Published OnlineFirst August 3, 2010; DOI: 10.1158/1078-0432.CCR-10-0891 Clinical Human Cancer Biology Cancer Research Genomic Profiling Reveals Alternative Genetic Pathways of Meningioma Malignant Progression Dependent on the Underlying NF2 Status Stéphane Goutagny1,2,4, Hong Wei Yang5, Jessica Zucman-Rossi1,3, Jennifer Chan6, Jonathan M. Dreyfuss7, Peter J. Park7, Peter M. Black5, Marco Giovannini1,8, Rona S. Carroll5, and Michel Kalamarides1,2,4 Abstract Purpose: Meningiomas are the most common central nervous system tumors in the population of age 35 and older. WHO defines three grades predictive of the risk of recurrence. Clinical data supporting histologic malignant progression of meningiomas are sparse and underlying molecular mechanisms are not clearly depicted. Experimental Design: We identified genetic alterations associated with histologic progression of 36 paired meningioma samples in 18 patients using 500K SNP genotyping arrays and NF2 gene sequencing. Results: The most frequent chromosome alterations observed in progressing meningioma samples are early alterations (i.e., present both in lower- and higher-grade samples of a single patient). In our series, NF2 gene inactivation was an early and frequent event in progressing meningioma samples (73%). Chro- mosome alterations acquired during progression from grade I to grade II meningioma were not recurrent. Progression to grade III was characterized by recurrent genomic alterations, the most frequent being CDKN2A/CDKN2B locus loss on 9p. Conclusion: Meningiomas displayed different patterns of genetic alterations during progression ac- cording to their NF2 status: NF2-mutated meningiomas showed higher chromosome instability during progression than NF2-nonmutated meningiomas, which had very few imbalanced chromosome seg- ments. This pattern of alterations could thus be used as markers in clinical practice to identify tumors prone to progress among grade I meningiomas. Clin Cancer Res; 16(16); 4155–64. ©2010 AACR. Meningiomas are the most common central nervous sys- suggesting that the NF2 gene might play a central role in tem tumors in the population of age 35 and older (1). They regulating leptomeningeal cell proliferation (2). Biallelic originate from the meningeal coverings of brain and spinal inactivation of the NF2 gene on chromosome 22 has been cord. The only well-established gene in meningioma gene- found in 30% to 70% of sporadic meningiomas, leading to sis is NF2 (Neurofibromatosis 2 OMIM 101000). Indivi- the loss of the NF2 gene product, merlin/schwannomin duals with neurofibromatosis type 2 (NF2) are at (3). In addition, NF2 inactivation is thought to be an early significantly elevated risk for developing meningiomas, event in sporadic meningioma pathogenesis and is ob- served as frequently in grade I tumors as in high-grade tu- mors (4). NF2 mouse modeling also supports a strong Authors' Affiliations: 1INSERM, U674; 2Université Paris 7-Denis Diderot, involvement of this gene in meningioma tumorigenesis. Institut Universitaire d'Hématologie; 3Université Paris 5-Descartes, Paris, Nf2 gene inactivation in meningeal cells using conditional France; 4AP-HP, Hôpital Beaujon, Service de Neurochirurgie, Clichy, France; Departments of 5Neurosurgery, 6Pathology, and 7Medicine, Nf2 knockout mice induces meningioma development (5). Brigham and Women's Hospital and Harvard Medical School, Boston, According to the 2000 WHO classification (6), about Massachusetts; and 8House Ear Institute, Los Angeles, California 80% of meningiomas are slow-growing grade I benign tu- Note: Supplementary data for this article are available at Clinical Cancer mors. Atypical grade II meningiomas constitute 15% to Research Online (http://clincancerres.aacrjournals.org/). 20% of meningiomas, and their incidence has been in- S. Goutagny and H.W. Yang are co-first authors. creasing since the WH0 2000 classification (7). One to Current address for J. Chan: Faculty of Medicine, University of Calgary, three percent of meningiomas are grade III tumors and be- Calgary, Alberta, Canada. have as true malignant neoplasms. Histologic progression Corresponding Author: Michel Kalamarides, Hôpital Beaujon, Neurochir- urgie, 100 Boulevard du General Leclerc, 92110 Clichy, France. Phone: of meningiomas has been recently depicted (8, 9): 17% to 33-1-4087-5164; Fax: 33-1-4739-6635; E-mail: michel.kalamarides@ 38% of grade II meningiomas derive from grade I tumors bjn.aphp.fr. and 54% to 70% of grade III meningiomas derive from doi: 10.1158/1078-0432.CCR-10-0891 grade I or II tumors. Several studies have been done to ©2010 American Association for Cancer Research. identify genes involved in meningioma progression using www.aacrjournals.org 4155 Downloaded from clincancerres.aacrjournals.org on October 1, 2021. © 2010 American Association for Cancer Research. Published OnlineFirst August 3, 2010; DOI: 10.1158/1078-0432.CCR-10-0891 Goutagny et al. instability during progression than NF2-nonmutated Translational Relevance meningiomas. Up to 25% of meningiomas show aggressive histo- Materials and Methods logic features and are characterized as grade II or III ac- cording to the WHO classification. A subset of these Patients and tumor samples tumors originate from grade I tumors that undergo A total of 18 progressing meningioma cases, corres- malignant progression. This study focuses on a unique ponding to 37 tumor samples, were selected (17 patients series of 36 paired meningioma samples (18 patients) with 2 samples and 1 patient with 3 samples). Clinical da- that showed tumor recurrence with histologic malig- ta are depicted in Table 1. Each sample had frozen mate- nant progression. High-density 500K SNP array geno- rial and paraffin-embedded blocks available to be typing allowed the fine cartography of genomic included in this study. All the patients were recruited in alterations by comparing samples and their subse- accordance with French law and consented under the In- quent progressing counterparts in the same patient. stitutional Review Board of Partners Healthcare. Patients The main finding of this study is that genomic altera- with multiple meningiomas or NF2 were excluded from tions associated to date with grade II or III meningio- the study. Two independent pathologic reviews by two mas, such as 1p, 6q, and 14q losses, are also observed neuropathologists were conducted on each case. A neuro- NF2 in grade I meningiomas with mutations that un- pathologist at the hospital where the surgery occurred re- dergo malignant progression. This chromosome insta- viewed each tumor specimen, and a neuropathologist at NF2 bility phenotype associated with status could be Brigham and Women's Hospital (J.C.) reviewed all paraf- used as a marker in clinical practice to identify grade I fin slides and confirmed the diagnosis, grade, and histo- meningiomas prone to progress. logic subtype (Supplementary Table S1). All tumors were graded according to the WHO 2000 grading scheme (6). Twenty 20-μm frozen sections were allocated for purifica- tion of genomic DNA with AllPrep DNA/RNA minikit (QIA- cDNA microarray and low-density comparative genomic GEN GmbH) following the manufacturer's protocol for each hybridization (10–14). A comparison of independent specimen. The tumor cell content was quantified by H&E cases from grade I, II, and III tumors identified homozy- staining of the frozen section. Only samples with a tumor gous CDKN2A/CDKN2B deletions in almost half of grade cell content >90% were retained for subsequent analysis. III meningiomas (15). In grade III meningiomas, 9p21 de- letions encompassing CDKN2A/CDKN2B are associated NF2 characterization with poorer survival (16). Very few studies including suc- The 17 NF2 exons were amplified by PCR and se- cessive, recurrent specimens from the same patient whose quenced using Big Dye terminator chemistry on an tumor had progressed to a more malignant histologic phe- ABI3110 (Applied Biosystems) following the manufac- notype have been conducted (17–20). They showed losses turer's recommendations. PCR protocols and primers se- on chromosome 22q in samples of all grades, associated quence are available on request. Sequences were with 1p, 9q, 10q, and 14q losses, sometimes solely ob- analyzed using Sequencher 4.7 software (Gene Codes). served in high-grade tumors. These studies were limited When no NF2 mutation was identified, DNA samples were by the small numbers of tumors involved (one to four further examined for large alterations using multiplex liga- progressing cases) and by the fact that analyses were con- tion-dependent probe amplification (MRC Holland) fol- ducted at the cytogenetic level. lowing the manufacturer's protocol. High-density SNP arrays are new powerful tools, en- abling the fine mapping of the tumor genome. Genomic Search for alterations in NF1 and CDH7 genes and transcriptional studies have been successfully con- Molecular investigation of NF1 gene was done at the ducted in a variety of cancers, such as lung cancer (21), cDNA level. Briefly, after reverse transcription of 1 μgof prostate cancer (22), and melanoma (23). In particular, total RNA, eight overlapping PCR products were se- high-density SNP arrays, such as 500K Single Nucleotide quenced (BDT Cycle Sequencing Ready Reaction Kit, Ap- Polymorphism array (500K SNP), enable detailed and ge- plied Biosystems) using a panel of 25 sequencing nome-wide identification of both loss of heterozygosity primers covering the entire 8,520-bp NF1 coding se- (LOH) events and copy number alterations in cancer