Meningioma Pathology, Genetics, and Biology

Journal of Neuropathology and Experimental Neurology Vol. 63, No. 4 Copyright 2004 by the American Association of Neuropathologists April, 2004 pp. 275 286 Meningioma Pathology, Genetics, and Biology KATRIN LAMSZUS, MD Abstract. Over the past 5 to 10 years, important advances were made in the understanding of meningioma biology. Progress in molecular genetics probably represents the most important accomplishment in the comprehensive knowledge of meningioma pathogenesis. Several genes could be identified as targets for mutation or inactivation. Additional chromosomal regions were found to be commonly deleted or amplified, suggesting the presence of further tumor suppressor genes or proto-oncogenes, respectively, in these regions. Histopathologically, the most important innovation is represented by the revised WHO classification in the year 2000. Meningioma grading criteria in the new classification scheme are more precise and objective, and should thus improve consistency in predicting tumor recurrence and aggressive behavior. This review focuses mainly on the advances in molecular biology that were achieved in recent years. It summarizes the most important aspects of meningioma classification as the basis to place biological observations into a correlative context, and, further, includes mechanisms of angiogenesis and edema formation as well as the role of hormone receptors in meningiomas. Key Words: Angiogenesis; Classification; Edema; Meningioma; Neurofibromatosis; Progesterone. INTRODUCTION Meningiomas have long been a subject of intense genetic and biological interest. They were among the first solid neoplasms studied using cytogenetic techniques. Frequent monosomy of chromosome 22 in meningiomas was reported as early as 1972 (1). Cytogenetically, meningioma has meanwhile become one of the best-studied neoplasms in humans. Over the past decade, advanced techniques such as automated sequencing, microsatellite analysis, fluorescence in situ hybridization (FISH), or comparative genomic hybridization (CGH) have further facilitated rapid identification of alterations at the molecular genetic level. For the first time, the revised WHO classification of 2000 includes genetic findings in addition to pathological and immunohistochemical features. For glial tumors, genetic alterations meanwhile have even become clinically relevant, insofar as deletions on chromosome arms 1p and 19q have been found associated with responsiveness to chemotherapy and therapeutic outcome of oligodendroglial tumors. For meningiomas, none of the typical genetic aberrations have yet attained clinical relevance. However, the comprehensive analysis of these aberrations in relation to histological grade has led to a model in which early alterations that are presumably involved in meningioma formation could be distinguished from later alterations that are associated with tumor progression (2). In addition to genetic alterations, other biological features of meningiomas have also been investigated. Since the late 1970s, hormone receptors have received great From Department of Neurosurgery, University Hospital Hamburg- Eppendorf, Hamburg, Germany. Correspondence to: Katrin Lamszus, MD, Laboratory for Brain Tumor Biology, Department of Neurosurgery, University Hospital Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany. E-mail: lamszus@uke.uni-hamburg.de attention, although, more recently, progress has somewhat stagnated as receptor expression profiles are now relatively well characterized and therapeutic efforts targeting these receptors have so far been largely disappointing. Another field that has attracted interest, especially since the advancement of imaging techniques in the 1990s, are mechanisms of edema formation. These appear to be closely related to tumor angioarchitecture, and a variety of growth factors have since been analyzed for their contribution to edema formation and meningioma angiogenesis. The purpose of this review is to outline (i) important aspects of meningioma classification and grading, (ii) common gene and chromosome alterations that form the basis for a tumor progression model, (iii) the current status in hormone receptor research, and (iv) mechanisms of edema formation and angiogenesis. CLINICOPATHOLOGICAL ASPECTS Incidence and Etiology Meningiomas are composed of neoplastic meningothelial (arachnoidal cap) cells. They constitute approximately 20% of all primary intracranial tumors, with an approximate annual incidence of 6 per 100,000 (2). The peak incidence is between the sixth and seventh decades of life. Particularly among middle-aged patients, meningiomas are significantly more frequent in woman than in men with a greater than 2:1 ratio. The majority of meningiomas are attached to the dura mater and arise within the intracranial cavity, the spinal canal, or, rarely, the orbit. Some meningiomas, especially those of the sphenoid wing, may arise primarily as intraosseous tumors. Approximately 40% of meningioma patients suffer from seizures; other clinical symptoms depend upon the location of the tumor. Several endogenous and exogenous factors predispose to meningioma development (2). The majority of patients 275

276 LAMSZUS who suffer from neurofibromatosis type 2 (NF2) develop meningiomas. In addition, families with an increased susceptibility to meningiomas but without NF2 have been reported. The clearest exogenous association exists for ionizing radiation, after which meningiomas develop with an average latency period of several decades. In addition, sex hormones have been implicated, mainly due to the overrepresentation of females among meningioma patients. Classification and Prognosis The most important factors that determine the likelihood of meningioma recurrence are extent of tumor resection and histological grade. Extent of resection is still classified according to a scheme proposed by Simpson in 1957, ranging from grade 1 (complete resection) to grade 5 (decompression only) (3). The histopathological meningioma classification received special attention by the working group that devised the 2000 WHO classification, and several substantial changes were introduced (4). These changes are mainly based on a series of studies from the Mayo Clinic in which histopathological parameters were correlated with clinical prognostic parameters (5, 6). Whereas the previous WHO classification that dates back to 1993 had ill-defined borders between benign, atypical, and anaplastic meningiomas, grading criteria are now much more stringent and objective. The majority of meningiomas (80% 90%) are benign and classified as WHO grade I (MI) (2, 5). A variety of histopathological subtypes fall into this category, including meningothelial, fibrous, and transitional meningiomas as the most common variants. Less common subtypes are psammomatous, angiomatous, microcystic, secretory, lymphoplasmacyte-rich, and metaplastic meningioma. MI carry a relatively low risk of recurrence (7% 20%) and of aggressive behavior. Occasional mitoses and pleomorphic nuclei are tolerable features within MI. However, the location in which these tumors arise has a critical impact on prognosis. Whereas tumors of the convexity are usually cured by surgical resection, skull base tumors often have an unfavorable outcome. In particular, meningiomas that arise in the petroclival region or involve the cavernous sinus or orbit often display slow but relentless growth, leading to widespread invasion and destruction of bony structures. These tumors may either remain histologically benign over many years or they may eventually progress towards a higher grade. Between 5% and 15% of meningiomas are classified as atypical, corresponding to WHO grade II (MII) (2, 5). Diagnostic criteria for atypical meningiomas are either increased mitotic activity (defined as 4 mitoses/10 highpower fields of 0.16 mm 2 ) or 3 or more of the following features: increased cellularity, small cells with high nucleus to cytoplasm ratio, prominent nucleoli, uninterrupted patternless or sheet-like growth, and necroses. These criteria correlate with higher recurrence rates (30% 40%). In addition to tumors that fulfill the criteria for atypia, chordoid and clear cell meningiomas are also associated with a high rate of recurrence and aggressive behavior and are therefore also classified as WHO grade II. Anaplastic (malignant) meningiomas, WHO grade III (MIII) are rare (1% 3% of meningiomas) (2). They display histological features of frank malignancy far in excess of the abnormalities present in atypical meningiomas. Such features comprise either obviously malignant cytology (e.g. resemblance to sarcoma, carcinoma, or melanoma) or high mitotic indices ( 20 mitoses/10 highpower fields). The rare variants of papillary or rhabdoid meningioma are also classified as WHO grade III due to their highly aggressive behavior. Malignant meningiomas have recurrence rates of 50% to 80%, and are usually fatal within less than 2 years following diagnosis. Notably, neither brain invasion nor proliferation indices (e.g. MIB-1 labeling) have been incorporated into the current WHO grading criteria. Brain invasive meningiomas have a higher chance of recurrence and behave like atypical meningiomas; therefore, some authors prefer to assign them to WHO grade II (5, 7). However, brain invasion is considered a feature more pertinent to staging than to malignancy grade, and molecular genetic investigations have failed to reveal alterations characteristic of high-grade meningiomas in MI with brain invasion (4). In addition, proliferation indices were not included into the grading criteria; due to high interinstitutional and interindividual variations, reliable cut-off levels for different grades cannot be defined. Nevertheless, brain invasion as well as proliferation indices provide valuable additional prognostic information. Therefore, the WHO working group recommended adding phrases such as with brain invasion or with high proliferative activity to the diagnosis if appropriate (4). Despite the incorporation of genetic and immunohistochemical findings, meningioma classification is still based entirely on conventional histological criteria. Immunohistochemistry mainly has a role in differential diagnosis, for example, when distinguishing meningioma from hemangiopericytoma or other mesenchymal tumors. Genetic analyses have also made important contributions to differential diagnostics, for example, when recognizing hemangiopericytoma as an entity distinct from meningioma (8); however, they currently have no role in routine meningioma diagnosis. The present WHO classification, with its more stringent criteria, should provide an improved framework for genetic studies and foster their comparability, which, hopefully, will lead to even more refined combined histologic-genetic models. Many of the genetic studies that are reviewed in the next sections were performed on the basis of the 1993 WHO classification. It should be kept in mind that the

MENINGIOMAS 277 reported findings may require some adjustment according to the current classification. GENETICS NF2 Gene Mutations in the NF2 tumor suppressor gene, located in the chromosomal region 22q12.2, represent the most frequent gene alteration in meningiomas. Early cytogenetic studies had already found monosomy of chromosome 22 in up to 70% of meningiomas (for review see Collins et al [9]). This observation correlated well with subsequent molecular genetic studies that identified loss of heterozygosity (LOH) at polymorphic markers on 22q as the most common molecular genetic alteration, present in 40% to 70% of all meningiomas (10, 11). Mutations in the NF2 gene were detected in up to 60% of sporadic meningiomas and are typically associated with LOH 22q (12 14). Most NF2 mutations have a truncating effect, indicating that inactivation of the NF2 gene product merlin (schwannomin) is functionally important for meningioma pathogenesis. Absent or strongly reduced immunoreactivity for merlin was found in the majority of meningiomas (15 17), and was strongly associated with LOH 22q (14). Merlin belongs to the 4.1 family of structural proteins that link the cytoskeleton to proteins of the cytoplasmic membrane (18). The mechanism by which merlin exerts a tumor suppressive activity is still poorly understood. The disruption of the signaling cascade that leads to cytoskeletal reorganization is considered to be critical to tumor formation. Overexpression of merlin in both NF2- negative and NF2-positive human meningioma cells significantly inhibited their proliferation in vitro, which provides further evidence for a role of merlin as a negative regulator of tumor growth (19). Molecular genetic as well as protein studies showed that the frequency of NF2/merlin alterations differs among the 3 most common benign meningioma variants. Whereas fibroblastic and transitional meningiomas harbor NF2 mutations in approximately 70% to 80% of cases, meningothelial meningiomas carry mutations in only 25% (13). A close correlation between the fibroblastic variant and LOH 22q was also found (20). Correspondingly, reduced merlin expression was observed in the majority of fibroblastic and transitional meningiomas, but rarely in meningothelial tumors (15, 17). The low frequency of NF2 alterations in the meningothelial variant suggests that the genetic origin of these tumors is largely independent of NF2 gene alterations. Moreover, the similar frequency of NF2 mutations in atypical and anaplastic as well as in benign fibroblastic and transitional meningiomas suggests that NF2 mutations are not involved in progression to higher-grade meningiomas. Rather, NF2 mutations appear to represent an early alteration that is Fig. 1. Hypothetic model of genomic alterations associated with the formation of benign meningiomas and progression towards atypia and anaplasia. Only those alterations that were found in more than 30% of tumors of a specific grade in the majority of studies (see text) are listed as contributing to the development of a particular grade (arrow). Alterations that increase in frequency by at least another 20% (averaged over several studies) in tumors of the next higher histological grade, despite already contributing to the previous grade, are listed again. involved in the formation of most benign meningiomas (Fig. 1). In some meningiomas no NF2 mutation or LOH 22q could be detected despite a lack of merlin. An alternative mechanism that involves merlin degradation by the protease -calpain was proposed to account for this phenomenon (21). Different studies demonstrated activation of -calpain in more than 50% of meningiomas, although no association between activation of -calpain and merlin status could be established (14, 17). Instead, concordance of LOH 22q and merlin loss suggested that other mechanisms, such as homozygous deletions or methylation as well as undetected NF2 mutations, may account for the loss of merlin (14). Meningiomas in NF2 Patients Most patients who suffer from NF2 develop meningiomas. NF2-associated meningiomas differ from sporadic ones in several respects: (i) they usually arise several decades earlier in life, (ii) they are frequently multiple, and (iii) they belong more often to the fibroblastic variant (22). NF2-associated meningiomas exhibit deletions on chromosome arm 22q in almost 100% of cases, a frequency that is significantly higher than in sporadic meningiomas (7, 23). Additional genetic alterations such as deletions on 1p, 6q, 9p, 10q, 14q, and 18q occur at similar frequency as in sporadic cases (23). Most investigations found that the frequency of atypical or malignant meningiomas was not increased among NF2-associated tumors (23 25). The MIB-1 labeling index in NF-2-associated meningiomas appeared to be

278 LAMSZUS slightly higher in 1 study (25), but a more detailed analysis of a larger number of samples revealed no difference compared with sporadic meningiomas (23). In a recent study, NF2-associated meningiomas together with pediatric meningiomas were found to contain a higher percentage of WHO grade II and grade III tumors than sporadic cases (7). However, the feature of brain invasiveness in an otherwise benign tumor was considered equivalent to WHO grade II. The experience in our institution is that NF2 patients rarely develop malignant meningiomas and that mortality in these patients is usually due to other causes. Multiple and Recurrent Meningiomas Multiple meningiomas are independently arising and spatially separate tumors. They are observed in 1% to 8% of meningioma patients. Multiple meningiomas are particularly common in NF2 patients and also in rare non-nf2 families with a hereditary meningioma predisposition. Several studies investigated whether multiple meningiomas represent metastatic meningeal seeding of 1 tumor or de novo formation of separate tumors. Identical NF2 mutations and patterns of X-chromosome inactivation revealed that the majority of multiple as well as recurrent meningiomas are of clonal origin, so that multiplicity most likely represents subarachnoid spread (26 29). Alternatively, NF2 mosaicism could underlie some cases. NF2 mutations and clonal origin were more frequently found in patients with a larger number of tumors than in patients with only 2 tumors, which is compatible with either mosaicism or with an increased ability of liquorigenic seeding caused by a mutant NF2 gene. A recent study focused specifically on differences between multiple meningiomas in sporadic versus familial non-nf2 cases (30). All NF2 mutations that were detected occurred in tumors from patients with no affected relatives, suggesting that the NF2 inactivation is frequently involved in multiple sporadic meningiomas but is rare in multiple meningioma kindreds. In agreement with this observation, linkage analysis of a family with multiple meningiomas showed no segregation with the NF2 locus (31). In another multiple meningioma kindred, immunoreactivity for merlin was detected, implying that the NF2 gene was not inactivated (32). Interestingly, most non- NF2 meningioma family members develop meningothelial meningiomas, which is in line with the observation that this variant seems to arise independently of NF2 inactivation. Other Genes on Chromosome 22 Several studies suggested that other tumor suppressor genes may lie outside the NF2 region on the long arm of chromosome 22. The frequency of LOH 22 exceeds that of NF2 mutations in meningiomas, and deletion mapping revealed interstitial deletions on 22q that did not include the NF2 locus in some tumors (20). Furthermore, a recent case report described monosomy for chromosome 22 but lack of NF2 mutation in multiple meningiomas from a single patient (33). Several candidate tumor suppressor genes on 22q were cloned in recent years, and some were screened for mutations and/or altered expression levels in meningiomas. Three of these genes map to the 22q12.2 region in relatively close proximity to the NF2 gene, namely ADTB1 ( -adaptin, BAM22), RRP22, and GAR22. The ADTB1 gene was cloned based on a 140-kb homozygous deletion in a sporadic meningioma. Expression analysis showed that 12% of sporadic meningiomas lacked ADTB1 transcripts. However, no mutations could be identified in 110 sporadic meningiomas, suggesting epigenetic mechanisms of gene inactivation (34). Similarly, no RRP22 or GAR22 mutations were detected in 12 meningiomas, of which half displayed LOH in the 22q12-q22 region, although none exhibited NF2 mutations (35). Several other candidate genes map outside the 22q12.2 region. The MN1 gene is located at the 22q12.1 segment. MN1 was found to be disrupted by a translocation in a meningioma (36), however, subsequent studies demonstrated that it acts as an oncogenic transcription coactivator rather than a tumor suppressor. The hsnf5/ini1 (SMARCB1) gene, which maps to 22q11.23, is frequently mutated in atypical teratoid/rhabdoid tumors. In 1 study, an identical missense mutation in the hsnf5/ini1 gene was identified in 4 of 126 meningiomas analyzed (37). Three of these 4 tumors also contained NF2 mutations, suggesting that loss of hsnf5/ INI1 function does not represent an alternative mechanism to NF2 inactivation in the pathogenesis of meningiomas, but that silencing of hsnf5/ini1 may co-operate with impairment of NF2 function. Expression of the CLTCL1/CLH-22 gene, which maps to 22q11.21, was found to be absent in 80% (37 of 46) meningiomas analyzed, suggesting that this gene may also be relevant to tumor development (38). Among tumors with absent gene expression were cases with and without chromosome 22 deletions. However, mutations of CLTCL1/CLH-22 have not been reported and the mechanism of its loss of expression is unclear. The LARGE gene maps distally to the NF2 region (22q12.3), and represents another interesting candidate gene (39). However, it is one of the largest human genes, and mutation or expression analyses have not been published. DAL-1 Gene and Other Alterations on Chromosome 18 The DAL-1 protein shares significant homology with merlin and also belongs to the 4.1 family of membraneassociated proteins. It has tumor suppressor properties and maps to chromosomal region 18p11.3. Recent immunohistochemical studies showed that DAL-1 expression is lost in 76% of sporadic meningiomas, a frequency

MENINGIOMAS 279 similar to that detected for loss of merlin (40, 41). Lack of DAL-1 protein was only slightly, and not significantly, more frequent in anaplastic meningiomas (87%) than in benign and atypical meningiomas (70% 76%), suggesting that it represents an early event in meningioma tumorigenesis (41). These immunohistochemical findings were largely reflected at the mrna expression level. Although LOH at 18p11.3 was detected in 71% of investigated cases (40), the mechanism of DAL-1 inactivation is still unresolved. Mutations were not detected, but screening had not included the full gene (40). Given the frequent LOH at the DAL-1 gene location, homozygous deletions are unlikely, so that epigenetic alterations remain a more likely possibility. Combined loss of DAL-1 and merlin was detected in 58% of investigated cases (41), suggesting that both are not part of a single growth regulatory pathway in which inactivation of either member causes the same effect. A tendency for more frequent combined DAL-1 and merlin loss was observed in anaplastic meningiomas (70%) compared to atypical (60%) and benign (50%) ones, suggesting that although both alterations are considered early changes, simultaneous loss of both proteins may provide a selective growth advantage (41). Cytogenetic and CGH analyses have failed to recognize 18p11.3 as a region of frequent chromosomal losses. Microsatellite analysis revealed that deletions in this region are centered around the DAL-1 gene locus and are too small to be detected by karyotyping or CGH (41). In contrast, CGH analysis frequently identified losses of genetic material from the long arm of chromosome 18. These losses were associated with increasing histological grade and therefore appear to be associated with meningioma progression (Fig. 1). Losses on 18q were detected in 67% of MIII and 40% of MII, but only 13% of MI (42, 43). Büschges et al investigated 37 meningiomas for mutation and expression of 4 tumor suppressor genes located at 18q21, namely MADH2, MADH4, APM-1, and DCC (43). However, only 1 missense mutation in the APM-1 gene was found in an atypical meningioma. No other mutations were identified and transcripts for all 4 genes were detectable in all tumors. These findings do not support a significant role for MADH2, MADH4, APM-1, and DCC alterations in meningioma pathogenesis. Based on CGH analysis, which identified a single meningioma in which loss was restricted to 18q22-qter (42), and on studies of non-cns tumors with losses on 18q, it was suggested that a putative meningioma progression-associated gene may be located distally to the 18q21 region (43). Chromosome 1 Deletions on 1p are the second most frequent chromosomal abnormality in meningiomas. The frequency of 1p deletions increases with tumor grade and occurs in 13% to 26% of MI, in 40% to 76% of MII, and 70% to 100% of MIII. Moreover, 1p deletions were found to be associated with tumor progression in several individual cases of recurrent meningiomas (42, 44 50). These findings suggest that loss of genomic information from 1p is relevant to meningioma progression rather than tumor formation (Fig. 1). Several genes on 1p were screened for alterations in meningiomas, including CDKN2C (p18 INK4c ), p73, RAD45L, and ALPL. In 3 different studies of more than 100 meningiomas only 1 mutation in the CDKN2C gene, which is located at 1p32, and 1 homozygous deletion were found (49, 51, 52). No loss of CDKN2C transcripts and no aberrant methylation were detected, suggesting that the CDKN2C gene is rarely altered in meningiomas (49, 51). The RAD54L gene also maps to 1p32. No RAD54L mutations were detected in a series of 29 meningiomas (53), however, a silent polymorphism at nucleotide 2,290 turned out to be associated with a higher frequency of meningioma development (54). The ALPL gene at 1p36.1-p34 encodes for alkaline phosphatase. Loss of alkaline phosphatase activity was reported to be strongly associated with loss of 1p in meningiomas, leading to the assumption that ALPL might have tumor suppressor function (55, 56). However, structural ALPL alterations have not been documented, and functional evidence for tumor suppressor properties of alkaline phosphatase is lacking. Another candidate meningioma suppressor gene is TP73, which maps to 1p36.32. However, only 1 mutation was found in more than 50 meningiomas analyzed (57, 58), which argues against a significant role of TP73 inactivation in meningioma progression. Expression of TP73 was found to increase with tumor grade, suggesting that TP73 might have a dominant oncogenic function rather than a classic tumor suppressor function (58). Taken together, the analyses of individual genes on 1p do not support a significant meningioma suppressor function of any of the genes investigated. Deletion mapping studies defined at least 2 different commonly deleted regions on 1p, suggesting that more than 1 tumor suppressor gene on 1p might be involved in meningioma progression. One of these regions was mapped to 1p32 (52, 54, 59, 60). However, sequence information that became available through the human genome project indicates that mapping data that were previously interpreted mainly on the basis of recombination events require revision in several instances. For example, Sulman et al had mapped the minimally deleted region at 1p32 between microsatellite markers D1S2713 distally and D1S2134 proximally (59). According to current databases, however, D1S2713 maps to 1p34.1, and D1S2134 maps to 1p33 (http:// genome.cse.ucsc.edu/cgi-bin/, and http://www.ncbi.nlm. nih.gov/mapview/maps.cgi). Similarly, Leraud et al had identified a commonly deleted region between D1S234

280 LAMSZUS distally and D1S2797 proximally, supposedly at 1p32, whereas current databases map D1S234 to 1p36.11 and D1S2797 to 1p33 (52). Even the order of microsatellite markers that were chosen in different studies is not always in agreement with current databases. Correct interpretation of previous mapping results would require an extensive re-analysis of the data on the basis of the available sequence information. Such an analysis is beyond the capacity and scope of this review. A second common region of deletion was mapped to 1pter-p34 distally to the D1S496 locus (46, 49). According to current databases, this microsatellite still maps to 1p34.3 and consequently there is less confusion for this region than for the more proximal region. A terminally located region of deletion at 1p36 was also reported by Bello et al (60). This group further identified up to 3 other commonly deleted regions, including a frequently affected region at 1p34-p32 and 2 less frequently deleted regions at 1p22 and 1p21.1-p13. Taken together, it appears that at least 2 common regions of deletion are present on chromosome arm 1p, namely one at 1p34-p32 and another at 1pter-p34. It remains to be shown whether re-analysis of previous studies based on genomic sequence information can clarify some of the confusing mapping data and can narrow down regions containing putative tumor suppressor genes. Naturally, the current confusion is not unique to meningiomas, since deletions on 1p are also common in oligodendrogliomas, neuroblastomas, and many epithelial cancers. Chromosome 14 Cytogenetically, loss of chromosome 14 represents the third most frequently detected abnormality in meningiomas after aberrations of chromosomes 22 and 1 (61). LOH and FISH analyses identified deletions on chromosome arm 14q in up to 31% of MI, 40% to 57% of MII, and 55% to 100% of MIII (42, 45, 47, 50, 62, 63). The strongly increased frequency of 14q deletions in tumors of higher grade suggests an involvement in meningioma progression (Fig. 1). A recent study demonstrated that deletions on this chromosome arm were an independent adverse prognostic parameter, which when combined with histological grade and patient age could identify patients at increased risk of relapse (64). A similar observation was made in another study in which losses of 14q in MI were predictive of tumor recurrence (50). No specific meningioma suppressor gene has yet been identified on chromosome 14. Deletion mapping studies have defined several commonly deleted regions on 14q; however, as already described for 1p, their interpretation requires caution since most studies were performed before completion of the Human Genome Project. Simon et al had mapped a critical region to 14q24.3-q32.33 between microsatellites D14S48 and D14S23 (45). Similarly, Menon et al had identified 14q24.3-q32.3 as the smallest commonly deleted region. Also Tse et al described a cluster region of deletion at 14q24.3-q31, but found another region at 14q32.1-q32.2 (63), whereas Leone et al suggested 2 different regions, one at 14q22-q24 and another at 14q32 (47). In contrast, a CGH study by Weber et al, 1 tumor defined a region of common deletion at 14q21 (42). It has to be concluded from these studies that no consistent commonly deleted region has yet been identified on 14q, and no particular tumor suppressor gene has evolved as prime candidate for a meningioma progression gene. Chromosome 10 Deletions on chromosome 10 have received almost as much attention in meningiomas as alterations on 14q. Rempel at al initially discovered an association between allelic losses on chromosome 10 with meningioma progression (65). Most subsequent LOH or CGH analyses detected deletions preferably on the long arm of chromosome 10. In several large studies, frequencies of 10q deletions were in the order of 5% to 12% for MI, 29% to 40% for MII, and 40% to 58% for MIII (42, 45, 66, 67). These percentages were even higher in 2 recent studies by Mihaila et al in which 11 different microsatellite loci on chromosome 10 in 208 meningiomas were analyzed (68, 69). Correlative analyses revealed an unfavorable prognostic significance for LOH at D10S179 (1p14) or D10S169 (1q26.3), which predicted higher tumor grade, and of D10S209 (10q26.12) and D10S169 (10q26.3), which may predict shorter survival and/or shorter time to recurrence, respectively (68). No specific tumor suppressor gene on chromosome 10 has yet been shown to be inactivated in a major fraction of meningiomas. The PTEN gene at 10q23.3 was analyzed in a large number of samples, however, mutations were only detected in 2 different MIII and no homozygous deletions were found (66, 70, 71). The DMBT1 gene, which maps to 10q26.11-q26.12, has also been investigated, but no homozygous deletions were found in atypical or malignant meningiomas (67). Mapping studies defined several different commonly deleted regions on chromosome 10. Simon et al had initially mapped such a region to 10q24-qter (45). However, according to current databases, the order and placement of microsatellite loci are slightly different, so that a partial deletion in one of the analyzed tumors would now place the minimally deleted region distally to a marker at 10q25.3. Peters et al observed LOH most often at microsatellite loci D10S676 (10q22.1) and D10S677 (10q23.33) (66). By CGH analysis, Weber et al obtained evidence for commonly deleted regions on both chromosome arms, namely one at 10p15 and one at 10q25- qter (42). The latest study by Mihaila et al defined 1

MENINGIOMAS 281 region on 10p between 10pter and D10S89 (p12.1), and 3 regions on 10q, namely between D10S109 (10q22.3) and D10S215 (10q23.31), between D10S187 (10q25.3) and D10S209 (10q26.12), and between D10S169 (10q26.3) and 10qter. Taken together, these studies indicate that the pattern of allelic losses on chromosome 10 is so complex that no single consistent minimal region of deletion could yet be identified. Chromosome 9 Losses of genetic material on chromosome 9 were frequently found in malignant meningiomas, but only rarely in benign or atypical ones (42). The short arm of chromosome 9 has attracted particular attention, as it contains the tumor suppressor genes CDKN2A (p16 INK4a /MTS1), p14 ARF, and CDKN2B (p15 INK4b /MTS2) at 9p21, which are inactivated at a high frequency in a variety of human tumors. The p16 and p15 proteins regulate cell cycle progression at the G 1 /S-phase checkpoint by inhibiting cyclin-cdk complexes, which are involved in the transcriptional control and phosphorylation of prb. The p14 ARF protein blocks Mdm2-mediated degradation of p53. Losses on 9p, as determined by combined CGH and microsatellite analysis, were found in 38% of MIII, 18% of MII, and 5% of MI (49). By FISH analysis the frequencies of deletion at 9p21 or monosomy 9 were 2- fold to 3-fold higher, most likely because FISH analysis on tumor sections also detects alterations that are only focal (72). Boström et al found homozygous deletions of CDKN2A, p14 ARF, and CDKN2B in 46% of MIII, and 3% of MII but never in MI (49). Two of 13 MIII carried somatic point mutations in CDKN2A and p14 ARF. In addition, 5 tumors without homozygous loss or mutation lacked detectable transcripts for at least 1 of the 3 genes (5% of MI, 9% of MII, and 8% of MIII). Thus, the majority of malignant meningiomas displayed alterations of CDKN2A, p14 ARF, and CDKN2B, whereas these were infrequent in benign meningiomas. Similarly, in an expression analysis by Simon et al, only 26% of MI and MII, but 57% of MIII lacked both p16 and p15 protein expression. In addition, 36% of non-malignant but 71% of the anaplastic meningiomas lacked p14 protein (73). These studies show that both the prb and p53 pathways are disrupted in the majority of MIII, and that inactivation of cell cycle regulation at the G1/S-phase checkpoint may be critical to the malignant progression of meningiomas. Interestingly, a recent study demonstrated a prognostic relevance for CDKN2A alterations, showing that among patients with anaplastic meningiomas, those with CDKN2A deletions had a significantly shorter survival (72). Chromosome 17 The tumor suppressor gene TP53, which is located on the short arm of chromosome 17, is one of the most frequently mutated genes in human cancer in general and in astrocytomas in particular. However, TP53 mutations are rare in meningiomas. Several relatively large studies reported either no or only single meningiomas with sporadic TP53 mutations (49, 71, 74, 75). Mutation of TP53 frequently results in increased stability of the p53 protein, which, in contrast to wild-type p53, then becomes detectable by immunohistochemistry. Other stabilization mechanisms may also lead to p53 accumulation and thus detectability. Immunohistochemical analyses of p53 in meningiomas have yielded contradictory results, and there seems to be a substantial degree of overlap between different malignancy grades (76). In essence, most (but not all) studies demonstrated an association between p53 accumulation and malignancy grade (75 78). The biological significance of this p53 accumulation in meningiomas is not clear. More recently, the long arm of chromosome 17 has also attracted interest since CGH analysis demonstrated high-level amplification on 17q in 42% of anaplastic meningiomas but in almost none of the non-malignant meningiomas (42). A subsequent study identified copy number increases at 17q microsatellite markers in 61% of MIII in contrast to only 21% in MII and 14% MI (79). However, only 2 MIII in this series displayed high-level amplification defined as a 5-fold increase in the copy number of 1 allele. The amplification patterns defined several common regions of increased allele copy number, one of which encompassed the putative proto-oncogene PS6K at 17q23. However, in contrast to another recent study by Cai et al, which identified PS6K amplification in 3/22 anaplastic meningiomas (80), only low level copy number increases were detected in 13/44 tumors (i.e. 4/ 19 MII and 9/18 MIII), despite high-level amplification of adjacent loci in 2 MIII. This discrepancy could have technical reasons, because Cai et al used FISH analysis, which recognizes also only focal areas with high-level PS6K amplification, whereas focal high-level amplifications may have appeared as low-level amplification in the real-time PCR analysis performed by Büschges et al (79). Taken together, these studies suggest that PS6K may not be the major target gene for high-level amplification detected by microsatellite analyses in malignant meningiomas, but that its amplification in tumor cell subpopulations may still be important for progression. Mapping results define at least a bipartite common region of amplification at 17q22-q23, between markers D17S790 and D17S1607 as well as between D17S1160 and PS6K, suggesting the possibility of more than one relevant protooncogene (79). Alterations on Other Chromosomes Numerous other chromosomal alterations have also been identified in meningiomas, albeit less consistently than those described in the previous paragraphs. Relatively frequent among them are losses on 3p, 6q, X, and

282 LAMSZUS Y, as well as gains on 1q, 9q, 12q, 15q, and 20q (42, 81 83). Very few studies have searched for specific alterations on these chromosome arms. For example, some studies showed that the CDK4 and MDM2 genes, which are located on chromosome arm 12q, are rarely amplified in meningiomas (42, 49, 84, 85). Similarly, no other specific gene alterations on these less commonly altered chromosomes could be identified in a significant number of cases. Radiation-Induced Meningiomas Ionizing radiation to the skull is a well-established risk factor for meningioma development. Most of the knowledge about this association was obtained from immigrants to Israel who had been treated with low-dose cranial irradiation for tinea capitis between 1948 and 1960. Usually, patients who were treated during childhood developed meningiomas after a latency period of 20 to 40 years. Radiation-induced meningiomas (RIM) are often clinically and histologically more aggressive (corresponding to MII or MIII), are often multiple, and have a higher proliferative activity than their sporadic counterparts (2). A few studies addressed the genetic mechanisms involved in RIM development. Mutation analyses of altogether more than 30 RIM showed that NF2 mutations are relatively rare and occur in less than 25% of RIM compared to more than 50% in sporadic control cases (71, 86). Likewise, allelic losses on 22q were only detected in 2/7 RIM (86), and a recent CGH study found monosomy for chromosome 22 in only 1/5 cases (87). Thus, NF2 alterations appear to play a less important role in the pathogenesis of RIM than in sporadic meningiomas. Several other genes, including some that are prone to developing radiation-associated mutations, were also analyzed in RIM. Mutations in the TP53 and PTEN genes were confined to single cases, and no mutations were observed in the HRAS, KRAS and NRAS genes (71). Allelic losses on 1p were detected at a slightly higher frequency in RIM than in sporadic meningiomas and CGH analysis identified losses on 7p, which are uncommon in sporadic meningiomas, in 67% of RIM (87). Therefore, putative tumor suppressor genes on 1p and 7p could be relevant to the development of RIM, although the available series are too small to draw reliable conclusions. Telomerase Telomeres consist of stretches of repetitive DNA sequences that maintain chromosomal stability. Telomeric DNA is progressively shortened with each mitosis. When telomeres reach a critical length, cells ultimately become senescent. Moreover, shortened telomeres can initiate aberrant fusion or recombination of chromosome ends and thus contribute to cancer development. Telomerase is a reverse transcriptase that stabilizes telomere length and is necessary for unlimited cell proliferation. In contrast to germ-line cells and most embryonic cells, telomerase is not active in most normal adult tissues. However, it is frequently reactivated in cancer. Telomerase activity has been detected in only 3% to 21% of benign meningiomas, but in 58% to 92% of MII and 100% of MIII (88 90). Langford et al described that telomerase activity was strongly associated with poor outcome in benign meningiomas, suggesting that it may provide a prognostic marker (88). Two essential components were identified in human telomerase: an RNA subunit termed htr (telomerase RNA) that contains a template sequence for telomeric repeat synthesis, and a reverse transcriptase subunit htert (telomerase reverse transcriptase). Generally, expression of htert correlates best with telomerase activity, whereas htr can be present even when enzyme activity is repressed. Simon et al showed that the detection rate for htert expression in meningiomas was even higher than that for telomerase activity and that all telomerase-positive tumors also expressed htert, but not vice versa (89). In addition, some tumors that subsequently recurred expressed htert but lacked detectable telomerase activity. These findings suggest that htert might even be a more sensitive marker for an aggressive clinical course than telomerase activity. BIOLOGY Angiogenesis, Edema, and Growth Factors Meningiomas derive their blood supply predominantly from meningeal vessels originating in the external carotid circulation. Additional supply from cerebral-pial vessels is present in approximately 60% of patients (91). Meningiomas are of variable vascularity, ranging from sparsely vascularized to highly vascular angiomatous meningiomas. They further display variable degrees of peritumoral brain edema, ranging from absent to life-threatening conditions. In contrast to gliomas, there is no obvious association between edema and histological grade in meningiomas. Vascular endothelial growth factor-a (VEGF-A), which has also been termed vascular permeability factor, is considered a key regulator of angiogenesis and edema formation. The VEGF-A mrna is expressed by meningioma cells (92, 93). Several studies demonstrated that VEGF-A levels in meningiomas are associated with the extent of peritumoral edema (92 94). Moreover, meningiomas with striking VEGF-A expression usually appeared to receive some blood supply through cerebralpial arteries, and both VEGF-A expression as well as cerebral-pial blood supply were associated with the extent of brain edema (94). Two small studies suggested that VEGF-A mrna expression may correlate with meningioma vascularity (93, 95). However, when determining VEGF-A protein levels

MENINGIOMAS 283 in a relatively large number of 69 meningiomas, no association with microvessel density could be confirmed (96). Moreover, and in contrast to gliomas, there was no association between microvessel density and histological grade. Nevertheless, VEGF-A levels were increased 10- fold in anaplastic meningiomas and 2-fold in atypical meningiomas compared to benign ones. VEGF-A contained in protein extracts of human meningiomas induced capillary-like tube formation and migration of endothelial cells in vitro, indicating biological activity in this context (96). Another recent study reported a correlation between VEGF-A protein expression and recurrence of benign meningiomas (97). Taken together, these findings suggest that VEGF-A is probably involved in vascular remodeling and angiogenesis in meningiomas which does, however, not result in a net increase in vessel number with increasing histological grade. Supposing that the more malignant meningiomas have a higher oxygen and metabolic demand, VEGF-A might facilitate adaptation by modulating vascular permeability. Several other growth factors, including VEGF-B, placenta growth factor, scatter factor/hepatocyte growth factor, and fibroblast growth factor-2 have also been analyzed in meningiomas. However, no clear correlation between either of these factors and meningioma angiogenesis or malignancy grade has yet been established. Moreover, expression of several other growth factors and their receptors, including epidermal growth factor, platelet-derived growth factor, and insulin-like growth factor and others has been analyzed in meningiomas. Their detailed discussion is beyond the scope of this article; for reviews see Black et al (98) and Sanson and Cornu (99). Hormones Meningiomas are more than 2-fold more frequent in woman than in men (2, 99). Accelerated meningioma growth has been observed during pregnancy and during the luteal phase of the menstrual cycle. Moreover, meningiomas seem to arise at a slightly increased rate in breast cancer patients. Taken together, these observations suggest an etiological role for sex hormones in the growth of these tumors. Most hormone receptor studies in meningiomas were performed in the 1990s using immunohistochemistry. These studies showed that estrogen receptors are only expressed in approximately 10% of meningiomas and only at very low levels (reviewed in Black et al [98], McCutcheon [100], and Sanson and Cornu [99]). In contrast, progesterone and androgen receptors are present in approximately two thirds of meningiomas. Both are more frequently expressed in women than in men (101 103). Progesterone receptors have attracted the greatest interest. Their expression is inversely associated with histological grade, and a study on 70 meningioma patients showed that the presence of progesterone receptors was a favorable prognostic factor (103). Meningioma cell proliferation was found to be inhibited by the progesterone receptor antagonist mifepristone (RU486) in vitro, suggesting a treatment modality. However, several small clinical trials that were performed reported only marginal responses (reviewed in Sanson and Cornu [99] and Ragel and Jensen [104]), and some prospective trials are still awaiting completion. Anti-androgens also have antiproliferative effects on meningioma cells in vitro, but their clinical usefulness has not yet been investigated. Meningiomas also express non-steroid hormone receptors, including growth hormone, somatostatin and dopamine receptors. Several in vitro or experimental in vivo studies demonstrated antiproliferative effects of the growth hormone receptor antagonist pegvisomant, the somatostatin agonist octreotide, and the dopamine D2 receptor agonist bromocriptine on meningiomas (99, 104). However, full-scale clinical trials with these substances have not been performed. OUTLOOK The completion of the Human Genome Project together with the development of increasingly more sophisticated molecular research techniques have set the stage for future high resolution genome-wide studies. The example of the DAL-1 tumor suppressor gene, where allelic deletions were too small to be picked up by conventional screening methods, underscores the need for more sensitive techniques. A great improvement in sensitivity is achieved by matrix-cgh (array-cgh), which has an approximately 100-fold higher resolution than conventional CGH analysis and is able to narrow chromosomal gains or losses to 75,000 bp segments. Another expanding field is that of high-density cdna arrays, which have so far been applied to meningiomas in only a few studies (105, 106). Gene expression profiling by microarrays revealed characteristic profiles for different meningioma grades and can thus provide prognostic information. Patterns of gene over- or underexpression may also lead to discovery of unknown tumor suppressor genes or oncogenes. However, for several genes that lack expression in a major fraction of meningiomas no structural gene alterations can be identified. Inactivation of these genes apparently does not follow the classic scheme described for tumor suppressor genes. Instead, epigenetic inactivation by aberrant methylation or suppression of transcription by other mechanisms most likely accounts for the absence of gene expression. Recently, array technology was expanded also to methylation analysis, so that high-throughput screening may soon generate comprehensive profiles of methylated genes. The investigation to what extent aberrant methylation can explain inactivation of genes relevant to meningioma formation or progression remains

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