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Glioblastoma is the most common glioma with high mortality and poor prognosis. Radiation resistance is one of the large challenges in the treatment of glioma. The study aimed to identify whether DNA polymerase ζ affects glioma cell radiosensitivity. The mRNA and protein levels of REV3L and REV7 were examined using quantitative real-time PCR and western blot. After REV3L and REV7 knockdown in a GBM cell line (A172), we assessed cell viability, colonies, apoptosis, and immune escape. The underlying mechanisms were evaluated using western blot and were confirmed using rescue experiments. The results showed that REV3L and REV7 levels were increased in glioma and related to poor survival. γ-ray treatment inhibited cell viability, survival fraction, and immune escape, and induced apoptosis of glioma cells from a GBM cell line, whereas knockdown of REV3L or REV7 enhanced these effects. Mechanically, silencing of REV3L or REV7 inactivated the PI3K/AKT/mTOR pathway. IGF-1 treatment abrogated the effects on cell viability, colonies, and apoptosis induced by REV3L or REV7 knocking down. Taken together, silencing of REV3L and REV7 inhibited radiation resistance via inactivating the PI3K/AKT/mTOR pathway, suggesting that targeting DNA polymerase ζ may be a new strategy to reduce the radiotherapy resistance of glioma.

Purpose: Ferroptosis is a type of regulatory cell death, caused by excessive lipid peroxidation This study aimed to explore whether ionizing radiation could induce ferroptosis in glioma cells and whether carbonic anhydrase 9 (CA9) knockdown could enhance the killing effect of ionizing radiation on hypoxic glioma cells through ferroptosis., Materials and Methods: The protein levels of Acyl-CoA Synthetase Long Chain Family Member 4 (ACSL4) were detected by Western blot in glioma cells irradiated by different doses of X-ray. The relative mRNA levels of ferroptosis markers and intracellular iron-associated proteins were detected by Real-time qPCR. Lipid peroxidation of glioma cells was detected by oxidation-sensitive probe C11-BODIPY581/591 staining. CCK-8 Assay was used to detect cell viability after X-ray irradiation. Cloning formation assay was used to assess the radiosensitivity of glioma cells. The exposure of cell surface calreticulin was measured by immunofluorescence staining., Results: X-ray induced lipid peroxidation and ferroptosis markers expression in U251 and GL261 glioma cells. Knockdown of CA9 in hypoxic glioma cells significantly altered the expression of iron regulation-related proteins and enhanced X-ray-induced ferroptosis and radiosensitivity. The ferroptosis inhibitor significantly improved the survival of cells irradiated by X-ray, while ferroptosis inducers (FINs) enhanced the lethal effect of X-ray on cells. Enhancing ferroptosis in glioma cells promoted the exposure and release of damage-associated molecular patterns (DAMPs)., Conclusions: Ionizing radiation can induce ferroptosis in glioma cells. CA9 knockdown can enhance the radiosensitivity of hypoxic glioma cells and overcome the resistance of ferroptosis under hypoxia. Enhancing ferroptosis will become a new idea to improve the efficacy of radiotherapy for glioma.

Purpose: After irradiation, double-stranded DNA leaked into the cytoplasm activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, leading to the production of type I interferon (IFNI). In this study, we sought to probe the effect of ionizing radiation on activity of cGAS-STING-IFNI pathway in normoxic or hypoxic glioma cells and explore a more effective method to activate the signaling pathway, thereby activating the anti-tumor immune response and improving the therapeutic effect of radiotherapy for glioma., Materials and Methods: Human glioma cells U251 and T98G cultured in normoxia or hypoxia (1% O 2 ) were irradiated with different doses of X-ray. The relative expressions of cGAS, IFN-I stimulated genes (ISGs), and three-prime repair exonuclease 1 (TREX1) were detected by qPCR. The expression levels of interferon regulatory factor 3 (IRF3) and p-IRF3 proteins were detected by Western blot. The production of cGAMP and IFN-β in the supernatant was detected by ELISA assay. U251 and T98G cell lines with stable knockdown of TREX1 were established after transfection with lentivirus vectors. EdU cell proliferation assay was used to screen suitable metal ions concentrations. The phagocytosis of DCs was observed by immunofluorescence microscope. The phenotype of DCs was detected by flow cytometry. The migration ability of DCs was detected by a transwell experiment., Results: We found that cytosolic dsDNA, 2'3'-cGAMP, cGAS and ISGs expression, and IFN-β in cell supernatant were all increased with the doses of X-ray in the range of 0-16 Gy in normoxic glioma cells. Nevertheless, hypoxia significantly inhibited the radiation-induced dose-dependent activation of cGAS-STING-IFNI pathway. Furthermore, manganese (II) ion (Mn 2+ ) significantly improved cGAS-STING-IFNI pathway activation induced by X-ray in both normoxic and hypoxic glioma cells, thereby promoting the maturation and migration of DCs., Conclusions: The responses of cGAS-STING-IFNI pathway to ionizing radiation were mainly investigated under normoxic condition, but the experiments described here indicated that hypoxia could hinder the pathway activation. However, Mn 2+ showed radiosensitizing effects on the pathway under either normoxic or hypoxic conditions demonstrating its potential as a radiosensitizer for glioma through activating an anti-tumor immune response.

Radiotherapy has been established as a major therapeutic modality for glioma, whereas new therapeutic targets are needed to prevent tumor recurrence. This study intends to explore the regulatory role of magnesium transporter 1 (MAGT1) in radiotherapy resistance of glioma through modulating ERK and programmed death-1-ligand 1 (PD-L1). Our bioinformatics analysis identified differentially expressed MAGT1 in glioma, expression of which was subsequently determined in cohort data of TCGA database and microarray dataset as well as glioma cell lines. Artificial modulation of MAGT1, ERK, and PD-L1 expression was performed to examine their effects on glioma cell proliferation and radioresistance, as reflected by MTT and colony formation assays under irradiation. Mouse glioma cells with manipulated MAGT1 and ERK inhibitors were further injected into mice to assess the in vivo tumor formation ability of glioma cells. It was noted that MAGT1 expression was highly expressed in glioma tissues of TCGA data and microarray dataset, which was then validated in glioma cell lines. Ectopic expression of MAGT1 was revealed to promote the proliferation and radioresistance of glioma cells, which was attributed to the MAGT1-mediated activation of the ERK/MAPK signaling pathway. It was illuminated that MAGT1 stimulated PD-L1 expression through the ERK/MAPK pathway and thus facilitated glioma cell growth. Additionally, MAGT1 overexpression accelerated the in vivo tumor formation of glioma cells, while the ERK inhibitor negated its effect. In conclusion, MAGT1 enhances the growth and radioresistance of glioma cells through the ERK/MAPK signaling pathway-mediated upregulation of PD-L1 expression.

Background/aim: Upregulation of Forkhead box G1 (FOXG1) has recently been observed in many cancers, while its effect on radiosensitivity in glioma is still unclear. In this study, we hypothesized that FOXG1 be a major player in radioresistance of glioma as well as the underlying mechanism., Methods: Immunohistochemistry (IHC) was conducted to assess FOXG1 expression in glioma tissues and glioma-adjacent tissues. Western Blot was implemented to detect the expression of autophagy-related proteins. CCK-8, colony formation and flow cytometry assays were implemented to assess cell viability, proliferation and apoptosis, respectively. Transmission electron microscope (TEM) was used to observe autophagic vesicles. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assay was applied to detect the expression of FOXG1., Results: The present study demonstrated that FOXG1 was highly expressed in glioma tissues. FOXG1 expression level was up-regulated in glioma cells following exposure to X-ray irradiation. FOXG1 can attenuate radiosensitivity of glioma cells. Moreover, it revealed that FOXG1 attenuate radiosensitivity of glioma cells by promoting autophagy., Conclusions: The present study suggests that FOXG1 is a pivotal molecule for circumventing radiation-induced cell death in malignant glioma cells through the regulation of autophagy, and it may be a target for the treatment of human brain glioma.

Purpose: Despite being a major treatment modality for brain cancer due to its efficiency in achieving cancer control, radiotherapy has long been known to cause long-term side effects, including radiation-induced cognitive impairment (RICI). Neurogenesis inhibition due to radiation-induced damage in neural stem cells (NSCs) has been demonstrated to be an important mechanism underlying RICI. Radiation-induced bystander effects (RIBEs) denote the biological responses in non-targeted cells after their neighboring cells are irradiated. We have previously demonstrated that RIBEs could play an important role in the skin wound healing process. Therefore, we aimed to investigate whether RIBEs contribute to RICI in this study., Materials and Methods: The transwell co-culture method was used to investigate bystander effects in mouse NSCs induced by irradiated GL261 mouse glioma cells in vitro. The proliferation, neurosphere-forming capacity and differentiation potential of NSCs were determined as the bystander endpoints. The exosomes were extracted from the media used to culture GL261 cells and were injected into the hippocampus of C57BL/6 mice. Two months later, the neurogenesis of mice was assessed using BrdU incorporation and immunofluorescence microscopy, and cognitive function was evaluated by the Morris Water Maze., Results: After co-culture with GL261 glioma cells, mouse NSCs displayed inhibited proliferation and reduced neurosphere-forming capacity and differentiation potential. The irradiated GL261 cells caused greater inhibition and reduction in NSCs than unirradiated GL261 cells. Moreover, adding the exosomes secreted by GL261 cells into the culture of NSCs inhibited NSC proliferation, suggesting that the cancer cell-derived exosomes may be critical intercellular signals. Furthermore, injection of the exosomes from GL261 cells into the hippocampus of mice caused significant neurogenesis inhibition and cognitive impairment two month later, and the exosomes from irradiated GL261 cells induced greater inhibitory effects., Conclusion: RIBEs mediated by the exosomes from irradiated cancer cells could contribute to RICI and, therefore, could be a novel mechanism underlying RICI.

Macroautophagy/autophagy is a natural intracellular process that maintains cellular homeostasis and protects cells from death under stress conditions. Autophagy sustains tumor survival and growth when induced by common cancer treatments, including IR and cytotoxic chemotherapy, thereby contributing to therapeutic resistance of tumors. In this study, we report that the expression of MIR93, noted in two clinically relevant tumor subtypes of GBM, influenced GSC phenotype as well as tumor response to therapy through its effects on autophagy. Our mechanistic studies revealed that MIR93 regulated autophagic activities in GSCs through simultaneous inhibition of multiple autophagy regulators, including BECN1/Beclin 1, ATG5, ATG4B, and SQSTM1/p62. Moreover, two first-line treatments for GBM, IR and temozolomide (TMZ), as well as rapamycin (Rap), the prototypic MTOR inhibitor, decreased MIR93 expression that, in turn, stimulated autophagic processes in GSCs. Inhibition of autophagy by ectopic MIR93 expression, or via autophagy inhibitors NSC (an ATG4B inhibitor) and CQ, enhanced the activity of IR and TMZ against GSCs. Collectively, our findings reveal a key role for MIR93 in the regulation of autophagy and suggest a combination treatment strategy involving the inhibition of autophagy while administering cytotoxic therapy. Abbreviations: ACTB: actin beta; ATG4B: autophagy related 4B cysteine peptidase; ATG5: autophagy related 5; BECN1: beclin 1; CL: classical; CQ: chloroquine diphosphate; CSCs: cancer stem cells; GBM: glioblastoma; GSCs: glioma stem-like cells; HEK: human embryonic kidney; IB: immunoblotting; IF: immunofluorescent staining; IR: irradiation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MES: mesenchymal; MIR93: microRNA 93; MIRC: a control miRNA; miRNA/miR: microRNA; MTOR: mechanistic target of rapamycin kinase; NSC: NSC185085; PN: proneural; qRT-PCR: quantitative reverse transcription-polymerase chain reaction; Rap: rapamycin; SQSTM1/p62: sequestosome 1; TCGA: the cancer genome atlas; TMZ: temozolomide; WT: wild type; ZIP93: lentiviral miRZIP targeting MIR93; ZIPC: lentiviral miRZip targeting control miRNA.

Objectives: The use of X-ray for therapeutics always raises the problem of radiation hazards to living beings. In this research, we explored the radioprotective activity of glutathione (GSH) on cognitive ability of X-ray radiated tumor-bearing mice., Methods: Forty C57BL/6 mice were chosen to establish the GL261 glioma model and randomly divided into four groups: Model group, X-ray group, Pre-GSH group and Pos-GSH group. Morris water maze test was used to test cognitive ability. Moreover, histopathological observation of hippocampus was observed by hematoxylin and eosin (HE) staining. The protein expression of choline acetyl transferase (ChAT) was measured by western blot, simultaneously the contents of acetylcholinesterase (Ach), superoxide dismutase (SOD), methane dicarboxylic aldehyde (MDA),TNF-α and IL-6 were detected by the respective kit., Results: There was a significant difference in X-ray group of the escape latency from the Model group (P<0.05). Besides, HE staining revealed that nucleus in hippocampus cells were pyknotic, glial cells were hyperplastic and the nerve cells were swelling in X-ray group. In X-ray group the expression of ChAT and Ache were decreased versus Model group. Finally, the cognitive ability in Pre-GSH and Pos-GSH group was enhanced than X-ray group, in which the cognitive ability of Pos-GSH group was higher than the Pre-GSH group., Discussion: X-ray impaired the brain tissues and cognitive ability of tumor-bearing mice. The damages of brain tissues were alleviated by Pre-GSH and Pos-GSH protection and the efficacy of Pos-GSH protection was superior to Pre-GSH protection. Abbreviation Ach: Acetylcholinesterase; GSH: Glutathione; HE: Hematoxylin and eosin; MDA: methane dicarboxylic aldehyde; SOD: Superoxide dismutase; TV: Tumor volume; TW: Tumor weight.

Primary malignant glioma cell lines are being used for initial screening of anticancer agents. We utilized a simple mechanical disaggregation method for deriving cell lines from tumor tissues; and a Coverslip Culture-Acridine Orange Staining method to study cellular damage. Cell lines could be grown for up to three passages within three weeks after surgery. Cell proliferation, total cellular damage, and MTT assay were studied as parameters of cytotoxic response. Frequencies of damaged cells varied in different cell lines; and increased after cytotoxic treatments under clinically relevant conditions. These methods could contribute to preclinical evaluation of treatment response before commencement of radio-chemotherapy.

Radiotherapy is one of the most widely used treatments for therapy of malignant tumors, but resistance to radiation of hypoxic cells in tumor tissues is still a serious concern. Previous studies have demonstrated that silver nanoparticles (AgNPs) enhance the radiosensitivity of human glioma cells in vitro, but the effect of AgNPs on hypoxic glioma cells has not been investigated in detail. The main purpose of this study is to evaluate the radiosensitizing efficacy of AgNPs on hypoxic glioma cells. The half maximal inhibitory concentration (IC50) values of AgNPs for the hypoxic U251 cells and C6 cells were 30.32 μg/mL and 27.53 μg/mL, respectively. The sensitization enhancement ratio (SER) demonstrated that AgNPs exhibit higher capacity in radiosensitization in hypoxic cells (U251: 1.78; C6: 1.84) than that in normoxic cells (U251: 1.34; C6: 1.45). The underlying mechanism of AgNPs' radiosensitization in hypoxic cells is through the promotion of apoptosis and enhanced destructive autophagy. There is evidence of crosstalk between apoptosis and autophagy in AgNPs-radiosensitized hypoxic cells where inhibition of autophagy results in decreased apoptosis. These findings suggest that AgNPs can be used as a highly effective nano-radiosensitizer for the treatment of hypoxic glioma.

It was reported that γ-irradiation had a controversial therapeutic effect on glioma cells. We aimed to investigate the cytotoxic effect on the glioma cells induced by γ-irradiation and explore the treatment to rescue the phenotype alteration of remaining cells. We used transwell assay to detect the glioma cell invasion and migration capacity. Cell proliferation and apoptosis were tested by the CCK-8 assay and flow cytometry respectively. Western Blot was used to detect the activity of Hedgehog signaling pathway and Epithelial-to-Mesenchymal Transition (EMT) status. γ-irradiation showed cytotoxic effect on LN229 cells in vitro, whereas this contribution was limited in U251 cells. However, it could significantly stimulated EMT process in both LN229 and U251. Curcumin (CCM) could rescue EMT process induced by γ-irradiation via the suppression of Gli1 and the upregulation of Sufu. The location and expression of EMT markers were also verified by Immunofluorescence. Immunohistochemistry assay was used on intracranial glioma tissues of nude mice. The capacities of cell migration and invasion were suppressed with combined therapy. This research showed Curcumin could rescue the EMT process induced by γ-irradiation via inhibiting the Hedgehog signaling pathway and potentiate the cell cytotoxic effect in vivo and in vitro.

Purpose: To uncover the role of EZH2 and its opponent ASHL2, a polycomb and trithorax group protein, respectively, on the radioresponsiveness of glioma cell lines., Materials and Methods: Expression of EZH2 and ASHL2 was inhibited by siRNA in glioma cell lines. The effect on histone methylation, gene expression, DNA damage repair signaling, cell cycle checkpoints, apoptosis and tumor control were evaluated., Results: Inhibition of EZH2 (EZH2i) led to a transcriptional dysregulation with upregulation of 544 and downregulation of 445 genes. In comparison, ASH2L inhibition (ASH2Li) had an opposed effect with upregulation of 289 and downregulation of 970 genes. EZH2i and ASH2Li significantly reduced methylation of H3K27 and increased methylation of H3K9, respectively. EZH2i and ASH2Li significantly increased and decreased the number of residual γH2AX foci at 24 h after IR, respectively. The former significantly increased radiation-induced cell cycle arrest in G2/M and apoptotic cell death, while ASH2Li decreased both. In addition, a significant shift of the radioresponse curve by -1.22 + 0.23 Gy (p < 0.0001) in the plaque monolayer assay was found after EZH2i in A7 but not in M059K., Conclusion: Overall, epigenetic modulation is a promising approach to evaluate the role of chromatin structure for the radioresponsiveness of glioma cell lines.

Purpose: Irradiation of brain stimulates the expression of inflammatory mediators, some of which can modify the ability of cancer cells to infiltrate the brain. In the present study, the time window during which this stimulation occurs was determined., Materials and Methods: Brain of Fischer rat was irradiated (15 Gy) and expression of pro-inflammatory mediators IL-1β, IL-6 and TNF-α was measured from 4 h to 20 days post-irradiation. Level of the matrix metalloproteinase 2 (MMP-2) and prostaglandin E2 (PGE2) which can favor cancer cell infiltration were also measured. The F98 glioma cells were implanted either during (4 h post-irradiation) or after (10 days post-irradiation) the pro-inflammatory phase. Infiltration distance of F98 cells in brain parenchyma and the median survival time of the animals were determined., Results: Expression of IL-1β, IL-6 and TNF-α was significantly increased in the irradiated brains with a peak at 4 h post-irradiation. Implantation of F98 glioma cells 4 h post-irradiation reduced the median survival time of Fischer rats to 18 days, compared to 25 days when the F98 were implanted in non-irradiated brain. Irradiation of the brain increased the distance of infiltration of F98 cells and was associated with increased levels of MMP-2 and PGE2. Conversely, F98 cells implanted 10 days post-irradiation have infiltrated the brain over a shorter distance and the median survival time of rats was increased to 35 days., Conclusions: Cancer recurrence is frequently observed in GBM patients. A better understanding of the inflammatory response observed in irradiated brain could contribute to develop new therapeutic modalities to further increase the efficiency of radiotherapy.

Objective: This review article summarizes in vitro, in vivo, and clinical evidence pertaining to temozolomide (TMZ) and bevacizumab (BEV) efficacy and mechanism of action in gliomas., Methods: Relevant publications published before June 2013 in PubMed database were reviewed., Results: Temozolomide and BEV are current chemotherapeutic agents treating patients with high-grade glioma, including glioblastoma. In vitro and in vivo studies have proposed discordant cell death pathways for TMZ as either apoptosis or autophagy using different experimental setting details or cell lines. In addition, BEV may cause cell death through hypoxia-induced autophagy or unspecific indirect effects on cancer cells. The complexity of cancer cells in glioma has contributed to their resistance of both chemotherapies. In clinical trials, overall survival duration in glioma patients with recurrence (8-9 months) is lower than that in newly diagnosed patients (12-15 months)., Conclusion: Our collected data support the addition of radiotherapy, BEV, and other targeted agents to TMZ treatment, indicating prolonged survival duration in newly diagnosed patients. However, the optimal regimen for treating high-grade glioma cannot be concluded without more clinical trials.

Background: The purpose of this study was to assess the efficacy and toxicity of radiotherapy (RT) with concurrent temozolomide (TMZ) chemotherapy followed by adjuvant TMZ in children with diffuse intrinsic pontine glioma (DIPG)., Methods: Patients younger than 18 years with newly diagnosed DIPG were enrolled. Children were treated with focal RT along with concurrent daily TMZ. Four weeks after completing the initial RT-TMZ schedule, adjuvant TMZ was given every 28 days up to 12 cycles or progression disease., Results: Fifteen children with a median age of 9 years were enrolled. Fourteenth out of the 15 patients completed the chemoradiotherapy. The toxicity associated with TMZ was primarily haematopoietic. At a median follow-up of 15 months 13 children had died and 2 children were alive with progressive disease. No patient experienced complete response (CR). The median time to progression was 7.15 months., Conclusion: Chemoradiotherapy with TMZ followed by adjuvant TMZ did not improve the poor prognosis associated with DIPG in children.

Purpose: Synchrotron microbeam radiation therapy (MRT) is a radiosurgery concept in the preclinical stage, developed mainly for brain tumor treatment. Experimental studies suggest that with MRT a better therapeutic index can be obtained than with homogenous field radiotherapy, but the underlying cellular mechanisms need further understanding. The aim of this study was to investigate the dynamics of radiation-induced bystander effects (RIBE) in rats after exposing one brain hemisphere to either MRT or homogenous synchrotron radiation (HSR)., Materials and Methods: Healthy and tumor-bearing Wistar rats were exposed to doses of 17.5, 35, 70 or 350 Gy, applied either as MRT or HSR to the right cerebral hemisphere. Rats were euthanized at 4, 8 and 12 hours after irradiation to assess the release of bystander signals. Brains and urinary bladders were dissected, and explants for bystander clonogenic reporter assays were set up., Results: Clonogenic survival showed that RIBE occurred in both the non-irradiated brain hemisphere and in bladder of normal and tumor-bearing rats, while the irradiated hemisphere showed the direct effects of radiation., Conclusion: The RIBE observed in our reporter cells shows that both MRT and HSR yield a demonstrable abscopal effect after high doses of irradiation; presumably as part of a systemic response.

Purpose: Severe treatment-related lymphopenia (TRL) occurs in 40% of patients with high grade gliomas (HGG) receiving glucocorticoids, temozolomide, and radiation. This occurs following radiation, persists for months, and is associated with reduced survival. As all three treatment modalities are lymphotoxic, this study was conducted to estimate the radiation dose that lymphocytes receive passing through the radiation field and if this could explain the observed TRL., Materials and Methods: A typical glioblastoma plan (8-cm tumor, 60 Gy/30 fractions) was constructed using the Pinnacle™ radiation planning system. Radiation doses to circulating cells (DCC) were analyzed using MatLab™. The primary endpoints were mean DCC and percent of circulating cells receiving ≥0.5 Gy. The model was also used to study how changes in target volumes (PTV), dose rates, and delivery techniques affect DCC., Results: The modeling determined that while a single radiation fraction delivered 0.5 Gy to 5% of circulating cells, after 30 fractions 99% of circulating blood had received ≥0.5 Gy. The mean DCC was 2.2 Gy and was similar for IMRT, 3D-conformal techniques, and different dose rates. Major changes in PTV size affected mean DCC and percent of circulating cells receiving ≥0.5 Gy., Conclusions: Standard treatment plans for brain tumors deliver potentially lymphotoxic radiation doses to the entire circulating blood pool. Altering dose rates or delivery techniques are unlikely to significantly affect DCC by the end of treatment. Novel approaches are needed to limit radiation to circulating lymphocytes given the association of lymphopenia with poorer survival in patients with HGG.

Background: Primary brain tumors have always been associated with high morbidity and mortality. Glioma is the most common type of malignant brain tumors,with a high probability of recurrence after surgical excision and with poor prognosis.The purpose of this study was to compare the therapeutic efficacy of computed tomography (CT)-guided interstitial (125)I seed implantation with traditional radiochemotherapy for treatment of recurrent gliomas., Results: The response rate at 1, 3, 6 and 12 months after (125)I seed implantation was 68.6, 74.3, 77.1 and 62.8% respectively, which was significantly higher than the group treated with the conventional chemoradiation protocol (p < 0.05). Patients exposed to (125)I seed implantation had a median survival of 29.0 months, whereas the median survival of those treated with traditional radiochemotherapy was 19.0 months. The difference observed between the two groups was significant. There were no severe complications or mortality associated with either treatment, except for one case of intracerebral hemorrhage around the tumor area in the (125)I seed implants group., Methods: From November 2002 to May 2010, 73 consecutive patients with recurrent gliomas were treated with CT-guided (125)I seed implantation (35 cases) or traditional radiochemotherapy (38 cases). Patients were followed up after treatment and the therapeutic effect was evaluated by comparing the response and survival rates of the two groups. In particular, patients treated with (125)I seed implantation were monitored for adverse side effects., Conclusions: CT-guided (125)I seed implantation is safe and well-tolerated and more importantly, shows superior efficacy compared with conventional radiochemotherapy. This suggests that CT-guided (125)I seed implantation could be an alternative approach for recurrent gliomas.

Background: Multidisciplinary team (MDT) working in oncology aims to improve outcomes for patients with cancer. One role is to ensure the implementation of best practice and National Institute for Health and Clinical Excellence (NICE) guidance. In this study, we have assessed the role of MDT in implementing the TA121 appraisal of the use of carmustine wafers in high grade gliomas., Methods: 296 patients with high-grade glioma suitable for maximal resection were recruited from 17 Neurosurgical Centres. The number of patients treated with carmustine wafers and reasons for not using this were recorded. Complications at 48 hours post-operatively and at 6 weeks post-radiotherapy were recorded., Results: 94/296 (32%) of suitable patients received carmustine wafers. In 55% of cases carmustine was not used due to either surgeon preference or a lack of an MDT decision. There was no increased complication rate with carmustine use at either 48 hours post-surgery or at 6 weeks post radiotherapy. Use of carmustine wafers did not decrease access to and use of chemoradiotherapy., Conclusions: One third of patients suitable for carmustine wafers received them. Their use was neither associated with more frequent complications, nor decreased use of chemoradiotherapy. Implementation of NICE TA121 Guidance is extremely variable in different MDTs across the United Kingdom.

Purpose: Our aim was to study calcium overload-induced apoptosis and its relation to reactive oxygen species (ROS) in rat C6 glioma cells after sonodynamic treatment (SDT)., Materials and Methods: Hematoporphyrin monomethyl ether (HMME) was used as the sonosensitizer. The concentration of intracellular Ca(2+) ([Ca(2+)](i)) was measured by fluorometry. Apoptosis and necrosis rates were evaluated by a flow cytometry. Moreover, sarcoplasmic reticulum Ca(2+) -ATPase (SERCA(2)), cytochrome c (cyto-c) and cleaved caspase-3 were investigated by immunoblotting., Results: Our study indicated that [Ca(2 +)](i) and ROS increased in cells of SDT group, the apoptosis rate, quantity of cyto-c and cleaved caspase-3 markedly increased after SDT. Furthermore, N-Acetyl-L-cysteine (NAC) or 1,2-bisethane-N,N,N',N'-tetraacetic acid tetrakis ester (BAPTA-AM) could decrease the apoptosis rate, the release of cyto-c and cleaved caspase-3 in SDT group, SERCA(2) degradation was found in SDT group and could also be prevented by the addition of NAC., Conclusions: Our results show that HMME-SDT can induce C6 cell death through both necrosis and apoptosis. ROS in C6 cells play a decisive role in HMME-SDT-induced cell death. The endoplasmic reticulum (ER) may be a major target of HMME-SDT, ROS can induce SERCA(2) degradation, causing the elevation of [Ca(2+)](i).

A human glioblastoma multiforme cell line (U87) and its derived-spheroids were irradiated either using a conventional irradiation (CIR) or a CK-like irradiation (IIR) in which the 8 Gy was delivered intermittently over a period of 40 minutes. The ability of glioma cells to migrate into a matrigel matrix was evaluated on days 1-8 from irradiation. Irradiation with CK-driven IIR significantly increased the invasion potential of U87 cells in a matrigel-based assay. In contrast to CIR, IIR was associated with increased levels of TGF-β at four days (Real time PCR), β1-integrin at 4-5 days (real-time PCR and western blot) and no elevation in phosphorylated AKT at days 4 and 5 (western blot). Our data suggests that glioma cell invasion as well as elevations of TGF-β and β1-integrin are associated with IIR and not CIR.

Purpose: To investigate the possible cytotoxic interactions between the chemotherapeutic drug temozolomide (TMZ) and the cyclooxygenase-2 inhibitor meloxicam (MLC) or of both drugs combined with X-rays in three human glioma cell lines (D384, Hs 683 and U251)., Materials and Methods: Cells were exposed to TMZ (96 hours) and MLC was co-incubated during the last 24 h. Thereafter, cells were irradiated with X-rays and plated for a clonogenic assay. Total cell numbers and the numbers of surviving cells were determined to study the recovery of the cell populations (up until 19 days) following different combinations of TMZ, MLC and X-rays., Results: The combination of MLC and TMZ caused an enhanced cytotoxic effect in D384 and Hs 683. Various treatment combinations demonstrated significant radiation enhancement in all three cell lines. Long-term observations of D384 cells demonstrated that the repopulation rates of the surviving cells are far less affected by the various treatment protocols than those from the non-surviving cells., Conclusions: The present study demonstrates that a combination of TMZ and MLC resulted in a significant potentiation of their cytotoxicity in D384 and Hs683. The combination of these two drugs can also cause considerable enhancement of the radiation response in human glioma cell lines, although only D384 cells benefit from trimodal over bimodal treatment.

Temozolomide-based chemotherapy represents an incremental improvement in the treatment of patients with high-grade gliomas. Notwithstanding a survival benefit in a subset of patients with high-grade gliomas, temozolomide (TMZ; Temodar®, Schering-Plough Pharmaceuticals, NJ, USA) is the primarily palliative treatment for the vast majority of patients. Indeed, for patients with newly diagnosed glioblastoma, the median increase in survival for treatment with TMZ and radiotherapy is only 2.5 months compared with radiotherapy alone. Additionally, recent studies suggest that 60-75% of patients with glioblastoma derive no benefit from treatment with TMZ. For the treatment of recurrent anaplastic gliomas, more than 50% of patients fail TMZ treatment with cancer progression at 6 months, demonstrating that TMZ is only a modestly effective chemotherapy. In addition, 15-20% of patients treated with TMZ develop clinically significant toxicity, which can leave further treatment unsafe. Despite the availability of TMZ, there is still a substantial need for a chemotherapeutic agent that is more effective and safe. In fact, there still remains a significant unmet need for more effective treatments of high-grade gliomas (improved palliation or cure), whether that treatment be by surgery, radiotherapy, chemotherapy or any yet to be developed type of treatment, such as 'targeted therapies'.

Presently, cancer response is measured by imaging assessment of tumor volumes or by repeated biopsy to analyze pharmacodynamics. These methods of monitoring cancer response are inefficient because volume changes typically require therapy for prolonged time intervals and neoplasms within the brain are less amenable to sequential biopsies. Peptide ligands selected from phage-displayed peptide libraries can rapidly differentiate responding from resistant gliomas. These peptides, in turn, can be labeled with internal emitters to provide a means of noninvasive assessment of glioma susceptibility to radiotherapy within 24 h of therapy. This is platform technology and could allow for ineffective therapy to be modified or switched so that patients are not subjected to a delayed reassessment (2 months) of response to therapy.

Malignant gliomas are the commonest primary brain tumours in adults and harbour a dismal prognosis. So far, established parameters with predictive significance for prognosis do not include the neurological functional assessment of patients. The aim of this study was to investigate the prognostic value of parameters assessing neurological status in malignant glioma patients for outcome and survival. Evaluation of neurological status included the Sawaya Functional Grade (SFG) and the Neurological Performance Scale (NPS). Moreover, the Karnofsky Performance Status (KPS), medical risk factors, extension of resection, postoperative complications, and the predictive value of these variables for outcome and survival were studied. For this purpose, we have analysed 110 consecutive patients with malignant gliomas who were treated at our institution. As expected, age (p <0.001), KPS (p = 0.003) and extent of resection (p <0.001) were good predictors of survival. Strikingly, SFG and NPS showed the highest significant correlation with survival (both p <0.0001) and, furthermore, they were the only predictors of the relapse-free time (p = 0.035 and p = 0.004, respectively). Our data strongly suggests that SFG and NPS accurately predict both survival and relapse-free time, which may add a valuable tool for tailoring individual therapy strategies.

Radioimmunotherapy (RIT) may improve the management of malignant gliomas. A Phase I clinical trial was performed to evaluate, for the first time, the toxicity and clinical effect of an intracavitary administration of a single dose of Nimotuzumab (h-R3) labeled wit (188)Re. Nimotuzumab is a humanized monoclonal antibody directed against epidermal growth factor receptors. Three patients with anaplastic astrocytoma (AA) and 8 with glioblastoma multiforme (GBM) were intended to be treated with 3 mg of mAb labelled with 10 or 15 mCi of (188)Re. In patients treated with 10 mCi (n=6) transitory worsening of pre-existing neurological symptoms were observed. Two patients treated with 15 mCi (n=4) developed early severe neurological symptoms and one also developed late severe toxicity (radionecrosis). In the group treated with 10 mCi, 1 GBM patient died in progression 6 months after the treatment, 2 patients (1 GBM and 1 AA) developed stable disease during 3 months. One GBM patient had partial response for more than 1 year and 2 patients (1 GBM and 1 AA) were asymptomatic and in complete response after 3 years of treatment. Maximal tolerated dose of the radioimmunoconjugate (188)Re-Nimotuzumab was 3 mg of the h-R3 labelled with 10 mCi of (188)Re. The radioimmunoconjugate showed a high retention in the surgical created resection cavity and the brain adjacent tissues with a mean value of 85.5 % of the injected dose one hour post-administration. This radioimmunoconjugate may be relatively safe and a promising therapeutic approach for treating high grade gliomas.

High-grade gliomas are the most commonly diagnosed malignant brain tumor in adults. Prognosis can be estimated by examining risk factors, including histology, age and performance status. Postoperative radiation therapy is associated with improved survival and standard treatment includes external beam radiotherapy to a dose of 60 Gy in 30-33 fractions. Patients with poor prognostic features have a more limited benefit from radiotherapy. This article reviews the current state of knowledge on risk stratification and analyzes strategies that can be employed to better individualize treatment for poor-prognosis patients.

Recently completed trials suggest the addition of nitrosourea-based chemotherapy to radiotherapy increases the progression-free but not overall survival of grade II and III gliomas. Temozolomide has proven benefit in grade II/III gliomas progressive following standard therapy and when added to radiation for glioblastoma. Newly launched and planned phase III trials will explore whether the addition of temozolomide to radiotherapy improves overall survival in grade II/III as well as the prognostic and predictive value of 1p/19q analyses and MGMT promotor methylation status. Additionally, they will measure cognition and quality of life to determine if improvements in time to progression translate into better functional status and patient satisfaction.

Purpose: The influence of X-ray and (12)C heavy ion irradiation on tumor cell migration and of beta(3) and beta(1) integrin expression was investigated., Material and Methods: Two different tumor cell lines (U87 glioma and HCT116 colon carcinoma cells) were irradiated with 1, 3, or 10 Gy X-rays or (12)C heavy ions. 24 h after irradiation a standardized Boyden Chamber assay for migration analysis was performed and cells were lysed for Western blotting., Results: Radiation-induced influences were cell line- and radiation type-dependent. X-rays decreased HCT116 migration at higher doses and appear to increase U87 migration after 3 Gy. Heavy ions decreased migration of both cell lines dose-dependently. A trend of increased beta(3) and beta(1) integrin expression in U87 cells after both radiation types was observed. beta(1) integrin expression in HCT116 cells was increased after X-rays but decreased after heavy ion irradiation., Conclusions: Results suggest that irradiation of tumor cells can modulate their migratory behavior. An increased migration, as shown with U87, leaves a higher probability of metastatic induction after irradiation of solid tumors in vivo, whereas an invariably reduced tumor cell migration, as shown after heavy ion treatment, could diminish the hazard of radiation-induced metastasis. As integrin expression and migration were only partially correlated, other migration-related surface molecules may be more relevant for radiation effects on tumor cell motility.

Malignant gliomas represent an heterogeneous group of brain tumors both in terms of natural history and response to treatment. The standard therapeutic approach for treating glioblastomas is a combination of radiotherapy and concomitant/adjuvant temozolomide, and methylguanine-DNA methyltransferase promoter methylation is now recognized as an important factor for predicting both prognosis and response to alkylating agents. In the future, the discovery of targeted therapies will increasingly allow personalized medical treatments. Anaplastic oligodendroglial tumors display a better prognosis and are more chemosensitive than glioblastomas; the discovery of molecular factors of prognostic significance, such as 1p/19q codeletion, will lead to different treatment strategies for different subgroups of patients. Gliomatosis cerebri is a rare diffuse glioma, and upfront chemotherapy is increasingly being employed instead of whole-brain radiotherapy to avoid/delay cognitive defects in long surviving patients, despite the lack of data to support this.

Adults with primary malignant glioma have an unacceptably poor outcome. Most of these tumors recur at or adjacent to the site of origin, which indicates that failure to eradicate local tumor growth is a major factor contributing to poor outcome. Therefore, locoregional therapies may improve local control and overall outcome for malignant glioma patients. Malignant gliomas selectively express several factors that are not present on normal CNS tissue. Regional administration of radiolabeled monoclonal antibodies targeting tumor-specific antigens expressed by malignant gliomas offers an innovative therapeutic strategy that has recently demonstrated encouraging antitumor activity and acceptable toxicity in clinical trials at a number of centers. Most studies have utilized monoclonal antibodies against tenascin-C, an extracellular matrix glycoprotein ubiquitously expressed by malignant gliomas. This review summarizes clinical trials performed using radiolabeled antitenascin-C monoclonal antibodies for malignant glioma patients to date and highlights future plans to further develop this therapeutic strategy.

The identification of molecular markers associated with tumor but not with normal tissue has allowed the development of highly specific, targeted therapies for the treatment of cancer. Over the last several years, tremendous advances in our understanding of the genetic and molecular changes involved in the progression of malignant gliomas have triggered a large effort in the development of targeted therapies to treat these tumors. However, to date only a modest clinical benefit, limited to subsets of patients, has been demonstrated. Furthermore, despite a high degree of target selectivity, the use of targeted therapies often has systemic toxicity. The reasons behind this limited clinical success are complex and include the intricacy of the signaling pathways in gliomas and the heterogeneity of the disease process, compounded by existing limitations in assessing the efficacy of these novel agents when conventional end points and clinical trial designs are utilized. However, despite these difficulties targeted therapies remain a very attractive avenue of treatment for malignant gliomas. Three basic approaches are needed to overcome the hurdles associated with targeted therapies: first, further development of genetic profiling techniques will help to better determine the genetic changes and molecular pathways involved in gliomas and will potentially allow the design of individualized therapies based on the genetic and molecular signature of each tumor. Second, there is a need for the development of better combination strategies (complementary targeted agents or targeted agents with chemotherapy drugs) directed towards disease heterogeneity. Third, we need to optimize the design of preclinical and clinical trials to obtain the maximum amount of information in the shortest period of time.

Objective: To study histopathologic changes and the role of the microglia/macrophage cell in the therapeutic effect of I-125 interstitial brachytherapy on the cerebral gliomas., Methods: Out of a series of 60 cases with cerebral astrocytomas and other brain tumors treated with I-125 interstitial brachytherapy, autopsy materials were available in ten cases 0.75 and 60 months after irradiation. The patients were treated with the maximum dosage (60 Gy) on the tumor periphery. Besides the routine hematoxylin-eosine and Mallory's PTAH trichrome staining, immunohistochemical reactions were carried out for CD15, CD31, CD34, CD45, CD68, CPM, HAM56 and HLR-DR antigens on paraffin sections to study immunologic phenotypic characteristics of the reaction cell population around gliomas after I-125 treatment., Result: One month after irradiation, a necrotic zone developed around the I-125 seeds within the 72 Gy isodose curve. Histologically, there was a fresh coagulation necrosis in the center of the lesion. Reactive zone has not yet developed but scattered interstitial and perivascular CD68 positive macrophages were present in the surrounding brain tissues. Six months after the I-125 isotope treatment, a reactive zone developed: a microglial rim around the necrosis tissue, and a broad area of proliferating vessels and glial fibrillary acidic protein (GFAP) positive astroglial cells which contained CD68 positive activated microglial and macrophage cells. Fifty-four months after I-125 interstitial irradiation, the necrotic center became colliquative and cystic. The microglial rim was replaced by round end stage (HLR-DR and CD31 positive) macrophages. The reactive zone was characterized by astrocytic gliosis but vascular proliferation and macrophages were lacking., Conclusion: Results of the present immunohistochemical study suggest that the early lesions are characterized by migrating macrophages apparently concerned with the removal of necrotic debris. The established phase of reactive zone around the necrotic center is characterized by a narrow inner rim of microglial accumulation and a broad outer area characterized by astrocytic gliosis, vascular proliferation, activated microglia and infiltration by macrophages. In the burned-out phases of I-125 interstitial brachytherapy of gliomas, the necrosis undergoes liquefaction and the microglial rim is replaced by astrocytic gliosis which can be considered as equivalent to the scar tissue formed around necrosis outside the central nervous system.

In vitro studies using monolayer cultures of human tumor cell lines have shown that 2-DG selectively inhibits energy-dependent DNA repair and cellular recovery processes in cancer cells. However, monolayer cultures differ greatly from the complex environmental conditions generated in solid tumors that develop inhomogeneous hypoxic and necrotic regions. In contrast, multicellular spheroids mimic heterogeneous cellular behavior and the consequent functional characteristics of in vivo solid tumors, and serve as important in vitro model to investigate tumor biology and responses to potential therapeutic agents. The present study compares the radiomodification by 2-DG in monolayer cultures and spheroids of a human glioma cell line (BMG-1) to gain insight into the effects in solid tumors. In spheroids, the glucose consumption (2.1 p mole/cell/h) and lactate production (3.67 p mole/cell/h) was nearly 2-3 fold higher than in monolayer cells (0.83 and 1.43 p mole/cell/h respectively). Presence of 2-DG (5 mM) for 2-4 h inhibited the glucose usage and lactate production by 70% in spheroids, while a 35% reduction was observed in monolayer cells. Under these conditions, 2-DG drastically enhanced the radiation-induced cell death of spheroids (by 2-3 folds); while a 40% increase was observed in monolayer cells. Radiosensitization by 2-DG in monolayer cells was primarily due to an increase in mitotic death (23%) linked to cytogenetic damage (micronuclei), whereas a profound induction of apoptosis (40%) accounted for the sensitization in spheroids. Although the Bcl-2 and Bax levels were significantly higher in spheroids, Bcl-2/Bax ratio was similar in monolayers and spheroids. Comet assay revealed a late onset of DNA breaks in the presence of 2- DG following irradiation only in spheroids, which corroborated well with the late onset of oxidative stress. 2-DG did not induce a significant cell cycle delay in monolayers, while a transient G(2) delay was apparent in spheroids.

The aim of this study was to examine the cytotoxicity and mechanism of apoptosis induction of verotoxin-1 (VT-1) in human glioma cell lines. VT-1 is a member of the shiga-toxin family expressed by some serotypes of Escherichia coli and Shigella dysenteriae. Shiga-toxins have been shown to induce apoptosis by binding to its membrane receptor Gb3. The human glioma cell lines SF-767, U-343 MG, and U-251 MG were studied together with BT4C, a rat glioma cell line. Cells were first screened for Gb3 expression by flow cytometry. Fluorescein diacetate was used to determine cell viability after VT-1 and irradiation exposure and apoptosis was studied by TUNEL staining, a mitochondrial membrane potential assay, and caspase activity assays. SF-767 and U-343 MG cells were found to express Gb3 and were also sensitive to VT-1-induced cytotoxicity, whereas nonGb3-expressing U-251 MG and BT4C glioma cells were not. VT-1 depolarized the mitochondrial membrane and activated caspase-9 and -3 of SF-767 and U-343 MG cells. VT-1 exposure for 72 h resulted in approx. 60 and 90% TUNEL-stained cells, respectively. D, L-Threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) an inhibitor of glucosylceramide synthesis was used to block Gb3 synthesis. Two mumol/L PPMP for 72 h abolished SF-767 and U-343 MG expression of Gb3 and made the cells completely resistant to VT-1 induced apoptosis. Key components of MAP kinase signalling pathways that control BAX and mitochondrial function were investigated. VT-1 induced JNK phosphorylation in both cell lines, suggesting that survival signal pathways were overruled by VT-1-induced JNK activation leading to mitochondrial depolarization, caspase-9 activation and apoptosis. Immunohistochemistry of cryostat section from glioma biopsies demonstrated expression of Gb3 was in the vascular endothelial cells as well as tumor cells, but not in astrocytes. The high specificity and apoptosis inducing properties of verotoxin-1 indicates that the toxin may be a potential anti-neoplastic agent for Gb3-expressing gliomas.

The glycolytic inhibitor 2-deoxy-D-glucose (2-DG) has been shown to enhance the cell death induced by radiation and other DNA damaging agents selectively in cells with high rates of glycolysis, like cancer cells. While energy linked modification of DNA and cellular repair processes have been suggested as possible mechanisms of sensitization, other effects such as global stress response cannot be excluded. In this pilot study, we have investigated the effect of 2-DG and radiation on the transcriptome in an attempt to elucidate how 2-DG impacts gene expression in undamaged verses irradiation (IR) damaged cells using a human malignant glioma cell line, U-87. Exponentially growing U-87 cells were exposed to various combinations of 2-DG and X-rays and total RNA was isolated four hours after exposure. Gene expression changes were elucidated using Affymetrix GeneChips. As expected, U-87 cells treated with 2-DG showed activation of several endoplasmic reticulum stress response genes. Selective RT-PCR and Western blotting confirmed these gene alterations. Given that glucose deprivation leads to p53 activation and 2-DG led to activation of p53 response genes in our present study (e.g., PMAIP1 and GADD45A), we examined the impact of transient p53 knockdown and observed that induction of PMAIP1 and GADD45A appear to be via p53-independent mechanisms. The majority of gene alterations seen with IR-treatment alone were consistent with previous reports. While most gene alterations seen with 2-DG and IR dual treatment were confirmed in the gene profiles seen with individual (2-DG or IR) treatments, several genes appeared differentially regulated between IR and 2-DG (e.g., DUSP8, IL8, GADD45B). Additionally, gene expression patterns suggested alterations in cell cycle regulation, apoptosis, and cytokine signaling pathways. Taken together, this study provides new insights into how the transcriptome of tumor cells are likely to be affected by a combined stress caused by IR and 2-DG.

Malignant gliomas are one of the most difficult tumors to treat, with only modest advances being made in the past few decades. Surgery and radiation have had the greatest impact, increasing survival. Chemotherapy modestly increases survival. The use of chemotherapy in the treatment of malignant gliomas is the focus of this paper and the more commonly used agents at diagnosis and relapse are reviewed. Since most patients fail first-, second- and even third-line agents that are commercially available, some of the more relevant new biological compounds will also be discussed. As treatments for brain tumors evolve, it is likely that optimal therapies will come from combination therapies that incorporate target-specific and chemotherapeutic agents.

Response of a solid tumor to radiation treatment depends, in part, on the intrinsic radiosensitivity of tumor cells, the proliferation rate of tumor cells between radiation treatments and the hypoxic state of the tumor cells. A successful radiosensitizing agent would target S-phase cells and hypoxia. Recently, we demonstrated the anti-tumor effects of flavopiridol in the GL261 murine glioma model might involve 1) recruitment of tumor cells to S-phase (Newcomb et al Cell Cycle 2004; 3:230-234) and 2) an anti-angiogenic effect on the tumor vasculature by downregulation of hypoxia-inducible factor -1alpha (HIF-1alpha) (Newcomb et al Neuro-Oncology 2005; 7:225-235). Given that flavopiridol has demonstrated radiosensitizing activity in several murine tumor models, we tested whether it would enhance the response of GL261 tumors to radiation. In the present study, we evaluated the intrinsic radiation sensitivity of the GL261 glioma model using the tumor control/cure dose of radiation assay (TCD(50)). We found that a single dose of 65 Gy (CI 57.1-73.1) was required to cure 50% of the tumors locally. Using the tumor growth delay assay, fractionated radiation (5 fractions of 5 Gy over 10 days) combined with flavopiridol (5 mg/kg) given three times weekly for 3 cycles produced a significant growth delay. Our results indicate that the GL261 murine glioma model mimics the radioresistance encountered in human gliomas, and thus should prove useful in identifying promising new investigational radiosensitizers for use in the treatment of glioma patients.

Progress in the biological and molecular characterization of gliomas and studies of factors associated with tumor growth and progression have led to translational research projects and the development of rational new approaches regarding prognostic models, better prediction of response to treatment and innovative therapeutic strategies. This review summarizes the available data on established and emerging prognostic factors and prognostic scores, and discusses their limitations as well as their potential influence on future therapeutic efforts. Recent developments in standard treatment options (i.e., surgery, radiotherapy and chemotherapy) are reviewed. Experimental data indicate that inhibition of several signaling pathways (e.g., epidermal growth factor, transforming growth factor-beta and phosphatidylinositol 3 kinase) may represent a promising therapeutic strategy. Some inhibitory agents (i.e., drugs, antibodies and antisense oligonucleotides) have now entered clinical trials, mainly for recurrent gliomas and a small number are being tested in combination with radiotherapy. Early results of such approaches are presented.

Randomized trials have demonstrated Gliadel improves survival for appropriately selected patients with newly diagnosed malignant glioma. As only limited information is available to guide the management of patients who have Gliadel controlled-release BCNU wafers implanted in the cranial resection cavity prior to radiotherapy (RT), this retrospective review was conducted to describe clinical course, toxicity, and pathologic findings after this therapy for newly diagnosed malignant glioma. Forty-six consecutive patients receiving Gliadel (3.8% BCNU impregnated wafers) followed by radiotherapy for newly diagnosed malignant glioma at Johns Hopkins Hospital from 1990 to August 1999 were identified, although one was lost to follow up and is excluded. Patients were evaluated for postoperative infection, pathology at reoperation, and survival. Twenty-eight patients received radiotherapy at Johns Hopkins and these patients are also evaluable for toxicity experienced during and one month after completion of RT. The median age of all patients is 57 years. Eighty-nine percent had glioblastoma, and median follow-up of surviving glioblastoma patients is 16.8 (12-20) months. Postoperative infection or need for reoperation within 30 days was uncommon after Gliadel placement. Full-dose radiotherapy was tolerable after Gliadel implantation. Five patients (19%) developed neurologic symptoms during radiotherapy responding to increased steroids and/or anticonvulsants, whereas an additional 8 of 27 (30%) developed neurologic symptoms during dexamethasone taper that responded to increases in dexamethasone dose. At one month after RT, 58% of patients were still on dexamethasone despite attempted taper. Fifteen of 45 patients, 33% underwent reoperation or biopsy for a new local contrast-enhancing lesion. In five of 15 (33%) the reoperation revealed necrosis or treatment effect without active tumor. Two of five patients with treatment/effect necrosis has a third surgery 2.9 and 3.2 months after the initial reoperation, and treatment effect/necrosis without tumor was demonstrated in both cases. The Kaplan-Meier median survival for all the glioblastoma patients is 12.8 (95% CI 9.6, 15.9) months. For glioblastoma patients under 55 years old, median survival is 15.9 (95% CI 13.5, too few events) months whereas for older patients it is 9.6 (7.7, 14.4) months. We conclude that Gliadel followed by full-dose standard radiotherapy is acutely well tolerated, although, close supervision should be emphasized during dexamethasone taper. Median survival in excess of one year suggests that there are not complications that result in overall premature death. The finding of necrosis/treatment effect was noted in five of 45 (11%) of all patients and five of 15 (33%) of those undergoing reoperation. Therefore, the possibility of necrosis/treatment effect should be considered for each patient with radiographic findings suspicious for local recurrence.

According to epidemiological estimations, the elderly are going to constitute an increasing proportion of patients with gliomas in the near future. Predominantly glioblastoma histology with invariably fatal outcome, disabling comorbidities and presumed low tolerability of radiochemotherapeutic treatments are the main reasons why elderly patients have been under-represented in the majority of neuro-oncological clinical trials conducted so far. Some small retrospective studies have reported that patients with good performance status receiving surgery plus radiotherapy, and sometimes chemotherapy, may achieve a survival comparable with that of younger patients, however, in the absence of randomized studies, the balance of benefits and adverse effects of aggressive treatments remains controversial. Multidisciplinary evaluation of prognostic factors, such as performance status, cognitive functions, tumor operability and burden of comorbidities, appears to be mandatory in order to choose which patients must not be deprived of an integrated treatment with surgery, full-dose radiotherapy and chemotherapy, and which patients may reasonably be given a shorter radiotherapy plan, or even no treatment at all due to the rapidly fatal course of their disease. Peculiar features of malignant gliomas in the elderly and some practical recommendations of management will be presented and discussed in this review.

Purpose: To investigate and compare the ability of DNA-dependent protein kinase (DNA-PK)-deficient and -proficient cells to undergo apoptosis after exposure to low and high linear energy transfer (LET) radiation., Materials and Methods: A human glioma cell line M059J lacking the catalytic subunit of DNA-PK (DNA-PKcs) and its DNA-PKcs-proficient counterpart, M059K, were exposed to 1 and 4 Gy of accelerated nitrogen ions (14N, 140 eV nm(-1), 8-12 Gy min(-1)) or 60Co gamma-rays (0.2 eV nm(-1), 0.7 Gy min(-1)). The induction of apoptosis was studied up to 144 h post-irradiation using two different methods: morphological characterization of apoptotic cells after fluorescent staining and cell size distribution analysis to detect apoptotic bodies. In parallel, protein expression of DNA-PKcs and poly(ADP-ribose) polymerase (PARP) as well as DNA-PK and caspase-3 activity were investigated., Results: Low and high LET radiations (4 Gy) induced a time-dependent apoptotic response in both cell lines. Low LET radiation induced a significantly elevated apoptotic response in M059J as compared with M059K cells at 144 h post-irradiation. Following high LET radiation exposure, there was no difference between the cell lines at this time. PARP cleavage was detected in M059J cells following both low and high LET irradiation, while only high LET radiation induced PARP cleavage in M059K cells. These cleavages occurred in the absence of caspase-3 activation., Conclusions: M059J and M059K cells both display radiation-induced apoptosis, which occur independently of caspase-3 activation. The apoptotic course differs between the two cell lines and is dependent on the quality of radiation.

Despite therapeutic interventions including surgery, chemotherapy and radiotherapy, glioblastoma multiforme (GBM) has a very poor prognosis and novel therapies are required. MDA-7 (IL-24), when expressed via a recombinant replication defective adenovirus, Ad.mda-7, has profound anti-proliferative and cytotoxic effects in a variety of tumor cells, but not in non-transformed cells. The present studies examined the combined impact of Ad.mda-7 and ionizing radiation on the proliferation and survival of GBM cells. Ad.mda-7 reduced the proliferation of rodent and human glioma cells in MTT assays and in colony formation assays. The anti-proliferative effects of Admda-7 were enhanced by radiation in a greater than additive fashion. In vitro, this cellular change correlated with enhanced cell numbers in G1/G0 and G2/M phases of the cell cycle, implying Ad.mda-7 radiosensitizes tumor cells in a cell cycle-independent manner. The radiosensitizing effects were not observed in cultures of non-transformed primary astrocytes. The enhanced reduction in growth correlated with increased necrosis and DNA degradation. Ad.mda-7 enhanced p38 and ERK1/2 activity but did not alter JNK or Akt activity. Irradiation of cells expressing MDA-7 suppressed ERK1/2 activity and dramatically enhanced JNK1/2 activity without altering either Akt or p38 activity. Inhibition of JNK1/2, but not p38, signaling abolished the radiosensitizing properties of MDA-7. Inhibition of neither ERK1/2 nor PI3K signaling enhanced the anti-proliferative effects of Ad.mda-7, whereas combined inhibition of both pathways enhanced cell killing, suggesting that ERK and PI3K signaling can be protective against MDA-7 lethality.

Purpose: Low-dose hyperradiosensitivity (HRS) has been demonstrated in numerous cell lines in vitro, including a number of radioresistant human malignant glioma cell lines such as A7. The aim of our experiment was to show whether HRS can be exploited by using ultrafractionated irradiation (UF) to improve local control of A7 tumours growing in nude mice. Extrapolation of the in vitro results predict a 3.7-fold difference in the efficacy of UF compared with conventional fractionation (CF)., Material and Methods: Subcutaneuously growing A7 tumours were irradiated either with UF (126 fractions in 6 weeks, 0.4 Gy per fraction) or CF (30 fractions in 6 weeks, 1.68 Gy per fraction). The total dose was 50.4 Gy in both experimental arms. Fractionated irradiations were given under ambient conditions and followed by graded top-up doses under clamp hypoxia. Endpoints were tumour growth delay and local tumour control 180 days after the end of treatment., Results: UF resulted in a significant decrease of tumour growth delay and in a significant increase of the top-up TCD(50) compared with CF (40.0 Gy [95% CI 29; 61 Gy] versus 28.3 Gy [24; 35 Gy], p=0.047)., Conclusions: Despite a pronounced HRS phenomenon in vitro, UF was significantly less effective than CF in A7 human malignant glioma in nude mice. These results neither disprove the existence of HRS nor do they exclude a possible clinical value of UF. The findings rather indicate that simplistic extrapolation from results obtained after single-dose exposure or few fractions in vitro is not sufficient to predict outcome of UF in vivo and that comprehensive evaluation of novel treatment options in animal models continues to be an essential requirement for clinical translation.

Purpose: Human gliomas are known to be radioresistant and the aim was to determine if this resistance in part could be due to an adaptive response., Materials and Methods: Human U-87MG glioma cells were used. Three different radiation regimens that could be related to clinical treatments were tested for their ability to cause an adaptive response. Cell survival and DNA double-strand breakage were the measured endpoints., Results: All three regimens caused an adaptive response in terms of cell survival when given priming doses of radiation. The DNA double-strand break endpoint also showed fewer breaks when the adaptive response occurred., Conclusions: Using irradiation regimens that closely resembled clinical applications, in vitro data are presented that show an adaptive response in human glioma cells. This effect in part could be responsible for the radioresistance of human gliomas.

Purpose: Following acute irradiation, excess radiosensitivity is generally seen at doses <1 Gy, a phenomenon termed "low-dose hyper-radiosensitivity" (HRS). A very strong, HRS-like inverse dose-rate effect has also been described following continuous low dose-rate (LDR) irradiation at <30 cGy h(-1). We report on the sequential irradiation of a cell line by such LDR exposures followed by low acute doses, where either treatment individually would elicit a hypersensitive response. The aim was to determine if a prior LDR exposure would remove the HRS normally seen in response to very small acute radiation doses., Materials and Methods: T98G human glioma cells were given single continuous LDR exposures of 5-60 cGy h(-1) using a (60)Co gamma-source. At intervals of 0 or 4 h following LDR irradiation, cells were further irradiated with a range of acute doses using 240-kVp X-rays. The response to the combined treatment was assessed using high-precision clonogenic cell survival assays, and the amount of HRS at acute doses <1 Gy was determined., Results: LDR at > or = 60 cGy h(-1) to total doses up to 5 Gy in asynchronously growing cells did not remove HRS in the subsequent acute-dose survival curve. In confluent cultures, subsequent acute-dose HRS was not present after an LDR dose of 5 Gy at either 60 or 30 cGy h(-1), but returned if a 4-h interval was left between LDR and acute-dose irradiation. In confluent cultures, acute-dose HRS remained for LDR treatments at 5 or 10 cGy h(-1) or if the total dose was 2 Gy. Taking all cultures and dose-rates together, the "degree" of acute-dose HRS, as measured by alpha(s), was significantly greater in cells irradiated at LDR to a total dose of 2 than of 5Gy., Conclusions: Initial LDR exposure can affect a subsequent HRS response. HRS is reduced after LDR exposures at greater dose intensity, but can recover again within 4 h of completion of LDR exposure. This suggests that processes determining increased resistance to small acute doses (removal of HRS) might be governed by the level of repairable DNA lesions.

Treatment of low-grade gliomas is one of the most challenging management dilemmas in neuro-oncology. Young age of onset and low rate of growth theoretically favor minimally invasive treatments. Yet, local recurrence and conversion to malignant glioma are the expected outcome within 4-8 years from diagnosis and impose the use of additional therapies, such as radiotherapy and chemotherapy. Due to low incidence and paucity of randomized trials, the correct timing and dosage of radiotherapy are still controversial, as well as the indications and possible benefits of the administration of cytotoxic drugs. Current concepts and future perspectives in the treatment of low-grade gliomas drawn from recent scientific literature are here summarized and discussed.

Many clinical trials have shown that the most important prognostic variable in patients with malignant glioma is advanced age. However, can some patients aged >60 years still have relatively good outcomes with conventional surgical and radiotherapeutic treatment? A previous audit of practice (1983-89) suggested that functional status was an important prognostic variable in the elderly. We have reviewed a further cohort (1989-96) to evaluate changes in practice and outcomes given advances in neuroimaging, neurosurgery and radiotherapy. The major findings in this series of 80 patients aged over 60 years with a histological diagnosis of supratentorial malignant glioma were: (i) There was a relationship between management undertaken and clinical status of the patients (p < 0.01), i.e. patients in good grade generally had tumour debulking and radiotherapy, whilst those in poor grade generally had only biopsy. (ii) There was a significant increase in survival of patients in the second period who received surgical debulking and post-operative radiotherapy (from a median of 23 to 41 weeks (p < 0.05). (iii) It is likely that case selection accounted for much of this improvement since there was a direct relationship between median survival time and good clinical grade using the WHO performance scale. (iv) A shorter radiotherapy course (30 Gy in six fractions) was as efficacious as a conventional course (60 Gy in 30 fractions), and those patients having radiotherapy survived significantly longer than those not having this treatment (p = 0.001). This study has again demonstrated the importance of preoperative clinical grade and radiotherapy treatment in determining outcomes in patients >60 years. To put these data in a societal context a recent prospective multicentre audit of patients with malignant glioma in Scotland, and another audit from our unit, showed that between 24 and 65% of patients aged >60 years, with a CT diagnosis of malignant glioma do not undergo either surgery or radiotherapy. Advanced age per se should not be a bar to interventional treatment in patients aged >60 years with suspected malignant glioma.