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SummaryGlioblastomas (GBMs) are aggressive primary malignant brain tumors characterized by extensive levels of inter- and intra-tumor genetic and phenotypic heterogeneity. Patient-derived organoids (PDOs) have recently emerged as useful models to study such heterogeneity. Here, we present bulk exome as well as single-cell genome and transcriptome profiles of primary IDH wild type GBMs from ten patients, including two recurrent tumors, as well as PDOs and brain tumor-initiating cell (BTIC) lines derived from these patients. We find that PDOs are genetically similar to and variably retain gene expression characteristics of their parent tumors. At the phenotypic level, PDOs appear to exhibit similar levels of transcriptional heterogeneity as their parent tumors, whereas BTIC lines tend to be enriched for cells in a more uniform transcriptional state. The datasets introduced here will provide a valuable resource to help guide experiments using GBM-derived organoids, especially in the context of studying cellular heterogeneity.

Despite the discovery and widespread use of the chemotherapeutic drug temozolomide (TMZ), glioblastoma (GBM) remains a fatal cancer. TMZ, a DNA alkylating agent, provides a moderate survival benefit to patients whose tumours do not express the O6-methylguanine-methyltransferase (MGMT) gene. However, even these TMZ-sensitive GBMs recur and upon doing so, many are resistant to TMZ. The development of TMZ resistance is commonly associated with mutations in mismatch repair (MMR) and the re-expression of MGMT. Upregulation of homologous recombination (HR) and base excision repair (BER) have also been implicated as mechanisms of acquired TMZ resistance. To better characterize these mechanisms and to develop strategies to prevent or overcome resistance, our laboratory has implemented an in vitro model of inducible resistance in which frequent exposure to TMZ (100µM) yields multiple resistant colonies in the MGMT-methylated GBM cell line U251N (Yip et. al). These colonies displayed varying methods of resistance to TMZ, including those that have been clinically observed in recurrent, TMZ-treated GBMs. Several colonies harboured mutations in MMR genes MSH6, MSH2, and MLH1 with low or absent expression of their respective proteins. In addition, some MMR wild-type colonies had increased expression of poly-ADP ribose (a polymer required for the recognition of DNA breaks by the BER machinery), suggesting that upregulation of BER may be driving resistance. Furthermore, Western Blot analysis revealed that occasional colonies re-expressed MGMT. Interestingly, a few colonies did not possess these alterations, suggesting that their resistance may result from further downstream modifications of MMR or BER, or by mutations in HR. With a more comprehensive characterization of these U251 colonies, we hope to learn more about TMZ resistance in GBM, and refine treatments or preventative therapies for molecularly distinct, TMZ-resistant, recurrent tumours.

Objectives Beryllium salts (here, beryllium sulphate) can produce a cytostatic effect in some cell types. The basis for this effect may include increased expression of proliferation inhibitors, reduced expression of proliferation promoters, or both. This study sought to determine the role of p53, the tumour-suppressing transcription factor, in mediating beryllium-induced cytostasis. Materials and methods Human A172 glioma cells express wild-type TP53 gene. Activity of p53 was experimentally manipulated using siRNA and related approaches. Key elements of the beryllium-response were compared in normal and p53-knockdown A172 cells using RT-PCR and Western blotting. Results In A172 cells, 10 μm BeSO4 caused 300% increase in CDKN1A (cyclin-dependent kinase inhibitor p21) mRNA and 90% reduction of CCNE2 (cyclin E2) mRNA. The increased p21 mRNA and reduced cyclin E2 mRNA were each dependent on presence of functional p53. For p21, increased mRNA led to commensurately increased protein levels. In contrast, reduction in cyclin E2 mRNA levels did not lead to corresponding reductions in cyclin E2 protein. The proteasomal inhibitor MG-132 caused p53 protein to increase, but it had no effect on cyclin E2 protein levels. Cycloheximide time course studies indicated that the cyclin E2 protein half-life was more than 12 hours in these cells. Conclusions Beryllium elicited p53-dependent changes in mRNA levels of key determinants of cell proliferation such as p21 and cyclin E2. However, cyclin E2 protein appeared to be aberrantly regulated in this cell type, as its turnover was unexpectedly slow.

Glioblastomas (GBMs) account for nearly half of all primary malignant brain tumours, and current therapies are often only marginally effective. Our understanding of the underlying biology of these tumours and the development of new therapies have been complicated in part by widespread inter- and intratumoural heterogeneity. To characterize this heterogeneity, we performed regional subsampling of primary glioblastomas and derived organoids from these tissue samples. We then performed single-cell RNA-sequencing (scRNA-seq) on these primary regional subsamples and 1-3 matched organoids per sample. We have profiled samples from six tumour sets to date and have obtained sequencing data for 21,234 primary tissue cells and 14,742 organoid cells. While the most apparent differences in gene expression appear to be between individual tumours, we were also able to identify similar cellular subpopulations across tissue samples and across organoids. Importantly, organoids derived from the same tissue sample appeared to be composed of similar cellular subpopulations and were highly comparable to each other, indicating that replicate organoids faithfully represent the original tumour tissue. Overall, our scRNA-seq approach will help evaluate the utility of tumour-derived organoids as model systems for GBM and will aid in identifying cellular subpopulations defined by gene expression patterns, both in primary GBM regional subsamples and their associated organoids. These analyses will allow for the characterization of clonal or subclonal populations that are likely to respond to different therapeutic approaches and may also uncover novel therapeutic targets previously unrevealed through bulk analyses.

Objective: This analysis was performed to assess whether antiepileptic drugs (AEDs) modulate the effectiveness of temozolomide radiochemotherapy in patients with newly diagnosed glioblastoma. Methods: The European Organization for Research and Treatment of Cancer (EORTC) 26981-22981/National Cancer Institute of Canada (NCIC) CE.3 clinical trial database of radiotherapy (RT) with or without temozolomide (TMZ) for newly diagnosed glioblastoma was examined to assess the impact of the interaction between AED use and chemoradiotherapy on survival. Data were adjusted for known prognostic factors. Results: When treatment began, 175 patients (30.5%) were AED-free, 277 (48.3%) were taking any enzyme-inducing AED (EIAED) and 135 (23.4%) were taking any non-EIAED. Patients receiving valproic acid (VPA) only had more grade 3/4 thrombopenia and leukopenia than patients without an AED or patients taking an EIAED only. The overall survival (OS) of patients who were receiving an AED at baseline vs not receiving any AED was similar. Patients receiving VPA alone (97 [16.9%]) appeared to derive more survival benefit from TMZ/RT (hazard ratio [HR] 0.39, 95% confidence interval [CI] 0.24-0.63) than patients receiving an EIAED only (252 [44%]) (HR 0.69, 95% CI 0.53-0.90) or patients not receiving any AED (HR 0.67, 95% CI 0.49-0.93). Conclusions: VPA may be preferred over an EIAED in patients with glioblastoma who require an AED during TMZ-based chemoradiotherapy. Future studies are needed to determine whether VPA increases TMZ bioavailability or acts as an inhibitor of histone deacetylases and thereby sensitizes for radiochemotherapy in vivo. Neurology (R) 2011;77:1156-1164

Adult Glioblastomas (GBMs) remain one of the least curable brain cancers despite the discovery and use of DNA alkylating agent Temozolomide (TMZ). TMZ provides a moderate survival benefit to sensitive patients whose O6-methylguanine-methyltransferase (MGMT) gene is silenced by promoter methylation. Unfortunately, TMZ potential is stunted because of the rapid onset of tumour recurrence and acquired resistance believed to result from the upregulation of DNA damage repair by the base excision repair (BER), mismatch repair (MMR), or homologous recombination (HR) systems. Our laboratory previously demonstrated that cell lines obtained from recurrent, TMZ-resistant GBMs could be re-sensitized to TMZ when treated with an inhibitor of poly (ADP-ribose) polymerase-1 (PARP-1) – a protein instrumental in the recruitment of BER machinery. From this preliminary research, we postulate that PARP-1 inhibition may not only be used to overcome established resistance in GBM but may also be used to prevent its emergence altogether. To test this hypothesis, we utilized the MGMT-methylated GBM cell line U251N and developed an in vitro model of inducible TMZ resistance. We verified that prolonged treatment of U251N cells with TMZ resulted in the emergence of resistant colonies that resembled recurrent GBM clinically observed in TMZ-treated patients. However, when the parental U251N line was co-treated with TMZ and PARP-1 inhibitor ABT-888, resistant colonies failed to appear. Therefore, PARP-1 inhibition may possess the potential to maintain tumour sensitivity to TMZ as well as evade the otherwise inevitable development of resistance in GBM.

To determine the response rate, time to disease progression, survival, and toxicity of intravenous carboplatin and chronic oral high-dose tamoxifen in patients with recurrent malignant gliomas. Patients with histological confirmation of recurrent malignant gliomas were eligible for this multicenter phase II trial. Treatment consisted of 400 mg/m2 carboplatin intravenously every 4 weeks and oral high dose chronic tamoxifen (80 mg bid in women and 100 mg bid in men). Twenty seven patients met the eligibility criteria and were evaluable for response. The histological subtypes were: 16 (59%) glioblastoma multiforme (GBM), malignant astrocytoma (5 patients), malignant mixed glioma (5 patients), and glioblastoma/gliosarcoma (1 patient). Twenty-two patients (82%) had an ECOG performance status of 0 or 1. No complete responses were observed, 4 patients (15%) achieved a partial response, and 14 patients (52%) had stable disease. Median time to progression was 3.65 months (95%CI 2.56, 4.83). Median overall survival was 14.09 months (95%CI 7.06, 19.91). One patient with a recurrent GBM had a sustained partial response and is progression free 81 months since starting treatment. Another patient with mixed malignant oligoastrocytoma also had a prolonged partial response (lasting 63 months) and is alive 84 months after treatment for recurrence. The most frequently reported grade 3 or 4 toxicities were fatigue (19%), nausea (11%) and anorexia (11%). Carboplatin and high dose tamoxifen has similar response rates to other regimens for recurrent malignant gliomas and are probably equivalent to those found using tamoxifen as monotherapy. Long-lasting periods of disease free survival in some patients (particularly those with malignant mixed oligo astrocytomas) were found.

Purpose To prospectively compare standard radiation therapy (RT) with an abbreviated course of RT in older patients with glioblastoma multiforme (GBM). Patients and Methods One hundred patients with GBM, age 60 years or older, were randomly assigned after surgery to receive either standard RT (60 Gy in 30 fractions over 6 weeks) or a shorter course of RT (40 Gy in 15 fractions over 3 weeks). The primary end point was overall survival. The secondary end points were proportionate survival at 6 months, health-related quality of life (HRQoL), and corticosteroid requirement. HRQoL was assessed using the Karnofsky performance status (KPS) and Functional Assessment of Cancer Therapy-Brain (FACT-Br). Results All patients had died at the time of analysis. Overall survival times measured from randomization were similar at 5.1 months for standard RT versus 5.6 months for the shorter course (log-rank test, P = .57). The survival probabilities at 6 months were also similar at 44.7% for standard RT versus 41.7% for the shorter course (lower-bound 95% CI, −13.7). KPS scores varied markedly but were not significantly different between the two groups (Wilcoxon test, P = .63). Low completion rates of the FACT-Br (45%) precluded meaningful comparisons between the two groups. Of patients completing RT as planned, 49% of patients (standard RT) versus 23% required an increase in posttreatment corticosteroid dosage (χ2 test, P = .02). Conclusion There is no difference in survival between patients receiving standard RT or short-course RT. In view of the similar KPS scores, decreased increment in corticosteroid requirement, and reduced treatment time, the abbreviated course of RT seems to be a reasonable treatment option for older patients with GBM.

In this clinical and histopathological study, the frequency of long-term glioblastoma multiforme (GBM) survivors (LTGBMSs) was determined in a population-based study. The Alberta Cancer Registry was used to identify all patients diagnosed with GBM in Alberta between January 1, 1975, and December 31, 1991. Patient charts were reviewed and histology reexamined. LTGBMSs were defined as GBM patients surviving 3 years after diagnosis. Each LTGBMS was compared with 3 age-, sex-, and year of diagnosis-matched controls, and patient/treatment or tumor characteristics that predicted long-term survival were determined. There were 689 GBMs diagnosed in the study period; 15 (2.2%) of these patients survived 3 years. LTGBMSs (average age, 43.5 +/- 3.3 years) were significantly younger when compared with all GBM patients (average age, 53.0 +/- 0.56 years). LTGBMSs had a higher Karnofsky Performance Status score at diagnosis compared with controls. LTGBMSs were much more likely to have had a gross total resection and adjuvant chemotherapy than control GBM patients. Tumors from LTGBMSs tended to have fewer mitoses and a significantly lower Ki-67 cellular proliferation index compared with controls. Radiation-induced dementia was common and disabling in LTG-BMSs. In conclusion, conventionally treated GBM patients in an unselected population have a very small chance of long-term survival. The use of aggressive surgical resection and adjuvant chemotherapy may make long-term survival more likely in GBM patients if their performance status is high at diagnosis.

Background:Long-term glioblastoma multiforme survivors (LTGBMS) are uncommon. The frequency which these occur in an unselected population and factors which produce these unusually long survivors are unknown.Objectives:To determine in a population- based study 1) the frequency of LTGBMS in a population and 2) identify which patient, treatment or tumor characteristics would predict which glioblastoma (GBM) patient would become a LTGBMS.Methods:The Alberta Cancer Registry was used to identify all patients diagnosed with GBM in southern Alberta between 1/1/75 - 12/31/91. Patient charts were reviewed and histology re-examined by a blinded neuropathologist. LTGBMS were defined as GBM patients surviving ≥ 3 years after diagnosis. Each LTGBMS was compared to three age-, gender-, and year of diagnosis-matched controls to compare patient, treatment, and tumor factors to GBM patients without long-term survival.Results:There were 279 GBMs diagnosed in the study period. Five (1.8%) survived ≥ three years (range, 3.2-15.8 years). Seven additional long-term survivors, who carried a diagnosis of GBM, were excluded after neuropathologic review; the most common revised diagnosis was malignant oligodendroglioma. LTGBMS (avg. age = 45 years) were significantly younger when compared to all GBM patients (avg. age = 59 years, p - 0.0001) diagnosed in the study period. LTGBMS had a higher KPS at diagnosis (p = 0.001) compared to controls. Tumors from LTGBMS tended to have fewer mitoses and a lower Ki-67 cellular proliferative index compared to controls. Radiation-induced dementia was common and disabling in LTGBMS.Conclusions:These data highlight the dismal prognosis for GBM patients who have both a short median survival and very small chance (1.8%) of long-term survival. The LTGBMS were younger, had a higher performance status, and their tumors tended to proliferate less rapidly than control GBM patients. When long-term survival does occur it is often accompanied by severe treatment-induced dementia.

✓ In a pilot study, two groups of patients with malignant glioma underwent sequential neuropsychological evaluations after successful tumor treatment. Group 1 included nine patients treated from 1981 to 1985; all patients received irradiation and eight underwent chemotherapy. The baseline neuropsychological assessment was performed 1 to 63 months after tumor diagnosis, with follow-up evaluations at irregular intervals over the next 3 to 7 years. Six patients in Group 1 exhibited impairment on most measures at baseline; subsequently, two patients developed profound cognitive impairment. Initially, three patients functioned in the average range on most tasks; thereafter, two deteriorated on one measure each. Group 2 was ascertained prospectively and included 16 patients treated from 1985 to 1987, all of whom received irradiation and chemotherapy. The first evaluation was performed 18 months after diagnosis, then every 6 months for 2 years, and then yearly. Compared to a control group, those in Group 2 had significant cognitive impairment at baseline. Cognitive performance did not change over the next 12 months in 10 patients who remained free of tumor, but within 2 years of baseline testing, deterioration on specific tasks was evident in two of seven disease-free survivors. When last tested, five of six disease-free survivors had deteriorated on one or more measures. Unlike Group 1, severe global cognitive impairment was not seen, perhaps because Group 2 was followed for a shorter time. Verbal and nonverbal composite scores derived from intelligence quotient (IQ) tests showed less impairment at baseline than did other measures and were more likely to remain stable subsequently. Verbal memory and sustained attention were the most impaired at baseline, and verbal learning and flexibility in thinking showed the greatest tendency to decline over time. Cognitive functioning in survivors of high-grade glioma is best measured and monitored by tests that probe a broader spectrum of abilities than IQ. Neuropsychological measures used in this analysis lacked sensitivity at the lower end of the impaired range. Future studies should use tests better able to discern cognitive differences at low performance levels. Based on this experience, the authors conclude that most long-term survivors of high-grade glioma will have significant cognitive difficulties, usually evident by the first assessment; some patients will develop profound impairment years later, and few are capable of fully independent living.

PURPOSE We conducted a phase III trial comparing intravenous (IV) diaziquone (AZQ) and carmustine (BCNU) as single agents in patients with cerebral anaplastic gliomas who had received surgery and radiotherapy. Its purpose was to compare the efficacy of AZQ with that of BCNU, the standard agent for brain tumor chemotherapy. PATIENTS AND METHODS Randomization between the two regimens occurred 8 weeks after completion of radiotherapy. A total of 251 patients were randomized to receive either AZQ or BCNU, and there were no significant differences between the two treatment arms in any of the known prognostic variables, including age, histologic grade, and Karnofsky performance status (KPS). RESULTS There was no significant difference in either time to tumor progression or survival between the two treatment arms. Age and histology were strong predictors of outcome, whereas KPS had relatively less effect. Three groups of patients with distinctly different outcomes could be identified: (1) older age (45+) and glioblastoma/gliosarcoma (GBM/GS) patients had a median survival of 37 weeks after randomization; (2) patients with either older age or GBM/GS had a median survival of 61 weeks; and (3) younger age (< 45) and non-GBM/GS (usually anaplastic astrocytoma) patients had a median survival of 147 weeks. Toxicity was primarily hematologic, although acute gastrointestinal toxicity and chronic pulmonary toxicity were more common with BCNU. Patients randomized to AZQ who had significant hematologic toxicity that required dose reduction after the first treatment cycle had significantly longer time to tumor progression and survival than those who did not require dose reduction (P = .011 and .016, respectively). CONCLUSION There was no significant difference in efficacy between AZQ and BCNU in patients with anaplastic gliomas as tested in this study, although AZQ was somewhat better tolerated.

This is one of the few studies that have explored the value of baseline symptoms and health-related quality of life (HRQOL) in predicting survival in brain cancer patients. Baseline HRQOL scores (from the EORTC QLQ-C30 and the Brain Cancer Module (BN 20)) were examined in 490 newly diagnosed glioblastoma cancer patients for the relationship with overall survival by using Cox proportional hazards regression models. Refined techniques as the bootstrap re-sampling procedure and the computation of C-indexes and R(2)-coefficients were used to try and validate the model. Classical analysis controlled for major clinical prognostic factors selected cognitive functioning (P=0.0001), global health status (P=0.0055) and social functioning (P, Clinical Trial, Phase III, Journal Article, Randomized Controlled Trial, Research Support, N.I.H. Extramural, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Molecular genetic subsets of anaplastic oligodendroglioma behave in biologically distinct ways, in both their rates of growth and their responses to standard therapies. In a series of 64 cases, we evaluated whether allelic loss of chromosomal arms 1p and 19q, an early molecular event in the genesis of chemosensitive oligodendrogliomas, is related to tumor location and extent of tumor spread in the brain. We observed that tumor genotype was closely associated with tumor location (P0.001). Anaplastic oligodendrogliomas located in the frontal, parietal, and occipital lobes were significantly more likely to harbor allelic loss of chromosomal arms 1p and 19q than histologically indistinguishable tumors arising in the temporal lobe, insula, and diencephalon (P0.001). In addition, loss of heterozygosity for 1p and 19q was significantly associated with a bilateral pattern of growth (P = 0.037); all seven bilaterally distributed anaplastic oligodendrogliomas had 1p and 19q allelic loss. We conclude, therefore, that molecular subtypes of oligodendrogliomas may arise preferentially in certain lobes of the brain and have differential patterns of growth, with tumors having allelic loss of chromosomes 1p and 19q occurring most frequently in the frontal lobes and having a tendency for widespread growth across the midline. These findings encourage inquiries into the biological basis of such marked differences and already have implications for the current management of these neoplasms.

We examined the effect of p53 inactivation on the response of U87MG glioma cells to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). These studies were motivated by three observations: (a) some human astrocytomas are sensitive to BCNU and some are resistant; (b) chemosensitive astrocytomas are more likely to be found in young adults whose tumors are more likely to harbor a p53 mutation; and (c) mouse astrocytes lacking the p53 gene are more sensitive to BCNU than wild-type cells. Here, we observed that p53 inactivation by transfection with pCMV-E6 sensitized U87MG cells to BCNU. Compared with control U87MG-neo cells with intact p53 function, the clonogenic survival of U87MG-E6 cells exposed to BCNU was reduced significantly. In U87MG-E6 cells, sensitization to BCNU was associated with failure of p21(WAF1) induction, transient cell cycle arrest in S phase, accumulation of polyploid cells, and significant cell death. In contrast, resistance to BCNU in U87MG-neo cells was associated with up-regulation of p53, prolonged induction of p21(WAF1), sustained cell cycle arrest in S phase, and enhancement of DNA repair. U87MG cells with disrupted p53 function were less able to repair BCNU-induced DNA damage and survive this chemotherapeutic insult. The question arises of whether p53 dysfunction might be a chemosensitizing genetic alteration in human astrocytic gliomas.

In a prior study of anaplastic oligodendrogliomas treated with chemotherapy at diagnosis or at recurrence after radiotherapy, allelic loss of chromosome 1p correlated with better chemotherapeutic response and overall survival. However, in this group of patients in whom therapeutic management was not uniform, loss of 1p did not identify all chemosensitive tumors, nor did all patients whose tumors harbor a 1p loss have long survival.To clarify the clinical relevance of molecular genetic testing at the time of diagnosis for patients with anaplastic oligodendrogliomas, we studied a larger, more homogeneous group of 50 patients with histologically defined anaplastic oligodendrogliomas treated with a chemotherapeutic regimen as the principal initial therapy.We demonstrate that these tumors can be divided genetically into four therapeutically and prognostically relevant subgroups. Patients whose tumors have combined but isolated losses of 1p and 19q have marked and durable responses to chemotherapy associated with long survival, with or without postoperative radiation therapy. Other tumors with chromosome 1p alterations also respond to chemotherapy, but with shorter duration of response and patient survival. Tumors lacking 1p loss can also be divided into two subgroups: those with TP53 mutations, which may also respond to chemotherapy but recur quickly, and those without TP53 mutations, which are poorly responsive, aggressive tumors that are clinically and genotypically similar to glioblastomas.These data raise the possibility, for the first time, that therapeutic decisions at the time of diagnosis might be tailored to particular genetic subtypes of anaplastic oligodendroglioma.

Allelic loss of the short arm of chromosome 1 predicts radiographic response to chemotherapy and long overall survival times in patients with anaplastic oligodendrogliomas. Using a database of patients with oligodendrogliomas in whom chromosome 1p status was known, we explored whether allelic loss of 1p also predicted longer duration of tumor control when radiotherapy was part of the initial treatment of these patients.We measured progression-free survival following radiotherapy in a cohort of patients with World Health Organization (WHO) Grade II and WHO Grade III oligodendrogliomas. The effects on progression-free survival of patient age, Karnofsky performance score (KPS), tumor grade when irradiated and chromosome 1p status were examined by univariate and multivariate statistical analyses. For the subset of patients with newly diagnosed anaplastic oligodendrogliomas, relationships between use of chemotherapy, chromosome 1p status and progression-free survival were also examined.Fifty-five patients (29 male, 26 female; ages 18-75 years; median, 44 years; KPS 50-90, median 80) were irradiated for either a WHO Grade II (n = 19) or Grade III (n = 36) oligodendroglioma. Twenty-eight patients had chemotherapy immediately prior to radiotherapy, and 27 had chemotherapy at progression following radiotherapy. The median radiation dose was 54 Gy in 30 fractions. Loss of heterozygosity (LOH) at chromosome 1p was evident in 36 tumors and absent in 19. Overall median progression-free survival after radiotherapy was 40.4 months. Median progression-free survival was 55.0 months for patients whose tumors harbored 1p loss vs. 6.2 months for those patients whose tumors retained both copies of chromosome 1p (p0.001). On both univariate and multivariate analyses, chromosome lp loss was the principal independent predictor of longer progression-free survival for patients with Grade II and III oligodendrogliomas. For Grade III oligodendrogliomas, chemotherapy as an adjunct to radiotherapy prolonged tumor control for those patients whose tumors harbored allelic loss of chromosome 1p (p = 0.004).These data suggest allelic loss of chromosome 1p in patients with oligodendroglial neoplasms predicts longer progression-free survival among patients receiving radiotherapy +/- chemotherapy as part of their initial treatment. Chromosome 1p loss may be an important stratification variable in future therapeutic trials of oligodendroglioma.

The two principal subtypes of glial neoplasms, astrocytomas and oligodendrogliomas, exhibit striking differences in response to chemotherapy. This differential chemosensitivity might be explained by the specific genetic alterations causing gliomas but could also be attributable to specific properties intrinsic to the cells from which gliomas arise. To examine the possibility that chemosensitivity might be associated with lineage-specific properties of potential ancestors of these tumors, we explored: (a) the expression of drug resistance genes in rat glial cells; (b) the sensitivity of rat glial subtypes to the bifunctional alkylating agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU); and (c) the effect of O6-methylguanine-DNA methyltransferase (MGMT) and glutathione modulation on resistance to BCNU. Astrocytes, O-2A progenitors, and oligodendrocytes each displayed a unique pattern of expression of six drug resistance genes: MGMT, GST mu, GST pi,p53, MDR, and MT. Oligodendrocytes were more sensitive to BCNU than either astrocytes or O-2A progenitors. The increased resistance of astrocytes in comparison to oligodendrocytes was modulated, at least in part, by both O6-benzylguanine (BG) and DL-buthionine-(S,R)-sulfoximine, suggesting a role for both MGMT and glutathione in the resistance of astrocytes to BCNU. The sensitivity of O-2A progenitors to BCNU following BG pretreatment is virtually indistinguishable from that of oligodendrocytes depleted of MGMT, suggesting that the down-regulation of MGMT is sufficient to account for the increased sensitivity of oligodendrocyte lineage cells to BCNU as they differentiate. These experiments provide support for the hypothesis that properties of glial cells retained in gliomas may contribute to the differential chemosensitivity of glial neoplasms.

The Quality Standards Subcommittee seeks to develop scientifically sound, clinically relevant practice parameters for the practice of neurology. Practice parameters are strategies for patient management that assist physicians in clinical decision making. A practice parameter is one or more specific recommendations based on analysis of evidence on a specific clinical problem. These might include diagnosis, symptoms, treatment, or procedure evaluation. American Academy of Neurology (AAN) members have requested the publication of a practice parameter on the use of prophylactic anticonvulsants in patients with primary and metastatic brain tumors. Physicians often administer anticonvulsant medication prophylactically to patients with brain tumors, despite the lack of definitive evidence that prophylactic anticonvulsant therapy is effective in preventing first seizures.1-4 If anticonvulsant medications were free of side effects, their prophylactic use might be attractive even without such evidence. However, discomfort, expense, and inconvenience result from drug treatment and periodic monitoring of serum drug concentrations. Typical anticonvulsant-induced side effects, including cognitive impairment, myelosuppression, liver dysfunction, and dermatologic reactions (ranging from minor rashes to life-threatening Stevens–Johnson syndrome), appear to occur more frequently in patients with brain tumors than in other patient groups,3,5-16 although direct comparison studies have not been published. A spectrum of side effects unique to patients with brain tumors must also be considered. Phenytoin, carbamazepine, and phenobarbital reduce the efficacy of corticosteroids,17-21 which are administered almost universally to brain tumor patients. In addition, the ability of these anticonvulsants to stimulate the cytochrome P450 enzyme system results in markedly accelerated metabolism of a wide spectrum of chemotherapeutic agents including nitrosoureas, paclitaxel, cyclophosphamide, topotecan, irinotecan, thiotepa, 9-aminocampothecin, adriamycin, and methotrexate.22-34 As a result, inadequate chemotherapeutic dosing of brain tumor patients has been identified recently as a widespread and critically important problem.35 Conversely, …

A decade ago, neuro-oncologists began asking important questions about the optimal management of patients with low-grade gliomas of the cerebral hemispheres.1 Should all patients be exposed to the risks of surgery, simply for diagnosis? Does radiotherapy prolong tumor control and patient survival? Is early radiotherapy preferable to delayed treatment? Will better tumor control by radiation be negated by cognitive impairment or other toxicities? Is high-dose radiotherapy more effective than lower dose treatment? Is there a role for chemotherapy in the treatment of low-grade glioma? In this issue of Neurology , Olson et al.2 address many of these important questions as they pertain to patients with oligodendrogliomas and oligoastrocytomas, two subtypes of hemispheric low-grade glioma with similar clinical features and molecular origins. Their analysis shows that patients with oligodendrogliomas and oligoastrocytomas have remarkably favorable prognoses. The median progression-free survival time in this series was 5 years, and the median overall survival time was 16.3 years. These good outcomes, among the best “on-record,” attest to the slow growth of oligodendrogliomas and mixed gliomas and to earlier diagnosis in the era of modern neuroimaging. Since the introduction of CT and …

Object. The authors conducted a retrospective review to examine and document the frequency, degree, and timing of the radiologically confirmed response to radiotherapy of low-grade gliomas in children. Methods. Between 1963 and 1995, 80 patients 17 years of age or younger were referred to the London Regional Cancer Centre in London, Ontario after diagnosis of a low-grade glioma. All patients underwent surgical resection or biopsy procedures and 47 underwent radiotherapy (40 postoperatively and seven at the time of tumor progression). Nineteen patients with residual measurable lesions who received radiation therapy were selected for volumetric analysis of tumor response to this treatment. The extent and timing of response to radiation were determined by the process of comparing postoperative, preirradiation computerized tomography (CT) scans with postirradiation, follow-up CT scans. For one patient the comparison was made by using serial magnetic resonance images. Residual tumor was found on postoperative CT scans in all cases. The mean preradiotherapy tumor volume was 17.1 cm3, and the postradiotherapy volume was reduced to a mean of 11.5 cm3. A reduction in tumor volume was demonstrated in eight patients by the time of their first postirradiation follow-up CT scan and in two patients a slower reduction in volume over time was shown, bringing the total number of “responders” to 10. In five of these 10 patients the tumor had shown a maximum response by the time of the first postirradiation CT scan; the median time to response was 3.3 months. A 25% or greater reduction in tumor volume was seen in eight (42%) of the 19 patients. A 50% or greater reduction was noted in five (26%) of the patients. A complete response was demonstrated at 7, 12, and 15 months, and 5 years, respectively, in four patients (21%). One responder's tumor eventually increased in size after radiotherapy and he died of his disease. The magnitude of the radiographically demonstrated response to radiation did not correlate significantly with clinical outcome (that is, survival or symptom improvement). Conclusions. On the basis of this CT scan analysis of the response of low-grade gliomas in children to radiotherapy, the authors suggest that these lesions respond to radiation, as demonstrated by tumor shrinkage on serial imaging. Major or complete responses occur occasionally. However, low-grade gliomas in children mimic other benign brain tumors such as pituitary adenomas and meningiomas in that, although growth is frequently arrested after radiotherapy, residual tumor can persist for many years, illustrating that tumor shrinkage may not be a good measure of treatment efficacy. Nevertheless, radiation therapy can result in improvement of clinical symptomatology in association with or independent of visible tumor reduction. As radiation treatment techniques become increasingly conformal and because studies indicate that lower doses of radiation may be equally effective, improvement of symptoms may be an important consideration when weighing treatment options, particularly in patients with residual or unresectable disease.

Traditionally, cytotoxic drugs have played a limited role in the treatment of brain tumors, but important advances in chemotherapy have occurred during the past decade. Certain central nervous system (CNS) malignancies are remarkably chemosensitive. These include primary CNS lymphoma, medulloblastoma, oligodendroglioma, and intracranial germ-cell tumors. This review focuses on advances in the chemotherapy of these chemosensitive tumors and also discusses the potential use of chemotherapeutic agents, both cytotoxic and cytostatic, in other brain tumors, such as glioblastomas and anaplastic astrocytomas. In addition, a brief description of future directions that may hold promise, including high-dose chemotherapy with stem-cell rescue, blood-brain barrier disruption, and regional treatment using controlled-release biodegradable polymers, is included.

Recurrence of malignant glioma following radiotherapy most commonly occurs in close proximity to the original contrast enhancing CT/MRI tumor volume. For this reason current radiation planning favors focal radiotherapy fields designed to cover the preoperative tumor contrast enhancing volume +/- surrounding edema with a 2-4 centimetre margin. Two patients with bifrontal malignant gliomas treated with such radiotherapy fields experienced out of field tumor progression while on treatment. Posterior extension along the corpus callosum, not evident on pretreatment imaging, was hypothesized as the cause of the geographic miss. The literature documenting recurrence patterns of malignant glioma following radiotherapy support focal field radiotherapy fields for most patients with malignant glioma. Reporting bias may exist in the literature, however, due to the whole brain radiotherapy used in older series reporting recurrence patterns and exclusion of patients with bihemispheric or more locally extensive tumors in more modern series. Tumor location and pattern of growth at presentation may be important factors in predicting patterns of spread and relapse after radiotherapy.

Twenty years ago a phase III trial by the Brain Tumor Study Group demonstrated that surgery and radiation were better treatment for malignant glioma than surgery alone.1 Maximal surgical resection and conventional radiotherapy quickly became the standard of care for patients with high-grade glioma. Since then many other treatments, most often cytotoxic drugs,2 have been added to surgery and radiation, and compared with standard therapy in randomized controlled trials(RCTs). None has unequivocally prolonged tumor control or patient survival.3 While these failures speak largely to the resilient nature of malignant glioma, certain issues surrounding the initiation and conduct of phase III trials of brain tumor treatment have surfaced in recent years and warrant discussion before the next generation of antiglioma therapies are tested. Some RCTs were based on inconclusive phase II data and may have been initiated prematurely, while others had divergent entry criteria, inadequate control for prognostic factors, and different outcome measures. Moreover, few studies evaluated quality of life (QOL), toxicity, or cost in a comprehensive way and none considered patient preferences for treatment. In this report we describe methods of assessing the results of phase II trials as a means of selecting treatments that most warrant phase III evaluation, and discuss challenges in the design and conduct of RCTs of new therapies for malignant glioma. When is an RCT warranted? When are the results of a phase II study sufficiently compelling to warrant a randomized trial? Randomized controlled trials are initiated when new treatments are found to have antiglioma activity in phase II evaluation. Phase II trials are of two types:(1) those that measure tumor response and (2) those that measure tumor control. In studies of response , treatment is given to patients with visible tumor, and the rate and duration of the response is recorded. …

PURPOSE We studied the natural history of postoperative enhancement on magnetic resonance (MR) scans in patients with malignant glioma to determine the following: (1) when a postoperative MR scan most accurately shows residual enhancing tumor; and (2) whether repeated doses of the contrast agent gadopentetate dimeglumine (Gd-DTPA) were well tolerated. PATIENTS AND METHODS Seventeen patients with malignant glioma underwent tumor resection; four (24%) had nonenhancing tumors preoperatively. Serial MR scans were performed on postoperative days 1, 3, 5, 7, 14, and 21 and were analyzed qualitatively and quantitatively. The evolution of enhancement and subacute hemorrhage were described and measured. A uniform schedule of postoperative dexamethasone administration was used in all but four patients (24%) (each required higher doses to maintain neurologic function). RESULTS Nontumoral, marginal (i.e., postsurgical) enhancement, potentially mimicking residual tumor, developed in eight patients (53%), including tumors that were nonenhancing preoperatively, and was maximal from days 5 to 14. Tumor enhancement was optimally visualized on postoperative days 3 to 5. Nine of 10 patients (90%) with gross residual enhancing tumor showed an increase of enhancing tumor size during the study. Methemoglobin was detected at some time in all patients (100%) and was usually minor, but in six (35%) it interfered with residual tumor assessment. The 97 doses of Gd-DTPA, administered in 17 patients, were well tolerated. CONCLUSION When accurate assessment of residual enhancing tumor is needed in patients with malignant glioma, an MR scan performed on postoperative days 3 to 5 should minimize the confounding effects of postsurgical enhancement and methemoglobin. The repeated administration of Gd-DTPA over several weeks is well tolerated.

We examined the response of normal and p53-deficient mouse astrocytes to the alkylating agent 1,3-bis(2-chloroethyl)-l-nitrosourea (BCNU), a clinically useful DNA-damaging drug to which some human astrocytomas are resistant and some are sensitive. Astrocyte cultures were isolated from the cerebrums of wild-type, heterozygous, and knockout p53 neonatal mice and treated with various concentrations of BCNU. Wild-type p53 astrocytes were significantly more resistant to BCNU than were knockout p53 astrocytes, with heterozygous astrocytes exhibiting an intermediate level of resistance, Cell cycle analysis showed that wild-type p53 astrocytes treated with BCNU demonstrated a decline in the percentage of cells in G1 and an increase in the percentage of cells in G2. Similar cell cycle responses to BCNU occurred in knockout p53 astrocytes, suggesting that this effect was p53 independent. In contrast, G1 arrest was observed in wild-type astrocytes exposed to ionizing radiation and was not observed in knockout astrocytes, indicating a p53-dependent response. Our findings point to an as yet uncharacterized p53-associated mechanism of resistance to BCNU in mouse astrocytes.

This study was designed to evaluate strategies to overcome the resistance of anaplastic gliomas of the brain to external beam radiotherapy (ERT) plus carmustine (BCNU). Patients wereor = 15 years of age, had a histologic diagnosis of malignant glioma, and a Karnofsky performance status (KPS)or = 60%.In Randomization 1, patients were assigned to receive either ERT alone (61.2 Gy) or ERT plus mitomycin C (Mito, IV 12.5 mg/m(2)) during the first and fourth week of ERT. After this treatment, patients went on to Randomization 2, where they were assigned to receive either BCNU (i.v. 200 mg/m(2)) given at 6-week intervals or 6-mercaptopurine (6- MP, 750 mg/m(2) IV daily for 3 days every six weeks), with BCNU given on the third day of the 6-MP treatment. Three hundred twenty-seven patients underwent Randomization 1. One hundred sixty-four received ERT alone, and 163 received ERT + Mito [average 52.7 years; 63% male; 69% glioblastoma multiforme (GBM); 66% had a resection; 56% KPSor = 90%]. Step-wise analysis of survival from Randomization 1 or 2 indicates that survival was significantly diminished by: (a) ageor = 45 years (b) KPS90%; (c) GBM/gliosarcoma histology; (d) stereotactic biopsy as opposed to open biopsy or resection. Median survival from Randomization 1 in both arms (ERT + Mito) was 10.8 months. Median survival from Randomization 2 was 9.3 months for BCNU/6MP vs. 11.4 months for the BCNU group (p = 0.35). Carmustine/6-MP showed a possible survival benefit for histologies other than GBM/GS. Two hundred and thirty-three patients underwent Randomization 2. The proportion of patients in the ERT group who terminated study prior to Randomization 2 was significantly less in the ERT group than in the ERT + Mito group (20 vs. 37%, p0.001).(a) The addition of Mito to ERT had no impact on survival; (b) patients treated with ERT + Mito were at greater risk of terminating therapy prior to Randomization 2; (c) there was not a significant survival benefit to the addition of 6-MP to BCNU.

Although oligodendrogliomas cannot be considered benign tumors, many are slow-growing indolent tumors for which prolonged life expectancy is the rule. Others behave in a more aggressive, malignant fashion, as would be predicted by their histologic characteristics. The treatment for oligodendrogliomas is evolving, with conservative management still reserved for nonanaplastic tumors, and increasingly aggressive systemic therapies being developed for more aggressive or recurrent tumors.

PURPOSE We studied corticosteroid-induced magnetic resonance (MR) scan changes in patients with recurrent malignant glioma to determine if corticosteroid therapy started concurrently with investigational treatment might yield false-positive responses. PATIENTS AND METHODS Ten symptomatic patients not on corticosteroids when malignant glioma recurred had a baseline MR scan performed before corticosteroid treatment, followed by serial scans at weekly intervals for 1 month while on dexamethasone (16 mg/d). The maximum cross-sectional areas and volumes of the gadolinium-enhancing regions (tumor) and T2-weighted abnormalities (tumor plus edema) were compared quantitatively and qualitatively for each series of scans. RESULTS Nine of 10 patients (90%) had a measurable reduction in the size of the gadolinium-enhancing region or T2-weighted abnormality with corticosteroid treatment. The maximum cross-sectional area and volume of the gadolinium-enhancing region decreased by at least 25% in three of 10 patients (30%). The maximum cross-sectional area and volume of the T2-weighted abnormality decreased by at least 25% in five of 10 patients (50%). Maximum measurable radiologic improvement was evident within 2 weeks in most patients. MR scans were judged improved by the reporting neuroradiologist in seven of 10 (70%). These subjective visual improvements were also evident within 2 weeks, but generally described as slight or modest. CONCLUSION Corticosteroid-induced MR scan reductions in tumor size may confound the assessment of response of recurrent malignant gliomas to investigational agents. For patients who start corticosteroids for symptom control, investigational treatment should be delayed until a new baseline MR image is established 2 weeks later. Response is then judged by comparing subsequent MR scans with the new corticosteroid-influenced baseline image.

Primary of the brain and meninges are uncommon tumors. Information regarding optimum treatment is limited due to their rarity, and the best form of therapy is not yet known.Nine patients between the ages of 22 and 61 years with primary fibrosarcomas confined to the brain and meninges were studied clinicopathologically. Tumors were superficially located in five patients and intracerebrally or deep in four patients. Treatment consisted of maximum feasible surgical resection (seven patients, gross total; one, subtotal; one, no surgery before death) and radiation therapy (45-60 Gy conventional fractionation) in eight of nine patients.Fibrosarcomas were moderate or high grade in seven of nine patients (78%). Immunohistochemical reactions with antisera to glial fibrillary acid protein (GFAP), cytokeratin, vimentin, desmin, and S-100 demonstrated vimentin positivity in five of nine patients and cytokeratin positivity in two of nine patients. Individual GFAP-positive cells were seen in two cases, thought to represent trapped reactive astrocytes. None were S-100-positive or desmin-positive. Eight patients have died, with a median survival time of 7.5 months (range, 1 day-96 months). Local recurrence developed in eight patients and distant recurrence in six patients. Systemic metastases developed in four patients (50%) and meningeal seeding in four patients (50%). Longer survival was observed in superficially located tumors (range, 7.5 months-96 months) compared with intracerebral tumors (range, 1 day-9 months).Primary fibrosarcomas of the brain are uncommon tumors, usually of high histologic grade, with a high rate of local recurrence. Their propensity for meningeal and distant relapse distinguishes them from tumors of glial origin. Immunohistochemistry is of limited diagnostic value, although it may facilitate exclusion of other diagnoses. Because the prognosis after conventional surgery with 50-60 Gy external beam radiation therapy is relatively poor, the authors recommend more aggressive therapy with maximal feasible resection followed by external beam radiation therapy to doses of 64-66 Gy. When effective chemotherapy is established for soft tissue sarcomas of the extremities, this should be evaluated in view of the high incidence of distant metastases.

This is a descriptive study of 19 families with glial tumors. Twelve were identified prospectively from 178 consecutive, unrelated adults and children with newly diagnosed gliomas seen at a regional cancer center between 01 Jan 89 and 31 Mar 91 (6.7%). There were 45 affected members (42 confirmed); 30 males, 15 females, ages 4 months-78 years (median, 44.5 years; mean, 38.9 years). Two families had four affected members, three families had three, and the others two. All confirmed tumors were supratentorial and all, save one, contained an astrocytic element. Three additional members of two families had other brain or neuroectodermal tumors. These families were not unusually cancer prone and did not appear to have neurofibromatosis, tuberous sclerosis, or colonic polyposis. There was no consistent pattern of inheritance.

2055 Background: Treatment of anaplastic oligodendroglioma (AO) and anaplastic oligoastrocytomas (AOA) is controversial. Radiotherapy (RT) remains the standard therapy, but not without toxicity. Exploiting the chemosensitivity of these tumors using myeloablative chemotherapy with APBPCR is a potential strategy to defer RT. We previously reported results using induction PCV chemotherapy; subsequently the induction regimen was changed to temozolomide (TMZ) which is reported here. Methods: Patients were treated with six cycles of TMZ at 200mg/m2 on standard 5/28 day schedule. MRI was performed after three cycles and then after six cycles. Patients with surgical gross total resection who maintained response or patients who responded to temozolomide (CR or PR defined as >50% reduction in tumor) were eligible for myeloablative chemotherapy with thiotepa 250mg/m2/day for three days followed by busulfan 3.2mg/kg/day for three days, followed by APBPCR. 1p19q status was analyzed prospectively; however, patients were enrolled without regard to deletion status. Results: 19 patients (16 AO, 2 AOA, 1 low-grade oligodendroglioma with radiographic features suggestive of high-grade tumor) with a median age of 42 (28–56) and KPS of 90 (70–100) were enrolled. 13 patients had co-deletion of 1p/19q, 2 had intact 1p/19q, 1 pt had biopsy without enough tissue, and pending in 3. Eleven patients were eligible for APBPCR: 10 patients either maintained surgical CR (9) or had a response to TMZ (1) and went on to transplant; one surgical CR pt refused transplant. Six patients were ineligible for transplant because best response of SD (2), PD (2, both 1p19q intact), insurance denial (2). Two patients are still receiving induction TMZ. Median progression-free (PFS) and overall survivals (OS) have not been reached at a median follow-up of 20 months. 2 of the 10 patients who underwent APBPCR recurred, one at 16.1 and one at 34.2 months. No veno-occlusive disease was observed during transplant and no treatment-related deaths occurred. Conclusions: TMZ followed by myeloablative chemotherapy with APBPCR can be safely administered to newly diagnosed AO patients. [Table: see text]

Stereotactic biopsy of CNS tumors provides a small amount of tissue for pathologic diagnosis. This potentially leads to inaccurate grading of gliomas because of their histologic heterogeneity. We compared histologic diagnoses in a consecutive series of 329 patients with newly diagnosed anaplastic gliomas whose diagnoses were established by either stereotactic biopsy or open resection. Of 262 patients undergoing resection, 214 (82%) had glioblastomas and 48 (18%) had anaplastic astrocytomas (AAs). Of 67 patients undergoing stereotactic biopsy, 33 (49%) had glioblastomas and 34 (51%) had AAs. This difference suggests that some AAs diagnosed by stereotactic biopsy are actually glioblastomas and has important implications for the design and interpretation of clinical trials.

2054 Background: Elderly patients have glioblastomas (GBM) that are aggressive and poorly responsive to treatment. They are also prone to the side effects of treatment of GBM. Methods: To shed light on the treatment of elderly patients with GBM, we reviewed the treatment toxicities and survival of patients 65 years of age or older who were treated with chemoradiotherapy, which is the new standard of care for GBM in younger patients. Results: Thirty-nine patients at a single cancer center in Canada met the eligibility criteria for this retrospective study. Nineteen patients were treated initially with TMZ and radiotherapy and 20 others were treated with radiotherapy alone (only two had TMZ subsequently). Eight patients in the chemoradiotherapy group (42%) experienced grade III or IV toxicity versus none in the radiotherapy group. The median overall survival in the chemoradiotherapy group was 8.5 months (range, 2.0–24.7 months) versus 5.2 months (range, 1.5–14.2 months) in the radiotherapy group, an apparen...

We suggest "new" response criteria for phase II studies of supratentorial malignant glioma and favor rigorous criteria similar to those in medical oncology, with important modifications. Four response categories are proposed: complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). Response in this scheme is based on major changes in tumor size on the enhanced computed tomographic (CT) or magnetic resonance imaging (MRI) scan. Scan changes are interpreted in light of steroid use and neurologic findings. We advocate careful patient selection, emphasize pitfalls in the assessment of response, and suggest guidelines to minimize misinterpretations of response.