To identify critical tumor suppressor loci that are associated with the development of medulloblastoma, we performed a comprehensive genome-wide allelotype analysis in a series of 12 medulloblastomas. Non-random allelic imbalances were identified on chromosomes 7q (58.3%), 8p (66.7%), 16q (58.3%), 17p (58.3%) and 17q (66.7%). Comparative genomic hybridization analysis confirmed that allelic imbalances on 8p, 16q and 17p were due to loss of genetic materials. Finer deletion mapping in an expanded series of 23 medulloblastomas localized the common deletion region on 8p to an interval of 18.14 cM on 8p22–23.2. We then searched within the region of loss on 8p for loci that might contain homozygous deletion using comparative duplex PCR. An overlapping homozygous deletion region was identified in a 1.8 cM interval on 8p22–23.1, between markers D8S520 and D8S1130, in two medulloblastomas. This region of homozygous deletion also encompasses the 1.4 cM minimal deletion region detected on 8p in ductal carcinoma in situ of breast. In conclusion, we reported for the first time a detailed deletion mapping on 8p in medulloblastoma and have identified a region of homozygous deletion on 8p22–23.1 that is likely to contain a critical tumor suppressor gene involved in the development of medulloblastoma.
Medulloblastoma is a malignant, invasive embryonal tumor that predominantly arises in the cerebellum. It is the most common brain tumor in children and constitutes 10–20% of pediatric central nervous system neoplasms. Medulloblastoma has a propensity for cerebrospinal dissemination, which is present in one-third of patients at presentation. Significant advances in a multimodal therapy, including surgery, radiotherapy and chemotherapy, have greatly improved the outcomes of patients. In the past 30 years, the 5-year survival rate has increased from 10 to about 50%. However, long-term survival in children with advanced disease is only about 30% (Giangaspero et al., 2000; Heideman et al., 1997). Further enhancement of survival will rely on a better understanding of the biology of this malignant disease to improve current treatments or to develop novel therapy.
Non-random loss of genetic material from chromosomal loci is a common feature in the development of tumors and is often indicative of the presence of critical tumor suppressor genes in the given tumor. Inactivation of multiple tumor suppressor genes is typically required for tumor formation. In medulloblastoma, deletion in the short arm of chromosome 17 is the most common genetic abnormality seen in up to 50% of tumors (Bigner et al., 1988). This result suggests that loss of a tumor suppressor gene located on 17p plays an important role in the pathogenesis of this malignant tumor. The TP53 gene was found to be infrequently mutated in medulloblastoma, indicating that TP53 is not the target on 17p (Saylors et al., 1991). Using comparative genomic hybridization technique, several groups have demonstrated the involvement of multiple chromosomes in medulloblastoma (Avet-Loiseau et al., 1999; Gilhuis et al., 2000; Nicholson et al., 1999; Reardon et al., 1997). Of those chromosomes that show deletions, losses of chromosomes 10q and 8p are recurrent genetic alterations, with about 30% of tumors showing such abnormalities. Other common genetic losses occurring at a lower frequency include deletion of chromosomes 3, 6, 9, 11, 16 and 22. We have previously performed a deletion mapping study on chromosomes 10q, 11 and 16. Three regions of frequent loss were localized to the Deleted in Malignant Brain Tumor 1 (DMBT1) locus on 10q25–26, 11p13–15.1 and 16q24 (Yin et al., 2001). So far, no medulloblastoma-associated tumor suppressor genes have been identified in any of these aberrant chromosomes.
In this study, we aimed to search for critical tumor suppressor loci that are important for the development of medullolastoma. We used high-resolution genome-wide allelotype analysis followed by finer deletion mapping to localize chromosomal regions with frequent deletion. Non-random allelic deletions were identified on 8p, 16q and 17p. Further mapping has revealed a homozygous deletion region of 1.8 cM on 8p22–23.1, which is likely to contain a tumor suppressor involved in the tumorigenesis of medulloblastoma. In addition, the Deleted in Liver Cancer 1 (DLC1) gene, a recently cloned candidate tumor suppressor gene located on 8p21.3–22, was also assessed for its involvement in medulloblastoma.
Clinical data of patients
In the series, there were 24 classical, two desmoplastic and one anaplastic medulloblastomas. All tumors were cerebellar. No history of Gorlin or Turcot syndrome was associated with this series of tumors. There were three adult and 21 pediatric patients and their ages ranged from 2–69 years with a mean of 12.5 years. The male/female ratio was 2.8 : 1. One child (case M1) experienced two relapses over a period of 4 years.
To localize critical tumor suppressor loci involved in the tumorigenesis of medulloblastoma, we performed a comprehensive genome-wide allelotype analysis on 12 medulloblastomas. All 22 autosomes were examined for allelic imbalances. An average of 238 (62.3%) informative loci/case was detected in our series. Allelic imbalances were seen in all 39 autosomal arms. The highest frequencies of allelic imbalance were detected on chromosomal arms 8p and 17q (66.7%), whereas the arms with the lowest frequencies of allelic imbalances were seen on chromosomes 5p, 9p, 9q, 11q, 16p, 18q, 19p, and 21p (8.3%). The mean percentage of allelic imbalances was 26.7%±16.6%. Figure 1 summarizes the frequency of allelic imbalances at each autosomal arm. In the present study, 43.3% (mean percentage+s.d., 26.7+16.6%) was chosen to be a significant percentage of allelic imbalances in our series. This represents the 99% confidence upper limit for the overall rate for random chromosome loss or imbalance in tumors. Non-random allelic imbalances above the baseline (43.3%) were identified on chromosomal arms 7q (58.3%), 8p (66.7%), 16q (58.3%), 17p (58.3%), and 17q (66.7%). Representative results of allelic imbalances at selected microsatellite loci are illustrated in Figure 2a. Using CGH analysis, we confirmed that allelic imbalances detected by allelotyping on chromosomes 8p, 16q, and 17p were due to chromosome loss, whereas those of 7q and 17q referred to allelic gain (Figure 2b). We have also delineated the common regions of deletion on chromosomes that are frequently lost in medulloblastoma. These regions of loss on chromosomes 8p, 16q and 17p were mapped to 8p12-pter (37 cM), 16q22.3-pter (46.6 cM) and 17p13.1-pter (15 cM), respectively. In addition, chromosomal arms with frequencies of allelic imbalances higher than the mean percentage of LOH were identified on 3p (33.3%), 3q (33.3%), 4q (41.7%), 7p (33.3%), 8q (41.7%), 10q (41.7%), 13q (33.3%), 14q (33.3%), and 20q (33.3%).
The fractional allelic loss (FAL) for each tumor was also determined. FAL is defined as the fraction of the total number of informative chromosomal arms that displays allelic loss. In the series, the FAL values ranged from 0.08 to 0.77. Interestingly, the tumor with the highest FAL (0.77) was a desmoplastic variant (case M15). The mean FAL was 0.26±0.19. There was no correlation between FAL and patient age, sex, or tumor type.
Localization of common deletion region on 8p
To refine the common deletion region on the short arm of chromosome 8, we evaluated the LOH pattern in an expanded series of 21 primary and two recurrent medulloblastomas using a separate panel of 19 microsatellite markers mapping to 8p. Nineteen of 23 (83%) tumors showed allelic loss on at least two loci (Figure 3). The overall frequency of LOH on 8p was 51% (124 of 243 informative markers), comparable to that observed in allelotype analysis (58.8%, 30 of 51 informative markers). Two cases (M15 and M16) revealed LOH on all informative markers examined, suggesting the presence of monosomy 8 in these tumors. The other 21 cases showed partial or interstitial deletion on 8p. Combining all LOH data, a common region of deletion was narrowed down from 37 cM on 8p12–23, as determined by allelotyping, to an interval of 18.14 cM on 8p22–23.2, between markers D8S262 and D8S1130. The frequency of LOH at this region was 70.7%. In addition, one tumor (M3) revealed microsatellite instability on the marker D8S1721. Case M1 and its two recurrent tumors showed the same allelic loss profile in all informative markers examined.
Detection of homozygous deletion on 8p22–23.1
Identification of homozygous deletion region is important for positional cloning of tumor suppressor gene. We therefore screened chromosomal loci, at which the heterozygous alleles were flanked by lost alleles, on 8p for possible homozygous deletion using comparative duplex PCR. Two tumors were found to have homozygous deletion: M2 at D8S520 and M3 at D8S1130 (Figure 4). These two tumors shared an overlapping deletion region, with the two markers being only 1.8 cM apart. The homozygous deletion region was mapped to the junction of 8p22 and 8p23.1. Thus we have further refined the common region of deletion on 8p to an interval of 1.8 cM.
Molecular genetic investigations of DLC1 gene
A recently identified candidate tumor suppressor gene termed DLC1, that is located on 8p21.3–22, was investigated for its involvement in medulloblastoma. Using duplex PCR, we did not detect homozygous deletion of the DLC1 gene in the tumor series (data not shown). We then employed a highly sensitive method, CSGE, to detect somatic mutations in the DLC1 gene. Twenty-six medulloblastomas were screened. No heteroduplexes with aberrant gel shift were seen, indicating the absence of somatic mutations in the DLC1 gene in medulloblastoma (data not shown).
In this study, we performed a high-resolution genome-wide allelotype analysis on medulloblastoma with an aim to localize putative tumor suppressor loci involved in this malignant neoplasm. Non-random allelic losses were identified on chromosomes 8p (66.7%), 16q (58.3%) and 17p (58.3%), strongly indicating that alterations of these chromosomes play critical roles in medulloblastoma. Further detailed mapping unveiled on 8p22–23.1 a novel region of homozygous deletion in an interval of 1.8 cM, flanked by markers D8S520 and D8S1130.
To compare our findings with those of previous investigations, we have reviewed genetic data from six genome-wide studies covering a total of 107 medulloblastomas (Blaeker et al., 1996; Reardon et al., 1997; Avet-Loiseau et al., 1999; Nicholson et al., 1999; Gilhuis et al., 2000; von Deimling et al., 2000). The summarized results revealed that deletions of 17p (39%), 10q (35%), 8p (32%), 11p (28%), 11q (28%), and 16q (20%) are the most frequent genetic abnormalities involving loss of genetic materials. Although the number of medulloblastomas allelotyped in the present study is limited (12 cases), our results are consistent with the published data. We have previously mapped the regions of frequent loss on chromosomes 11 and 16 to 11p13–15.1 and 16q24.1–24.3, respectively (Yin et al., 2001). The localization of these critical loci is important for future fine mapping and cloning of candidate tumor suppressor genes. The DMBT1 locus on 10q25–26 is also found to be closely associated with the development of medulloblastoma (Yin et al., 2001).
The most striking finding in the present study is the high incidence (67%) of allelic deletion on chromosome 8p in medulloblastoma. This result is in agreement with previous low-resolution allelotyping study (50%) but is about twofold higher than those determined by CGH studies (32%) (Blaeker et al., 1996; Reardon et al., 1997; Avet-Loiseau et al., 1999; Nicholson et al., 1999; Gilhuis et al., 2000). The use of high-density markers in the present microsatellite analysis probably explains the increased sensitivity of detecting allelic loss. Frequent deletion of the chromosomal arm 8p has also been detected in a variety of tumors, such as prostate, breast, lung, colorectal, bladder, head and neck, liver, ovarian and gastric cancers (Sun et al., 1999; Wang et al., 1999; Wistuba et al., 1999; Farrington et al., 1996; Takle and Knowles, 1996; Sunwoo et al., 1999; Pineau et al., 1999; Wright et al., 1998; Baffa et al., 2000). Deletion mapping has identified several sub-chromosomal regions on 8p that are important for cancer development. These regions are localized to 8p12, 8p21, 8p22, and 8p23-pter. Within the deletion loci, critical regions with homozygous deletion have been identified on 8p21, 8p22, and 8p23 (Kagan et al., 1995; Prasad et al., 1998; Iswad et al., 1999; Levy et al., 1999; Sun et al., 1999; Arbieva et al., 2000). These results suggest that multiple tumor suppressor genes are located on the short arm of chromosome 8. In addition, loss of 8p has been found to be associated with clinicopathological findings. For instance, frequent deletion of 8p was correlated with invasive behavior in breast cancer and with poor outcome in prostate cancer. (Yaremko et al., 1996; Jenkins et al., 1998). Functional studies demonstrated that restoration of normal chromosome 8p suppressed tumorigenecity and metastasis in colon and prostate tumor cells respectively, further supporting the involvement of 8p loss in tumorigenesis (Nihei et al., 1996; Tanaka et al., 1996). In this study, we have for the first time reported a detailed deletion mapping on chromosome 8p in medulloblastoma and have identified a homozygous deletion region that encompasses an interval of 1.8 cM on 8p22–23.1. Losses of 8p22 and 8p23.1 subregions are recurrent genetic aberrations in a number of tumors such as prostate, pancreas, breast, liver, gastric, ovarian and colorectal cancers (Arbieva et al., 2000; Pineau et al., 1999; Levy et al., 1999; Wang et al., 1999; Baffa et al., 2000; Wright et al., 1998; Fujiwara et al., 1993; Lassus et al., 2001). Three critical deletion regions and several candidate tumor suppressor genes have been identified within the locus of 8p22–23.1 and their relative genetic locations are shown in Figure 4. One deletion region is located in an interval of 1.4 cM, defined by markers D8S520 and D8S261, in ductal carcinoma in situ of breast (Wang et al., 1999). This deletion region lies within the homozygously deleted region identified in medulloblastoma, suggesting the presence of a novel tumor suppressor gene at this locus. It is likely that the same tumor suppressor gene from this region is operative in both breast cancer and medulloblastoma. A sequence-ready YAC/BAC clone contig has been constructed for ∼1.7 cM in this region and at least 10 known genes and expressed sequence tags are mapped (Wang et al., 1999). It remains to be determined whether any of these genes is the candidate tumor suppressor in the interval flanked by D8S520 and D8S1130. Another critical deletion region that maps to 8p22 is located proximal to the 1.8 cM interval identified in this study. An overlapping region of homozygous deletion of 1–1.5 cM was seen in a metastatic prostate tumor and a pancreatic tumor cell line (Arbieva et al., 2000; Bova et al., 1996; Levy et al., 1999). No candidate tumor suppressor genes have yet been identified from this region. The third deletion region is located on 8p23.1 between D8S262 and D8S520. This region is found deleted in about 60% of ovarian tumors (Lassus et al., 2001).
Recently, a novel tumor suppressor gene named DLC1, which is associated with hepatocellular carcinoma, is isolated from 8p21.3–p22 (Yuan et al., 1998). Recent draft human genome sequence has mapped the DLC1 gene to 8p22. The DLC1 encoded product is a member of the Rho family GTPases, which function as important regulators in the organization of actin cytoskeleton. In tumors, dysregulation of the RhoGTPases-associated signaling pathways may lead to motile and invasive phenotype (Schmitz et al., 2000). Wilson et al. (2000) detected one missense mutation of the DLC1 gene in a series of 126 colorectal and 33 ovarian tumors and cell lines. Ng et al. (2000) detected homozygous deletion in two of six hepatocellular carcinomas and two hepatoma cell lines. More importantly, they demonstrated that overexpression of DLC1 in DLC1-deficient hepatoma cells resulted in suppressed growth (Ng et al., 2000). In the present study, we evaluated whether DLC1 plays a role in the development of medulloblastoma. We examined the DLC1 gene by comparative duplex PCR and mutation analyses. No homozygous deletion and somatic mutation of the DLC1 gene sequence were detected in the series. Our results suggest that DLC-1 is not involved in medulloblastoma.
Another candidate tumor suppressor gene termed LZTS1/FEZ1 (Leucine Zipper. Putative Tumor Suppressor 1) is mapped to 8p22. Somatic mutation and altered transcript expression of LZTS1/FEZ1 have been detected in several cancer including esophageal, prostate and gastric tumors (Ishii et al., 1999; Vecchione et al., 2001). A functional approach demonstrated that overexpression of LZTS1/FEZ1 in LZTS1/FEZ1-negative cancer cells resulted in suppression of tumorigenicity and reduced cell growth. Further investigation showed that LZTS1/FEZ1 is involved in regulation of mitosis (Ishii et al., 2001). These results strongly suggest that LZTS1/FEZ1 is a potential tumor suppressor involved in various human cancer.
In conclusion, our comprehensive and extensive deletion mapping analysis has unveiled a region of homozygous deletion on chromosome 8p22–23.1 in medulloblastoma. This region is likely to contain a critical tumor suppressor important for the development of this malignant neoplasm. The recent availability of physical map and gene sequence within the homozygous deletion region will facilitate the identification of the putative tumor suppressor gene on 8p22–23.1.
Materials and methods
Patients and specimens
Twenty-seven medulloblastoma samples of 25 primary and two recurrent tumors were collected from the Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, Hong Kong and four hospitals in China (An Hui Medical University, Nan Jin Medical University, Hang Zhou Hospital, and Shan Xi Hospital). Informed consent was also obtained from the patients. Each tumor was classified according to current World Health Organization (WHO) criteria (Giangaspero et al., 2000). All investigations in this study were performed on frozen tissues, except for two cases in which formalin-fixed paraffin-embedded materials were used. Samples were considered suitable for the study if tumor cell content was histologically confirmed to be greater than 80%. Tumor-matched blood samples were also collected as constitutional controls. High molecular weight DNA was isolated from both tumor and blood samples using the conventional proteinase K-phenol/chloroform extraction method.
Polymerase chain reaction (PCR) based-microsatellite analysis was used to evaluate allelic imbalances or loss of heterozygosity (LOH) in medulloblastoma. A high-resolution genome-wide allelotype analysis was performed according to reported protocols (Tong et al., 2001). Briefly, 382 microsatellite loci derived from 22 autosomes were examined for allelic imbalances. The average interval of these loci is about 10 cM. The polymorphic microsatellite markers were obtained from the ABI Prism Linkage Mapping Set V. 2 (Applied Biosystems, Foster City, CA, USA) and were originally selected from the Généthon human linkage map. The set consists of primer pairs end-labeled with either one of three fluorescent dyes, FAM, HEX, or NED. PCR was performed in a final volume of 7.5 μl containing two primer pairs (2.5 pmoles of each primer), 60 ng of DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.2 mM deoxyribonucleoside triphosphates, 2.5 mM MgCl2 and 0.6 unit of AmpliTaq Gold DNA polymerase (Applied Biosystems). To facilitate high throughput of samples, all liquid handling and thermal cycling were carried out in an ABI Prism 877 robotic workstation (Applied Biosystems). PCR was started, according to manufacturer's recommendation, with 95°C for 15 min, followed by 10 cycles composed of 94°C for 15 s, 55°C for 15 s and 72°C for 30 s, and another 22 cycles composed of 89°C for 15 s, 55°C for 15 s and 72°C for 30 s. Amplified PCR products of multiple loci were pooled and electrophoresed in denaturing 5% polyacrylamide gels on an ABI Prism 377 automated DNA sequencer (Applied Biosystems). The data collected were analysed using GeneScan Analysis software version 3.1 (Applied Biosystems). Allelic imbalance was defined by calculating the allelic ratio (AR) of both normal (N) and tumor (T) DNA, where AR was the ratio of peak height of the longer allele (N2 or T2) to that of the shorter allele (N1 or T1), i.e., AR=(N2/N1)/(T2/T1). Allelic imbalance was indicated when the ratio was greater than 1.5 or smaller than 0.5, representing loss of longer or shorter allele respectively. As both allelic loss and gain exhibited LOH pattern in microsatellite analysis, we therefore used comparative genomic hybridization (CGH) to verify the allelic status. The CGH analysis was performed according to protocol reported (Yin et al., 2000).
To narrow down the deletion region on the short arm of chromosome 8, we performed finer deletion mapping using radioactively labeled microsatellite markers as described previously (Yin et al., 2001). A separate set of 19 polymorphic markers mapping to 8p was used. The order of markers was arranged according to the genetic map compiled by Broman et al. (1998) and is available at the website http://research.marshfieldclinic.org/genetics.
Comparative duplex PCR for detection of homozygous deletion
Homozygous deletion was investigated by comparative duplex PCR. The PCR reaction was optimized in a panel of five normal DNA samples, until equivalent levels of amplification were detected in the target and reference loci. The PCR mix was prepared as described in the microsatellite analysis. The amplification conditions included an enzyme activation step at 95°C for 10 min, followed by 26–28 cycles composed of 94°C for 30 s, 56–58°C for 30 s, and 72°C for 1 min. Chromosomal loci were considered homozygously deleted when the peak height (in the fluorescence method) or the band intensity (in the radioactive method) of the target loci was less than 80% of that of the reference loci. Each locus of homozygous deletion was verified with at least two reference markers.
To assess homozygous deletion in the DLC1 gene, tumor DNA was amplified with primers specific for exon 3 or exon 9 of DLC1 and D5S816, D5S1480 or the STS marker SGC33915 that are located on chromosome 5. The latter markers were chosen as reference loci because genetic aberrations on chromosome 5 are rarely detected in medulloblastoma.
Conformation-sensitive gel electrophoresis
All 13 exons and splice junctions of the DLC1 gene were screened for somatic mutations by conformation-sensitive gel electrophoresis (CSGE) as described previously (Leung et al., 2001). Seventeen pairs of primers were designed and their sequences are listed in Table 1. DNA was amplified by PCR with annealing temperature optimized at 50–56°C for 38 cycles. PCR products were resolved on nondenaturing 15% polyacrylamide gels at 40 W for 4–6 h, stained with 1 μg/ml SYBR Gold solution (Molecular Probes, Eugene, OR, USA), and visualized under UV illumination.
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This study was supported by a Direct Grant from the Research Grant Council of Hong Kong.
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