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[Cancer Research 65, 703-707, February 1, 2005]
© 2005 American Association for Cancer Research

Priority Reports

Genomic Amplification of Orthodenticle Homologue 2 in Medulloblastomas

Kathy Boon1, Charles G. Eberhart2 and Gregory J. Riggins1

Departments of 1 Neurosurgery and 2 Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland

Request for reprints: Gregory J. Riggins, Department of Neurosurgery, 5th Floor, Mason F. Lord Building, Center Tower, 5200 Eastern Avenue, Baltimore, MD 21224. Phone: 410-550-9686; Fax: 410-550-9689; E-mail: griggin1{at}jhmi.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To better understand the genetic basis of medulloblastoma development, we sought genomic amplifications and deletions in these tumors using digital karyotyping in combination with expression analysis. Five medulloblastoma genomes were karyotyped by sequencing an average of 195,745 genomic DNA tags for each analysis. Tags were tallied at unique positions and mapped to the human genome to determine DNA copy numbers in high resolution along each chromosome. Genomic alterations normally associated with medulloblastomas, including MYC amplification and isochromosome 17q, were easily detected. Surprisingly, analysis of only five genomes revealed novel amplicons on chromosome 14q, one of which contained the orthodenticle homologue 2 (OTX2) homeobox gene. DNA copy number analysis showed that OTX2 had undergone genomic amplification in 2 of 11 medulloblastoma cell lines and 8 of 42 primary tumors. The three genes and a predicted open reading frame flanking OTX2 in the 14q amplicon were not amplified in at least one of the other nine amplicons, implicating OTX2 as the gene target conferring a selective advantage. The degree of OTX2 amplification ranged from 8 copies to over 50 copies of the gene. OTX2 transcript was highly and specifically expressed in medulloblastoma or developing cells. Serial analysis of gene expression of 240 different human tumors or normal tissues revealed that 96% of all 783 OTX2 transcripts sequenced were in medulloblastomas or embryonic stem cells. OTX2 functions to specify the fate of neuroectoderm in various regions of the developing brain. This developmental role is consistent with the evidence suggesting that OTX2 is a medulloblastoma oncogene.

Key Words: Medulloblastoma • Genomic Amplification • Homeobox Gene • Digital Karyotyping


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Medulloblastomas are a frequently studied brain cancer occurring in about 1 in 200,000 children annually. These tumors are a high-grade embryonal tumor of the cerebellum, with a small round blue cell appearance similar to other tumors arising from primitive neuroectoderm (1).

The molecular basis of medulloblastoma formation is beginning to be understood. For example, the sonic hedgehog and the Wnt pathway can be activated in a percentage of medulloblastomas by mutation of critical pathway regulators (2, 3). Larger chromosomal alterations, most frequently isochromosome 17q with loss of a 17p, have been documented. Genomic amplification via double-minute chromosomes is a mechanism where the MYC oncogene is activated in about 5% of medulloblastomas (4). Comparative genomic hybridization studies have identified large genomic regions of amplification and deletion, but typically only large megabase regions of alteration can be found, making it difficult to identify the target of gain or loss. However, higher-resolution techniques for looking at DNA copy number have recently been developed, such as array-based comparative genomic hybridization. A recent study of 58 known oncogenes in medulloblastomas showed genomic gains of several genes including PIK3CA, PGY1, MET, ERBB2, and CSE1L (5). Another high-resolution method called digital karyotyping was recently developed based on serial analysis of gene expression (SAGE) technology (6), and employed to implicate new genes in colon cancer (7, 8). Digital karyotyping works by counting 21 bp sequence tags generated from specific locations in the genome; normally the 21 bp adjacent to the first NlaIII site next to a SacI restriction enzyme site. Approximately 200,000 tags are counted by high-throughput sequencing and the tags are mapped to the genome and counted to identify chromosomal changes such as amplifications and deletions.

In this study, digital karyotyping was used to determine genome-wide DNA copy number for five medulloblastomas. A novel amplification located on 14q22.3 was validated by quantitative real-time PCR (Q-OCR) in 2 of 11 (18%) medulloblastoma cell lines and in 8 of 42 (19%) primary tumors. The amplicon included the gene orthodenticle homologue 2 (OTX2), a homeobox gene. A combination of gene expression analysis and genomic mapping was used to implicate only this gene. OTX2 plays an important role in specification and regionalization of the forebrain and midbrain in early embryogenesis (9), suggesting its possible functional role in medulloblastoma development.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Primary Tumors and Cell Lines. Medulloblastoma tissue was snap frozen in liquid nitrogen. A neuropathologist gave an unambiguous diagnosis of medulloblastoma for all cases, and confirmed that the sample margins were tumor cells and not normal tissue. Thirty samples from Johns Hopkins Neuropathology, eight from the National Cancer Institute Cooperative Human Tissue network, and four from the Duke Brain Tumor Bank were used in these Institutional Review Board-approved studies. The following medulloblastoma cell lines were used in this study: D283 Med (10), D341 Med, D425 Med, D556 Med, MCD1, UW228-2, D721 Med, D487 Med, D581 Med, and MHH-Med-1 (11).

Digital Karyotyping. Genomic DNA was isolated using a DNeasy kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer from five medulloblastoma cell lines: D487 Med, D556 Med, D721 Med, D283 Med, and MHH-Med-1. For each library 1 µg of genomic DNA was digested 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, Brown Deer, WI) and ligated to linkers including recognition sites for MmeI. The 21 bp sequence tags were released by digestion with MmeI as has been described for Long SAGE (6). A detailed protocol can be obtained at http://www.digitalkaryotyping.org (7). The isolated tags are self-ligated, PCR amplified concatenated, cloned in pZero (Invitrogen, Carlsbad, CA), and sequenced. The SAGE 2000 software package enables the extraction of the genomic tags from the sequence files. The virtual genomic tags were extracted from the human genome sequence (UCSC Genome Bioinformatics, July 2003 assembly, http://genome.ucsc.edu/) and downloaded from http://www.digitalkaryotyping.org. DNA from plasmid inserts containing serial genomic tags were purified and sequenced at Agencourt Bioscience Corporation (Beverly, MA) as part of the Cancer Genome Anatomy Project.

Quantitative Real-Time PCR. Copy number changes between normal human DNA and medulloblastoma cell lines or primary tumors were determined by Q-PCR on an iCycler apparatus (Bio-Rad, Hercules, CA). The repetitive element Line-1, which has an equivalent number in cancer and normal genomes, was used for the normalization of DNA content. Calculations and PCR conditions were all done as previously described (7, 8). All PCRs were done in triplicates and threshold cycle numbers were averaged. PCR primers were designed using Primer 3 http://www.genome. wi.mit.edu/cgi-bin/primer/primer3_www.cgi) and are available upon request. For transcript analysis total RNA was extracted from 11 medulloblastoma cell lines using the RNAgents total RNA isolation system (Promega, Madison, WI). Synthesis of cDNA and quantitative PCR were done as previously described (12).

Immunoblotting. Cells from various medulloblastoma cell lines were lysed in 50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 1% NP40, 0.25% Na-deoxycholate, 1 mmol/L EDTA, and protease inhibitors (Protease cocktail, Roche Diagnostics, Indianapolis, IN). Total protein concentration was assayed with a Bradford Protein assay kit (Bio-Rad). Equal amounts of total protein were separated on an SDS-polyacrylamide gel and electro-blotted onto nitrocellulose membrane. Blocking of membrane and incubation with antibodies was done according to standard procedures and visualized with the enhanced chemiluminescence plus detection system (Amershan Biosciences, Piscataway, NJ). Monoclonal antibodies raised against OTX2 (R&D systems, Minneapolis, MN) were used at a 1:50 dilution overnight at 4°C.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Digital Karyotyping of Medulloblastomas. DNA from five established medulloblastoma cell lines was purified for digital karyotyping (7). Cultured cell lines were used to avoid contaminating normal cells. The majority of 21 bp genomic tags produced by this method contain sufficient information to identify a unique genomic position. We sequenced a total of 978,724 tags with an average of 195,745 tags per library Table 1). The experimental tags were filtered to remove repetitive elements and compared with virtual tag sequences extracted from the public genome sequence. An average of 104,030 filtered tags per library (Table 1) was aligned across each chromosome. Tag densities were analyzed in windows ranging from 50 to 1,000 virtual tags (7). These densities were calculated for each window by summing the experimental tags observed and dividing the sum by the average tag count for all the same-sized windows across the genome.


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  		Table 1. Summary digital karyotyping libraries and copy number amplifications

 
The MYC gene was used as an internal positive control. More than 25 copies at the genomic DNA level were found for D487 and D556 Med and between 2 and 6 copies for D721 and D283 Med. Digital karyotyping accurately predicted the published genomic copy numbers for all five cell lines previously determined by fluorescent in situ hybridization (13). MYC diploid genome copy numbers for all five cell lines were also corroborated by Q-PCR (Table 1). Further analysis of the digital karyotypes of D556 Med, D721 Med, D283 Med, and MHH-Med-1 did not reveal genomic alterations other than those already known (14, 15). Analysis of chromosome 17 provided a second internal control. D487 Med had a digital karyotype that produced an average of three copies of 17q and one copy of 17p. This karyotype was consistent with one isochromosome 17q and one normal chromosome 17, as previously described for D487 Med (14) and observed in many medulloblastomas (1).

Of most interest were two novel chromosome 14q amplifications found in D487 Med (Fig. 1A). Amplicon A, on chromosomal band 14q22.3, extended from genomic map positions 53.7 to 55.5 Mb from pter. Tag densities ranged from ~8 to 13 copies per diploid genome within this region (Fig. 1A). The second amplified region (Amplicon B) has tag densities between 8 and 12.4 and is located between 71.5 and 73.0 Mb on chromosomal band 14q24.3. Four known genes and one open reading frame are located in Amplicon A and 14 genes in Amplicon B. Previous cytogenetic studies of D487 Med had failed to detect this amplification and indicated that chromosome 14 was present with no gross rearrangements (14).



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  		Figure 1. A, low-resolution tag density maps of three regions in cell line D487 Med generated by digital karyotyping using a sliding window of 300. Top, chromosome 17 data for a genome with one normal chromosome, an isochromosome 17q, and loss of 17p. Bottom, two chromosome 14 amplicons found on 14q22.3 (A) and 14q24.3 (B). MYC amplification on chromosome 8 is shown as a reference. Y axis, copy numbers per haploid genome; X axis, chromosomal position extending over the whole chromosome. B, graphic representation of the genes located within amplicon (A) on 14q22.3. Arrows, indication whether the gene is transcribed from the positive (->) or negative (<-) strand. A detailed view of the corresponding region on 14q22.3 can be obtained at http://genome.ucsc.edu/ using the coordinates 53.7 and 55.8 Mb. C, display of messenger RNA/genomic DNA alignment for OTX2. The exons are indicated by the boxes along the genomic DNA (line in bold). Striped boxes, coding sequences. Solid black arrows, primer positions for genomic amplification detection (two sets); open gray arrows, primers used to amplify exons for mutation detection; dotted arrows, primers used to confirm expression of the gene transcripts using Q-PCR.

 
OTX2 Transcript Expression. We observed that for the 19 genes in two 14q amplicons, only one gene, OTX2, had high messenger RNA transcript levels specific to medulloblastoma. SAGE data from 20 primary medulloblastomas and a variety of malignant and normal tissues totaling 240 different cell and tumor types were surveyed (refs. 12, 16; Fig. 2A, http://cgap.nci.nih.gov/SAGE). The 10 bp OTX2 SAGE tag (ACCAACTGGT) occurs 783 times in the National Cancer Institute SAGE Genie database of 15.7 million human tags, with 96% of the OTX2 transcript tags being found highly expressed in medulloblastomas or embryonic stem cells. There was little or no expression of OTX2 in the large variety of normal adult tissues or other cancer types. Two alternative transcripts of OTX2 are known with an identical 3' end. The longer transcript (a) contains all five exons and the shorter transcript (b) lacks the first two exons (Fig. 1C). Both transcripts encode for the same protein with the exception of the first eight amino acids (encoded by exon 3) that are missing in transcript b. Q-PCR of OTX2 transcription in the medulloblastoma cell line panel also showed overexpression in 7 of 11 samples (Fig. 2B). In particular, the cell lines D487 and D425 showed respectively 15- and 45-fold increase in transcript levels when compared with normal cerebellum. The longer transcript (a) was detected in 6 of 11 medulloblastoma cell lines using conventional PCR (Fig. 2B). High protein expression was also found in D487 and D425 Med using a monoclonal OTX2 antibody. A clearly predominant band at a slightly higher molecular weight than predicted, but thought to represent a posttranslationally modified OTX2 protein, correlated with the transcript levels with two exceptions, D721 Med and D581 Med (Fig. 2B). High protein expression was found in the two cell lines with OTX2 genomic amplification.



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  		Figure 2. A, expression of OTX2 in 240 different SAGE libraries indicating specificity of expression for medulloblastoma and embryonic stem cells. These "Digital Northern" results for OTX2 (SAGE tag: ACCAACTGGT) were obtained from SAGE Genie(http://cgap.nci.nih.gov/SAGE). B, Q-PCR analysis of OTX2 transcript levels in medulloblastoma cell lines. The graph shows the relative expression level when compared with normal cerebellum (2x). Bottom, end-point analysis of the Q-PCR, detection of the full-length transcript using PCR primers starting at exon 1 or exon 2, and a control for cDNA quality. OTX2 monoclonal antibodies identify a similar band in cell lysates from the various medulloblastoma cell lines. The protein is detectable in most cell lines with a high transcript expression like D487 and D425 Med and barely or not detectable in cell lines with low or no transcript expression like MHH-1-Med, UW228-2, and MCD1.

 
14q Minimal Amplified Region. The transcript expression pattern of OTX2 and its role in brain development prompted us to further analyze Amplicon A on 14q22.3. We used Q-PCR and a genomic DNA template to determine copy number for the genes in the amplicon from the five original plus six additional medulloblastoma cell lines.

For OTX2 two independent sets of genomic primers were used, one set flanking exon 1 and the second set between exon 4 and the exon 4/5 intron (Fig. 1C). The OTX2 copy numbers for all five original cell lines were confirmed (Table 1) and a new OTX2 25-copy amplicon was found in D425 Med. In contrast, previous cytogenetic studies by Aldorsi et al. (15) showed for D425 Med two copies of intact chromosome 14 without amplification.

The other known and/or predicted genes in this region, KTN1, PELI2, C14orf101, and SEC10L1, were also tested for genomic amplification in medulloblastoma cell lines (Fig. 3). However, in D425 Med the amplification was observed only in OTX2 and not in the flanking genes (Fig. 3). Furthermore, expression analysis by Q-PCR in D425 Med showed a very high level of OTX2 transcript and a barely detectable level of C14orf101 (Fig. 2B). Barring an unidentified gene hiding within the D425 Med amplicon, the limited amplified region implicated only OTX2 as a gene that might provide a selective advantage to tumor cells when replicated.



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  		Figure 3. Extent of genomic amplification in 10 medulloblastomas with OTX2 amplification and three controls. Relative genomic amplification levels are indicated as copies per diploid genome and generated by Q-PCR at each gene where a bar is shown. Copy numbers were not determined for KTN1 and SEC10L1 in primary tumor samples Med 6, 7, and 8. Also PELI2 was not determined in samples Med 7 and Med 8. The relative copy number of the predicted open reading frame C14orf101 was only determined in the three depicted medulloblastoma cell line samples. D425 Med amplification is limited to OTX2 and flanking gene Sec10L1 is excluded in D487 Med.

 
Genomic Amplification in Primary Tumors. We extended the analysis of OTX2 gene copy number to 42 primary human medulloblastoma samples. In 8 samples (19%), OTX2 was genomically amplified (Fig. 3). The size of these amplicons was consistent with the 14q amplicon A found in D487 Med (Fig. 1B) and generally contained flanking genes. The relative copy numbers for these eight samples are shown in Fig. 3 and varied from 8 to 56 copies.

OTX2 Mutation Analysis. Some amplified oncogenes, such as the epidermal growth factor receptor, contain further rearrangements or point mutations beyond genomic amplification. In an attempt to further implicate OTX2, we sought such activating mutations in 23 medulloblastoma genomic DNA samples. Exons 3, 4, and 5 (Fig. 1C), which include the complete coding sequence, were amplified for sequencing. However, a wild-type sequence was observed in each case.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Digital karyotyping was used to analyze genomic copy number in five medulloblastoma cell lines and identified two novel chromosome 14 amplicons. The amplicon on 14q22.3 included the OTX2 gene and was shown to be present in both medulloblastoma cell lines and primary tumors at a similar incidence of ~19%. Although the amplicon typically contained flanking genes, one cell line had only OTX2 amplified, suggesting OTX2 as the target of amplification. Of the five possible genes in the amplicon, only OTX2 had an expression pattern consistent with pathologic transcriptional activation of the gene in medulloblastoma.

We found the combined analysis of high-resolution copy number and gene expression specificity to be useful in this case. OTX2 had previously been reported to be overexpressed at the transcript level in medulloblastomas when compared with fetal brain or cerebellum (17, 18), but not implicated through genomic alteration. Our identification of a genomically amplified transcription factor in medulloblastoma is not without precedent, as this is the case for the MYC oncogene.

Whereas increased gene dosage should account for elevated OTX2 expression in ~19% of medulloblastomas, a much higher percentage of medulloblastomas express the transcript and protein at high levels, suggesting additional mechanism(s) for pathway activation. We did not locate mutations in OTX2 coding sequence, and if they exist, they are a rare event. However, activation of the OTX2 promoter by mutations or upstream transcription factors remains a possibility for activating transcription. In embryogenesis OTX2 expression is tightly regulated by cis-acting elements including TCF/Lef binding sites which would place the Wnt signaling pathway upstream of OTX2 (9). Wnt signaling is activated in approximately one quarter of medulloblastomas, and it may drive OTX2 expression in a significant fraction of cases.

Development of the forebrain, midbrain, and anterior hindbrain depend on proper OTX2 levels (19). OTX2 controls the forebrain and midbrain development in a dose-dependent manner by regulating Fgf8 (along the anterior-posterior axis) and SHH (along the dorsal-ventral axis; ref. 20). Ectopically expressing the OTX2 gene in the anterior hindbrain of developing mice using a knock-in strategy prevented development of the cerebellar vermis, but produced an enlarged inferior colliculus (21). OTX2 also physically interacts with the translational initiation factor 4E, providing another mechanism by which OTX2 could modulate tumor cell behavior (22). The role of OTX2 in the development of the brain is consistent with its proposed role as a medulloblastoma oncogene. Further studies are required to determine if inhibition of OTX2 function can repress medulloblastoma growth.


   Acknowledgments
 
Grant support: Ludwig Trust, the Cancer Genome Anatomy Project (NIH 23X-S073), and NIH grants (U01 CA88128 and K08 NS43279).

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 with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Victor E. Velculescu and Jordan M. Cummins (Johns Hopkins University) for digital karyotyping technical support and Daniela Gerhard for CGAP administrative support. C.G. Eberhart is recipient of a Burroughs Wellcome Fund Career Award. G.J. Riggins is the recipient of the Irving J. Sherman, M.D. Research Professorship in Neurosurgery.

Received 9/22/04. Revised 11/23/04. Accepted 12/ 3/04.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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