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The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an iron-mimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors.

Purpose: Immunologic targeting of tumor-specific gene mutations may allow precise eradication of neoplastic cells without toxicity. Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively activated and immunogenic mutation not expressed in normal tissues but widely expressed in glioblastoma multiforme (GBM) and other neoplasms., Patients and Methods: A phase II, multicenter trial was undertaken to assess the immunogenicity of an EGFRvIII-targeted peptide vaccine and to estimate the progression-free survival (PFS) and overall survival (OS) of vaccinated patients with newly diagnosed EGFRvIII-expressing GBM with minimal residual disease. Intradermal vaccinations were given until toxicity or tumor progression was observed. Sample size was calculated to differentiate between PFS rates of 20% and 40% 6 months after vaccination., Results: There were no symptomatic autoimmune reactions. The 6-month PFS rate after vaccination was 67% (95% CI, 40% to 83%) and after diagnosis was 94% (95% CI, 67% to 99%; n = 18). The median OS was 26.0 months (95% CI, 21.0 to 47.7 months). After adjustment for age and Karnofsky performance status, the OS of vaccinated patients was greater than that observed in a control group matched for eligibility criteria, prognostic factors, and temozolomide treatment (hazard ratio, 5.3; P = .0013; n = 17). The development of specific antibody (P = .025) or delayed-type hypersensitivity (P = .03) responses to EGFRvIII had a significant effect on OS. At recurrence, 82% (95% CI, 48% to 97%) of patients had lost EGFRvIII expression (P < .001)., Conclusion: EGFRvIII-targeted vaccination in patients with GBM warrants investigation in a phase III, randomized trial.

Background: DNA strand breaks pose the greatest threat to genomic stability. Genetically determined mutagen sensitivity predisposes individuals to a variety of cancers, including glioma. However, polymorphisms in DNA strand break repair genes that may determine mutagen sensitivity are not well studied in cancer risk, especially in gliomas., Methods: We correlated genotype data for tag single-nucleotide polymorphisms (tSNPs) of DNA strand break repair genes with a gamma-radiation-induced mutagen sensitivity phenotype [expressed as mean breaks per cell (B/C)] in samples from 426 glioma patients. We also conducted analysis to assess joint and haplotype effects of single-nucleotide polymorphisms (SNPs) on mutagen sensitivity. We further validate our results in an independent external control group totaling 662 subjects., Results: Of the 392 tSNPs examined, we found that mutagen sensitivity was modified by one tSNP in the EME2 gene and six tSNPs in the RAD51L1 gene (P < 0.01). Among the six RAD51L1 SNPs tested in the validation set, one (RAD51L1 rs2180611) was significantly associated with mutagen sensitivity (P = 0.025). Moreover, we found a significant dose-response relationship between the mutagen sensitivity and the number of adverse tSNP genotypes. Furthermore, haplotype analysis revealed that RAD51L1 haplotypes F-A (zero adverse allele) and F-E (six adverse alleles) exhibited the lowest (0.42) and highest (0.93) mean B/C values, respectively. A similar dose-response relationship also existed between the mutagen sensitivity and the number of adverse haplotypes., Conclusion: These results suggest that polymorphisms in and haplotypes of the RAD51L1 gene, which is involved in the double-strand break repair pathway, modulate gamma-radiation-induced mutagen sensitivity.

Glioblastoma multiforme (GBM) is a severe brain malignancy with limited treatment and dismal prognosis. The tumor suppressor PTEN, a major inhibitor of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway, is frequently deleted in GBM tumors. PTEN antagonizes PI3K by dephosphorylating PI3K phosphoinositide substrates at the plasma membrane. The PTEN binding adapter protein NHERF1/EBP50 is overexpressed in GBM but its effects on tumorigenesis have yet to be determined. Here, we show that NHERF1 is localized to the plasma membrane in normal astrocytes and to the cytoplasm of GBM tumor cells. This cytoplasmic shift paralleled an altered membrane distribution of wild-type PTEN with consecutive Akt activation. Membrane re-targeting of NHERF1 in GBM cells recruited PTEN to the membrane and suppressed Akt activation and cell proliferation. Conversely, NHERF1 depletion in GBM cells with membrane-localized NHERF1 increased cell proliferation and Akt activation. Our findings define a tumor suppressor role for NHERF1 at the plasma membrane, and reveal a novel mechanism for PI3K/Akt activation through PTEN inactivation caused by a loss of membrane-localized NHERF1.

Brain metastases are highly resistant to chemotherapy. Metastatic tumor cells are known to exploit the host microenvironment for their growth and survival. We report here that melanoma brain metastases are surrounded and infiltrated by activated astrocytes, and we hypothesized that these astrocytes can play a role similar to their established ability to protect neurons from apoptosis. In coculture experiments, astrocytes, but not fibroblasts, reduced apoptosis in human melanoma cells treated with various chemotherapeutic drugs. This chemoprotective effect was dependent on physical contact and gap junctional communication between astrocytes and tumor cells. Moreover, the protective effect of astrocytes resulted from their sequestering calcium from the cytoplasm of tumor cells. These data suggest that brain tumors can, in principle, harness the neuroprotective effects of reactive astrocytes for their own survival and implicate a heretofore unrecognized mechanism for resistance in brain metastasis that might be of relevance in the clinic.

Angiogenesis involves a complex set of cell-cell and cell-extracellular matrix (ECM) interactions that coordinately promote and inhibit blood vessel growth and sprouting. Although many factors that promote angiogenesis have been characterized, the identities and mechanisms of action of endogenous inhibitors of angiogenesis remain unclear. Furthermore, little is known about how cancer cells selectively circumvent the actions of these inhibitors to promote pathological angiogenesis, a requisite event for tumor progression. Using mosaic mouse models of the malignant brain cancer, astrocytoma, we report that tumor cells induce pathological angiogenesis by suppressing expression of the ECM protein receptor alphavbeta8 integrin. Diminished integrin expression in astrocytoma cells leads to reduced activation of latent TGFbetas, resulting in impaired TGFbeta receptor signaling in tumor-associated endothelial cells. These data reveal that astrocytoma cells manipulate their angiogenic balance by selectively suppressing alphavbeta8 integrin expression and function. Finally, these results show that an adhesion and signaling axis normally involved in developmental brain angiogenesis is pathologically exploited in adult brain tumors.

Object: Despite a favorable outcome for most patients with WHO Grade I meningiomas, a subset of these patients will have recurrent or progressive disease that advances to a higher grade and requires increasingly aggressive therapy. The goal of this study was to identify clinical characteristics associated with the recurrence of benign meningiomas and their acceleration to atypical and malignant histological types., Methods: Records of 216 patients with WHO Grade I, II, or III meningioma that were initially treated between 1965 and 2001 were retrospectively reviewed. Median follow-up was 7.2 years., Results: Patients with non-skull base cranial meningiomas (82 of 105 [78%]) were more likely to have undergone a gross-total resection than patients with skull base meningiomas (32 of 78 [41%]; p < 0.001). Consequently, patients with Grade I non-skull base cranial meningiomas had better 5-year recurrence-free survival (69%) than patients with Grade I skull base meningiomas (56%) or Grade II or III tumors at any site (50%; p = 0.005). Unexpectedly, patients with non-skull base tumors who experienced a recurrence (8 of 22 [36%]) were more likely than patients with skull base tumors (1 of 19 [5%]) to have a higher grade tumor at recurrence (p = 0.024). Furthermore, the median MIB-1 labeling index of Grade I non-skull base cranial meningiomas (2.60%) was significantly higher than that of Grade I skull base tumors (1.35%; p = 0.016)., Conclusions: Cranial meningiomas that occur outside of the skull base are more likely to have a higher MIB-1 labeling index and recur with a higher grade than those within the skull base, suggesting that non-skull base cranial tumors may have a more aggressive biology than skull base tumors.

Our recent studies have shown that the FoxM1B transcription factor is overexpressed in human glioma tissues and that the level of its expression correlates directly with glioma grade. However, whether FoxM1B plays a role in the early development of glioma (i.e., in transformation) is unknown. In this study, we found that the FoxM1B molecule causes cellular transformation and tumor formation in normal human astrocytes (NHA) immortalized by p53 and pRB inhibition. Moreover, brain tumors that arose from intracranial injection of FoxM1B-expressing immortalized NHAs displayed glioblastoma multiforme (GBM) phenotypes, suggesting that FoxM1B overexpression in immortalized NHAs not only transforms the cells but also leads to GBM formation. Mechanistically, our results showed that overexpression of FoxM1B upregulated NEDD4-1, an E3 ligase that mediates the degradation and downregulation of phosphatase and tensin homologue (PTEN) in multiple cell lines. Decreased PTEN in turn resulted in the hyperactivation of Akt, which led to phosphorylation and cytoplasmic retention of FoxO3a. Blocking Akt activation with phosphoinositide 3-kinase/Akt inhibitors inhibited the FoxM1B-induced transformation of immortalized NHAs. Furthermore, overexpression of FoxM1B in immortalized NHAs increased the expression of survivin, cyclin D1, and cyclin E, which are important molecules for tumor growth. Collectively, these results indicate that overexpression of FoxM1B, in cooperation with p53 and pRB inhibition in NHA cells, promotes astrocyte transformation and GBM formation through multiple mechanisms.

Hypermethylation of the O(6)-methylguanine-DNA-methyltransferase (MGMT) gene has been shown to be associated with improved outcome in glioblastoma (GBM) and may be a predictive marker of sensitivity to alkylating agents. However, the predictive utility of this marker has not been rigorously tested with regard to sensitivity to other therapies, namely radiation. To address this issue, we assessed MGMT methylation status in a cohort of patients with GBM who underwent radiation treatment but did not receive chemotherapy as a component of adjuvant treatment. Formalin-fixed, paraffin-embedded tumor samples from 225 patients with newly diagnosed GBM were analyzed via methylation-specific, quantitative real-time polymerase chain reaction following bisulfite treatment on isolated DNA to assess MGMT promoter methylation status. In patients who received radiotherapy alone following resection, methylation of the MGMT promoter correlated with an improved response to radiotherapy. Unmethylated tumors were twice as likely to progress during radiation treatment. The median time interval between resection and tumor progression of unmethylated tumors was also nearly half that of methylated tumors. Promoter methylation was also found to confer improved overall survival in patients who did not receive adjuvant alkylating chemotherapy. Multivariable analysis demonstrated that methylation status was independent of age, Karnofsky performance score, and extent of resection as a predictor of time to progression and overall survival. Our data suggest that MGMT promoter methylation appears to be a predictive biomarker of radiation response. Since this biomarker has also been shown to predict response to alkylating agents, perhaps MGMT promoter methylation represents a general, favorable prognostic factor in GBM.

HOXA genes encode critical transcriptional regulators of embryonic development that have been implicated in cancer. In this study, we documented functional relevance and mechanism of activation of HOXA9 in glioblastoma (GBM), the most common malignant brain tumor. Expression of HOXA genes was investigated using reverse transcription-PCR in primary gliomas and glioblastoma cell lines and was validated in two sets of expression array data. In a subset of GBM, HOXA genes are aberrently activated within confined chromosomal domains. Transcriptional activation of the HOXA cluster was reversible by a phosphoinostide 3-kinase (PI3K) inhibitor through an epigenetic mechanism involving histone H3K27 trimethylation. Functional studies of HOXA9 showed its capacity to decrease apoptosis and increase cellular proliferation along with tumor necrosis factor-related apoptosis-including ligand resistance. Notably, aberrant expression of HOXA9 was independently predictive of shorter overall and progression-free survival in two GBM patient sets and improved survival prediction by MGMT promoter methylation. Thus, HOXA9 activation is a novel, independent, and negative prognostic marker in GBM that is reversible through a PI3K-associated epigenetic mechanism. Our findings suggest a transcriptional pathway through which PI3K activates oncogenic HOXA expression with implications for mTOR or PI3K targeted therapies.

The objective of this phase I study was to determine the maximal tolerated dose (MTD) of erlotinib in patients with recurrent malignant gliomas (MGs) or recurrent meningiomas on enzyme-inducing antiepileptic drugs (EIAEDs). Dose escalation was by a standard 3 x 3 design. The initial starting dose of erlotinib was 150 mg daily. If no dose-limiting toxicity (DLT) was observed, then dose escalation occurs as follows: 200 mg/day, 275 mg/day, and then increased in 125 mg increments until the MTD was reached. The MTD was defined as the dose where < or = 1 of 6 patients experienced a DLT and the dose above had 2 or more DLTs. The MTD was 650 mg/day; the observed DLTs were grade 3 rash in 2 patients at 775 mg/day. Pharmacokinetic analysis showed a significant influence of EIAEDs on the metabolism of erlotinib when compared with our phase II data published separately. Primary toxicities were rash and diarrhea. The MTD of erlotinib in patients receiving EIAEDs is substantially higher than the standard dose of 150 mg. This has important implications for further development of this drug in the treatment of MG as well as the optimal management of patients with other malignancies such as NSCLC who are on enzyme-inducing drugs.

Purpose: To compare the characteristics of deregulation of HER receptors and their ligands between primary tumor and corresponding brain metastases of non-small cell lung carcinoma (NSCLC)., Experimental Design: Fifty-five NSCLC primary tumors and corresponding brain metastases specimens were examined for the immunohistochemical expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR, Her2, Her3, and phosphorylated Her3, and their ligands EGF, transforming growth factor-alpha, amphiregulin, epiregulin, betacellulin, heparin-binding EGFR-like growth factor, neuregulin (NRG) 1, and NRG2. Analysis of EGFR copy number using fluorescence in situ hybridization and mutation by PCR-based sequencing was also done., Results: Metastases showed significantly higher immunohistochemical expression of EGF (membrane: brain metastases 66.0 versus primary tumors 48.5; P = 0.027; nucleus: brain metastases 92.2 versus 67.4; P = 0.008), amphiregulin (nucleus: brain metastases 53.7 versus primary tumors 33.7; P = 0.019), phosphorylated EGFR (membrane: brain metastases 161.5 versus primary tumors 76.0; P < 0.0001; cytoplasm: brain metastases 101.5 versus primary tumors 55.9; P = 0.014), and phosphorylated Her3 (membrane: brain metastases 25.0 versus primary tumors 3.7; P = 0.001) than primary tumors did. Primary tumors showed significantly higher expression of cytoplasmic transforming growth factor-alpha(primary tumors 149.8 versus brain metastases 111.3; P = 0.008) and NRG1 (primary tumors 158.5 versus brain metastases 122.8; P = 0.006). In adenocarcinomas, a similar high frequency of EGFR copy number gain (high polysomy and amplification) was detected in primary (65%) and brain metastasis (63%) sites. However, adenocarcinoma metastases (30%) showed higher frequency of EGFR amplification than corresponding primary tumors (10%). Patients whose primary tumors showed EGFR amplification tended to develop brain metastases at an earlier time point., Conclusions: Our findings suggest that NSCLC brain metastases have some significant differences in HER family receptor-related abnormalities from primary lung tumors.

The fact that glioblastomas, which are one of the most devastating cancers, frequently express the Delta-EGFR (epithelial growth factor receptor) also called mutant variant III of EGFR (EGFRvIII) suggests that this cancer cell-specific receptor might serve as an ideal target for cancer therapy. To assess its potential as such a target, we constructed an oncolytic adenovirus with Retargeted Infectivity Via EGFR (Delta-24-RIVER) on the backbone of Delta-24. This new oncolytic adenovirus targets, as Delta-24 does, the disrupted Rb pathway in cancer cells; in addition, this adenovirus has also been retargeted through the abrogation of CAR binding (Y477A mutation in adenoviral fiber protein) and insertion of an EGFRvIII-specific binding peptide in the HI loop of the fiber protein. As compared with Delta-24, Delta-24-RIVER induced EGFRvIII-selective cytotoxicity in U-87 MG isogenic cell lines and in tetracycline-inducible EGFRVIII expressing U-251 MG cells. Accordingly, by tittering the viral progeny and examining fiber protein expression in the above cells, we showed that the replication of this new construct also correlated with EGFRvIII expression. Consistently, immunohistochemistry staining of the adenoviral capsid protein hexon in the virus-treated tumors revealed that the virus replicated more efficiently in EGFRvIII-expressing U-87 MG.DeltaEGFR xenografts than in the tumors grown from U-87 MG cells. Importantly, treatment with Delta-24-RIVER prolonged the survival of animals with intracranial xenografts derived from U-87 MG.DeltaEGFR cells. Therefore, our results constitute the first proof of the direct targeting of a cancer-specific receptor using an oncolytic adenovirus.

It is generally accepted that glioma develops through accumulation of genetic alterations. We hypothesized that polymorphisms of candidate genes involved in the DNA repair pathways may contribute to susceptibility to glioma. To address this possibility, we conducted a study on 373 Caucasian glioma cases and 365 cancer-free Caucasian controls to assess associations between glioma risk and 18 functional single-nucleotide polymorphisms in DNA repair genes. We evaluated potential gene-gene and gene-environment interactions using a multianalytic strategy combining logistic regression, multifactor dimensionality reduction and classification and regression tree approaches. In the single-locus analysis, six single-nucleotide polymorphisms [ERCC1 3' untranslated region (UTR), XRCC1 R399Q, APEX1 E148D, PARP1 A762V, MGMT F84L, and LIG1 5'UTR] showed a significant association with glioma risk. In the analysis of cumulative genetic risk of multiple single-nucleotide polymorphisms, a significant gene-dosage effect was found for increased glioma risk with increasing numbers of adverse genotypes involving the aforementioned six single-nucleotide polymorphisms (P(trend) = 0.0004). Furthermore, the multifactor dimensionality reduction and classification and regression tree analyses identified MGMT F84L as the predominant risk factor for glioma and revealed strong interactions among ionizing radiation exposure, PARP1 A762V, MGMT F84L, and APEX1 E148D. Interestingly, the risk for glioma was dramatically increased in ionizing radiation exposure individuals who had the wild-type genotypes of MGMT F84L and PARP1 A762V (adjusted odds ratios, 5.95; 95% confidence intervals, 2.21-16.65). Taken together, these results suggest that polymorphisms in DNA repair genes may act individually or together to contribute to glioma risk.

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

Purpose: Previous literature provides some evidence that atopic diseases, IgE levels, and inflammatory gene polymorphisms may be associated with risk of glioblastoma. The purpose of this study was to investigate the effects of certain inflammatory gene single nucleotide polymorphisms (SNP) on patient survival. Malignant gliomas are the most common type of primary brain tumor in adults, however, few prognostic factors have been identified., Experimental Design: Using 694 incident adult glioma cases identified between 2001 and 2006 in Harris County, TX, we examined seven SNPs in the interleukin (IL)-4, IL-13, and IL-4 receptor (IL4R) genes. Cox proportional hazards regression was used to examine the association between the SNPs and overall and long-term survival, controlling for age at diagnosis, time between diagnosis and registration, extent of surgical resection, radiation therapy, and chemotherapy., Results: We found that among high-grade glioma cases, IL4R rs1805016 (TT versus GT/GG) was significantly protective against mortality over time [hazard ratios (HR), 0.59; 95% confidence intervals (CI), 0.40-0.88]. The IL4R rs1805016 and rs1805015 TT genotypes were both found to be significantly associated with survival beyond 1 year among patients with high-grade glioma (HR, 0.44; 95% CI, 0.27-0.73 and HR, 0.63; 95% CI, 0.44-0.91, respectively). Furthermore, the IL4R haplotype analysis showed that SNPs in the IL4R gene may be interacting to affect long-term survival among high-grade glioma cases., Conclusions: These findings indicate that polymorphisms in inflammation pathway genes may play an important role in glioma survival. Further research on the effects of these polymorphisms on glioma prognosis is warranted.

Here we show that in contrast to other cancer types, tumor necrosis factor (TNF)-alpha suppresses YKL-40 expression in glioma cell lines in a nuclear factor kappaB (NF-kappaB) dependent manner. Even though TNF-alpha causes recruitment of p65 and p50 subunits of NF-kappaB to the YKL-40 promoter in all cell types, recruitment of histone deacetylases (HDAC)-1 and -2, and a consequent deacetylation of histone H3 at the YKL-40 promoter occurs only in glioma cells. Importantly, using chromatin immunoprecipitation assays in frozen glioblastoma multiforme tissues, we show that YKL-40 levels decrease consistent with HDAC1 recruitment despite high levels of nuclear p-p65. This study presents a paradigm for NF-kappaB regulation of one of its targets in a strict cell type specific manner.

The association between human cytomegalovirus (HCMV) infection and glioblastoma has been a source of debate in recent years because of conflicting laboratory reports concerning the presence of the virus in glioma tissue. HCMV is a ubiquitous herpesvirus that exhibits tropism for glial cells and has been shown to transform cells in vitro. Using sensitive immunohistochemical and in situ hybridization methods in 50 glioma samples, we detected HCMV antigen and DNA in 21/21 cases of glioblastoma, 9/12 cases of anaplastic gliomas and 14/17 cases of low-grade gliomas. Reactivity against the HCMV IE1 antigen (72 kDa) exhibited histology-specific patterns with more nuclear staining for anaplastic and low-grade gliomas, while GBMs showed nuclear and cytoplasmic staining that likely occurs with latent infection. Using IHC, the number of HCMV-positive cells in GBMs was 79% compared to 48% in lower grade tumors. Non-tumor areas of the tissue contained only four and 1% of HCMV-positive cells for GBMs and lower grade tumors, respectively. Hybridization to HCMV DNA in infected cells corresponded to patterns of immunoreactivity. Our findings support previous reports of the presence of HCMV infection in glioma tissues and advocate optimization of laboratory methods for the detection of active HCMV infections. This will allow for detection of low-level latent infections that may play an important role in the initiation and/or promotion of malignant gliomas.

Focal necrosis is a key pathologic feature that distinguishes glioblastoma from lower grade glioma. The presence of necrosis in a glioblastoma could promote its rapid growth and clinical progression. Focal necrosis of glioblastoma seems to be associated with thrombosis that result from hyper-coagulability. In the present study, we found that glioblastoma cells had a high level of constitutive nuclear factor (NF)-kappaB activity, which was directly correlated with necrosis in glioblastomas. We also found a direct correlation between NF-kappaB activity and the expression of tissue factor (TF), a potent procoagulant factor in gliomas. Inhibition of TF by an inhibitory antibody prevented the procoagulant activity of glioblastoma cells, indicating a TF-dependent mechanism. Blockade of NF-kappaB activation significantly inhibited TF expression and the procoagulant activity of glioblastoma cells in vitro. Blockade of NF-kappaB activation also significantly inhibited in vivo expression of TF, which was directly correlated with decreased necrosis formation and tumor growth of glioblastoma cells in nude mice. Collectively, these results suggest that elevated NF-kappaB activity in glioblastomas cells plays a critical role in necrosis formation of glioblastoma and that inhibition of NF-kappaB activity in glioblastoma can suppress necrosis formation and progressive growth.

Background: The purpose of this retrospective study was to determine, in a cohort of patients with breast cancer and central nervous system (CNS) metastases, the effect of trastuzumab in patients with human epidermal growth factor receptor 2 (HER2)-positive disease and to compare this with that of patients with HER2-negative disease., Methods: Five hundred and ninety-eight patients with invasive breast cancer, CNS metastases and known HER2 status were identified. Time to CNS metastases and survival after CNS metastases were estimated by the Kaplan-Meier method, and Cox models were fitted to determine the association between HER2 status, trastuzumab treatment and outcomes after adjustment for other patient characteristics., Results: In the multivariable model, patients with HER2-negative disease [Hazard ratio (HR) 1.50, 95% confidence interval (CI) 1.15-1.95, P = 0.003] and patients with HER2-positive disease who did not receive trastuzumab (HR 2.13, 95% CI 1.51-3.00, P < 0.0001) had shorter times to CNS metastases compared with patients with HER2-positive disease who had received trastuzumab as first-line therapy for metastases. Furthermore, patients with HER2-negative disease (HR 1.66, 95% CI 1.31-2.12, P < 0.0001) and patients with HER2-positive disease who had never received trastuzumab (HR 1.34, 95% CI 0.78-2.30, P = 0.28) had an increased hazard of death compared with patients with HER2-positive disease who had received trastuzumab before or at the time of CNS metastases diagnosis., Conclusion: In our cohort of patients with breast cancer and CNS metastases, patients with HER2-positive disease treated with trastuzumab had longer times to development of and better survival from CNS metastases compared with patients with HER2-positive disease who had never received trastuzumab and patients with HER2-negative breast cancer.

Background: Tumor-predominant splice isoforms were identified during comparative in silico sequence analysis of EST clones, suggesting that global aberrant alternative pre-mRNA splicing may be an epigenetic phenomenon in cancer. We used an exon expression array to perform an objective, genome-wide survey of glioma-specific splicing in 24 GBM and 12 nontumor brain samples. Validation studies were performed using RT-PCR on glioma cell lines, patient tumor and nontumor brain samples., Results: In total, we confirmed 14 genes with glioma-specific splicing; seven were novel events identified by the exon expression array (A2BP1, BCAS1, CACNA1G, CLTA, KCNC2, SNCB, and TPD52L2). Our data indicate that large changes (> 5-fold) in alternative splicing are infrequent in gliomagenesis (< 3% of interrogated RefSeq entries). The lack of splicing changes may derive from the small number of splicing factors observed to be aberrantly expressed., Conclusion: While we observed some tumor-specific alternative splicing, the number of genes showing exclusive tumor-specific isoforms was on the order of tens, rather than the hundreds suggested previously by in silico mining. Given the important role of alternative splicing in neural differentiation, there may be selective pressure to maintain a majority of splicing events in order to retain glial-like characteristics of the tumor cells.

A personal history of asthma or allergy has been associated with a reduced risk for adult malignant gliomas. Recent reports on the use of nonsteroidal anti-inflammatory drugs (NSAID) and the presence of risk alleles in asthma susceptibility genes showed similar inverse associations. To further explore the relationship between immune mediators and gliomas, we examined the use of NSAID and antihistamines, history of asthma or allergy, and infection in 325 glioma cases and 600 frequency-matched controls from the metropolitan area of Houston, TX (2001-2006). The regular use of NSAID was associated with a 33% reduction in the risk for glioma, suggestive of possible antitumor activity. Surprisingly, regular long-term antihistamine use among those reporting a history of asthma or allergies was significantly associated with a 3.5-fold increase in the risk for glioma. Similar to previous reports, cases in our study were less likely to have reported asthma, allergy, or a history of a number of viral infections (chickenpox or shingles, oral herpes, and mononucleosis) than controls. We therefore speculate that the observed positive association with antihistamine use may reflect an alteration of protective immune factors in susceptible individuals. Our results lend additional support for an important but unknown link between malignant brain tumors and immune mediators.

The North American Brain Tumor Consortium (NABTC) uses 6-month progression-free survival (6moPFS) as the efficacy end point of therapy trials for adult patients with recurrent high-grade gliomas. In this study, we investigated whether progression status at 6 months predicts survival from that time, implying the potential for prolonged survival if progression could be delayed. We also evaluated earlier time points to determine whether the time of progression assessment alters the strength of the prediction. Data were from 596 patient enrollments (159 with grade III gliomas and 437 with grade IV tumors) in NABTC phase II protocols between February 1998 and December 2002. Outcome was assessed statistically using Kaplan-Meier curves and Cox proportional hazards models. Median survivals were 39 and 30 weeks for patients with grade III and grade IV tumors, respectively. Twenty-eight percent of patients with grade III and 16% of patients with grade IV tumors had progression-free survival of >26 weeks. Progression status at 9, 18, and 26 weeks predicted survival from those times for patients with grade III or grade IV tumors (p < 0.001 and hazard ratios < 0.5 in all cases). Including KPS, age, number of prior chemotherapies, and response in a multivariate model did not substantively change the results. Progression status at 6 months is a strong predictor of survival, and 6moPFS is a valid end point for trials of therapy for recurrent malignant glioma. Earlier assessments of progression status also predicted survival and may be incorporated in the design of future clinical trials.

Background: The Cancer Genome Atlas project (TCGA) has initiated the analysis of multiple samples of a variety of tumor types, starting with glioblastoma multiforme. The analytical methods encompass genomic and transcriptomic information, as well as demographic and clinical data about the sample donors. The data create the opportunity for a systematic screening of the components of the molecular machinery for features that may be associated with tumor formation. The wealth of existing mechanistic information about cancer cell biology provides a natural reference for the exploratory exercise., Methodology/principal Findings: Glioblastoma multiforme DNA copy number data was generated by The Cancer Genome Atlas project for 167 patients using 227 aCGH experiments, and was analyzed to build a catalog of aberrant regions. Genome screening was performed using an information theory approach in order to quantify aberration as a deviation from a centrality without the bias of untested assumptions about its parametric nature. A novel Cancer Genome Browser software application was developed and is made public to provide a user-friendly graphical interface in which the reported results can be reproduced. The application source code and stand alone executable are available at (http://code.google.com/p/cancergenome) and (http://bioinformaticstation.org), respectively., Conclusions/significance: The most important known copy number alterations for glioblastoma were correctly recovered using entropy as a measure of aberration. Additional alterations were identified in different pathways, such as cell proliferation, cell junctions and neural development. Moreover, novel candidates for oncogenes and tumor suppressors were also detected. A detailed map of aberrant regions is provided.

Purpose: To determine the maximum tolerated dose of irinotecan when administrated with temozolomide every 28 days, in patients with recurrent malignant glioma who were also receiving CYP450 enzyme-inducing antiepileptic drugs (EIAED), and to characterize the pharmacokinetics of irinotecan and its metabolites. The study was also intended to assess whether temozolomide affects the conversion of irinotecan to SN-38., Design: Patients with recurrent malignant glioma received a fixed dose of temozolomide (150 mg/m(2)) daily for 5 days from days 1 to 5 every 28 days, and an i.v. infusion of irinotecan on days 1 and 15 of each cycle. The starting dose of irinotecan was 350 mg/m(2), which was escalated to 550 mg/m(2) in 50-mg/m(2) increments. The plasma pharmacokinetics of irinotecan and its active metabolite, SN-38, were determined during the infusion of irinotecan on cycle 1, day 1., Results: Thirty-three patients were enrolled into the study and treated. Thirty-one patients were evaluable for both tumor response and toxicity and two patients were evaluable for toxicity only. Common toxicities included neutropenia and thrombocytopenia, nausea, vomiting, and diarrhea. Dose-limiting toxicities were grade 3 diarrhea and nausea/vomiting. The maximum tolerated dose for irinotecan was determined to be 500 mg/m(2)., Conclusions: The recommended phase II dose of irinotecan in combination with temozolomide for patients receiving EIAEDs is 500 mg/m(2), administrated every 15 days on a 28-day schedule. This study also confirmed that concomitant administration of EIAEDs increases irinotecan clearance and influences SN-38 disposition. No pharmacokinetic interaction was observed between temozolomide and irinotecan.

Purpose: Glioblastoma is the most common primary malignant tumor in the brain. It aggressively invades the surrounding parenchyma, often allowing the tumor to progress after surgery. Accumulating evidence has shown that phosphorylated p21-activated kinase 1 (Pak1), a mediator of small guanosine triphosphatases, plays a role in the proliferation, survival, and invasiveness of cancer cells. Thus, we examined patterns of Pak1 expression in glioblastoma and sought to determine whether the level of phosphorylated Pak1 in glioblastoma cells is associated with patient survival time., Experimental Design: We carried out immunohistochemical staining for phosphorylated Pak1 in tumor specimens from 136 patients with glioblastoma; the tumors were classified according to Pak1 protein levels in the cytoplasm and nucleus. We compared the patients' overall survival times using Kaplan-Meier analysis and estimated the effects of levels of cytoplasmic or nuclear phosphorylated Pak1. We then down-regulated Pak1 by using small interfering RNA to knock down Pak1 in two glioblastoma cell lines to determine whether Pak1 contributed to cell viability and invasion., Results: Median overall survival was significantly shorter in patients with tumors showing a moderate or high level of cytoplasmic phosphorylated Pak1 than in patients with tumors showing no cytoplasmic phosphorylated Pak1. The level of nuclear phosphorylated Pak1 was not related to survival time. Knockdown of Pak1 suppressed the invasion, but not the viability, of U87-MG and U373-MG cells., Conclusions: The presence of phosphorylated Pak1 in the cytoplasm of glioblastoma cells is associated with shorter survival, and Pak1 plays a role in the invasiveness of glioblastoma. These data suggest that Pak1 might be a potential target for the management of glioblastoma.

The purpose of this study was to assess the relationship between progression-free survival (PFS) in patients treated with DFMO + PCV (procarbazine, CCNU, vincristine) chemotherapy for malignant gliomas with tumor cell ornithine decarboxylase (ODC) activity. Formalin-fixed slides were obtained for study patients with anaplastic gliomas (AGs) and glioblastoma treated on protocol DM92-035. ODC levels were measured using an antibody to ODC coupled to Alexa 647 dye (Ab-ODC-Alexa 647). Ab-ODC-Alexa 647 intensity in transgenic murine hearts of differing ODC activity was used to calculate ODC activity in tumor cell nucleoplasm. In total, tumor specimens for 31 of 114 (27%) patients treated on the AG strata and 10 patients from the GBM strata were obtained. We found that tumor ODC level heterogeneity increased with increasing tumor malignancy. In a Cox proportional hazards model, PFS was found to be inversely related to median tumor ODC activity, with an unadjusted hazard ratio for median ODC group (>3.3 vs. 3.3 nmol/30 min/mug protein. Of AG tumors in which ODC activity was evaluated, 26% had ODC levels > 3.3 nmol/30 min/mug protein. This study shows that Ab-ODC-Alexa 647 fluorescence intensity can be used as a surrogate marker of ODC biochemical activity in AGs and can predict PFS to DFMO-based chemotherapy., ((c) 2007 Wiley-Liss, Inc.)

Background: Previous studies have been inconclusive in estimating the risk of different cancer sites among close relatives of glioma patients; however, malignant melanoma has consistently been described., Methods: We obtained family history information from 1,476 glioma patients under age 75 years who registered at M. D. Anderson Cancer Center between June 1992 and June 2006. The number of observed cancers (N=1,001) among 8,746 first-degree relatives (FDR) was compared with the number expected from age-, sex-, and calendar year-specific rates from the Surveillance, Epidemiology, and End Results Program using standardized incidence ratios (SIR)., Results: The overall SIR for any cancer was 1.21 (95% confidence interval, 1.14-1.29). Among FDRs under 45 years the overall SIR was 5.08, and for relatives >45 years the overall SIR was 0.95. The SIRs were significantly elevated for brain tumors (2.14), melanoma (2.02), and sarcoma (3.83). We observed an excess of pancreatic cancer, which was significantly higher only among mothers., Conclusion: We observed an overall 21% increase in cancer among the FDRs of glioma patients including excess cases of brain tumors and melanoma, which could point to similar genetic contributions to these two malignancies. A large international linkage study is under way to examine potential genomic regions important for familial glioma.

We recently showed that FoxM1 is overexpressed in human glioblastomas and that forced FoxM1B expression in anaplastic astrocytoma cells leads to the formation of highly invasive glioblastoma multiforme (GBM) in nude mice. However, the molecular mechanisms by which FoxM1 enhances glioma invasion are unknown. In this study, we found that FoxM1 overexpression increased matrix metalloproteinase (MMP)-2 expression in glioma cells, whereas blockade of FoxM1 expression suppressed MMP-2 expression. Transfection of FoxM1 into glioma cells directly activated the MMP-2 promoter, whereas inhibition of FoxM1 expression by FoxM1-siRNA suppressed its activation. We identified a FoxM1-binding site in the MMP-2 promoter and demonstrated that FoxM1 protein bound directly to it. Mutation of this FoxM1-binding site significantly attenuated MMP-2 promoter activity. Furthermore, FoxM1 overexpression increased the invasiveness of glioma cells, whereas inhibition of FoxM1 expression suppressed the invasiveness of GBM cells. Inhibition of MMP-2 by a specific MMP-2 inhibitor reversed the invasive phenotype of glioma cells overexpressing FoxM1. Finally, immunohistochemical analysis of 45 human GBM specimens showed a significant correlation between FoxM1 overexpression and elevated MMP-2 expression. Collectively, these findings provide evidence that FoxM1 contributes to glioma progression by enhancing MMP-2 gene transcription and thus tumor-cell invasion.

Human cancer genome and epigenome projects aim to identify new cancer genes and targets for therapy that have been overlooked by conventional approaches. Here we integrated large-scale genomics and epigenomics of 31 human infiltrative gliomas and identified low-frequency deletion and highly recurrent epigenetic silencing of WNK2, encoding a putative serine/threonine kinase. Prior cancer genome sequencing projects also identified point mutations in WNK1-4, suggesting that WNK family genes may have a role in cancers. We observed consistent gene silencing in tumors with dense aberrant methylation across 1.3 kb of the CpG island but more variable expression when the 5'-most region remained unmethylated. This primary tumor data fit well with WNK2 promoter analysis, which showed strong promoter activity in the 5'-most region, equivalent to the simian virus 40 promoter, but no activity in the 3' region. WT WNK2 exhibited autophosphorylation and protein kinase activity that was enhanced in cells exposed to hypertonic conditions, similar to WNK1. WNK2 inhibited up to 78% of colony formation by glioma cells but in an unexpectedly kinase-independent manner. The WNK2 silencing by epigenetic mechanisms was significantly associated (P < 0.01) with a known genetic signature of chemosensitive oligodendroglial tumors, 1p and 19q deletion, in two small but independent tumor sets. Taken together, the epigenetic silencing, occasional deletion and point mutation, and functional assessment suggest that aberrations of WNK2 may contribute to unregulated tumor cell growth. Thus, our integrated genetic and epigenetic approach might be useful to identify genes that are widely relevant to cancer, even when genetic alterations of the locus are infrequent.

The Rb/E2F pathway is deregulated in most human brain tumors, and the finding that loss of E2F1 reduced pituitary tumorigenesis in Rb(+/-) mice suggests that loss of pRb induces brain tumors by activating E2F1. We therefore investigated the role of E2F1 in the development and maintenance of brain cancer using a transgenic mouse model engineered to express E2F1 specifically within glial cells (GFAP-tgE2F1). GFAP-tgE2F1 mice developed a highly penetrant phenotype characterized by neurologic defects, and examination of the brains revealed the presence of brain tumors in 20% of these animals. Importantly, the distribution of tumors according to mouse age suggests the existence of a bimodal pattern of tumor development, forcing a comparison with the human disease. Mice, at an early age, with deregulated E2F1 show the formation of embryonal brain tumors such as medulloblastoma, choroid plexus carcinoma, and primary neuroectodermal tumor. Conversely, at an older age, mice escaping embryonal tumor formation present with malignant gliomas, which are typically identified in the human adult population. Thus, this study offers the first evidence for a global role of E2F1 in the formation and maintenance of multilineage brain tumors, irrefutably establishing E2F1 as an oncogene in the brain.

Purpose: Glioblastoma multiforme (GBM) is a devastating brain tumor with a median survival of 6 months after recurrence. Cintredekin besudotox (CB) is a recombinant protein consisting of interleukin-13 (IL-13) and a truncated form of Pseudomonas exotoxin (PE38QQR). Convection-enhanced delivery (CED) is a locoregional-administration method leading to high-tissue concentrations with large volume of distributions. We assessed the use of intracerebral CED to deliver CB in patients with recurrent malignant glioma (MG)., Patients and Methods: Three phase I clinical studies evaluated intracerebral CED of CB along with tumor resection. The main objectives were to assess the tolerability of various concentrations and infusion durations; tissue distribution; and methods for optimizing delivery. All patients underwent tumor resection followed by a single intraparenchymal infusion (in addition to the intraparenchymal one following resection), with a portion of patients who had a preresection intratumoral infusion., Results: A total of 51 patients with MG were treated including 46 patients with GBM. The maximum tolerated intraparenchymal concentration was 0.5 microg/mL and tumor necrosis was observed at this concentration. Infusion durations of up to 6 days were well tolerated. Postoperative catheter placement appears to be important for optimal drug distribution. CB- and procedure-related adverse events were primarily limited to the CNS. Overall median survival for GBM patients is 42.7 weeks and 55.6 weeks for patients with optimally positioned catheters with patient follow-up extending beyond 5 years., Conclusion: CB appears to have a favorable risk-benefit profile. CED is a complex delivery method requiring catheter placement via a second procedure to achieve accurate catheter positioning, better drug distribution, and better outcome.

Pilocytic astrocytomas (PAs) are the most common glioma in children. Whereas many PAs are slow-growing or clinically indolent, others exhibit more aggressive features with tumor recurrence and death. To identify genetic signatures that might predict PA clinical behavior, we did gene expression profiling on 41 primary PAs arising sporadically and in patients with neurofibromatosis type 1 (NF1). Whereas no expression signature was found that could discriminate clinically aggressive or recurrent tumors from more indolent cases, PAs arising in patients with NF1 did exhibit a unique gene expression pattern. In addition, we identified a gene expression signature that stratified PAs by location (supratentorial versus infratentorial). Lastly, we also identified a gene expression pattern common to PAs and normal mouse astrocytes and neural stem cells from these distinct brain regions as well as a gene expression pattern shared between PAs and another human glial tumor (ependymoma) arising supratentorially compared with those originating in the posterior fossa. These results suggest that glial tumors share an intrinsic, lineage-specific molecular signature that reflects the brain region in which their nonmalignant predecessors originated.

Global protein analysis of treated and untreated glioblastoma cell lines was performed. Proteomic analysis revealed the identity of proteins that were significantly modulated by the treatment with wild-type TP53 and the cytotoxic chemotherapy SN38. In particular, galectin-1 was found to be negatively regulated by transfection with TP53 and further down-regulated by SN38. Expression level changes were confirmed by Western blot. Subsequent analysis of several high-grade glioma cell lines demonstrated very high levels of galectin-1, regardless if the cell lines contained mutant or wild-type TP53. High expression of galectin-1 in a human orthotopic murine tumor model was also detected by immunohistochemistry and revealed a consistent pattern of preferential expression in peripheral or leading tumor edges. Further examination of galectin-1 expression through microarray analysis in tumor materials from patients confirmed galectin-1 as a valuable biomarker and possible therapeutic target. These results demonstrate the utility of using proteomic approaches to interrogate and identify potential useful targets for cancer therapy by evaluating specific tumor responses, either positive or negative, to various therapies.

The abnormal function of tyrosine kinase receptors is a hallmark of malignant gliomas. Tie2 receptor tyrosine kinase is a specific endothelial cell receptor whose function is positively regulated by angiopoietin 1 (Ang1). Recently, Tie2 has also been found in the nonvascular compartment of several tumors, including leukemia as well as breast, gastric, and thyroid cancers. There is, however, little information on the function of the Ang1/Tie2 pathway in the non-stromal cells within human tumors. We found that surgical glioblastoma specimens contained a subpopulation of Tie2+/CD31- and Tie2+/GFAP+ cells, suggesting that Tie2 is indeed expressed outside the vascular compartment of gliomas. Furthermore, analysis of a tissue array consisting of 116 human glioma samples showed that Tie2 expression in the neoplastic glial cells was significantly associated with progression from a lower to higher grade. Importantly, Ang1 stimulation of Tie2+ glioma cells resulted in increased adherence of the cells to collagen I and IV, suggesting that Tie2 regulates glioma cell adhesion to the extracellular matrix. Conversely, the down-regulation of Tie2 levels by small interference RNA or the addition of soluble Tie2 abrogated the Ang1-mediated effect on cell adhesion. In studying the expression of cell adhesion molecules, we found that Tie2 activation was related to the up-regulation of integrin beta1 levels and the formation of focal adhesions. These results, together with the reported fact that malignant gliomas express high levels of Ang1, suggest the existence of an autocrine loop in malignant gliomas and that a Tie2-dependent pathway modulates cell-to-extracellular matrix adhesion, providing new insights into the highly infiltrative phenotype of human gliomas.

Purpose: Since anaplastic gliomas (AG) depend on matrix metalloproteinases for tumor cell invasion and angiogenesis, we undertook this phase II study to evaluate the matrix metalloproteinase inhibitor marimastat (MT), combined with the alkylator temozolomide (TMZ) in patients with recurrent AG, looking for improved outcomes., Patients and Methods: Patients were treated every 28 days with TMZ at 150-200 mg/m2 once daily on days 1-5 and MT at 25 mg twice daily on days 8-28. Results were compared to our database of anaplastic glioma patients treated at recurrence. A subanalysis of the relationship between MT-induced joint-related toxicity and anticonvulsant status was carried out., Results: Forty-nine patients were enrolled; all were assessable for toxicity, and 46 were included in the efficacy analysis. Joint and muscle complaints occurred in 32 patients (65%); 1 patient was removed from the study due to toxicity. The best protocol response was a complete response in 1 patient and a partial response in 2 patients (3/46=7%, 95% confidence interval (CI)=1, 18). Median time to progression was 24 weeks (95% CI=16, 56), and the progression-free survival (PFS) rate was 48% at 6 months (95% CI=35%, 65%), which surpassed the protocol objective of 40%. Sub-analysis showed a positive impact of joint-related toxicity due to MT on PFS whether or not patients were taking CYP450 enzyme-inducing anticonvulsants., Conclusions: Even though this regimen is more efficacious than our comparator of historical controls in recurrent AG, the regimen was roughly equivalent to single-agent TMZ and was associated with additional toxicity. The sub-analysis suggests pharmacokinetic and drug-drug interactions which may positively impact responses to MT.

Gliomas account for almost 80% of primary malignant brain tumors, and they result in more years of life lost than do any other tumors. Glioblastoma, the most common type of glioma, is associated with very poor survival, so glioma epidemiology has focused on identifying factors that can be modified to prevent this disease. Only two relatively rare factors have so far been conclusively shown to affect glioma risk--exposure to high doses of ionizing radiation, and inherited mutations of highly penetrant genes associated with rare syndromes. In addition, preliminary evidence points to a lower glioma risk among people with allergic conditions and high levels of serum IgE. Recent research has focused on identifying germline polymorphisms associated with risk of glioma, and using molecular markers to classify glial tumors into more-homogenous groups. Because gene products probably interact with environmental factors or developmental signals to produce gliomas, large studies are needed to analyze associations between polymorphisms and glioma. Cohort studies of immune factors and glioma risk are being undertaken to validate the results of case-control studies. Studies of polymorphisms of genetic pathways with strong prior hypotheses are being planned, and whole-genome scans are being proposed to study high-risk families and case-control series. The Brain Tumor Epidemiology Consortium has been formed to co-ordinate these studies.

Purpose: It has previously been reported that the patient response to gefitinib depends on the presence of mutations within the kinase domain of epidermal growth factor receptor (EGFR) or the expression of its truncated form, EGFR variant III (EGFRvIII). The focus of this study was to determine if these alterations are present within the tyrosine kinase and ligand-binding domain of EGFR in urothelial carcinoma., Experimental Design: The kinase domain found within exons 18 to 21 of the EGFR from 11 bladder cancer cell lines and 75 patient tumors were subjected to automated sequencing. EGFRvIII expression was determined by immunohistochemistry using a urothelial carcinoma tissue microarray, and its expression was subsequently verified by reverse transcription PCR, real-time PCR, and Western blot analysis, using an EGFRvIII-transfected glioblastoma cell line and glioblastoma tumors as positive controls., Results: Our analysis failed to detect mutations within the tyrosine kinase domain of EGFR in the 11 cell lines and 75 patients tested. The initial analysis of EGFRvIII expression by immunohistochemistry revealed that at least 50% of the patient tumors expressed EGFRvIII in a urothelial carcinoma tissue microarray. Conflicting reports exist, however, regarding the extent of EGFRvIII expression in tissues owing to the specificity of the antibodies and the methodologies used. Therefore, we sought to validate this observation by reverse transcription PCR, real-time PCR, and Western blot analysis. In these assays, none of the samples were positive for EGFRvIII except for control transfectants and glioblastomas., Conclusions: When our results are taken together, we conclude that alterations within the tyrosine kinase domain and expression of EGFRvIII are rare events in bladder cancer. The present study has clinical implications in selecting tyrosine kinase inhibitors for the therapy of urothelial carcinoma.

Purpose: Activation of mitogen-activated protein kinase (MAPK) and members of the Akt pathway have been shown to promote cell proliferation, survival, and resistance to radiation. This study was conducted to determine whether any of these markers are associated with survival time and response to radiation in glioblastoma., Experimental Design: The expression of phosphorylated (p-)Akt, mammalian target of rapamycin (p-mTOR), p-p70S6K, and p-MAPK were assessed by immunohistochemical staining in 268 cases of newly diagnosed glioblastoma. YKL-40, a prognostic marker previously examined in these tumors, was also included in the analysis. Expression data were tested for correlations with response to radiation therapy in 131 subtotally resected cases and overall survival (in all cases). Results were validated in an analysis of 60 patients enrolled in clinical trials at a second institution., Results: Elevated p-MAPK expression was most strongly associated with poor response to radiotherapy, a finding corroborated in the validation cohort. For survival, higher expressions of p-mTOR, p-p70S6K, and p-MAPK were associated with worse outcome (all P < 0.03). YKL-40 expression was associated with the expressions of p-MAPK, p-mTOR, and p-p70S6K (all P < 0.02), with a trend toward association with p-Akt expression (P = 0.095). When known clinical variables were added to a multivariate analysis, only age, Karnofsky performance score, and p-MAPK expression emerged as independent prognostic factors., Conclusions: p-MAPK and activated members of the Akt pathway are markers of outcome in glioblastoma. Elevated expression of p-MAPK is associated with increased radiation resistance and represents an independent prognostic factor in these tumors.

Systematic analyses of the expression of angiogenic regulators in cancer models should yield useful information for the development of novel therapies for malignant gliomas. In this study, we analyzed tumor growth, vascularization, and angiopoietin-2 (Ang2) expression during the development of U-87 MG xenografts. We found that tumoral angiogenesis in this model follows a multistage process characterized by avascular, prolific peripheral angiogenesis, and late vascular phases. On day 4, we observed an area of central necrosis, a peripheral ring of Ang2-positive glioma cells, and reactive Ang2-positive vascular structures in the tumor/brain interface. When the tumor had developed a vascular network, Ang2 was expressed only in peripheral vascular structures. Because Ang2 expression was downmodulated in the late stages of development, probably to maintain a stable tumoral vasculature, we next studied whether sustained Ang2 expression might impair vascular development and, ultimately, tumor growth. Ang2 prevented the formation of capillary-like structures by and impaired angiogenesis in a chorioallantoic membrane chicken model. Finally, we tested the effect of sustained Ang2 expression on U-87 MG xenograft development. Ang2 significantly prolonged the survival of intracranial U-87 MG tumor-bearing animals. Examination of Ang2-treated xenografts revealed areas of tumor necrosis and vascular damage. We therefore conclude that deregulated Ang2 expression during gliomagenesis hindered successful angiogenesis and that therapies that sustain Ang2 expression might be effective against malignant gliomas.

Background: We previously identified brain type fatty acid-binding protein (FABP7) as a prognostic marker for patients with glioblastoma (GBM). Increased expression of FABP7 is associated with reduced survival. To investigate possible molecular mechanisms underlying this association, we compared the expression and subcellular localization of FABP7 in non-tumor brain tissues with different types of glioma, and examined the expression of FABP7 and epidermal growth factor receptor (EGFR) in GBM tumors., Methods: Expression of FABP7 in non-tumor brain and glioma specimens was examined using immunohistochemistry, and its correlation to the clinical behavior of the tumors was analyzed. We also analyzed the association between FABP7 and EGFR expression in different sets of GBM specimens using published DNA microarray datasets and semi-quantitative immunohistochemistry. In vitro migration was examined using SF763 glioma cell line., Results: FABP7 was present in a unique population of glia in normal human brain, and its expression was increased in a subset of reactive astrocytes. FABP7 immunoreactivity in grade I pilocytic astrocytoma was predominantly cytoplasmic, whereas nuclear FABP7 was detected in other types of infiltrative glioma. Nuclear, not cytoplasmic, FABP7 immunoreactivity was associated with EGFR overexpression in GBM (N = 61, p = 0.008). Expression of the FABP7 gene in GBM also correlated with the abundance of EGFR mRNA in our previous microarray analyses (N = 34, p = 0.016) and an independent public microarray dataset (N = 28, p = 0.03). Compared to those negative for both markers, nuclear FABP7-positive/EGFR-positive and nuclear FABP7-positive/EGFR-negative GBM tumors demonstrated shortest survival, whereas those only positive for EGFR had intermediate survival. EGFR activation increased nuclear FABP7 immunoreactivity in a glioma cell line in vitro, and inhibition of FABP7 expression suppressed EGF-induced glioma-cell migration. Our data suggested that in EGFR-positive GBM the presence of nuclear FABP7 immunoreactivity increases the risk of poor prognosis, Conclusion: In this study, we identified a possible mechanism as the basis of the association between nuclear FABP7 and poor prognosis of GBM. FABP7 expression can be found in all grades of astrocytoma, but neoplastic cells with nuclear FABP7 were only seen in infiltrative types of tumors. Nuclear FABP7 may be induced by EGFR activation to promote migration of GBM tumor cells. Positive nuclear FABP7 and EGFR overexpression correlated with short survival in EGFR-positive GBM patients. Therefore, nuclear FABP7 immunoreactivity could be used to monitor the progression of EGFR-overexpressed GBM.

The transcription factor Forkhead box M1 (FoxM1) is overexpressed in malignant glioma. However, the functional importance of this factor in human glioma is not known. In the present study, we found that FoxM1B was the predominant FoxM1 isoform expressed in human glioma but not in normal brain tissue. The level of FoxM1 protein expression in human glioma tissues was directly correlated with the glioma grade. The level of FoxM1 protein expression in human glioblastoma tissues was inversely correlated with patient survival. Enforced FoxM1B expression caused SW1783 and Hs683 glioma cells, which do not form tumor xenografts, to regain tumorigenicity in nude mouse model systems. Moreover, gliomas that arose from FoxM1B-transfected anaplastic astrocytoma SW1783 cells displayed glioblastoma multiforme phenotypes. Inhibition of FoxM1 expression in glioblastoma U-87MG cells suppressed their anchorage-independent growth in vitro and tumorigenicity in vivo. Furthermore, we found that FoxM1 regulates the expression of Skp2 protein, which is known to promote degradation of the cell cycle regulator p27(Kip1). These results showed that FoxM1 is overexpressed in human glioblastomas and contributes to glioma tumorigenicity. Therefore, FoxM1 might be a new potential target of therapy for human malignant gliomas.

Purpose: Glioblastoma multiforme (GBM) is the most common and aggressive glioma with the poorest survival. Use of biomarkers for screening patients with GBM may be used to modify treatments and improve outcomes. The level of human telomerase (hTERT) expression is an independent predictor of outcome of many cancers, and a functional variant of hTERT MNS16A (shorter tandem repeats or short [S] allele) is associated with increased hTERT mRNA expression. We investigated whether hTERT MNS16A variant genotype predicted survival in GBM patients., Patients and Methods: We genotyped hTERT MNS16A in 299 GBM patients using polymerase chain reaction and determined hTERT genotype by classifying the DNA band of 243 or 272 base pairs (bp) as S allele and 302 or 333 bp as long (L) allele. We compared overall survival using Kaplan-Meier estimates and equality of survival distributions using the log-rank test, and we computed univariate and multivariate Cox proportional hazards models to estimate the effects of selected variables., Results: Overall survival differed significantly by hTERT MNS16A genotype, with median survivals of 25.1, 14.7, and 14.6 months for the SS, SL, and LL genotypes, respectively. Compared with the SS genotype, the hazard ratios for the SL and LL genotypes were 1.69 and 1.87, respectively, after adjustment for other factors. Multivariate Cox regression analysis showed an independent statistically significant association between the hTERT MNS16A variant genotype and outcome., Conclusion: A functional hTERT MNS16A genotype is a potential biomarker for assessment of survival outcome of GBM. Larger studies are needed to verify these findings.

Brain metastasis is a major cause of morbidity and mortality in patients with melanoma. The molecular changes that lead to brain metastasis remain poorly understood. In this study, we developed a model to study human melanoma brain metastasis and found that Stat3 activity was increased in human brain metastatic melanoma cells when compared with that in cutaneous melanoma cells. The expression of activated Stat3 is also increased in human brain metastasis specimens when compared with that in the primary melanoma specimens. Increased Stat3 activation by transfection with a constitutively activated Stat3 enhanced brain metastasis, whereas blockade of Stat3 activation by transfection with a dominant-negative Stat3 suppressed brain metastasis of human melanoma cells in animal models. Furthermore, altered Stat3 activity profoundly affected melanoma angiogenesis in vivo and melanoma cell invasion in vitro and significantly affected the expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-2 (MMP-2) in vivo and in vitro. Finally, Stat3 activity transcriptionally regulated the promoter activity of bFGF in addition to VEGF and MMP-2 in human melanoma cells. These results indicated that Stat3 activation plays an important role in dysregulated expression of bFGF, VEGF, and MMP-2 as well as angiogenesis and invasion of melanoma cells and contributes to brain metastasis of melanoma. Therefore, Stat3 activation might be a new potential target for therapy of human melanoma brain metastases.

In this paper the authors highlight recent findings from molecular epidemiology studies of glioma origin and prognosis and suggest promising paths for future research. The reasons for variation in glioma incidence according to time period of diagnosis, sex, age, ancestry and ethnicity, and geography are poorly understood, as are factors that affect prognosis. High-dose therapeutic ionizing irradiation and rare mutations in highly penetrant genes associated with certain rare syndromes--the only two established causes of glioma--can be called upon to explain few cases. Both familial aggregation of gliomas and the inverse association of allergies and immune-related conditions with gliomas have been shown consistently, but the explanations for these associations are inadequately developed or unknown. Several biomarkers do predict prognosis, but only evaluation of loss of 1p and 19q in oligodendroglial tumors are incorporated in clinical practice. Ongoing research focuses on classifying homogeneous groups of tumors on the basis of molecular markers and identifying inherited polymorphisms that may influence survival or risk. Because most cases of glioma have yet to furnish either an environmental or a genetic explanation, the greatest potential for discovery may lie in genomic studies in conjunction with continued evaluation of environmental and developmental factors. Large sample sizes and multidisciplinary teams with expertise in neuropathology, genetics, epidemiology, functional genomics, bioinformatics, biostatistics, immunology, and neurooncology are required for these studies to permit exploration of potentially relevant pathways and modifying effects of other genes or exposures, and to avoid false-positive findings. Improving survival rates for patients harboring astrocytic tumors will probably require many randomized clinical trials of novel treatment strategies.

Purpose: YKL-40 is a secreted protein that has been reported to be overexpressed in epithelial cancers and gliomas, although its function is unknown. Previous data in a smaller sample set suggested that YKL-40 was a marker associated with a poorer clinical outcome and a genetically defined subgroup of glioblastoma. Here we test these findings in a larger series of patients with glioblastoma, and in particular, determine if tumor YKL-40 expression is associated with radiation response., Experimental Design: Patients (n=147) with subtotal resections were studied for imaging-assessed changes in tumor size in serial studies following radiation therapy. An additional set (n=140) of glioblastoma patients who underwent a gross-total resection was tested to validate the survival association and extend them to patients with minimal residual disease., Results: In the subtotal resection group, higher YKL-40 expression was significantly associated with poorer radiation response, shorter time to progression and shorter overall survival. The association of higher YKL-40 expression with poorer survival was validated in the gross-total resection group. In multivariate analysis with both groups combined (n = 287), YKL-40 was an independent predictor of survival after adjusting for patient age, performance status, and extent of resection. YKL-40 expression was also compared with genetically defined subsets of glioblastoma by assessing epidermal growth factor receptor amplification and loss at chromosome 10q, two of the common recurring aberrations in these tumors, using fluorescent in situ hybridization. YKL-40 was significantly associated with 10q loss., Conclusions: The findings implicate YKL-40 as an important marker of therapeutic response and genetic subtype in glioblastomas and suggest that it may play an oncogenic role in these tumors.

Glioblastoma multiforme (GBM) is the most common form of malignant glioma, characterized by genetic instability, intratumoral histopathological variability, and unpredictable clinical behavior. We investigated global gene expression in surgical samples of brain tumors. Gene expression profiling revealed large differences between normal brain samples and tumor tissues and between GBMs and lower-grade oligodendroglial tumors. Extensive differences in gene expression were found among GBMs, particularly in genes involved in angiogenesis, immune cell infiltration, and extracellular matrix remodeling. We found that the gene expression patterns in paired specimens from the same GBM invariably were more closely related to each other than to any other tumor, even when the paired specimens had strikingly divergent histologies. Survival analyses revealed a set of approximately 70 genes more highly expressed in rapidly progressing tumors that stratified GBMs into two groups that differed by >4-fold in median duration of survival. We further investigated one gene from the group, FABP7, and confirmed its association with survival in two unrelated cohorts totaling 105 patients. Expression of FABP7 enhanced the motility of glioma-derived cells in vitro. Our analyses thus identify and validate a prognostic marker of both biologic and clinical significance and provide a series of putative markers for additional evaluation.

Glioblastoma, the most aggressive primary brain tumor in humans, exhibits a large degree of molecular heterogeneity. Understanding the molecular pathology of a tumor and its linkage to behavior is an important foundation for developing and evaluating approaches to clinical management. Here we integrate array-comparative genomic hybridization and array-based gene expression profiles to identify relationships between DNA copy number aberrations, gene expression alterations, and survival in 34 patients with glioblastoma. Unsupervised clustering on either profile resulted in similar groups of patients, and groups defined by either method were associated with survival. The high concordance between these separate molecular classifications suggested a strong association between alterations on the DNA and RNA levels. We therefore investigated relationships between DNA copy number and gene expression changes. Loss of chromosome 10, a predominant genetic change, was associated not only with changes in the expression of genes located on chromosome 10 but also with genome-wide differences in gene expression. We found that CHI3L1/YKL-40 was significantly associated with both chromosome 10 copy number loss and poorer survival. Immortalized human astrocytes stably transfected with CHI3L1/YKL-40 exhibited changes in gene expression similar to patterns observed in human tumors and conferred radioresistance and increased invasion in vitro. Taken together, the results indicate that integrating DNA and mRNA-based tumor profiles offers the potential for a clinically relevant classification more robust than either method alone and provides a basis for identifying genes important in glioma pathogenesis.

Purpose: Angiogenic behavior is a critical aspect of cancer biology and subject to regulation by multiple molecular pathways. Because the signal transducer and activator of transcription 3 (Stat3) transcription factor regulates multiple genes important to angiogenesis, we sought to determine whether Stat3 expression is related to vascular endothelial growth factor (VEGF) expression and microvessel density (MVD) in gastric cancer and whether these factors predict survival in gastric cancer patients., Experimental Design: The expression of Stat3 and VEGF was determined by immunohistochemistry using archival tissues from 86 cases of resected human gastric cancer and confirmed by Western blot analysis. Angiogenic phenotype was determined by CD34 staining and microvessel counting., Results: Stat3 expression correlated with VEGF expression and MVD. In univariate survival analyses, Stat3 expression (P = 0.013) and MVD (P = 0.036) were associated with inferior survival. However, when Stat3 expression, VEGF expression, MVD, stage, completeness of resection, Lauren's histologic classification, and age were entered into a Cox proportional hazards model, only strong Stat3 expression (P = 0.049) and advanced stage (P < 0.01) were independently prognostic of poor survival. Furthermore, genetically enforced alterations of activated Stat3 expression led to altered VEGF expression and angiogenic potential in human gastric cancer cells., Conclusion: Dysregulated Stat3 activation may play an important role in VEGF overexpression and elevated angiogenic phenotype in gastric cancer and contribute to gastric cancer development and progression.

Background: Small cell glioblastoma (GBM) is a variant with monomorphous, deceptively bland nuclei that often is misdiagnosed as anaplastic oligodendroglioma., Methods: To elucidate its clinicopathologic and genetic features, the authors studied 71 adult patients (median age, 57 years), including 22 patients who were identified from a set of 229 GBMs (10%) that had been characterized previously by epidermal growth factor receptor (EGFR)/EGFR-vIII variant immunohistochemistry. Tumors also were analyzed by fluorescence in situ hybridization for 1p, 19q, 10q, and EGFR copy numbers., Results: Radiologically, 37% of tumors that were not selected for grade showed minimal to no enhancement. Similarly, 33% of tumors had no endothelial hyperplasia or necrosis histologically, qualifying only as anaplastic astrocytoma (Grade III) using World Health Organization criteria. Nevertheless, such tumors progressed rapidly, with mortality rates that were indistinguishable from their Grade IV counterparts. The median survival for 37 patients who were followed until death was 11 months. Oligodendroglioma-like histology included chicken-wire vasculature (86%), haloes (73%), perineuronal satellitosis (58%), and microcalcifications (45%), although mucin-filled microcystic spaces were lacking. No small cell astrocytomas had 1p/19q codeletions, whereas EGFR amplification and 10q deletions were present in 69% and 97% of small cell astrocytomas, respectively. The tumors expressed EGFR and EGFR-vIII more commonly than nonsmall cell GBMs (83% vs. 35% [P < 0.001]; 50% vs. 21% [P < 0.001] respectively)., Conclusions: Small cell astrocytoma is an aggressive histologic variant that behaved like primary GBM, even in the absence of endothelial hyperplasia and necrosis. Despite considerable morphologic overlap with anaplastic oligodendroglioma, clinicopathologic and genetic features were distinct. Fifty percent of small cell astrocytomas expressed the constitutively activated vIII mutant form of EGFR, and molecular testing for 10q deletion improved the diagnostic sensitivity over EGFR alone., ((c) 2004 American Cancer Society)