The molecular genetics of central nervous system tumors

Pathology (1998) 30, pp. 196-202

THE MOLECULAR GENETICS OF CENTRAL NERVOUS SYSTEM TUMORS HO-KEUNG NG AND PAULA Y.P. LAM

Department of Anatomical & Cellular Pathology, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong

Summary Over the past few years, although much has been learned about the molecular genetics of central nervous system (CNS) tumors, researchers and pathologists are only beginning to understand the scientific basis of the development of these tumors. Data accumulated so far support the division of gtioblastoma into two clinical and molecular subsets. Primary or de novo glioblastomas occur in older patients, are clinically aggressive and exhibit epidermal growth factor receptor amplification or overexpression. Secondary glioblastomas develop from pre-existing low-grade astrocytomas, have a more protracted clinical course, and frequently contain p53 mutations. Both types of tumors show deletions of chromosome !0 and possibly mutations of the PTEN/MMAC1 gene as an endstage event. Oligoden,~rogliomas have been shown to have genetic abnormalities distinct from those of the astrocytic tumors, commonly involving chromosomes lp and 19q. As regards meningiomas, loss of chromosome 22q and mutations of the neurofibromatosis type 2 gene are frequent events and loss of chromosome 14q and 10q may be seen in atypical or malignant transformation. Such genetic findings, apart from providing a better understanding of neoplastic transformation in brain tumors, are beginning to form the basis of a new approach to neuro-oncology.

Key words: Astrocytictumor, glioblastoma, meningioma,molecular tumorigenesis. Abbreviations: EGFR: Epidermal growth factor receptor; LOH, loss of hetemzygosity;PDGF: Platelet-derivedgrowth factor. Accepted 18 December 1997

INTRODUCTION The advent of molecular biologic techniques in studying brain tumors has provided scientists and pathologists with new information about the pathogenesis of central nervous system tumors. As in most human cancers, genetic abnormalities found so far involve amplification of oncogenes, mutations of tumor suppressor genes or deletions of chromosomal segments harboring potential tumor suppressor sites. The recent revision of the WHO classification grades the astrocytic tumors into pilocytic astrocytoma (grade I), fibrillary astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma multiforme (grade IV). 1 Pitocytic astrocytomas, with a much more benign behavior, are biologically distinct from the spectrum of diffuse astrocytomas. For the latter group, the majority of low-grade tumors will progress to malignant forms with the passage

of time. 2 In this review, we will focus on the diffuse astrocytomas, with brief discussions on the other gliomas and meningiomas. Emphasis will be on the molecular mechanisms of tumorigenesis. TOWARDS A MOLECULAR CLASSIFICATION OF ASTROCYTIC TUMORS Glioblastoma multiforme, the commonest primary brain tumor, may develop de novo (primary gliOblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). The patient with a primary glioblastoma is usually older, the clinical history is short, tumor growth is rapid and the prognosis dismal. For secondary glioblastomas, there should be clinical, radiologic or histologic evidence of an evolution from a less malignant precursor lesion, ie; low-grade or anaplastic astrocytoma, over an interval of at least six months. 3 Patients with secondary glioblastomas are usually younger, the tumors progress more slowly and the chance of survival following treatment is better. 3 Although these two groups are generally indistinguishable histologically, a vast amount of evidence has accumulated that they follow different pathways of molecular tumorigenesis, culminating in a common terminal phenotype (Fig. 1). yon Deimling, Louis and co-workers use the terms type I and type II glioblastomas, which are roughly equivalent to secondary and primary glioblastomas, respectively, as used by Kleihues and co-workers.4"5 Primary" and secondary gliobtastomas describe tumors by their clinical characteristics, and these two groups also exhibit different genetic properties, whereas type I and type II gliobtastomas emphasize the different molecular properties rather than clinical features. Type I tumors are characterized by loss of heterozygosity (LOH) of chromosome t7p closely associated with p53 mutations and absence of epidermal growth factor (EGFR) gene amplification. Type II tumors are characterized by EGFR amplification and lack of 17p LOH/p53 muation. In this article, the terms primary and secondary glioblastomas will be used. It has also become clear recently that another subgroup of glioblastoma multiforme, the giant cell glioblastoma, can be delineated from the other glioblastomas. Although giant cell glioblastomas develop clinically de novo, they contain genetic alterations similar to those observed in secondary glioblastomas. This may account for their generally favorable prognosis. 6

EPIDERMAL GROWTH FACTOR RECEPTOR The EGFR, a transmembrane receptor tyrosine kinase, is overexpressed in about 40% of gtioblastomas and the

0031-3025/98/020196-07 © 1998 Royal College of Pathologists of Australasia

MOLECULAR GENET1CS OF CNS TUMORS

I Precursor ] LOH 17q (NF1)

[ Precursor I

[ Precursor [ PDGF

overexpression

LOH 17p (p53) LOH 22q

[ Astrocytoma I

197

MDM2

gene amplification

CDK4

gene amplification LOH 13q (Rb) LOH 9p (p15, p16) LOH 19q .....Astrocytoma II1 I I LOH 10 Pilocytie

astrocytomas

t Secondary

GBM

EGFR

gene amplification

LOH 10

t Primary GBM

Fig. 1 Potential molecular pathways of aslrocytomas (modified from I Kleihues and Ohgaki 3)

EGFR gene may be amplified or rearranged, producing truncated, constitutively active mutants] -t° In our experience, transfection of an antisense EGFR construct results in loss of anchorage-independent growth and a reduced rate of proliferation m malignant glioma cell lines] ~ Aberrations of EGFR are usually associated with a loss of chromosome 10] ° It has been suggested that EGFR abnormalities are prevalent in primary glioblastomas and to an extent are mutually exclusive with p53 mutations in astrocytic tumors) 2 The latter is much more commonly found in secondary than primary glioblastomas. 4's EGFR amplification and overexpression, with resultant growth stimulatory, antiapoptotic and angiogenic effects, are believed to be the primary reasons for the poorer outcome of primary glioblastomas over secondary glioblastomas, s

OTHER ONCOGENES INVOLVED IN ASTROCYTIC TUMORS Other oncogenes reported to be involved in the development of astrocytic tumors are listed in Table 1. Platelet derived growth factor (PDGF) is a dimer of combinations of A and B chains. The ligands are recognized by two types of cell surface receptors, PDGFR-c~ and PDGF-/?, which belong to the tyrosine kinase family of receptors. Studies have shown that PDGF-A chain and PDGF-c~ receptors are overexpressed in astrocytomas though gene amplifications are uncommon. 13'14 On the other hand, PDGF-B chain and PDGF-/~ receptor are overexpressed in endothelial cells which often proliferate exuberantly in glioblastomas) 50verexpression of PDGFR-c~ is strongly associated with loss of heterozygosity of chromosome 17p and p53 mutations, suggesting that PDGF abnormality is typical of the pathway leading to the lbrmation of secondary glibolastoma. 14 The MDM2 oncogene contains a p53 DNA-binding site and the transcription of MDM2 is induced by wild-type p53. The MDM2 protein, in turn, forms a complex with p53 abolishing the latter's transcription activity. Thus, an autoregulatory feedback loop regulates the activity of p53 and MDM2 and amplification or overexpression of MDM2

provides a mechanism to escape from the p53 regulated control of cell growth) 6 Amplification of MDM2 is present in fewer than 10% of glioblastom&s and is apparently more commonly found in primary glioblastomas) v' is The relationship between MDM2 and p53 protein accumulation will be discussed in later sections. Other oncogenes that have been reported to be involved in astrocytic tumors are tabulated in Table 1.

p53 GENE IN ASTROCYTIC TUMORS The p53 gene on chromosome 17p has a major role in apoptosis, cell cycle arrest, and response to DNA damage. Loss of heterozygosity of chromosome 17p occurs in about 35% of astrocytic tumors regardless of histological grades19, 2o and p53 mutations also occur in approximately one-third of all grades of astrocytic tumors. 21 This suggests that p53 mutation occurs early on in the pathogenesis of astrocytic tumors, although in other authors' experience, p53 mutations may be infrequent in low-grade astrocytomas and they have also been found in malignant transformation of fibrillary astrocytomas to high-grade lesions. 22-2s There are no brain specific mutations but high frequencies of mutations are found in the evolutionarily conserved domains of exons 5-8, in particular codons 175, 245, 248, 273 and 282. 4 Mutations are primarily missense mutations. 21 These codons are located within the DNAbinding domain and mutations at these sites would lead to loss of p53-mediated transcriptional activity. ~6 p53 mutations or LOH of 17p are almost never found in GBMs with EGFR amplification, leading researchers to propose the two types of GBMs alluded to earlier: the type I or secondary glioblastomas have frequent p53 mutations but no EGFR gene amplification, whereas the type II or primary glioblastomas frequently have amplified or overexpressed EGFR but no p53 muations. 3' 4, 12,27 In tumors in which multiple tumor biopsies are examined by molecular methods, progression of low-grade astrocytomas to anaplastic astrocytoma or glioblastoma seems to occur at a similar frequency in lesions with and without p53 mutations but the time interval until progression in anaplastic

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Pathology (1998), 30, May

TABLE 1 Oncogenes involved in the development of" astrocytomas Oncogenes Growth factors sis" (PDGF)

Chromosome location 22ql 2.3-13.1

Aberrations found

References

Amplification, rearrangement, or overexpression

See text

Cell surface receptors with protein-tyrosin kinase activity erB 7p12-13 Amplification, rearrangement

See text

overexpression Rearrangement

88

lntracetlular transducers met 7q31 H- ras 11p 1 5 . 5 N-ras lpl 3

Amplification Overexpression Amplification, rearrangement

89 90 91

Nuclear proteins fos N-myc gli

14q24.3 2p24.1 12q 13-14.3

Overexpression Amplification Amplification

92 93 94, 95

MDM2

12q 13-14

Amplification

See text

rosl

6q21-22

astrocytoma or glioblastoma tends to be shorter for towgrade astrocytomas carrying a p53 mutation. 28 Again in these tumors identical mutations of p53 are found with multiple biopsy samples. 25'z8 Immunohistochemical studies of p53 protein often show a higher percentage of positive staining than mutations. 2~'29 The mechanism of this discrepancy between immunoreactivity and mutation-is not always apparent. The MDM2 oncogene, the product of which binds to and inactivates p53 protein, has been examined and does not appear to be amplified in astrocytomas with p53 protein accumulation. 29-31 Similarly, none of the gliomas with MDM2 amplifications shows p53 mutations. ~s Investigations of the downstream regulator CIP1 gene have not revealed alterations in those tumors. 32 The p53 protein that has been accumulated in some tumors has been shown to be wildtype and may reflect a physiological response by p53 to the increased DNA damage or deregulated proliferation that is associated with high-grade tumors. 2~'3o On the other hand, the finding of p53 mutations in some astrocytic tumors with 17p LOH suggests the presence of another as yet unidentified tumor suppressor gene residing on 17p. 2~'33-35 A recent report has shown LOH at another locus at 17p13.3 that is independent of the p53 locus but so far no gene has yet been identified. 36

CHROMOSOME 10 AND PTEN/MMACl TUMOR SUPPRESSOR GENE More than 70% of glioblastomas show LOH on chromosome 10 and many cases show loss of the entire chromosome 10. 5,37 As this genetic abnormality is not found in low-grade astrocytomas and only rarely in anaplastic astrocytomas, it is considered that this is a late event during astrocytoma progression. Amplification of EGFR in glioblastoma is always associated with loss of chromosome 10, in particular 10q, suggesting that inactivation of a chromosome 10 gene may precede EGFR gene amplification. 1°'38 The mechanism of this relationship is unclear but it has been suggested that loss of chromosome 10 genes may foster a degree of genomic instability that allows EGFR amplification. 4 The recently identified tumor suppressor gene PTEN/MMAC1 on chromosome 10q23.3

was found to be mutated in 30% of glioblastoma cell lines and xenografts. 39'4° At the time of writing, analyses of PTEN/MMAC1 mutations in gliomas have not yet been published. It has also been believed for some time that tumor suppressor genes for glioblastomas other than PTEN/MMAC1 exist in chromosome 10q, possibly in the region of 10q25 as well as another site at 10p. 4' 5'10'41~43 Recently, a new tumor suppressor gene, DMB121, has been identified on chromosome 10q25.3-26.1 and intragenic deletions are found in 23% of glioblastomas. 44 As can be seen from Fig. 1, LOH10 occurs in both primary and secondary glioblastomas and may be a common endstage event for both groups of tumors.

p16, CDK4 AND Rb ABNORMLAITIES Transition from fibrillary astrocytoma to anaplastic astrocytoma is believed to be associated with inactivation of tumor suppressor genes on chromosomes 9p and 13q. The pl6 gene located on 9p21 has been shown to inhibit specifically the binding of CDK4 to cyclin D, thus preventing phosphorylation of the Rb protein and subsequent progression of the cell cycle. Inactivation of the pl6 gene by deletion has been reported in about 40% of glioblastomas and 20% of anaplastic astrocytomas. 4s'46 However, such deletions have not been observed in low-grade astrocytomas, indicating that loss of functions in these genes may be responsible for the malignant progression of astrocytomas.45.47-49 Deletions of the pl6 gene are much more commonly seen in primary than secondary glioblastoma. 3 Astrocytic tumors with homozygous pl6 deletions also have higher Ki-67 proliferation indices than those without pl6 deletions9 The prevalence of pl6 deletion, together with the frequent EGFR amplification and overexpression, is regarded as the main reason for a primary glioblastoma's unfavorable prognosis. Other than homozygous deletions, point mutations and methylation of the 5 CpG islands are rare alternative mechanisms of inactivation of the pl6 gene. 51-53 Fig. 2 shows a simplified scheme of the molecular control of the cell cycle. Other than deletion of the p16 gene, the next most common alteration of the pl6-CDK4cyclin D1/pRb cell cycle pathway is pRb inactivation,

MOLECULARGENETICSOF CNS TUMORS 199

f GO

Cyclin D-CDK4/6 complex

G1

,/ ~Rb~,,~ p2/

\

"----MDM2 ,~

P04

I p53

DNA damage

p15

p16

Fig. 2 Schematicrepresentationof some cell-cycleregulatory genes that have been implicatedin the formationof astrocytomas.Arrows represent activation; blunt-ended bars represent inhibition. See text for details of involvementof these genes in CNS tumors.

occurring in about 20-30% of high-grade astrocytomas. 46's3-55 Amplification and overexpression of the CDK4 gene on chromosome 12 have been observed in 10-15% of anaplastic astrocytomas and glioblastomas. 45'54'56'57 In general, an inverse relationship exists between genetic events o f p l 6 and pRb in gliomas and p16 deletion, pRb inactivation and CDK4 amplification rarely occur together in the same tumors. 45' s3, 54 This suggests that alterations of any individual component of the pathway have a similar effect on cell cycle deregulation. 4 Disruption of the cell cycle can occur through loss of suppressor activity of the p53 protein, amplification of the MDM2 gene, or point mutation of the p21 WAF-1/CIP1 gene, which has been discussed in the previous section.

LOSS OF OTHER POTENTIAL TUMOR SUPPRESSOR SITES Allelic loss on chromosome 19q has been observed in up to 40% of gliomas. This region is involved not only in astrocytomas of all grades, but also in oligodendrogliomas and oligoastrocytomas.58-6° Chromosome 19q loss is the only genetic alteration shared by tumors of astrocytic and otigodendroglial lineage. A putative tumor suppressor gene has been mapped to 19q13.3 and although a number of candidate genes have been cloned, the responsible tumor suppressor gene has not yet been identified. 6~'62 However, other workers have produced contradictory results in that loss of the 19q marker was undetectable in astrocytomas; instead, loss of the short arm on chromosome 19 appears to be associated with astrocytomas. 63 Allelic loss of chromosome 22q, which contains the neurofibromatosis type 2 tumor suppressor gene (NF2) is seen in 20-30% of astrocytomas. 38 However, studies so far have failed to detect any mutation in the entire coding sequence of the NF2 gene, raising the possibility that another gene nearby may be involved in the tumorigenesis of astrocytomas. 64 Loss of heterozygosity of sites on chromosome 11 has been found in about 30% of high-grade astrocytic tumors, suggesting a putative tumor suppressor gene at 11p15.5 but so far no candidate gene has been cloned. 6s

MOLECULAR ALTERATIONS IN OTHER BRAIN TUMORS Only a brief discussion of the molecular alterations in brain tumors other than the diffuse astrocytomas will be carried out here, and they have been less intensely studied. The pilocytic astrocytomas are typifed by allelic loss on chromosomes 17q, a phenomenon not observed in the diffuse astrocytomas and pilocytic astrocytomas are commonly found in the neurofibromatosis type 1 (NF1) syndrome. 66 However, in contrast to other tumor suppressor genes, mutations of NF1 are not found in pilocytic astrocytomas; instead, there is an upregulation of the NF1 transcript. 67 Similarly, p53 mutations are distinctly uncommon in pilocytic astrocytomas. ~9'68 Oligodendroglial tumors are generally believed to develop from molecular pathways distinct from those of astrocytomas. One of the most frequent genetic alteration in oligodendrogtomas is LOH of 19q, which occurs in about 50-80% of oligodendroglial tumors. 59'69-71 Another important genetic alteration is LOH of the short arm of chromosome 1, which is found in 40-90% of oligodendroglial tumors in different series. 70-72 All oligodendrogliomas with lp LOH also had LOH on 19q, suggesting LOH of Ip to be an essential step in the evolution of these tumors. 70 Unlike the astrocytic tumors, aberrations of the p53 and MDM2 genes are uncommon events in o l i g o d e n d r o g l i a l t u m o r s . 25'73'74 Curiously, in contrast to astrocytic tumors in which EGFR overexpression is commonly found in the high-grade tumors, 50% of oligodendrogliat tumors, generally a low-grade lesion, overexpress

EGFR. 75 As regards ependymomas, most of the genetic changes described for astrocytic and otigodendroglial tumors are not present and in general, specific chromosomal aberrations and cancer genes for ependymomas remain to be identified. Ependymoma is a known complication of the neurofibromatosis type 2 (NF2) syndrome. Allelic loss of 22q, where the NF2 gene resides at 22q12, occurs in about 30% of ependymomas but some investigators have found mutations of the NF2 gene in ependymomas while others have not. 76-78 Mutations of p53 gene are uncommon in ependymomas.73' 74, 79

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In meningiomas, cytogenetic studies have established that up to 70% of meningiomas show complete or partial loss of chromosome 22 and deletions of chromosomes 1 and 14 are also common, s° Similarly to ependymomas, meningiomas are also frequently found in type 2 neurofibromatosis. Allelic loss of 22q occurs in about 60% of meningiomas and mutations of the N F 2 gene in about 40% of cases. 64"81-83 Both LOH and N F 2 mutations occur in the same tumor in many cases, clearly fulfilling Knudson's hypothesis for tumor suppressor genes. Multiple specific segments of chromosome 14q are also potential tumor suppressor sites for meningiomas, which may be related to atypical and anaplastic changes in meningiomas. 84 Similarly, loss of chromosome 10 loci have been described to be associated with malignant changesY Fine deletion mapping and specific cloning of genes in these chromosomes have yet to be performed.

THE FUTURE Over the decades, brain tumors, in particular glioblastomas, have retained their dismal prognosis despite advances in neurosurgical techniques and chemotherapy. While the classification of CNS tumors continues to rely on morphological features,: it has become increasingly clear that there are subsets within the glioblastomas, characterized by their molecular genetic aberrations as well as differential clinical behaviors. At present, only a fraction of the genetic abnormalities of astrocytic tumors has been unraveled. It is conceivable that in the future, clinicopathologic as well as therapeutic studies of gliomas may require molecular characterization of tumors, eg; p 5 3 mutation. Apart from providing an understanding of tumorigenesis, molecular studies in the future may yield information useful for management and prognostication for astrocytic tumors. While there is no specific immunohistochemical marker for oligodendrogliomas, information is emerging that these tumors follow genetic pathways distinct from those of astrocytomas. Such information may aid in the precise identification of oligodendroglial tumors, which is essential as there is mounting evidence that oligodendroglial tumors respond to chemotherapy and can be treated differently from other gtiomas, s6, sv As regards meningiomas, although the key factors for recurrence remain the completeness of surgical excision and tumor grading, genetic aberrations in chromosomes 14q and 10q may provide an adjunct to clinical prediction in the future. ACKNOWLEDGEMENT This study was supported by a grant from the Hong Kong Research Grant Council. Address for correspondence: H-K.N., Departmentof Anatomical& Cellu-

lar Pathology, Prince of Wales ttospital, Shatin, Hong Kong.

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