Glioblastoma Multiforme Cohort Research Articles

Abstract 1351: Alternative splicing of neurofibromin 1 is associated with elevated MAPK activity and poor prognosis in glioma

Abstract High-grade gliomas (HGG) are invasive with poor prognosis regardless of age: diffuse intrinsic pontine gliomas (DIPG), arising in the brainstem, are almost universally fatal and the leading cause of brain-tumor death in children; while adult anaplastic astrocytoma and glioblastoma multiforme (GBM) have median survivals of 1-3 years. The mutational spectra of adult and pediatric HGG differ, with pediatric tumours containing recurrent mutations of H3F3A and HIST1H3B. However, alterations leading to RAS/MAPK/PI3K pathway activation, including PDGFRA amplification, EGFRvIII, BRAF-V600E, NF1 deletion, are frequently found, although not all tumours will have mutations in this pathway. The neurofibromin 1 (NF1) gene negatively regulates RAS signalling by stimulating RAS-GTP turnover, thereby leading to RAS-inactivation. The two major isoforms, NF1-I and NF1-II, differ only by inclusion of the 21 aa exon23a in the GAP-related domain of NF1-II. Exon23a-inclusion has been shown to render NF1 10 times less active towards RAS, leading to elevated MAPK signalling. The brain expresses predominantly NF1-I, while the major isoform elsewhere is NF1-II. Here we used RNA-Seq to identify genes alternatively spliced between DIPG and normal brain, identifying an isoform switch from NF1-I in normal brain to NF1-II; we additionally found the same isoform switch in the TCGA adult GBM and LGG cohorts. For both GBM and LGG, RAS/MAPK/PI3K wild-type tumors with elevated NF1-II conferred significantly reduced patient survival compared to RAS/MAPK/PI3K mutant tumors. NF1-exon23a inclusion is known to be repressed in cell model systems by the CELF and ELAV-like families of splice regulators. We further show that members of these gene families are downregulated in HGG. Together, our results indicate a novel mechanism by which gliomas can activate signaling downstream from RAS independent of mutations and tumor grade, which promotes tumorigenesis by regulating pathways such as proliferation and invasion. Citation Format: Robert Siddaway, Arun Ramani, Man Yu, Michael Brudno, Cynthia Hawkins. Alternative splicing of neurofibromin 1 is associated with elevated MAPK activity and poor prognosis in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1351. doi:10.1158/1538-7445.AM2017-1351

Membrane Proteome Analysis of Glioblastoma Cell Invasion

Glioblastoma multiforme (GBM) tumor invasion is facilitated by cell migration and degradation of the extracellular matrix. Invadopodia are actin-rich structures that protrude from the plasma membrane in direct contact with the extracellular matrix and are proposed to participate in epithelial-mesenchymal transition. We characterized the invasiveness of 9 established GBM cell lines using an invadopodia assay and performed quantitative mass spectrometry-based proteomic analyses on enriched membrane fractions. All GBM cells produced invadopodia, with a 65% difference between the most invasive cell line (U87MG) and the least invasive cell line (LN229) (p = 0.0001). Overall, 1,141 proteins were identified in the GBM membrane proteome; the levels of 49 proteins correlated with cell invasiveness. Ingenuity Pathway Analysis predicted activation "cell movement" (z-score = 2.608, p = 3.94E(-04)) in more invasive cells and generated a network of invasion-associated proteins with direct links to key regulators of invadopodia formation. Gene expression data relating to the invasion-associated proteins ITGA5 (integrin α5), CD97, and ANXA1 (annexin A1) showed prognostic significance in independent GBM cohorts. Fluorescence microscopy demonstrated ITGA5, CD97, and ANXA1 localization in invadopodia assays, and small interfering RNA knockdown of ITGA5 reduced invadopodia formation in U87MG cells. Thus, invasion-associated proteins, including ITGA5, may prove to be useful anti-invasive targets; volociximab, a therapeutic antibody against integrin α5β1, may be useful for treatment of patients with GBM.

Cyclic nucleotide phosphodiesterase-1C (PDE1C) drives cell proliferation, migration and invasion in glioblastoma multiforme cells in vitro.

Cyclic nucleotides (cAMP & cGMP) are critical intracellular second messengers involved in the transduction of a diverse array of stimuli and their catabolism is mediated by phosphodiesterases (PDEs). We previously detected focal genomic amplification of PDE1C in >90 glioblastoma multiforme (GBM) cells suggesting a potential as a novel therapeutic target in these cells. In this report, we show that genomic gain of PDE1C was associated with increased expression in low passage GBM-derived cell cultures. We demonstrate that PDE1C is essential in driving cell proliferation, migration and invasion in GBM cultures since silencing of this gene significantly mitigates these functions. We also define the mechanistic basis of this functional effect through whole genome expression analysis by identifying down-stream gene effectors of PDE1C which are involved in cell cycle and cell adhesion regulation. In addition, we also demonstrate that Vinpocetine, a general PDE1 inhibitor, can also attenuate proliferation with no effect on invasion/migration. Up-regulation of at least one of this gene set (IL8, CXCL2, FOSB, NFE2L3, SUB1, SORBS2, WNT5A, and MMP1) in TCGA GBM cohorts is associated with worse outcome and PDE1C silencing down-regulated their expression, thus also indicating potential to influence patient survival. Therefore we conclude that proliferation, migration, and invasion of GBM cells could also be regulated downstream of PDE1C.

Tumor suppressor miR-181c attenuates proliferation, invasion, and self-renewal abilities in glioblastoma.

Glioblastoma multiforme (GBM) is well known for its aggressiveness, but the underlying mechanisms are unclear, limiting the treatment. In the present study, we showed that miR-181c, a commonly downregulated miRNA in GBM reported by several miRNA profiles, was associated with the mesenchymal subtype of GBM and predicted the outcome for patients from a GBM cohort (n=518) obtained from The Cancer Genome Atlas database. A multivariate analysis showed that miR-181c was an independent prognostic indicator for GBM patients. Quantitative reverse transcription PCR showed that miR-181c was expressed poorly in neurospheres of glioma cells that resemble glioma stem cells. Proliferation and invasion assays showed that miR-181c also blocked the proliferation and invasion abilities of glioma cells. Limiting dilution and colony formation assays showed that miR-181c attenuated the self-renewal ability of glioma cells. Finally, investigation of the mechanism defined Notch2, a key molecular of Notch signaling, as the functional downstream target of miR-181c. An inverse correlation was found between miR-181c and Notch2 in glioma cells and verified in fresh glioma samples. Taken together, the present study showed that miR-181c can be considered a valuable indicator for the outcome of GBM patients. miR-181c acts as a tumor suppressor that attenuates proliferation, invasion, and self-renewal capacities by downregulation of Notch2 in glioma cells.

Statin use and survival following glioblastoma multiforme

AimWhile some studies indicate a potential chemopreventive effect of statin use on the risk of glioma, the effect of statins on the prognosis of brain tumours has not yet been examined. We thus conducted a cohort study evaluating the influence of statin use on survival in patients with glioblastoma multiforme (GBM). MethodsWe identified 1562 patients diagnosed with GBM during 2000–2009 from the Danish Cancer Registry and linked this cohort to Danish nationwide demographic and health registries. Within the GBM cohort, each patient recorded as using statins prior to diagnosis (defined as ≥2 redeemed prescriptions) was matched to two statin non-users (<2 redeemed prescriptions) by propensity scores based on age, gender, year of diagnosis, comorbidity, and use of selected drugs. Cox proportional hazard models were used to compute hazard ratios (HRs) and 95% confidence intervals (CI) for all-cause death associated with prediagnostic statin use. ResultsA total of 339 GBM patients were included in the analyses. Of these, 325 died during median follow-up of 6.9 months (interquartile range: 3.8–13.4 months). Prediagnostic statin use was associated with a reduced HR of death (0.79; 95% CI: 0.63–1.00). The HRs decreased with increasing duration or intensity of prediagnostic statin use [long-term (≥5 years) statin use: HR 0.75 (95% CI: 0.47–1.20); high-intensity statin use: HR 0.66 (95% CI: 0.44–0.98)]. Additional adjustment for oncotherapeutic modalities yielded similar results (overall HR 0.80, 95% CI: 0.63–1.01). ConclusionLong-term prediagnostic statin use may improve survival following GBM.

P06 * SUPRATENTORIAL PNET IN ADULTS: MRI CAN HELP DISTINGUISH FROM GLIOBLASTOMA - DWI IS KEY

INTRODUCTION: Extra-pineal supratentorial primitive neuro-ectodermal tumours (sPNETs) are considered rare in adults and little is known about their radiological appearances. The most clinically relevant pre-surgical differential diagnosis is glioblastoma multiforme (GBM). We present a series of patients with sPNET over a 6 year period (2008-2014), and provide an imaging update with a review of the existing literature. METHOD: Retrospective review of CT and MRI characteristics of 7 patients with histologically proven sPNETs. Comparisons were made with similarly located and histopathologically confirmed WHO Grade 4 GBMs. RESULTS: 6 of the 7 sPNETs occurred de novo. One arose within the resection bed of a WHO Grade 1 pilocytic astrocytoma. There were 4 frontal and 3 temporal lobe incidences. Mean age was 42.8yrs (range 23 - 73 yrs, median 36 yrs). On CT the solid component of the tumour was usually hyperdense. Some of the small sPNETs contained central haemorrhage, confirmed on MRI. Larger tumours were solid/cystic. On MRI the solid component of all sPNETs was T2 hypointense, showed avid enhancement and marked restricted diffusion, typical of a high grade tumour. The degree of restricted diffusion was notably greater in the sPNET group when compared to the GBM cohort. CONCLUSION: The extreme rarity of sPNETs in adults makes for difficult interpretation of existing imaging modalities. In non-histopathologically confirmed cases, imaging could be misinterpreted as high grade glioma. However, greater restricted diffusion in sPNETs may help distinguish them from GBMs. Advanced MRI techniques and correlation with spectroscopy and molecular markers will also help in the future.

SOCS3 Promoter Hypermethylation Is a Favorable Prognosticator and a Novel Indicator for G-CIMP-Positive GBM Patients

BackgroundHypermethylation of the suppressor of cytokine signaling 3(SOCS3) promoter has been reported to predict a poor prognosis in several cancers including glioblstoma multiforme (GBM). We explored the function of SOCS3 promoter hypermethylation in GBM cohorts, including analysis of the CpG island methylator phenotype (CIMP), when a large number of gene loci are simultaneously hypermethylated.MethodsA whole genome promoter methylation profile was performed in a cohort of 33 GBM samples, with 13 long-term survivors (LTS; overall survival ≥ 18 months) and 20 short-term survivors (STS; overall survival ≤ 9 months). The SOCS3 promoter methylation status was compared between the two groups. In addition, we investigated the relationship of SOCS3 promoter methylation and G-CIMP status.ResultsInterestingly, in our present study, we found that SOCS3 promoter methylation was statistically significantly higher in the 13 LTS than that in the 20 STS. Furthermore, high SOCS3 promoter methylation detected via pyro-sequencing predicted a better prognosis in an independent cohort containing 62 GBM patients. This correlation was validated by the dataset from the Cancer Genome Atlas(TCGA) and the Chinese Cancer Genome Atlas(CGGA). In addition, we found that hypermethylation of the SOCS3 promoter was tightly associated with the G-CIMP-positive GBM patients.ConclusionsUsing a total of 359 clinical samples, we demonstrate that SOCS3 promoter hypermethylation status has a favorable prognostic value in GBM patients because of whole genome methylation status. Particularly, the hypermethylation of the SOCS3 promoter indicates positive G-CIMP status.

MiR-331-3p regulates expression of neuropilin-2 in glioblastoma

Aberrant expression of microRNAs (miRNAs), a class of small non-coding regulatory RNAs, has been implicated in the development and progression of high-grade gliomas. However, the precise mechanistic role of many miRNAs in this disease remains unclear. Here, we investigate the functional role of miR-331-3p in glioblastoma multiforme (GBM). We found that miR-331-3p expression in GBM cell lines is significantly lower than in normal brain, and that transient overexpression of miR-331-3p inhibits GBM cell line proliferation and clonogenic growth, suggesting a possible tumor suppressor role for miR-331-3p in this system. Bioinformatics analysis identified neuropilin-2 (NRP-2) as a putative target of miR-331-3p. Using transfection studies, we validated NRP-2 mRNA as a target of miR-331-3p in GBM cell lines, and show that NRP-2 expression is regulated by miR-331-3p. RNA interference (RNAi) to inhibit NRP-2 expression in vitro decreased the growth and clonogenic growth of GBM cell lines, providing further support for an oncogenic role for NRP-2 in high-grade gliomas. We also show that miR-331-3p inhibits GBM cell migration, an effect due in part to reduced NRP-2 expression. Finally, we identified a significant inverse correlation between miR-331-3p and NRP-2 expression in The Cancer Genome Atlas GBM cohort of 491 patients. Together, our results suggest that a loss of miR-331-3p expression contributes to GBM development and progression, at least in part via upregulating NRP-2 expression and increasing cell proliferation and clonogenic growth.

Network Signatures of Survival in Glioblastoma Multiforme

To determine a molecular basis for prognostic differences in glioblastoma multiforme (GBM), we employed a combinatorial network analysis framework to exhaustively search for molecular patterns in protein-protein interaction (PPI) networks. We identified a dysregulated molecular signature distinguishing short-term (survival<225 days) from long-term (survival>635 days) survivors of GBM using whole genome expression data from The Cancer Genome Atlas (TCGA). A 50-gene subnetwork signature achieved 80% prediction accuracy when tested against an independent gene expression dataset. Functional annotations for the subnetwork signature included “protein kinase cascade,” “IκB kinase/NFκB cascade,” and “regulation of programmed cell death” – all of which were not significant in signatures of existing subtypes. Finally, we used label-free proteomics to examine how our subnetwork signature predicted protein level expression differences in an independent GBM cohort of 16 patients. We found that the genes discovered using network biology had a higher probability of dysregulated protein expression than either genes exhibiting individual differential expression or genes derived from known GBM subtypes. In particular, the long-term survivor subtype was characterized by increased protein expression of DNM1 and MAPK1 and decreased expression of HSPA9, PSMD3, and CANX. Overall, we demonstrate that the combinatorial analysis of gene expression data constrained by PPIs outlines an approach for the discovery of robust and translatable molecular signatures in GBM.

Functional Profiling of Precursor MicroRNAs Identifies MicroRNAs Essential for Glioma Proliferation

Cancer initiation and progression involve microRNAs that can function like tumor suppressors and oncogenes. The functional significance of most miRNAs is currently unknown. To determine systematically which microRNAs are essential for glioma growth, we screened a precursor microRNA library in three human glioblastoma and one astroglial cell line model systems. The most prominent and consistent cell proliferation–reducing hits were validated in secondary screening with an additional apoptosis endpoint. The functional screening data were integrated in the miRNA expression data to find underexpressed true functional tumor suppressor miRNAs. In addition, we used miRNA-target gene predictions and combined siRNA functional screening data to find the most probable miRNA-target gene pairs with a similar functional effect on proliferation. Nine novel functional miRNAs (hsa-miR-129, -136, -145, -155, -181b, -342-5p, -342-3p, -376a/b) in GBM cell lines were validated for their importance in glioma cell growth, and similar effects for six target genes (ROCK1, RHOA, MET, CSF1R, EIF2AK1, FGF7) of these miRNAs were shown functionally. The clinical significance of the functional hits was validated in miRNA expression data from the TCGA glioblastoma multiforme (GBM) tumor cohort. Five tumor suppressor miRNAs (hsa-miR-136, -145, -342, -129, -376a) showed significant underexpression in clinical GBM tumor samples from the TCGA GBM cohort further supporting the role of these miRNAs in vivo. Most importantly, higher hsa-miR-145 expression in GBM tumors yielded significantly better survival (p<0.005) in a subset of patients thus validating it as a genuine tumor suppressor miRNA. This systematic functional profiling provides important new knowledge about functionally relevant miRNAs in GBM biology and may offer new targets for treating glioma.

Invariant delineation of nuclear architecture in glioblastoma multiforme for clinical and molecular association.

Automated analysis of whole mount tissue sections can provide insights into tumor subtypes and the underlying molecular basis of neoplasm. However, since tumor sections are collected from different laboratories, inherent technical and biological variations impede analysis for very large datasets such as The Cancer Genome Atlas (TCGA). Our objective is to characterize tumor histopathology, through the delineation of the nuclear regions, from hematoxylin and eosin (H&E) stained tissue sections. Such a representation can then be mined for intrinsic subtypes across a large dataset for prediction and molecular association. Furthermore, nuclear segmentation is formulated within a multi-reference graph framework with geodesic constraints, which enables computation of multidimensional representations, on a cell-by-cell basis, for functional enrichment and bioinformatics analysis. Here, we present a novel method, multi-reference graph cut (MRGC), for nuclear segmentation that overcomes technical variations associated with sample preparation by incorporating prior knowledge from manually annotated reference images and local image features. The proposed approach has been validated on manually annotated samples and then applied to a dataset of 377 Glioblastoma Multiforme (GBM) whole slide images from 146 patients. For the GBM cohort, multidimensional representation of the nuclear features and their organization have identified 1) statistically significant subtypes based on several morphometric indexes, 2) whether each subtype can be predictive or not, and 3) that the molecular correlates of predictive subtypes are consistent with the literature. Data and intermediaries for a number of tumor types (GBM, low grade glial, and kidney renal clear carcinoma) are available at: http://tcga.lbl.gov for correlation with TCGA molecular data. The website also provides an interface for panning and zooming of whole mount tissue sections with/without overlaid segmentation results for quality control.

Radiotherapy plus the anti-EGFR mAb nimotuzumab or placebo for the treatment of high-grade glioma patients.

2515 Background: Despite remarkable advances in multimodal therapy, high grade glioma (HGG) patients still face a poor prognosis. EGFR is well validated as a primary contributor of HGG initiation and progression. Nimotuzumab is a humanized monoclonal antibody (mAbs) that recognizes the EGFR extracellular domain. While it has similar preclinical and clinical activity when compared to other anti-EGFR mAbs, it does not induce skin toxicity or hypomagnesemia. Methods: A randomized, double blind, multicentric clinical trial was conducted in 70 anaplastic astrocytoma (AA) and glioblastoma multiforme (GBM) patients that received radiotherapy (RT) plus nimotuzumab or placebo. Patients received 6 weekly doses of nimotuzumab or placebo together with radiotherapy. Treatment was maintained every 3 weeks, until completing 1 year of treatment. GBM patients did not receive temozolomide since the drug cannot be sold to Cuba. The objectives of this study were to assess the overall survival, progression free survival (PFS), response rate, immunogenicity and safety in both treatment groups. Results: Seventy patients were included in the study: 41 AA and 29 GBM. The median cumulative dose was 3600 mg of nimotuzumab and the median antibody number of doses was 16. The combination of nimotuzumab and radiotherapy was very safe. The most prevalent related adverse events included grade 1-2 nausea, fever, tremors and anorexia. There was no increasing toxicity with repeated drug exposure. No anti-idyotipic response was detected. The mean and median survival time for subjects treated with nimotuzumab and RT was 31.06 and 17.76 months while the mean and median survival time for controls was 21.07 and 12.63 months, respectively. For the evaluable patients of the AA stratum, the median survival time was 44.56 months (active drug) vs. 14.6 months (control). For the evaluable patients in the GBM cohort, the median survival time was 16.06 months (nimotuzumab arm) vs. 8.36 months (placebo arm). Median PFS was 18.23 vs. 6.25 months. Conclusions: In this randomized trial, nimotuzumab continues showing an excellent safety profile and positive efficacy results in patients with high grade glioma in combination with irradiation.

A phase I trial of surgical resection with biodegradable carmustine (BCNU) wafer placement followed by vaccination with dendritic cells pulsed with tumor lysate for patients with malignant glioma

2033 Background: Our prior immunotherapy trials demonstrated efficacy in generating a tumor specific immune response in malignant glioma and the potential for high tumor-specific toxicity and sustained tumoricidal activity. Immunotherapy may synergize with chemotherapy and biodegradable carmustine (BCNU) wafers extend overall survival from 11.6 to 13.9 months. Methods: We exploited this synergistic effect to maintain a cytotoxic environment around the tumor milieu. Patients with high-grade glioma were eligible after maximal resection with biodegradable carmustine (BCNU) wafer placement. Screening leukapheresis is used to isolate mononuclear cells which are differentiated into dendritic cells, pulsed with tumor lysate, and then 3 intradermal vaccines are administered at 2-week intervals. Patients continued systemic chemotherapy after vaccine or at progression. Results: Eighteen patients have been enrolled (7 Male, 11 Female) between April 2007 and February 2009 with one screen failure and two patients with clinical progression prior to vaccination. The median patient age was 57 years (26 to 74 ) and median Karnofsky performance status was 90% (80–100). The histology included 3 newly diagnosed glioblastoma multiforme (GBM), 8 recurrent GBM, 2 newly diagnosed anaplastic astrocytoma (AA), and 2 recurrent AA. 15 patients were successfully treated by vaccine injections with 12 patients receiving vaccine every 2 weeks x 3 followed by adjuvant chemotherapy. Our preliminary data on 15 patients and 39 courses of Dendritic Cell vaccines demonstrate one grade 3 toxicity of fever/chest pain. A stable disease interval of 13 to 90 weeks was observed for patients who received vaccine. The 3 newly diagnosed GBM patients have stable disease (18 to 71 weeks). In the recurrent GBM cohort 7/8 patients had progression within 6 months from the post-vaccination MRI. Conclusions: This phase I study demonstrates the safety, feasibility of dendritic cell vaccination with biodegradable carmustine (BCNU) wafers with one grade 3 AE. Immunological data is pending to determine potential synergy of dendritic cell vaccination with intracranial chemotherapy. No significant financial relationships to disclose.

Abstract LB-240: Clustering of phospho-proteins targeted by enzastaurin identifies significant biomarker association with patient outcome and novel associations between biomarker groupings in a glioblastoma multiforme cohort

Abstract Introduction: Enzastaurin (LY317615.HCl) is a novel acyclic bisindolylmaleimide currently in Phase II clinical trials in combination with temozolomide and radiation for the front-line treatment of glioblastoma multiforme. Enzastaurin was identified as an ATP-competitive inhibitor of PKC-β subsequently as an inhibitor of other AGC-family kinases, including other PKC isoforms, p90RSK, GSK3β, and p70S6K. In a wide array of human cancer cell lines, including glioblastoma cell lines, enzastaurin treatment blocks signaling through the PI3 kinase/AKT/mTOR pathway. Accordingly, enzastaurin suppresses the phosphorylation of GSK3Bser9, AKTser473, CREBser133, and the S6 ribosomal protein at ser235/236 and ser240/244. We therefore sought to evaluate the levels of these key phosphorylation events in a series of primary human glioblastoma multiforme tissues in order to determine whether any or all of these phosphorylation events might be related to patient survival. Methods: A retrospective glioblastoma multiforme cohort of 115 patients was evaluated by quantitative immunofluoresence using AQUA® analysis for PhosphoCREB ser133, PhosphoS6 ser240/244, PhosphoGSK3B ser9, and total GSK3B. AQUA® technology quantitatively measured protein expression in patient samples, allowing for relative concentrations of the phosphorylated markers to be calculated and grouping of patients by clustering of AQUA® scores. Results: Spearman-Rho analysis identified strong direct correlations between PhosphoCREB ser133 and PhosphoS6 ser240/244 and between PhosphoCREB ser133 and PhosphoGSK3Bser9 expression in this cohort of patients. Univariate Kaplan Meier survival analysis of these patients based on clustered AQUA® scores revealed that these markers were indeed inversely related to disease-specific survival (phosphoGSK3B ser9 P&amp;lt;0.05). Multiparametric Correlative Discovery™ analysis also indicated a strong association of the cluster of patients with low levels of phosphoprotein expression and better survival. Conclusions: These data reveal that the signaling pathways targeted by enzastaurin were activated specifically in patients with the poorest survival and also suggest that these phosphomarkers, alone or in concert, may be useful for patient stratification.

Bromodeoxyuridine labeling index in glioblastoma multiforme: Relation to radiation response, age, and survival

Various measures of the rate of tumor cell proliferation have been found to predict survival in patients with intracerebral gliomas. We correlated the bromodeoxyuridine labeling index (BrdUrd LI) with the response to radiation therapy, survival, and known prognostic factors in a series of patients with glioblastoma multiforme (GM) to test its utility as a prognostic factor. The BrdUrd LI was determined in 200 newly diagnosed intracranial GMs. Age and sex were known for all patients. The response to radiation therapy was determined in 116 patients by comparing neuroimaging studies obtained before and after external beam radiation therapy. Survival was analyzed in 64 patients who were treated according to two consecutive prospective clinical protocols. The median BrdUrd LI was 6.5% (mean, 7.2%; range, 1.1-25.4%). The BrdUrd LI did not correlate significantly with age, sex, radiation response, or survival. Age and Karnofsky performance score were independent prognostic factors in our cohort. The proliferative rate as measured by BrdUrd LI was not a prognostic factor in our GM cohort. The BrdUrd LI did not correlate significantly with known prognostic factors in GM. There was no significant relationship between BrdUrd LI and radiation response.