Title: Molecular and Genomic Alterations in Glioblastoma Multiforme
Abstract: In recent years, important advances have been achieved in the understanding of the molecular biology of glioblastoma multiforme (GBM); thus, complex genetic alterations and genomic profiles, which recurrently involve multiple signaling pathways, have been defined, leading to the first molecular/genetic classification of the disease. In this regard, different genetic alterations and genetic pathways appear to distinguish primary (eg, EGFR amplification) versus secondary (eg, IDH1/2 or TP53 mutation) GBM. Such genetic alterations target distinct combinations of the growth factor receptor–ras signaling pathways, as well as the phosphatidylinositol 3-kinase/phosphatase and tensin homolog/AKT, retinoblastoma/cyclin-dependent kinase (CDK) N2A-p16INK4A, and TP53/mouse double minute (MDM) 2/MDM4/CDKN2A-p14ARF pathways, in cells that present features associated with key stages of normal neurogenesis and (normal) central nervous system cell types. This translates into well-defined genomic profiles that have been recently classified by The Cancer Genome Atlas Consortium into four subtypes: classic, mesenchymal, proneural, and neural GBM. Herein, we review the most relevant genetic alterations of primary versus secondary GBM, the specific signaling pathways involved, and the overall genomic profile of this genetically heterogeneous group of malignant tumors. In recent years, important advances have been achieved in the understanding of the molecular biology of glioblastoma multiforme (GBM); thus, complex genetic alterations and genomic profiles, which recurrently involve multiple signaling pathways, have been defined, leading to the first molecular/genetic classification of the disease. In this regard, different genetic alterations and genetic pathways appear to distinguish primary (eg, EGFR amplification) versus secondary (eg, IDH1/2 or TP53 mutation) GBM. Such genetic alterations target distinct combinations of the growth factor receptor–ras signaling pathways, as well as the phosphatidylinositol 3-kinase/phosphatase and tensin homolog/AKT, retinoblastoma/cyclin-dependent kinase (CDK) N2A-p16INK4A, and TP53/mouse double minute (MDM) 2/MDM4/CDKN2A-p14ARF pathways, in cells that present features associated with key stages of normal neurogenesis and (normal) central nervous system cell types. This translates into well-defined genomic profiles that have been recently classified by The Cancer Genome Atlas Consortium into four subtypes: classic, mesenchymal, proneural, and neural GBM. Herein, we review the most relevant genetic alterations of primary versus secondary GBM, the specific signaling pathways involved, and the overall genomic profile of this genetically heterogeneous group of malignant tumors. Glioblastoma multiforme (GBM) is a World Health Organization grade IV astrocytoma, which represents the most common and aggressive primary brain tumor. Most GBMs are primary tumors that arise de novo as aggressive, highly invasive neoplasias, usually in the absence of clinical, radiological, or histopathological evidence of prior disease and precedent lower-grade lesions; thus, approximately two-thirds of patients with primary GBM have a clinical history of <3 months,1Ohgaki H. Dessen P. Jourde B. Horstmann S. Nishikawa T. Di Patre P.L. Burkhard C. Schuler D. Probst-Hensch N.M. Maiorka P.C. Baeza N. Pisani P. Yonekawa Y. Yasargil M.G. Lutolf U.M. Kleihues P. Genetic pathways to glioblastoma: a population-based study.Cancer Res. 2004; 64: 6892-6899Crossref PubMed Scopus (1046) Google Scholar, 2Brennan C.W. Verhaak R.G. McKenna A. Campos B. Noushmehr H. Salama S.R. et al.TCGA Research NetworkThe somatic genomic landscape of glioblastoma.Cell. 2013; 155: 462-477Abstract Full Text Full Text PDF PubMed Scopus (3052) Google Scholar with rapid development of clinical symptoms. By contrast, secondary GBMs are much less common and they derive from the transformation/progression of lower-grade astrocytomas.1Ohgaki H. Dessen P. Jourde B. Horstmann S. Nishikawa T. Di Patre P.L. Burkhard C. Schuler D. Probst-Hensch N.M. Maiorka P.C. Baeza N. Pisani P. Yonekawa Y. Yasargil M.G. Lutolf U.M. Kleihues P. Genetic pathways to glioblastoma: a population-based study.Cancer Res. 2004; 64: 6892-6899Crossref PubMed Scopus (1046) Google Scholar Interestingly, these two subtypes of glioblastoma also affect patients at different ages: primary GBM is more common in older patients, whereas secondary GBM tends to occur among patients <45 years. Primary and secondary GBMs are usually indistinguishable on histological grounds, but they show clearly different genetic alterations and genomic profiles (Table 1), supporting the notion that the two groups of GBM arise through different genetic pathways.23Mao H. Lebrun D.G. Yang J. Zhu V.F. Li M. Deregulated signaling pathways in glioblastoma multiforme: molecular mechanisms and therapeutic targets.Cancer Invest. 2012; 30: 48-56Crossref PubMed Scopus (183) Google Scholar Herein, we review current knowledge about the signaling pathways most commonly involved in GBM, the molecular and genetic alterations of primary and secondary GBM, including the clinical impact of such alterations, and the most relevant gene expression profiling subgroups of these tumors.Table 1Epigenetic and Genetic Alterations as Well as Gene/Protein Expression Profiles Typically Found in Primary versus Secondary GlioblastomasVariablePrimary glioblastoma, % (95%)Secondary glioblastoma, % (5%)ReferencePromoter methylation MGMT36753Nakamura M. Watanabe T. Yonekawa Y. Kleihues P. Ohgaki H. Promoter methylation of the DNA repair gene MGMT in astrocytomas is frequently associated with G: C--> A: t mutations of the TP53 tumor suppressor gene.Carcinogenesis. 2001; 22: 1715-1719Crossref PubMed Scopus (221) Google Scholar TIMP-328714Nakamura M. Ishida E. Shimada K. Kishi M. Nakase H. Sakaki T. Konishi N. Frequent LOH on 22q12.3 and TIMP-3 inactivation occur in the progression to secondary glioblastomas.Lab Invest. 2005; 85: 165-175Crossref PubMed Scopus (72) Google Scholar RB14435Nakamura M. Yonekawa Y. Kleihues P. Ohgaki H. Promoter hypermethylation of the RB1 gene in glioblastomas.Lab Invest. 2001; 81: 77-82Crossref PubMed Scopus (150) Google Scholar CDKN2A-p14ARF6316Nakamura M. Watanabe T. Klangby U. Asker C. Wiman K. Yonekawa Y. Kleihues P. Ohgaki H. p14ARF deletion and methylation in genetic pathways to glioblastomas.Brain Pathol. 2001; 11: 159-168Crossref PubMed Scopus (209) Google Scholar CDKN2A-p16INK4a3196Nakamura M. Watanabe T. Klangby U. Asker C. Wiman K. Yonekawa Y. Kleihues P. Ohgaki H. p14ARF deletion and methylation in genetic pathways to glioblastomas.Brain Pathol. 2001; 11: 159-168Crossref PubMed Scopus (209) Google ScholarGenetic alterations IDH1 mutation567–857Jiao Y. Killela P.J. Reitman Z.J. Rasheed A.B. Heaphy C.M. de Wilde R.F. Rodriguez F.J. Rosemberg S. Oba-Shinjo S.M. Nagahashi Marie S.K. Bettegowda C. Agrawal N. Lipp E. Pirozzi C. Lopez G. He Y. Friedman H. Friedman A.H. Riggins G.J. Holdhoff M. Burger P. McLendon R. Bigner D.D. Vogelstein B. Meeker A.K. Kinzler K.W. Papadopoulos N. Diaz L.A. Yan H. Frequent ATRX, CIC, FUBP1 and IDH1 mutations refine the classification of malignant gliomas.Oncotarget. 2012; 3: 709-722Crossref PubMed Scopus (447) Google Scholar, 8Yan H. Parsons D.W. Jin G. McLendon R. Rasheed B.A. Yuan W. Kos I. Batinic-Haberle I. Jones S. Riggins G.J. Friedman H. Friedman A. Reardon D. Herndon J. Kinzler K.W. Velculescu V.E. Vogelstein B. Bigner D.D. 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Genetic pathways to glioblastoma: a population-based study.Cancer Res. 2004; 64: 6892-6899Crossref PubMed Scopus (1046) Google Scholar TERT mutation58281Ohgaki H. Dessen P. Jourde B. Horstmann S. Nishikawa T. Di Patre P.L. Burkhard C. Schuler D. Probst-Hensch N.M. Maiorka P.C. Baeza N. Pisani P. Yonekawa Y. Yasargil M.G. Lutolf U.M. Kleihues P. Genetic pathways to glioblastoma: a population-based study.Cancer Res. 2004; 64: 6892-6899Crossref PubMed Scopus (1046) Google Scholar, 10Fujisawa H. Reis R.M. Nakamura M. Colella S. Yonekawa Y. Kleihues P. Ohgaki H. Loss of heterozygosity on chromosome 10 is more extensive in primary (de novo) than in secondary glioblastomas.Lab Invest. 2000; 80: 65-72Crossref PubMed Scopus (151) Google Scholar CDKN2A-p16INK4a deletion31–78191Ohgaki H. Dessen P. Jourde B. Horstmann S. Nishikawa T. Di Patre P.L. Burkhard C. Schuler D. Probst-Hensch N.M. Maiorka P.C. Baeza N. Pisani P. Yonekawa Y. Yasargil M.G. Lutolf U.M. Kleihues P. Genetic pathways to glioblastoma: a population-based study.Cancer Res. 2004; 64: 6892-6899Crossref PubMed Scopus (1046) Google Scholar TP53 mutation28651Ohgaki H. Dessen P. Jourde B. Horstmann S. Nishikawa T. Di Patre P.L. Burkhard C. Schuler D. Probst-Hensch N.M. Maiorka P.C. Baeza N. Pisani P. Yonekawa Y. Yasargil M.G. Lutolf U.M. Kleihues P. Genetic pathways to glioblastoma: a population-based study.Cancer Res. 2004; 64: 6892-6899Crossref PubMed Scopus (1046) Google Scholar PTEN mutation2541Ohgaki H. Dessen P. Jourde B. Horstmann S. Nishikawa T. Di Patre P.L. Burkhard C. Schuler D. Probst-Hensch N.M. Maiorka P.C. Baeza N. Pisani P. Yonekawa Y. Yasargil M.G. Lutolf U.M. Kleihues P. Genetic pathways to glioblastoma: a population-based study.Cancer Res. 2004; 64: 6892-6899Crossref PubMed Scopus (1046) Google Scholar LOH 10p47810Fujisawa H. Reis R.M. Nakamura M. Colella S. Yonekawa Y. Kleihues P. Ohgaki H. 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Alexe G. Lawrence M. O'Kelly M. Tamayo P. Weir B.A. Gabriel S. Winckler W. Gupta S. Jakkula L. Feiler H.S. Hodgson J.G. James C.D. Sarkaria J.N. Brennan C. Kahn A. Spellman P.T. Wilson R.K. Speed T.P. Gray J.W. Meyerson M. Getz G. Perou C.M. Hayes D.N. Integrated 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 (4982) Google Scholar ERCC6†Two-dimensional protein gel electrophoresis.LowHigh16Furuta M. Weil R.J. Vortmeyer A.O. Huang S. Lei J. Huang T.N. Lee Y.S. Bhowmick D.A. Lubensky I.A. Oldfield E.H. Zhuang Z. Protein patterns and proteins that identify subtypes of glioblastoma multiforme.Oncogene. 2004; 23: 6806-6814Crossref PubMed Scopus (77) Google Scholar DUOX2†Two-dimensional protein gel electrophoresis.LowHigh16Furuta M. Weil R.J. Vortmeyer A.O. Huang S. Lei J. Huang T.N. Lee Y.S. Bhowmick D.A. Lubensky I.A. Oldfield E.H. Zhuang Z. Protein patterns and proteins that identify subtypes of glioblastoma multiforme.Oncogene. 2004; 23: 6806-6814Crossref PubMed Scopus (77) Google Scholar HNRPA3†Two-dimensional protein gel electrophoresis.LowHigh16Furuta M. Weil R.J. Vortmeyer A.O. Huang S. Lei J. Huang T.N. Lee Y.S. Bhowmick D.A. Lubensky I.A. Oldfield E.H. Zhuang Z. Protein patterns and proteins that identify subtypes of glioblastoma multiforme.Oncogene. 2004; 23: 6806-6814Crossref PubMed Scopus (77) Google Scholar WNT-11 protein precursor†Two-dimensional protein gel electrophoresis.LowHigh16Furuta M. Weil R.J. Vortmeyer A.O. Huang S. Lei J. Huang T.N. Lee Y.S. Bhowmick D.A. Lubensky I.A. Oldfield E.H. Zhuang Z. Protein patterns and proteins that identify subtypes of glioblastoma multiforme.Oncogene. 2004; 23: 6806-6814Crossref PubMed Scopus (77) Google Scholar Cadherin-related tumor-suppressor homolog precursor†Two-dimensional protein gel electrophoresis.LowHigh16Furuta M. Weil R.J. Vortmeyer A.O. Huang S. Lei J. Huang T.N. Lee Y.S. Bhowmick D.A. Lubensky I.A. Oldfield E.H. Zhuang Z. Protein patterns and proteins that identify subtypes of glioblastoma multiforme.Oncogene. 2004; 23: 6806-6814Crossref PubMed Scopus (77) Google Scholar ADAMTS-19†Two-dimensional protein gel electrophoresis.LowHigh16Furuta M. Weil R.J. Vortmeyer A.O. Huang S. Lei J. Huang T.N. Lee Y.S. Bhowmick D.A. Lubensky I.A. Oldfield E.H. Zhuang Z. Protein patterns and proteins that identify subtypes of glioblastoma multiforme.Oncogene. 2004; 23: 6806-6814Crossref PubMed Scopus (77) Google ScholarModified from Ohgaki and Kleihues22Ohgaki H. Kleihues P. Genetic pathways to primary and secondary glioblastoma.Am J Pathol. 2007; 170: 1445-1453Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar with permission from the American Society for Investigative Pathology.ADAMTS-19, ADAM metallopeptidase with thrombospondin type 1 motif, 19; APO-1, apoptosis-mediating cell membrane protein; ASCL1, achaete-scute complex-like 1; DUOX2, dual oxidase 2; EGFR, epidermal growth factor receptor; ERCC6, excision repair cross-complementation group 6; HNRPA3, heterogeneous nuclear ribonucleoprotein A3; IGFBP, insulin-like growth factor binding protein; LOH, loss of heterozygosity; MDM, mouse double minute; MMP, matrix metalloproteinases; PDGFR, platelet-derived growth factor receptor; TIMP, tissue inhibitor of metalloproteinases; VEGF, vascular endothelial growth factor; WNT-11, wingless-type MMTV integration site family, member 11.∗ Immunohistochemistry.† Two-dimensional protein gel electrophoresis.‡ Enzyme-linked immunosorbent assay.§ cDNA array.¶ RT-PCR. Open table in a new tab Modified from Ohgaki and Kleihues22Ohgaki H. Kleihues P. Genetic pathways to primary and secondary glioblastoma.Am J Pathol. 2007; 170: 1445-1453Abstract Full Text Full Text PDF PubMed Scopus (1075) Google Scholar with permission from the American Society for Investigative Pathology. ADAMTS-19, ADAM metallopeptidase with thrombospondin type 1 motif, 19; APO-1, apoptosis-mediating cell membrane protein; ASCL1, achaete-scute complex-like 1; DUOX2, dual oxidase 2; EGFR, epidermal growth factor receptor; ERCC6, excision repair cross-complementation group 6; HNRPA3, heterogeneous nuclear ribonucleoprotein A3; IGFBP, insulin-like growth factor binding protein; LOH, loss of heterozygosity; MDM, mouse double minute; MMP, matrix metalloproteinases; PDGFR, platelet-derived growth factor receptor; TIMP, tissue inhibitor of metalloproteinases; VEGF, vascular endothelial growth factor; WNT-11, wingless-type MMTV integration site family, member 11. The many different genetic and molecular alterations present in GBM lead to modifications of several major signaling pathways that result in brain tumor growth and progression24The Cancer Genome Atlas (TCGA) Research NetworkComprehensive genomic characterization defines human glioblastoma genes and core pathways.Nature. 2008; 455: 1061-1068Crossref PubMed Scopus (5794) Google Scholar, 25Dunn G.P. Rinne M.L. Wykosky J. Genovese G. Quayle S.N. Dunn I.F. Agarwalla P.K. Chheda M.G. Campos B. Wang A. Brennan C. Ligon K.L. Furnari F. Cavenee W.K. Depinho R.A. Chin L. Hahn W.C. Emerging insights into the molecular and cellular basis of glioblastoma.Genes Dev. 2012; 26: 756-784Crossref PubMed Scopus (417) Google Scholar (Figure 1A). Although the involvement of several well-known pathways in gliomagenesis is indubitable, there are complex interactions among them, including interactions with additional unknown players, which potentially contribute to the initiation and transformation of GBM.26Nakada M. Kita D. Watanabe T. Hayashi Y. Teng L. Pyko I.V. Hamada J. Aberrant signaling pathways in glioma.Cancers (Basel). 2011; 3: 3242-3278Crossref PubMed Scopus (138) Google Scholar The most relevant signaling pathways involved in GBM include, among others, growth factor tyrosine kinase receptor (TKR)–triggered pathways, including the Ras sarcoma (Ras) pathway, as well as the phosphatidylinositol 3-kinase (PI3K)/phosphatase and tensin homolog (PTEN)/AKT, retinoblastoma (RB)/cyclin-dependent kinase (CDK) N2A-p16INK4a, and the TP53/mouse double minute 2 (MDM2)/MDM 4/CDKN2A-p14ARF pathways. Both the platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) play an important role in normal and tumoral gliogenesis, through activation of complex intracellular cascades modulated by G-protein–coupled receptors and second messengers that converge at multiple sites. Overexpression of PDGF and EGF receptor (EGFR) in GBM suggests that these TKR-signaling pathways are critical targets in gliomagenesis.27Zheng Y. McFarland B.C. Drygin D. Yu H. Bellis S.L. Kim H. Bredel M. Benveniste E.N. Targeting protein kinase CK2 suppresses prosurvival signaling pathways and growth of glioblastoma.Clin Cancer Res. 2013; 19: 6484-6494Crossref PubMed Scopus (112) Google Scholar The PDGF family consists of four different ligands (PDGF-A, PDGF-B, PDGF-C, and PDGF-D) that signal through the PDGF receptor (PDGFR) α and PDGFRβ.28Fredriksson L. Li H. Eriksson U. The PDGF family: four gene products form five dimeric isoforms.Cytokine Growth Factor Rev. 2004; 15: 197-204Abstract Full Text Full Text PDF PubMed Scopus (596) Google Scholar Both the PDGF ligands and receptors are often co-expressed in glioma cell lines and primary GBM tissues, suggesting the establishment of both autocrine and paracrine signaling loops, which may contribute to tumor formation and progression. PDGFRA and PDGFA are expressed in tumor cells, whereas PDGFB and PDGFRB have been typically found in glioma-associated endothelial cells. Studies on the two new PDGFR ligands PDGFC and PDGFD indicate that they may also play a role in the development of brain tumors. Because co-expression of PDGF and PDGFR has been observed in astrocytomas of all grades, PDGF autocrine signaling may be considered as an early event, with additional secondary alterations in cell signaling being potentially required for progression to GBM. A subset of gliomas characterized by dysregulated PDGFR activity (due to amplification and rearrangement of the PDGFRA gene locus and/or overexpression of the PDGF ligand) has been described,29Alentorn A. Marie Y. Carpentier C. Boisselier B. Giry M. Labussiére M. Mokhtari K. Hoang-Xuan K. Sanson M. Delattre J.Y. Idbaih A. Prevalence clinico-pathological value, and co-occurrence of PDGFRA abnormalities in diffuse gliomas.Neuro Oncol. 2012; 14: 1393-1403Crossref PubMed Scopus (36) Google Scholar and characterized by The Cancer Genome Atlas (TCGA; see below).21Verhaak R.G. Hoadley K.A. Purdom E. Wang V. Qi Y. Wilkerson M.D. Miller C.R. Ding L. Golub T. Mesirov J.P. Alexe G. Lawrence M. O'Kelly M. Tamayo P. Weir B.A. Gabriel S. Winckler W. Gupta S. Jakkula L. Feiler H.S. Hodgson J.G. James C.D. Sarkaria J.N. Brennan C. Kahn A. Spellman P.T. Wilson R.K. Speed T.P. Gray J.W. Meyerson M. Getz G. Perou C.M. Hayes D.N. 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EGFR gene mutations/rearrangements and expression of their aberrant protein products are frequently observed in GBM so far; seven common variants have been identified, from which variant 3 (EGFRvIII or del2-7 EGFR, ΔEGFR), which lacks a sequence of 267 amino acids in the extracellular ligand-binding domain leading to a constitutively activated EGFR and pathway, is the most frequent one (it is present in 20% to 50% of GBMs that carry EGFR amplification). The introduction of this truncated receptor into glioma cells dramatically enhances their tumorigenicity in vivo through both increased cellular proliferation and reduced apoptosis.33Hatanpaa K.J. Burma S. Zhao D. Habib A.A. Epidermal growth factor receptor in glioma: signal transduction, neuropathology, imaging, and radioresistance.Neoplasia. 2010; 12: 675-684Abstract Full Text PDF PubMed Scopus (321) Google Scholar Ligand-activated receptors trigger downstream signal transduction pathways, including the Ras/rapidly accelerated fibrosarcoma (Raf)/mitogen-activated protein kinase (MAPK) pathway, the PI3K/AKT pathway, the protein kinase C pathway, and the STAT pathway, together with vascular endothelial growth factor production, with an impact on cell proliferation, migration, invasion, resistance to apoptosis, and tumor neovascularization.34Patil C.G. Nuno M. Elramsisy A. Mukherjee D. Carico C. Dantis J. Hu J. Yu J.S. Fa