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Genomic Amplification of Orthodenticle Homologue 2 in Medulloblastomas

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Genomic Amplification of Orthodenticle Homologue 2 in Medulloblastomas Priority Reports Genomic Amplification of Orthodenticle Homologue 2 in Medulloblastomas Kathy Boon,1 Charles G. Eberhart,2 and Gregory J. Riggins1 Departments of 1Neurosurgery and 2Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland Abstract activated in about 5% of medulloblastomas (4). Comparative To better understand the genetic basis of medulloblastoma genomic hybridization studies have identified large genomic development, we sought genomic amplifications and dele- regions of amplification and deletion, but typically only large tions in these tumors using digital karyotyping in combina- megabase regions of alteration can be found, making it difficult to tion with expression analysis. Five medulloblastoma genomes identify the target of gain or loss. However, higher-resolution were karyotyped by sequencing an average of 195,745 techniques for looking at DNA copy number have recently been genomic DNA tags for each analysis. Tags were tallied at developed, such as array-based comparative genomic hybridiza- unique positions and mapped to the human genome to tion. A recent study of 58 known oncogenes in medulloblastomas determine DNA copy numbers in high resolution along each showed genomic gains of several genes including PIK3CA, PGY1, chromosome. Genomic alterations normally associated with MET, ERBB2, and CSE1L (5). Another high-resolution method medulloblastomas, including MYC amplification and isochro- called digital karyotyping was recently developed based on serial analysis of gene expression (SAGE) technology (6), and employed to mosome 17q, were easily detected. Surprisingly, analysis of only five genomes revealed novel amplicons on chromosome implicate new genes in colon cancer (7, 8). Digital karyotyping 14q, one of which contained the orthodenticle homologue 2 works by counting 21 bp sequence tags generated from specific (OTX2) homeobox gene. DNA copy number analysis showed locations in the genome; normally the 21 bp adjacent to the first that OTX2 had undergone genomic amplification in 2 of 11 NlaIII site next to a SacI restriction enzyme site. Approximately medulloblastoma cell lines and 8 of 42 primary tumors. The 200,000 tags are counted by high-throughput sequencing and the three genes and a predicted open reading frame flanking tags are mapped to the genome and counted to identify OTX2 in the 14q amplicon were not amplified in at least one chromosomal changes such as amplifications and deletions. of the other nine amplicons, implicating OTX2 as the gene In this study, digital karyotyping was used to determine genome- target conferring a selective advantage. The degree of OTX2 wide DNA copy number for five medulloblastomas. A novel amplification ranged from 8 copies to over 50 copies of the amplification located on 14q22.3 was validated by quantitative real- gene. OTX2 transcript was highly and specifically expressed in time PCR (Q-OCR) in 2 of 11 (18%) medulloblastoma cell lines and medulloblastoma or developing cells. Serial analysis of gene in 8 of 42 (19%) primary tumors. The amplicon included the gene expression of 240 different human tumors or normal tissues orthodenticle homologue 2 (OTX2), a homeobox gene. A combina- revealed that 96% of all 783 OTX2 transcripts sequenced were tion of gene expression analysis and genomic mapping was used to in medulloblastomas or embryonic stem cells. OTX2 functions implicate only this gene. OTX2 plays an important role in to specify the fate of neuroectoderm in various regions of the specification and regionalization of the forebrain and midbrain in developing brain. This developmental role is consistent with early embryogenesis (9), suggesting its possible functional role the evidence suggesting that OTX2 is a medulloblastoma in medulloblastoma development. oncogene. (Cancer Res 2005; 65(3): 703-7) Materials and Methods Introduction Primary Tumors and Cell Lines. Medulloblastoma tissue was snap frozen in liquid nitrogen. A neuropathologist gave an unambiguous Medulloblastomas are a frequently studied brain cancer occur- diagnosis of medulloblastoma for all cases, and confirmed that the sample ring in about 1 in 200,000 children annually. These tumors are a margins were tumor cells and not normal tissue. Thirty samples from Johns high-grade embryonal tumor of the cerebellum, with a small round Hopkins Neuropathology, eight from the National Cancer Institute blue cell appearance similar to other tumors arising from primitive Cooperative Human Tissue network, and four from the Duke Brain Tumor neuroectoderm (1). Bank were used in these Institutional Review Board-approved studies. The The molecular basis of medulloblastoma formation is beginning following medulloblastoma cell lines were used in this study: D283 Med (10), to be understood. For example, the sonic hedgehog and the Wnt D341 Med, D425 Med, D556 Med, MCD1, UW228-2, D721 Med, D487 Med, pathway can be activated in a percentage of medulloblastomas by D581 Med, and MHH-Med-1 (11). mutation of critical pathway regulators (2, 3). Larger chromosomal Digital Karyotyping. Genomic DNA was isolated using a DNeasy kit alterations, most frequently isochromosome 17q with loss of a 17p, (Qiagen, Valencia, CA) according to the instructions of the manufacturer have been documented. Genomic amplification via double-minute from five medulloblastoma cell lines: D487 Med, D556 Med, D721 Med, D283 Med, and MHH-Med-1. For each library 1 Ag of genomic DNA was digested chromosomes is a mechanism where the MYC oncogene is with mapping enzyme SacI ligated to biotinylated SacI linkers (Integrated DNA Technologies, Coralville, IA) and digested with the fragmenting enzyme NlaIII. DNA fragments containing the biotinylated linkers were isolated using streptavidin-coated magnetic beads (Dynal Biotech, Request for reprints: Gregory J. Riggins, Department of Neurosurgery, 5th Floor, Brown Deer, WI) and ligated to linkers including recognition sites for Mason F. Lord Building, Center Tower, 5200 Eastern Avenue, Baltimore, MD 21224. Phone: 410-550-9686; Fax: 410-550-9689; E-mail: [email protected]. MmeI. The 21 bp sequence tags were released by digestion with MmeIas I2005 American Association for Cancer Research. has been described for Long SAGE (6). A detailed protocol can be obtained www.aacrjournals.org 703 Cancer Res 2005; 65: (3). February 1, 2005 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2005 American Association for Cancer Research. Cancer Research at http://www.digitalkaryotyping.org (7). The isolated tags are self-ligated, An average of 104,030 filtered tags per library (Table 1) was aligned PCR amplified concatenated, cloned in pZero (Invitrogen, Carlsbad, CA), across each chromosome. Tag densities were analyzed in windows and sequenced. The SAGE 2000 software package enables the extraction of ranging from 50 to 1,000 virtual tags (7). These densities were the genomic tags from the sequence files. The virtual genomic tags were calculated for each window by summing the experimental tags extracted from the human genome sequence (UCSC Genome Bioinfor- observed and dividing the sum by the average tag count for all the matics, July 2003 assembly, http://genome.ucsc.edu/) and downloaded from http://www.digitalkaryotyping.org. DNA from plasmid inserts containing same-sized windows across the genome. serial genomic tags were purified and sequenced at Agencourt Bioscience The MYC gene was used as an internal positive control. More Corporation (Beverly, MA) as part of the Cancer Genome Anatomy Project. than 25 copies at the genomic DNA level were found for D487 and Quantitative Real-Time PCR. Copy number changes between normal D556 Med and between 2 and 6 copies for D721 and D283 Med. human DNA and medulloblastoma cell lines or primary tumors were Digital karyotyping accurately predicted the published genomic determined by Q-PCR on an iCycler apparatus (Bio-Rad, Hercules, CA). The copy numbers for all five cell lines previously determined by repetitive element Line-1, which has an equivalent number in cancer and fluorescent in situ hybridization (13). MYC diploid genome copy normal genomes, was used for the normalization of DNA content. numbers for all five cell lines were also corroborated by Q-PCR Calculations and PCR conditions were all done as previously described (Table 1). Further analysis of the digital karyotypes of D556 Med, (7, 8). All PCRs were done in triplicates and threshold cycle numbers were D721 Med, D283 Med, and MHH-Med-1 did not reveal genomic averaged. PCR primers were designed using Primer 3 http://www.genome. alterations other than those already known (14, 15). Analysis of wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and are available upon request. For transcript analysis total RNA was extracted from 11 medullo- chromosome 17 provided a second internal control. D487 Med had blastoma cell lines using the RNAgents total RNA isolation system a digital karyotype that produced an average of three copies of 17q (Promega, Madison, WI). Synthesis of cDNA and quantitative PCR were and one copy of 17p. This karyotype was consistent with one done as previously described (12). isochromosome 17q and one normal chromosome 17, as Immunoblotting. Cells from various medulloblastoma cell lines previously described for D487 Med (14) and observed in many were lysed in 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% NP40, medulloblastomas (1). 0.25% Na-deoxycholate, 1 mmol/L EDTA, and protease inhibitors (Protease Of most interest were two novel chromosome 14q amplifica- cocktail, Roche
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