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Journal of Clinical Oncology, Vol 23, No 34 (December 1), 2005: pp. 8853-8862 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.02.8589 Genomic and Protein Expression Profiling Identifies CDK6 As Novel Independent Prognostic Marker in MedulloblastomaFrom the Division of Molecular Genetics, German Cancer Research Center; Central Unit Biostatistics, German Cancer Research Center, Heidelberg; Department of Neuropathology, Heinrich Heine University, Duesseldorf, Germany; Wellcome Trust Genome Campus, The Wellcome Trust Sanger Institute, Hinxton, United Kingdom; and Department of Neuropathology, Burdenko Neurosurgical Institute, Moscow, Russia. Address reprint requests to Peter Lichter, PhD, German Cancer Research Center, Division of Molecular Genetics (B060), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany; e-mail: m.macleod{at}dkfz.de
PURPOSE: Medulloblastoma is the most common malignant brain tumor in children. Despite multimodal aggressive treatment, nearly half of the patients die as a result of this tumor. Identification of molecular markers for prognosis and development of novel pathogenesis-based therapies depends crucially on a better understanding of medulloblastoma pathomechanisms. PATIENTS AND METHODS: We performed genome-wide analysis of DNA copy number imbalances in 47 medulloblastomas using comparative genomic hybridization to large insert DNA microarrays (matrix-CGH). The expression of selected candidate genes identified by matrix-CGH was analyzed immunohistochemically on tissue microarrays representing medulloblastomas from 189 clinically well-documented patients. To identify novel prognostic markers, genomic findings and protein expression data were correlated to patient survival. RESULTS: Matrix-CGH analysis revealed frequent DNA copy number alterations of several novel candidate regions. Among these, gains at 17q23.2-qter (P < .01) and losses at 17p13.1 to 17p13.3 (P = .04) were significantly correlated to poor prognosis. Within 17q23.2-qter and 7q21.2, two of the most frequently gained chromosomal regions, confined amplicons were identified that contained the PPM1D and CDK6 genes, respectively. Immunohistochemistry revealed strong expression of PPM1D in 148 (88%) of 168 and CDK6 in 50 (30%) of 169 medulloblastomas. Overexpression of CDK6 correlated significantly with poor prognosis (P < .01) and represented an independent prognostic marker of overall survival on multivariate analysis (P = .02). CONCLUSION: We identified CDK6 as a novel molecular marker that can be determined by immunohistochemistry on routinely processed tissue specimens and may facilitate the prognostic assessment of medulloblastoma patients. Furthermore, increased protein-levels of PPM1D and CDK6 may link the TP53 and RB1 tumor suppressor pathways to medulloblastoma pathomechanisms.
Medulloblastoma is a highly malignant embryonal tumor of the cerebellum that accounts for 20% to 25% of all primary brain tumors in children; the incidence peaks at 7 years of age.1 Several clinical, histologic, and molecular parameters have been identified as predictive factors for patient outcome.2-6 Despite aggressive multimodal therapy, including surgery, irradiation, and chemotherapy, disease dissemination is common and the 5-year survival rate is only 50% to 60%.7 To develop novel therapeutic strategies for the improvement of clinical outcome, it is of paramount importance to increase our understanding of the cellular and molecular basis of this disease. Previous molecular studies showed that the sonic hedgehog signaling pathway is aberrantly activated by genetic alterations in the PTCH, SMOH, or SUFU genes in up to 20% of medulloblastomas, affecting mostly tumors of the desmoplastic subtype.8 A smaller fraction of medulloblastomas demonstrate alterations in genes involved in wingless/WNT (wingless-type MMTV integration site family) signaling, such as CNNTB1, APC, and AXIN.8,9 Studies using conventional karyotyping or comparative genomic hybridization (CGH) identified trisomy 7 and isochromosome 17 [i(17q)] as frequent chromosomal aberrations in medulloblastoma.10-16 Furthermore, amplification of the proto-oncogenes MYC or MYCN is common in large-cell anaplastic medulloblastoma—a rare but highly malignant subtype of medulloblastoma.15-19 To overcome the restricted resolution of chromosomal CGH,20 matrix-based CGH (matrix-CGH) was developed using hybridization targets immobilized on microarrays.21-23 For genome-wide profiling of copy number imbalances, we constructed a DNA microarray consisting of approximately 6,000 large-insert genomic clones covering the whole genome with an average resolution of 0.5 megabases (Mb).24 In this study, we investigated 47 sporadic medulloblastomas using this microarray to identify recurrent DNA copy number imbalances and novel medulloblastoma-associated candidate genes. The matrix-CGH findings were compared with real-time quantitative reverse transcriptase polymerase chain reaction (RQ-PCR), interphase fluorescent in situ hybridization (FISH), and immunohistochemistry (IHC) using a tissue microarray (TMA) composed of medulloblastomas from 189 patients with available clinical follow-up. Genomic imbalances were clustered, combined with protein expression data, and correlated to clinical outcome to search for novel prognostic markers.
Tumor Material and Patient Characteristics All samples used in this study were collected at the Department of Neuropathology, Burdenko Neurosurgical Institute (Moscow, Russia), between 1993 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 WHO classification.25 The tumors were classified by histology as classic, desmoplastic, or anaplastic medulloblastomas; the latter diagnosis was made only when signs of moderate or severe anaplasia were present.3 All samples were provided from an existing tumor bank for research purposes and approval to link laboratory data to clinical data was obtained by the institutional review board. This procedure is according to the official statement of the National Ethics Council of the federal government of Germany (March 2004). Tumor and patient characteristics are summarized in \?\Appendix Table 1. In the matrix-CGH and TMA study, 53 and 189 tumor samples were analyzed, respectively. All patients received craniospinal irradiation after surgery consisting of 36 Gy to the craniospinal axis and a boost of 53 to 56 Gy to the posterior fossa. Adjuvant chemotherapy using lomustine, cisplatin, and vincristine7,26 was administered routinely to all patients treated after 1995, including the patients of the matrix-CGH study and 110 patients (58%) in the TMA study. Before 1995, no chemotherapy had been applied. On May 1, 2004, the 189 medulloblastoma patients included in the TMA study had a median follow-up time of 43 months (range, 3 to 130 months), estimated according to Korn.27 There were 118 survivors and 71 patients who died during follow-up. Sixty-two deceased patients exhibited disease dissemination along the neuroaxis, whereas the remaining nine patients displayed isolated local tumor progression. The 53 medulloblastoma patients included in the matrix-CGH study had a median follow-up time of 30 months (range, 3 to 50 months).
Labeling and Hybridization to Microarrays Selection of genomic clones, isolation of BAC DNA, performance of degenerate oligonucleotide primer-PCR, and preparation of microarrays were described previously.24,28,29 Genomic DNA from tumor tissue and blood of healthy donors was isolated using the Blood and Cell Culture Kit (Qiagen, Hilden, Germany) following the instructions of the suppliers. Labeling, hybridization, and washing procedure were performed as reported previously.24
Image and Data Analysis
Preparation of TMAs
FISH
RNA Isolation
RQ-PCR
IHC
Statistical Analysis
Genomic Imbalances in Medulloblastomas In this study, 47 tumors were analyzed by high-resolution matrix-CGH (Fig 1). The most recurrent aberrations affecting entire chromosome bands were gains of 17q23.2-qter (51%), 7q11.21-qter (32%), chromosome 7 (30%), and 1q21.1 to q44 (13%), as well as losses of 17p13.1 to 13.3 (43%), 11p12pter (21%), 10q24.33-qter (17%), and chromosome 8 (17%). Each tumor displayed DNA copy number imbalances in at least one region. On median, 2.5 imbalances were scored per patient in desmoplastic tumors (n = 8), seven in classic tumors (n = 27), and six in anaplastic tumors (n = 12).
Hot Spots of DNA Copy Number Aberrations By superimposing the genomic profiles of the 47 medulloblastomas, minimally overlapping regions of DNA copy number imbalances were identified. Regions smaller than 3 Mb, which were present in at least five tumors, and all loci exhibiting amplification or homozygous deletion are listed in Table 1. Homozygous deletions were detected once each at 1p36.21 and 2q37.1 to 37.3. Recurrent amplifications were found at 8q24.21 (MYC) in two tumors, 2p24.3 (MYCN) in three tumors, and 7q21.2 in three tumors. MYC and MYCN amplifications (n = 5) were associated with metastasis (n = 5; 0 of 31 M0 v 5/16 M1-3) and poor clinical outcome (estimated survival probability 12 months after surgery 0.20 v 0.86). The novel amplicon at 7q21.2 spanned a minimally overlapping region of 250 kb and contained CDK6 as the only known gene. Furthermore, previously reported amplicons17,37 at 3p21 to 23, 5p15, and 12p13 were narrowed in size to 1.18, 2.30, and 9.65 Mb, respectively, and new amplicons were detected once each at 1q32.1, 7q36.2 to 36.3, 8q21.3 to 22.2, and 17q23.2.
Results of FISH Analyses To independently confirm the matrix-CGH data, we performed FISH analyses for the MYCN and CDK6 loci on the TMA. Figure 2A shows high-level amplification of MYCN in the tumor sample 1m10. Of the 15 tumors that showed single-copy gain of the CDK6 locus in matrix-CGH analysis, 11 were represented on the TMA. In each of these tumors, FISH analysis confirmed single-copy gains. The three tumors showing CDK6 amplification were not represented on the TMA; however, in tumor 1m26 the high-level amplification could be confirmed using a cytospin preparation (Fig 2B). CDK6 copy number was estimated as more than 20 copies per cell (range, 24 to 66 copies per cell). Two more tumors carrying CDK6 amplifications were identified on the TMA by FISH screening of 60 tumors, which were not in the series of samples analyzed by matrix-CGH. The amplifications in these tumors were present over large tumor areas and focal amplifications were not detectable (Fig 2C).
Correlation of Chromosome 17 Aberrations With Poor Survival When log-rank tests were used, genomic imbalances on chromosome 17 were found to be related significantly to poor overall survival in the 47 patients included in the matrix-CGH study (Fig 2D). The loss of about 9 Mb on 17p and gain of about 23.8 Mb on 17q cover three of the previously identified hot spots of DNA copy number aberration (Table 1). Unsupervised clustering divided our tumor set into two about equally large groups (Fig 3A). The most striking difference between these groups was the almost complete absence (group I) or presence (group II) of imbalances on chromosome 17 including an apparent i(17q), gain of chromosome 17 and isolated gain on 17q. The most common of these imbalances was gain of 17q23.3-qter, occurring in 24 of 25 group II tumors. Other imbalances more frequently observed in group II than group I included gain of 7q11.21-qter, loss of 10q, and loss of 11p12-qter. Groups I and II again could be divided into two subgroups each. Tumors in groups Ia and Ib were characterized by the presence or absence of chromosome 6. Group IIa showed a gain of 8q13.2-qter and contained the two MYC amplifications, whereas tumors of group IIb frequently displayed loss of chromosome 8 and included all CDK6 (n = 3) and MYCN (n = 2) amplifications. Correlation analyses of clinical and DNA copy number data identified significant differences between the subgroups (Fig 3B). Patients with group II tumors showed significantly worse overall survival compared with patients with group I tumors. Furthermore, the tumors of group II were more often of anaplastic and less commonly of desmoplastic histology, and had more multicopy number aberrations (amplifications/homozygous deletions).
High Protein Expression Levels of PPM1D and CDK6 in Medulloblastomas The strong correlation between the presence of multicopy number imbalances and adverse prognosis lead us to investigate more closely the novel DNA amplicons at 17q23.2 (n = 1) and 7q21.2 (n = 3). These amplicons were located within the two most frequently gained chromosomal regions in our study (Table 1), and potentially indicate the map position of proto-oncogenes that might be relevant for medulloblastoma pathogenesis. These particular amplicons are defined by three (17q23.2; Fig 4A) and two (7q21.2; Fig 4B) clones, which contain the APPBP2 and PPM1D genes or the CDK6 gene. To evaluate the potential pathogenic importance of these amplicons, we analyzed mRNA and protein expression of PPM1D and CDK6. When compared with normal cerebellum reference, PPM1D mRNA was moderately overexpressed in seven of the nine tumors (Fig 4C), whereas CDK6 mRNA was strongly overexpressed in nine of the nine tumors (Fig 4D). The mean of two individual experiments was plotted and error bars represent 1 x SD. Tumors were selected to include different histologic subtypes and varying complexity of genetic aberrations. Protein expression was determined by IHC analysis of a large series of tumors using TMAs (Appendix Table 1). Strong and widespread immunoreactivity, defined as nuclear staining in more than 30% of the tumor cells, was observed for PPM1D in 148 of 168 (88%) tumors (Figs 4E and 4F) and for CDK6 in 50 of 169 (30%) tumors (Figs 4G and 4H).
CDK6 Expression Status Is an Independent Predictor of Prognosis Comparison of tumors with no or low CDK6 staining versus tumors with moderate or high staining (Fig 5A) identified CDK6 overexpression as a prognostic marker that was associated significantly with poor clinical outcome using univariate analysis (Fig 5B; log-rank test, P < .01). The proportional hazards regression model identified four significant prognostic factors: chemotherapy (P < .01), stage of metastasis (P < .01), histology (P = .02), and CDK6 overexpression (P = .02). Age (P = .57), sex (P = .09), and level of resection (P = .49) were not statistically significant. Results are illustrated in Figure 5C.
The relationships of CDK6 overexpression and the presence of the potential molecular prognostic markers loss 17p13.3 to 13.1 and amplification of MYC or MYCN also were evaluated using informative tumor samples of the matrix-CGH study (n = 29). CDK6 overexpression is not associated with loss of 17p (P = .41) and tumors with MYC or MYCN amplification did not necessarily show CDK6 overexpression (one of two and one of three, respectively).
Matrix-CGH analysis identified DNA copy number alterations of several novel candidate genes that may play a role in medulloblastoma pathogenesis. The PPM1D gene at 17q23.2 was amplified in one medulloblastoma and showed a lower copy number gain in 24 other tumors, making it the most frequent genomic aberration detected in our tumor series (25 of 47; 53%). Notably, the region 7q21.2, containing CDK6, was gained or amplified in 32% (15 of 47) of the tumors. Regarding the frequent losses at 17p13.1, our findings suggest DLG4 as an interesting candidate gene, in addition to other known candidates, such as TP53, LIS1, HIC1, and REN.38-43 Candidate tumor suppressor genes located in the deleted regions on chromosome arm 10q include UTF1, the gene product of which seems to function as transcriptional regulator of DNA-polymerase II promoters, as well as PTEN, which encodes a known tumor suppressor that functions as an inhibitor of the phosphoinositol-3-kinase/AKT signaling pathway.44 Detailed mutational analyses and functional studies will be required to elucidate the role that each of these genes might play in medulloblastoma pathogenesis. In a previous study,45 expression analysis was performed on a subset (n = 26) of the tumor samples used here. When correlating candidate genes from genomic amplicons identified by matrix-CGH with their respective expression ratios, gene dosage effects are observed for three of four loci (CDK6, MYCN, and CCND2). Only MYC amplification did not result in increased gene expression. The high frequency of aberrations on chromosome 17 supports its central importance in medulloblastoma pathogenesis. We confirmed frequent loss on 17p and identified a region of recurrent copy number gain on 17q, both of which were correlated with poor overall survival in univariate analyses. These analyses have to be viewed with caution, however, given that loss of 17p and gain of 17q are not independent events in tumors with i(17q). Chromosome 17 aberrations also had prominent impact in the unsupervised cluster analysis (ie, characterized the tumors of a group of patients with poor prognosis [group II]). Molecular classification correlated significantly with histology mainly because of seven of eight desmoplastic tumors clustering in the group with good prognosis (group I) and eight of 12 anaplastic tumors clustering in the group with poor prognosis. Tumors with classic histology were evenly distributed between groups I and II. Amplifications of MYC and MYCN, which were found to be mutually exclusive in a recent study of medulloblastoma,19 clustered in subgroups IIa and IIb, respectively. In addition to all MYCN amplifications, group IIb tumors contained all of the amplifications involving the CDK6 locus. Recently, MYCN and the CDK6 partner cyclin D1 were identified as targets of the sonic hedgehog pathway involved in the proliferation of neuronal precursors in medulloblastoma.46 Finally, group IIa, which only contained pediatric tumors, represented a clinically defined group of patients with poor prognosis and anaplastic tumors carrying MYC amplifications.6 The amplicon detected at 17q23.2 contained the genes APPBP2 and PPM1D, which were also reported to be coamplified in neuroblastoma47 as well as breast48 and ovarian carcinoma.49 Overexpression of PPM1D but not APPBP2 was reported to cooperate with rat sarcoma viral oncogene homolog in transforming primary mouse fibroblasts.48 Recently, it was shown that overexpression of PPM1D in mouse embryo fibroblasts abolished TP53 tumor-suppressor activity,50 whereas PPM1D deficiency led to resistance against oncogenic transformation mediated by the p16INK4A and p19ARF (p14ARF in human) pathways.51 Although increased PPM1D protein is not associated with poor prognosis, it might contribute to escape from the TP53-dependent control of cell cycle regulation, apoptosis, and DNA repair. PPM1D, which itself is induced by TP53, takes part in a negative-feedback loop downregulating TP53 directly via p38 MAPK.52 Our results, together with published data,53 indicate that different levels of deregulation of the TP53 pathway might exist in medulloblastoma, including amplification or gain and strong expression of PPM1D, copy number gain/amplification of MDM4, frequent monoallelic loss of TP53 and, less commonly, TP53 mutation. The novel amplicon detected at 7q21.2 contained CDK6 as the only gene, and genomic amplification was associated with increased CDK6 protein levels. However, CDK6 overexpression was not restricted to cases with gene amplification, thus suggesting that additional mechanisms exist, resulting in high protein levels of CDK6 in approximately 30% of medulloblastomas. At least in part, a direct connection between PPM1D and CDK6 protein expression in medulloblastoma could be mediated by p38 MAPK52,54 (Fig 6). Overexpression of CDK6 was reported previously in T-cell lymphoma55 and a small subset of malignant gliomas.56 Similar to CDK4, the CDK6 protein is activated by binding to D-type cyclins. The cyclin D/CDK6 complexes phosphorylate the retinoblastoma protein RB1 and the related proteins RBL1 and RBL2, leading to the release of E2F transcription factors.57 In line with our findings, data from mouse models support the importance of retinoblastoma pathway alterations in medulloblastoma development.58-61 High levels of CDK6 protein may result in constitutive phosphorylation of RB1, which may override inhibition by p16INK4A, as shown in murine embryonal stem cells.62 Recent studies also showed that CDK6 overexpression blocks differentiation in leukemic cells63 and osteoblasts,54 suggesting that CDK6 is a key regulator of the RB1 pathway affecting proliferation and differentiation in medulloblastoma and other tumors.
For better risk stratification and the planning of individual therapeutic regimens, it is important to have reliable markers for the assessment of prognosis and response to therapy. At present, prognostic markers for medulloblastoma patients include certain clinical factors; the histologic subtype; and a few molecular parameters, such as loss of 17p, amplification and overexpression of MYC or MYCN, and expression levels of TRKC, ERBB2, and PDGFRB.64-68 In this study, we identified a novel prognostic marker for medulloblastoma patients, namely overexpression of CDK6. In multivariate regression analysis, we found protein overexpression to be an independent prognostic factor for poor overall survival of medulloblastoma patients. Given its higher frequency as compared with the occurrence of DNA amplifications, we consider CDK6 overexpression to be a more informative marker than either MYC or MYCN amplification. No statistically significant overlap was observed between CDK6 expression and 17p13.3 to 13.1 loss in the matrix-CGH data set, suggesting that these are independent prognostic factors that can complement each other in diagnostics. Given that CDK6 expression can be determined by means of IHC on formalin-fixed paraffin sections, this marker is well suited for the routine diagnostic setting as well as an evaluation in controlled clinical studies. Moreover, therapeutic approaches specifically targeting cyclin-dependent kinases are under development and may provide a novel option for the treatment of this tumor.69
The Appendix is included in the full-text version of this article, available online at www.jco.org. It is not included in the PDF (via Adobe® Acrobat Reader®) version.
The authors indicated no potential conflicts of interest.
We thank Stefanie Hofmann, Andrea Wittmann, Frauke Devens, Björn Tews, and Gunnar Wrobel for excellent technical support. We also thank the Mapping Groups of the Wellcome Trust Sanger Institute for initial clone supply and verification.
Supported by Bundesministerium f  r Bildungund Forschung Grants No. 01GS0460 and 01GRO417 (P.L.), and by a Kekulé-fellowship of the Fonds der Chemischen Industrie (F.M.). Both F.M. and B.R. contributed equally to this work. Authors' disclosures of potential conflicts of interest are found at the end of this article.
1. Gurney JG, Kadan-Lottick N: Brain and other central nervous system tumors: Rates, trends, and epidemiology. Curr Opin Oncol 13:160-166, 2001[CrossRef][Medline]
2. Fisher PG, Burger PC, Eberhart CG: Biologic risk stratification of medulloblastoma: The real time is now. J Clin Oncol 22:971-974, 2004 3. Eberhart CG, Kepner JL, Goldthwaite PT, et al: Histopathologic grading of medulloblastomas: A Pediatric Oncology Group study. Cancer 94:552-560, 2002[CrossRef][Medline] 4. Giangaspero F, Perilongo G, Fondelli MP, et al: Medulloblastoma with extensive nodularity: A variant with favorable prognosis. J Neurosurg 91:971-977, 1999[Medline] 5. Giangaspero F, Rigobello L, Badiali M, et al: Large-cell medulloblastomas: A distinct variant with highly aggressive behavior. Am J Surg Pathol 16:687-693, 1992[Medline] 6. Brown HG, Kepner JL, Perlman EJ, et al: "Large cell/anaplastic" medulloblastomas: A Pediatric Oncology Group Study. J Neuropathol Exp Neurol 59:857-865, 2000[Medline]
7. Zeltzer PM, Boyett JM, Finlay JL, et al: Metastasis stage, adjuvant treatment, and residual tumor are prognostic factors for medulloblastoma in children: Conclusions from the Children's Cancer Group 921 randomized phase III study. J Clin Oncol 17:832-845, 1999 8. Gilbertson R: Paediatric embryonic brain tumours: Biological and clinical relevance of molecular genetic abnormalities. Eur J Cancer 38:675-685, 2002
9. Wechsler-Reya RJ: Analysis of gene expression in the normal and malignant cerebellum. Recent Prog Horm Res 58:227-248, 2003 10. Bayani J, Zielenska M, Marrano P, et al: Molecular cytogenetic analysis of medulloblastomas and supratentorial primitive neuroectodermal tumors by using conventional banding, comparative genomic hybridization, and spectral karyotyping. J Neurosurg 93:437-448, 2000[Medline]
11. Pan E, Pellarin M, Holmes E, et al: Isochromosome 17q is a negative prognostic factor in poor-risk childhood medulloblastoma patients. Clin Cancer Res 11:4733-4740, 2005 12. Nicholson JC, Ross FM, Kohler JA, et al: Comparative genomic hybridization and histological variation in primitive neuroectodermal tumours. Br J Cancer 80:1322-1331, 1999[CrossRef][Medline]
13. Reardon DA, Michalkiewicz E, Boyett JM, et al: Extensive genomic abnormalities in childhood medulloblastoma by comparative genomic hybridization. Cancer Res 57:4042-4047, 1997 14. Avet-Loiseau H, Venuat AM, Terrier-Lacombe MJ, et al: Comparative genomic hybridization detects many recurrent imbalances in central nervous system primitive neuroectodermal tumours in children. Br J Cancer 79:1843-1847, 1999[CrossRef][Medline] 15. Schutz BR, Scheurlen W, Krauss J, et al: Mapping of chromosomal gains and losses in primitive neuroectodermal tumors by comparative genomic hybridization. Genes Chromosomes Cancer 16:196-203, 1996[CrossRef][Medline] 16. Gilhuis HJ, Anderl KL, Boerman RH, et al: Comparative genomic hybridization of medulloblastomas and clinical relevance: Eleven new cases and a review of the literature. Clin Neurol Neurosurg 102:203-209, 2000[CrossRef][Medline] 17. Eberhart CG, Kratz JE, Schuster A, et al: Comparative genomic hybridization detects an increased number of chromosomal alterations in large cell/anaplastic medulloblastomas. Brain Pathol 12:36-44, 2002[Medline]
18. Hui AB, Takano H, Lo KW, et al: Identification of a novel homozygous deletion region at 6q23.1 in medulloblastomas using high-resolution array comparative genomic hybridization analysis. Clin Cancer Res 11:4707-4716, 2005
19. Lamont JM, McManamy CS, Pearson AD, et al: Combined histopathological and molecular cytogenetic stratification of medulloblastoma patients. Clin Cancer Res 10:5482-5493, 2004 20. Lichter P, Joos S, Bentz M, et al: Comparative genomic hybridization: Uses and limitations. Semin Hematol 37:348-357, 2000[CrossRef][Medline] 21. Solinas-Toldo S, Lampel S, Stilgenbauer S, et al: Matrix-based comparative genomic hybridization: Biochips to screen for genomic imbalances. Genes Chromosomes Cancer 20:399-407, 1997[CrossRef][Medline] 22. Pinkel D, Segraves R, Sudar D, et al: High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat Genet 20:207-211, 1998[CrossRef][Medline]
23. Schwaenen C, Nessling M, Wessendorf S, et al: Automated array-based genomic profiling in chronic lymphocytic leukemia: Development of a clinical tool and discovery of recurrent genomic alterations. Proc Natl Acad Sci U S A 101:1039-1044, 2004 24. Zielinski B, Gratias S, Toedt G, et al: Detection of chromosomal imbalances in retinoblastoma by matrix-based comparative genomic hybridization. Genes Chromosomes Cancer 43:294-301, 2005[CrossRef][Medline] 25. Giangaspero F, Bigner SH, Kleihues P, et al: Medulloblastoma, in Cavanee WK, Kleihues P (eds): Pathology and Genetics of Tumours of the Nervous System. Lyons, France, IARC Press, 2000, pp 129-137 26. Kortmann RD, Kuhl J, Timmermann B, et al: Postoperative neoadjuvant chemotherapy before radiotherapy as compared to immediate radiotherapy followed by maintenance chemotherapy in the treatment of medulloblastoma in childhood: Results of the German prospective randomized trial HIT '91. Int J Radiat Oncol Biol Phys 46:269-279, 2000[CrossRef][Medline] 27. Korn EL: Censoring distributions as a measure of follow-up in survival analysis. Stat Med 5:255-260, 1986[Medline] 28. Fiegler H, Carr P, Douglas EJ, et al: DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones. Genes Chromosomes Cancer 36:361-374, 2003[CrossRef][Medline] 29. Knight SJ, Lese CM, Precht KS, et al: An optimized set of human telomere clones for studying telomere integrity and architecture. Am J Hum Genet 67:320-332, 2000[CrossRef][Medline]
30. Freier K, Joos S, Flechtenmacher C, et al: Tissue microarray analysis reveals site-specific prevalence of oncogene amplifications in head and neck squamous cell carcinoma. Cancer Res 63:1179-1182, 2003
31. Waye JS, England SB, Willard HF: Genomic organization of alpha satellite DNA on human chromosome 7: Evidence for two distinct alphoid domains on a single chromosome. Mol Cell Biol 7:349-356, 1987
32. Bubendorf L, Kononen J, Koivisto P, et al: Survey of gene amplifications during prostate cancer progression by high-throughout fluorescence in situ hybridization on tissue microarrays. Cancer Res 59:803-806, 1999 33. Lichter P, Cremer T, Borden J, et al: Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries. Hum Genet 80:224-234, 1988[CrossRef][Medline]
34. Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45, 2001 35. Harrell FE Jr, Shih YC: Using full probability models to compute probabilities of actual interest to decision makers. Int J Technol Assess Health Care 17:17-26, 2001[CrossRef][Medline]
36. de Hoon MJ, Imoto S, Nolan J, et al: Open source clustering software. Bioinformatics 20:1453-1454, 2004
37. Fan X, Wang Y, Kratz J, et al: HTERT gene amplification and increased mRNA expression in central nervous system embryonal tumors. Am J Pathol 162:1763-1769, 2003
38. Adesina AM, Nalbantoglu J, Cavenee WK: p53 gene mutation and mdm2 gene amplification are uncommon in medulloblastoma. Cancer Res 54:5649-5651, 1994
39. Biegel JA, Burk CD, Barr FG, et al: Evidence for a 17p tumor related locus distinct from p53 in pediatric primitive neuroectodermal tumors. Cancer Res 52:3391-3395, 1992
40. Di Marcotullio L, Ferretti E, De Smaele E, et al: REN(KCTD11) is a suppressor of Hedgehog signaling and is deleted in human medulloblastoma. Proc Natl Acad Sci U S A 101:10833-10838, 2004 41. Koch A, Tonn J, Kraus JA, et al: Molecular analysis of the lissencephaly gene 1 (LIS-1) in medulloblastomas. Neuropathol Appl Neurobiol 22:233-242, 1996[CrossRef][Medline] 42. McDonald JD, Daneshvar L, Willert JR, et al: Physical mapping of chromosome 17p13.3 in the region of a putative tumor suppressor gene important in medulloblastoma. Genomics 23:229-232, 1994[CrossRef][Medline] 43. Ohgaki H, Eibl RH, Schwab M, et al: Mutations of the p53 tumor suppressor gene in neoplasms of the human nervous system. Mol Carcinog 8:74-80, 1993[Medline]
44. Wu X, Senechal K, Neshat MS, et al: The PTEN/MMAC1 tumor suppressor phosphatase functions as a negative regulator of the phosphoinositide 3-kinase/Akt pathway. Proc Natl Acad Sci U S A 95:15587-15591, 1998
45. Neben K, Korshunov A, Benner A, et al: Microarray-based screening for molecular markers in medulloblastoma revealed STK15 as independent predictor for survival. Cancer Res 64:3103-3111, 2004
46. Oliver TG, Grasfeder LL, Carroll AL, et al: Transcriptional profiling of the Sonic hedgehog response: A critical role for N-myc in proliferation of neuronal precursors. Proc Natl Acad Sci U S A 100:7331-7336, 2003
47. Saito-Ohara F, Imoto I, Inoue J, et al: PPM1D is a potential target for 17q gain in neuroblastoma. Cancer Res 63:1876-1883, 2003 48. Li J, Yang Y, Peng Y, et al: Oncogenic properties of PPM1D located within a breast cancer amplification epicenter at 17q23. Nat Genet 31:133-134, 2002[CrossRef][Medline]
49. Hirasawa A, Saito-Ohara F, Inoue J, et al: Association of 17q21-q24 gain in ovarian clear cell adenocarcinomas with poor prognosis and identification of PPM1D and APPBP2 as likely amplification targets. Clin Cancer Res 9:1995-2004, 2003 50. Bulavin DV, Demidov ON, Saito S, et al: Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity. Nat Genet 31:210-215, 2002[CrossRef][Medline] 51. Bulavin DV, Phillips C, Nannenga B, et al: Inactivation of the Wip1 phosphatase inhibits mammary tumorigenesis through p38 MAPK-mediated activation of the p16(Ink4a)-p19(Arf) pathway. Nat Genet 36:343-350, 2004[CrossRef][Medline] 52. Takekawa M, Adachi M, Nakahata A, et al: p53-inducible wip1 phosphatase mediates a negative feedback regulation of p38 MAPK-p53 signaling in response to UV radiation. Embo J 19:6517-6526, 2000[CrossRef][Medline] 53. Portwine C, Chilton-MacNeill S, Brown C, et al: Absence of germline and somatic p53 alterations in children with sporadic brain tumors. J Neurooncol 52:227-235, 2001[CrossRef][Medline] 54. Moro T, Ogasawara T, Chikuda H, et al: Inhibition of Cdk6 expression through p38 MAP kinase is involved in differentiation of mouse prechondrocyte ATDC5. J Cell Physiol 204:927-933, 2005[CrossRef][Medline] 55. Chilosi M, Doglioni C, Yan Z, et al: Differential expression of cyclin-dependent kinase 6 in cortical thymocytes and T-cell lymphoblastic lymphoma/leukemia. Am J Pathol 152:209-217, 1998[Abstract]
56. Costello JF, Plass C, Arap W, et al: Cyclin-dependent kinase 6 (CDK6) amplification in human gliomas identified using two-dimensional separation of genomic DNA. Cancer Res 57:1250-1254, 1997 57. Ortega S, Malumbres M, Barbacid M: Cyclin D-dependent kinases, INK4 inhibitors and cancer. Biochim Biophys Acta 1602:73-87, 2002[Medline]
58. Zindy F, Nilsson LM, Nguyen L, et al: Hemangiosarcomas, medulloblastomas, and other tumors in Ink4c/p53-null mice. Cancer Res 63:5420-5427, 2003
59. Wetmore C, Eberhart DE, Curran T: Loss of p53 but not ARF accelerates medulloblastoma in mice heterozygous for patched. Cancer Res 61:513-516, 2001
60. Marino S, Vooijs M, van Der Gulden H, et al: Induction of medulloblastomas in p53-null mutant mice by somatic inactivation of Rb in the external granular layer cells of the cerebellum. Genes Dev 14:994-1004, 2000
61. Dyer MA: Mouse models of childhood cancer of the nervous system. J Clin Pathol 57:561-576, 2004 62. Faast R, White J, Cartwright P, et al: Cdk6-cyclin D3 activity in murine ES cells is resistant to inhibition by p16(INK4a). Oncogene 23:491-502, 2004[CrossRef][Medline] 63. Matushansky I, Radparvar F, Skoultchi AI: CDK6 blocks differentiation: Coupling cell proliferation to the block to differentiation in leukemic cells. Oncogene 22:4143-4149, 2003[CrossRef][Medline] 64. Eberhart CG, Kratz J, Wang Y, et al: Histopathological and molecular prognostic markers in medulloblastoma: C-myc, N-myc, TrkC, and anaplasia. J Neuropathol Exp Neurol 63:441-449, 2004[Medline]
65. Gajjar A, Hernan R, Kocak M, et al: Clinical, histopathologic, and molecular markers of prognosis: Toward a new disease risk stratification system for medulloblastoma. J Clin Oncol 22:984-993, 2004
66. Grotzer MA, Hogarty MD, Janss AJ, et al: MYC messenger RNA expression predicts survival outcome in childhood primitive neuroectodermal tumor/medulloblastoma. Clin Cancer Res 7:2425-2433, 2001 67. Gilbertson RJ, Clifford SC: PDGFRB is overexpressed in metastatic medulloblastoma. Nat Genet 35:197-198, 2003[CrossRef][Medline] 68. MacDonald TJ, Brown KM, LaFleur B, et al: Expression profiling of medulloblastoma: PDGFRA and the RAS/MAPK pathway as therapeutic targets for metastatic disease. Nat Genet 29:143-152, 2001[CrossRef][Medline]
69. Fry DW, Harvey PJ, Keller PR, et al: Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts. Mol Cancer Ther 3:1427-1438, 2004 Submitted May 26, 2005; accepted September 9, 2005. This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
16/< 16 years), M (metastatic stage, M0/M1-3), S (overall survival, alive/deceased), and O (occurrence of amplifications/homozygous deletions, yes/no).
