Darell D. Bigner

Cancers arise owing to the accumulation of mutations in critical genes that alter normal programmes of cell proliferation, differentiation and death. As the first stage of a systematic genome-wide screen for these genes, we have prioritized for analysis signalling pathways in which at least one gene is mutated in human cancer. The RAS–RAF–MEK–ERK–MAP kinase pathway mediates cellular responses to growth signals1. RAS is mutated to an oncogenic form in about 15% of human cancer. The three RAF genes code for cytoplasmic serine/threonine kinases that are regulated by binding RAS1,2,3. Here we report BRAF somatic missense mutations in 66% of malignant melanomas and at lower frequency in a wide range of human cancers. All mutations are within the kinase domain, with a single substitution (V599E) accounting for 80%. Mutated BRAF proteins have elevated kinase activity and are transforming in NIH3T3 cells. Furthermore, RAS function is not required for the growth of cancer cell lines with the V599E mutation. As BRAF is a serine/threonine kinase that is commonly activated by somatic point mutation in human cancer, it may provide new therapeutic opportunities in malignant melanoma.

Glioblastoma multiforme (GBM) is the most common and lethal type of brain cancer. To identify the genetic alterations in GBMs, we sequenced 20,661 protein coding genes, determined the presence of amplifications and deletions using high-density oligonucleotide arrays, and performed gene expression analyses using next-generation sequencing technologies in 22 human tumor samples. This comprehensive analysis led to the discovery of a variety of genes that were not known to be altered in GBMs. Most notably, we found recurrent mutations in the active site of isocitrate dehydrogenase 1 ( IDH1 ) in 12% of GBM patients. Mutations in IDH1 occurred in a large fraction of young patients and in most patients with secondary GBMs and were associated with an increase in overall survival. These studies demonstrate the value of unbiased genomic analyses in the characterization of human brain cancer and identify a potentially useful genetic alteration for the classification and targeted therapy of GBMs.

A recent genomewide mutational analysis of glioblastomas (World Health Organization [WHO] grade IV glioma) revealed somatic mutations of the isocitrate dehydrogenase 1 gene (IDH1) in a fraction of such tumors, most frequently in tumors that were known to have evolved from lower-grade gliomas (secondary glioblastomas).

We describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

Abstract Cancer is driven by genetic change, and the advent of massively parallel sequencing has enabled systematic documentation of this variation at the whole-genome scale 1–3 . Here we report the integrative analysis of 2,658 whole-cancer genomes and their matching normal tissues across 38 tumour types from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). We describe the generation of the PCAWG resource, facilitated by international data sharing using compute clouds. On average, cancer genomes contained 4–5 driver mutations when combining coding and non-coding genomic elements; however, in around 5% of cases no drivers were identified, suggesting that cancer driver discovery is not yet complete. Chromothripsis, in which many clustered structural variants arise in a single catastrophic event, is frequently an early event in tumour evolution; in acral melanoma, for example, these events precede most somatic point mutations and affect several cancer-associated genes simultaneously. Cancers with abnormal telomere maintenance often originate from tissues with low replicative activity and show several mechanisms of preventing telomere attrition to critical levels. Common and rare germline variants affect patterns of somatic mutation, including point mutations, structural variants and somatic retrotransposition. A collection of papers from the PCAWG Consortium describes non-coding mutations that drive cancer beyond those in the TERT promoter 4 ; identifies new signatures of mutational processes that cause base substitutions, small insertions and deletions and structural variation 5,6 ; analyses timings and patterns of tumour evolution 7 ; describes the diverse transcriptional consequences of somatic mutation on splicing, expression levels, fusion genes and promoter activity 8,9 ; and evaluates a range of more-specialized features of cancer genomes 8,10–18 .

The prognosis for patients with recurrent glioblastoma multiforme is poor, with a median survival of 3 to 6 months. We performed a phase II trial of bevacizumab, a monoclonal antibody to vascular endothelial growth factor, in combination with irinotecan.This phase II trial included two cohorts of patients. The initial cohort, comprising 23 patients, received bevacizumab at 10 mg/kg plus irinotecan every 2 weeks. The dose of irinotecan was based on the patient's anticonvulsant: Patients taking enzyme-inducing antiepileptic drugs (EIAEDs) received 340 mg/m2, and patients not taking EIAEDs received 125 mg/m2. After this regimen was deemed safe and effective, the irinotecan schedule was changed to an accepted brain tumor regimen of four doses in 6 weeks, in anticipation of a phase III randomized trial of irinotecan versus irinotecan and bevacizumab. The second cohort, comprising 12 patients, received bevacizumab 15 mg/kg every 21 days and irinotecan on days 1, 8, 22, and 29. Each cycle was 6 weeks long and concluded with patient evaluations, including magnetic resonance imaging.The 6-month progression-free survival among all 35 patients was 46% (95% CI, 32% to 66%). The 6-month overall survival was 77% (95% CI, 64% to 92%). Twenty of the 35 patients (57%; 95% CI, 39% to 74%) had at least a partial response. One patient developed a CNS hemorrhage, which occurred in his 10th cycle. Four patients developed thromboembolic complications (deep venous thrombosis and/or pulmonary emboli).Bevacizumab and irinotecan is an effective treatment for recurrent glioblastoma multiforme and has moderate toxicity.

Malignant gliomas are highly lethal cancers dependent on angiogenesis. Critical tumor subpopulations within gliomas share characteristics with neural stem cells. We examined the potential of stem cell-like glioma cells (SCLGC) to support tumor angiogenesis. SCLGC isolated from human glioblastoma biopsy specimens and xenografts potently generated tumors when implanted into the brains of immunocompromised mice, whereas non-SCLGC tumor cells isolated from only a few tumors formed secondary tumors when xenotransplanted. Tumors derived from SCLGC were morphologically distinguishable from non-SCLGC tumor populations by widespread tumor angiogenesis, necrosis, and hemorrhage. To determine a potential molecular mechanism for SCLGC in angiogenesis, we measured the expression of a panel of angiogenic factors secreted by SCLGC. In comparison with matched non-SCLGC populations, SCLGC consistently secreted markedly elevated levels of vascular endothelial growth factor (VEGF), which were further induced by hypoxia. In an in vitro model of angiogenesis, SCLGC-conditioned medium significantly increased endothelial cell migration and tube formation compared with non-SCLGC tumor cell-conditioned medium. The proangiogenic effects of glioma SCLGC on endothelial cells were specifically abolished by the anti-VEGF neutralizing antibody bevacizumab, which is in clinical use for cancer therapy. Furthermore, bevacizumab displayed potent antiangiogenic efficacy in vivo and suppressed growth of xenografts derived from SCLGC but limited efficacy against xenografts derived from a matched non-SCLGC population. Together these data indicate that stem cell-like tumor cells can be a crucial source of key angiogenic factors in cancers and that targeting proangiogenic factors from stem cell-like tumor populations may be critical for patient therapy.

Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked ( ATRX ) or death-domain associated protein ( DAXX ) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase ( TERT ) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.

Abstract Purpose: Recurrent grade III-IV gliomas have a dismal prognosis with minimal improvements in survival seen following currently available salvage therapy. This study was conducted to determine if the combination of a novel antiangiogenic therapy, bevacizumab, and a cytotoxic agent, irinotecan, is safe and effective for patients with recurrent grade III-IV glioma. Experimental Design: We conducted a phase II trial of bevacizumab and irinotecan in adults with recurrent grade III-IV glioma. Patients with evidence of intracranial hemorrhage on initial brain magnetic resonance imaging were excluded. Patients were scheduled to receive bevacizumab and irinotecan i.v. every 2 weeks of a 6-week cycle. Bevacizumab was administered at 10 mg/kg. The dose of irinotecan was determined based on antiepileptic use: patients taking enzyme-inducing antiepileptic drugs received 340 mg/m2, whereas patients not taking enzyme-inducing antiepileptic drugs received 125 mg/m2. Toxicity and response were assessed. Results: Thirty-two patients were assessed (23 with grade IV glioma and 9 with grade III glioma). Radiographic responses were noted in 63% (20 of 32) of patients (14 of 23 grade IV patients and 6 of 9 grade III patients). The median progression-free survival was 23 weeks for all patients (95% confidence interval, 15-30 weeks; 20 weeks for grade IV patients and 30 weeks for grade III patients). The 6-month progression-free survival probability was 38% and the 6-month overall survival probability was 72%. No central nervous system hemorrhages occurred, but three patients developed deep venous thromboses or pulmonary emboli, and one patient had an arterial ischemic stroke. Conclusions: The combination of bevacizumab and irinotecan is an active regimen for recurrent grade III-IV glioma with acceptable toxicity.

Chromosome tips seem to be maintained by an unusual mechanism in tumors that have mutations in chromatin remodeling genes.

Primary malignant gliomas from 63 patients were analyzed to determine the relationship between amplification of the gene encoding the epidermal growth factor receptor (EGFR) and expression of the corresponding mRNA. Twenty-four tumors were found to have amplified the EGFR gene and amplification of other genes occurred in three additional tumors. Hybridization with synthetic RNA probes was used to quantitate mRNA levels in situ. All 24 tumors with amplification of the EGFR gene had high levels of expression of this gene, while none of the 39 tumors without amplification had increased levels. This shows that, in human gliomas, large increases in the expression of the EGFR gene are invariably associated with alterations in gene structure.

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.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.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).EGFRvIII-targeted vaccination in patients with GBM warrants investigation in a phase III, randomized trial.

To evaluate the efficacy and tolerability of gefitinib (ZD1839, Iressa; AstraZeneca, Wilmington, DE), a novel epidermal growth factor receptor tyrosine kinase inhibitor, in patients with recurrent glioblastoma.This was an open-label, single-center phase II trial. Fifty-seven patients with first recurrence of a glioblastoma who were previously treated with surgical resection, radiation, and usually chemotherapy underwent an open biopsy or resection at evaluation for confirmation of tumor recurrence. Each patient initially received 500 mg of gefitinib orally once daily; dose escalation to 750 mg then 1,000 mg, if a patient received enzyme-inducing antiepileptic drugs or dexamethasone, was allowed within each patient.Although no objective tumor responses were seen among the 53 assessable patients, only 21% of patients (11 of 53 patients) had measurable disease at treatment initiation. Seventeen percent of patients (nine of 53 patients) underwent at least six 4-week cycles, and the 6-month event-free survival (EFS) was 13% (seven of 53 patients). The median EFS time was 8.1 weeks, and the median overall survival (OS) time from treatment initiation was 39.4 weeks. Adverse events were generally mild (grade 1 or 2) and consisted mainly of skin reactions and diarrhea. Drug-related toxicities were more frequent at higher doses. Withdrawal caused by drug-related adverse events occurred in 6% of patients (three of 53 patients). Although the presence of diarrhea positively predicted favorable OS from treatment initiation, epidermal growth factor receptor expression did not correlate with either EFS or OS.Gefitinib is well tolerated and has activity in patients with recurrent glioblastoma. Further study of this agent at higher doses is warranted.

Medulloblastoma (MB) is the most common malignant brain tumor of children. To identify the genetic alterations in this tumor type, we searched for copy number alterations using high-density microarrays and sequenced all known protein-coding genes and microRNA genes using Sanger sequencing in a set of 22 MBs. We found that, on average, each tumor had 11 gene alterations, fewer by a factor of 5 to 10 than in the adult solid tumors that have been sequenced to date. In addition to alterations in the Hedgehog and Wnt pathways, our analysis led to the discovery of genes not previously known to be altered in MBs. Most notably, inactivating mutations of the histone-lysine N-methyltransferase genes MLL2 or MLL3 were identified in 16% of MB patients. These results demonstrate key differences between the genetic landscapes of adult and childhood cancers, highlight dysregulation of developmental pathways as an important mechanism underlying MBs, and identify a role for a specific type of histone methylation in human tumorigenesis.

// Yuchen Jiao 1,* , Patrick J. Killela 2,* , Zachary J. Reitman 2,* , B. Ahmed Rasheed 2 , Christopher M. Heaphy 1 , Roeland F. de Wilde 1 , Fausto J. Rodriguez 1 , Sergio Rosemberg 3 , Sueli Mieko Oba-Shinjo 3 , Suely Kazue Nagahashi Marie 3 , Chetan Bettegowda 1 , Nishant Agrawal 1 , Eric Lipp 2 , Christopher J. Pirozzi 2 , Giselle Y. Lopez 2 , Yiping He 2 , Henry S. Friedman 2 , Allan H. Friedman 2 , Gregory J. Riggins 1 , Matthias Holdhoff 1,4 , Peter Burger 1 , Roger E. McLendon 2 , Darell D. Bigner 2 , Bert Vogelstein 1 , Alan K. Meeker 1 , Kenneth W. Kinzler 1 , Nickolas Papadopoulos 1 , Luis A. Diaz Jr 1,4 , Hai Yan 2 1 Ludwig Center for Cancer Genetics and Howard Hughes Medical Institutions, The Johns Hopkins Kimmel Cancer Center, the Department of Oncology, the Department of Pathology, the Department of Neurosurgery, the Johns Hopkins Medical Institutions, Baltimore, Maryland, USA 2 The Preston Robert Tisch Brain Tumor Center at Duke, The Pediatric Brain Tumor Foundation Institute, the Department of Pathology, the Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA 3 The Department of Pathology, the Department of Neurology, School of Medicine, University of Sao Paulo, Sao Paulo, Sao Paulo, Brazil 4 The Swim Across America Laboratory at Johns Hopkins, The Johns Hopkins Medical Institutions, Baltimore, Maryland, USA * Denotes equal contribution Correspondence: Hai Yan, email: // Luis Diaz, email: // Keywords : ALT, IDH1, IDH2, Mixed Gliomas Received : July 31, 2012, Accepted : August 2, 2012, Published : August 3, 2012 Abstract Mutations in the critical chromatin modifier ATRX and mutations in CIC and FUBP1 , which are potent regulators of cell growth, have been discovered in specific subtypes of gliomas, the most common type of primary malignant brain tumors. However, the frequency of these mutationsin many subtypes of gliomas, and their association with clinical features of the patients, is poorly understood. Here we analyzed these loci in 363 brain tumors. ATRX is frequently mutated in grade II-III astrocytomas (71%), oligoastrocytomas (68%), and secondary glioblastomas (57%), and ATRX mutations are associated with IDH1 mutations and with an alternative lengthening of telomeres phenotype. CIC and FUBP1 mutations occurred frequently in oligodendrogliomas (46% and 24%, respectively) but rarely in astrocytomas or oligoastrocytomas (&lt;10%). This analysis allowed us to define two highly recurrent genetic signatures in gliomas: IDH1/ATRX (I-A) and IDH1/CIC/FUBP1 (I-CF). Patients with I-CF gliomas had a significantly longer median overall survival (96 months) than patients with I-A gliomas (51 months) and patients with gliomas that did not harbor either signature (13 months). The genetic signatures distinguished clinically distinct groups of oligoastrocytoma patients, which usually present a diagnostic challenge, and were associated with differences in clinical outcome even among individual tumor types. In addition to providing new clues about the genetic alterations underlying gliomas, the results have immediate clinical implications, providing a tripartite genetic signature that can serve as a useful adjunct to conventional glioma classification that may aid in prognosis, treatment selection, and therapeutic trial design.

Oligodendrogliomas are the second most common malignant brain tumor in adults and exhibit characteristic losses of chromosomes 1p and 19q. To identify the molecular genetic basis for this alteration, we performed exomic sequencing of seven tumors. Among other changes, we found that the CIC gene (homolog of the Drosophila gene capicua) on chromosome 19q was somatically mutated in six cases and that the FUBP1 gene [encoding far-upstream element (FUSE) binding protein] on chromosome 1p was somatically mutated in two tumors. Examination of 27 additional oligodendrogliomas revealed 12 and 3 more tumors with mutations of CIC and FUBP1, respectively, 58% of which were predicted to result in truncations of the encoded proteins. These results suggest a critical role for these genes in the biology and pathology of oligodendrocytes.

Anna Lasorella, Raul Rabadan, Antonio Iavarone and colleagues report an integrated analysis of genomic alterations in glioblastoma. They identify and functionally validate several new driver events, including loss-of-function mutations in CTNND2 and recurrent EGFR fusions. Glioblastoma is one of the most challenging forms of cancer to treat. Here we describe a computational platform that integrates the analysis of copy number variations and somatic mutations and unravels the landscape of in-frame gene fusions in glioblastoma. We found mutations with loss of heterozygosity in LZTR1, encoding an adaptor of CUL3-containing E3 ligase complexes. Mutations and deletions disrupt LZTR1 function, which restrains the self renewal and growth of glioma spheres that retain stem cell features. Loss-of-function mutations in CTNND2 target a neural-specific gene and are associated with the transformation of glioma cells along the very aggressive mesenchymal phenotype. We also report recurrent translocations that fuse the coding sequence of EGFR to several partners, with EGFR-SEPT14 being the most frequent functional gene fusion in human glioblastoma. EGFR-SEPT14 fusions activate STAT3 signaling and confer mitogen independence and sensitivity to EGFR inhibition. These results provide insights into the pathogenesis of glioblastoma and highlight new targets for therapeutic intervention.

Epidermal growth factor receptor (EGFR) is overexpressed in head and neck squamous cell carcinoma (HNSCC) where expression levels correlate with decreased survival. Therapies that block EGFR have shown limited efficacy in clinical trials and primarily when combined with standard therapy. The most common form of mutant EGFR (EGFRvIII) has been described in several cancers, chiefly glioblastoma. The present study was undertaken to determine the incidence of EGFRvIII expression in HNSCC and the biological consequences of EGFRvIII on tumor growth in response to EGFR targeting.Thirty-three HNSCC tumors were evaluated by immunostaining and reverse transcription-PCR for EGFRvIII expression. A representative HNSCC cell line was stably transfected with an EGFRvIII expression construct. EGFRvIII-expressing cells and vector-transfected controls were compared for growth rates in vitro and in vivo as well as chemotherapy-induced apoptosis and the consequences of EGFR inhibition using the chimeric monoclonal antibody C225/cetuximab/Erbitux.EGFRvIII expression was detected in 42% of HNSCC tumors where EGFRvIII was always found in conjunction with wild-type EGFR. HNSCC cells expressing EGFRvIII showed increased proliferation in vitro and increased tumor volumes in vivo compared with vector-transfected controls. Furthermore, EGFRvIII-transfected HNSCC cells showed decreased apoptosis in response to cisplatin and decreased growth inhibition following treatment with C225 compared with vector-transfected control cells.EGFRvIII is expressed in HNSCC where it contributes to enhanced growth and resistance to targeting wild-type EGFR. The antitumor efficacy of EGFR targeting strategies may be enhanced by the addition of EGFRvIII-specific blockade.

Point mutations of the NADP + -dependent isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) occur early in the pathogenesis of gliomas. When mutated, IDH1 and IDH2 gain the ability to produce the metabolite ( R )-2-hydroxyglutarate (2HG), but the downstream effects of mutant IDH1 and IDH2 proteins or of 2HG on cellular metabolism are unknown. We profiled &gt;200 metabolites in human oligodendroglioma (HOG) cells to determine the effects of expression of IDH1 and IDH2 mutants. Levels of amino acids, glutathione metabolites, choline derivatives, and tricarboxylic acid (TCA) cycle intermediates were altered in mutant IDH1- and IDH2-expressing cells. These changes were similar to those identified after treatment of the cells with 2HG. Remarkably, N -acetyl-aspartyl-glutamate (NAAG), a common dipeptide in brain, was 50-fold reduced in cells expressing IDH1 mutants and 8.3-fold reduced in cells expressing IDH2 mutants. NAAG also was significantly lower in human glioma tissues containing IDH mutations than in gliomas without such mutations. These metabolic changes provide clues to the pathogenesis of tumors associated with IDH gene mutations.

The epidermal growth factor receptor variant III deletion mutation, EGFRvIII, is expressed in ∼30% of primary glioblastoma and linked to poor long-term survival. Rindopepimut consists of the unique EGFRvIII peptide sequence conjugated to keyhole limpet hemocyanin. In previous phase II trials (ACTIVATE/ACT II), rindopepimut was well tolerated with robust EGFRvIII-specific immune responses and promising progression-free and overall survival. This multicenter, single-arm phase II clinical trial (ACT III) was performed to confirm these results.Rindopepimut and standard adjuvant temozolomide chemotherapy were administered to 65 patients with newly diagnosed EGFRvIII-expressing (EGFRvIII+) glioblastoma after gross total resection and chemoradiation.Progression-free survival at 5.5 months (∼8.5 mo from diagnosis) was 66%. Relative to study entry, median overall survival was 21.8 months, and 36-month overall survival was 26%. Extended rindopepimut vaccination (up to 3.5+ years) was well tolerated. Grades 1-2 injection site reactions were frequent. Anti-EGFRvIII antibody titers increased ≥4-fold in 85% of patients, and increased with duration of treatment. EGFRvIII was eliminated in 4/6 (67%) tumor samples obtained after >3 months of therapy.This study confirms, in a multicenter setting, the preliminary results seen in previous phase II trials of rindopepimut. A pivotal, double-blind, randomized, phase III trial ("ACT IV") is under way.

Patients with incomplete surgical resection of medulloblastoma are controversially regarded as having a marker of high-risk disease, which leads to patients undergoing aggressive surgical resections, so-called second-look surgeries, and intensified chemoradiotherapy. All previous studies assessing the clinical importance of extent of resection have not accounted for molecular subgroup. We analysed the prognostic value of extent of resection in a subgroup-specific manner.We retrospectively identified patients who had a histological diagnosis of medulloblastoma and complete data about extent of resection and survival from centres participating in the Medulloblastoma Advanced Genomics International Consortium. We collected from resections done between April, 1997, and February, 2013, at 35 international institutions. We established medulloblastoma subgroup affiliation by gene expression profiling on frozen or formalin-fixed paraffin-embedded tissues. We classified extent of resection on the basis of postoperative imaging as gross total resection (no residual tumour), near-total resection (<1·5 cm(2) tumour remaining), or sub-total resection (≥1·5 cm(2) tumour remaining). We did multivariable analyses of overall survival and progression-free survival using the variables molecular subgroup (WNT, SHH, group 4, and group 3), age (<3 vs ≥3 years old), metastatic status (metastases vs no metastases), geographical location of therapy (North America/Australia vs rest of the world), receipt of chemotherapy (yes vs no) and receipt of craniospinal irradiation (<30 Gy or >30 Gy vs no craniospinal irradiation). The primary analysis outcome was the effect of extent of resection by molecular subgroup and the effects of other clinical variables on overall and progression-free survival.We included 787 patients with medulloblastoma (86 with WNT tumours, 242 with SHH tumours, 163 with group 3 tumours, and 296 with group 4 tumours) in our multivariable Cox models of progression-free and overall survival. We found that the prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. We identified a progression-free survival benefit for gross total resection over sub-total resection (hazard ratio [HR] 1·45, 95% CI 1·07-1·96, p=0·16) but no overall survival benefit (HR 1·23, 0·87-1·72, p=0·24). We saw no progression-free survival or overall survival benefit for gross total resection compared with near-total resection (HR 1·05, 0·71-1·53, p=0·8158 for progression-free survival and HR 1·14, 0·75-1·72, p=0·55 for overall survival). No significant survival benefit existed for greater extent of resection for patients with WNT, SHH, or group 3 tumours (HR 1·03, 0·67-1·58, p=0·89 for sub-total resection vs gross total resection). For patients with group 4 tumours, gross total resection conferred a benefit to progression-free survival compared with sub-total resection (HR 1·97, 1·22-3·17, p=0·0056), especially for those with metastatic disease (HR 2·22, 1·00-4·93, p=0·050). However, gross total resection had no effect on overall survival compared with sub-total resection in patients with group 4 tumours (HR 1·67, 0·93-2·99, p=0·084).The prognostic benefit of increased extent of resection for patients with medulloblastoma is attenuated after molecular subgroup affiliation is taken into account. Although maximum safe surgical resection should remain the standard of care, surgical removal of small residual portions of medulloblastoma is not recommended when the likelihood of neurological morbidity is high because there is no definitive benefit to gross total resection compared with near-total resection.Canadian Cancer Society Research Institute, Terry Fox Research Institute, Canadian Institutes of Health Research, National Institutes of Health, Pediatric Brain Tumor Foundation, and the Garron Family Chair in Childhood Cancer Research.

Mutations in the chromatin remodeling gene ARID1A have recently been identified in the majority of ovarian clear cell carcinomas (OCCCs). To determine the prevalence of mutations in other tumor types, we evaluated 759 malignant neoplasms including those of the pancreas, breast, colon, stomach, lung, prostate, brain, and blood (leukemias). We identified truncating mutations in 6% of the neoplasms studied; nontruncating somatic mutations were identified in an additional 0.4% of neoplasms. Mutations were most commonly found in gastrointestinal samples with 12 of 119 (10%) colorectal and 10 of 100 (10%) gastric neoplasms, respectively, harboring changes. More than half of the mutated colorectal and gastric cancers displayed microsatellite instability (MSI) and the mutations in these tumors were out-of-frame insertions or deletions at mononucleotide repeats. Mutations were also identified in 2-8% of tumors of the pancreas, breast, brain (medulloblastomas), prostate, and lung, and none of these tumors displayed MSI. These findings suggest that the aberrant chromatin remodeling consequent to ARID1A inactivation contributes to a variety of different types of neoplasms.

Frequent mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) and the promoter of telomerase reverse transcriptase (TERT) represent two significant discoveries in glioma genomics. Understanding the degree to which these two mutations co-occur or occur exclusively of one another in glioma subtypes presents a unique opportunity to guide glioma classification and prognosis. We analyzed the relationship between overall survival (OS) and the presence of IDH1/2 and TERT promoter mutations in a panel of 473 adult gliomas. We hypothesized and show that genetic signatures capable of distinguishing among several types of gliomas could be established providing clinically relevant information that can serve as an adjunct to histopathological diagnosis. We found that mutations in the TERT promoter occurred in 74.2% of glioblastomas (GBM), but occurred in a minority of Grade II-III astrocytomas (18.2%). In contrast, IDH1/2 mutations were observed in 78.4% of Grade II-III astrocytomas, but were uncommon in primary GBM. In oligodendrogliomas, TERT promoter and IDH1/2 mutations co-occurred in 79% of cases. Patients whose Grade III-IV gliomas exhibit TERT promoter mutations alone predominately have primary GBMs associated with poor median OS (11.5 months). Patients whose Grade III-IV gliomas exhibit IDH1/2 mutations alone predominately have astrocytic morphologies and exhibit a median OS of 57 months while patients whose tumors exhibit both TERT promoter and IDH1/2 mutations predominately exhibit oligodendroglial morphologies and exhibit median OS of 125 months. Analyzing gliomas based on their genetic signatures allows for the stratification of these patients into distinct cohorts, with unique prognosis and survival.

In the year 2000, an estimated 1220 100 new cases of invasive cancer will be diagnosed in the United States, and about 552 200 people are expected to die of it (Greenlee et al. 2000). Treatment of cancer has conventionally consisted of surgery, radiation and chemotherapy. Despite improved cancer therapy involving early detection, the surgical removal of solid tumor masses, and the use of radiotherapy, cytotoxic agents, or both, truly effective therapeutic approaches are still lacking and thus urgently needed. Extensive analyses have defined tumor-associated and, less frequently, tumor-specific, surface antigens displayed on the malignant cell surface. Targeting cells selectively through these surface antigens by molecular probes is inherently different from surgery, radiation, and chemotherapy and has been considered as a favored modality for cancer therapy. A considerable effort has been made over the past decade to develop new immunotherapeutic strategies against cancer; recently, such strategies have shown promise (Waksal 1999, Yang et al. 1999, Kreitman et al. 2000). Targeted therapy can be accomplished by using tumor-specific monoclonal antibodies (Mabs) alone or Mabs armed with radionuclides, prodrugs, or toxins, which selectively kill tumor cells while not destroying normal cells. Many growth factors and their receptors play important roles in modulating cell division, proliferation and differentiation, and the possibility of disrupting these processes has led to the development of novel therapeutic agents for cancer treatment. Therefore, growth factor receptors are attractive candidates for targeted therapy, as they are often overexpressed on the surface of cancer cells. Among these, the type I epidermal growth factor (EGF)-related family of tyrosine kinase growth factor receptors are expressed in a broad spectrum of tumor types, which classifies them as one of the most frequently implicated cell-surface markers for human cancers. This family consists of four members: EGF receptor (EGFR), ErbB-2, ErbB-3 and ErbB-4 (Gullick 1998). EGFR was the first cell-surface glycoprotein identified to be amplified and rearranged in glioblastoma multiforme (GBM) and to act

Despite the strikingly grave prognosis for older patients with glioblastomas, significant variability in patient outcome is experienced. To explore the potential for developing improved prognostic capabilities based on the elucidation of potential biological relationships, we did analyses of genes commonly mutated, amplified, or deleted in glioblastomas and DNA microarray gene expression data from tumors of glioblastoma patients of age >50 for whom survival is known. No prognostic significance was associated with genetic changes in epidermal growth factor receptor (amplified in 17 of 41 patients), TP53 (mutated in 11 of 41 patients), p16INK4A (deleted in 15 of 33 patients), or phosphatase and tensin homologue (mutated in 15 of 41 patients). Statistical analysis of the gene expression data in connection with survival involved exploration of regression models on small subsets of genes, based on computational search over multiple regression models with cross-validation to assess predictive validity. The analysis generated a set of regression models that, when weighted and combined according to posterior probabilities implied by the statistical analysis, identify patterns in expression of a small subset of genes that are associated with survival and have value in assessing survival risks. The dominant genes across such multiple regression models involve three key genes-SPARC (Osteonectin), Doublecortex, and Semaphorin3B-which play key roles in cellular migration processes. Additional analysis, based on statistical graphical association models constructed using similar computational analysis methods, reveals other genes which support the view that multiple mediators of tumor invasion may be important prognostic factor in glioblastomas in older patients.

Oligodendroglial neoplasms are a subgroup of gliomas with distinctive morphological characteristics. In the present study we have evaluated a series of these tumors to define their molecular profiles and to determine whether there is a relationship between molecular genetic parameters and histological pattern in this tumor type. Loss of heterozygosity (LOH) for 1p and 19q was seen in 17/23 (74%) well-differentiated oligodendrogliomas, in 18/23 (83%) anaplastic oligodendrogliomas, and in 3/8 (38%) oligoastrocytomas grades II and III. LOH for 17p and/or mutations of the TP53 gene occurred in 14 of these 55 tumors. Only one of the 14 cases with 17p LOH/TP53 gene mutation also had LOH for 1p and 19q, and significant astrocytic elements were seen histologically in the majority of these 14 tumors. LOH for 9p and/or deletion of the CDKN2A gene occurred in 15 of these 55 tumors, and 11 of these cases were among the 24 (42%) anaplastic oligodendrogliomas. Comparative genomic hybridization (CGH) identified the majority of cases with 1p and 19q loss and, in addition, showed frequent loss of chromosomes 4, 14, 15, and 18. These findings demonstrate that oligodendroglial neoplasms usually have loss of 1p and 19q whereas astrocytomas of the progressive type frequently contain mutations of the TP53 gene, and that 9p loss and CDKN2A deletions are associated with progression from well-differentiated to anaplastic oligodendrogliomas.

Temozolomide (Temodar; Schering-Plough Corp, Kenilworth, NJ) is an imidazole tetrazinone that undergoes chemical conversion to the active methylating agent 5-(3-methyltriazen-1yl)imidazole-4-carboximide under physiologic conditions. Previous studies have confirmed activity of Temodar in the treatment of progressive and newly diagnosed malignant gliomas. We have extended these results, and now we report results of a phase II trial of Temodar for patients with progressive, low-grade glioma.Temodar was administered orally once a day for five consecutive days (in a fasting state) at a starting dose of 200 mg/m(2)/d. Treatment cycles were repeated every 28 days following the first daily dose of Temodar. Response criteria used a combination of magnetic resonance imaging and physical examination to evaluate activity.Forty-six patients with low-grade glioma have been treated to date. The objective response rate was 61% (24% complete response and 37% partial response), with an additional 35% of patients having stable disease. Median progression-free survival (PFS) was 22 months (95% confidence interval [CI], 15 to infinity months) with a 6-month PFS of 98% (95% CI, 94% to 100%) and a 12-month PFS of 76% (95% CI, 63% to 92%). Toxicity observed during the study was limited to only six patients. Three patients experienced grade 3 neutropenia, with a duration greater than 3 weeks in one patient, and two patients experienced grade 3 thrombocytopenia. One patient experienced > or = grade 4 toxicity, with intracerebral hemorrhage, neutropenia, thrombocytopenia, sepsis, and death.Initial results indicate that Temodar may be active in the treatment of low-grade glioma, and thus, further evaluation of this agent in the treatment of these tumors is warranted.

The PTEN gene, located on 10q23, has recently been implicated as a candidate tumor suppressor gene in brain, breast and prostate tumors. In the present study, 123 brain tumors, including various grades and histological types of gliomas occurring in children and adults, were analyzed for PTEN mutations by SSCP assay and sequencing. Mutations in the PTEN gene were found in 13 of 42 adult glioblastomas and 3 of 13 adult anaplastic astrocytomas, whereas none of the 21 low-grade adult gliomas or the 22 childhood gliomas of all grades showed mutations. The single medulloblastoma with a mutation was a recurrent tumor that also possessed a p53 mutation. High-grade adult gliomas with PTEN mutations included cases that also contained gene amplification or p53 gene mutations, as well as cases that did not contain either of these abnormalities. There was no obvious relationship between presence of PTEN mutation and survival; however, there was a tendency for PTEN mutations to occur in older age group patients. This analysis suggest that PTEN gene mutations are restricted to high-grade adult gliomas and that this abnormality is independent of the presence or absence of gene amplification or p53 gene mutation in these tumors.

De novo glioblastomas develop in older patients without prior clinical history of less malignant tumors. Progressive glioblastomas are common among younger patients and arise through progression from lower-grade astrocytomas. CDKN2A deletions, PTEN alterations, and EGFR amplification are more prevalent among de novo glioblastomas, whereas p53 mutations are more common among progressive glioblastomas. Loss of heterozygosity (LOH) for chromosome 10 is seen uniformly among both de novo and progressive high-grade astrocytomas. The inactivation of the PTEN gene is found in approximately 30% to 40% of astrocytomas with chromosome 10 loss, and LOH pattern in the remaining astrocytomas strongly supports the presence of another yet unidentified tumor suppressor gene telomeric to PTEN. More than 80% of oligodendrogliomas exhibit LOH for 1p and 19q alleles. Oligoastrocytomas with 1p/19q LOH are related to oligodendrogliomas, and those with p53 mutations are related to astrocytomas.

The purpose of this study is to determine the maximum tolerated dose (MTD), dose-limiting toxicity (DLT), and intracerebral distribution of a recombinant toxin (TP-38) targeting the epidermal growth factor receptor in patients with recurrent malignant brain tumors using the intracerebral infusion technique of convection-enhanced delivery (CED). Twenty patients were enrolled and stratified for dose escalation by the presence of residual tumor from 25 to 100 ng/ml in a 40-ml infusion volume. In the last eight patients, coinfusion of 123I-albumin was performed to monitor distribution within the brain. The MTD was not reached in this study. Dose escalation was stopped at 100 ng/ml due to inconsistent drug delivery as evidenced by imaging the coinfused 123I-albumin. Two DLTs were seen, and both were neurologic. Median survival after TP-38 was 28 weeks (95% confidence interval, 26.5–102.8). Of 15 patients treated with residual disease, two (13.3%) demonstrated radiographic responses, including one patient with glioblastoma multiforme who had a nearly complete response and remains alive >260 weeks after therapy. Coinfusion of 123I-albumin demonstrated that high concentrations of the infusate could be delivered >4 cm from the catheter tip. However, only 3 of 16 (19%) catheters produced intraparenchymal infusate distribution, while the majority leaked infusate into the cerebrospinal fluid spaces. Intracerebral CED of TP-38 was well tolerated and produced some durable radiographic responses at doses ⩽100 ng/ml. CED has significant potential for enhancing delivery of therapeutic macromolecules throughout the human brain. However, the potential efficacy of drugs delivered by this technique may be severely constrained by ineffective infusion in many patients.

A new continuous cell line and transplantable xenograft, D283 Med, was derived from the peritoneal implant and ascitic fluid of a child with metastatic medulloblastoma and grew in vitro in suspension culture with spontaneous macroscopic spheroid formation. The in vitro population doubling time was 52.55 hours. Mean colony forming efficiency in an agarose medium was 1.83 ± 0.56%. The cell line, D283 Med, grew in athymic mice as serially transplantable intracranial and subcutaneous xenografts. Intracranial tumors grew as masses of small cells with scant cytoplasm and abundant mitotic figures and prominent anuclear zones resembling neuroblastic rosettes. Subcutaneous (SQ) tumors were markedly cellular neoplasms but did not contain rosettes. They expressed glutamine synthetase, neuron-specific enolase and neurofilament protein. Glial fibrillary acidic protein and S-100 protein were not detected. The SQ tumors grew to 500 mm3 with a latency of 52.55 ± 12.5 days and a doubling time of 9.33 ± 2.39 days. The stemline karyotypes of the peritoneal implant and ascitic fluid cells contained an extra copy of chromosome number 11 and three marker chromosomes (8q+, 17p+, 20q+). The cultured cell line and subcutaneous and intracranial xenografts retained the three marker chromosomes and differed from the original karyotype only in that they lacked the additional copy of chromosome number 11. This cell line and transplantable xenograft may allow further analysis of the biological properties and therapeutic sensitivity of human medulloblastoma.

Abstract Purpose: Although patients with newly diagnosed WHO grade 3 malignant glioma have a more favorable prognosis than those with WHO grade 4 malignant glioma, salvage therapies following recurrence offer essentially palliative benefit. We did a phase II trial of bevacizumab, a monoclonal antibody to vascular endothelial growth factor, in combination with irinotecan for patients with recurrent grade 3 malignant glioma. Experimental Design: Upon documentation of adequate safety among an initial cohort of nine patients treated with bevacizumab (10 mg/kg) and irinotecan every 14 days, a second cohort (n = 24) was treated with bevacizumab (15 mg/kg) every 3 weeks with irinotecan on days 1, 8, 22, and 29 of each 42-day cycle. For both cohorts, the dose of irinotecan was 340 mg/m2 for patients on enzyme-inducing antiepileptic drugs (EIAED) and 125 mg/m2 for patients not on EIAEDs. After each 6-week cycle, patients were evaluated with a physical examination and magnetic resonance imaging. Results: The 6-month progression-free survival was 55% (95% confidence interval, 36-70%). The 6-month overall survival was 79% (95% confidence interval, 61-89%). Twenty patients (61%) had at least a partial response. Outcome did not differ between the two treatment cohorts. Significant adverse events were infrequent and included a central nervous system hemorrhage in one patient, and one patient who developed thrombotic thrombocytopenic purpura. Conclusion: Bevacizumab and irinotecan is an active regimen with acceptable toxicity for patients with recurrent WHO grade 3 malignant glioma.

Abstract Temozolomide is a DNA-methylating agent used in the treatment of malignant gliomas. In this study, we have examined if inhibition of poly(ADP-ribose) polymerase (PARP) could increase the cytotoxicity of temozolomide, particularly in cells deficient in DNA mismatch repair. Athymic mice, transplanted with mismatch repair–proficient [D-245 MG] or deficient [D-245 MG (PR)] xenografts, were treated with a combination of temozolomide and the PARP inhibitor, INO-1001. For the tumors deficient in mismatch repair, the most effective dose of INO-1001 was found to be 150 mg/kg, given i.p. thrice at 4-hour intervals with the first injection in combination with 262.5 mg/kg temozolomide (0.75 LD10). This dose of temozolomide by itself induced no partial regressions and a 4-day growth delay. In two separate experiments, the combination therapy increased the growth delay by 21.6 and 9.7 days with partial regressions observed in four of eight and three of nine mice, respectively. The addition of INO-1001 had a more modest, yet statistically significant, increase in tumor growth delay in the mismatch repair–proficient xenografts. In these experiments, mice were treated with a lower amount of temozolomide (88 mg/kg), which resulted in growth delays of 43.1 and 39.2 days. When the temozolomide treatment was in combination with 200 mg/kg INO-1001, there was an increase in growth delay to 48.9 and 45.7 days, respectively. These results suggest that inhibition of PARP may increase the efficacy of temozolomide in the treatment of malignant gliomas, particularly in tumors deficient in DNA mismatch repair.

Monoallelic point mutations of the NADP(+)-dependent isocitrate dehydrogenases IDH1 and IDH2 occur frequently in gliomas, acute myeloid leukemias, and chondromas, and display robust association with specific DNA hypermethylation signatures. Here we show that heterozygous expression of the IDH1(R132H) allele is sufficient to induce the genome-wide alterations in DNA methylation characteristic of these tumors. Using a gene-targeting approach, we knocked-in a single copy of the most frequently observed IDH1 mutation, R132H, into a human cancer cell line and profiled changes in DNA methylation at over 27,000 CpG dinucleotides relative to wild-type parental cells. We find that IDH1(R132H/WT) mutation induces widespread alterations in DNA methylation, including hypermethylation of 2010 and hypomethylation of 842 CpG loci. We demonstrate that many of these alterations are consistent with those observed in IDH1-mutant and G-CIMP+ primary gliomas and can segregate IDH wild-type and mutated tumors as well as those exhibiting the G-CIMP phenotype in unsupervised analysis of two primary glioma cohorts. Further, we show that the direction of IDH1(R132H/WT)-mediated DNA methylation change is largely dependent upon preexisting DNA methylation levels, resulting in depletion of moderately methylated loci. Additionally, whereas the levels of multiple histone H3 and H4 methylation modifications were globally increased, consistent with broad inhibition of histone demethylation, hypermethylation at H3K9 in particular accompanied locus-specific DNA hypermethylation at several genes down-regulated in IDH1(R132H/WT) knock-in cells. These data provide insight on epigenetic alterations induced by IDH1 mutations and support a causal role for IDH1(R132H/WT) mutants in driving epigenetic instability in human cancer cells.

The permeability of different brain tumor models to horseradish peroxidase (HRP) was examined by determining the fraction of tumor that contained HRP after intravenous administration. The intracerebral tumor models studied were Avian Sarcoma Virus (ASV)-induced tumors and tumors from transplanted RG-2, S69-C1-5, and 9L cell lines. The average fraction of RG-2 tumors permeable to HRP was .95; of S69-C1-5 tumors, .699; of ASV-induced tumors. .63; and of 9L tumors, .52. Except for the RG-2 tumors, there was considerable regional variation in HRP permeability, which was most marked in the ASV-induced tumors. In ASV-induced tumors, HRP permeability did not correlate with tumor histological classification, size, or anatomic location within the brain. The subcutaneous tumor models studied were RG-2-, S69-C1-5, and 9L-transplanted tumors in rats, and human glioblastoma cell lines transplanted into nude mice. All were completely permeable to HRP. These results indicate that significant differences in permeability to HRP exist among brain tumor models when the tumors are intracerebral, and that all subcutaneous tumors from transplanted glial cell lines are completely permeable to HRP. These variables must be considered in future studies of permeability in experimental brain tumors. Care must be exercised in extrapolating results about permeability from one brain tumor model to another.

Abstract Transforming growth factor-β (TGF-β) is a multifunctional cytokine that promotes malignant glioma invasion, angiogenesis, and immunosuppression. Antisense oligonucleotide suppression of TGF-β2 ligand expression has shown promise in preclinical and clinical studies but at least two ligands mediate the effects of TGF-β in gliomas. Therefore, we examined the effects of SB-431542, a novel, small molecule inhibitor of the type I TGF-β receptor, on a panel of human malignant glioma cell lines. SB-431542 blocked the phosphorylation and nuclear translocation of the SMADs, intracellular mediators of TGF-β signaling, with decreased TGF-β–mediated transcription. Furthermore, SB-431542 inhibited the expression of two critical effectors of TGF-β-vascular endothelial growth factor and plasminogen activator inhibitor-1. SB-431542 treatment of glioma cultures inhibited proliferation, TGF-β–mediated morphologic changes, and cellular motility. Together, our results suggest that small molecule inhibitors of TGF-β receptors may offer a novel therapy for malignant gliomas by reducing cell proliferation, angiogenesis, and motility.

Hai Yan, Zachary Reitman and colleagues report exome sequencing of resected tumor tissue from brainstem gliomas and thalamic gliomas and identify mutations in PPM1D in brainstem gliomas. Gliomas arising in the brainstem and thalamus are devastating tumors that are difficult to surgically resect. To determine the genetic and epigenetic landscape of these tumors, we performed exomic sequencing of 14 brainstem gliomas (BSGs) and 12 thalamic gliomas. We also performed targeted mutational analysis of an additional 24 such tumors and genome-wide methylation profiling of 45 gliomas. This study led to the discovery of tumor-specific mutations in PPM1D, encoding wild-type p53–induced protein phosphatase 1D (WIP1), in 37.5% of the BSGs that harbored hallmark H3F3A mutations encoding p.Lys27Met substitutions. PPM1D mutations were mutually exclusive with TP53 mutations in BSG and attenuated p53 activation in vitro. PPM1D mutations were truncating alterations in exon 6 that enhanced the ability of PPM1D to suppress the activation of the DNA damage response checkpoint protein CHK2. These results define PPM1D as a frequent target of somatic mutation and as a potential therapeutic target in brainstem gliomas.

Telomerase reverse transcriptase (TERT) promoter mutations were recently shown to drive telomerase activity in various cancer types, including medulloblastoma. However, the clinical and biological implications of TERT mutations in medulloblastoma have not been described. Hence, we sought to describe these mutations and their impact in a subgroup-specific manner. We analyzed the TERT promoter by direct sequencing and genotyping in 466 medulloblastomas. The mutational distributions were determined according to subgroup affiliation, demographics, and clinical, prognostic, and molecular features. Integrated genomics approaches were used to identify specific somatic copy number alterations in TERT promoter-mutated and wild-type tumors. Overall, TERT promoter mutations were identified in 21 % of medulloblastomas. Strikingly, the highest frequencies of TERT mutations were observed in SHH (83 %; 55/66) and WNT (31 %; 4/13) medulloblastomas derived from adult patients. Group 3 and Group 4 harbored this alteration in <5 % of cases and showed no association with increased patient age. The prognostic implications of these mutations were highly subgroup-specific. TERT mutations identified a subset with good and poor prognosis in SHH and Group 4 tumors, respectively. Monosomy 6 was mostly restricted to WNT tumors without TERT mutations. Hallmark SHH focal copy number aberrations and chromosome 10q deletion were mutually exclusive with TERT mutations within SHH tumors. TERT promoter mutations are the most common recurrent somatic point mutation in medulloblastoma, and are very highly enriched in adult SHH and WNT tumors. TERT mutations define a subset of SHH medulloblastoma with distinct demographics, cytogenetics, and outcomes.

Abstract Purpose: More brain tumor markers are required for prognosis and targeted therapy. We have identified and validated promising molecular therapeutic glioblastoma multiforme (GBM) targets: human transmembrane glycoprotein nonmetastatic melanoma protein B (GPNMBwt) and a splice variant form (GPNMBsv, a 12-amino-acid in-frame insertion in the extracellular domain). Experimental Design: We have done genetic and immunohistochemical evaluation of human GBM to determine incidence, distribution, and pattern of localization of GPNMB antigens in brain tumors as well as survival analyses. Results: Quantitative real-time PCR on 50 newly diagnosed GBM patient tumor samples indicated that 35 of 50 GBMs (70%) were positive for GPNMBwt+sv transcripts and 15 of 50 GBMs (30%) were positive for GPNMBsv transcripts. Normal brain samples expressed little or no GPNMB mRNA. We have isolated and characterized an anti-GPNMB polyclonal rabbit antiserum (2640) and two IgG2b monoclonal antibodies (mAb; G11 and U2). The binding affinity constants of the mAbs ranged from 0.27 × 108 to 9.6 × 108 M−1 measured by surface plasmon resonance with immobilized GPNMB, or 1.7 to 2.1 × 108 M−1 by Scatchard analyses with cell-expressed GPNMB. Immunohistochemical analysis detected GPNMB in a membranous and cytoplasmic pattern in 52 of 79 GBMs (66%), with focal perivascular reactivity in ∼27%. Quantitative flow cytometric analysis revealed GPNMB cell surface molecular density of 1.1 × 104 to 7.8 × 104 molecules per cell, levels sufficient for mAb targeting. Increased GPNMB mRNA levels correlated with elevated GPNMB protein expression in GBM biopsy samples. Univariate and multivariate analyses correlated expression of GPNMB with survival of 39 GBM patients using RNA expression and immunohistochemical data, establishing that patients with relatively high mRNA GPNMB transcript levels (wt+sv and wt), &amp;gt;3-fold over normal brain, as well as positive immunohistochemistry, have a significantly higher risk of death (hazard ratios, 3.0, 2.2, and 2.8, respectively). Conclusions: Increased mRNA and protein levels in GBM patient biopsy samples correlated with higher survival risk; as a detectable surface membrane protein in glioma cells, the data indicate that GPNMB is a potentially useful tumor-associated antigen and prognostic predictor for therapeutic approaches with malignant gliomas or any malignant tumor that expresses GPNMB.

Effects of epidermal growth factor (EGF) and an antibody (Ab-528) reactive against the binding site for EGF on human EGF receptors were studied on multicellular tumor spheroids obtained from three human glioma cell lines with high (D-37 MG), medium (D-247 MG), and low (D-263 MG) levels of EGF receptor expression. The D-247 MG and D-263 MG spheroids grew slowly or not at all in the absence of EGF, while in the presence of EGF they were growth stimulated. Tumor cell migration, as measured by the spread of cells from spheroids on a plastic substratum, was increased by the addition of EGF for all three cell lines. Stimulation of migration could be blocked by a subsequent addition of Ab-528 to the medium at a concentration of 50 micrograms/ml. Invasiveness of glioma cell spheroids into fetal rat brain aggregates was related to EGF receptor expression; the two lines with medium to high receptor expression (D-247 MG and D-37 MG) were invasive, while the line with low EGF receptor expression (D-263 MG) was noninvasive, as assessed by an in vitro coculture assay. In the D-247 MG cell line, morphometry revealed EGF-enhanced invasiveness of the tumor cells. The addition of the Ab-528 to EGF-treated cocultures reduced invasion in both D-247 MG and D-37 MG cell lines. Antibody Ab-528 alone did not affect glioma cell growth or migration but did inhibit invasiveness. The present study suggests that, in brain tumors with an increased number of normal-sized Mr 170,000 EGF receptors, EGF or an EGF-like ligand such as transforming growth factor-alpha may selectively facilitate expansive tumor growth and tumor cell invasion. This effect may in part be blocked or retarded by specific antibodies to the EGF receptor.

Mutations of the isocitrate dehydrogenase (IDH) metabolic enzymes IDH1 and IDH2 have been found to be frequent and early genetic alterations in astrocytomas and oligodendrogliomas. All mutations identified to date affect a single amino acid located within the isocitrate binding site (R132 of IDH1 and the analogous R172 residue of IDH2). IDH1 and IDH2 mutations define a specific subtype of gliomas and may have significant utility for the diagnosis, prognosis, and treatment of patients with these tumors.

Abstract Purpose: To determine if the addition of bevacizumab to radiation therapy and temozolomide, followed by bevacizumab, temozolomide, and irinotecan, for newly diagnosed glioblastoma patients is safe and effective. Experimental Design: A total of 75 patients with newly diagnosed glioblastoma were enrolled in the phase II trial that investigated the addition of bevacizumab to standard radiation therapy and daily temozolomide followed by the addition of bevacizumab and irinotecan to adjuvant temozolomide. The bevacizumab was given at 10 mg/kg every 14 days beginning a minimum of 4 weeks postcraniotomy. Two weeks after radiation therapy, the patients began 6 to 12 cycles of 5-day temozolomide with bevacizumab and irinotecan every 14 days. The primary endpoint was the proportion of patients alive 16 months after informed consent. Results: The therapy had moderate toxicity. Three patients, one of whom had a grade 2 central nervous system hemorrhage, came off study during radiation therapy. Seventy patients started the postradiation therapy, and 16 (23%) terminated this adjuvant therapy early because of toxicity. The median overall survival was 21.2 months (95% CI: 17.2–25.4), and 65% of the patients were alive at 16 months (95% CI: 53.4–74.9). The median progression-free survival was 14.2 months (95% CI: 12–16). Conclusion: The addition of bevacizumab to standard radiation therapy and temozolomide, followed by bevacizumab, irinotecan, and temozolomide, for the treatment of newly diagnosed glioblastoma has moderate toxicity and may improve efficacy compared with historical controls. The results from phase III trials are required before the role of bevacizumab for newly diagnosed glioblastoma is established. Clin Cancer Res; 17(12); 4119–24. ©2011 AACR.

Sorafenib, an oral VEGFR-2, Raf, PDGFR, c-KIT and Flt-3 inhibitor, is active against renal cell and hepatocellular carcinomas, and has recently demonstrated promising activity for lung and breast cancers. In addition, various protracted temozolomide dosing schedules have been evaluated as a strategy to further enhance its anti-tumor activity. We reasoned that sorafenib and protracted, daily temozolomide may provide complementary therapeutic benefit, and therefore performed a phase 2 trial among recurrent glioblastoma patients. Adult glioblastoma patients at any recurrence after standard temozolomide chemoradiotherapy received sorafenib (400 mg twice daily) and continuous daily temozolomide (50 mg/m²/day). Assessments were performed every eight weeks. The primary endpoint was progression-free survival at 6 months (PFS-6) and secondary end points were radiographic response, overall survival (OS), safety and sorafenib pharmacokinetics. Of 32 enrolled patients, 12 (38%) were on CYP3-A inducing anti-epileptics (EIAEDs), 17 (53%) had 2 or more prior progressions, 15 had progressed while receiving 5-day temozolomide, and 12 (38%) had failed either prior bevacizumab or VEGFR inhibitor therapy. The most common grade ≥ 3 toxicities were palmer-planter erythrodysesthesia (19%) and elevated amylase/lipase (13%). Sorafenib pharmacokinetic exposures were comparable on day 1 regardless of EIAED status, but significantly lower on day 28 for patients on EIAEDs (P = 0.0431). With a median follow-up of 93 weeks, PFS-6 was 9.4%. Only one patient (3%) achieved a partial response. In conclusion, sorafenib can be safely administered with daily temozolomide, but this regimen has limited activity for recurrent GBM. Co-administration of EIAEDs can lower sorafenib exposures in this population.

Abstract The majority of glioblastomas can be classified into molecular subgroups based on mutations in the TERT promoter ( TERTp ) and isocitrate dehydrogenase 1 or 2 ( IDH ). These molecular subgroups utilize distinct genetic mechanisms of telomere maintenance, either TERTp mutation leading to telomerase activation or ATRX- mutation leading to an alternative lengthening of telomeres phenotype (ALT). However, about 20% of glioblastomas lack alterations in TERTp and IDH . These tumors, designated TERTp WT - IDH WT glioblastomas, do not have well-established genetic biomarkers or defined mechanisms of telomere maintenance. Here we report the genetic landscape of TERTp WT - IDH WT glioblastoma and identify SMARCAL1 inactivating mutations as a novel genetic mechanism of ALT. Furthermore, we identify a novel mechanism of telomerase activation in glioblastomas that occurs via chromosomal rearrangements upstream of TERT . Collectively, our findings define novel molecular subgroups of glioblastoma, including a telomerase-positive subgroup driven by TERT -structural rearrangements ( IDH WT - TERT SV ), and an ALT-positive subgroup ( IDH WT -ALT) with mutations in ATRX or SMARCAL1 .

Glioblastoma, the most common primary malignant brain tumor among adults, is a highly angiogenic and deadly tumor. Angiogenesis in glioblastoma, driven by hypoxia-dependent and independent mechanisms, is primarily mediated by vascular endothelial growth factor (VEGF), and generates blood vessels with distinctive features. The outcome for patients with recurrent glioblastoma is poor because of ineffective therapies. However, recent encouraging rates of radiographic response and progression-free survival, and adequate safety, led the FDA to grant accelerated approval of bevacizumab, a humanized monoclonal antibody against VEGF, for the treatment of recurrent glioblastoma in May 2009. These results have triggered significant interest in additional antiangiogenic agents and therapeutic strategies for patients with both recurrent and newly diagnosed glioblastoma. Given the potent antipermeability effect of VEGF inhibitors, the Radiologic Assessment in Neuro-Oncology (RANO) criteria were recently implemented to better assess response among patients with glioblastoma. Although bevacizumab improves survival and quality of life, eventual tumor progression is the norm. Better understanding of resistance mechanisms to VEGF inhibitors and identification of effective therapy after bevacizumab progression are currently a critical need for patients with glioblastoma.

KMT2D (lysine (K)-specific methyltransferase 2D), formerly named MLL2 (myeloid/lymphoid or mixed-lineage leukemia 2, also known as ALR/MLL4), is a histone methyltransferase that plays an important role in regulating gene transcription. In particular, it targets histone H3 lysine 4 (H3K4), whose methylations serve as a gene activation mark. Recently, KMT2D has emerged as one of the most frequently mutated genes in a variety of cancers and in other human diseases, including lymphoma, medulloblastoma, gastric cancer, and Kabuki syndrome. Mutations in KMT2D identified thus far point to its loss-of-function in pathogenesis and suggest its role as a tumor suppressor in various tissues. To determine the effect of a KMT2D deficiency on neoplastic cells, we used homologous recombination- and nuclease-mediated gene editing approaches to generate a panel of isogenic colorectal and medulloblastoma cancer cell lines that differ with respect to their endogenous KMT2D status. We found that a KMT2D deficiency resulted in attenuated cancer cell proliferation and defective cell migration. Analysis of histone H3 modifications revealed that KMT2D was essential for maintaining the level of global H3K4 monomethylation and that its enzymatic SET domain was directly responsible for this function. Furthermore, we found that a majority of KMT2D binding sites are located in regions of potential enhancer elements. Together, these findings revealed the role of KMT2D in regulating enhancer elements in human cells and shed light on the tumorigenic role of its deficiency. Our study supports that KMT2D has distinct roles in neoplastic cells, as opposed to normal cells, and that inhibiting KMT2D may be a viable strategy for cancer therapeutics.

Abstract Point mutations at Arg132 of the cytoplasmic NADP+-dependent isocitrate dehydrogenase 1 (IDH1) occur frequently in gliomas and result in a gain of function to produce the “oncometabolite” D-2-hydroxyglutarate (D-2HG). The mutated IDH1 allele is usually associated with a wild-type IDH1 allele (heterozygous) in cancer. Here, we identify 2 gliomas that underwent loss of the wild-type IDH1 allele but retained the mutant IDH1 allele following tumor progression from World Health Organization (WHO) grade III anaplastic astrocytomas to WHO grade IV glioblastomas. Intratumoral D-2HG was 14-fold lower in the glioblastomas lacking wild-type IDH1 than in glioblastomas with heterozygous IDH1 mutations. To characterize the contribution of wild-type IDH1 to cancer cell D-2HG production, we established an IDH1-mutated astrocytoma (IMA) cell line from a WHO grade III anaplastic astrocytoma. Disruption of the wild-type IDH1 allele in IMA cells by gene targeting resulted in an 87-fold decrease in cellular D-2HG levels, showing that both wild-type and mutant IDH1 alleles are required for D-2HG production in glioma cells. Expression of wild-type IDH1 was also critical for mutant IDH1-associated D-2HG production in the colorectal cancer cell line HCT116. These insights may aid in the development of therapeutic strategies to target IDH1-mutated cancers. Cancer Res; 73(2); 496–501. ©2012 AACR.

Gliomas frequently contain mutations in the cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (IDH1) or the mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2). Several different amino acid substitutions recur at either IDH1 R132 or IDH2 R172 in glioma patients. Genetic evidence indicates that these mutations share a common gain of function, but it is unclear whether the shared function is dominant negative activity, neomorphic production of (R)-2-hydroxyglutarate (2HG), or both.We show by coprecipitation that five cancer-derived IDH1 R132 mutants bind IDH1-WT but that three cancer-derived IDH2 R172 mutants exert minimal binding to IDH2-WT. None of the mutants dominant-negatively lower isocitrate dehydrogenase activity at physiological (40 µM) isocitrate concentrations in mammalian cell lysates. In contrast to this, all of these mutants confer 10- to 100-fold higher 2HG production to cells, and glioma tissues containing IDH1 R132 or IDH2 R172 mutations contain high levels of 2HG compared to glioma tissues without IDH mutations (54.4 vs. 0.1 mg 2HG/g protein).Binding to, or dominant inhibition of, WT IDH1 or IDH2 is not a shared feature of the IDH1 and IDH2 mutations, and thus is not likely to be important in cancer. The fact that the gain of the enzymatic activity to produce 2HG is a shared feature of the IDH1 and IDH2 mutations suggests that this is an important function for these mutants in driving cancer pathogenesis.

Lymphodepletion augments adoptive cell transfer during antitumor immunotherapy, producing dramatic clinical responses in patients with malignant melanoma. We report that the lymphopenia induced by the chemotherapeutic agent temozolomide (TMZ) enhances vaccine-driven immune responses and significantly reduces malignant growth in an established model of murine tumorigenesis. Unexpectedly, despite the improved antitumor efficacy engendered by TMZ-induced lymphopenia, there was a treatment related increase in the frequency of immunosuppressive regulatory T cells (T(Regs); P = .0006). Monoclonal antibody (mAb)-mediated inhibition of the high-affinity IL-2 receptor α (IL-2Rα/CD25) during immunotherapy in normal mice depleted T(Regs) (73% reduction; P = .0154) but also abolished vaccine-induced immune responses. However, during lymphodepletion, IL-2Rα blockade decreased T(Regs) (93% reduction; P = .0001) without impairing effector T-cell responses, to augment therapeutic antitumor efficacy (66% reduction in tumor growth; P = .0024). Of clinical relevance, we also demonstrate that anti-IL-2Rα mAb administration during recovery from lymphodepletive TMZ in patients with glioblastoma reduced T(Reg) frequency (48% reduction; P = .0061) while permitting vaccine-stimulated antitumor effector cell expansion. To our knowledge, this is the first report of systemic antibody-mediated T(Reg) depletion during lymphopenia and the consequent synergistic enhancement of vaccine-driven cellular responses, as well as the first demonstration that anti-IL-2Rα mAbs function differentially in nonlymphopenic versus lymphopenic contexts.

Myeloid/lymphoid or mixed-lineage leukemia (MLL)-family genes encode histone lysine methyltransferases that play important roles in epigenetic regulation of gene transcription. MLL genes are frequently mutated in human cancers. Unlike MLL1, MLL2 (also known as ALR/MLL4) and its homolog MLL3 are not well-understood. Specifically, little is known regarding the extent of global MLL2 involvement in the regulation of gene expression and the mechanism underlying its alterations in driving tumorigenesis. Here we profile the global loci targeted by MLL2. A combinatorial analysis of the MLL2 binding profile and gene expression in MLL2 wild-type versus MLL2-null isogenic cell lines identified direct transcriptional target genes and revealed the connection of MLL2 to multiple cellular signaling pathways, including the p53 pathway, cAMP-mediated signaling, and cholestasis signaling. In particular, we demonstrate that MLL2 participates in retinoic acid receptor signaling by promoting retinoic acid-responsive gene transcription. Our results present a genome-wide integrative analysis of the MLL2 target loci and suggest potential mechanisms underlying tumorigenesis driven by MLL2 alterations.

Glial tumors of all grades and histological types from 72 adults and 48 children were analyzed for mutations of the TP53 gene, loss of heterozygosity (LOH) for 17p, and accumulation of TP53 protein to determine whether the incidence and type of TP53 alterations differ among tumors of different histological type and between tumors from adults and children. These tumors were also evaluated for LOH for chromosome 10 and for amplification of the epidermal growth factor receptor, C-MYC, N-MYC, GLI, platelet-derived growth factor receptor-alpha, and murine double minute 2 genes to determine the patterns of molecular alterations involved in the progression of these neoplasms. Seventeen of the 120 tumors contained mutations of the TP53 gene. One of the tumors with TP53 gene mutation was from one of the 48 patients less than 18 years of age. Twelve of the 17 tumors with mutations occurred among the 27 patients in the 18-45-year age group, while 4 tumors with mutations were among the 45 patients more than 45 years old. There was also an increased incidence of TP53 mutation in patients with anaplastic astrocytoma histology. However, no significant association between presence of TP53 mutation and patient survival was observed. These studies demonstrate that TP53 gene mutations are a common mechanism for glial cell neoplasms in the 18-45-year age group but are unrelated to progression and advanced histological grade. LOH for chromosome 10 and gene amplification, however, occurring in 82 and 40%, respectively, of glioblastoma multiforme, whether seen alone or along with TP53 gene alterations, are related to advanced histological grade of the tumor. In childhood gliomas, in contrast, TP53 gene alterations, LOH for 17p and 10q, and gene amplification are uncommon in tumors of all grades, suggesting that presently unknown mechanisms are responsible for the genesis and progression of these tumors.

We employed a genetically defined human cancer model to investigate the contributions of two genes up-regulated in several cancers to phenotypic changes associated with late stages of tumorigenesis. Specifically, tumor cells expressing two structurally unrelated bone-related genes, osteonectin and osteoactivin, acquired a highly invasive phenotype when implanted intracranially in immunocompromised mice. Mimicking a subset of gliomas, tumor cells invaded brain along blood vessels and developed altered vasculature at the brain-tumor interface, suggesting that production of those two proteins by tumor cells may create a complex relationship between invading tumor and vasculature co-opted during tumor invasion. Interestingly, the same tumor cells formed massive spontaneous metastases when implanted subcutaneously. This dramatic alteration in tumor phenotype indicates that cellular microenvironment plays an important role in defining the specific effects of those gene products in tumor behavior. In vitro examination of tumor cells expressing either osteonectin or osteoactivin revealed that there was no impact on cellular growth or death but increased invasiveness and expression of MMP-9 and MMP-3. Specific pharmacologic inhibitors of MMP-2/9 and MMP-3 blocked the increased in vitro invasion associated with osteoactivin expression, but only MMP-3 inhibition altered the invasive in vitrophenotype mediated by osteonectin. Results from this genetically defined model system are supported by similar findings obtained from several established tumor cell lines derived originally from human patients. In sum, these results reveal that the expression of a single bone-related gene can dramatically alter or modify tumor cell behavior and may confer differential growth characteristics in different microenvironments. Genetically defined human cancer models offer useful tools in functional genomics to define the roles of specific genes in late stages of carcinogenesis. We employed a genetically defined human cancer model to investigate the contributions of two genes up-regulated in several cancers to phenotypic changes associated with late stages of tumorigenesis. Specifically, tumor cells expressing two structurally unrelated bone-related genes, osteonectin and osteoactivin, acquired a highly invasive phenotype when implanted intracranially in immunocompromised mice. Mimicking a subset of gliomas, tumor cells invaded brain along blood vessels and developed altered vasculature at the brain-tumor interface, suggesting that production of those two proteins by tumor cells may create a complex relationship between invading tumor and vasculature co-opted during tumor invasion. Interestingly, the same tumor cells formed massive spontaneous metastases when implanted subcutaneously. This dramatic alteration in tumor phenotype indicates that cellular microenvironment plays an important role in defining the specific effects of those gene products in tumor behavior. In vitro examination of tumor cells expressing either osteonectin or osteoactivin revealed that there was no impact on cellular growth or death but increased invasiveness and expression of MMP-9 and MMP-3. Specific pharmacologic inhibitors of MMP-2/9 and MMP-3 blocked the increased in vitro invasion associated with osteoactivin expression, but only MMP-3 inhibition altered the invasive in vitrophenotype mediated by osteonectin. Results from this genetically defined model system are supported by similar findings obtained from several established tumor cell lines derived originally from human patients. In sum, these results reveal that the expression of a single bone-related gene can dramatically alter or modify tumor cell behavior and may confer differential growth characteristics in different microenvironments. Genetically defined human cancer models offer useful tools in functional genomics to define the roles of specific genes in late stages of carcinogenesis. matrix metalloproteinase epidermal growth factor receptor platelet-derived growth factor receptor insulin-like growth factor-1 receptor severe combined immune deficiency Gene expression analyses of human cancers have yielded tremendous quantities of data. Unfortunately, the phenotypic consequences of many changes in gene expression pattern between cancers and their corresponding normal tissues are largely unclear. To address this problem, we took a functional genomics approach by using a genetically defined glioma model system to investigate genes involved in the acquisition of malignant phenotype associated with late stages of tumorigenesis. Particularly, we were interested in genes whose expression is not associated with normal brain tissues or astrocyte cultures but which are nevertheless overexpressed in gliomas. Among those candidate genes, several bone-related genes were noticeably overexpressed in a high proportion of gliomas. Of note, two structurally unrelated genes, osteonectin and osteoactivin, have been found previously to be overexpressed in several other types of cancers, but their precise contribution to the development of specific cancer phenotype has yet to be elucidated. Osteonectin, also known as secreted protein, acidic and rich in cysteine (SPARC) or BM-40, is a 43-kDa extracellular matrix protein. Osteonectin was originally discovered as one of the most abundant non-collagenous components of bone, but it is also expressed in a number of other cell types that are involved in active remodeling of tissues (1Bradshaw A.D. Sage E.H. J. Clin. Invest. 2001; 107: 1049-1054Crossref PubMed Scopus (524) Google Scholar). Thus, the primary physiological role of osteonectin has been postulated to be an important modulator of cell-extracellular matrix interactions during the processes of tissue remodeling (2Brekken R.A Sage E.H. Matrix Biol. 2001; 19: 816-827Crossref PubMed Scopus (57) Google Scholar, 3Porter P.L. Sage E.H. Lane T.F. Funk S.E. Gown A.M. J. Histochem. Cytochem. 1995; 43: 791-800Crossref PubMed Scopus (195) Google Scholar). Osteonectin is also abnormally expressed in many cancers, including gliomas (4Rempel S.A. Golembieski W.A. Ge S. Lemke N. Elisevich K. Mikkelsen T. Gutierrez J.A. J. Neuropathol. Exp. Neurol. 1998; 57: 1112-1121Crossref PubMed Scopus (120) Google Scholar, 5Lal A. Lash A.E. Altschul S.F. Velculescu V. Zhang L. McLendon R.E. Marra M.A. Prange C. Morin P.J. Polyak K. Papadopoulos N. Vogelstein B. Kinzler K.W. Strausberg R.L. Riggins G.J. Cancer Res. 1999; 59: 5403-5407PubMed Google Scholar), medulloblastomas (6Rempel S.A. Ge S. Gutierrez J.A. Clin. Cancer Res. 1999; 5: 237-241PubMed Google Scholar), meningiomas (7MacDonald T.J. Brown K.M. LaFleur B. Peterson K. Lawlor C. Chen Y. Packer R.J. Cogen P. Stephan D.A. Nat. Genet. 2001; 29: 143-152Crossref PubMed Scopus (391) Google Scholar), and cancers of the gastrointestinal tract, breast, lung, kidney, adrenal cortex, prostate, and bladder (3Porter P.L. Sage E.H. Lane T.F. Funk S.E. Gown A.M. J. Histochem. Cytochem. 1995; 43: 791-800Crossref PubMed Scopus (195) Google Scholar, 8Ledda F. Bravo A.I. Adris S. Bover L. Mordoh J. Podhajcer O.L. J. Invest. Dermatol. 1997; 108: 210-214Abstract Full Text PDF PubMed Scopus (149) Google Scholar, 9Thomas R. True L.D. Bassuk J.A. Lange P.H. Vessella R.L. Clin. Cancer Res. 2000; 6: 1140-1149PubMed Google Scholar, 10Le Bail B. Faouzi S. Boussarie L. Guirouilh J. Blanc J.F. Carles J. Bioulac-Sage P. Balabaud C. Rosenbaum J. J. Pathol. 1999; 189: 46-52Crossref PubMed Scopus (90) Google Scholar, 11Porte H. Chastre E. Prevot S. Nordlinger B. Empereur S. Basset P. Chambon P. Gespach C. Int. J. Cancer. 1995; 64: 70-75Crossref PubMed Scopus (164) Google Scholar). In gliomas, osteonectin is expressed in all tumor grades, usually at the tumor-brain margin and sites of neoangiogenesis (4Rempel S.A. Golembieski W.A. Ge S. Lemke N. Elisevich K. Mikkelsen T. Gutierrez J.A. J. Neuropathol. Exp. Neurol. 1998; 57: 1112-1121Crossref PubMed Scopus (120) Google Scholar), suggesting that osteonectin expression may be involved in tumor cell invasion (14Golembieski W.A. Ge S. Nelson K. Mikkelsen T. Rempel S.A. Int. J. Dev. Neurosci. 1999; 17: 463-472Crossref PubMed Scopus (80) Google Scholar). In contrast, increased osteonectin expression in other tumor types is associated with a conversion to invasive and metastatic tumors and a correlation in some instances with elevated expression of matrix metalloproteinases (MMPs)1that is linked to increased tumor malignancy (11Porte H. Chastre E. Prevot S. Nordlinger B. Empereur S. Basset P. Chambon P. Gespach C. Int. J. Cancer. 1995; 64: 70-75Crossref PubMed Scopus (164) Google Scholar, 12Gilles C. Bassuk J.A. Pulyaeva H. Sage E.H. Foidart J.M. Thompson E.W. Cancer Res. 1998; 58: 5529-5536PubMed Google Scholar, 13Sternlicht M. Werb Z. Annu. Rev. Cell Dev. Biol. 2001; 17: 463-516Crossref PubMed Scopus (3256) Google Scholar). Importantly, reduced expression of osteonectin by an antisense approach was found to correlate with a reduction in tumor formation by melanoma cells (15Ledda M.F. Adris S. Bravo A.I. Kairiyama C. Bover L. Chernajovsky Y. Mordoh J. Podhajcer O.L. Nat. Med. 1997; 3: 171-176Crossref PubMed Scopus (209) Google Scholar). Thus, although the precise role of osteonectin in the pathological process of carcinogenesis remains to be elucidated, osteonectin overexpression is intimately correlated with the progression of tumorigenesis of multiple types of human cancers. Osteoactivin, also known as GPNMB or dendritic cell-associated, heparan sulfate proteoglycan-integrin ligand (DC-HIL), is a type I transmembrane glycoprotein that is localized to the cell surface and lysosomal membranes (16Shikano S. Bonkobara M. Zukas P.K. Ariizumi K. J. Biol. Chem. 2001; 276: 8125-8134Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), as well as in a secreted form (17Safadi F.F. Xu J. Smock S.L. Rico M.C. Owen T.A. Popoff S.N. J. Cell. Biochem. 2001; 84: 12-26Crossref PubMed Scopus (136) Google Scholar). Highly expressed in bone, the physiological function of osteoactivin is postulated to be involved in the regulation of osteoblast maturation (16Shikano S. Bonkobara M. Zukas P.K. Ariizumi K. J. Biol. Chem. 2001; 276: 8125-8134Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Interestingly, osteoactivin is found to be overexpressed in melanomas (18Weterman M.A. Ajubi N. van Dinter I.M. Degen W.G. van Muijen G.N. Ruitter D.J. Bloemers H.P. Int. J. Cancer. 1995; 60: 73-81Crossref PubMed Scopus (213) Google Scholar), gliomas (19Loging W.T. Lal A. Siu I.M. Loney T.L. Wikstrand C.J. Marra M.A. Prange C. Bigner D.D. Strausberg R.L. Riggins G.J. Genome Res. 2000; 10: 1393-1402Crossref PubMed Scopus (88) Google Scholar), and cancers of the breast, stomach, and pancreas. 2NCBI SAGE Genie web site. Although the precise role of osteoactivin in cancer development remains unknown, it is tempting to suggest that the significant elevation of this bone-related protein in gliomas has a role in brain tumor progression. To determine the specific contributions of osteonectin and osteoactivin expression to human cancer development, we employed a genetically defined model system in which the genetic effects of increased gene expression could be directly linked to phenotypic changes of tumorigenesis. In this system, transformed human astrocytes by the sequential introduction of the simian virus-40 large T antigen, the human telomerase catalytic subunit (hTERT), and oncogenic Ha-Ras display a phenotype mimicking that of low grade gliomas in the formation of non-invasive tumor mass in immune-compromised animals (20Rich J.N. Guo C. McLendon R.E. Bigner D.D. Wang X.F. Counter C.M. Cancer Res. 2001; 61: 3556-3560PubMed Google Scholar). The characteristics of this human tumor model system allowed us to test specifically the phenotypic changes associated with tumor progression derived from the expression of genes under investigation. Consequently, we found that expression of osteonectin or osteoactivin was associated with angiocentric intracranial invasion and increased production of MMPs by the tumor cells. Furthermore, osteonectin and osteoactivin expression caused the development of spontaneous metastases systemically when the tumor cells were implanted subcutaneously. Our results suggest that the expression of bone-related genes in advanced human cancers may represent a novel mechanism by which tumor cells acquire capabilities that are associated with the phenotypic changes in late stages of tumorigenesis dependent on tumor microenvironment. Moreover, the data strongly support the notion that the genetically defined model of human cancers (20Rich J.N. Guo C. McLendon R.E. Bigner D.D. Wang X.F. Counter C.M. Cancer Res. 2001; 61: 3556-3560PubMed Google Scholar, 21Hahn W.C. Counter C.M. Lundberg A.S. Beijersbergen R.L. Brooks M.W. Weinberg R.A. Nature. 1999; 400: 464-468Crossref PubMed Scopus (1991) Google Scholar, 22Elenbaas B. Spirio L. Koerner F. Fleming M.D. Zimonjic D.B. Donaher J.L. Popescu N.C. Hahn W.C. Weinberg R.A. Genes Dev. 2001; 15: 50-65Crossref PubMed Scopus (689) Google Scholar, 23Sonoda Y. Ozawa T. Hirose Y. Aldape K.D. McMahon M. Berger M.S. Pieper R.O. Cancer Res. 2001; 61: 4956-4960PubMed Google Scholar) offers a useful tool for functional genomics in defining the pathological contributions of specific genes to late stages of tumorigenesis. A genetically defined human glioma cell line was generated as described previously (20Rich J.N. Guo C. McLendon R.E. Bigner D.D. Wang X.F. Counter C.M. Cancer Res. 2001; 61: 3556-3560PubMed Google Scholar). A 1.5-kb cDNA fragment (a generous gift from Sandra Rempel, Henry Ford Hospital) and a full-length cDNA fragment of GPNMB (a generous gift from H. P. Bloemers, University of Nijmegen, The Netherlands) were each cloned into a retroviral backbone with a bleo selection marker. Cells underwent a positive selection with Zeocin (400 μg/ml, Invitrogen). U87MG, U251MG (American Type Culture Collection, Manassas, VA), and D54MG (Duke University Medical Center) were infected with a retrovirus expressing either a puromycin resistance gene or osteonectin and puromycin resistance. Selection was undertaken with puromycin (1 μg/ml). Early passage polyclonal cultures were used for all experiments. Cells were analyzed for the expression of osteonectin, growth factor receptors, and MMPs by Western analysis. A 10-cm plate was lysed, and 50 μg of total cellular protein was used for each sample. Samples were subjected to SDS-PAGE analysis and transferred to a PDVF membrane. The membrane was blocked in Tris-buffered saline with 0.05% Tween 20 and 5% albumin. Primary antibodies for anti-osteonectin (Hematologic Technologies, Essex Junction, VT), anti-GPNMB (gift of Carol Wikstrand, Duke University), anti-actin (Santa Cruz Biotechnology, Santa Cruz, CA), anti-tubulin (Sigma), anti-MMP-9 (Calbiochem), anti-MMP-3 (Calbiochem), anti-MMP-2 (Calbiochem), anti-epidermal growth factor receptor (EGFR) (gift of Carol Wikstrand, Duke University), anti-platelet-derived growth factor receptor β (PDGFRβ) (Santa Cruz Biotechnology), anti-PDGFRα (Santa Cruz Biotechnology), or anti-insulin-like growth factor-1 receptor (IGF-1R) (Calbiochem) antibodies were used. Secondary antibodies were either goat anti-rabbit (Bio-Rad) or sheep anti-rabbit antibodies (Amersham Biosciences). Cells were plated into 12-well plates at a density of 2 × 104 cells per well and labeled for the last 6 h with 4 μCi of [3H]thymidine, fixed in 10% trichloroacetic acid, and lysed in 0.2 N NaOH. [3H]thymidine incorporation into the DNA was measured with a scintillation counter. Each measurement was performed in triplicate. Cells were plated into 10-cm plates at a density of 5 × 105cells per well, serum-starved overnight after attachment, and then fed with media containing serum for 24 h. Cells were then trypsinized, fixed in 70% ethanol, washed once in phosphate buffered saline, and resuspended in RNaseA (100 μg/ml) and propidium iodide (50 μg/ml). Samples were analyzed on a FACScan (BD Biosciences) flow cytometer. Each experiment was performed in triplicate. For these assays, 35-mm plates were prepared with a base layer of Dulbecco's minimal essential media with 10% calf serum (Invitrogen) and 0.6% bacto agar (BD Biosciences). Cells were plated at a density of 5 × 104 cells per plate in a mix of Dulbecco's minimal essential media with 10% calf serum and 0.4% bacto agar. Plated cells were fed once a week with 0.5 ml of Dulbecco's minimal essential media plus 10% calf serum and 0.4% bacto agar. After 14 days, the plates were stained with 0.5 ml of 0.005% crystal violet. On each plate, colonies with more than 30 cells were counted. Each measurement was performed in triplicate. Intracranial tumor formation was tested with SCID-beige mice injected with 1 × 106glioma cells in 10 μl of Methocel (Dow Chemical Co., Midland, IL). Mice were sacrificed when they developed any neurological abnormalities. Brains were serially sectioned and examined by histopathology. Immunohistochemistry was performed on all tumors with hematoxylin and eosin, factor VIII, collagen IV, and Ki-67. Vascular characteristics of tumors were compared through an examination of vessel number and size in selected high power fields. Fields were selected to represent maximal tumor diameters without significant areas of necrosis. Internal organs were completely removed and evaluated grossly for macroscopic metastases. Selected livers and lungs were examined for microscopic metastases. SCID-beige mice (Taconic, Germantown, NY) were subcutaneously injected in the flank with 10 × 106 glioma cells per mouse in 100 μl of Matrigel (BD Biosciences) (20Rich J.N. Guo C. McLendon R.E. Bigner D.D. Wang X.F. Counter C.M. Cancer Res. 2001; 61: 3556-3560PubMed Google Scholar). Mice were regularly checked for tumor formation. Tumor volume was calculated with the formula 0.5 × (length) × (width)2. Tumors were removed and examined by immunohistopathology. Mice that were noted to have significant weight loss were sacrificed regardless of primary tumor size. All internal organs including the brain were removed at the time of sacrifice and grossly examined for metastases. Selected lungs, livers, and brains were sectioned and examined for microscopic metastases. Kits were purchased from BD Biosciences and used according to instructions. Briefly, 2.5 × 104 tumor cells were incubated in selected conditions (serum-free media, 1% Me2SO control, 100 μm MMP-2 Inhibitor I (Calbiochem), 100 μmMMP-2/9 Inhibitor I (Calbiochem), or 100 μm MMP-3 Inhibitor II (Calbiochem)) and then allowed to attach and invade. Cells were fixed 22 h after plating. The invasion was calculated as a ratio of that shown by control (uncoated) inserts to that shown by Matrigel-coated inserts. Experiments were performed in triplicate. Wilcoxon rank sum test was used in all analysis Differential gene expression analyses of primary brain tumors and normal brain by the SAGE technology have permitted the identification of numerous genes that are increased in expression in malignant gliomas, including osteonectin and osteoactivin (4Rempel S.A. Golembieski W.A. Ge S. Lemke N. Elisevich K. Mikkelsen T. Gutierrez J.A. J. Neuropathol. Exp. Neurol. 1998; 57: 1112-1121Crossref PubMed Scopus (120) Google Scholar, 19Loging W.T. Lal A. Siu I.M. Loney T.L. Wikstrand C.J. Marra M.A. Prange C. Bigner D.D. Strausberg R.L. Riggins G.J. Genome Res. 2000; 10: 1393-1402Crossref PubMed Scopus (88) Google Scholar). To understand the role of these two genes in the pathogenesis of gliomas, we determined the effects of overexpressing osteonectin or osteoactivin by using a genetically defined human glioma cell line (hereafter referred to as the THR glioma line) developed in our laboratory (20Rich J.N. Guo C. McLendon R.E. Bigner D.D. Wang X.F. Counter C.M. Cancer Res. 2001; 61: 3556-3560PubMed Google Scholar). When implanted intracranially in immune-compromised mice, THR cells display a non-invasive phenotype. This phenotype is in contrast to that of gliomas in patients that are universally invasive, suggesting that additional genetic alterations beyond those already present in this cell line are required to develop an invasive phenotype in vivo. Thus, the THR cells represent an ideal model system in which to study the contributions of specific genetic changes to late stages of glioma development. Through the use of a retroviral system with an independent selectable marker, we generated polyclonal THR glioma cells that ectopically expressed either osteonectin (Fig. 1 A) or osteoactivin (Fig. 1 B). Early passage cells were used in all experiments to minimize genetic changes associated with simian virus 40 (SV40) T antigen expression. To determine the potential impact of osteonectin or osteoactivin expression on tumor development, we implanted these engineered THR cell lines intracranially in suspensions. In this nature environment, the genetically defined glioma cells formed large extra-axial masses with histologic features consistent with a malignant neural tumor including pseudopallisading necrosis (Fig. 1 C), regardless of whether the tumors expressed the vector, osteonectin, or osteoactivin. Tumors derived from vector control cells were located predominantly within the subarachnoid space, and rare foci superficially invaded the Virchow-Robin spaces but extended no deeper than the molecular layer of the superficial cerebral cortex. The Virchow-Robin spaces and vasculature remained delicate without expansion by tumor, increased numbers of vessels, or hypertrophy of the endothelium (not shown). However, in 23 of 33 mice implanted with the osteonectin-expressing glioma cells, tumors were located within the subarachnoid space and displayed a striking phenotype of invasion (Fig. 1 D), as well as expansion of the perivascular Virchow-Robin spaces by masses of tumor cells that extended into the deep layers of the cerebral cortex (Fig. 1 E) similar to previous reports of glioma invasion in mouse models (24Laws Jr., E.R. Goldberg W.J. Bernstein J.J. Int. J. Dev. Neurosci. 1993; 11: 691-697Crossref PubMed Scopus (64) Google Scholar). In sharp contrast to the tumors derived from vector control glioma cells, blood vessels associated with the tumors were increased in size and number (Fig. 1 F). These features readily contrasted with adjacent brain tissues not invaded by the tumor. Neoplastic cells in rare nests and single cells were seen adjacent to the Virchow-Robin spaces in the parenchyma, a finding that may represent either true invasion of brain parenchyma or formation of an angiocentric perivascular zone outside the limits of the pia-glial membrane. The prominent tumor associated vasculature was readily detected by immunohistochemical staining of the factor VIII antigen, which showed enhanced proliferation and endothelial hypertrophy of the osteonectin-expressing glioma tumors in comparison with the normal vasculature seen in adjacent brain, as well as the tumors derived from the vector control cells. Osteoactivin-expressing cells formed intracranial tumors in a fashion similar to those expressing osteonectin but with a lower rate of brain invasion (Fig. 1, D and G) and fewer associated vascular alterations (Fig. 1 H). Taken together, our data suggest that the expression of osteonectin and osteoactivin induce glioma invasion in the context of brain environment associated with penetrating blood vessels. Having documented the dramatic effects of osteonectin and osteoactivin expression on tumor invasion in xenograph experiments, we next investigated the mechanism by which these genes exert tumorigenic effects. Although in vitro systems are incomplete models of tumor invasion and metastasis as they lack normal stromal interactions, they allow selected analysis of potential changes at the cellular level, including cell proliferation, apoptosis, and invasion. Previously, it was reported that osteonectin inhibits cell proliferation (25Bradshaw A.D. Francki A. Motamed K. Howe C. Sage E.H. Mol. Biol. Cell. 1999; 10: 1569-1579Crossref PubMed Scopus (88) Google Scholar) and induces apoptosis in some ovarian carcinoma cell lines (26Yiu G.K. Chan W.Y. Ng S.W. Chan P.S. Cheung K.K. Berkowitz R.S. Mok S.C. Am. J. Pathol. 2001; 159: 609-622Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). However, we find that expression of osteonectin or osteoactivin in our THR glioma cells did not cause any significant changes in cell proliferation (Fig.2 A), apoptosis (Fig.2 A), or DNA synthesis (Fig. 2 B). Disruption of osteonectin expression has been linked to loss of IGF-1R expression (27Basu A. Rodeck U. Prendergast G.C. Howe C.C. Cell Growth & Differ. 1999; 10: 721-728PubMed Google Scholar), but we found no differences in the expression levels of IGF-1R, PDGFR-α or -β, or EGFR between control and osteonectin-expressing cells (Fig. 2 C). Osteoactivin expression was associated with an increase in EGFR expression but a decrease in PDGFR-α expression and no change in PDGFR-β or IGF-1R expression (Fig. 2 C). In addition, the ectopic expression of osteonectin or osteoactivin in human astrocytes expressing SV40 T antigen and human telomerase catalytic subunit failed to provide a mitogenic stimulus to transform these cells in the absence of oncogenic Ras as measured by soft agar colony formation assays (Fig. 2 D). These results suggest that osteonectin and osteoactivin expression is mainly associated with the acquired abilities by the tumor cells in invasion rather than tumor initiation. To test this hypothesis, we examined the impact of osteonectin and osteoactivin expression on tumor cell invasion in vitro. As shown in Fig.3, A and B, osteonectin or osteoactivin expression significantly increased cell invasion in all cell lines tested as measured by the Matrigel invasion assay. Constitutive expression of osteonectin or osteoactivin appeared to increase the ability of tumor cells to degrade components of the extracellular matrix and increased motility, a critical aspect of invasive cancers.Figure 3Osteonectin and osteoactivin expression is associated with increased invasion and expression of MMP-9 and MMP-3. As shown in A, Matrigel invasion assays were performed with the genetically defined THR glioma cell line, D54MG, U87MG, and U251MG, each engineered to express osteonectin (ON). The expression of osteonectin was clearly linked to increased invasion relative to vector control (VEC). *,p = 0.01. As shown in B, Matrigel analysis of glioma cells expressing osteoactivin with specific MMP-2, MMP-2/9, and MMP-3 inhibitors ablated the increase in invasion associated with osteoactivin expression (OA) relative to vector controls. CON, control. *, p = 0.01 relative to vector control; **, p = 0.01 relative to untreated osteoactivin-expressing tumor. As shown in C, Western analysis of a genetically defined glioma cell line with vector control (V), osteonectin, or osteoactivin expression revealed increased expression of gelatinase B (MMP-9) and stromelysin-1 (MMP-3) but minimal change of gelatinase A (MMP-2). As shown inD, Matrigel analysis of glioma cells expressing either vector control or osteonectin showed no change in invasion with an MMP-2/9 inhibitor, but an MMP-3 inhibitor ablated the increase in invasion associated with osteonectin expression (*, p = 0.01 relative to untreated). As shown in E, parental genetically defined THR glioma cells were treated with 50 μg/ml purified human osteonectin (Hematologic Technologies, Essex, VT). Conditioned media were collected simultaneously with either no osteonectin or after treatment with osteonectin for specific times. The media (50 μl) was resolved by SDS-PAGE and immunoblotted for stromelysin-1 (MMP-3).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further explore the mechanisms that mediate the invasive effects of osteonectin and osteoactivin, we examined the expression profiles of proteins intimately associated with tumor invasion, specifically the MMPs. As shown in Fig. 3 C, we found that osteonectin and osteoactivin expression significantly increased the production of MMP-9 and MMP-3 by the tumor cells with minimal changes in MMP-2 expression. To test whether this up-regulation of MMPs is directly linked to the invasive behavior of tumors expressing osteonectin and osteoactivin, we examined the effect of specific MMP inhibitors in the Matrigel invasion assay. Treatment of cells expressing osteonectin with an MMP-2/9 inhibitor had no effect (Fig. 3 D), suggesting that the increase in MMP-9 expression was not a major contributing factor to the changes in invasive behavior by the osteonectin-expressing tumor cells. However, MMP-3 inhibitor treatment substantially reduced invasion through Matrigel by the same tumor cells (Fig. 3 D), suggesting an important role for MMP-3 in mediating the in vitro invasive behavior of these cells. Consistent with this observation, treatment of parental THR cells with exogenous osteonectin induced the production of MMP-3 within 4 h (Fig. 3 E), whereas the expression of MMP-2 and MMP-9 did not change (data not shown). Similarly, invasion of Matrigel by THR cells expressing osteoactivin was also most sensitive to a blockage of MMP-3 activity, although this activity was partially inhibited by inhibitors specific for MMP-2 and MMP-2/9 (Fig.3 B). Taken together, the data suggest that osteonectin and osteoactivin may promote tumor invasion, at least in part, by increasing the production of specific MMPs. Since osteonectin and osteoactivin are expressed in other types of advanced cancers, we investigated the potential effects of these two genes on tumor progression in a different growth microenvironment. To do this, osteonectin-expressing and osteoactivin-expressing THR glioma cells were subcutaneously injected into the flanks of SCID-beige mice. The primary subcutaneous tumors derived from the glioma cells expressing osteonectin have an identical latency period (∼29 days) to a vector control cell line but grow to a larger volume (Fig.4 A). Tumors from the osteoactivin-expressing cell line display a longer latency (∼39 days) but subsequently grow to, on average, a larger size than vector controls (Fig. 4 A). Tumors expressing osteonectin and osteoactivin did not exhibit a difference in angiogenic features from the control cells (data not shown). Strikingly, both types of tumor cells developed massive spontaneous intrathoracic and/or intraperitoneal metastases (Fig. 4, B and C). Metastases were found in 50% (16 of 32) of mice injected with osteonectin-expressing glioma cells and 14% (3 of 15) of osteoactivin-expressing glioma cells (Fig. 4 D). In contrast, the THR vector control cell line exhibited a single small, discrete metastasis in only 1 of 15 mice, and no metastases developed in 25 mice implanted with the THR parental cell line (not shown). Interestingly, the development of metastases was independent of primary tumor size. Three of 16 mice that developed metastases from osteonectin-expressing cells had no grossly evident subcutaneous tumors at the initial site of injection at the time of euthanasia. Therefore, the data suggest that the development of a metastatic phenotype from osteonectin-expressing glioma cells is associated with an early stage of tumor development inside the animals. Upon close pathological examination, we found that tumors derived from osteonectin-expressing glioma cells display a phenotype of solid and circumscribed neoplasms capable of invading the pancreas, peripancreatic soft tissues, subcutaneous tissues, and liver (Fig.4, E and F). Examination of hematoxylin- and eosin-stained sections of tumors derived from osteonectin-expressing cells revealed a malignant phenotype of poorly differentiated neoplasm. Within the tumor mass, there were numerous areas of atypical mitoses and necrosis pseudopalisaded by neoplastic cells. Taken together, histological analyses of tumors derived from osteonectin-expressing cells revealed typical pathological features that are consistent with metastatic disease. Interestingly, we found that the metastasized tumors did not possess the features of prominently increased vasculature and hypertrophic endothelium that are often associated with the invasive brain tumors. Consistent with this observation, lymphatic/vascular invasion and perineural extension were not detected in the metastatic sites. To test whether the effects of osteonectin described above were cell line-specific, we investigated the behavior of tumor development by the human glioma cell lines D54MG, U87MG, and U251MG engineered to express higher levels of osteonectin (Fig. 4 G). Importantly, we found that ectopic expression of osteonectin by these glioma cell lines also induced spontaneous metastases when they were injected subcutaneously in the flanks of SCID-beige mice (Fig. 4 D). These results suggest that osteonectin expression can induce a non-metastatic cancer cell line to adopt a metastatic phenotype with the alteration in the expression of only a single gene. Technological improvements now permit comprehensive analysis of gene expression patterns in cancer specimens. The resulting data have created a wealth of information to be mined, but utilization of the data remains difficult due to a lack of functional information for specific genes. Here we demonstrate the use of a genetically defined human cancer model system to investigate the function of specific genes differentially expressed in human cancers. Previously developed model systems of cancer have included cell lines created from human tumors and murine models. Although each model system has been of great benefit to our understanding of the contributions that specific genetic alterations play in the development of specific phenotypes of cancer, each system has significant drawbacks. Human cell lines are generally derived from advanced cancers that have widespread genetic changes and genomic instability as well as changes from long passages in cell culture. Murine models permit genetic control but are labor-intensive and suffer from potential species-specific differences in the process of transformation and gene function (28Hamad N.M. Elconin J.H. Karnoub A.E. Bai W. Rich J.N. Abraham R.T. Der C.J. Counter C.M. Genes Dev. 2002; 16: 2045-2057Crossref PubMed Scopus (349) Google Scholar). Thus, a genetically defined human cancer model system offers a useful tool to determine the roles of specific genes in carcinogenesis, particularly the late stages of this multistep process. Using this system, we investigated genes that normally appear to be highly expressed in bone without presence in brain tissues but have significantly increased expression in human gliomas. Although the expression of both osteonectin and osteoactivin has been linked to tumor progression in previous studies, their precise contributions to late stages of tumorigenesis remain unclear. Through an ectopic expression strategy in this model system, we revealed that osteonectin expression induced a highly malignant phenotype with significantly increased brain invasion associated with vascular proliferation and spontaneous systemic metastasis, whereas osteoactivin expression resulted in a more modest increase in malignant phenotype with less frequent metastases and lower degrees of vascular change. Although the highly metastatic characteristics displayed by the tumor cells in our study do not fully replicate the precise behavior of gliomas in humans, the data strongly support the notion that expression of osteonectin and osteoactivin could significantly alter or modify tumor behavior. Considering the fact that these genes are also overexpressed in other types of human cancers that do display a metastatic phenotype, this finding is clearly relevant and significant to our understanding of the mechanism underlying metastasis associated with those types of human cancers. Furthermore, our results indicate that the cellular microenvironment potently modulates the phenotypic behavior of cancer cells that express those two genes since glioma cells expressing osteonectin, and to a lesser extent, osteoactivin, formed large spontaneous metastases with subcutaneous implantation but remained localized to the brain with intracranial implantation. The vascular and extracellular environment in the brain is radically different from that of other organs, with the presence of the blood-brain barrier, absence of lymphatics, and brain-specific extracellular matrix and cell-cell interactions associated with neuronal and glial migration during development. These differences may partially account for the differential behavior of cells expressing osteonectin implanted in the brain versus other parts of the body. This finding strongly suggests that contributions to the development of specific tumorigenic phenotypes by the expression of specific genes are dependent on the specific characteristics of the microenvironment associated with tumor progression, adding another layer of complexity to the molecular mechanisms underlying late stages of tumorigenesis. Another potentially important finding is that osteonectin and osteoactivin appear to promote a specific invasive phenotype intracranially involving tumor cell invasion along pre-existent blood vessels in the Virchow-Robin spaces and development of altered vasculature at the brain-tumor interface. This phenotype mimics the behavior of a subset of human gliomas and many invasive medulloblastomas. Osteonectin has been reported to have an effect on angiogenesis through regulation of VEGF activity (29Vajkoczy P. Menger M.D. Goldbrunner R. Ge S. Fong T.A. Vollmar B. Schilling L. Ullrich A. Hirth K.P. Tonn J.C. Schmiedek P. Rempel S.A. Int. J. Cancer. 2000; 87: 261-268Crossref PubMed Scopus (69) Google Scholar). In our studies, neither osteonectin nor osteoactivin induced significant changes in the vasculature as measured by vessel number or diameter within the primary tumors, whether formed subcutaneously or intracranially. In contrast, intracranial tumor cells expressing osteonectin and osteoactivin grow along penetrating blood vessels, and the blood vessels at the invasive front were markedly abnormal with vessel hypertrophy and hyperplasia. These results suggest that the production of osteonectin and osteoactivin by glioma cells may create a complex relationship between invading tumor and normal vasculature that may be co-opted during tumor invasion, consequently allowing expansion of the tumor mass without the induction of angiogenesis at a significant level. The malignant phenotypes mediated by osteonectin and osteoactivin likely involve multiple mechanisms at the molecular level, with the induction of MMP expression as an important component. The rapid increase in MMP-3 protein levels in response to osteonectin treatment by the THR tumor cells strongly suggests that osteonectin may regulate the production of this specific MMP through a more direct mechanism. Taken together, our results validate the use of a genetically defined human cancer model system in investigating the contributions of specific genes to late stages of tumorigenesis. This in vivosystem permits the application of a functional genomics approach in defining the specific activities of genes that have been identified to be abnormally expressed in human cancers, particularly those associated with tumor invasion, metastasis, and angiogenesis. We thank G. J. Riggins, A. Hjelmeland, J. Herndon, and R. McLendon for helpful discussions, S. Rempel for osteonectin cDNA, C. Wikstrand for GPNMB antibody, and S. Keir, Y. Yu, and R. Nelson for technical assistance. J. Parsons provided editorial support.

The development of novel immunotherapy strategies for non-small cell lung cancer (NSCLC) will be facilitated by the identification of tumor-specific targets. Although the epidermal growth factor receptor (EGFR) is overexpressed in many cases of NSCLC, its wide distribution in normal tissue may limit its suitability as an immunotherapeutic target. However, mutations within the EGFR that are unique to malignancies may provide specific targets for immunotherapeutic intervention. For example, one mutant form, the type III deletion mutant of the EGFR, that has been identified in glioblastomas contains a novel peptide sequence in its extracellular domain which is detectable by anti-peptide antisera. In this study, the prevalence of this type of mutation of the EGFR in NSCLC was determined. Thirty-two frozen sections of primary NSCLC were examined by immunocytochemistry to determine the presence of native and mutated EGFR. Native EGFR was overexpressed in 12 of the 32 sections and a diffuse cellular distribution of the EGFR type III deletion mutation was identified in five (16%) of the specimens (2 of 13 squamous, 2 of 2 mixed, 0 of 10 adenocarcinoma, and 1 of 7 undifferentiated). This mutated EGFR was not detected in sections of normal breast, lung, skin, ovary, colon, kidney, endometrium, and placenta. The type III EGFR deletion mutant, expressed in some cases of NSCLC, may be a molecularly defined, tumor-specific antigen in lung cancer.

To determine the maximum-tolerated dose (MTD) of iodine-131 ((131)I)-labeled 81C6 antitenascin monoclonal antibody (mAb) administered clinically into surgically created resection cavities (SCRCs) in malignant glioma patients and to identify any objective responses with this treatment.In this phase I trial, newly diagnosed patients with malignant gliomas with no prior external-beam therapy or chemotherapy were treated with a single injection of (131)I-labeled 81C6 through a Rickham reservoir into the resection cavity. The initial dose was 20 mCi and escalation was in 20-mCi increments. Patients were observed for toxicity and response until death or for a minimum of 1 year after treatment.We treated 42 patients with (131)I-labeled 81C6 mAb in administered doses up to 180 mCi. Dose-limiting toxicity was observed at doses greater than 120 mCi and consisted of delayed neurotoxicity. None of the patients developed major hematologic toxicity. Median survival for patients with glioblastoma multiforme and for all patients was 69 and 79 weeks, respectively.The MTD for administration of (131)I-labeled 81C6 into the SCRC of newly diagnosed patients with no prior radiation therapy or chemotherapy was 120 mCi. Dose-limiting toxicity was delayed neurologic toxicity. We are encouraged by the survival and toxicity and by the low 2.5% prevalence of debulking surgery for symptomatic radiation necrosis.

An additional tumor suppressor gene on chromosome 9p telomeric to the CDKN2A/B locus has long been postulated to exist. Using Affymetrix 250K single nucleotide polymorphism arrays to screen for copy number changes in glioblastoma multiforme (GBM), we detected a high frequency of deletions of the PTPRD gene, which encodes a receptor protein tyrosine phosphatase at chromosome 9p23-24.1. Missense and nonsense mutations of PTPRD were identified in a subset of the samples lacking deletions, including an inherited mutation with somatic loss of the wild-type allele. We then sequenced the gene in melanoma and identified 10 somatic mutations in 7 of 57 tumors (12%). Reconstitution of PTPRD expression in GBM and melanoma cells harboring deletions or mutations led to growth suppression and apoptosis that was alleviated by both the somatic and constitutional mutations. These data implicate PTPRD in the pathogenesis of tumors of neuroectodermal origin and, when taken together with other recent reports of PTPRD mutations in adenocarcinoma of the colon and lung, suggest that PTPRD may be one of a select group of tumor suppressor genes that are inactivated in a wide range of common human tumor types.

Convection-enhanced delivery (CED) is a novel drug delivery technique that uses positive infusion pressure to deliver therapeutic agents directly into the interstitial spaces of the brain. Despite the promise of CED, clinical trials have demonstrated that target-tissue anatomy and patient-specific physiology play a major role in drug distribution using this technique. In this study, we retrospectively tested the ability of a software algorithm using MR diffusion tensor imaging to predict patient-specific drug distributions by CED. A tumor-targeted cytotoxin, cintredekin besudotox (interleukin 13-PE38QQR), was coinfused with iodine 123-labeled human serum albumin (123I-HSA), in patients with recurrent malignant gliomas. The spatial distribution of 123I-HSA was then compared to a drug distribution simulation provided by the software algorithm. The algorithm had a high sensitivity (71.4%) and specificity (100%) for identifying the high proportion (7 of 14) of catheter trajectories that failed to deliver drug into the desired anatomical region (p = 0.021). This usually occurred when catheter trajectories crossed deep sulci, resulting in leak of the infusate into the subarachnoid cerebrospinal fluid space. The mean concordance of the volume of distribution at the 50% isodose level between the actual 123I-HSA distribution and simulation was 65.75% (95% confidence interval [CI], 52.0%-79.5%), and the mean maximal inplane deviation was less than 8.5 mm (95% CI, 4.0-13.0 mm). The use of this simulation algorithm was considered clinically useful in 84.6% of catheters. Routine use of this algorithm, and its further developments, should improve prospective selection of catheter trajectories, and thereby improve the efficacy of drugs delivered by this promising technique.

Abstract Cytogenetic and RFLP studies have shown that chromosome 10 is frequently lost in tumor cells from glioblastomas, suggesting that a suppressor gene important in tumorigenesis is present on this chromosome. Forty‐one tumors were examined for loss of heterozygosity at 23 loci on chromosome 10 to determine the smallest common deletion interval on this chromosome. Seven tumors did not lose heterozygosity for any of the markers. Twenty‐three tumors lost an allele for all the informative loci. In 11 tumors heterozygosity was maintained at some loci and lost at other loci, indicating partial deletion of chromosome 10. The common region of deletion in these 11 tumors was located in 10q24‐q26 between the markers pHUK‐8 and pMCT122.2.

The purpose of this study was to determine the feasibility and assess the efficacy and toxicity, among newly diagnosed malignant glioma patients, of administering (131)I-labeled murine antitenascin monoclonal antibody 81C6 ((131)I-81C6) into a surgically created resection cavity (SCRC) to achieve a patient-specific, 44-Gy boost to the 2-cm SCRC margin. A radioactivity dose of (131)I-81C6 calculated to achieve a 44-Gy boost to the SCRC was administered, followed by conventional external beam radiotherapy (XRT) and chemotherapy. Twenty-one patients were enrolled in the study: 16 with glioblastoma multiforme (GBM) and 5 with anaplastic astrocytoma. Twenty patients received the targeted 44-Gy boost (+/-10%) to the SCRC. Attributable toxicity was mild and limited to reversible grade 3 neutropenia or thrombocytopenia (n = 3; 14%), CNS wound infections (n = 3; 14%), and headache (n = 2; 10%). With a median follow-up of 151 weeks, median overall survival times for all patients and those with GBM are 96.6 and 90.6 weeks, respectively; 87% of GBM patients are alive at 1 year. It is feasible to consistently achieve a 44-Gy boost dose to the SCRC margin with patient-specific dosing of (131)I-81C6. Our study regimen ((131)I-81C6 + XRT + temozolomide) was well tolerated and had encouraging survival. To determine if selection of good-prognosis patients affects outcome associated with this approach, the U.S. Food and Drug Administration has approved a trial randomizing newly diagnosed GBM patients to either our study regimen or standard XRT plus temozolomide.

Epidermal growth factor receptor variant III (EGFRvIII) is an oncogenic, constitutively active mutant form of the EGFR that is commonly expressed in glioblastoma and is also detected in a number of epithelial cancers. EGFRvIII presents a unique antigenic target for anti-EGFRvIII vaccines and it has been shown to modulate response to EGFR kinase inhibitor therapy. Thus, detection in clinical samples may be warranted. Existing patents preclude the use of anti-EGFRvIII antibodies for clinical detection. Further, frozen tissue is not routinely available, particularly for patients treated in the community. Thus, detection of EGFRvIII in formalin-fixed paraffin-embedded (FFPE) clinical samples is a major challenge.We developed a real-time reverse transcription-PCR (RT-PCR) assay for detecting EGFRvIII in FFPE samples and analyzed 59 FFPE glioblastoma clinical samples with paired frozen tissue from the same surgical resection. We assessed EGFRvIII protein expression by immunohistochemistry using two distinct specific anti-EGFRvIII antibodies and examined EGFR gene amplification by fluorescence in situ hybridization.The FFPE RT-PCR assay detected EGFRvIII in 16 of 59 (27%) samples, exclusively in cases with EGFR amplification, consistent with the expected frequency of this alteration. The FFPE RT-PCR assay was more sensitive and specific for detecting EGFRvIII than either of the two antibodies alone, or in combination, with a sensitivity of 93% (95% confidence interval, 0.78-1.00) and a specificity of 98% (95% confidence interval, 0.93-1.00).This assay will facilitate accurate assessment of EGFRvIII in clinical samples and may aid in the development of strategies for stratifying patients for EGFRvIII-directed therapies.

The glioblastoma genome displays remarkable chromosomal aberrations, which harbor critical glioblastoma-specific genes contributing to several oncogenetic pathways.To identify glioblastoma-targeted genes, we completed a multifaceted genome-wide analysis to characterize the most significant aberrations of DNA content occurring in glioblastomas.We performed copy number analysis of 111 glioblastomas by Digital Karyotyping and Illumina BeadChip assays and validated our findings using data from the TCGA (The Cancer Genome Atlas) glioblastoma project.From this study, we identified recurrent focal copy number alterations in 1p36.23 and 4p16.3.Expression analyses of genes located in the two regions revealed genes which are dysregulated in glioblastomas.Specifically, we identify EGFR negative regulator, ERRFI1, within the minimal region of deletion in 1p36.23.In glioblastoma cells with a focal deletion of the ERRFI1 locus, restoration of ERRFI1 expression slowed cell migration.Furthermore, we demonstrate that TACC3, an Aurora-A kinase substrate, on 4p16.3, displays gain of copy number, is overexpressed in a glioma-grade-specific pattern, and correlates with Aurora kinase overexpression in glioblastomas.Our multifaceted genomic evaluation of glioblastoma establishes ERRFI1 as a potential candidate tumor suppressor gene and TACC3 as a potential oncogene, and provides insight on targets for oncogenic pathway-based therapy.

Abstract Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant ( K d ) of 33×10 -9 m , and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with various modifications, as well as dissecting signal transduction pathways.

Bevacizumab improves outcome for most recurrent glioblastoma patients, but the duration of benefit is limited and survival after initial bevacizumab progression is poor. We evaluated bevacizumab continuation beyond initial progression among recurrent glioblastoma patients as it is a common, yet unsupported practice in some countries. We analysed outcome among all patients (n=99) who received subsequent therapy after progression on one of five consecutive, single-arm, phase II clinical trials evaluating bevacizumab regimens for recurrent glioblastoma. Of note, the five trials contained similar eligibility, treatment and assessment criteria, and achieved comparable outcome. The median overall survival (OS) and OS at 6 months for patients who continued bevacizumab therapy (n=55) were 5.9 months (95% confidence interval (CI): 4.4, 7.6) and 49.2% (95% CI: 35.2, 61.8), compared with 4.0 months (95% CI: 2.1, 5.4) and 29.5% (95% CI: 17.0, 43.2) for patients treated with a non-bevacizumab regimen (n=44; P=0.014). Bevacizumab continuation was an independent predictor of improved OS (hazard ratio=0.64; P=0.04). The results of our retrospective pooled analysis suggest that bevacizumab continuation beyond initial progression modestly improves survival compared with available non-bevacizumab therapy for recurrent glioblastoma patients require evaluation in an appropriately randomised, prospective trial.

Anaplastic astrocytoma WHO grade III (A3) is a lethal brain tumor that often occurs in middle aged patients. Clinically, it is challenging to distinguish A3 from glioblastoma multiforme (GBM) WHO grade IV. To reveal the genetic landscape of this tumor type, we sequenced the exome of a cohort of A3s (n=16). For comparison and to illuminate the genomic landscape of other glioma subtypes, we also included in our study diffuse astrocytoma WHO grade II (A2, n=7), oligoastrocytoma WHO grade II (OA2, n=2), anaplastic oligoastrocytoma WHO grade III (OA3, n=4), and GBM (n=28). Exome sequencing of A3s identified frequent mutations in IDH1 (75%, 12/16), ATRX (63%, 10/16), and TP53 (82%, 13/16). In contrast, the majority of GBMs (75%, 21/28) did not contain IDH1 or ATRX mutations, and displayed a distinct spectrum of mutations. Finally, our study also identified novel genes that were not previously linked to this tumor type. In particular, we found mutations in Notch pathway genes (NOTCH1, NOTCH2, NOTCH4, NOTCH2NL), including a recurrent NOTCH1-A465Tmutation, in 31% (5/16) of A3s. This study suggests genetic signatures will be useful for the classification of gliomas.

The unfolded protein response (UPR) is an endoplasmic reticulum (ER)-based cytoprotective mechanism acting to prevent pathologies accompanying protein aggregation. It is frequently active in tumors, but relatively unstudied in gliomas. We hypothesized that UPR stress effects on glioma cells might protect tumors from additional exogenous stress (ie, chemotherapeutics), postulating that protection was concurrent with altered tumor cell metabolism. Using human brain tumor cell lines, xenograft tumors, human samples and gene expression databases, we determined molecular features of glioma cell UPR induction/activation, and here report a detailed analysis of UPR transcriptional/translational/metabolic responses. Immunohistochemistry, Western and Northern blots identified elevated levels of UPR transcription factors and downstream ER chaperone targets in gliomas. Microarray profiling revealed distinct regulation of stress responses between xenograft tumors and parent cell lines, with gene ontology and network analyses linking gene expression to cell survival and metabolic processes. Human glioma samples were examined for levels of the ER chaperone GRP94 by immunohistochemistry and for other UPR components by Western blotting. Gene and protein expression data from patient gliomas correlated poor patient prognoses with increased expression of ER chaperones, UPR target genes, and metabolic enzymes (glycolysis and lipogenesis). NMR-based metabolomic studies revealed increased metabolic outputs in glucose uptake with elevated glycolytic activity as well as increased phospholipid turnover. Elevated levels of amino acids, antioxidants, and cholesterol were also evident upon UPR stress; in particular, recurrent tumors had overall higher lipid outputs and elevated specific UPR arms. Clonogenicity studies following temozolomide treatment of stressed or unstressed cells demonstrated UPR-induced chemoresistance. Our data characterize the UPR in glioma cells and human tumors, and link the UPR to chemoresistance possibly via enhanced metabolism. Given the role of the UPR in the balance between cell survival and apoptosis, targeting the UPR and/or controlling metabolic activity may prove beneficial for malignant glioma therapeutics.

Of the 4 medulloblastoma subgroups, Group 3 is the most aggressive but the importance of angiogenesis is unknown. This study sought to determine the role of angiogenesis and identify clinically relevant biomarkers of tumor vascularity and survival in Group 3 medulloblastoma.VEGFA mRNA expression and survival from several patient cohorts were analyzed. Group 3 xenografts were implanted intracranially in nude rats. Dynamic susceptibility weighted (DSC) MRI and susceptibility weighted imaging (SWI) were obtained. DSC MRI was used to calculate relative cerebral blood volume (rCBV) and flow (rCBF). Tumor vessel density and rat vascular endothelial growth factor alpha (VEGFA) expression were determined.Patient VEGFA mRNA levels were significantly elevated in Group 3 compared with the other subgroups (P < 0.001) and associated with survival. Xenografts D283, D341, and D425 were identified as Group 3 by RNA hierarchical clustering and MYC amplification. The D283 group had the lowest rCBV and rCBF, followed by D341 and D425 (P < 0.05). These values corresponded to histological vessel density (P < 0.05), rat VEGFA expression (P < 0.05), and survival (P = 0.002). Gene set enrichment analysis identified 5 putative genes with expression profiles corresponding with these findings: RNH1, SCG2, VEGFA, AGGF1, and PROK2. SWI identified 3 xenograft-independent categories of intratumoral vascular architecture with distinct survival (P = 0.004): organized, diffuse microvascular, and heterogeneous.Angiogenesis plays an important role in Group 3 medulloblastoma pathogenesis and survival. DSC MRI and SWI are clinically relevant biomarkers for tumor vascularity and overall survival and can be used to direct the use of antivascular therapies for patients with Group 3 medulloblastoma.

We sought to characterize the pathway by which the multifunctional cytokine transforming growth factor-β (TGF-β) inhibits the proliferation of normal astrocytes, and we analyzed the alterations in the TGF-β pathway in human glioma cell lines. Upon TGF-β treatment, primary rat astrocytes showed a significant decrease in DNA synthesis upon thymidine incorporation with a cell cycle arrest in the G₁ phase. Western analysis of the astrocytes revealed that the expression of the cyclin-dependent kinase inhibitor (CdkI) p15^INK4B was significantly up-regulated upon TGF-β treatment without a change in other CdkI levels. The retinoblastoma protein (Rb) became hypophosphorylated, and Cdk2 activity decreased. Analysis of <i>Smad3</i> null mouse astrocytes showed a significant loss of both TGF-β-mediated growth inhibition and p15^INK4B induction compared with wild-type mouse astrocytes. Infection of rat astrocytes by <i>SMAD3</i>and<i>SMAD4</i>adenoviruses failed to induce increased expression of p15^INK4B, implying indirect transcriptional regulation of p15^INK4B by SMAD3. High-grade human gliomas secrete TGF-β, yet are resistant to its growth inhibitory effects. Analysis of the effects of TGF-β on 12 human glioma cell lines showed that TGF-β mildly inhibited the growth of six lines, had no effect on four lines, and stimulated the growth of two lines. The majority of glioma lines had homozygous deletions of the p15^INK4B gene, except for two lines that expressed p15^INK4B protein, which was induced further upon TGF-β treatment. Three lines mildly induced CdkI p21^WAF1 expression in response to TGF-β. Most tumor lines retained other TGF-β-mediated responses, including extracellular matrix protein and angiogenic factor secretion, which may contribute to increased malignant behavior. This suggests that the loss of p15^INK4B may explain, in part, the selective loss of growth inhibition by TGF-β in gliomas to form a more aggressive tumor phenotype.

Genes expressed specifically in malignant tissue may have potential as therapeutic targets but have been difficult to locate for most cancers. The information hidden within certain public databases can reveal RNA transcripts specifically expressed in transformed tissue. To be useful, database information must be verified and a more complete pattern of tissue expression must be demonstrated. We tested database mining plus rapid screening by fluorescent-PCR expression comparison (F-PEC) as an approach to locate candidate brain tumor antigens. Cancer Genome Anatomy Project (CGAP) data was mined for genes highly expressed in glioblastoma multiforme. From 13 mined genes, seven showed potential as possible tumor markers or antigens as determined by further expression profiling. Now that large-scale expression information is readily available for many of the commonly occurring cancers, other candidate tumor markers or antigens could be located and evaluated with this approach. [The expression data described in this paper have been submitted to the NCBI SAGEmap database under library name SAGE_Duke_GBM_H1110, SAGE_pooled_GBM, SAGE_BB542_whitematter, and SAGE_normal_pool(6 th ).]

Although chemotherapy offers promise of increased survival for children with medulloblastoma and glioblastoma multiforme, drug resistance occurs frequently, resulting in tumor progression and death. Resistance to nitrosoureas and methylating agents, which damage DNA, can be mediated by a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGAT). Depletion of this protein with alkylguanines or methylating agents, however, restores tumor cell sensitivity to the cytotoxicity of chloroethylnitrosoureas (e.g., carmustine [BCNU]).This study was designed to determine whether resistance to the activity of nitrosourea (the drug BCNU) in BCNU-resistant human medulloblastoma (D341 Med) and human glioblastoma multiforme (D-456 MG) can be reversed by the methylating agent streptozocin and the O6-substituted guanines O6-methylguanine and O6-benzylguanine.Xenografts were grown subcutaneously in athymic BALB/c mice. BCNU was administered as a single intraperitoneal injection at doses of 100 mg/m2, 75 mg/m2, or 38 mg/m2--i.e., 1.0, 0.75, or 0.38, respectively, of the dose lethal to 10% of treated animals (LD10). Mice were treated intraperitoneally with a single dose of O6-benzylguanine or O6-methylguanine (240 mg/m2) or with streptozocin (600 mg/m2) daily for 4 days. Response was assessed by tumor growth delay and tumor regression. AGAT activity in the xenografts was measured at 1 and 6 hours after pretreatment, at the time tumors were excised.Pretreatment with O6-benzylguanine, O6-methylguanine, or streptozocin reduced AGAT activity to 4%, 25%, and 95% of control values, respectively, in D341 Med and 0%, 0%, and 25% of control values, respectively, in D-456 MG 1 hour after injection. After 6 hours, levels changed to 7%, 61%, and 116% of control values in D341 Med and 0%, 79%, and 21% of control values in D-456 MG, respectively. Both D341 Med and D-456 MG xenografts were completely resistant to BCNU at its LD10. Pretreatment with O6-benzylguanine increased BCNU sensitivity in both types of xenograft. In contrast, treatment with BCNU plus O6-methylguanine or streptozocin did not produce growth delays substantially different from those produced by BCNU alone, reflecting the more efficient depletion of AGAT by O6-benzylguanine. Following therapy with BCNU plus O6-benzylguanine at 0.38 LD10, tumor regressions were seen in eight of 10 D341 Med and in all 10 D-456 MG xenografts.We recommend comprehensive clinical toxicologic evaluation of combination therapy with O6-benzylguanine plus BCNU, which would allow subsequent design of phase I clinical trials.

Promoter hypermethylation of the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT) has been associated with an enhanced response to chloroethylating and methylating agents in patients with malignant glioma. The purpose of this study was to compare three distinct yet related indices for measuring AGT to determine if these assays could be used interchangeably when AGT status is to be used to guide chemotherapeutic decisions. Real-time methylation-specific PCR (MSP), assessed as the ratio of methylated AGT copies to internal beta-actin control, was used to quantitate AGT hypermethylation in 32 glioma samples. Data were compared with AGT enzyme activity as well as immunohistochemical detection of AGT protein from the same samples. Hypermethylation of the AGT promoter was detected in 19 of 31 (61%) samples evaluable by MSP. Low-level AGT, defined as <20% nuclear AGT staining by immunohistochemistry, was found in 10 of 32 samples (31%), whereas 12 of 32 (38%) had low levels of AGT activity. Correlation of immunohistochemistry to AGT activity was statistically significant (P = 0.014) as was the correlation of immunohistochemistry to MSP (P = 0.043), whereas MSP compared with AGT activity (P = 0.246) was not significant. Cross-tabulation of immunohistochemistry and MSP data based on prognostic groups, where good prognosis was represented by an immunohistochemistry of <20% and an MSP ratio >12, showed no significant relationship (P = 0.214), suggesting that one assay cannot be used interchangeably for another. The observed discordance between respective measures of AGT based on prognosis supports further standardization of AGT assays designed to guide therapeutic practice. The data also suggest that consideration be given to the large population of AGT-expressing cells within samples when therapeutic strategies based on tumor methylation are used.

The immunohistochemical detection of factor VIII/von Willebrand factor antigen (FVIII/vWF-AG) in formalin-fixed paraffin-embedded tissues was investigated using the peroxidase-antiperoxidase (PAP) method. Highly purified human FVIII/vWF was used to raise rabbit anti-FVIII/vWF-AG serum. In addition to anti-FVIII/vWF-AG activity, the unabsorbed antiserum had anti-IgG, anti-IgM, and anti-alpha2-macroglobulin specificities. Following exhaustive absorption with these proteins, the antiserum reacted monospecifically for FVIII/vWF-AG in immunodiffusion, immunoelectrophoresis, and PAP immunohistochemistry. Sections of normal tissues from six patients and a total of 43 neoplasms were examined. Treatment of the tissue sections with trypsin prior to application of the antiserum markedly increased the sensitivity of FVIII/vWF-AG detection. The positive staining for FVIII/vWF-AG was restricted to endothelial cells in both neoplastic and nonneoplastic tissue. In general, the hyperplastic endothelia in neoplastic and reactive tissues stained more intensely than those in normal tissues. Expression of FVIII/vWF-AG by nonendothelial neoplastic cells was not observed. FVIII/vWF-AG is a reliable marker for endothelial cells.

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

A major mechanism of resistance to methylating agents, including temozolomide, is the DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (AGT). Preclinical data indicates that defective DNA mismatch repair (MMR) results in tolerance to temozolomide regardless of AGT activity. The purpose of this study was to determine the role of MMR deficiency in mediating resistance in samples from patients with both newly diagnosed malignant gliomas and those who have failed temozolomide therapy.The roles of AGT and MMR deficiency in mediating resistance in glioblastoma multiforme were assessed by immunohistochemistry and microsatellite instability (MSI), respectively. The mutation status of the MSH6 gene, a proposed correlate of temozolomide resistance, was determined by direct sequencing and compared with data from immunofluorescent detection of MSH6 protein and reverse transcription-PCR amplification of MSH6 RNA.Seventy percent of newly diagnosed and 78% of failed-therapy glioblastoma multiforme samples expressed nuclear AGT protein in > or = 20% of cells analyzed, suggesting alternate means of resistance in 20% to 30% of cases. Single loci MSI was observed in 3% of patient samples; no sample showed the presence of high MSI. MSI was not shown to correlate with MSH6 mutation or loss of MSH6 protein expression.Although high AGT levels may mediate resistance in a portion of these samples, MMR deficiency does not seem to be responsible for mediating temozolomide resistance in adult malignant glioma. Accordingly, the presence of a fraction of samples exhibiting both low AGT expression and MMR proficiency suggests that additional mechanisms of temozolomide resistance are operational in the clinic.

BACKGROUND: Convection-enhanced delivery (CED) permits site-specific therapeutic drug delivery within interstitial spaces at increased dosages through circumvention of the blood-brain barrier. CED is currently limited by suboptimal methodologies for monitoring the delivery of therapeutic agents that would permit technical optimization and enhanced therapeutic efficacy. OBJECTIVE: To determine whether a readily available small-molecule MRI contrast agent, gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA), could effectively track the distribution of larger therapeutic agents. METHODS: Gd-DTPA was coinfused with the larger molecular tracer, 124I-labeled human serum albumin (124I-HSA), during CED of an EGFRvIII-specific immunotoxin as part of treatment for a patient with glioblastoma. RESULTS: Infusion of both tracers was safe in this patient. Analysis of both Gd-DTPA and 124I-HSA during and after infusion revealed a high degree of anatomical and volumetric overlap. CONCLUSION: Gd-DTPA may be able to accurately demonstrate the anatomic and volumetric distribution of large molecules used for antitumor therapy with high resolution and in combination with fluid-attenuated inversion recovery (FLAIR) imaging, and provide additional information about leaks into cerebrospinal fluid spaces and resection cavities. Similar studies should be performed in additional patients to validate our findings and help refine the methodologies we used.

Temozolomide (TMZ) is an alkylating agent shown to prolong survival in patients with high grade glioma and is routinely used to treat melanoma brain metastases. A prominent side effect of TMZ is induction of profound lymphopenia, which some suggest may be incompatible with immunotherapy. Conversely, it has been proposed that recovery from chemotherapy-induced lymphopenia may actually be exploited to potentiate T-cell responses. Here, we report the first demonstration of TMZ as an immune host-conditioning regimen in an experimental model of brain tumor and examine its impact on antitumor efficacy of a well-characterized peptide vaccine. Our results show that high-dose, myeloablative (MA) TMZ resulted in markedly reduced CD4(+), CD8(+) T-cell and CD4(+)Foxp3(+) TReg counts. Adoptive transfer of naïve CD8(+) T cells and vaccination in this setting led to an approximately 70-fold expansion of antigen-specific CD8(+) T cells over controls. Ex vivo analysis of effector functions revealed significantly enhanced levels of pro-inflammatory cytokine secretion from mice receiving MA TMZ when compared to those treated with a lower lymphodepletive, non-myeloablative (NMA) dose. Importantly, MA TMZ, but not NMA TMZ was uniquely associated with an elevation of endogenous IL-2 serum levels, which we also show was required for optimal T-cell expansion. Accordingly, in a murine model of established intracerebral tumor, vaccination-induced immunity in the setting of MA TMZ-but not lymphodepletive, NMA TMZ-led to significantly prolonged survival. Overall, these results may be used to leverage the side-effects of a clinically-approved chemotherapy and should be considered in future study design of immune-based treatments for brain tumors.

Object Recurrent malignant gliomas have inherent resistance to traditional chemotherapy. Novel therapies target specific molecular mechanisms involved in abnormal signaling and resistance to apoptosis. The proteasome is a key regulator of multiple cellular functions, and its inhibition in malignant astrocytic lines causes cell growth arrest and apoptotic cell death. The proteasome inhibitor bortezomib was reported to have very good in vitro activity against malignant glioma cell lines, with modest activity in animal models as well as in clinical trials as a single agent. In this paper, the authors describe the multiple effects of bortezomib in both in vitro and in vivo glioma models and offer a novel explanation for its seeming lack of activity. Methods Glioma stem-like cells (GSCs) were obtained from resected glioblastomas (GBMs) at surgery and expanded in culture. Stable glioma cell lines (U21 and D54) as well as temozolomide (TMZ)-resistant glioma cells derived from U251 and D54-MG were also cultured. GSCs from 2 different tumors, as well as D54 and U251 cells, were treated with bortezomib, and the effect of the drug was measured using an XTT cell viability assay. The activity of bortezomib was then determined in D54-MG and/or U251 cells using apoptosis analysis as well as caspase-3 activity and proteasome activity measurements. Human glioma xenograft models were created in nude mice by subcutaneous injection. Bevacizumab was administered via intraperitoneal injection at a dose of 5 mg/kg daily. Bortezomib was administered by intraperitoneal injection 1 hour after bevacizumab administration in doses of at a dose of 0.35 mg/kg on days 1, 4, 8, and 11 every 21 days. Tumors were measured twice weekly. Results Bortezomib induced caspase-3 activation and apoptotic cell death in stable glioma cell lines and in glioma stem-like cells (GSCs) derived from malignant tumor specimens Furthermore, TMZ-resistant glioma cell lines retained susceptibility to the proteasome inhibition. The bortezomib activity was directly proportional with the cells' baseline proteasome activity. The proteasome inhibition stimulated both hypoxia-inducible factor (HIF)–1α and vascular endothelial growth factor (VEGF) production in malignant GSCs. As such, the VEGF produced by GSCs stimulated endothelial cell growth, an effect that could be prevented by the addition of bevacizumab (VEGF antibody) to the media. Similarly, administration of bortezomib and bevacizumab to athymic mice carrying subcutaneous malignant glioma xenografts resulted in greater tumor inhibition and greater improvement in survival than administration of either drug alone. These data indicate that simultaneous proteasome inhibition and VEGF blockade offer increased benefit as a strategy for malignant glioma therapy. Conclusions The results of this study indicate that combination therapies based on bortezomib and bevacizumab might offer an increased benefit when the two agents are used in combination. These drugs have a complementary mechanism of action and therefore can be used together to treat TMZ-resistant malignant gliomas.

Abstract Inactivating mutations in the transcriptional repression factor Capicua (CIC) occur in approximately 50% of human oligodendrogliomas, but mechanistic links to pathogenesis are unclear. To address this question, we generated Cic-deficient mice and human oligodendroglioma cell models. Genetic deficiency in mice resulted in a partially penetrant embryonic or perinatal lethal phenotype, with the production of an aberrant proliferative neural population in surviving animals. In vitro cultured neural stem cells derived from Cic conditional knockout mice bypassed an EGF requirement for proliferation and displayed a defect in their potential for oligodendrocyte differentiation. Cic is known to participate in gene suppression that can be relieved by EGFR signal, but we found that cic also activated expression of a broad range of EGFR-independent genes. In an orthotopic mouse model of glioma, we found that Cic loss potentiated the formation and reduced the latency in tumor development. Collectively, our results define an important role for Cic in regulating neural cell proliferation and lineage specification, and suggest mechanistic explanations for how CIC mutations may impact the pathogenesis and therapeutic targeting of oligodendroglioma. Cancer Res; 77(22); 6097–108. ©2017 AACR.

IDH1 mutations occur in the majority of low-grade gliomas and lead to the production of the oncometabolite, D-2-hydroxyglutarate (D-2HG). To understand the effects of tumor-associated mutant IDH1 (IDH1-R132H) on both the neural stem cell (NSC) population and brain tumorigenesis, genetically faithful cell lines and mouse model systems were generated. Here, it is reported that mouse NSCs expressing Idh1-R132H displayed reduced proliferation due to p53-mediated cell-cycle arrest as well as a decreased ability to undergo neuronal differentiation. In vivo, Idh1-R132H expression reduced proliferation of cells within the germinal zone of the subventricular zone (SVZ). The NSCs within this area were dispersed and disorganized in mutant animals, suggesting that Idh1-R132H perturbed the NSCs and the microenvironment from which gliomas arise. In addition, tumor-bearing animals expressing mutant Idh1 displayed a prolonged survival and also overexpressed Olig2, features consistent with IDH1-mutated human gliomas. These data indicate that mutant Idh1 disrupts the NSC microenvironment and the candidate cell-of-origin for glioma; thus, altering the progression of tumorigenesis. In addition, this study provides a mutant Idh1 brain tumor model that genetically recapitulates human disease, laying the foundation for future investigations on mutant IDH1-mediated brain tumorigenesis and targeted therapy.Implications: Through the use of a conditional mutant mouse model that confers a less aggressive tumor phenotype, this study reveals that mutant Idh1 impacts the candidate cell-of-origin for gliomas. Mol Cancer Res; 15(5); 507-20. ©2017 AACR.

Gliomas remain one of the deadliest forms of cancer. Improved therapeutics will require a better understanding of the molecular nature of these tumors. We, therefore, mimicked the most common genetic changes found in grade III-IV gliomas, disruption of the p53 and RB pathways and activation of telomere maintenance and independence from growth factors, through the ectopic expression of the SV40 T/t-Ag oncogene, an oncogenic form of H-ras (H-ras(V12G)), and the human telomerase catalytic subunit hTERT in normal human astrocytes. The resulting cells displayed many of the hallmarks of grade III-IV gliomas, including greatly expanded life span and growth in soft agar and, most importantly, were tumorigenic with pathology consistent with grade III-IV neuroectodermal tumors in mice. This model system will, for the first time, allow the biological significance of selected genetic alterations to be studied in human gliomas.

N-succinimidyl-3-(tri-n-butylstannyl)benzoate (m-BuATE), N-succinimidyl-3-(tri-methylstannyl)benzoate (m-MeATE) and N-succinimidyl-4-(tri-n-butylstannyl)benzoate (p-BuATE) were synthesized and radioiodinated using either N-chlorosuccinimide (NCS) or t-butylhydroperoxide (TBHP) as the oxidant. Radiohalogenation of m-MeATE proceeded more rapidly than m-BuATE. NCS was the more efficient oxidant at reaction times less than 15 min; use of both TBHP and NCS resulted in nearly quantitative yields after 15 min when m-MeATE was used. Using NCS, achieving optimal antibody coupling and specific binding required purification of the active ester by HPLC; in contrast, with TBHP, only Sep-Pak purification was needed.

The distribution of type VI collagen was examined immunohistochemically in normal tissues and in 24 human gliomas and six medulloblastomas. Its localization in the neoplasms was compared with that of fibronectin and glioma-mesenchymal extracellular matrix (GMEM) glycoprotein. In normal non-neural tissues type VI collagen was demonstrated in the interstitial connective tissue and in some basement membranes. In normal brain it was localized to the vasculature, leptomeninges, and pial-glial membrane. In neoplasms type VI collagen and fibronectin codistributed in the vasculature and stromal connective tissue. The GMEM glycoprotein, as identified by monoclonal antibody (MAb) 81C6, and a related glioma-mesenchymal matrix antigen identified by MAb 2A6, were expressed not only in the tumor vasculature and connective tissue, but also within the tumor parenchyma in association with glioma cells. The staining intensity was variable in 20 malignant gliomas and weak to absent in two pilocytic astrocytomas and six medulloblastomas. An oligodendroglioma and ependymoma both expressed the 2A6 epitope, but staining with MAb 81C6 was weak to absent. The antigens identified by MAb 81C6 and MAb 2A6 represent the only recognized extracellular matrix components, other than proteoglycans, that are associated with glioma cells in vivo. As prominent constituents of the pericellular matrix, they may be involved in recognized matrix functions such as the modulation of cell adhesion and migration.

Abstract Purpose: This phase II trial was designed to define the efficacy of Gliadel wafers in combination with an infusion of O6-benzylguanine (O6-BG) that suppresses tumor O6-alkylguanine-DNA alkyltransferase (AGT) levels in patients with recurrent glioblastoma multiforme for 5 days and to evaluate the safety of this combination therapy. Experimental Design: This was a phase II, open-label, single center trial. On gross total resection of the tumor, up to eight Gliadel wafers were implanted. Bolus infusion of O6-BG was administered at 120 mg/m2 over 1 hour on days 1, 3, and 5, along with a continuous infusion at 30 mg/m2/d. The primary end points were 6-month overall survival (OS) and safety, and the secondary end points were 1-year, 2-year, and median OS. Results: Fifty-two patients were accrued. The 6-month OS was 82% [95% confidence interval (95% CI), 72-93%]. The 1- and 2-year OS rates were 47% (95% CI, 35-63%) and 10% (95% CI, 3-32%), respectively. The median OS was 50.3 weeks (95% CI, 36.1-69.4 weeks). Treatment-related toxicity with this drug combination included grade 3 hydrocephalus (9.6%), grade 3 cerebrospinal fluid (CSF) leak (19.2%), and grade 3 CSF/brain infection (13.4%). Conclusion: The efficacy of implanted Gliadel wafers may be improved with the addition of O6-BG. Although systemically administered O6-BG can be coadministered with Gliadel wafers safely, it may increase the risk of hydrocephalus, CSF leak, and CSF/brain infection. Future trials are required to verify that inhibition of tumor AGT levels by O6-BG results in increased efficacy of Gliadel wafers without added toxicity.

The epidermal growth factor receptor variant III (EGFRvIII) is a consistent tumor-specific mutation that is widely expressed in glioblastoma multiforme (GBM) and other neoplasms. As such it represents a truly tumor-specific target for antitumor immunotherapy. Although endogenous humoral responses to EGFRvIII have been reported in patients with EGFRvIII-expressing breast cancer, it is not known whether de novo responses can be generated or endogenous responses enhanced with an EGFRvIII-specific vaccine. To assess this in clinical trials, we have developed and validated an immunoassay to measure and isolate anti-EGFRvIII and anti-KLH antibodies from the serum of patients vaccinated with an EGFRvIII-specific peptide (PEPvIII) conjugated to keyhole limpet hemocyanin (KLH). Using magnetic beads with immobilized antigen we captured and detected anti-EGFRvIII and anti-KLH antibodies in serum from patients before and after vaccinations. Using this assay, we found that significant levels of antibody for tumor-specific antigen EGFRvIII (> 4 µg/mL) and KLH could be induced after vaccination with PEPvIII-KLH.

Isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2) are enzymes which convert isocitrate to α-ketoglutarate while reducing nicotinamide adenine dinucleotide phosphate (NADP + to NADPH). IDH1/2 were recently identified as mutated in a large percentage of progressive gliomas. These mutations occur at IDH1R132 or the homologous IDH2R172. Melanomas share some genetic features with IDH1/2-mutated gliomas, such as frequent TP53 mutation. We sought to test whether melanoma is associated with IDH1/2 mutations. Seventy-eight human melanoma samples were analyzed for IDH1R132 and IDH2R172 mutation status. A somatic, heterozygous IDH1 c.C394T (p.R132C) mutation was identified in one human melanoma metastasis to the lung. Having identified this mutation in one metastasis, we sought to test the hypothesis that certain selective pressures in the brain environment may specifically favor the cell growth or survival of tumor cells with mutations in IDH1/2, regardless of primary tumor site. To address this, we analyzed IDH1R132 and IDH2R172 mutation status 53 metastatic brain tumors, including nine melanoma metastases. Results revealed no mutations in any samples. This lack of mutations would suggest that mutations in IDH1R132 or IDH2R172 may be necessary for the formation of tumors in a cell-lineage dependent manner, with a particularly strong selective pressure for mutations in progressive gliomas; this also suggests the lack of a particular selective pressure for growth in brain tissue in general. Studies on the cell-lineages of tumors with IDH1/2 mutations may help clarify the role of these mutations in the development of brain tumors.

Adult malignant brainstem gliomas (BSGs) are poorly characterized due to their relative rarity. We have examined histopathologically confirmed cases of adult malignant BSGs to better characterize the patient and tumor features and outcomes, including the natural history, presentation, imaging, molecular characteristics, prognostic factors, and appropriate treatments. A total of 34 patients were identified, consisting of 22 anaplastic astrocytomas (AAs) and 12 glioblastomas (GBMs). The overall median survival for all patients was 25.8 months, with patients having GBMs experiencing significantly worse survival (12.1 vs. 77.0 months, p = 0.0011). The majority of tumors revealed immunoreactivity for EGFR (93.3 %) and MGMT (64.7 %). Most tumors also exhibited chromosomal abnormalities affecting the loci of epidermal growth factor receptor (92.9 %), MET (100 %), PTEN (61.5 %), and 9p21 (80 %). AAs more commonly appeared diffusely enhancing (50.0 vs. 27.3 %) or diffusely nonenhancing (25.0 vs. 0.0 %), while GBMs were more likely to exhibit focal enhancement (54.6 vs. 10.0 %). Multivariate analysis revealed confirmed histopathology for GBM to significantly affect survival (HR 4.80; 95 % CI 1.86–12.4; p = 0.0012). In conclusion, adult malignant BSGs have an overall poor prognosis, with GBM tumors faring significantly worse than AAs. As AAs and GBMs have differing imaging characteristics, tissue diagnosis may be necessary to accurately determine patient prognosis and identify molecular characteristics which may aid in the treatment of these aggressive tumors.

Poliovirus oncolytic immunotherapy is a putatively novel approach to treat pediatric brain tumors. This work sought to determine expression of the poliovirus receptor (PVR), CD155, in low-grade and malignant pediatric brain tumors and its ability to infect, propagate, and inhibit cell proliferation. CD155 expression in pleomorphic xanthoastrocytoma (PXA), medulloblastoma, atypical teratoid rhabdoid tumor, primitive neuroectodermal tumor, and anaplastic ependymoma specimens was assessed. The ability of the polio: rhinovirus recombinant, PVSRIPO, to infect PXA (645 [BRAF V600E mutation], 2363) and medulloblastoma (D283, D341) cells were determined by viral propagation measurement and cell proliferation. PVR mRNA expression was evaluated in 763 medulloblastoma and 1231 normal brain samples. CD155 was expressed in all 12 patient specimens and in PXA and medulloblastoma cell lines. One-step growth curves at a multiplicity of infection of 10 demonstrated productive infection and peak plaque formation units at 5–10 hours. PVSRIPO infection significantly decreased cellular proliferation in 2363, 645, and D341 cell lines at 48 hours (p < 0.05) and resulted in cell death. PVR expression was highest in medulloblastoma subtypes Group 3γ, WNTα, and WNTβ (p < 0.001). This proof-of-concept in vitro study demonstrates that PVSRIPO is capable of infecting, propagating, prohibiting cell proliferation, and killing PXA and Group 3 medulloblastoma.

Hotspot mutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in a number of human cancers and confer a neomorphic enzyme activity that catalyzes the conversion of α-ketoglutarate (αKG) to the oncometabolite D-(2)-hydroxyglutarate (D2HG). In malignant gliomas, IDH1R132H expression induces widespread metabolic reprogramming, possibly requiring compensatory mechanisms to sustain the normal biosynthetic requirements of actively proliferating tumor cells. We used genetically engineered mouse models of glioma and quantitative metabolomics to investigate IDH1R132H-dependent metabolic reprogramming and its potential to induce biosynthetic liabilities that can be exploited for glioma therapy. In gliomagenic neural progenitor cells, IDH1R132H expression increased the abundance of dipeptide metabolites, depleted key tricarboxylic acid cycle metabolites, and slowed progression of murine gliomas. Notably, expression of glutamate dehydrogenase GDH2, a hominoid-specific enzyme with relatively restricted expression to the brain, was critically involved in compensating for IDH1R132H-induced metabolic alterations and promoting IDH1R132H glioma growth. Indeed, we found that recently evolved amino acid substitutions in the GDH2 allosteric domain conferred its nonredundant, glioma-promoting properties in the presence of IDH1 mutation. Our results indicate that among the unique roles for GDH2 in the human forebrain is its ability to limit IDH1R132H-mediated metabolic liabilities, thus promoting glioma growth in this context. Results from this study raise the possibility that GDH2-specific inhibition may be a viable therapeutic strategy for gliomas with IDH mutations.Significance: These findings show that the homonid-specific brain enzyme GDH2 may be essential to mitigate metabolic liabilities created by IDH1 mutations in glioma, with possible implications to leverage its therapeutic management by IDH1 inhibitors. Cancer Res; 78(1); 36-50. ©2017 AACR.

Abstract Homozygous deletion of methylthioadenosine phosphorylase (MTAP) is one of the most frequent genetic alterations in glioblastoma (GBM), but its pathologic consequences remain unclear. In this study, we report that loss of MTAP results in profound epigenetic reprogramming characterized by hypomethylation of PROM1/CD133–associated stem cell regulatory pathways. MTAP deficiency promotes glioma stem-like cell (GSC) formation with increased expression of PROM1/CD133 and enhanced tumorigenicity of GBM cells and is associated with poor prognosis in patients with GBM. As a combined consequence of purine production deficiency in MTAP-null GBM and the critical dependence of GSCs on purines, the enriched subset of CD133+ cells in MTAP-null GBM can be effectively depleted by inhibition of de novo purine synthesis. These findings suggest that MTAP loss promotes the pathogenesis of GBM by shaping the epigenetic landscape and stemness of GBM cells while simultaneously providing a unique opportunity for GBM therapeutics. Significance: This study links the frequently mutated metabolic enzyme MTAP to dysregulated epigenetics and cancer cell stemness and establishes MTAP status as a factor for consideration in characterizing GBM and developing therapeutic strategies.

A methylator-resistant human glioblastoma multiforme xenograft, D-245 MG (PR), in athymic nude mice was established by serially treating the parent xenograft D-245 MG with procarbazine. D-245 MG xenografts were sensitive to procarbazine, temozolomide, N-methyl-N-nitrosourea, 1,3-bis(2-chloroethyl)-1-nitrosourea, 9-aminocamptothecin, topotecan, CPT-11, cyclophosphamide, and busulfan. D-245 MG (PR) xenografts were resistant to procarbazine, temozolomide, N-methyl-N-nitrosourea, and busulfan, but they were sensitive to the other agents. Both D-245 MG and D-245 MG (PR) xenografts displayed no O6-alkylguanine-DNA alkyltransferase activity, and their levels of glutathione and glutathione-S-transferase were similar. D-245 MG xenografts expressed the human mismatch repair proteins hMSH2 and hMLH1, whereas D-245 MG (PR) expressed hMLH1 but not hMSH2.

Phosphoramide mustard-induced DNA interstrand cross-links were studied both in vitro and by computer simulation. The local determinants for the formation of phosphoramide mustard-induced DNA interstrand cross-links were defined by using different pairs of synthetic oligonucleotide duplexes, each of which contained a single potentially cross-linkable site. Phosphoramide mustard was found to cross-link dG to dG at a 5'-d(GAC)-3'. The structural basis for the formation of this 1,3 cross-link was studied by molecular dynamics and quantum chemistry. Molecular dynamics indicated that the geometrical proximity of the binding sites also favored a 1,3 dG-to-dG linkage over a 1,2 dG-to-dG linkage in a 5'-d(GCC)-3' sequence. While the enthalpies of 1,2 and 1,3 mustard cross-linked DNA were found to be very close, a 1,3 structure was more flexible and may therefore be in a considerably higher entropic state.

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that promotes malignant glioma invasion, angiogenesis, and immunosuppression. Antisense oligonucleotide suppression of TGF-beta(2) ligand expression has shown promise in preclinical and clinical studies but at least two ligands mediate the effects of TGF-beta in gliomas. Therefore, we examined the effects of SB-431542, a novel, small molecule inhibitor of the type I TGF-beta receptor, on a panel of human malignant glioma cell lines. SB-431542 blocked the phosphorylation and nuclear translocation of the SMADs, intracellular mediators of TGF-beta signaling, with decreased TGF-beta-mediated transcription. Furthermore, SB-431542 inhibited the expression of two critical effectors of TGF-beta-vascular endothelial growth factor and plasminogen activator inhibitor-1. SB-431542 treatment of glioma cultures inhibited proliferation, TGF-beta-mediated morphologic changes, and cellular motility. Together, our results suggest that small molecule inhibitors of TGF-beta receptors may offer a novel therapy for malignant gliomas by reducing cell proliferation, angiogenesis, and motility.

Convection-enhanced delivery (CED) is a novel technique used to deliver agents to the brain parenchyma for treatment of neoplastic, infectious, and degenerative conditions. The purpose of this study was to determine if CED would provide a larger volume of distribution (Vd) of a radiolabeled monoclonal antibody (mAb) than a bolus injection.Patients harboring a recurrent glioblastoma multiforme that reacted with the antitenascin mAb 81C6 during immunohistochemical analysis were randomized to receive an intratumoral injection of the human-murine chimeric mAb Ch81C6, which had been labeled with the 123I tracer. The mAb was administered by either a bolus injection or CED via a stereotactically placed catheter; between 48 and 72 hours later the mAb was again administered using the other technique. Injections of escalating doses of a 131I-labeled therapeutic mAb were then delivered using the technique shown to produce the largest Vd by single-photon emission computerized tomography.Convection-enhanced delivery has enormous potential for administering drugs to sites within the central nervous system. For the relatively small volumes injected in this study, however, CED did not provide a significant increase in the Vd when compared with the bolus injection. Nevertheless, a clear cross-over effect was seen, which was probably related to the temporal proximity of the two infusions.

Genomic alterations leading to aberrant activation of cyclin/cyclin-dependent kinase (cdk) complexes drive the pathogenesis of many common human tumor types. In the case of glioblastoma multiforme (GBM), these alterations are most commonly due to homozygous deletion of p16(INK4a) and less commonly due to genomic amplifications of individual genes encoding cyclins or cdks. Here, we describe deletion of the p18(INK4c) cdk inhibitor as a novel genetic alteration driving the pathogenesis of GBM. Deletions of p18(INK4c) often occurred in tumors also harboring homozygous deletions of p16(INK4a). Expression of p18(INK4c) was completely absent in 43% of GBM primary tumors studied by immunohistochemistry. Lentiviral reconstitution of p18(INK4c) expression at physiologic levels in p18(INK4c)-deficient but not p18(INK4c)-proficient GBM cells led to senescence-like G(1) cell cycle arrest. These studies identify p18(INK4c) as a GBM tumor suppressor gene, revealing an additional mechanism leading to aberrant activation of cyclin/cdk complexes in this terrible malignancy.

Epidermal growth factor receptor (EGFR) gene amplification, mutations, and/or aberrant activation are frequent abnormalities in malignant gliomas and other human cancers and have been associated with an aggressive clinical course and a poor therapeutic outcome. Elevated glutathione S-transferase P1 (GSTP1), a major drug-metabolizing and stress response signaling protein, is also associated with drug resistance and poor clinical outcome in gliomas and other cancers. Here, we provide evidence that GSTP1 is a downstream EGFR target and that EGFR binds to and phosphorylates tyrosine residues in the GSTP1 protein in vitro and in vivo. Mass spectrometry and mutagenesis analyses in a cell-free system and in gliomas cells identified Tyr-7 and Tyr-198 as major EGFR-specific phospho-acceptor residues in the GSTP1 protein. The phosphorylation increased GSTP1 enzymatic activity significantly, and computer-based modeling showed a corresponding increase in electronegativity of the GSTP1 active site. In human glioma and breast cancer cells, epidermal growth factor stimulation rapidly increased GSTP1 tyrosine phosphorylation and decreased cisplatin sensitivity. Lapatinib, a clinically active EGFR inhibitor, significantly reversed the epidermal growth factor-induced cisplatin resistance. These data define phosphorylation and activation of GSTP1 by EGFR as a novel, heretofore unrecognized component of the EGFR signaling network and a novel mechanism of tumor drug resistance, particularly in tumors with elevated GSTP1 and/or activated EGFR.

Frequent somatic hotspot mutations in isocitrate dehydrogenase 1 (IDH1) have been identified in gliomas, acute myeloid leukemias, chondrosarcomas, and other cancers, providing a likely avenue for targeted cancer therapy.However, whether mutant IDH1 protein is required for maintaining IDH1 mutated tumor cell growth remains unknown.Here, using a genetically engineered inducible system, we report that selective suppression of endogenous mutant IDH1 expression in HT1080, a fibrosarcoma cell line with a native IDH1 R132C heterozygous mutation, significantly inhibits cell proliferation and decreases clonogenic potential.Our findings offer insights into changes that may contribute to the inhibition of cell proliferation and offer a strong preclinical rationale for utilizing mutant IDH1 as a valid therapeutic target.

Abstract Glioblastoma is universally fatal because of its propensity for rapid recurrence due to highly migratory tumor cells. Unraveling the genomic complexity that underlies this migratory characteristic could provide therapeutic targets that would greatly complement current surgical therapy. Using multiple high-resolution genomic screening methods, we identified a single locus, adherens junctional associated protein 1 (AJAP1) on chromosome 1p36 that is lost or epigenetically silenced in many glioblastomas. We found AJAP1 expression absent or reduced in 86% and 100% of primary glioblastoma tumors and cell lines, respectively, and the loss of expression correlates with AJAP1 methylation. Restoration of AJAP1 gene expression by transfection or demethylation agents results in decreased tumor cell migration in glioblastoma cell lines. This work shows the significant loss of expression of AJAP1 in glioblastoma and provides evidence of its role in the highly migratory characteristic of these tumors. Mol Cancer Res; 10(2); 208–17. ©2012 AACR.