Neuro-Oncology Advance Access published November 15, 2012 Neuro-Oncology doi:10.1093/neuonc/nos269 NEURO-ONCOLOGY MicroRNA profiling in pediatric pilocytic astrocytoma reveals biologically relevant targets, including PBX3, NFIB, and METAP2 Cheng-Ying Ho, Eli Bar, Caterina Giannini, Luigi Marchionni, Matthias A. Karajannis, David Zagzag, David H. Gutmann, Charles G. Eberhart, and Fausto J. Rodriguez Division of Neuropathology, Department of Pathology, Johns Hopkins University, Baltimore, Maryland (C.-Y.H., E.B., C.G.E., F.J.R.); Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota (C.G.); Downloaded from Cancer Biology Program, the Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, Maryland (L.M.); Division of Pediatric Hematology/Oncology, Department of Pediatrics (M.A.K.); Division of Neuropathology, Department of Pathology, NYU Langone Medical Center, New York, New York (D.Z.); Department of Neurology, Washington University School of Medicine, St. Louis, Missouri (D.H.G.) http://neuro-oncology.oxfordjournals.org/ Pilocytic astrocytoma (PA) is a World HealthOrganization the mRNA and/or protein level in PA included PBX3, grade I glioma that occurs most commonly in children METAP2, and NFIB. A unique miRNA profile exists and young adults. Specific genetic alterations have in PA, compared with brain tissue. These miRNAs and been described in PA, but the pathogenesis remains their targets may play a role in the pathogenesis of PA. poorly understood. We studied microRNA (miRNA) al- terations in a large cohort of patients with PA. A total of Keywords: BRAF, glioma, microRNA, 43 PA, including 35 sporadic grade I PA, 4 neurofibro- neurofibromatosis, pilocytic astrocytoma. matosis-1 (NF1)–associated PA, and 4 PA with pilo- myxoid features, as well as 5 nonneoplastic brain by guest on November 16, 2012 ilocytic astrocytoma (PA) is a World Health controls were examined. BRAF fusion status was as- Organization (WHO) grade I neoplasm represent- sessed in most cases. RNA was examined using the ing the most frequent primary glioma among chil- Agilent Human miRNA Microarray V3 platform. P dren and young adults. Most PAs have an excellent Expression of miRNA subsets was validated using quan- outcome after gross total resection, particularly when titative real-time PCR (qRT-PCR) with Taqman probes. they arise in accessible anatomic locations, such as the Validation of predicted protein targets was performed cerebellum. However, a subset may behave in a more ag- on tissue microarrays with the use of immunohistochem- gressive fashion and clinically progress despite the use of istry. We identified a subset of miRNAs that were differ- conventional treatments. Histologic features associated entially expressed in pediatric PAs versus normal brain with a more aggressive course include the presence of tissue: 13 miRNAs were underexpressed, and 20 monomorphous pilomyxoid features (ie, pilomyxoid miRNAs were overexpressed in tumors. Differences variant)1 and anaplasia in the form of brisk mitotic were validated by qRT-PCR in a subset, with mean activity with or without necrosis.2 fold change in tumor versus brain of -17 (miR-124), Recent studies have highlighted novel genetic alter- -15 (miR-129), and 19.8 (miR-21). Searching for pre- ations associated with PA. Tandem duplications of the dicted protein targets in Targetscan, we identified a BRAF kinase domain, leading to a BRAF:KIAA1549 number of known and putative oncogenes that were pre- fusion, are present in most PAs.3–10 Asubsetoftumors dicted targets of miRNA sets relatively underexpressed have other, usually mutually exclusive alterations, includ- in PA. Predicted targets with increased expression at ing RAF1 rearrangements, a FAM131B-BRAF fusion me- diated by a small interstitial deletion, or small BRAF insertions.10 –12 In addition, PA is the most frequent Received March 22, 2012; accepted September 12, 2012. glioma in patients with neurofibromatosis type 1,13 result- Corresponding Author: Fausto J. Rodriguez, MD, Department of ing from germline mutations in the NF1 gene and homozy- Pathology, Division of Neuropathology, Johns Hopkins Hospital, gous inactivation in associated tumors, leading to RAS Sheikh Zayed Tower, Rm M2101, 1800 Orleans St, Baltimore, MD activation. All these genetic alterations lead to down- 14 21231 ([email protected]). stream activation of the MAPK signaling pathway. # The Author(s) 2012. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: [email protected]. Ho et al.: MicroRNA profiles of pilocytic astrocytoma In recent years, an increasing role for noncoding small WHO grade I PAs, 4 NF1-associated PAs, and 4 PAs RNA (ie, microRNA) has been uncovered in carcinogen- with pilomyxoid features. All patients (except for 2) esis. Mature microRNAs are small, single-stranded RNA were ≤18 years of age at the time of surgery (median, molecules that bind to regulatory sequences of key 10 years). Nonneoplastic tissue controls included fetal mRNAs, promoting their degradation and/or inhibiting cerebellum (n ¼ 1) and pediatric cerebellum (n ¼ 1) ob- translation. In the process of carcinogenesis, the upregu- tained by autopsy and cerebral cortex obtained during lation of particular microRNAs may affect tumor sup- seizure surgery (n ¼ 2) or biopsy for a nonspecific neuro- pressors, whereas downregulation may allow logic disorder with gliosis (n ¼ 1). Nine additional overexpression of oncoproteins, resulting in deregula- autopsy-derived cerebellar tissue samples were included tion of cell proliferation and survival. for quantitative real-time polymerase chain reaction Several microRNAs have been shown to be involved in (qRT-PCR) experiments. brain tumorigenesis, including miR-21, miR-7, miR-181a/ Anatomic locations for tumor samples included optic b, miR-221, and miR-222,15 –19 and seem to regulate onco- pathways (n ¼ 10) and supratentorial (n ¼ 11) and genic signaling pathways in diffuse gliomas, such as glio- infratentorial compartments (n ¼ 22). Most tumors blastoma.20,21 PTEN in particular is a key tumor were previously evaluated for BRAF alterations as part suppressor gene frequently inactivated in diffusely infiltrat- of a separate study and by PCR and sequencing as re- ing gliomas and may in fact be targeted, albeit not exclu- ported.7 Tumors were classified as aggressive if they pro- Downloaded from sively, by specific microRNAs, such as miR-21.22 Because gressed significantly within a year despite conventional microRNAs may have .1 target, microRNA profiling therapies or recurred after gross total resections. Five pa- may stratify biological and clinically relevant subgroups tients met these criteria. Patient and tumor data are sum- more accurately than conventional mRNA profiling.23 marized in Table 1. All studies were approved by the Low-grade pediatric gliomas, such as PAs in particular, Johns Hopkins, NYU, and Mayo Clinic Institutional are attractive for microRNA study because they lack Review Boards. http://neuro-oncology.oxfordjournals.org/ gross genomic alterations,24 suggesting that undiscovered epigenetic and subtle genetic changes may contribute to their pathogenesis. In addition, microRNA profiling may MicroRNA Profiling lead to further, biology-based refinements in classifying histopathologically ambiguous low-grade gliomas that Snap frozen fresh tumor tissue samples were used for the defy traditional classification schemes and may have prog- microRNA studies. Sample quality assessment and nostic or therapeutic significance. microarray analyses were performed at the Sidney Because of the low level of genetic instability observed Kimmel Comprehensive Cancer Center Microarray in PAs, we hypothesized that epigenetic or posttranscrip- Core Facility at the Johns Hopkins University tional regulation may play an important role in their (Baltimore, MD). In brief, total RNA was isolated pathogenesis, as described in other low-grade neuroecto- using miRNeasy Mini kits (Qiagen), followed by 25– 27 dermal tumors, such as schwannomas. Some studies quality checks of both total RNA and small RNA with by guest on November 16, 2012 have also highlighted a possible role for microRNAs in use of a 2100 Bioanalyzer and software that detects pediatric brain tumors,28,29 including medulloblastoma 28S and 18S ribosomal RNA ratio, total RNA and ependymoma.30 A small number of PAs have also Integrity Number, and small RNA and miRNA concen- been tested for microRNA levels, which may be differen- trations in the total RNA isolated. Only samples with tially expressed in this tumor type.29 Furthermore, over- adequate total and microRNA were used in the study. expression of specific microRNAs has been observed in Samples were hybridized to a Human miRNA papillary carcinoma of the thyroid, another tumor char- Microarray V3 kit (G4470C, Agilent Technologies) acterized by BRAF aberrations.31 Identification of key platform previously proven to produce reproducible microRNAs also provides a rationale for developing results as described elsewhere.33 This array contains inhibitory RNA strategies for therapeutic purposes in 866 human and 89 human viral microRNAs from patients with cancer.32 In the current study, we investigat- the Sanger database, version 12.0. (http://microrna. ed global microRNA expression in a large series of genet- sanger.ac.uk/sequences/). Each miRNA species is ically characterized PAs, including various pathologic printed 20 times with replicate