + RT (median 5.0 y) over CT (2.2 y, P = .02) or RT (1.9 y, P < .0001) alone. In codeleted cases, CT alone did not appear to shorten OS in comparison with CT + RT, and PCV appeared to offer longer disease control than temozolomide but without a clear survival advantage. Combined CT + RT led to longer disease control and survival than did CT or RT alone in cases with no 1p19q deletion. Ongoing trials will address these issues prospectively.
 
One of the biggest challenges in glioma management is accurately tracking tumor response to treatment. While direct tissue assessment might be ideal, repeat surgery is not always feasible and subject to sampling error. Consequently, MRI (or less ideally CT) is the surrogate tool for tracking changes in tumor in response to therapy. However, there are clear limitations to MRI which, in reality, is just a picture and we do not always know what biological process or processes that picture symbolizes.
 
Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft tissue sarcomas accounting for 3%-10% of all soft tissue sarcomas. Neurofibromatosis type 1 (NF1) is the most important known risk factor. MPNSTs are often diagnosed at an advanced stage when distant metastases have developed. Although surgical resection remains the main treatment for MPNSTs, complete surgical resection is rarely possible. The prognosis for patients with MPNSTs is poor. There is an urgent need for improved therapies. To this end, we investigated whether microRNA (miR), specifically miR-204, might be implicated in MPNSTs because it is located at a cancer-associated genomic region exhibiting high frequency of loss of heterozygosity in tumors. We show that miR-204 expression is downregulated in NF1 and non-NF1 MPNST tumor tissues and in tumor cell lines. Restoring miR-204 expression in MPNST cell lines STS26T (non-NF1), ST88-14 (NF1), and T265p21 (NF1) significantly reduces cellular proliferation, migration, and invasion in vitro. Restoring miR-204 expression in STS26T decreases tumor growth and malignant progression in vivo. We also report that miR-204 inhibits Ras signaling and expression of high mobility group gene A2. These findings support the hypothesis that miR-204 plays critical roles in MPNST development and tumor progression. miR-204 may represent a novel biomarker for diagnosis and a candidate target with which to develop effective therapies for MPNSTs.
 
Progression-free survival (PFS) per central assessment (A) and overall survival (B). Tumor assessments that were taken after initiation of any non–AMG 102 antitumor therapy, tumor resection, or first tumor progression were excluded from PFS. All deaths were included. PFS was defined as the time in weeks from the first dose of AMG 102 to first progression by Macdonald criteria or death from any cause, whichever occurred first. Patients who did not progress or die were censored at the last evaluable radiograph. If a patient had no evaluable postbaseline radiograph and did not die, then the patient was censored at day 1. 
Demographics and Key Baseline Characteristics
Analysis of hepatocyte growth factor / scatter factor (HGF / SF) and c-Met as biomarkers. Plasma log total HGF / SF (A) and soluble c-Met (B) concentrations from week 1 until the end of treatment (EOT). (C) Representative images of high (maximum intensity scale of 3) versus low (maximum intensity scale of 1) cytoplasmic c-Met expression assessed by immunohistochemistry staining of patient archival tumor samples. 
Patient Incidence of Treatment-Related Adverse Events a
This phase II study evaluated the efficacy and safety of AMG 102 (rilotumumab), a fully human monoclonal antibody against hepatocyte growth factor/scatter factor (HGF/SF), in patients with recurrent glioblastoma (GBM). Patients with histologically confirmed, measurable recurrent GBM or gliosarcoma (World Health Organization grade 4) and ≤3 relapses or prior systemic therapies received AMG 102 (10 or 20 mg/kg) by infusion every 2 weeks. The primary endpoint was best confirmed objective response rate (central assessment) per Macdonald criteria. Of the 61 patients who enrolled, 60 received AMG 102. Twenty-nine patients (48%) had previously received bevacizumab. There were no objective responses per central assessment, but 1 patient had an objective response per investigator assessment. Median overall survival (95% CI) in the 10- and 20-mg/kg cohorts was 6.5 months (4.1-9.8) and 5.4 months (3.4-11.4), respectively, and progression-free survival (PFS) per central assessment was 4.1 weeks (4.0-4.1) and 4.3 weeks (4.1-8.1), respectively. PFS was similar among patients who had previously received bevacizumab compared with bevacizumab-naive patients. The most common adverse events were fatigue (38%), headache (33%), and peripheral edema (23%). AMG 102 serum concentrations increased approximately dose-proportionally with 2-fold accumulation at steady state. Plasma total HGF/SF and soluble c-Met concentrations increased 12.05- and 1.12-fold, respectively, from baseline during AMG 102 treatment. AMG 102 monotherapy at doses up to 20 mg/kg was not associated with significant antitumor activity in heavily pretreated patients with recurrent GBM.
 
Overall survival data among recursive partitioning analysis (RPA) classes for patients from the 5-aminolevulinic acid (ALA) study ALA Study Database a 
The benefit of cytoreductive surgery for glioblastoma multiforme (GBM) is unclear, and selection bias in past series has been observed. The 5-aminolevulinic acid (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 Radiation Therapy Oncology Group recursive partitioning analysis (RTOG-RPA) would predict survival of these patients and whether there was any benefit from extensive resections depending on RPA class. A total of 243 per-protocol patients with newly diagnosed GBM were operated on with or without ALA and treated by radiotherapy. Postoperative MRI was obtained in all patients. Patients were allocated into RTOG-RPA classes III-V based on age, KPS, neurological condition, and mental status (as derived from the NIH Stroke Scale). 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 clearly longer in RPA classes IV and V (17.7 months vs. 12.9 months, p = 0.0015, and 13.7 months vs. 10.4 months, p = 0.0398; 2-year rates: 21.0% vs. 4.4% and 11.1% vs. 2.6%, respectively), but was not in the small subgroup of RPA class III patients (19.3 vs. 16.3 months, p = 0.14). 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.
 
A growing body of work suggests that astrocytomas and glioblastoma multiforme will require carefully tailored, molecularly targeted therapy for successful treatment. Recent efforts to comprehensively identify mutations and gene expression changes in glioblastoma have shown that mutation of NF1 is a common alteration in human glioblastoma. We have developed and characterized a panel of 14 tumor lines from grades II through IV astrocytomas developed from our Nf1-/+;Trp53-/+cis mouse model and have used this panel to characterize signal transduction pathways and inhibitors that are candidate therapeutic targets for astrocytoma and glioblastoma. We show that these tumors express platelet-derived growth factor receptor-α, epidermal growth factor receptor, and their respective ligands to varying degrees. We find that both the MEK and PI3K signaling pathways downstream of epidermal growth factor receptor and platelet-derived growth factor receptor-α are necessary for full proliferation of astrocytoma cells; however, inhibition of the PI3K pathway is more effective than inhibition of MEK at blocking cell growth. We have examined inhibitors of the PI3K/Akt/mTOR signaling pathway and find that PI-103 and TCN show particular promise for inhibiting growth in Nf1 and Trp53 mutant astrocytoma cells.
 
Evidence that granule exocytosis occurs during the interaction between TALL-104 and glioma cells. A. Calcein-loaded 04-11-MG glioma cells before (upper panel) and then at 5000 s after the addition of TALL-104 cells (lower panel). The arrows in each of the panels point to the locations of adherent fluorescent cells initially present that lost fluorescence during the experiment. B. Fluorescence traces from individual cells shown in A. The upper trace is an example of a cell that did not release calcein; the second, third, and bottom three panels monitor individual cells that displayed calcein release at 900 s, 1500 s, and 3100 s, respectively, after the addition of the TALL-104 cells. C. Time course of hitting for the experiment shown in A and B. Approximately 25% of cells were hit over the 5000-s (83-min) duration of the experiment.
TALL-104 is a human leukemic T cell line that expresses markers characteristic of both cytotoxic T lymphocytes and natural killer cells. TALL-104 cells are potent tumor killers, and the use of lethally irradiated TALL-104 as cellular therapy for a variety of tumors has been explored. We investigated the interactions of TALL-104 cells with human brain tumor cells. TALL-104 cells mediated increased lysis of a panel of brain tumor cells at low effector-to-target ratios over time. We obtained evidence that TALL-104 cells injured glioma cells by both apoptotic and necrotic pathways. A 7-amino actinomycin D flow cytometry assay revealed that the percentages of both apoptotic and necrotic glioma cells increased after TALL-104 cell/glioma cell coincubations. Fluorescent microscopy studies and a quantitative morphologic assay confirmed that TALL-104 cell/glioma cell interactions resulted in tumor cell apoptosis. Cytokines are secreted when TALL-104 cells are coincubated with brain tumor cells; however, morphologic analysis assays revealed that the soluble factors contained within clarified supernates obtained from 4 h coincubates added back to brain tumor cell cultures did not trigger the glioma apoptosis. TALL-104 cells do not express Fas ligand, even upon coincubation with glioma targets, which suggests that the Fas/Fas ligand apoptotic pathway is not likely responsible for the cell injury observed. We obtained evidence that cell injury is calcium dependent and that lytic granule exocytosis is triggered by contact of TALL-104 cells with human glioma cells, suggesting that this pathway mediates glioma cell apoptosis and necrosis.
 
Signaling pathways involved in the development of the brain and pathogenesis of medulloblastoma and ependymoma. Deregulation of these pathways is important in the pathogenesis of medulloblastoma and ependymoma. Interactions among these pathways are multiple and complex. 
Differentially expressed genetic and proteomic markers identified in medulloblastomas and ependymomas (continued) 
Differentially expressed genetic and proteomic markers identified in medulloblastomas and ependymomas (continued) 
Differentially expressed genetic and proteomic markers identified in medulloblastomas and ependymomas 
Balanced chromosomal translocations identified in medulloblastomas and ependymomas. 
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 outcome for these patients. It has become clear that the deregulation of signaling pathways essential in brain development, for example, sonic hedgehog (SHH), Wnt, and Notch pathways, plays an important role in pathogenesis and biological behavior, especially for medulloblastomas. More recently, data have become available about the cells of origin of brain tumors and 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.
 
Correlation of miR-106a expression between matched FFPE and fresh frozen GBM samples evaluated by qRT-PCR (n ¼ 24). (A and B) Correlation of Ct values of U6 (A) and miR-106a (B) in FFPE samples compared with corresponding fresh frozen tissues. (C) Correlation of fold changes of miR-106a expression between matched FFPE and fresh frozen samples. The fold changes of miR-106a were calculated as the difference between average expression levels of GBM samples and 4 corresponding normal brain tissues. Abbreviation: FLAIR, fluid attenuated inversion recovery. 
The stability of miR-106a expression in FFPE samples stored at different time points. (A and B) The Ct values of U6 (A) and miR-106a (B) in 1-, 3-, and 7-year-old FFPE GBM samples. (C) The fold changes of miR-106a expression in 1-, 3-, and 7-year-old FFPE GBM samples. The fold changes of miR-106a were calculated as the difference between average expression levels of GBM samples and 4 corresponding normal brain tissues. 
Kaplan-Meier curves for OS of the second cohort of GBM patients for miR-106a expression. The miR-106a expression levels were converted into discrete variables by discriminating the samples into 2 classes (high and low), under or over median value. Kaplan-Meier survival curves for miR-106a expression detected by qRT-PCR in the second cohort of GBM patients (A), patients with standard postoperative radiotherapy (B), patients in the GTR group (C), and patients in the STR group (D). 
Background Very little is known regarding correlation of micro RNA (miR)-106a with clinical outcomes of patients with glioblastoma multiforme (GBM). This study determined whether miR-106a could be used as an independent prognostic biomarker in those patients.MethodsA total of 156 GBM patients were divided into 2 cohorts. In the first cohort, matched fresh frozen and formalin-fixed paraffin-embedded (FFPE) samples were collected from 24 GBM patients, while in the second cohort, only FFPE samples were collected from 132 GBM patients. MiR-106a expression levels were examined by quantitative real-time PCR in the 2 cohorts and further validated by in situ hybridization assay in the second cohort. The correlation between miR-106a expression levels and overall survival was evaluated in the second cohort of 114 GBM patients available for follow-up by a log-rank test and a multivariate Cox proportional hazards model.ResultsOur data showed a very good correlation of miR-106a or U6 expression between fresh frozen and FFPE GBM specimens, with Pearson's correlation coefficients of 0.849 and 0.823, respectively (P < .001). Their expression levels in archival FFPE samples were quite stable for at least 7 years when stored at room temperature. Multivariate analysis revealed that the expression level of miR-106a was an independent and significant predictor of overall survival in GBM patients (P = .011).Conclusions MiR-106a expression was relatively abundant and stable in a large cohort of archival FFPE GBM specimens and could be used as an independent prognostic biomarker in those patients. Thus, miR-106a can be used to predict prognosis and treatment response in individual GBM patients.
 
The purpose of this study was to determine whether chromosome 10q loss is a predictor of tumor aggressiveness and poor clinical outcome in patients with oligodendroglial tumors alone or together with loss of heterozygosity (LOH) on chromosomes 1p and 19q. A microsatellite analysis was performed on sections from 130 patients with grade II and grade III oligodendroglial tumors to assess the allelic status of chromosomes 1p, 19q, and 10q, plus detailed clinical and radiological information was taken prospectively. Median age at diagnosis was 45.5 years. Seventy-eight patients had disease progression after initial therapy; median progression-free survival (PFS) was 27.5 months. Age <47 years, postoperative Karnofsky performance score >65, no contrast enhancement on MRI, grade II, and complete removal on surgery were significantly correlated with a better PFS. Median overall survival (OS) was 40.5 months. Pure oligodendroglioma and temozolomide chemotherapy were correlated with better OS. 10q LOH was correlated with anaplastic grade and 1p19q LOH correlated with pure oligodendroglioma. There was a significant association between LOH status and the tumors' response to chemotherapy: 92.3% with 1p19q LOH, 83.3% without allelic losses, 50% with 1p19q10q LOH, and 14.5% with 10q LOH. Patients with 10q LOH alone had PFS of 6 months and a 3-year survival rate of 1%, when compared with 36 months and 85%, respectively, in patients with 1p19q LOH but without 10q LOH. 1p loss was correlated with better PFS (P < .005) and OS (P = .0007), whereas 10q loss was correlated with decreased PFS (P < .0001) and OS (P < .0001). 10q LOH predicted a survival disadvantage in patients with oligodendroglial tumors irrespective of 1p/19q LOH status.
 
The prognostic advantage conferred by deletion of chromosomes 1p-19q on PFS is lost in patients with combined chromosome 9p loss. 
Correlations between clinical or molecular factors and PFS or OS: univariate analysis. (*: p , 0.05) 
The loss of chromosomes 1p–19q is the only prognostic molecular alteration identified in low-grade gliomas (LGGs) to date. Search for loss of heterozygosity (LOH) on chromosomes 1p, 9p, 10q, and 19q was performed in a series of 231 LGGs. Loss of chromosomes 1p–19q was strongly correlated with prolonged progression-free survival (PFS) and overall survival (OS) in univariate and multivariate analyses. LOH on 9p and 10q were associated with shortened PFS (P = .01 and .03, respectively) on univariate analysis. On multivariate analysis, LOH on 9p remained significant for PFS (P = .05), whereas LOH on 10q had a significant effect on OS (P = .02). Search for LOH 9p and 10q appears to be a useful complement to analysis of chromosomes 1p–19q in LGGs.
 
Association of 1p deletions and 19q deletions with survival group among patients with high-grade astrocytomas 
Representative FISH hybridizations performed on tissue microarrays. A. GBM with a normal disomic complement of 1p. Most cells have 2 green (1p32) and 2 red (1q42) signals. Some appear to have fewer than 2 copies because of the truncation artifact encountered in thin tissue sections (i.e., incomplete DNA complement in sectioned nuclei). Some signals are out of the plane of focus represented in the photograph. B. Pattern of 19q deletion in GBM showing 2 green (19p13) signals and 1 red (19q13.4) signal in most nuclei. C. Pattern of chromosome 10 deletion in a GBM. Only 1 green (PTEN) signal and 1 red (DMBT1) signal are detected in each nucleus, most likely representing either a large 10q deletion or the loss of an entire chromosome 10. D. Pattern of homozygous deletion of 9p21 (p16/CDKN2A) in a GBM. Only green signals from the centromeric probe are noted in the majority of nuclei. Signals from 9p21 (red) are almost entirely absent.
Association of 9p deletions and 10q deletions with survival group among patients with high-grade astrocytomas 
Survival periods vary considerably for patients with high-grade astrocytomas, and reliable prognostic markers are not currently available. We therefore investigated whether genetic losses from chromosomes 1p, 19q, 9p, or 10q were associated with survival in 89 high-grade astrocytomas using tissue microarrays (TMAs) derived from Radiation Therapy Oncology Group clinical trials. Cases included 15 anaplastic astrocytomas (AAs) and 74 glioblastomas (GBMs) selected on the basis of survival times significantly shorter or longer than the expected median. Genetic analysis was performed by TMA-fluorescence in situ hybridization (FISH) on array sections using 8 DNA probes, including those directed at 1p32, 19q13.4, 9p21 (p16/CDKN2A), and 10q (PTEN and DMBT1). Genetic status for each locus was correlated with patient survival group, and data were analyzed by using Fisher's exact test of association (adjusted P = 0.025). Losses of chromosome 1p, either alone or in combination with 19q, were encountered in only 2 cases, both AAs. This contrasts with oligodendrogliomas, in which combined 1p and 19q losses are frequent and predictive of prolonged survival. Solitary 19q loss was noted in 3/15 AAs and in 7/70 GBMs and was more frequent in the long-term survival group (P = 0.041, AA and GBM combined). Chromosome 9p loss was seen in 5/8 AAs and 39/57 GBMs, whereas chromosome 10q loss was detected in 4/15 AAs and 48/68 GBMs. The 9p and 10q deletions were slightly more frequent in short-term survivors, though none of the comparisons achieved statistical significance. Long-term and short-term survival groups of high-grade astrocytomas appear to have dissimilar frequencies of 19q, 9p, and 10q deletions. TMA-FISH is a rapid and efficient way of evaluating genetic alterations in such tumors.
 
Cell cycle profiles of U87MG cells examined by flow cytometry. U87MG cells were exposed to drug-free liposomes (control), Doxil (0.2 mg/ml), CPT-11 nanoliposomes (nLs-CPT-11) (5 mg/ml), or a combination of Doxil (0.2 mg/ml) and nLs-CPT-11 (5 mg/ml) for 24 h. Cells were harvested and analyzed by fluorescence-activated cell sorting as described in Materials and Methods.  
Cell cycle profiles of U251MG cells examined by flow cytometry. U251MG cells were exposed to drug-free liposomes (control), Doxil (0.2 mg/ml), CPT-11 nanoliposomes (nLs-CPT-11; 5 mg/ml), or a combination of Doxil (0.2 mg/ml) and nLs-CPT-11 (5 mg/ml) for 24 h. Cells were harvested and analyzed by fluorescence-activated cell sorting as described in Materials and Methods.  
Animals implanted with U251MG tumor cells. (A) Animals received liposome CED on day 7 and day 14 after tumor cell implantation. CED treatment on day 7 with nanoliposomal CPT-11 (nLs-CPT-11) and Doxil combination was able to eradicate all U251MG tumors in rodent striatum (see Fig. 5 caption). Each agent alone yielded only partial survival when delivered by CED on day 7. No animal in the day 14 combination therapy CED group survived longer than 78 days after tumor cell implantation. (B) Animals implanted with U87MG tumor cells received liposome CED on day 10 after tumor cell implantation. Three of six animals in the combination therapy group survived until termination of the study at day 70 after tumor cell implantation. Only one animal in the Doxil group survived until day 70. No animals in the nLs-CPT-11 group survived to the projected end of this survival study.  
Synergistic induction of cell death by Doxil and nanoliposomal CPT-11 (nLs-CPT-11) in U251MG glioma cells. (A) U87MG cells were treated for 24 h with increasing nLs-CPT-11 (0–2 mg/ml) concentrations and 24 h with increasing Doxil (0–2 mg/ml) concentrations. No synergy between the two agents was found by analysis of an isobologram (points above dotted line). (B) U251MG cells were treated identically to the U87MG cells with increasing Doxil and nLs-CPT-11 concentrations for 24 h. Synergy was determined between the two agents in the U251MG cell line as seen in the isobologram analysis (points below dotted line).  
Representative histology of animals used in this study. (A and B) Brain sections of animals used for toxicity study, euthanized 60 days after receiving the combination of Doxil (2.0 mg, 0.1 mg/ml) and nanoliposomal CPT-11 (nLs-CPT-11; 0.8 mg, 40 mg/ml). (C) Representative section of animals bearing U251MG xenografts. (D) Section from nLs-CPT-11 survivor of day 7 convection-enhanced delivery (CED) still bearing small U251MG tumor. (E) Survivor of day 7 CED combination therapy in U251MG at day 100. (F) Animal bearing U87MG xenografts. (G) Survivor of day 10 CED combination therapy in U87MG at day 70. (H and I) Representative sections showing DiIC 18 (3) fluorescent liposome distribution in U87MG (H) and U251MG (I) intracranial xenografts after 20 ml CED. White line represents infusion catheter placement; dotted line delineates tumor margin on histology sections; white arrows show necrotic areas in U251MG brain tumor xenografts.  
We have previously shown that convection-enhanced delivery (CED) of highly stable nanoparticle/liposome agents encapsulating chemotherapeutic drugs is effective against intracranial rodent brain tumor xenografts. In this study, we have evaluated the combination of a newly developed nanoparticle/liposome containing the topoisomerase I inhibitor CPT-11 (nanoliposomal CPT-11 [nLs-CPT-11]), and PEGylated liposomal doxorubicin (Doxil) containing the topoisomerase II inhibitor doxorubicin. Both drugs were detectable in the CNS for more than 36 days after a single CED application. Tissue half-life was 16.7 days for nLs-CPT-11 and 10.9 days for Doxil. The combination of the two agents produced synergistic cytotoxicity in vitro. In vivo in U251MG and U87MG intracranial rodent xenograft models, CED of the combination was also more efficacious than either agent used singly. Analysis of the parameters involved in this approach indicated that tissue pharmacokinetics, tumor microanatomy, and biochemical interactions of the drugs all contributed to the therapeutic efficacy observed. These findings have implications for further clinical applications of CED-based treatment of brain tumors.
 
Contrast-enhanced MRI scan of the brain of a 6-year-old female with GBM of the midbrain (Patient no. 3) before (A) and after (B) 5 courses of CPT-11.  
Clinical characteristics, tumor histology, and prior treatment of 22 patients with malignant brain tumors treated with CPT-11
A phase II study of irinotecan (CPT-11) was conducted at Duke University Medical Center, Durham, NC, to evaluate the activity of this agent in children with high-risk malignant brain tumors. A total of 22 children were enrolled in this study, including 13 with histologically verified recurrent malignant brain tumors (glioblastoma multiforme [GBM] 4, anaplastic astrocytoma 1, ependymoma 5, and medulloblastoma/primitive neuroectodermal tumor 3), 5 with recurrent diffuse pontine glioma, and 4 with newly diagnosed GBM. All patients with recurrent tumor had prior chemotherapy and/or irradiation. Each course of CPT-11 consisted of 125 mg/m ( 2 ) per week given i.v. for 4 weeks followed by a 2-week rest period. Patients with recurrent tumors received therapy until disease progression or unacceptable toxicity. Patients with newly diagnosed tumors initially received 3 cycles of treatment to assess tumor response and then were allowed radiotherapy at physician's choice; patients who demonstrated a response to CPT-11 prior to radiotherapy were allowed to continue the drug after radiation until disease progression or unacceptable toxicity. A 25% to 50% dose reduction was made for grade III-IV toxicity. Responses were assessed after every course by gadolinium-enhanced MRI of the brain and spine. Twenty-two patients received a median of 2 courses of CPT-11 (range, 1-16). Responses were seen in 4 of 9 patients with GBM or anaplastic astrocytoma (44%; 95% confidence interval, 11%-82%) (complete response in 2 patients with recurrent GBM lasting 9 months and 48+ months; partial response in one patient with a newly diagnosed midbrain GBM lasting 18 months prior to radiotherapy; and partial response lasting 11 months in 1 patient with recurrent anaplastic astrocytoma), 1 of 5 patients with recurrent ependymoma (partial response initially followed by stable disease lasting 11 months), and none of 5 patients with recurrent diffuse pontine glioma. Two of 3 patients with medulloblastoma/primitive neuroectodermal tumor had stable disease for 9 and 13 months. Toxicity was mainly myelosuppression, with 12 of 22 patients (50%) suffering grade II-IV neutropenia. Seven patients required dose reduction secondary to neutropenia. CPT-11, given in this schedule, appears to be active in children with malignant glioma, medulloblastoma, and ependymoma with acceptable toxicity. Ongoing studies will demonstrate if activity of CPT-11 can be enhanced when combined with alkylating agents, including carmustine and temozolomide.
 
Patient characteristics 
The purpose of this study was to determine the response to CPT-11 administered every three weeks to adults with progressive malignant glioma, treated with or without enzyme-inducing antiepileptic drug (EIAED) therapy, at the recommended phase 2 dose determined from a previous phase 1 study. Adult patients age 18 or older with a KPS of 60 or higher who had measurable recurrent grade III anaplastic glioma (AG) or grade IV glioblastoma multiforme (GBM) were eligible. No more than one prior chemotherapy was allowed, either as adjuvant therapy or for recurrent disease. The CPT-11 dose was 350 mg/m(2) i.v. every three weeks in patients not on EIAED and 750 mg/m(2) in patients on EIAED therapy. Patients with stable or responding disease could be treated until tumor progression or a total of 12 months of therapy. The primary end point of the study was to determine whether CPT-11 could significantly delay tumor progression, using the rate of six-month progression-free survival (PFS-6). The trial was sized to be able to discriminate between a 15% and 35% rate for the GBM group alone and between a 20% and 40% rate for the entire cohort. There were 51 eligible patients, including 38 GBM and 13 AG patients, enrolled. The median age was 52 and 42 years, respectively. PFS-6 for the entire cohort was 17.6%. PFS-6 was 15.7% (95% confidence interval [CI], 0.07-0.31) for the GBM patients and 23% (95% CI, 0.07-0.52) for AG patients. Toxicity for the group included diarrhea and myelosuppression. We conclude that the recommended phase 2 dose of CPT-11 for patients with or without EIAED was ineffective on this schedule, in this patient population.
 
Kaplan–Meier estimates for overall survival (A) and progression-free survival (B) stratified by vorinostat dose levels. 
Dose-escalation schema
Plasma Proteomic Profiles. ( A ) A heat map was generated to represent relative expression of plasma proteins from pretreatment samples. Ratios were normalized to the median value of the entire cohort and color intensity was assigned to ratios of protein expression; shades of red, proteins that are upregulated; shades of green, proteins that are downregulated; black, proteins that are unchanged. ( B ) Ratios of plasma protein expression following recurrence (or last available plasma sample for patients currently on study) were normalized to their pretreatment values. 
Patient characteristics
A phase I study was conducted to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) for the combination of vorinostat with bevacizumab and CPT-11 in recurrent glioblastoma. Vorinostat was combined with bevacizumab and CPT-11 and was escalated using a standard 3 + 3 design. Vorinostat was escalated up to 2 actively investigated doses of this compound or until the MTD was identified on the basis of DLTs. Correlative science involving proteomic profiling of serial patient plasma samples was performed. Nineteen patients were treated. The MTD of vorinostat was established at 400 mg on days 1-7 and 15-21 every 28 days when combined with bevacizumab and CPT-11. Common toxicities were fatigue and diarrhea. DLTs included fatigue, hypertension/hypotension, and central nervous system ischemia. Although the MTD was established, CPT-11 dose reductions were common early in therapy. High-dose vorinostat had an improved progression-free survival and overall survival when compared with low-dose vorinostat. Serum proteomic profiling identified IGFBP-5 and PDGF-AA as markers for improved PFS and recurrence, respectively. A MTD for the combination of vorinostat with bevacizumab and CPT-11 has been established, although it has poor long-term tolerability. With the increased toxicities associated with CPT-11 coupled with its unclear clinical significance, investigating the efficacy of vorinostat combined with bevacizumab alone may represent a more promising strategy to evaluate in the context of a phase II clinical trial.
 
Pharmacokinetic comparisons between patients on EIAEDs and patients not on EIAEDs receiving CPT-11 at the 350-mg/m 2 dose level* 
This study was conducted to determine the maximum tolerated dose and dose-limiting toxicity of irinotecan (CPT-11) administered every 3 weeks to adults with progressive malignant glioma who were treated with enzyme inducing antiepileptic drug (EIAED) therapy, and to compare the pharmacokinetics with those in patients not on EIAED therapy treated at the recommended phase 2 dose for other cancers. The CPT-11 dose was 350 mg/m(2) i.v. every 3 weeks and remained fixed in patients not on EIAED therapy, but the dose was escalated by 50-mg/m(2) increments in patients on EIAED therapy. CPT-11 and its metabolites SN-38, SN-38 glucuronide (SN-38G), and APC (7-ethyl-10[4-N-(5 aminopentanoic acid)-1-piperidine]-carbonyloxycamptothecin) were characterized in both groups. Patients on EIAEDs received 350 to 800 mg/m(2) of CPT-11. Dose-limiting toxicity was due to grade 3 diarrhea despite maximal doses of loperamide. The systemic levels of CPT-11, APC, SN-38G, and SN-38 were all lower in the EIAED group. There was a moderate-to-fair relationship between CPT-11 dose and the area under the curve (AUC) for CPT-11 and APC over the 2, but no relationship dosage range of 350 to 800 mg/m between CPT-11 dose and the AUC for SN-38 or SN-38G. At the 750-mg/m(2) dose, the AUC for CPT-11 (21.6 microg x h/ml) matched the AUC (21.6 microg x h/ml) in the non-EIAED group treated with 350 mg/m(2) of CPT-11. We conclude that the recommended phase 2 dose of CPT-11 for patients on EIAEDs is 750 mg/m(2) when given every 3 weeks. A phase 2 study of patients with recurrent malignant glioma is ongoing to assess the efficacy of CPT-11 when the dose is stratified according to the use of EIAEDs.
 
18 F FDG (left side) and 11 C MET (right side) PET images (upper) with MRI registration (lower).  
Patient characteristics
SUV max measurements for FDG and MET PET
Qualitative tumor uptake grading
Background Fluorine-18 ((18)F) fluorodeoxyglucose (FDG) positron emission tomography (PET) is limited in its evaluation of brain tumors due to the high basal activity of the cerebral cortex and white matter. Carbon-11 methionine ((11)C MET) has little uptake under normal conditions. We prospectively investigated the uptake of (18)F FDG and (11)C MET PET in patients with craniopharyngioma prior to proton therapy.Methods Ten patients newly diagnosed with craniopharyngioma underwent PET imaging using (18)F FDG and (11)C MET. PET and MRI studies were registered to help identify tumor volume. Measurements of maximum standardized uptake value (SUV(max)) were taken of the tumor and compared with noninvolved left frontal background white matter using a paired t-test. Uptake was graded using a 4-point scale.ResultsMedian patient age was 9 years (range 5-19). Seven patients were diagnosed by pathology, 1 by cyst fluid aspiration, and 2 by neuroimaging. Median FDG SUV(max) for tumor and background were 2.65 and 3.2, respectively. Median MET SUV(max) for tumor and background were 2.2 and 1, respectively. There was a significant difference between MET tumor SUV(max) and MET background SUV(max) (P = .0001). The difference between FDG tumor SUV(max) and FDG background SUV(max) was not significant (P = .3672).Conclusion(11)C MET PET uptake is significantly greater within the tumor compared with noninvolved background white matter, making it more useful than FDG PET in identifying active tumor in patients with craniopharyngioma. Future work will focus on using (11)C MET PET to discriminate between active and inactive tumor after irradiation.
 
Trypan blue exclusion assay and flow cytometry 24 h after treatment with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), quercetin, or the combination of both. (A) Cell death in U87-MG, A172, U251, LN229, and U373 cells upon treatment with quercetin. (B–D) Effect of quercetin on TRAIL-induced cytotoxicity in U87-MG, A172, U251, LN229, and U373 cells. (E) Effect of quercetin on TRAIL-induced apoptosis in U87-MG and U251 cells analyzed by flow cytometry. Control, not treated; Q50/100/200, quercetin 50/100/200 mM; TR100/500, TRAIL 100/500 ng/ml. *Significantly different from the respective control (t-test, *p , 0.05).  
Immunoblot demonstrating the effect of quercetin on tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)-induced proteolytic cleavage of poly(ADp-ribose) polymerase (pARp), caspase-7/-8/-9, and Bid in U87-MG, A172, U251, LN229, and U373 cells. (A) U87-MG, (B) A172, (C) U251, (D) LN229, and (E) U373 cells were treated for 8 h with quercetin (200 µM) in the presence or absence of TRAIL (100 ng/ml). TR100, TRAIL 100 ng/ml; Q200, quercetin 200 µM; CF, cleaved fragment. Immunoblots are representative of at least three independent experiments.  
proteasomal degradation of survivin is enhanced in glioma cells upon treatment with quercetin. Immunoblots show inhibition of quercetin-mediated downregulation of survivin by MG132 in U251 (A) and U87-MG (B) cells. Q0/200, quercetin 0/200 µM; MG0/5, MG132 0/5 mM.  
Quercetin-mediated suppression of survivin is regulated by Akt. (A and B) Immunoblots show reduced phosphorylated Akt (ph- Akt) and survivin levels in U87-MG (A) and U251 (B) cells following treatment with quercetin for 8 h. (C) Immunoblot shows that overexpression of constitutively active Akt inhibits quercetin-mediated suppression of survivin in U87-MG cells 72 h after transfection and 24 h after treatment with quercetin. Immunoblots are representative of at least three independent experiments. Q0/50/100/200, quercetin 0/50/100/200 mM.  
The flavonoid quercetin has been reported to inhibit the proliferation of cancer cells, whereas it has no effect on nonneoplastic cells. U87-MG, U251, A172, LN229, and U373 malignant glioma cells were treated with quercetin (50–200 μM). Quercetin did not cause cytotoxicity 24 h after treatment. Combining quercetin with tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) strongly augmented TRAIL-mediated apoptosis in U87-MG, U251, A172, and LN229 glioma cells; U373 cells could not be sensitized by quercetin to TRAIL-mediated apoptosis. TRAIL-induced apoptosis was 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, and A172 but not in U373 glioma cells. Quercetin exposure resulted in proteasomal degradation of survivin. TRAIL-quercetin–induced apoptosis was markedly reduced by overexpression of survivin. In addition, upon treatment with quercetin, downregulation of survivin was also regulated by the Akt pathway. Taken together, the results of the present study suggest that quercetin sensitizes glioma cells to death-receptor–mediated apoptosis by suppression of inhibitor of the apoptosis protein survivin.
 
Axial (L) and coronal (R) MRI images of the 1 patient who had a complete response. Top Panels. Prechemotherapy, contrast-enhanced, T1- weighted axial and coronal magnetic resonance images demonstrate heterogeneous enhancement of the tumor in the left temporal lobe. Bot- tom Panels. The enhancement has resolved after CPT-11 therapy, consistent with a complete response. 
The primary objective of this study was to determine the proportion of patients exhibiting a radiographic response in a cohort of patients with recurrent malignant glioma who were treated with irinotecan. Secondary objectives were to determine progression-free survival, overall survival, and toxicity. The trial was terminated after the first 18 patients were enrolled in this multicenter, 2-stage, phase 2 study. Twelve patients received concurrent enzyme-inducing antiepileptic drugs, and 6 did not. Each cycle consisted of a 90-min i.v. infusion of irinotecan every week for 4 consecutive weeks, followed by 2 weeks off. One patient had a complete response, 5 patients had stable disease, 5 patients had radiographic progression, 6 patients were removed from the study because of toxicity, and 1 patient refused further therapy and was removed from the study. The response rate in this study was 6% (1/18), and 28% (5/18) of these patients progressed while receiving irinotecan. Dose-limiting toxicities consisted of diarrhea in 5 patients, neutropenia in 1 patient, infection in 1 patient, and respiratory failure in 1 patient. Irinotecan had minimal efficacy in this cohort of 18 patients with recurrent malignant glioma. Toxicity was significant but similar to that reported in other patient populations.
 
Examples of T1-weighted MRIs (left panel), methionine uptake on MET-PET (middle panel), and coregistered MRI and PET data (right panel) for the 3 major tumor types. (A) Left mesial frontal DNT, which was not associated with a visually detectable increased methionine uptake (patient #9). (B) Right neocortical (temporal) DNT observed on T1-weighted MRI with a moderately increased methionine uptake (patient #6). (C) Left temporal ganglioglioma associated with a markedly increased methionine uptake (patient #30). (D) Right insular low-grade glioma associated with a moderately increased methionine uptake (patient #34). 
Correlation between qualitative MET-PET data and MRI data
Correlation between qualitative MET-PET data and pathology (P , .001)
Performance characteristics of TC r and TO r to discriminate DNT from other tumor types
Dysembryoplastic neuroepithelial tumors (DNTs) represent a prevalent cause of epileptogenic brain tumors, the natural evolution of which is much more benign than that of most gliomas. Previous studies have suggested that [(11)C]methionine positron emission tomography (MET-PET) could help to distinguish DNTs from other epileptogenic brain tumors, and hence optimize the management of patients. Here, we reassessed the diagnostic accuracy of MET-PET for the differentiation between DNT and other epileptogenic brain neoplasms in a larger population. We conducted a retrospective study of 77 patients with focal epilepsy related to a nonrapidly progressing brain tumor on MRI who underwent MET-PET, including 52 with a definite histopathology. MET-PET data were assessed by a structured visual analysis that distinguished normal, moderately abnormal, and markedly abnormal tumor methionine uptake and by semiquantitative ratio measurements. Pathology showed 21 DNTs (40%), 10 gangliogliomas (19%), 19 low-grade gliomas (37%), and 2 high-grade gliomas (4%). MET-PET visual findings significantly differed among the various tumor types (P < .001), as confirmed by semiquantitative analyses (P < .001 for all calculated ratios), regardless of gadolinium enhancement on MRI. All gliomas and gangliogliomas were associated with moderately or markedly increased tumor methionine uptake, whereas 9/21 DNTs had normal methionine uptake. Receiver operating characteristics analysis of the semiquantitative ratios showed an optimal cutoff threshold that distinguished DNTs from other tumor types with 90% specificity and 89% sensitivity. Normal MET-PET findings in patients with an epileptogenic nonrapidly progressing brain tumor are highly suggestive of DNT, whereas a markedly increased tumor methionine uptake makes this diagnosis unlikely.
 
Anti-EGFRvIII antibody titers. Antibody titers to EGFRvIII were measured by enzyme-linked immunosorbent assay as described under Methods. Each point represents an individual patient titer, while horizontal lines delineate the mean for that time point.  
Baseline demographic and clinical characteristics All Treated Patients (N ¼ 65) 
Kaplan– Meier estimates of PFS and OS. Survival durations are calculated from study entry, representing a median of 3.0 (range, 2.4–4.4) months from diagnosis (as shown in Table 1). Line markers represent censored data.  
Kaplan – Meier estimates of PFS and OS, by MGMT promoter methylation status. (A) PFS and (B) OS are calculated from study entry, representing a median of 3.0 (range, 2.4– 4.4) months from diagnosis (as shown in Table 1). Line markers represent censored data. HR, hazard ratio; NE, not estimated.  
Kaplan–Meier estimates of PFS and OS for phase II rindopepimut studies. (A) PFS and (B) OS are calculated from diagnosis. Line markers represent censored data. In the 3 rindopepimut studies (ACTIVATE, ACT II, and ACT III), rindopepimut vaccinations began 3 mo after diagnosis.  
The epidermal growth factor receptor variant III deletion mutation, EGFRvIII, is expressed in ∼30% of primary glioblastoma and linked to poor long-term survival. Rindopepimut consists of the unique EGFRvIII peptide sequence conjugated to keyhole limpet hemocyanin. In previous phase II trials (ACTIVATE/ACT II), rindopepimut was well tolerated with robust EGFRvIII-specific immune responses and promising progression-free and overall survival. This multicenter, single-arm phase II clinical trial (ACT III) was performed to confirm these results. Rindopepimut and standard adjuvant temozolomide chemotherapy were administered to 65 patients with newly diagnosed EGFRvIII-expressing (EGFRvIII+) glioblastoma after gross total resection and chemoradiation. Progression-free survival at 5.5 months (∼8.5 mo from diagnosis) was 66%. Relative to study entry, median overall survival was 21.8 months, and 36-month overall survival was 26%. Extended rindopepimut vaccination (up to 3.5+ years) was well tolerated. Grades 1-2 injection site reactions were frequent. Anti-EGFRvIII antibody titers increased ≥4-fold in 85% of patients, and increased with duration of treatment. EGFRvIII was eliminated in 4/6 (67%) tumor samples obtained after >3 months of therapy. This study confirms, in a multicenter setting, the preliminary results seen in previous phase II trials of rindopepimut. A pivotal, double-blind, randomized, phase III trial ("ACT IV") is under way. © The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
 
Ependymoma case information. Summary of clinic cal information of tumor samples used in the (A) micro- array and (B) Q-PCR sections of this study 
Fold change expression ratios derived from microarray analysis and Q-PCR of six genes found to be abnormally expressed by microarray analysis a 
Ependymomas are glial cell-derived tumors characterized by varying degrees of chromosomal abnormalities and variability in clinical behavior. Cytogenetic analysis of pediatric ependymoma has failed to identify consistent patterns of abnormalities, with the exception of monosomy of 22 or structural abnormalities of 22q. In this study, a total of 19 pediatric ependymoma samples were used in a series of expression profiling, quantitative real-time PCR (Q-PCR), and loss of heterozygosity experiments to identify candidate genes involved in the development of this type of pediatric malignancy. Of the 12,627 genes analyzed, a subset of 112 genes emerged as being abnormally expressed when compared to three normal brain controls. Genes with increased expression included the oncogene WNT5A; the p53 homologue p63; and several cell cycle, cell adhesion, and proliferation genes. Underexpressed genes comprised the NF2 interacting gene SCHIP-1 and the adenomatous polyposis coli (APC)-associated gene EB1 among others. We validated the abnormal expression of six of these genes by Q-PCR. The subset of differentially expressed genes also included four underexpressed transcripts mapping to 22q12.313.3. By Q-PCR we show that one of these genes, 7 CBX7(22q13.1), was deleted in 55% of cases. Other genes mapping to cytogenetic hot spots included two overexpressed and three underexpressed genes mapping to 1q31-41 and 6q21-q24.3, respectively. These genes represent candidate genes involved in ependymoma tumorigenesis. To the authors' knowledge, this is the first time microarray analysis and Q-PCR have been linked to identify heterozygous/homozygous deletions.
 
Ependymomas account for 2% of all intracranial tumors in adults. Considerable controversy continues to exist with regard to their prognostic factors and therapeutic management due to the rarity and the heterogeneity of series reported so far. The authors report a retrospective study of a homogenous population of 114 adult patients harboring WHO grade II intracranial ependymomas from 32 French Neurosurgical Centers between 1990 and 2004. All clinico-radiological and follow-up data were analyzed, and a central pathologic review was performed by two confirmed neuropathologists. The 5- and 10-year overall survival (OS) rates were 86.1% and 81.0%, respectively; the 5- and 10-year progression-free survival (PFS) rates were 74.6% and 58.9%, respectively. On multivariate analysis, the OS rates were associated with preoperative KPS score (P = .027), extent of surgery (P = .008), and tumor location (supratentorial vs infratentorial, P = .012). The multivariate analysis also revealed that the risk of recurrence was associated with incomplete resection (P = .001) and supratentotrial location (P = .038). Moreover, adjuvant radiotherapy (RT) for patients with incompletely resected tumors is responsible for a significant improvement of both overall (P = .005) and progression-free (P = .002) survival. This study clearly supports the major prognostic impact of the extent of surgery in WHO grade II. Interestingly, tumor location also seems to have an actual impact on both OS and PFS. Finally, the prognostic impact of RT was found to be beneficial for incompletely resected tumors.
 
Top-cited authors
Quinn Ostrom
  • Baylor College of Medicine
Carol Kruchko
  • Case Western Reserve University
Mark Richard Gilbert
  • National Institutes of Health
Michael Prados
  • University of California, San Francisco
Wolfgang Wick
  • Universität Heidelberg