Identification of Gains on 1 q and Epidermal Growth Factor Receptor Overexpression as Independent PrognosticMarkers in Intracranial Ependymoma

Purpose: Pathogenesis of ependymomas is still poorly understood and molecular markers for risk-adapted patient stratification are not available. Our aim was to screen for novel genomic imbalances and prognostic markers in ependymal tumors. Experimental Design:We analyzed 68 sporadic tumors by matrix-based comparative genomic hybridizationusingDNAmicroarrays containing >6,400 genomicDNAfragments. Novel recurrent genomic gains were validated by fluorescence in situ hybridization using a tissue microarray consisting of 170 intracranial ependymomas. Candidate genes were also tested for mRNA expression by quantitative real-time PCR, and protein expressionwas determined by immunohistochemistry on the tissue microarray. Results: Chromosomal gain of 1q correlated with pediatric patients (P = 0.004), intracranial ependymomas (P =0.05), and tumors of grade III (P =0.002). Gainof1q21.1-32.1wasassociated with tumor recurrence in intracranial ependymomas (P < 0.001). Furthermore, gain of 1q25 as determined by fluorescence in situ hybridization represented an independent prognostic marker for either recurrence-free survival (P < 0.001) or overall survival (P = 0.003). Recurrent gains at 5p15.33 covering hTERT were validated by immunohistochemistry, and elevated protein levels correlated with adverse prognosis (P = 0.01). In addition to frequent gains and high-level amplification of epidermal growth factor receptor (EGFR) at 7p11.2, immunohistochemistry revealed protein overexpression to be correlated with poor prognosis (P = 0.002). EGFR protein status subdivides intracranial grade II ependymomas into two different risk groups (P = 0.03) as shown by multivariate analysis. Conclusions: Thus, the states of 1q25 and EGFR represent independent prognostic markers for intracranial ependymomas to identify patient subgroups with different risk profiles in further clinical investigations. Moreover, EGFR might serve as therapeutic target for more specific chemotherapy applications. Ependymomas originate from the ependymal monolayer of the cerebral ventricles or from the central canal of the spinal cord. These tumors represent the third most common brain tumors of childhood accounting for f10% of cases but may also occur in adults (1, 2). Intracranial tumors are predominant in pediatric patients (90%), whereas most adults show spinal manifestation (60%; refs. 3, 4). Spinal tumor localization is usually associated with a favorable prognosis after gross total resection and does not need to be treated with adjuvant therapy (5, 6). In contrast, intracranial ependymomas tend to local tumor progression, which is reflected by a 5-year overall survival of only f60% (7, 8). The influence of the recent histologic ependymoma classification (WHO 2000) on clinical outcome, and the relationship between ependymoma grade and specific chromosomal aberrations is controversially discussed (2, 4, 9–12). Further important aspects of intracranial tumors remain challenges that urgently need to be addressed, including tremendous clinical heterogeneity (13) and tumor recurrence, which is associated with poor survival (14). Little is known about the genetic mechanisms underlying ependymal neoplasms and no molecular marker of clinical relevance has been identified thus far. Cytogenetic studies by conventional comparative genomic hybridization (CGH) revealed numerous chromosomal aberrations in these heterogenous tumors, such as a 30% to 50% incidence of aberrations involving chromosome 22, including monosomy 22 and deletions of 22q (9, 12, 15). Neurofibromatosis type 2 is associated with spinal ependymomas in f5% of cases (16), Imaging, Diagnosis, Prognosis Authors’ Affiliations: Division of Molecular Genetics and Central Unit Biostatistics, German Cancer Research Center ; Department of Pediatric Oncology, University of Heidelberg, Heidelberg, Germany and Department of Neuropathology, Burdenko Neurosurgical Institute, Moscow, Russia Received10/28/05; revised12/9/05; accepted1/19/06. Grant support: Bundesministerium für Bildung und Forschung grants 01GRO417 and 01GS0460 (B. Radlwimmer and P. Lichter) and Fonds der Chemischen Industrie Kekulé fellowship (F. Mendrzyk). The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with18 U.S.C. Section1734 solely to indicate this fact. Requests for reprints: Peter Lichter, Division of Molecular Genetics (B060), German Cancer Research Center, Im Neuenheimer Feld 580 (TP III), 69120 Heidelberg, Germany. Phone: 49-6221-42-4619; Fax: 49-6221-42-4639; E-mail: m.macleod@dkfz.de. F2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-2363 www.aacrjournals. org Clin Cancer Res 2006;12(7) April 1, 2006 2070 Research. on January 6, 2018. © 2006 American Association for Cancer clincancerres.aacrjournals. org Downloaded from indicating a role of the NF2 tumor suppressor gene at 22q12.2 in these tumors (17, 18). RNA expression profiling using DNA microarrays identified differences in ependymoma subgroups and potential candidate genes on chromosome 22q (13, 19). About 40% of pediatric ependymomas are reported to display balanced profiles by conventional CGH in comparison with only 10% in adults (9, 10, 12). Therefore, it has been suggested that the development of ependymomas at younger age might often be independent of chromosomal instability. Because no genetic factors to predict patients’ outcome are available thus far, our aims were to identify novel genomic imbalances by means of high-resolution genomic profiling and to validate their prognostic potential in a large series of ependymal tumors. Accordingly, we did matrix-based CGH of 68 sporadic tumors using a DNA microarray consisting of 6,400 genomic fragments (6K array), which cover the human genome with an average resolution of 0.5 Mb (20–24). Selected candidate genes of frequently aberrant genomic regions were additionally tested for mRNA expression by quantitative real-time PCR (QRT-PCR) in 10 representative tumor samples. Confirmation of gained regions by fluorescence in situ hybridization (FISH) and validation of array-based data by immunohistochemistry were done using a tissue microarray consisting of 170 clinically well-characterized intracranial ependymomas. Potential markers were correlated with clinicopathologic data in univariate and multivariate analyses. Materials andMethods Tumor material and patient characteristics. All samples used in this study were randomly collected at the Department of Neuropathology, Burdenko Neurosurgical Institute (Moscow, Russia) between 1987 and 2003. All diagnoses were confirmed by histologic assessment of specimens obtained at surgery by at least two neuropathologists according to the criteria of the 2000 WHO classification (2). These tumors were classified by histology as myxopapillary (grade I), classic (grade II), or anaplastic (grade III) ependymomas, considering the criteria for nonmyxopapillary tumors presented by Merchant