Human Molecular Genetics, 2000, Vol. 9, No. 10 1495-1500
© 2000 Oxford University Press
Loss of DAL-1, a protein 4.1-related tumor suppressor, is an important early event in the pathogenesis of meningiomas
Departments of 1Neurology and 2Neuropathology, Washington University School of Medicine, Box 8111, 660 S. Euclid Avenue, St Louis, MO 63110, USA, Departments of 3Neurosurgery and 4Neuropathology, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202, USA and Departments of 5Anatomy and 6Pathology, Virginia Commonwealth University, PO Box 908709, Richmond, VA 23298, USA
Received 28 January 2000; Revised and Accepted 2 April 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Meningiomas are common nervous system tumors, whose molecular pathogenesis is poorly understood. To date, the most frequent genetic alteration detected in these tumors is loss of heterozygosity (LOH) on chromosome 22q. This finding led to the identification of the neurofibromatosis 2 (NF2) tumor suppressor gene on 22q12, which is inactivated in 40% of sporadic meningiomas. The NF2 gene product, merlin (or schwannomin), is a member of the protein 4.1 family of membrane-associated proteins, which also includes ezrin, radixin and moesin. Recently, we identified another protein 4.1 gene, DAL-1 (differentially expressed in adenocarcinoma of the lung) located on chromosome 18p11.3, which is lost in ~60% of non-small cell lung carcinomas, and exhibits growth-suppressing properties in lung cancer cell lines. Given the homology between DAL-1 and NF2 and the identification of significant LOH in the region of DAL-1 in lung, breast and brain tumors, we investigated the possibility that loss of expression of DAL-1 was important for meningioma development. In this report, we demonstrate DAL-1 loss in 60% of sporadic meningiomas using LOH, RT–PCR, western blot and immunohistochemistry analyses. Analogous to merlin, we show that DAL-1 loss is an early event in meningioma tumorigenesis, suggesting that these two protein 4.1 family members are critical growth regulators in the pathogenesis of meningiomas. Furthermore, our work supports the emerging notion that membrane-associated alterations are important in the early stages of neoplastic transformation and the study of such alterations may elucidate the mechanism of tumorigenesis shared by other tumor types.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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The genetic events that are important in the molecular pathogenesis and malignant progression of sporadic meningiomas are only partly characterized (1,2). One of the most common regions implicated in meningioma tumorigenesis is chromosome 22q, where the neurofibromatosis 2 (NF2) tumor suppressor gene resides (3,4). Individuals affected with NF2 develop various central nervous system tumors, including an increased frequency of meningiomas (5). Mutations in the NF2 gene are common not only in NF2-related meningiomas but also in sporadic meningiomas (6–10). Loss of NF2 protein (merlin or schwannomin) expression occurs in 30–70% of sporadic meningiomas, arguing that merlin is one of the critical growth regulators for meningeal cells relevant to the development of meningiomas (11,12).
Alignment of the predicted merlin 595 amino acid sequence demonstrated striking sequence similarity to a group of proteins related to the erythrocyte protein band 4.1 family (3,4). Other members of this family include talin as well as three closely related proteins, ezrin, radixin and moesin (ERM proteins) (13). Merlin shares the greatest sequence similarity with the ERM proteins that link the actin cytoskeleton to cell surface glycoproteins. In this regard, merlin contains three predicted domains, an N-terminal region (residues 1–302), an 
-helix region (residues 303–478) and a unique C-terminus (residues 479–595) that lacks the conventional actin binding sequence found in other ERM proteins.
Recently, we identified a novel member of the protein 4.1 family on chromosome 18p11.3 with growth-suppressor properties (14). This gene, named differentially expressed in adenocarcinoma of the lung (DAL-1), is frequently inactivated in primary non-small cell lung carcinoma (NSCLC) tumors. In addition, significant loss of heterozygosity (LOH) for the DAL-1 region at 18p11.3 in tumors of the lung, brain and breast has recently been reported (15). DAL-1 is 73% identical in its N-terminal domain to members of the protein 4.1 family and shares structural similarity to the Drosophila 4.1 homologue coracle (16). DAL-1 is normally expressed at high levels in brain with lower levels of expression in kidney, intestine and testes. Given the sequence similarity of DAL-1 to merlin and its high levels of expression in the brain, we tested the hypothesis that DAL-1 represents a second critical protein 4.1-related tumor suppressor relevant to nervous system tumor development by characterizing the loss of DAL-1 expression in schwannomas and meningiomas.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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DAL-1 loss is not observed in sporadic schwannomas
Initial experiments focused on sporadic schwannomas, as high levels of DAL-1 expression were detected in normal rat Schwann cells as well as rat schwannoma cell lines (not shown). Characterization of 20 sporadic schwannoma specimens by western analysis and RT–PCR using DAL-1-specific primer sets failed to detect any loss of DAL-1 expression (not shown). This is in striking contrast to merlin expression, which is absent in as many as 70–100% of all sporadic schwannomas (8,11). These results indicate that DAL-1, unlike merlin, may not function as a critical growth regulator for Schwann cells relevant to the development of schwannomas.
DAL-1 loss is a common feature of sporadic meningiomas
Previous studies have demonstrated NF2 inactivation and loss of merlin expression in 30–70% of sporadic meningiomas (8,11). We sought to determine whether DAL-1 expression was similarly reduced or absent in meningiomas by performing a detailed analysis at the DNA, RNA and protein levels in a series of sporadic meningiomas. Using a rabbit polyclonal DAL-1 antibody (3A1), DAL-1 protein was detected by immunohistochemistry (IHC) in non-neoplastic human leptomeningeal tissues (Fig. 1b and c), in blood vessel walls (Fig. 1) and in the LTAg2B leptomeningeal cell line (data not shown). In contrast to the results in sporadic schwannomas, four of nine sporadic meningiomas exhibited loss of DAL-1 expression using reverse transcribed RNA PCR and western blot analysis (Fig. 1a). Similarly, merlin expression was lost in four of these nine sporadic meningiomas by western blot. As the expression of DAL-1 in blood vessels might confound our analyses, these meningiomas were also examined by immunohistochemistry. Identical results were obtained by this second method, as shown in Figure 1. Moreover, immunohistochemical analysis of an independent series of meningiomas from the Mayo Clinic demonstrated that 16 of 26 tumors were deficient for DAL-1 expression (Table 1). Therefore, our results suggest that DAL-1 is inactivated as frequently in meningiomas as the NF2 gene, the only confirmed meningioma tumor suppressor identified thus far.
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To determine whether the absence of DAL-1 expression correlated with alterations at the DNA level, LOH analyses were performed on another independent series of sporadic meningiomas from Henry Ford Hospital using the D18S481 marker on chromosome 18p11.3. Similar to the results obtained by immunohistochemistry, we observed an overall LOH frequency of 71% (12 of 17 tumors). To address whether this LOH was specific to band 18p11.3, we measured allelic deletion using marker D18S452, located in the proximal region bordering 18p11.2–3. Eight tumors previously shown to have LOH at D18S481 were informative at D18S452. Of these eight, five were negative for LOH at D18S452 (data not shown), indicating that LOH selectively involves band 18p11.3 where DAL-1 resides.
We next correlated allelic deletion with loss of DAL-1 protein in 11 sporadic meningiomas where data on both were available. In six of 11 tumors, LOH correlated with loss of DAL-1 protein and in three of the 11 tumors, no LOH corresponded to the maintenance of DAL-1 protein expression (Fig. 2), supporting previous results at the RNA and protein level (Fig. 1). Of the remaining two discordant tumors, case HFH080 lacked DAL-1 expression but did not exhibit LOH while case HFH050 maintained normal DAL-1 protein expression in the presence of LOH at D18S481. A similar percentage of NSCLC tumors have shown such discordance (data not shown), suggesting that in these cases, DAL-1 protein might be translated but functionally impaired or lost by alternative mutational mechanisms.
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Although comprehensive mutational analysis of DAL-1 awaits further characterization of the genomic structure, sufficient DNA was available from seven tumors (four with 18p11.3 LOH and DAL-1 negative by IHC, one with 18p11.3 LOH and DAL-1 expression by IHC and two that were negative for DAL-1 expression by IHC for which 18p11.3 LOH data were unavailable) to perform SSCP analysis on the highly conserved N-terminal FERM domain shared by protein 4.1 family members. Fragments containing the NF2 FERM domain missense mutations E106G, K79E and L64P were used as positive controls for the SSCP analysis as DAL-1 shares 73% identity with merlin in this region. Individual PCRs representing all seven exons of the DAL-1 FERM domain were analyzed. No mutations were identified by SSCP analysis. Moreover, direct sequencing of these SSCP products confirmed the SSCP analysis and identified no mutations, even when LOH on 18p11.3 was present. Given that two-thirds of NF2 patient mutations occur within the FERM domain, our results suggest that DAL-1 is unlikely to be altered by the mutational mechanisms operative in NF2.
Several possibilities might account for these results. First, mutations may exist in the incompletely characterized C-terminal region of DAL-1. Second, small alterations may be masked by contamination with normal cells when whole tissue extracts are used. Third, DAL-1 may be inactivated by other mechanisms. The absence of point mutations in primary tumors, despite the presence of allelic deletion, is a hallmark of the 9p21 tumor suppressor, p16 (17). This led to the discovery of homozygous deletions and promoter methylation as common and stable allele-specific mechanisms for tumor suppressor gene inactivation (18,19). Whereas LOH would not be expected in the former mechanism, hypermethylation remains a viable possibility. Although the DAL-1 promoter has yet to be characterized, 400 bp of sequence 5' to the DAL-1 translational start site is >60% GC rich, suggesting the presence of a CpG island and the potential for regulation by methylation. Additional mutational analyses in meningiomas and other tumors should determine the mechanism(s) by which DAL-1 protein expression is lost.
DAL-1 loss is an early event in meningioma tumorigenesis
To determine whether loss of DAL-1 expression correlated with meningioma grade, we analyzed by immunohistochemistry 26 sporadic meningiomas that were classified according to the Mayo Clinic and newly adopted World Health Organization grading criteria (20,21). These included 11 benign meningiomas without recurrence after 10 years of follow-up, five benign meningiomas with recurrence despite gross total resection, five atypical meningiomas and five anaplastic meningiomas. As shown in Table 1, loss of DAL-1 expression is likely to be an early event given that it was identified in 56–80% of all meningiomas, regardless of tumor grade. The increased loss of DAL-1 expression in the atypical meningiomas is intriguing given that these tumors are characterized predominantly by increased proliferation. However, this series of tumors is too small to reach statistical significance. We are presently expanding our analysis to include 200 sporadic meningiomas to better determine whether DAL-1 expression in meningiomas correlates with specific clinicopathologic features, such as recurrence, proliferative index and brain invasion.
Protein 4.1 tumor suppressors and meningioma pathogenesis
The observation that two protein 4.1 family molecules, NF2 and DAL-1, are implicated in the molecular pathogenesis of meningiomas suggests an important growth regulatory role for these cytoskeletal linkers in meningeal cells. Although our study is small, the overlapping expression patterns for DAL-1 and merlin in sporadic meningiomas do not suggest a single growth regulatory pathway, with inactivation of either member leading to the same effect.
Merlin is expressed in association with F-actin in the ruffling membranes of meningioma cells (22) whereas DAL-1 is localized to cell–cell contact points in areas rich in catenin and cadherins (14). These localizations suggest that both proteins may be critical sensors of cell–cell contact and mediate contact inhibition growth arrest by signaling through actin cytoskeletal-associated proteins. Previous work from our laboratory has demonstrated weak binding of merlin to actin through a unique N-terminal domain also present in other protein 4.1 molecules (23). This actin cytoskeletal localization has functional consequences in that merlin-deficient schwannoma cells display abnormal actin cytoskeletal organization (24). In addition, overexpression of wild-type, but not mutant, merlin molecules in rat schwannoma cells in vitro impairs cell attachment, spreading and motility (25). Studies are presently underway to determine whether DAL-1 functions in an analogous fashion.
The presence of two protein 4.1 tumor suppressor genes relevant to meningioma development supports the emerging concept that membrane-associated alterations are important events in the early stages of tumor formation. Future experiments to address the functional similarities and differences between DAL-1 and merlin in leptomeningeal and meningioma cells will likely elucidate the mechanism(s) that underlies the function of these important protein 4.1 tumor suppressor genes and define novel membrane- or cytoskeletal-associated pathways leading to tumorigenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Human tissues
Tumor specimens (sporadic schwannomas and meningiomas) were obtained from the Henry Ford Hospital, the Mayo Clinic and Washington University Tumor Repositories and used in accordance with approved Human Studies Protocols for each institution. None of these tumors were derived from individuals with clinical or radiographic evidence of NF2.
The LTAg2B human leptomeningeal cell line was generously provided by Dr Donna George (The University of Pennsylvania, PA) and maintained according to established protocols in her laboratory (26).
Western blot
Selected fresh frozen tumor specimens were homogenized in RIPA buffer containing protease inhibitors and the protein concentration determined by the BCA method (Pierce Chemical Co. Rockford, IL). One hundred micrograms of total protein was separated by 8% SDS–PAGE as described previously (11). Proteins were transferred onto Immobilon membranes for western blotting with affinity-purified DAL-1 (3A1) (14) and merlin (WA30) (11) rabbit polyclonal antibodies at 1:2000 and 1:1000 dilutions, respectively. Western blots were developed using horseradish peroxidase-conjugated secondary antibodies and ECL chemiluminescence (Amersham, Piscataway, NJ).
RT–PCR
Reverse transcribed RNA PCR was performed according to established protocols (27). Briefly, 3 µg of total RNA extracted using the Trizol method (Gibco-BRL Life Sciences, Rockville, MD) was subjected to reverse transcription using RNase inhibitors and SuperScript II enzyme (Gibco BRL Life Sciences) for 60 min at 42°C. DAL-1 (500 bp fragment) was detected using the DAL-1 primer set, 4RR- 5'-CGAGGGAAAGACTGA-3' and 38R- 5'-GGCTGTCTCTTCCTGT-3'. A cyclophilin primer set was included to control for RNA quality and quantity (5'-ATGGTCAACCCCACC GTGTT-3' and 5'-CGTTGTAAGTCACCACCCT-3') (27). The annealing temperature and cycle number for the DAL-1 PCR were 59°C and 35 cycles, respectively. RT–PCR products were resolved by non-denaturing PAGE and visualized by ethidium bromide staining.
Immunohistochemistry
Immunohistochemistry was performed according to established protocols (28). Paraffin sections were deparaffinized by immersion in xylene and rehydrated in water. Endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide for 5 min followed by antigen retrieval with 0.4% pepsin in 0.01 N HCl for 30 min at 37°C. Sections were then blocked in 1% bovine serum albumin for 1 h at room temperature. DAL-1 primary antibody incubations were performed at a 1:500 dilution overnight at 4°C followed by Vectastain development with diaminobenzidine and counterstained with Gill hematoxylin. Sections were then dehydrated and mounted for visualization by light microscopy.
LOH analysis
LOH analysis was performed on laser microdissected tissue from representative sections of paraffin-embedded normal and meningioma tissue using the PixCell ARC-100 apparatus (Arcturus, Inc., Mountain View, CA). Amplification was performed on 10 ng of DNA as previously described (14) using a FAM fluorescent-dye-tagged forward primer. Products were resolved on a horizontal ultrathin electrophoretic gel using a high-throughput fluorescence-based DNA fragment analyzer (GTI-9600; Genesys Technologies, Inc., Sauk City, WI). Allelic ratios were calculated and expressed as a percentage of intensity loss for the tumor allele compared with the corresponding normal allele (D-value). LOH was considered present when the calculated loss ratio for a tumor allele was >40% (D > 0.40). LOH analyses were confirmed in several independent PCR experiments with identical results for each tumor.
PCR–SSCP FERM domain mutation analysis
DNA was isolated from frozen tumor tissue of seven meningioma samples (HFH036, HFH050, HFH108, HFH116, HFH092, 0932374 and 92355351) using the PureGene kit (Gentra Systems, Minneapolis, MN). Lymphocyte DNA from two unrelated individuals was used as normal controls.
Seven exon fragments representing >95% of the N-terminal FERM domain of DAL-1 were amplified using the following primer sets: a 174 bp FERM1 fragment (primers DAL28 5'-GTATTGACATGCTTTGCTATTCT-3' and DAL29 5'-GCCATCTTATTTTCGATTCGTTT-3', a 241 bp FERM2 fragment (primers DAL12A 5'-CCTGGGTGTTAATGCTTGGAGT-3' and DAL13A 5'-GGAGCATTTAAGAGAAGCGCAAC-3'), a 271 bp FERM3 fragment (primers DAL20 5'-CCTTCGTTGTGTTGTG-3' and DAL21 5'-ACACCTTTCTGCCTCT-3'), a 156 bp FERM4 fragment (primers DAL10 5'-GAAAAGCATCATTGT-3' and DAL11 5'-TTGAAAGTTTAGGGT-3'), a 240 bp FERM5 fragment (primers DAL14A 5'-TCTGTTGTTTCTTGCTTTTATTTTC-3' and DAL15A 5'-AATGCTTGCTTGCTATCAGG-3'), a 180 bp FERM6 fragment (primers DAL30 5'-TTGAACAATTTGAAAGCACC-3' and DAL31 5'-AAAAACCAAAATAATCTCTTCC-3') and a 265 bp FERM7 fragment (primers DAL16A 5'-GTCTTTATGCTGTTTTTCTTTATTC-3' and DAL17A 5'-TCACATTCTTTTTGGGGTAGG-3'). NF2 FERM domain fragments containing missense mutations (E106G, K79E and L64P) as well as the corresponding wild-type NF2 FERM domain fragment were used as positive controls for the chosen SSCP conditions. Primers for these control fragments were MUT-NF2-F 5'-TGAAGTGGAAAGGGAA-3' and MUT-NF2-R 5'-TCTGCTTCTTTACCTG-3'. PCRs were performed for 35 cycles in 25 µl volumes using 250–500 ng of DNA and platinum Taq DNA polymerase (Gibco-BRL Life Sciences). Annealing temperatures ranged from 50 to 71°C. Following amplification, 10 µl of PCR reaction was added to 2 x SSCP gel loading dye (95% formamide, 20 mM EDTA pH 8.0, 0.05% xylene cyanol and 0.05% bromphenol blue), heated to 95°C and iced prior to loading on a 7% (37.5:1) 20-cm polyacrylamide gel containing 5% glycerol. SSCP analysis was run in 0.5x TBE at 40 W for 3–4 h in the D Code Universal Mutation Detection System (Bio-Rad, Hercules, CA) at a constant 22°C. Following electrophoresis, gels were stained in 1 µg/ml ethidium bromide for 30 min, visualized on a UV transilluminator and photographed.
Direct sequencing was performed using the forward and reverse primers initially used for SSCP analysis. All seven exons contained within the DAL-1 FERM domain were analyzed in meningioma tumors that were positive for LOH and negative for DAL-1 by immunohistochemical analysis (HF70, HF192, HF361 and HF513). The amplified SSCP fragments (20–30 ng) were sequenced using 20 ng of primer and 1 µl of reaction mix from the Big Dye sequencing kit (Amersham). Forward and reverse primers were used to sequence each fragment in both directions. Reactions were analyzed on an ABI 373 sequencer.
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ACKNOWLEDGEMENTS |
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We thank Dr Alice Gardner and Ms Marissa Jaggars for technical support, Dr Ab Guha at the University of Toronto Tumor Bank and Dr Bernd Scheithauer at the Mayo Clinic for meningioma specimens as well as Oliver Bogler for critical reading of this manuscript. This work was supported by grants from the National Institutes of Health (NS35848 to D.H.G.), the National Cancer Institute (CA777300 to I.F.N.) and a Royal Thai Scholarship (to K.K.).
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 314 362 7149; Fax: +1 314 362 9462; Email: gutmannd@neuro.wustl.edu
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 C. Yi, J. H. McCarty, S. A. Troutman, M. S. Eckman, R. T. Bronson, and J. L. Kissil Loss of the Putative Tumor Suppressor Band 4.1B/Dal1 Gene Is Dispensable for Normal Development and Does Not Predispose to Cancer Mol. Cell. Biol., November 15, 2005; 25(22): 10052 - 10059. [Abstract] [Full Text] [PDF]
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E. A. Lusis, M. A. Watson, M. R. Chicoine, M. Lyman, P. Roerig, G. Reifenberger, D. H. Gutmann, and A. Perry Integrative Genomic Analysis Identifies NDRG2 as a Candidate Tumor Suppressor Gene Frequently Inactivated in Clinically Aggressive Meningioma Cancer Res., August 15, 2005; 65(16): 7121 - 7126. [Abstract] [Full Text] [PDF]
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 S. Kikuchi, D. Yamada, T. Fukami, M. Masuda, M. Sakurai-Yageta, Y. N. Williams, T. Maruyama, H. Asamura, Y. Matsuno, M. Onizuka, et al. Promoter Methylation of DAL-1/4.1B Predicts Poor Prognosis in Non-Small Cell Lung Cancer Clin. Cancer Res., April 15, 2005; 11(8): 2954 - 2961. [Abstract] [Full Text] [PDF]
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D G R Evans, C Watson, A King, A J Wallace, and M E Baser Multiple meningiomas: differential involvement of the NF2 gene in children and adults J. Med. Genet., January 1, 2005; 42(1): 45 - 48. [Abstract] [Full Text] [PDF]
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D. Mihaila, J. A. Gutierrez, M. L. Rosenblum, I. F. Newsham, O. Bogler, and S. A. Rempel Meningiomas: Analysis of Loss of Heterozygosity on Chromosome 10 in Tumor Progression and the Delineation of Four Regions of Chromosomal Deletion in Common with Other Cancers Clin. Cancer Res., October 1, 2003; 9(12): 4435 - 4442. [Abstract] [Full Text] [PDF]
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 M. A. Watson, D. H. Gutmann, K. Peterson, M. R. Chicoine, B. K. Kleinschmidt-DeMasters, H. G. Brown, and A. Perry Molecular Characterization of Human Meningiomas by Gene Expression Profiling Using High-Density Oligonucleotide Microarrays Am. J. Pathol., August 1, 2002; 161(2): 665 - 672. [Abstract] [Full Text] [PDF]
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