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Malignant Glioma: Lessons from Genomics, Mouse Models, and Stem Cells

2012; Cell Press; Volume: 149; Issue: 1 Linguagem: Inglês

10.1016/j.cell.2012.03.009

1097-4172

Jian Chen, Renée M. McKay, Luis F. Parada,

Cancer-related molecular mechanisms research

Eighty percent of malignant tumors that develop in the central nervous system are malignant gliomas, which are essentially incurable. Here, we discuss how recent sequencing studies are identifying unexpected drivers of gliomagenesis, including mutations in isocitrate dehydrogenase 1 and the NF-κB pathway, and how genome-wide analyses are reshaping the classification schemes for tumors and enhancing prognostic value of molecular markers. We discuss the controversies surrounding glioma stem cells and explore how the integration of new molecular data allows for the generation of more informative animal models to advance our knowledge of glioma's origin, progression, and treatment. Eighty percent of malignant tumors that develop in the central nervous system are malignant gliomas, which are essentially incurable. Here, we discuss how recent sequencing studies are identifying unexpected drivers of gliomagenesis, including mutations in isocitrate dehydrogenase 1 and the NF-κB pathway, and how genome-wide analyses are reshaping the classification schemes for tumors and enhancing prognostic value of molecular markers. We discuss the controversies surrounding glioma stem cells and explore how the integration of new molecular data allows for the generation of more informative animal models to advance our knowledge of glioma's origin, progression, and treatment. Although the total incidence of primary central nervous system (CNS) tumors is only about 18.7 per 100,000 persons in the United States, 80% of the malignant tumors in the CNS are malignant gliomas, which are essentially incurable (CBTRUS, 2009CBTRUS (Central Brain Tumor Registry of the United States) (2009). CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in Eighteen States in 2002–2006.Google Scholar). Gliomas display histological similarities to glial cells, including astrocytes and oligodendrocytes. According to the 2007 World Health Organization (WHO) classification, malignant gliomas can be classified according to which cell they most resemble, such as astrocytomas, oligodendrogliomas, or oligoastrocytomas. More than half of gliomas are glioblastoma multiforme (GBM, grade IV astrocytoma), one of the most aggressive cancers (Louis et al., 2007Louis D.N. Ohgaki H. Wiestler O.D. Cavenee W.K. Burger P.C. Jouvet A. Scheithauer B.W. Kleihues P. The 2007 WHO classification of tumours of the central nervous system.Acta. Neuropathol. 2007; 114: 97-109Crossref PubMed Scopus (2565) Google Scholar). Despite decades of concerted effort and advances in surgery, radiation, and chemotherapy, the overall 5 year survival rate of GBM remains less than 5% and is even worse for elderly patients (CBTRUS, 2009CBTRUS (Central Brain Tumor Registry of the United States) (2009). CBTRUS Statistical Report: Primary Brain and Central Nervous System Tumors Diagnosed in Eighteen States in 2002–2006.Google Scholar). This dismal clinical outcome makes glioma an urgent subject of cancer research. Here, we discuss current advances in genomic analysis and genetic modeling of glioma and how these developments influence strategies for therapeutic intervention in this deadly disease. In the past two decades, cytogenetic and molecular genetic studies have identified a number of recurrent chromosomal abnormalities and genetic alterations in malignant gliomas, particularly in GBM. Advances in molecular technologies, especially high-density microarray and genome sequencing, have made it possible to evaluate genetic and epigenetic changes in these tumors at the genome-wide level. In a comprehensive study carried out by The Cancer Genome Atlas (TCGA) project, 601 cancer-related candidate genes were sequenced in more than 200 human GBM samples (TCGA, 2008TCGA (The Cancer Genome Atlas Research Network)Comprehensive genomic characterization defines human glioblastoma genes and core pathways.Nature. 2008; 455: 1061-1068Crossref PubMed Scopus (2232) Google Scholar). The project also analyzed genome-wide DNA copy number changes, DNA methylation status, and protein-coding and noncoding RNA expression (TCGA, 2008TCGA (The Cancer Genome Atlas Research Network)Comprehensive genomic characterization defines human glioblastoma genes and core pathways.Nature. 2008; 455: 1061-1068Crossref PubMed Scopus (2232) Google Scholar). A similar but complementary study by Parsons et al. sequenced 20,661 protein-encoding genes in 22 GBM samples and integrated the genetic alteration information with DNA copy number and gene expression profiles (Parsons et al., 2008Parsons D.W. Jones S. Zhang X. Lin J.C. Leary R.J. Angenendt P. Mankoo P. Carter H. Siu I.M. Gallia G.L. et al.An integrated genomic analysis of human glioblastoma multiforme.Science. 2008; 321: 1807-1812Crossref PubMed Scopus (1938) Google Scholar). These integrative genomic studies provided a comprehensive view of the complicated genomic landscape of GBM, revealing a set of core signaling pathways commonly activated in GBM (Figure 1): the P53 pathway, the RB pathway, and the RTK pathway (TCGA, 2008TCGA (The Cancer Genome Atlas Research Network)Comprehensive genomic characterization defines human glioblastoma genes and core pathways.Nature. 2008; 455: 1061-1068Crossref PubMed Scopus (2232) Google Scholar, Parsons et al., 2008Parsons D.W. Jones S. Zhang X. Lin J.C. Leary R.J. Angenendt P. Mankoo P. Carter H. Siu I.M. Gallia G.L. et al.An integrated genomic analysis of human glioblastoma multiforme.Science. 2008; 321: 1807-1812Crossref PubMed Scopus (1938) Google Scholar). Furnari et al. have written a detailed review of these pathways in glioma (Furnari et al., 2007Furnari F.B. Fenton T. Bachoo R.M. Mukasa A. Stommel J.M. Stegh A. Hahn W.C. Ligon K.L. Louis D.N. Brennan C. et al.Malignant astrocytic glioma: genetics, biology, and paths to treatment.Genes Dev. 2007; 21: 2683-2710Crossref PubMed Scopus (753) Google Scholar). The majority of GBM tumors have genetic alterations in all three pathways, which helps to fuel cell proliferation and enhance cell survival while allowing the tumor cell to escape from cell-cycle checkpoints, senescence, and apoptosis. In addition to confirming known genetic events, the TCGA sequencing data also provided somatic mutation information at the base pair level, revealing potential new roles for known tumor suppressors/oncogenes in GBM as well as new cancer driver genes. For example, it has long been observed that patients with germline mutations in the tumor suppressor gene responsible for neurofibromatosis type 1 (NF1) have an increased incidence of malignant glioma (Gutmann et al., 2002Gutmann D.H. Rasmussen S.A. Wolkenstein P. MacCollin M.M. Guha A. Inskip P.D. North K.N. Poyhonen M. Birch P.H. Friedman J.M. Gliomas presenting after age 10 in individuals with neurofibromatosis type 1 (NF1).Neurology. 2002; 59: 759-761Crossref PubMed Google Scholar, Friedman, 1999Friedman J.M. Other Malignancies.in: Friedman J.M. Gutmann D.H. MacCollin M. Riccardi V.M. Neurofibromatosis: Phenotype, Natural History, and Pathogenesis. Johns Hopkins Press, Baltimore, MD1999Google Scholar). Studies in genetic mouse models have also strongly suggested a causal role for NF1 mutation in glioma tumorigenesis (Alcantara Llaguno et al., 2009Alcantara Llaguno S. Chen J. Kwon C.H. Jackson E.L. Li Y. Burns D.K. Alvarez-Buylla A. Parada L.F. Malignant astrocytomas originate from neural stem/progenitor cells in a somatic tumor suppressor mouse model.Cancer Cell. 2009; 15: 45-56Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, Kwon et al., 2008Kwon C.H. Zhao D. Chen J. Alcantara S. Li Y. Burns D.K. Mason R.P. Lee E.Y. Wu H. Parada L.F. Pten haploinsufficiency accelerates formation of high-grade astrocytomas.Cancer Res. 2008; 68: 3286-3294Crossref PubMed Scopus (109) Google Scholar, Zhu et al., 2005Zhu Y. Guignard F. Zhao D. Liu L. Burns D.K. Mason R.P. Messing A. Parada L.F. Early inactivation of p53 tumor suppressor gene cooperating with NF1 loss induces malignant astrocytoma.Cancer Cell. 2005; 8: 119-130Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). However, the involvement of NF1 mutation in sporadic human GBM remained underappreciated until the TCGA project reported that 47 of the 206 patient samples, or 23%, had NF1 mutations or deletions, ranking it as the third most frequently somatically mutated gene among the 601 genes sequenced (TCGA, 2008TCGA (The Cancer Genome Atlas Research Network)Comprehensive genomic characterization defines human glioblastoma genes and core pathways.Nature. 2008; 455: 1061-1068Crossref PubMed Scopus (2232) Google Scholar). In addition to the core signaling pathways identified through genome-wide screening studies, Harsh et al. recently reported that heterozygous deletion of the NF-κB inhibitor α (NFKBIA) gene was present in a quarter of GBM samples (Bredel et al., 2011Bredel M. Scholtens D.M. Yadav A.K. Alvarez A.A. Renfrow J.J. Chandler J.P. Yu I.L. Carro M.S. Dai F. Tagge M.J. et al.NFKBIA deletion in glioblastomas.N. Engl. J. Med. 2011; 364: 627-637Crossref PubMed Scopus (70) Google Scholar). The NFKBIA gene encodes the protein IκBα, a crucial negative regulator in the canonical NF-κB signaling pathway. Under basal conditions, IκBα sequesters the NF-κB transcription factor heterodimer (p50/p65) in the cytoplasm. Upon stimulation with ligand such as tumor necrosis factor α (TNF-α) or lipopolysaccharide (LPS), IκBα is phosphorylated by the signalosome (Karin, 2006Karin M. Nuclear factor-kappaB in cancer development and progression.Nature. 2006; 441: 431-436Crossref PubMed Scopus (1611) Google Scholar). This phosphorylation leads to rapid ubiquitination and degradation of IκBα, which releases the inhibition of NF-κB and allows translocation of p50/p65 into the nucleus to activate transcription of downstream target genes, including many cytokines that can promote tumor growth and infiltration (Karin, 2006Karin M. Nuclear factor-kappaB in cancer development and progression.Nature. 2006; 441: 431-436Crossref PubMed Scopus (1611) Google Scholar). In GBM, NFKBIA deletion and EGFR amplification are mutually exclusive, raising the possibility that the two genetic events converge on the same pathway. Indeed, overexpression of NFKBIA reduced the viability of primary glioma cells in which NFKBIA was downregulated or EGFR was upregulated (Bredel et al., 2011Bredel M. Scholtens D.M. Yadav A.K. Alvarez A.A. Renfrow J.J. Chandler J.P. Yu I.L. Carro M.S. Dai F. Tagge M.J. et al.NFKBIA deletion in glioblastomas.N. Engl. J. Med. 2011; 364: 627-637Crossref PubMed Scopus (70) Google Scholar). In addition, both genetic events were associated with similar prognostic outcome, which is inferior to that of patients with normal expression levels of these two genes. However, the detailed molecular mechanism for the role of NF-κB in glioma development and progression and its connection with EGFR signaling remain to be investigated. Among the various genomic efforts to characterize gliomas, the biggest surprise came from the genome-wide exon sequencing project, in which R132 mutations of isocitrate dehydrogenase 1 (IDH1) were observed in 12% of the GBM samples (Parsons et al., 2008Parsons D.W. Jones S. Zhang X. Lin J.C. Leary R.J. Angenendt P. Mankoo P. Carter H. Siu I.M. Gallia G.L. et al.An integrated genomic analysis of human glioblastoma multiforme.Science. 2008; 321: 1807-1812Crossref PubMed Scopus (1938) Google Scholar). Subsequent studies revealed that as many as 70%–90% of the grade II/III gliomas harbored this IDH1 mutation (Yan et al., 2009Yan H. Parsons D.W. Jin G. McLendon R. Rasheed B.A. Yuan W. Kos I. Batinic-Haberle I. Jones S. Riggins G.J. et al.IDH1 and IDH2 mutations in gliomas.N. Engl. J. Med. 2009; 360: 765-773Crossref PubMed Scopus (1238) Google Scholar). Other studies have demonstrated that some gliomas contain IDH2 R172 mutations, an analog to IDH1 R132, at a lower frequency (Yan et al., 2009Yan H. Parsons D.W. Jin G. McLendon R. Rasheed B.A. Yuan W. Kos I. Batinic-Haberle I. Jones S. Riggins G.J. et al.IDH1 and IDH2 mutations in gliomas.N. Engl. J. Med. 2009; 360: 765-773Crossref PubMed Scopus (1238) Google Scholar). Two additional point mutations, IDH1 R100 and IDH2 R140, have been reported in AML but have not been observed in human glioma samples (Green and Beer, 2010Green A. Beer P. Somatic mutations of IDH1 and IDH2 in the leukemic transformation of myeloproliferative neoplasms.N. Engl. J. Med. 2010; 362: 369-370Crossref PubMed Scopus (123) Google Scholar). Normally, IDH1 and IDH2 convert isocitrate to α-ketoglutarate (α-KG) while at the same time reducing NADP+ to NADPH in the cytosol and mitochondria, respectively (Figure 2) (Reitman et al., 2010Reitman Z.J. Parsons D.W. Yan H. IDH1 and IDH2: not your typical oncogenes.Cancer Cell. 2010; 17: 215-216Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). It was initially hypothesized that the IDH1 R132 and IDH2 R172 mutations reduced the enzyme's ability to generate α-KG (Yan et al., 2009Yan H. Parsons D.W. Jin G. McLendon R. Rasheed B.A. Yuan W. Kos I. Batinic-Haberle I. Jones S. Riggins G.J. et al.IDH1 and IDH2 mutations in gliomas.N. Engl. J. Med. 2009; 360: 765-773Crossref PubMed Scopus (1238) Google Scholar). Subsequent studies reported a dominant-negative role for IDH1 R132 and suggested a tumor suppressor function for IDH1/2 (Zhao et al., 2009Zhao S. Lin Y. Xu W. Jiang W. Zha Z. Wang P. Yu W. Li Z. Gong L. Peng Y. et al.Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha.Science. 2009; 324: 261-265Crossref PubMed Scopus (429) Google Scholar). However, human genetic data showed that the mutations were always observed on a specific residue and only in one allele of the gene. These apparently narrow constraints on the nature of the mutations raised the possibility of neomorphic (gain-of-function) mutations (Green and Beer, 2010Green A. Beer P. Somatic mutations of IDH1 and IDH2 in the leukemic transformation of myeloproliferative neoplasms.N. Engl. J. Med. 2010; 362: 369-370Crossref PubMed Scopus (123) Google Scholar, Reitman et al., 2010Reitman Z.J. Parsons D.W. Yan H. IDH1 and IDH2: not your typical oncogenes.Cancer Cell. 2010; 17: 215-216Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, Yan et al., 2009Yan H. Parsons D.W. Jin G. McLendon R. Rasheed B.A. Yuan W. Kos I. Batinic-Haberle I. Jones S. Riggins G.J. et al.IDH1 and IDH2 mutations in gliomas.N. Engl. J. Med. 2009; 360: 765-773Crossref PubMed Scopus (1238) Google Scholar). Follow-up studies discovered that the IDH1/2 mutations had an NADPH-dependent ability to convert α-KG to D-2-hydroxyglutarate (D-2HG) (Figure 2), supporting a pro-oncogenic role for IDH1/2 (Dang et al., 2010Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. et al.Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2010; 465: 966Crossref PubMed Scopus (55) Google Scholar, Ward et al., 2010Ward P.S. Patel J. Wise D.R. Abdel-Wahab O. Bennett B.D. Coller H.A. Cross J.R. Fantin V.R. Hedvat C.V. Perl A.E. et al.The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate.Cancer Cell. 2010; 17: 225-234Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Consistent with this model, knocking down wild-type IDH1 in a glioma cell line slowed cell growth, and levels of 2HG were found to be 10-fold higher in IDH1/2 mutated glioma or leukemia samples (Dang et al., 2010Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. et al.Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2010; 465: 966Crossref PubMed Scopus (55) Google Scholar, Ward et al., 2010Ward P.S. Patel J. Wise D.R. Abdel-Wahab O. Bennett B.D. Coller H.A. Cross J.R. Fantin V.R. Hedvat C.V. Perl A.E. et al.The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate.Cancer Cell. 2010; 17: 225-234Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). Despite this intriguing data, the mechanism by which IDH1/2 mutations transform cells is far from clear. The discovery of a neomorphic enzymatic function for IDH1/2 raises the possibility that D-2HG may act as an oncometabolite (Dang et al., 2010Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. et al.Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2010; 465: 966Crossref PubMed Scopus (55) Google Scholar, Ward et al., 2010Ward P.S. Patel J. Wise D.R. Abdel-Wahab O. Bennett B.D. Coller H.A. Cross J.R. Fantin V.R. Hedvat C.V. Perl A.E. et al.The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate.Cancer Cell. 2010; 17: 225-234Abstract Full Text Full Text PDF PubMed Scopus (455) Google Scholar). It has been reported that increased levels of D-2HG in cells caused oxidative stress in rat brains (Latini et al., 2003Latini A. Scussiato K. Rosa R.B. Llesuy S. Belló-Klein A. Dutra-Filho C.S. Wajner M. D-2-hydroxyglutaric acid induces oxidative stress in cerebral cortex of young rats.Eur. J. Neurosci. 2003; 17: 2017-2022Crossref PubMed Scopus (51) Google Scholar), which could potentially promote oncogenesis. In the clinic, high levels of 2HG have been linked to a rare neurometabolic disorder called D-2-hydroxyglutaric aciduria (Kranendijk et al., 2010Kranendijk M. Struys E.A. van Schaftingen E. Gibson K.M. Kanhai W.A. van der Knaap M.S. Amiel J. Buist N.R. Das A.M. de Klerk J.B. et al.IDH2 mutations in patients with D-2-hydroxyglutaric aciduria.Science. 2010; 330: 336Crossref PubMed Scopus (60) Google Scholar). A subset of patients with this disease was found to harbor IDH2 R140 mutations. Though the patients showed significantly higher levels of D-2HG compared to leukemia or glioma patients with IDH1/2 mutations, they did not develop gliomas, leukemia, or other malignancies (Kranendijk et al., 2010Kranendijk M. Struys E.A. van Schaftingen E. Gibson K.M. Kanhai W.A. van der Knaap M.S. Amiel J. Buist N.R. Das A.M. de Klerk J.B. et al.IDH2 mutations in patients with D-2-hydroxyglutaric aciduria.Science. 2010; 330: 336Crossref PubMed Scopus (60) Google Scholar). In glioma patients, IDH1/2 mutations usually coexist with TP53 mutations (Yan et al., 2009Yan H. Parsons D.W. Jin G. McLendon R. Rasheed B.A. Yuan W. Kos I. Batinic-Haberle I. Jones S. Riggins G.J. et al.IDH1 and IDH2 mutations in gliomas.N. Engl. J. Med. 2009; 360: 765-773Crossref PubMed Scopus (1238) Google Scholar). It is possible that, like other oncogenic stresses that trigger cell death and senescence, cells with extremely high levels of 2HG may be restrained from further malignant transformation by a similar checkpoint mechanism. In addition, other substrates/products affected by the IDH1/2 mutations may also contribute to oncogenesis. Disrupting the balance of NADP+/NADPH is likely to result in a broad spectrum of cellular reactions, and α-KG is a key component of multiple pathways. For example, α-KG is a key substrate for prolyl hydroxylase domain proteins (PHDs) to catalyze hydroxylation of hypoxia-inducible factor (HIF), a key regulator of angiogenesis (Figure 2) (Semenza, 2010Semenza G.L. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics.Oncogene. 2010; 29: 625-634Crossref PubMed Scopus (505) Google Scholar). This hydroxylation allows HIF to be recognized and targeted for ubiquitin-mediated protein degradation. It has been observed that overexpression of IDH1 R132 in glioma cell lines resulted in increased levels of HIF-1α (Zhao et al., 2009Zhao S. Lin Y. Xu W. Jiang W. Zha Z. Wang P. Yu W. Li Z. Gong L. Peng Y. et al.Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha.Science. 2009; 324: 261-265Crossref PubMed Scopus (429) Google Scholar). However, whether cells with IDH1/2 mutations have lower levels of α-KG is still controversial (Dang et al., 2010Dang L. White D.W. Gross S. Bennett B.D. Bittinger M.A. Driggers E.M. Fantin V.R. Jang H.G. Jin S. Keenan M.C. et al.Cancer-associated IDH1 mutations produce 2-hydroxyglutarate.Nature. 2010; 465: 966Crossref PubMed Scopus (55) Google Scholar, Zhao et al., 2009Zhao S. Lin Y. Xu W. Jiang W. Zha Z. Wang P. Yu W. Li Z. Gong L. Peng Y. et al.Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic activity and induce HIF-1alpha.Science. 2009; 324: 261-265Crossref PubMed Scopus (429) Google Scholar). Given the structural similarity of α-KG and D-2HG, it is also possible that the new product D-2HG can compete with α-KG. Such competition has been linked to the oncogenic mechanism of succinate dehydrogenase (SDH) and fumarate hydratase (FH) mutations, in which accumulated succinate and fumarate compete with α-KG to inhibit the activity of PHDs (Semenza, 2010Semenza G.L. Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics.Oncogene. 2010; 29: 625-634Crossref PubMed Scopus (505) Google Scholar). In addition, α-KG is a substrate for particular histone and DNA demethylation enzymes (Figure 2). Reducing α-KG levels or levels of competing substrate would likely affect global gene expression. Indeed, 2-HG can inhibit multiple α-KG-dependent dioxygenases, which are important for DNA/histone demethylation (Xu et al., 2011Xu W. Yang H. Liu Y. Yang Y. Wang P. Kim S.H. Ito S. Yang C. Wang P. Xiao M.T. et al.Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases.Cancer Cell. 2011; 19: 17-30Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). Consistent with this idea, gliomas with IDH1 mutations showed significantly higher frequency of the CpG island methylator (CIM) phenotype and increased histone demethylation (Noushmehr et al., 2010Noushmehr H. Weisenberger D.J. Diefes K. Phillips H.S. Pujara K. Berman B.P. Pan F. Pelloski C.E. Sulman E.P. Bhat K.P. et al.Cancer Genome Atlas Research NetworkIdentification of a CpG island methylator phenotype that defines a distinct subgroup of glioma.Cancer Cell. 2010; 17: 510-522Abstract Full Text Full Text PDF PubMed Scopus (565) Google Scholar). Subclassification of GBM by congruence of genomic features has taken precedence in the field. A detailed summary of the recent progress and problems related to this topic can be found in a recent review by Vitucci et al. (Vitucci et al., 2011Vitucci M. Hayes D.N. Miller C.R. Gene expression profiling of gliomas: merging genomic and histopathological classification for personalised therapy.Br. J. Cancer. 2011; 104: 545-553Crossref PubMed Scopus (46) Google Scholar). In general, genome-wide studies have revealed that tumor histology correlates with distinct gene expression signatures. Furthermore, molecular profiles can identify subclasses of tumors that would otherwise be indistinguishable by standard morphological methods. One such example is primary and secondary GBM. Although the histology of both types of GBM is identical, primary GBM is thought to arise de novo, and secondary GBM has a longer history of disease progression from lower-grade tumors (Ohgaki and Kleihues, 2007Ohgaki H. Kleihues P. Genetic pathways to primary and secondary glioblastoma.Am. J. Pathol. 2007; 170: 1445-1453Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). Recently, however, several groups have used genome-wide analyses to successfully categorize these two subtypes based on their gene expression profiles (Maher et al., 2006Maher E.A. Brennan C. Wen P.Y. Durso L. Ligon K.L. Richardson A. Khatry D. Feng B. Sinha R. Louis D.N. et al.Marked genomic differences characterize primary and secondary glioblastoma subtypes and identify two distinct molecular and clinical secondary glioblastoma entities.Cancer Res. 2006; 66: 11502-11513Crossref PubMed Scopus (119) Google Scholar, Tso et al., 2006Tso C.L. Freije W.A. Day A. Chen Z. Merriman B. Perlina A. Lee Y. Dia E.Q. Yoshimoto K. Mischel P.S. et al.Distinct transcription profiles of primary and secondary glioblastoma subgroups.Cancer Res. 2006; 66: 159-167Crossref PubMed Scopus (115) Google Scholar). Depending on the sample pool and analysis methods, different studies have reported different numbers of subclasses. For example, Li et al. published their molecular analysis of glioma using two different unsupervised methods; they reported two main types, oligodendroglioma-rich (O) and glioblastoma-rich (G), which could be further divided into six subtypes (Li et al., 2009aLi A. Walling J. Ahn S. Kotliarov Y. Su Q. Quezado M. Oberholtzer J.C. Park J. Zenklusen J.C. Fine H.A. Unsupervised analysis of transcriptomic profiles reveals six glioma subtypes.Cancer Res. 2009; 69: 2091-2099Crossref PubMed Scopus (105) Google Scholar). Verhaak et al. performed unsupervised clustering analysis of the TCGA GBM data set and grouped the tumors into four subtypes termed proneural (PN), neural (NL), mesenchymal (MES), and classical (CL) (Verhaak et al., 2010Verhaak R.G. Hoadley K.A. Purdom E. Wang V. Qi Y. Wilkerson M.D. Miller C.R. Ding L. Golub T. Mesirov J.P. et al.Cancer Genome Atlas Research NetworkIntegrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1.Cancer Cell. 2010; 17: 98-110Abstract Full Text Full Text PDF PubMed Scopus (1183) Google Scholar). The PN and MES subtypes shared significant overlap with previous studies. Integration of genetic alteration events revealed that PN, MES, and CL subtypes were associated with aberrant PDGFRA/IDH1, NF1, and EGFR status, respectively. Gravendeel et al. also used an unsupervised algorithm to classify profiles of 276 gliomas into 24 different molecular "clusters," or subtypes (Gravendeel et al., 2009Gravendeel L.A.M. Kouwenhoven M.C.M. Gevaert O. de Rooi J.J. Stubbs A.P. Duijm J.E. Daemen A. Bleeker F.E. Bralten L.B.C. Kloosterhof N.K. et al.Intrinsic gene expression profiles of gliomas are a better predictor of survival than histology.Cancer Res. 2009; 69: 9065-9072Crossref PubMed Scopus (131) Google Scholar). A similar proneural subtype (C17) was also identified. A large number of these classification studies have now been carried out and provide interesting insights into the molecular nature of these tumors, as well additional questions and problems to pursue (Vitucci et al., 2011Vitucci M. Hayes D.N. Miller C.R. Gene expression profiling of gliomas: merging genomic and histopathological classification for personalised therapy.Br. J. Cancer. 2011; 104: 545-553Crossref PubMed Scopus (46) Google Scholar). The prognostic value of the molecular subclassification has also been evaluated, with several studies suggesting that gliomas with expression of genes associated with neurogenesis (proneural subtype) generally correlate with marginally improved survival (Vitucci et al., 2011Vitucci M. Hayes D.N. Miller C.R. Gene expression profiling of gliomas: merging genomic and histopathological classification for personalised therapy.Br. J. Cancer. 2011; 104: 545-553Crossref PubMed Scopus (46) Google Scholar). In contrast, gliomas with mesenchymal gene expression (mesenchymal subtype) usually have a poorer outcome (1 year for proneural versus 0.6 years for mesenchymal) (Vitucci et al., 2011Vitucci M. Hayes D.N. Miller C.R. Gene expression profiling of gliomas: merging genomic and histopathological classification for personalised therapy.Br. J. Cancer. 2011; 104: 545-553Crossref PubMed Scopus (46) Google Scholar). In an early study by Phillips et al., the proneural subtype included a significant proportion of grade III gliomas, which have a more favorable outcome compared to the more aggressive GBM (Phillips et al., 2006Phillips H.S. Kharbanda S. Chen R. Forrest W.F. Soriano R.H. Wu T.D. Misra A. Nigro J.M. Colman H. Soroceanu L. et al.Molecular subclasses of high-grade glioma predict prognosis, delineate a pattern of disease progression, and resemble stages in neurogenesis.Cancer Cell. 2006; 9: 157-173Abstract Full Text Full Text PDF PubMed Scopus (1005) Google Scholar). Thus, it is possible that the difference in patient survival merely reflects the known survival differences associated with tumor grade. This problem was emphasized by the TCGA study in which only GBM samples were analyzed. No prognostic differences were observed among the four different subtypes revealed in the TCGA study (Verhaak et al., 2010Verhaak R.G. Hoadley K.A. Purdom E. Wang V. Qi Y. Wilkerson M.D. Miller C.R. Ding L. Golub T. Mesirov J.P. et al.Cancer Genome Atlas Research NetworkIntegrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR, and NF1.Cancer Cell. 2010; 17: 98-110Abstract Full Text Full Text PDF PubMed Scopus (1183) Google Scholar). However, the C17 (PN) subtype in the Gravendeel study was found to have prolonged survival in either all of the gliomas or in the pure GBM population, confirming that the previous prognostic value of the molecular markers was not just the consequence of the selection methods, where markers were chosen based on their association with outcome in the training data sets (Gravendeel et al., 2009Gravendeel L.A.M. Kouwenhoven M.C.M. Gevaert O. de Rooi J.J. Stubbs A.P. Duijm J.E. Daemen A. Bleeker F.E. Bralten L.B.C. Kloosterhof N.K. et al.Intrinsic gene expression profiles of gliomas are a better predictor of survival than histology.Cancer Res. 2009; 69: 9065-9072Crossref PubMed Scopus (131) Google Scholar). Although gliomas with proliferative or mesenchymal characteristics generally have a worse outcom