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Genetic Pathways to Primary and Secondary Glioblastoma

      Glioblastoma is the most frequent and most malignant human brain tumor. The prognosis remains very poor, with most patients dying within 1 year after diagnosis. Primary and secondary glioblastoma constitute distinct disease subtypes, affecting patients of different age and developing through different genetic pathways. The majority of cases (>90%) are primary glioblastomas that develop rapidly de novo, without clinical or histological evidence of a less malignant precursor lesion. They affect mainly the elderly and are genetically characterized by loss of heterozygosity 10q (70% of cases), EGFR amplification (36%), p16INK4a deletion (31%), and PTEN mutations (25%). Secondary glioblastomas develop through progression from low-grade diffuse astrocytoma or anaplastic astrocytoma and manifest in younger patients. In the pathway to secondary glioblastoma, TP53 mutations are the most frequent and earliest detectable genetic alteration, already present in 60% of precursor low-grade astrocytomas. The mutation pattern is characterized by frequent G:C→A:T mutations at CpG sites. During progression to glioblastoma, additional mutations accumulate, including loss of heterozygosity 10q25-qter (∼70%), which is the most frequent genetic alteration in both primary and secondary glioblastomas. Primary and secondary glioblastomas also differ significantly in their pattern of promoter methylation and in expression profiles at RNA and protein levels. This has significant implications, particularly for the development of novel, targeted therapies, as discussed in this review.
      The distinction of primary and secondary glioblastoma was first made by the German neuropathologist Hans-Joachim Scherer.
      • Peiffer J
      • Kleihues P
      Hans-Joachim Scherer (1906–1945), pioneer in glioma research.
      In 1940, while working as a political refugee at the Institute Bunge in Antwerp (Belgium), he wrote, “From a biological and clinical point of view, the secondary glioblastomas developing in astrocytomas must be distinguished from ‘primary’ glioblastomas. They are probably responsible for most of the glioblastomas of long clinical duration.”
      • Scherer HJ
      Cerebral astrocytomas and their derivatives.
      This was a remarkable observation at that time; as late as 1979, the World Health Organization (WHO) did not consider the glioblastoma as an astrocytic tumor, listing it instead in a group of poorly differentiated and embryonal tumors.
      With the introduction of immunohistochemistry, the glioblastoma was firmly categorized as astrocytic neoplasm,
      • Kleihues P
      • Burger PC
      • Scheithauer BW
      but the separation of primary and secondary glioblastoma remained conceptual, without being used as diagnostic terms largely because these subtypes are considered histopathologically indistinguishable. During the past decade, evidence accumulated that they constitute distinct disease entities that affect patients of different age, develop through different genetic pathways,
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.
      show different RNA and protein expression profiles,
      • Godard S
      • Getz G
      • Delorenzi M
      • Farmer P
      • Kobayashi H
      • Desbaillets I
      • Nozaki M
      • Diserens AC
      • Hamou MF
      • Dietrich PY
      • Regli L
      • Janzer RC
      • Bucher P
      • Stupp R
      • de Tribolet N
      • Domany E
      • Hegi ME
      Classification of human astrocytic gliomas on the basis of gene expression: a correlated group of genes with angiogenic activity emerges as a strong predictor of subtypes.
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
      • Tso CL
      • Freije WA
      • Day A
      • Chen Z
      • Merriman B
      • Perlina A
      • Lee Y
      • Dia EQ
      • Yoshimoto K
      • Mischel PS
      • Liau LM
      • Cloughesy TF
      • Nelson SF
      Distinct transcription profiles of primary and secondary glioblastoma subgroups.
      and may differ in their response to radio- and chemotherapy.

      Epidemiology and Clinical Features

      Glioblastoma (WHO grade IV) is the most frequent histological type of brain tumor, accounting for 69% of all incident cases of astrocytic and oligodendroglial tumors, with 3.55 new cases in Switzerland per 100,000 population per year, adjusted to the European Standard Population.
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.
      The incidence rate of glioblastomas in the United States, adjusted to the United States Standard Population, is 2.96 new cases per 100,000 population per year [Central Brain Tumor Registry of the United States: Central Brain Tumor Registry of the United States (CBTRUS); http://www.cbtrus.org]. These incident cases do not include secondary glioblastomas that progressed from low-grade or anaplastic gliomas because only the first diagnosis is considered as an incident case.
      Primary glioblastomas present at diagnosis as full-blown tumors, without clinical, radiological, or histopathological evidence of a less-malignant precursor lesion. They are also termed de novo glioblastoma, but this does not suggest a single-step transformation; like other human neoplasms, they result from the acquisition of multiple genetic alterations. Secondary glioblastomas develop slowly through progression from low-grade diffuse astrocytoma (WHO grade II) or anaplastic astrocytoma (WHO grade III). The diagnosis of secondary glioblastoma requires clinical (neuroimaging) or histological (bioptic) evidence of an evolution from a less malignant astrocytoma.
      At a population level, we found that only 5% of all cases were secondary glioblastomas with histopathological evidence of a precursor low-grade or anaplastic astrocytoma.
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.
      This is consistent with observations at the University of Alabama, in which 19 of 392 (5%) cases of glioblastomas had histologically proven prior low-grade gliomas.
      • Dropcho EJ
      • Soong SJ
      The prognostic impact of prior low grade histology in patients with anaplastic gliomas: a case-control study.
      The incidence rate of low-grade and anaplastic astrocytomas is approximately two to three times higher than that of secondary glioblastoma
      • Okamoto Y
      • Di Patre PL
      • Burkhard C
      • Horstmann S
      • Jourde B
      • Fahey M
      • Schuler D
      • Probst-Hensch NM
      • Yasargil MG
      • Yonekawa Y
      • Lutolf U
      • Kleihues P
      • Ohgaki H
      Population-based study on incidence, survival rates, and genetic alterations of low-grade astrocytomas and oligodendrogliomas.
      • Ohgaki H
      • Kleihues P
      Epidemiology and etiology of gliomas.
      (CBTRUS; http://www.cbtrus.org). This may be explained at least in part by the fact that a significant fraction of patients with low-grade or anaplastic astrocytoma succumb to the disease before progression to glioblastoma occurs. Some cases with very rapid progression from low-grade or anaplastic astrocytoma may have been misclassified as primary glioblastoma. Even when taking into account this possibility, secondary glioblastomas constitute a relatively rare disease when compared with primary glioblastomas.
      At the population level, the majority of patients with primary glioblastomas (68%) had a clinical history of less than 3 months. The mean period from first symptoms to histological diagnosis was 6.3 months (Table 1).
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.
      The mean time to progression from anaplastic glioma to glioblastoma was ∼2 years, and that from low-grade glioma to glioblastoma was ∼5 years (Table 1).
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.
      Table 1Incidence, Age, Survival, and Genetic, Epigenetic Changes, and Expression Profiles in Primary and Secondary Glioblastomas
      Primary glioblastomaSecondary glioblastomaReferences
      Incidence rate
      Adjusted to the European Standard Population (per 100,000 persons per year).
      3.5310.199
      Incidence rate
      Adjusted to the World Standard Population (per 100,000 persons per year).
      2.5750.167
      Mean age62 years45 years
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.
      M/F ratio1.330.65
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.
      Clinical history<3 months: 68%From grade II:
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.
      3 to 6 months: 16%5.3 years
      >6 months: 16%From grade III:
      (mean, 6.3 months)1.4 years
      SurvivalMedian, 4.7 monthsMedian, 7.8 months
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      Genetic alterations
      TP53 mutations28%65%
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      EGFR amplification36%8%
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      PTEN mutations25%4%
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      p16INK4adeletion31%19%
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
       LOH 1p12%15%
      • Nakamura M
      • Yang F
      • Fujisawa H
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Loss of heterozygosity on chromosome 19 in secondary glioblastomas.
       LOH 10p47%8%
      • Fujisawa H
      • Reis RM
      • 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.
       LOH 10q47%54%
      • Fujisawa H
      • Reis RM
      • 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.
      70%63%
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
       LOH 13q12%38%
      • Nakamura M
      • Yang F
      • Fujisawa H
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Loss of heterozygosity on chromosome 19 in secondary glioblastomas.
       LOH 19q6%54%
      • Nakamura M
      • Yang F
      • Fujisawa H
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Loss of heterozygosity on chromosome 19 in secondary glioblastomas.
       LOH 22q41%82%
      • Nakamura 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.
      Promoter methylation
      p14ARF6%31%
      • Nakamura M
      • Watanabe T
      • Klangby U
      • Asker CE
      • Wiman KG
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      P14Arf deletion and methylation in genetic pathways to glioblastomas.
      p16INK4a3%19%
      • Nakamura M
      • Watanabe T
      • Klangby U
      • Asker CE
      • Wiman KG
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      P14Arf deletion and methylation in genetic pathways to glioblastomas.
       RB114%43%
      • Nakamura M
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Promoter hypermethylation of the RB1 gene in glioblastomas.
       MGMT36%75%
      • Nakamura M
      • Watanabe T
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Promoter hypermethylation of the DNA repair gene MGMT in astrocytomas is frequently associated with G: C → A:T mutations of the TP53 tumor suppressor gene.
       TIMP-328%71%
      • Nakamura 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.
      Expression profiles
       Fas (APO-1/CD95)
      Immunohistochemistry,
      100%21%
      • Tohma Y
      • Gratas C
      • Van Meir EG
      • Desbaillets I
      • Tenan M
      • Tachibana O
      • Kleihues P
      • Ohgaki H
      Necrogenesis and Fas/APO-1(CD95) expression in primary (de novo) and secondary glioblastomas.
       Survivin
      Immunohistochemistry,
      83%46%
      • Xie D
      • Zeng YX
      • Wang HJ
      • Wen JM
      • Tao Y
      • Sham JS
      • Guan XY
      Expression of cytoplasmic and nuclear Survivin in primary and secondary human glioblastoma.
       MMP-9
      Immunohistochemistry,
      69%14%
      • Choe G
      • Park JK
      • Jouben-Steele L
      • Kremen TJ
      • Liau LM
      • Vinters HV
      • Cloughesy TF
      • Mischel PS
      Active matrix metalloproteinase 9 expression is associated with primary glioblastoma subtype.
       EGFR
      Immunohistochemistry,
      63%10%
      • Watanabe K
      • Tachibana O
      • Sato K
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas.
       EGFR
      2-DGE,
      HighLow
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
       MDM2
      Immunohistochemistry,
      31%0%
      • Biernat W
      • Kleihues P
      • Yonekawa Y
      • Ohgaki H
      Amplification and overexpression of MDM2 in primary (de novo) glioblastomas.
       VEGF
      enzyme-linked immunosorbent assay,
      HighLow
      • Karcher S
      • Steiner HH
      • Ahmadi R
      • Zoubaa S
      • Vasvari G
      • Bauer H
      • Unterberg A
      • Herold-Mende C
      Different angiogenic phenotypes in primary and secondary glioblastomas.
       VEGF fms-related tyrosine kinase 1
      cDNA array,
      HighLow
      • Godard S
      • Getz G
      • Delorenzi M
      • Farmer P
      • Kobayashi H
      • Desbaillets I
      • Nozaki M
      • Diserens AC
      • Hamou MF
      • Dietrich PY
      • Regli L
      • Janzer RC
      • Bucher P
      • Stupp R
      • de Tribolet N
      • Domany E
      • Hegi ME
      Classification of human astrocytic gliomas on the basis of gene expression: a correlated group of genes with angiogenic activity emerges as a strong predictor of subtypes.
       IGFBP2
      cDNA array,
      HighLow
      • Godard S
      • Getz G
      • Delorenzi M
      • Farmer P
      • Kobayashi H
      • Desbaillets I
      • Nozaki M
      • Diserens AC
      • Hamou MF
      • Dietrich PY
      • Regli L
      • Janzer RC
      • Bucher P
      • Stupp R
      • de Tribolet N
      • Domany E
      • Hegi ME
      Classification of human astrocytic gliomas on the basis of gene expression: a correlated group of genes with angiogenic activity emerges as a strong predictor of subtypes.
       Tenascin-X-precursor
      2-DGE,
      HighLow
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
       Enolase 1
      2-DGE,
      HighLow
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
       Centrosome-associated protein 350
      2-DGE,
      HighLow
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
       TP53
      Immunohistochemistry,
      37%97%
      • Watanabe K
      • Tachibana O
      • Sato K
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas.
       ASCL1
      reverse transcriptase-polymerase chain reaction.
      33%88%
      • Somasundaram K
      • Reddy SP
      • Vinnakota K
      • Britto R
      • Subbarayan M
      • Nambiar S
      • Hebbar A
      • Samuel C
      • Shetty M
      • Sreepathi HK
      • Santosh V
      • Hegde AS
      • Hegde S
      • Kondaiah P
      • Rao MR
      Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma.
       Loss of TIMP-3
      Immunohistochemistry,
      17%64%
      • Nakamura 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.
       PDGF-AB
      enzyme-linked immunosorbent assay,
      LowHigh
      • Karcher S
      • Steiner HH
      • Ahmadi R
      • Zoubaa S
      • Vasvari G
      • Bauer H
      • Unterberg A
      • Herold-Mende C
      Different angiogenic phenotypes in primary and secondary glioblastomas.
       ERCC6
      2-DGE,
      LowHigh
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
       DUOX2
      2-DGE,
      LowHigh
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
       HNRPA3
      2-DGE,
      LowHigh
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
       WNT-11 protein precursor
      2-DGE,
      LowHigh
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
       Cadherin-related tumor suppressor homolog precursor
      2-DGE,
      LowHigh
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
       ADAMTS-19
      2-DGE,
      LowHigh
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
      M/F, male/female; PDGF-AB, platelet-derived growth factor AB.
      Bold indicates significantly more frequent than the other glioblastoma subtype.
      * Adjusted to the European Standard Population (per 100,000 persons per year).
      Adjusted to the World Standard Population (per 100,000 persons per year).
      a Immunohistochemistry,
      b 2-DGE,
      c enzyme-linked immunosorbent assay,
      d cDNA array,
      e reverse transcriptase-polymerase chain reaction.
      There is a striking difference in the age distribution of patients with primary and secondary glioblastomas (Figure 1). The mean age of primary glioblastoma patients was 62 years, whereas secondary glioblastomas developed in younger patients (45 years).
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.
      At a population level, primary glioblastomas developed more frequently in men (male to female ratio, 1:33), whereas secondary glioblastomas were more frequent in women (male to female ratio, 0:65).
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      A review of several studies also showed a tendency toward a higher male to female ratio in primary than secondary glioblastomas.
      • Godard S
      • Getz G
      • Delorenzi M
      • Farmer P
      • Kobayashi H
      • Desbaillets I
      • Nozaki M
      • Diserens AC
      • Hamou MF
      • Dietrich PY
      • Regli L
      • Janzer RC
      • Bucher P
      • Stupp R
      • de Tribolet N
      • Domany E
      • Hegi ME
      Classification of human astrocytic gliomas on the basis of gene expression: a correlated group of genes with angiogenic activity emerges as a strong predictor of subtypes.
      • Xie D
      • Zeng YX
      • Wang HJ
      • Wen JM
      • Tao Y
      • Sham JS
      • Guan XY
      Expression of cytoplasmic and nuclear Survivin in primary and secondary human glioblastoma.
      • Choe G
      • Park JK
      • Jouben-Steele L
      • Kremen TJ
      • Liau LM
      • Vinters HV
      • Cloughesy TF
      • Mischel PS
      Active matrix metalloproteinase 9 expression is associated with primary glioblastoma subtype.
      • Watanabe K
      • Tachibana O
      • Sato K
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas.
      • Karcher S
      • Steiner HH
      • Ahmadi R
      • Zoubaa S
      • Vasvari G
      • Bauer H
      • Unterberg A
      • Herold-Mende C
      Different angiogenic phenotypes in primary and secondary glioblastomas.
      This corroborates a previous observation that glioblastomas with TP53 mutations (a genetic hallmark of secondary glioblastoma) are more common in women.
      • Louis DN
      • von Deimling A
      • Chung RY
      • Rubio MP
      • Whaley JM
      • Eibl RH
      • Ohgaki H
      • Wiestler OD
      • Thor AD
      • Seizinger BR
      Comparative study of p53 gene and protein alterations in human astrocytic tumors.
      This is surprising because in hospital-based
      • Kleihues P
      • Cavenee WK
      Pathology and genetics of tumours of the nervous system.
      • Peraud A
      • Ansari H
      • Bise K
      • Reulen HJ
      Clinical outcome of supratentorial astrocytoma WHO grade II.
      and population-based studies
      • Okamoto Y
      • Di Patre PL
      • Burkhard C
      • Horstmann S
      • Jourde B
      • Fahey M
      • Schuler D
      • Probst-Hensch NM
      • Yasargil MG
      • Yonekawa Y
      • Lutolf U
      • Kleihues P
      • Ohgaki H
      Population-based study on incidence, survival rates, and genetic alterations of low-grade astrocytomas and oligodendrogliomas.
      (CBTRUS; http://www.cbtrus.org), the incidence of precursor low-grade or anaplastic gliomas was reported to be similar to or higher in males than in females. The possibility exists that in female patients gliomas progress more frequently or more rapidly to glioblastoma.
      Figure thumbnail gr1
      Figure 1Secondary glioblastomas develop in younger patients than primary glioblastomas. TP53 mutations occur in patients of any age group, whereas EGFR amplification occurs in older patients. Note that there is no single case of glioblastoma with EGFR amplification in patients younger than 35 years of age. (Modified from Ohgaki et al
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      ).
      The median survival of secondary glioblastoma patients was 7.8 months, significantly longer than that of primary glioblastoma patients (4.7 months; P = 0.003). However, this difference is considered largely due to the younger age of secondary glioblastoma patients, because younger age is a consistent and significant predictive factor of longer survival of glioblastoma patients.
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.
      After age adjustment, multivariate analyses showed no significant difference in survival of patients with primary and secondary glioblastomas.
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.

      Genetic Pathways to Primary and Secondary Glioblastoma

      Genetic pathways to primary and secondary glioblastomas at a population level are summarized in Figure 2. LOH 10q is most frequent in both primary and secondary glioblastomas. Epidermal growth factor receptor (EGFR) amplification and PTEN mutations are genetic alterations typical of primary glioblastomas, where-as TP53 mutations are early and frequent genetic alterations in the pathway leading to secondary glioblastomas.
      Figure thumbnail gr2
      Figure 2Genetic pathways to primary (de novo) and secondary glioblastomas at the population level (Modified from Ohgaki et al
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      ). LOH 10q is frequent in both primary and secondary glioblastomas. TP53 mutations are early and frequent genetic alterations in the pathway leading to secondary glioblastomas. *Genetic alterations that are significantly different in frequency between primary and secondary glioblastomas.

      EGFR/PTEN/Akt/mTOR Pathway

      The EGFR/PTEN/Akt/mTOR pathway is a key signaling pathway in the development of primary glioblastomas.
      • Kita D
      • Yonekawa Y
      • Weller M
      • Ohgaki H
      PI3KCA alterations in primary (de novo) and secondary glioblastomas.
      Amplification of the EGFR occurs in ∼40% of primary glioblastomas
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Ekstrand AJ
      • Sugawa N
      • James CD
      • Collins VP
      Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails.
      but rarely in secondary glioblastomas.
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Watanabe K
      • Tachibana O
      • Sato K
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas.
      EGFR overexpression is also more common in primary glioblastomas (>60%) than in secondary glioblastomas (<10%).
      • Watanabe K
      • Tachibana O
      • Sato K
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas.
      All primary glioblastomas with EGFR amplification show EGFR overexpression, and 70 to 90% of those with EGFR overexpression have EGFR amplification.
      • Tohma Y
      • Gratas C
      • Biernat W
      • Peraud A
      • Fukuda M
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      PTEN (MMAC1) mutations are frequent in primary glioblastomas (de novo) but not in secondary glioblastomas.
      • Biernat W
      • Huang H
      • Yokoo H
      • Kleihues P
      • Ohgaki H
      Predominant expression of mutant EGFR (EGFRvIII) is rare in primary glioblastomas.
      The age distribution of cases with EGFR amplification closely follows that of primary glioblastoma patients. In a large, population-based study, EGFR amplification was not detected in any glioblastoma from patients younger than 35 years of age (Figure 1).
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      This confirms studies on pediatric glioblastomas in which EGFR amplification is either absent or very rare.
      • Kraus JA
      • Felsberg J
      • Tonn JC
      • Reifenberger G
      • Pietsch T
      Molecular genetic analysis of the TP53, PTEN, CDKN2A, EGFR, CDK4 and MDM2 tumour-associated genes in supratentorial primitive neuroectodermal tumours and glioblastomas of childhood.
      EGFR amplicons are often mutated; variant 3 (EGFRvIII) with deletion of exons 2 to 7 is the most frequent type. It is associated with constitutive activation of the receptor and failure to attenuate signaling by receptor down-regulation, causes mitogenic effects, and has more powerful transforming activity.
      • Huang HS
      • Nagane M
      • Klingbeil CK
      • Lin H
      • Nishikawa R
      • Ji XD
      • Huang CM
      • Gill GN
      • Wiley HS
      • Cavenee WK
      The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling.
      The constitutively active EGFRvIII can enhance cell proliferation in part by down-regulation of p27 through activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway.
      • Narita Y
      • Nagane M
      • Mishima K
      • Huang HJ
      • Furnari FB
      • Cavenee WK
      Mutant epidermal growth factor receptor signaling down-regulates p27 through activation of the phosphatidylinositol 3-kinase/Akt pathway in glioblastomas.
      This variant occurs only in glioblastomas with concurrent wild-type EGFR amplification,
      • Ekstrand AJ
      • Sugawa N
      • James CD
      • Collins VP
      Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails.
      ie, primary glioblastomas (Table 1).
      EGFR becomes activated through the binding of growth factors (epidermal growth factor, transforming growth factor-α) to its extracellular domain, resulting in recruitment of PI3K to the cell membrane (Figure 3). PI3K phosphorylates phosphatidynositol-4,5-bisphosphate to the respective 3-phosphate (PIP3), which activates downstream effector molecules such as AKT (protein kinase B) and mTOR, the mammalian target of rapamycin; this results in cell proliferation and increased cell survival by blocking apoptosis. PTEN inhibits the PIP3 signal,
      • Mellinghoff IK
      • Wang MY
      • Vivanco I
      • Haas-Kogan DA
      • Zhu S
      • Dia EQ
      • Lu KV
      • Yoshimoto K
      • Huang JH
      • Chute DJ
      • Riggs BL
      • Horvath S
      • Liau LM
      • Cavenee WK
      • Rao PN
      • Beroukhim R
      • Peck TC
      • Lee JC
      • Sellers WR
      • Stokoe D
      • Prados M
      • Cloughesy TF
      • Sawyers CL
      • Mischel PS
      Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors.
      thereby inhibiting cell proliferation (Figure 3). Response to EGFR kinase inhibitors requires coexpression of EGFRvIII and PTEN.
      • Mellinghoff IK
      • Wang MY
      • Vivanco I
      • Haas-Kogan DA
      • Zhu S
      • Dia EQ
      • Lu KV
      • Yoshimoto K
      • Huang JH
      • Chute DJ
      • Riggs BL
      • Horvath S
      • Liau LM
      • Cavenee WK
      • Rao PN
      • Beroukhim R
      • Peck TC
      • Lee JC
      • Sellers WR
      • Stokoe D
      • Prados M
      • Cloughesy TF
      • Sawyers CL
      • Mischel PS
      Molecular determinants of the response of glioblastomas to EGFR kinase inhibitors.
      Figure thumbnail gr3
      Figure 3Major signaling pathways involved in the pathogenesis of glioblastomas.
      The PTEN (phosphatase and tensin homology) gene, located at 10q23.3,
      • Li J
      • Yen C
      • Liaw D
      • Podsypanina K
      • Bose S
      • Wang SI
      • Puc J
      • Miliaresis C
      • Rodgers L
      • McCombie R
      • Bigner SH
      • Giovanella BC
      • Ittmann M
      • Tycko B
      • Hibshoosh H
      • Wigler MH
      • Parsons R
      PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer.
      • Steck PA
      • Pershouse MA
      • Jasser SA
      • Yung WK
      • Lin H
      • Ligon AH
      • Langford LA
      • Baumgard ML
      • Hattier T
      • Davis T
      • Frye C
      • Hu R
      • Swedlund B
      • Teng DH
      • Tavtigian SV
      Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers.
      encodes a central domain with homology to the catalytic region of protein tyrosine phosphatases, which is important in the function of protein phosphatase
      • Myers MP
      • Stolarov JP
      • Eng C
      • Li J
      • Wang SI
      • Wigler MH
      • Parsons R
      • Tonks NK
      P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase.
      and 3′-phosphoinositol phosphatase activities.
      • Maehama T
      • Dixon JE
      The tumor suppressor, PTEN/MMAC1, dephosphorylates the lipid second messenger, phosphatidylinositol 3,4,5-trisphosphate.
      The amino terminal domain of PTEN, with homology to tensin and auxilin, is important in regulating cell migration and invasion by directly dephosphorylating focal adhesion kinase.
      • Tamura M
      • Gu J
      • Matsumoto K
      • Aota S
      • Parsons R
      • Yamada KM
      Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN.
      The PTEN gene is mutated in 15 to 40% of glioblastomas
      • Dahia PL
      PTEN, a unique tumor suppressor gene.
      • Knobbe CB
      • Merlo A
      • Reifenberger G
      Pten signaling in gliomas.
      and almost exclusively in primary glioblastomas (Table 1).
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Tohma Y
      • Gratas C
      • Biernat W
      • Peraud A
      • Fukuda M
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      PTEN (MMAC1) mutations are frequent in primary glioblastomas (de novo) but not in secondary glioblastomas.

      TP53/MDM2/p14ARF Pathway

      The TP53 pathway plays a crucial role in the development of secondary glioblastomas. TP53 mutations are the first detectable genetic alteration in two-thirds of precursor low-grade diffuse astrocytomas; this frequency is similar to that in anaplastic astrocytomas and secondary glioblastomas derived thereof (Figure 2).
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Watanabe K
      • Tachibana O
      • Sato K
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas.
      • Watanabe K
      • Sato K
      • Biernat W
      • Tachibana O
      • von Ammon K
      • Ogata N
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Incidence and timing of p53 mutations during astrocytoma progression in patients with multiple biopsies.
      TP53 mutations also occur in primary glioblastomas, but at a lower frequency (<30% of cases)
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      (Figure 2).
      In secondary glioblastomas, 57% of mutations have been reported to be located in the two hotspot codons 248 and 273; however, in primary glioblastomas, mutations were more equally distributed through all exons, with only 17% occurring in codons 248 and 273.
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      Furthermore, G:C→A:T transitions at CpG sites, considered to result from deamination of 5-meC, were significantly more frequent in secondary than in primary glioblastomas.
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      Thus, G:C→A:T mutations at CpG sites, particularly in the hotspot codons 248 and 273, seem to be an early event directly associated with malignant transformation in the pathway to secondary glioblastoma. The less specific pattern of TP53 mutations in primary glioblastomas may constitute, at least in part, secondary events due to increasing genomic instability during tumor development.
      Amplification of MDM2 is present in <10% of glioblastomas,
      • Reifenberger G
      • Liu L
      • Ichimura K
      • Schmidt EE
      • Collins VP
      Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations.
      exclusively in primary glioblastomas that lack a TP53 mutation.
      • Reifenberger G
      • Liu L
      • Ichimura K
      • Schmidt EE
      • Collins VP
      Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations.
      • Biernat W
      • Kleihues P
      • Yonekawa Y
      • Ohgaki H
      Amplification and overexpression of MDM2 in primary (de novo) glioblastomas.
      Loss of p14ARF expression has frequently been observed in glioblastomas (76%), and this typically correlates with homozygous deletion or promoter methylation of the p14ARF gene.
      • Nakamura M
      • Watanabe T
      • Klangby U
      • Asker CE
      • Wiman KG
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      P14Arf deletion and methylation in genetic pathways to glioblastomas.
      Promoter methylation of p14ARF was more frequent in secondary than primary glioblastomas, but there was no significant difference in the overall frequency of p14ARF alterations (homozygous deletion and promoter methylation) between glioblastoma subtypes.
      • Nakamura M
      • Watanabe T
      • Klangby U
      • Asker CE
      • Wiman KG
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      P14Arf deletion and methylation in genetic pathways to glioblastomas.
      The analysis of multiple biopsies from the same patients revealed p14ARF methylation already in one-third of precursor low-grade astrocytomas.
      • Nakamura M
      • Watanabe T
      • Klangby U
      • Asker CE
      • Wiman KG
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      P14Arf deletion and methylation in genetic pathways to glioblastomas.
      The TP53 gene at 17p13.1 encodes a 53-kd protein that plays a role in several cellular processes (Figure 3), including the cell cycle, response of cells to DNA damage, cell death, cell differentiation, and neovascularization.
      • Bögler O
      • Huang HJ
      • Kleihues P
      • Cavenee WK
      The p53 gene and its role in human brain tumors.
      After DNA damage, TP53 is activated and induces transcription of genes such as p21Waf1/Cip1.
      • Sherr CJ
      • Roberts JM
      CDK inhibitors: positive and negative regulators of G1-phase progression.
      • Stott FJ
      • Bates S
      • James MC
      • McConnell BB
      • Starborg M
      • Brookes S
      • Palmero I
      • Ryan K
      • Hara E
      • Vousden KH
      • Peters G
      The alternative product from the human CDKN2A locus, p14ARF, participates in a regulatory feedback loop with p53 and MDM2.
      The MDM2 binds to mutant and wild-type TP53 proteins, thereby inhibiting the ability of wild-type TP53 to activate transcription from minimal promoter sequences.
      • Momand J
      • Zambetti GP
      • Olson DC
      • George D
      • Levine AJ
      The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation.
      • Oliner JD
      • Kinzler KW
      • Meltzer PS
      • George DL
      • Vogelstein B
      Amplification of a gene encoding a p53-associated protein in human sarcomas.
      Conversely, transcription of the MDM2 gene is induced by wild-type TP53.
      • Barak Y
      • Gottlieb E
      • Juven Gershon T
      • Oren M
      Regulation of mdm2 expression by p53: alternative promoters produce transcripts with nonidentical translation potential.
      • Zauberman A
      • Flusberg D
      • Haupt Y
      • Barak Y
      • Oren M
      A functional p53-responsive intronic promoter is contained within the human mdm2 gene.
      In normal cells, this autoregulatory feedback loop regulates both the activity of the TP53 protein and the expression of MDM2.
      • Picksley SM
      • Lane DP
      The p53-mdm2 autoregulatory feedback loop: a paradigm for the regulation of growth control by p53?.
      The p14ARF gene product binds to MDM2 and inhibits MDM2-mediated p53 degradation and transactivational silencing.
      • Kamijo T
      • Weber JD
      • Zambetti G
      • Zindy F
      • Roussel MF
      • Sherr CJ
      Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2.
      • Pomerantz J
      • Schreiber-Agus N
      • Liegeois NJ
      • Silverman A
      • Alland L
      • Chin L
      • Potes J
      • Chen K
      • Orlow I
      • Lee HW
      • Cordon-Cardo C
      • DePinho RA
      The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53.
      • Fleming TP
      • Saxena A
      • Clark WC
      • Robertson JT
      • Oldfield EH
      • Aaronson SA
      • Ali IU
      Amplification and/or overexpression of platelet-derived growth factor receptors and epidermal growth factor receptor in human glial tumors.
      • Nürnberg P
      • Zischler H
      • Fuhrmann E
      • Thiel G
      • Losanova T
      • Kinzel D
      • Nisch G
      • Witkowski R
      • Epplen JT
      Coamplification of simple repetitive DNA fingerprint fragments and the EGFR gene in human gliomas.
      • Mercer WE
      • Shields MT
      • Amin M
      • Sauve GJ
      • Appella E
      • Romano JW
      • Ullrich SJ
      Negative growth regulation in a glioblastoma tumor cell line that conditionally expresses human wild-type p53.
      Conversely, p14ARF expression is negatively regulated by TP53 and inversely correlates with TP53 function in human tumor cell lines.
      • Stott FJ
      • Bates S
      • James MC
      • McConnell BB
      • Starborg M
      • Brookes S
      • Palmero I
      • Ryan K
      • Hara E
      • Vousden KH
      • Peters G
      The alternative product from the human CDKN2A locus, p14ARF, participates in a regulatory feedback loop with p53 and MDM2.
      Thus, loss of normal TP53 function may result from altered expression of any of the TP53, MDM2, or p14ARF genes (Figure 3).

      p16INK4a/RB1 Pathway

      The p16INK4a/RB1 pathway seems to be important in pathways to both primary and secondary glioblastomas. Homozygous p16INK4a deletions were more frequent in primary than in secondary glioblastomas,
      • Nakamura M
      • Watanabe T
      • Klangby U
      • Asker CE
      • Wiman KG
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      P14Arf deletion and methylation in genetic pathways to glioblastomas.
      • Biernat W
      • Tohma Y
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Alterations of cell cycle regulatory genes in primary (de novo) and secondary glioblastomas.
      but there was no significant difference in the overall frequency of p16INK4a alterations (homozygous deletion and promoter methylation).
      • Nakamura M
      • Watanabe T
      • Klangby U
      • Asker CE
      • Wiman KG
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      P14Arf deletion and methylation in genetic pathways to glioblastomas.
      Promoter methylation of the RB1 gene was significantly more frequent in secondary (43%) than in primary glioblastomas (14%).
      • Nakamura M
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Promoter hypermethylation of the RB1 gene in glioblastomas.
      There was a significant correlation between loss of RB1 expression and promoter methylation of the RB1 gene in glioblastomas.
      • Nakamura M
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Promoter hypermethylation of the RB1 gene in glioblastomas.
      RB1 promoter methylation was not detected in low-grade and anaplastic astrocytoma, indicating that it is a late event during astrocytoma progression.
      • Nakamura M
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Promoter hypermethylation of the RB1 gene in glioblastomas.
      RB1 protein controls progression through G1 to S phase of the cell cycle. The CDK4/cyclin D1 complex phosphorylates the RB1 protein, thereby inducing release of the E2F transcription factor that activates genes involved in the G1→S transition.
      • Sherr CJ
      • Roberts JM
      CDK inhibitors: positive and negative regulators of G1-phase progression.
      p16INK4a binds to CDK4, inhibits the CDK4/cyclin D1 complex, and thus inhibits the G1→S transition.
      • Sherr CJ
      • Roberts JM
      CDK inhibitors: positive and negative regulators of G1-phase progression.
      Thus, loss of normal RB1 function may result from altered expression of any of the RB1, p16INK4a, or CDK4 genes (Figure 3).

      Loss of Heterozygosity (LOH)

      LOH 10q is the most frequent genetic alteration occurring in both primary and secondary glioblastomas at similar frequencies (60 to 80%),
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Fujisawa H
      • Reis RM
      • 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.
      • Rasheed BK
      • McLendon RE
      • Friedman HS
      • Friedman AH
      • Fuchs HE
      • Bigner DD
      • Bigner SH
      Chromosome 10 deletion mapping in human gliomas: a common deletion region in 10q25.
      • Ichimura K
      • Schmidt EE
      • Miyakawa A
      • Goike HM
      • Collins VP
      Distinct patterns of deletion on 10p and 10q suggest involvement of multiple tumor suppressor genes in the development of astrocytic gliomas of different malignancy grades.
      • Fults D
      • Pedone CA
      • Thompson GE
      • Uchiyama CM
      • Gumpper KL
      • Iliev D
      • Vinson VL
      • Tavtigian SV
      • Perry III, WL
      Microsatellite deletion mapping on chromosome 10q and mutation analysis of MMAC1, FAS, and MXI1 in human glioblastoma multiforme.
      with a common deletion at 10q25-qter. In contrast, LOH 10p is largely exclusively present in primary glioblastomas,
      • Fujisawa H
      • Reis RM
      • 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.
      and complete loss of the entire chromosome 10 is typical for primary glioblastomas. Several LOH studies identified at least three commonly deleted loci, ie, 10p14-p15, 10q23-24 (PTEN), and 10q25-pter, suggesting the presence of several tumor-suppressor genes that may play significant roles in the pathogenesis of glioblastomas.
      • Rasheed BK
      • McLendon RE
      • Friedman HS
      • Friedman AH
      • Fuchs HE
      • Bigner DD
      • Bigner SH
      Chromosome 10 deletion mapping in human gliomas: a common deletion region in 10q25.
      • Ichimura K
      • Schmidt EE
      • Miyakawa A
      • Goike HM
      • Collins VP
      Distinct patterns of deletion on 10p and 10q suggest involvement of multiple tumor suppressor genes in the development of astrocytic gliomas of different malignancy grades.
      • Fults D
      • Pedone CA
      • Thompson GE
      • Uchiyama CM
      • Gumpper KL
      • Iliev D
      • Vinson VL
      • Tavtigian SV
      • Perry III, WL
      Microsatellite deletion mapping on chromosome 10q and mutation analysis of MMAC1, FAS, and MXI1 in human glioblastoma multiforme.
      Because LOH 10q25-qter is associated with histologically recognized transition from low-grade or anaplastic astrocytoma to glioblastoma phenotypes
      • Fujisawa H
      • Kurrer M
      • Reis RM
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Acquisition of the glioblastoma phenotype during astrocytoma progression is associated with loss of heterozygosity on 10q25-qter.
      and is commonly deleted in primary and secondary glioblastomas
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Ohgaki H
      • Kleihues P
      Population-based studies on incidence, survival rates, and genetic alterations in astrocytic and oligodendroglial gliomas.
      (Figure 2), the tumor suppressor gene(s) at these loci seem to be involved in the pathogenesis of both glioblastoma subtypes. This locus contains several putative tumor suppressor genes, including LGI1 at 10q24,
      • Chernova OB
      • Somerville RP
      • Cowell JK
      A novel gene, LGI1, from 10q24 is rearranged and downregulated in malignant brain tumors.
      BUB3 at 10q24-q26,
      • Cahill DP
      • da Costa LT
      • Carson-Walter EB
      • Kinzler KW
      • Vogelstein B
      • Lengauer C
      Characterization of MAD2B and other mitotic spindle checkpoint genes.
      MXI1 at 10q25.1,
      • Eagle LR
      • Yin X
      • Brothman AR
      • Williams BJ
      • Atkin NB
      • Prochownik EV
      Mutation of the MXI1 gene in prostate cancer.
      h-neu at 10q25.1,
      • Nakamura H
      • Yoshida M
      • Tsuiki H
      • Ito K
      • Ueno M
      • Nakao M
      • Oka K
      • Tada M
      • Kochi M
      • Kuratsu J
      • Ushio Y
      • Saya H
      Identification of a human homolog of the Drosophila neuralized gene within the 10q25.1 malignant astrocytoma deletion region.
      abLIM or LIMAB1 at 10q25.1,
      • Roof DJ
      • Hayes A
      • Adamian M
      • Chishti AH
      • Li T
      Molecular characterization of abLIM, a novel actin-binding and double zinc finger protein.
      • Kim AC
      • Peters LL
      • Knoll JHM
      • van Huffel C
      • Ciciotte SL
      • Kleyn PW
      • Chishti AH
      Limatin (LIMAB1), an actin-binding LIM protein, maps to mouse chromosome 19 and human chromosome 10q25, a region frequently deleted in human cancers.
      and DMBT1 at 10q26.1
      • Mollenhauer J
      • Wiemann S
      • Scheurlen W
      • Korn B
      • Hayashi Y
      • Wilgenbus KK
      • von Deimling A
      • Poustka A
      DMBT1, a new member of the SRCR superfamily, on chromosome 10q25.3-26.1 is deleted in malignant brain tumours.
      genes, but their role remains unclear because mutations of these genes have been rarely detected in glioblastomas.
      LOH 22q is significantly more frequent in secondary glioblastomas (82%) than in primary glioblastomas (41%).
      • Nakamura 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.
      Characterization of the 22q deletions in primary glioblastomas identified two minimally deleted regions at 22q12.3-13.2 and 22q13.31. The small (957 kb) deletion was also present in 22 of 23 secondary glioblastomas, a region in which the human tissue inhibitor of metalloproteinases-3 (TIMP-3) is located. TIMP-3promoter methylation was observed with significantly higher frequency in secondary than in primary glioblastomas and correlated with loss of TIMP-3 expression. LOH 19q is more frequent in secondary glioblastomas (54%) than primary glioblastomas (6%),
      • Nakamura M
      • Yang F
      • Fujisawa H
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Loss of heterozygosity on chromosome 19 in secondary glioblastomas.
      whereas LOH 1p and 13q occurred at a similar frequency in primary and secondary glioblastomas (Table 1).
      • Nakamura M
      • Yang F
      • Fujisawa H
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Loss of heterozygosity on chromosome 19 in secondary glioblastomas.

      Co-Presence of Genetic Alterations

      LOH 10q typically co-presents with any of the other genetic alterations in glioblastomas.
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      In contrast, TP53 mutations, EGFR amplification, and PTEN mutations show inverse associations with each other,
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      whereas there is a positive correlation between p16INK4a deletion and EGFR amplification.
      • Ohgaki H
      • Dessen P
      • Jourde B
      • Horstmann S
      • Nishikawa T
      • Di Patre PL
      • Burkhard C
      • Schuler D
      • Probst-Hensch NM
      • Maiorka PC
      • Baeza N
      • Pisani P
      • Yonekawa Y
      • Yasargil MG
      • Lutolf UM
      • Kleihues P
      Genetic pathways to glioblastoma: a population-based study.
      • Hegi ME
      • zur Hausen A
      • Ruedi D
      • Malin G
      • Kleihues P
      Hemizygous or homozygous deletion of the chromosomal region containing the p16INK4a gene is associated with amplification of the EGF receptor gene in glioblastomas.
      • Hayashi Y
      • Ueki K
      • Waha A
      • Wiestler OD
      • Louis DN
      • von Deimling A
      Association of EGFR gene amplification and CDKN2 (p16/MTS1) gene deletion in glioblastoma multiforme.
      These observations suggest that LOH 10q plus at least one or two other genetic alterations may contribute to the development of both primary and secondary glioblastomas. In the pathway to secondary glioblastoma, TP53 mutations occur early, and LOH 10q is a late event (Figure 2). However, the sequence of genetic alterations during the development of primary glioblastomas is unknown.

      Correlation between Genetic Alterations and Histopathological Features

      Glioblastoma is heterogeneous, with remarkable variability of histological features.
      • Kleihues P
      • Cavenee WK
      Pathology and genetics of tumours of the nervous system.
      Some of these tend to be associated with glioblastoma subtypes. The small cell phenotype is often associated with EGFR amplification and is thus a typical component of primary glioblastomas.
      • Burger PC
      • Pearl DK
      • Aldape K
      • Yates AJ
      • Scheithauer BW
      • Passe SM
      • Jenkins RB
      • James CD
      Small cell architecture—a histological equivalent of EGFR amplification in glioblastoma multiforme?.
      Tumor necrosis, in particular large ischemic necroses, are significantly more frequent in primary glioblastomas,
      • Tohma Y
      • Gratas C
      • Van Meir EG
      • Desbaillets I
      • Tenan M
      • Tachibana O
      • Kleihues P
      • Ohgaki H
      Necrogenesis and Fas/APO-1(CD95) expression in primary (de novo) and secondary glioblastomas.
      • Homma T
      • Fukushima T
      • Vaccarella S
      • Yonekawa Y
      • Di Patre PL
      • Franceschi S
      • Ohgaki H
      Correlation among pathology, genotype, and patient outcomes in glioblastoma.
      whereas secondary glioblastomas more frequently contain an oligodendroglioma component.
      • Homma T
      • Fukushima T
      • Vaccarella S
      • Yonekawa Y
      • Di Patre PL
      • Franceschi S
      • Ohgaki H
      Correlation among pathology, genotype, and patient outcomes in glioblastoma.

      Promoter Methylation

      Promoter methylation of p16INK4a, p14ARF, RB1, TIMP-3, and MGMT genes has been comparatively assessed in primary and secondary glioblastomas. Overall, secondary glioblastomas showed a higher frequency of promoter methylation than primary glioblastomas (Table 1). O6-Methylguanine-DNA methyltransferase (MGMT) is a repair protein that specifically removes promutagenic alkyl groups from the O6 position of guanine in DNA. MGMT therefore protects cells against carcinogenesis induced by alkylating agents, and an inverse correlation has been reported between MGMT activity and tissue-specific tumorigenesis induced by alkylating agents in rats.
      • Margison GP
      • Kleihues P
      Chemical carcinogenesis in the nervous system. Preferential accumulation of O6-methylguanine in rat brain deoxyribonucleic acid during repetitive administration of N-methyl-N-nitrosourea.
      • Goth R
      • Rajewsky MF
      Persistence of O6-ethylguanine in rat-brain DNA: correlation with nervous system-specific carcinogenesis by ethylnitrosourea.
      Repair of O6-alkylguanine adducts by tumor cells has been implicated in drug resistance because it reduces the cytotoxicity of alkylating chemotherapeutic agents.
      • Belanich M
      • Pastor M
      • Randall T
      • Guerra D
      • Kibitel J
      • Alas L
      • Li B
      • Citron M
      • Wasserman P
      • White A
      • Eyre H
      • Jaeckle K
      • Schulman S
      • Rector D
      • Prados M
      • Coons S
      • Shapiro W
      • Yarosh D
      Retrospective study of the correlation between the DNA repair protein alkyltransferase and survival of brain tumor patients treated with carmustine.
      Loss of MGMT expression caused by methylation of promoter CpG islands
      • Watts GS
      • Pieper RO
      • Costello JF
      • Peng YM
      • Dalton WS
      • Futscher BW
      Methylation of discrete regions of the O6-methylguanine DNA methyltransferase (MGMT) CpG island is associated with heterochromatinization of the MGMT transcription start site and silencing of the gene.
      • Qian XC
      • Brent TP
      Methylation hot spots in the 5′ flanking region denote silencing of the O6-methylguanine-DNA methyltransferase gene.
      was detected in 75% of secondary glioblastomas, significantly more frequently than in primary glioblastomas (36%).
      • Nakamura M
      • Watanabe T
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Promoter hypermethylation of the DNA repair gene MGMT in astrocytomas is frequently associated with G: C → A:T mutations of the TP53 tumor suppressor gene.
      The difference in frequency of MGMT methylation between primary and secondary glioblastomas is clinically relevant because patients with glioblastoma containing a methylated MGMT promoter were shown to have a substantially greater benefit from adjuvant temozolomide treatment.
      • Hegi ME
      • Diserens AC
      • Gorlia T
      • Hamou MF
      • de Tribolet N
      • Weller M
      • Kros JM
      • Hainfellner JA
      • Mason W
      • Mariani L
      • Bromberg JE
      • Hau P
      • Mirimanoff RO
      • Cairncross JG
      • Janzer RC
      • Stupp R
      MGMT gene silencing and benefit from temozolomide in glioblastoma.
      A correlation between the presence of TP53 mutations and MGMT promoter methylation has been reported in lung cancer
      • Wolf P
      • Hu YC
      • Doffek K
      • Sidransky D
      • Ahrendt SA
      O6-Methylguanine-DNA methyltransferase promoter hypermethylation shifts the p53 mutational spectrum in non-small cell lung cancer.
      and between the presences of G→A mutations in the K-ras gene in colon carcinomas.
      • Esteller M
      • Toyota M
      • Sanchez-Cespedes M
      • Capella G
      • Peinado MA
      • Watkins DN
      • Issa JP
      • Sidransky D
      • Baylin SB
      • Herman JG
      Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is associated with G to A mutations in K-ras in colorectal tumorigenesis.
      Similarly, the majority of low-grade astrocytomas with MGMT methylation (92%) contained a TP53 mutation, whereas only 39% of cases without MGMT methylation carried a TP53 mutation.
      • Nakamura M
      • Watanabe T
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Promoter hypermethylation of the DNA repair gene MGMT in astrocytomas is frequently associated with G: C → A:T mutations of the TP53 tumor suppressor gene.
      Furthermore, G:C→A:T transition mutations at CpG sites were significantly more frequent in low-grade astrocytomas with MGMT methylation (58%) than in those without (11%).
      • Nakamura M
      • Watanabe T
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Promoter hypermethylation of the DNA repair gene MGMT in astrocytomas is frequently associated with G: C → A:T mutations of the TP53 tumor suppressor gene.
      These findings suggest the possibility that TP53 mutations at CpG sites in low-grade gliomas may result, at least in part, from exogenous or endogenous factors that produce DNA adducts at the O6 position of guanine. Various adducts at this position are substrates for repair by MGMT.
      • Pegg AE
      Repair of O6-alkylguanine by alkyltransferases.
      Such adducts typically result from exposure to N-nitrosamides and related alkylating agents that cause brain tumors in rats,
      • Kleihues P
      • Rajewsky MF
      Chemical neuro-oncogenesis: role of structural DNA modifications, DNA repair and neural target cell population.
      but there is currently no evidence indicating that alkylating carcinogens are involved in the etiology of human brain tumors.

      Gene Expression and Proteomic Profiles

      Different genetic alterations in primary and secondary glioblastomas reflect different expression patterns at the RNA and protein levels (Table 1). In a cDNA expression array analysis with 1176 cancer-related genes, low-grade astrocytomas showed rather specific and similar expression profiles, whereas primary glioblastomas showed larger variation, and secondary glioblastomas displayed features of both groups.
      • Godard S
      • Getz G
      • Delorenzi M
      • Farmer P
      • Kobayashi H
      • Desbaillets I
      • Nozaki M
      • Diserens AC
      • Hamou MF
      • Dietrich PY
      • Regli L
      • Janzer RC
      • Bucher P
      • Stupp R
      • de Tribolet N
      • Domany E
      • Hegi ME
      Classification of human astrocytic gliomas on the basis of gene expression: a correlated group of genes with angiogenic activity emerges as a strong predictor of subtypes.
      Prominent genes expressed at a significantly higher level in primary than in secondary glioblastomas include vascular endothelial growth factor (VEGF) fms-related tyrosine kinase 1 (involved in angiogenesis) and IGFBP2.
      • Godard S
      • Getz G
      • Delorenzi M
      • Farmer P
      • Kobayashi H
      • Desbaillets I
      • Nozaki M
      • Diserens AC
      • Hamou MF
      • Dietrich PY
      • Regli L
      • Janzer RC
      • Bucher P
      • Stupp R
      • de Tribolet N
      • Domany E
      • Hegi ME
      Classification of human astrocytic gliomas on the basis of gene expression: a correlated group of genes with angiogenic activity emerges as a strong predictor of subtypes.
      Because VEGF is induced by hypoxia-inducible factor (HIF-1), this difference explains the higher frequency and greater extent of necrosis in primary glioblastomas. Using an array containing 14,500 genes, Tso and colleagues
      • Tso CL
      • Freije WA
      • Day A
      • Chen Z
      • Merriman B
      • Perlina A
      • Lee Y
      • Dia EQ
      • Yoshimoto K
      • Mischel PS
      • Liau LM
      • Cloughesy TF
      • Nelson SF
      Distinct transcription profiles of primary and secondary glioblastoma subgroups.
      showed that secondary glioblastomas primarily include mitotic cell cycle components, suggesting the loss of function in prominent cell cycle regulators, whereas primary glioblastomas preferentially express genes typical of a stromal response, suggesting the importance of extracellular signaling. Immunohistochemical staining of glioblastoma tissue arrays confirmed expression differences.
      • Tso CL
      • Freije WA
      • Day A
      • Chen Z
      • Merriman B
      • Perlina A
      • Lee Y
      • Dia EQ
      • Yoshimoto K
      • Mischel PS
      • Liau LM
      • Cloughesy TF
      • Nelson SF
      Distinct transcription profiles of primary and secondary glioblastoma subgroups.
      EGFR and MDM2 overexpression detected by immunohistochemistry are frequent and typical in primary glioblastomas.
      • Watanabe K
      • Tachibana O
      • Sato K
      • Yonekawa Y
      • Kleihues P
      • Ohgaki H
      Overexpression of the EGF receptor and p53 mutations are mutually exclusive in the evolution of primary and secondary glioblastomas.
      • Biernat W
      • Kleihues P
      • Yonekawa Y
      • Ohgaki H
      Amplification and overexpression of MDM2 in primary (de novo) glioblastomas.
      Immunohistochemistry revealed active matrix metalloproteinase-9 (MMP-9) in 69% of primary glioblastomas but only 14% of secondary glioblastomas.
      • Choe G
      • Park JK
      • Jouben-Steele L
      • Kremen TJ
      • Liau LM
      • Vinters HV
      • Cloughesy TF
      • Mischel PS
      Active matrix metalloproteinase 9 expression is associated with primary glioblastoma subtype.
      Active MMP-9 expression was strongly correlated with EGFRvIII expression,
      • Choe G
      • Park JK
      • Jouben-Steele L
      • Kremen TJ
      • Liau LM
      • Vinters HV
      • Cloughesy TF
      • Mischel PS
      Active matrix metalloproteinase 9 expression is associated with primary glioblastoma subtype.
      which is also typical for primary glioblastomas.
      • Ekstrand AJ
      • Sugawa N
      • James CD
      • Collins VP
      Amplified and rearranged epidermal growth factor receptor genes in human glioblastomas reveal deletions of sequences encoding portions of the N- and/or C-terminal tails.
      Survivin (located on 17q25), which was initially identified as a gene with structural homology to a family of genes known as inhibitors of apoptosis,
      • Ambrosini G
      • Adida C
      • Altieri DC
      A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma.
      is expressed significantly more frequently in primary (83%) than in secondary glioblastomas (46%).
      • Xie D
      • Zeng YX
      • Wang HJ
      • Wen JM
      • Tao Y
      • Sham JS
      • Guan XY
      Expression of cytoplasmic and nuclear Survivin in primary and secondary human glioblastoma.
      Somasundaram and colleagues
      • Somasundaram K
      • Reddy SP
      • Vinnakota K
      • Britto R
      • Subbarayan M
      • Nambiar S
      • Hebbar A
      • Samuel C
      • Shetty M
      • Sreepathi HK
      • Santosh V
      • Hegde AS
      • Hegde S
      • Kondaiah P
      • Rao MR
      Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma.
      reported that ASCL1 is overexpressed in 86% of grade II diffuse astrocytomas and 88% of secondary glioblastomas, whereas the majority (67%) of primary glioblastomas expressed similar to or less than normal brain levels. ASCL1 up-regulation was accompanied by inhibition of Notch signaling as seen by uninduced levels of HES1, a transcriptional target of Notch1, and increased levels of HES6, a dominant-negative inhibitor of HES1-mediated repression of ASCL1.
      • Somasundaram K
      • Reddy SP
      • Vinnakota K
      • Britto R
      • Subbarayan M
      • Nambiar S
      • Hebbar A
      • Samuel C
      • Shetty M
      • Sreepathi HK
      • Santosh V
      • Hegde AS
      • Hegde S
      • Kondaiah P
      • Rao MR
      Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma.
      Increased levels of the Notch ligand Delta1 inhibits Notch signaling via formation of intracellular Notch ligand autonomous complexes, suggesting that inhibition of Notch signaling may be an important early event in the pathway to secondary glioblastomas.
      • Somasundaram K
      • Reddy SP
      • Vinnakota K
      • Britto R
      • Subbarayan M
      • Nambiar S
      • Hebbar A
      • Samuel C
      • Shetty M
      • Sreepathi HK
      • Santosh V
      • Hegde AS
      • Hegde S
      • Kondaiah P
      • Rao MR
      Upregulation of ASCL1 and inhibition of Notch signaling pathway characterize progressive astrocytoma.
      A recent proteomics approach identified additional proteins that are differentially expressed between glioblastoma subtypes (Table 1). Using two-dimensional protein gel electrophoresis (2-DGE) and protein sequencing, Furuta and colleagues
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
      identified distinct protein patterns in primary and secondary glioblastomas. Proteins unique for primary glioblastomas were tenascin-X precursor (6q21.3), enolase 1 (1pter-p36.13), centrosome-associated protein 350 (1p36.13-q41), and EGFR (7p12.3-p12.1), whereas those unique for secondary glioblastomas were ERCC6 (10q11), DUOX2 (15q15.3), HNRPA3 (10q11.1), WNT-11 protein precursor (11q13.5), cadherin-related tumor suppressor homolog precursor (chromosomal location unknown), and ADAMTS-19 (5q31).
      • Furuta M
      • Weil RJ
      • Vortmeyer AO
      • Huang S
      • Lei J
      • Huang TN
      • Lee YS
      • Bhowmick DA
      • Lubensky IA
      • Oldfield EH
      • Zhuang Z
      Protein patterns and proteins that identify subtypes of glioblastoma multiforme.
      Quantification of the cytokines in the supernatant of 30 tissue-correspondent glioma cultures revealed a predominant expression of VEGF-A in primary glioblastomas and a significantly higher expression level of platelet-derived growth factor AB in secondary glioblastomas.
      • Huang HS
      • Nagane M
      • Klingbeil CK
      • Lin H
      • Nishikawa R
      • Ji XD
      • Huang CM
      • Gill GN
      • Wiley HS
      • Cavenee WK
      The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling.
      This suggests that optimal anti-angiogenic therapy may require targeting of multiple angiogenic pathways that differ significantly between primary and secondary glioblastomas.
      • Karcher S
      • Steiner HH
      • Ahmadi R
      • Zoubaa S
      • Vasvari G
      • Bauer H
      • Unterberg A
      • Herold-Mende C
      Different angiogenic phenotypes in primary and secondary glioblastomas.

      Conclusions

      Primary and secondary glioblastomas are distinct disease entities that affect different age groups of patients and develop through distinct genetic pathways with different mRNA and protein expression profiles. These differences are important, especially because they may affect sensitivity to radio- and chemotherapy and should thus be considered in the identification of targets for novel therapeutic approaches.

      Future Directions

      There are several chromosomal regions with LOH that probably contain tumor suppressor genes that need to be identified. The most common and extensive deletions involve chromosome 10q, distal of PTEN. It has been suggested that 10q25-qter harbors a putative tumor suppressor that may play a key role in the development of both primary and secondary glioblastomas. Glioblastoma is the first cancer type in The Cancer Genome Atlas project initiated by the United States National Cancer Institute and the National Human Genome Research Institute, which aims at establishing a database of high-resolution expression profiles, LOH, chromosome copy numbers, and sequence alterations in a total of 500 cases. If both primary and secondary glioblastomas were included, this would greatly increase our understanding of their molecular basis and facilitate the development of drugs that specifically target tumor subtypes with divergent genetic profiles.

      Acknowledgements

      We thank the Foundation for Promotion of Cancer Research, Japan, and the Cancer League of the Canton of Zurich, Switzerland, for their support of population-based studies on glioma patients.

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