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CpG Island Methylator Phenotype Is a Strong Determinant of Poor Prognosis in Neuroblastomas

¹ Carcinogenesis Division, National Cancer Center Research Institute; ² Department of Oral and Maxillo Facial Surgery, University of Tokyo Graduate School of Medicine; ³ Biochemistry Division, Chiba Cancer Center Research Institute; ⁴ Information Division, Research Center for Cancer Prevention and Screening, National Cancer Center; and ⁵ Department of Pediatric Surgery, National Center for Child Health and Development, Tokyo, Japan

Requests for reprints: Toshikazu Ushijima, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Phone: 133-547-5240; Fax: 135-565-1753; E-mail: tushijim{at}ncc.go.jp.

	Abstract

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Neuroblastoma, one of the most common pediatric solid tumors, is characterized by two extreme disease courses, spontaneous regression and life-threatening progression. Here, we conducted a genome-wide search for differences in DNA methylation that distinguish between neuroblastomas of the two types. Three CpG islands (CGI) and two groups of CGIs were found to be methylated specifically in neuroblastomas with a poor prognosis. By quantitative analysis of 140 independent cases, methylation of all the five CGI (groups) was shown to be closely associated with each other, conforming to the CpG island methylator phenotype (CIMP) concept. The presence of CIMP was sensitively detected by methylation of the PCDHB CGIs and associated with significantly poor survival (hazard ratio, 22.1; 95% confidence interval, 5.3-93.4; P < 0.0001). Almost all cases with N-myc amplification (37 of 38 cases) exhibited CIMP. Even in 102 cases without N-myc amplification, the presence of CIMP (30 cases) strongly predicted poor survival (hazard ratio, 12.4; 95% confidence interval, 2.6-58.9; P = 0.002). Methylation of PCDHB CGIs, located in their gene bodies, did not suppress gene expression or induce histone modifications. However, CIMP was significantly associated with methylation of promoter CGIs of the RASSF1A and BLU tumor suppressor genes. The results showed that neuroblastomas with CIMP have a poor prognosis and suggested induction of silencing of important genes as an underlying mechanism.

Key Words: Neuroblastoma • Epigenetics • CIMP • MS-RDA • prognostic marker

	Introduction

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Epigenetic abnormalities, especially alterations in DNA methylation, are intimately involved in development of various human tumors (1). Aberrant methylation of promoter CpG islands (CGI) causes inactivation of tumor suppressor genes. Genomic instability is caused by genomic hypomethylation and is associated with hypermethylation (2, 3). Identification of epigenetic abnormalities in human cancers is expected to lead not only to discovery of novel disease mechanisms but also to development of new diagnostic markers. Therefore, we previously developed a genome-wide scanning method, methylation-sensitive representational difference analysis (MS-RDA), for detecting differences in DNA methylation (4, 5). This technique analyzes unmethylated, CpG-rich regions of the genome and has already identified genes silenced in human lung, stomach, breast, and pancreatic cancers (6–9).

Neuroblastoma derived from primitive cells of the sympathetic nervous system is one of the most common solid tumors in childhood, characterized by two extreme disease courses, spontaneous regression, and life-threatening progression (10, 11). The clinical outcome is associated with disease stage, age at diagnosis, histologic classification, N-myc amplification, DNA ploidy, and TrkA overexpression (10–12). These characteristics are therefore used to classify cases into low-, intermediate-, and high-risk groups. However, especially in the cases with intermediate risk, prediction of prognosis and therapeutic decision-making are still difficult, and development of new markers is an urgent priority. Moreover, the molecular bases underlying the two distinct clinical courses are still unknown, and their clarification is needed to allow development of novel therapeutics.

In the present study, considering the major involvement of epigenetic machinery in embryonic development (13, 14), we searched for differences in DNA methylation between neuroblastomas with a good prognosis and counterparts with a poor prognosis by MS-RDA.

	Materials and Methods

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Tissue Samples and Cell Lines. Tumor samples were obtained from 145 nonrecurrent cases between 1995 and 1999 and were used under approval of institutional review boards. The mean age at initial diagnosis was 27 months (range, 0-216 months). Their clinical stages were determined according to the International Neuroblastoma Staging System, and 40, 17, 20, 60, and 8 cases belonged to stages I, II, III, IV, and IVS, respectively. Normal adrenal medulla tissue was collected from a case undergoing nephrectomy for a renal cancer. Neuroblastoma cell lines were obtained from the American Type Culture Collection (Manassas, VA), the Japanese Collection of Research Bioresources (Tokyo, Japan), and the RIKEN Bio Resource Center (Tsukuba, Japan). GANB was established by A.N. and normal human bronchial epithelial cells were purchased from Cambrex (East Rutherford, NJ). High molecular weight DNA and total RNA were extracted as previously described (7). Total RNAs of brain and adrenal glands were purchased from Clontech (Palo Alto, CA).

MS-RDA and Database Search. MS-RDA was done as previously described (4, 5). Genomic DNA of primary neuroblastomas with a good prognosis (cases 92, 98, 104, 112, and 148) and neuroblastoma cell lines established from cases with a poor prognosis (CHP134, IMR32, GANB, NGP, and TGW) were digested with HpaII, and then two pooled DNA samples were prepared. Although use of cell lines is highly recommended for MS-RDA (5), no cell lines were available for neuroblastomas with a good prognosis, and therefore we used the primary samples. To isolate CGIs that were hypermethylated in the latter, the cell line pool was used as the tester, and the primary tumor pool as the driver. MS-RDA in the opposite direction was also done. For each series of MS-RDA, 96 clones were analyzed for redundancy, and nonredundant clones were sequenced. Their genomic origins were examined using BLASTN software http://www.ncbi.nlm.nih.gov/BLAST/).

Sodium Bisulfite Modification and Methylation-Specific PCR. One microgram of DNA underwent sodtlbium bisulfite modification (15), and was suspended in 20 µL of TE buffer. For methylation-specific PCR (MSP), 1 µL of the solution was used for PCR with primers specific to methylated or unmethylated sequences. Using DNA from normal human bronchial epithelial and DNA methylated with SssI methylase, annealing temperatures specific for methylated and unmethylated primers were determined. Quantitative MSP was done separately for methylated DNA molecules and for unmethylated DNA molecules. Standard DNA was prepared by cloning PCR products amplified by methylated and unmethylated primers into a vector, respectively. The numbers of methylated and unmethylated molecules in a test sample were determined by comparing their amplification with those of standard samples containing 10 to 10⁶ molecules. The "methylation index" was calculated as the fraction of methylated molecules in the total DNA molecules (no. methylated molecules + no. unmethylated molecules). Each sample was analyzed twice, blind to clinical information, and high reproducibility was confirmed (correlation coefficient = 0.98).

The protocadherin ß (PCDHB) family consists of 16 genes with single exons and three pseudogenes on 5q31, and their CGIs are located in the gene bodies. MSP primers were designed to recognize 17 of the 19 members (all except for the PCDHB1 gene and the PCDHB19 pseudogene). The protocadherin (PCDHA) family consists of 15 genes and one pseudogene having unique first exons and shared exons 2 to 4 on 5q31, and their CGIs are located in exon 1. MSP primers were designed to recognize 13 of the 16 members (all except for the PCDHAC1 and PCDHAC2 genes and the PCDHA14 pseudogene). The hepatocyte growth factor-like protein (HLP/MSP/MST1) gene is highly homologous to macrophage stimulating, pseudogene 9 (MSTP9), and MSP primers were designed to recognize both of these. For DKFZp451I127, FLJ37440, Zinc finger protein 297 (ZNF297), and Cytochrome p450 CYP26C1 (CYP26C1), MSP primers were designed to recognize each of them specifically. The primers and PCR conditions are shown in Supplementary Table 1.

Semiquantitative and Quantitative Reverse Transcription-PCR. cDNA was synthesized from 3 µg of total RNA treated with DNase using a Superscript II kit (Invitrogen Co., Carlsbad, CA). For semiquantitative reverse transcription-PCR (PCDHB1-PCDHB15), multiple cycles of PCR were tested for each gene, and numbers giving a wide dynamic range were determined. The primers and PCR conditions are shown in Supplementary Table 2. For quantitative reverse transcription-PCR (PCDHB16), the number of cDNA molecules was determined by quantitative PCR, as in quantitative MSP, and the copy number was normalized to that of GAPDH.

Chromatin Immunoprecipitation Assay. From 1 x 10⁶ cells, DNA/histone complexes were immunoprecipitated, and DNA was eluted in 30 µL of TE after reversing cross-linking. Copy numbers of DNA molecules of the PCDHB16 exon, RASSF1A promoter, and GAPDH promoter in 1 µL of the eluate were determined by quantitative PCR (primer sequences in Supplementary Table 3), and normalized to the copy numbers in the input. Anti-acetyl-histone H3 antibody (AcH3) and anti-dimethylated-histone H3 (lysine 9; MetH3K9) were purchased from Cell Signalling (Beverly, MA).

Statistical Analysis. Associations between methylation levels among CGI groups were examined using the Pearson correlation coefficient and Fisher's exact test. Survival time was measured from the date of initial diagnosis to the date of death or last contact. Kaplan-Meier analysis and log-rank tests were done to compare survival between the groups defined by methylation levels. Hazard ratio (HR) between groups and dose-response relationships between methylation levels and survival were estimated by the Cox proportional hazard model. Kaplan-Meier curves were drawn with the help of Aabel software (Gigawiz. Ltd. Co., Tulsa, OK) and other analyses were conducted using SAS version 8.2 (SAS Institute, Inc., Cary, NC).

	Results

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Genome-Scanning for Differentially Methylated CpG Islands. MS-RDA was done using five primary neuroblastomas with a good prognosis and five neuroblastoma cell lines established from cases with a poor prognosis. Seven DNA fragments, derived from CGIs of PCDHB16, PCDHA1, HLP, DKFZp451I127, FLJ37440, ZNF297, and CYP26C1, were isolated as methylated in the latter samples. No DNA fragments were isolated as methylated in the former samples. Methylation statuses of (i) 17 CGIs of the PCDHB family (detailed structure in Supplementary Fig. 1), (ii) 13 CGIs of the PCDHA family, (iii) HLP and its pseudogene, and (iv) other four unique CGIs were examined by MSP. This revealed that the PCDHB family (5q31), the PCDHA family (5q31), HLP (3p21) and its pseudogene (1p36), DKFZp451I127 (5q14), and CYP26C1 (10q23) were specifically methylated in the latter samples (Fig. 1A and B).

View larger version (56K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Five CGIs isolated by MS-RDA and their methylation statuses in the samples used for MS-RDA. A, genomic structures of the five CGIs. GpC, CpG, and HpaII recognition sites (5'-CCGG-3') are shown by ticks. Closed boxes, exons; open boxes, clones isolated by MS-RDA; shaded boxes, regions analyzed by MSP. B, methylation statuses analyzed by MSP. M, MSP using primers specific to methylated DNA; U, MSP using primers specific to unmethylated DNA. All the five CGIs were found to be differentially methylated between the two groups used for MS-RDA.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 2. The distribution of methylation indices among the 140 cases analyzed: (i) 17 CGIs of the PCDHB family, (ii) 13 CGIs of the PCDHA family, (iii) CGIs of HLP and its pseudogene, (iv) DKFZp451I127, and (v) CYP26C1.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Predictive powers of methylation of the five CGI (groups) identified, and their multiple methylation: (i) 17 CGIs of the PCDHB family, (ii) 13 CGIs of the PCDHA family, (iii) CGIs of HLP and its pseudogene, (iv) DKFZp451I127, (v) CYP26C1, and (vi) methylation of three of these or more were analyzed by the Kaplan-Meier method using 140 primary samples. The PCDHB family, HLP, DKFZp451I127, CYP26C1, and methylation of multiple CGI (groups) had significant influence on survival.

Existence of the CpG Island Methylator Phenotype in Neuroblastomas. Methylation of the different CGI (groups) had shown close associations with each other Table 1). When correlation was analyzed as a continuous value, Pearson correlation coefficients between PCDHB and PCDHA, HLP, DKFZp451I127 and CYP26C1 were 0.55, 0.70, 0.26 and 0.77, respectively. This showed that multiple CGIs were simultaneously methylated in neuroblastomas with a poor prognosis (Supplementary Fig. 2A). The simultaneous methylation of (i) 17 CGIs of the PCDHB family, (ii) 13 CGIs of the PCDHA family, (iii) CGIs of HLP and its pseudogene, (iv) DKFZp451I127 CGI, and (v) CYP26C1 CGI conformed with the concept of the CpG island methylator phenotype (CIMP; ref. 16).

Predictive Power of CIMP, Compared with Known Prognostic Factors. Univariate analyses showed that N-myc amplification, low TrkA expression, DNA diploidy, and an age no younger than 1 year gave HRs of 9.5 (95% CI, 4.4-20.5), 3.9 (95% CI, 1.7-9.3), 4.2 (95% CI, 1.65-10.8), and 12.3 (95% CI, 3.7-41.7). Cases were stratified by these known factors Table 2). In those without N-myc amplification, CIMP also showed an influence with a HR of 12.4 (95% CI, 2.6-58.9), but almost all cases with N-myc amplification (37 of the 38 cases) showed CIMP. It was suggested that cases with N-myc amplification were contained in the cases with CIMP. CIMP was independent from TrkA overexpression, DNA ploidy, and age at diagnosis. Stage seemed to be a stronger prognostic factor. Notably, even when limited to cases in stages III and IV without N-myc amplification, which are classified into the intermediate risk group and clinically important, CIMP gave a HR of 4.8 (95% CI, 1.0-23.0; P = 0.048).

Effects of PCDHB Methylation on Gene Expression and Chromatin Structure. The CGIs of the PCDHB family were located in their gene bodies, whose methylation generally does not block gene transcription (17). The actual effects of methylation on expression were examined for 16 genes of the PCDHB family using 10 primary neuroblastomas with low methylation and five primary neuroblastomas with high methylation. The methylation was not associated with loss of expression a (representative result is shown in Fig. 4A). The effect of methylation of the PCDHB16 CGI on the histone modification was further examined by chromatin immunoprecipitation assay. It was found that DNA methylation of the PCDHB16 CGI did not induce histone H3 lysine 9 methylation or histone H3 deacetylation (data not shown).

View larger version (20K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Effects of methylation of the PCDHB family and DKFZp451I127 on gene expression. A, PCDHB16 expression was analyzed by quantitative RT-PCR in 10 primary samples with low methylation (open columns) and five primary samples with high methylation (closed columns), and no difference was observed between the two groups. B, silencing of DKFZp451I127 by methylation of its promoter CGI. The CGI was methylated in four cell lines, TGW, NB-1, SK-N-SH, and GOTO, whereas it was unmethylated in one cell line, SK-N-MC. DKFZp451I127 was expressed in SK-N-MC, but not expressed at all in the four cell lines with the promoter methylation.

Among the five CGI (groups) identified in this study, only that of DKFZp451I127 was located in a promoter region. Although its methylation was infrequent, the methylation was observed only in neuroblastomas with CIMP (Table 1), and was associated with expression loss (Fig. 4B). To make the association clearer, methylation statuses were analyzed for eight additional CGIs in promoter regions. It was shown that methylation of promoter CGIs of RASSF1A (3p21) and BLU (3p21) was far more frequently observed in neuroblastomas with CIMP (Table 1, P < 0.0001). At the same time, there was a preference for CGIs affected by CIMP among CGIs in promoter regions, and also among those outside promoter regions (Table 2).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Extensive methylation of multiple CGIs, conforming with the concept of CIMP, was here found specifically present in neuroblastomas with a poor prognosis and could be sensitively detected by focusing on the PCDHB family. PCDHB methylation did not suppress gene expression or induce histone modification. However, CIMP was associated with promoter methylation of RASSF1A and BLU genes and one of the mechanisms underlying the poor prognosis of neuroblastomas seemed to be silencing of these and possibly other tumor suppressor genes and genes important for differentiation.

CIMP was originally identified in colon cancers (16), but there has been some dispute over its presence (20). The clear correlation between CIMP and a poor prognosis found here for neuroblastomas was unequivocal and presumably reflects an intrinsic tendency for methylation of CGIs. This is because, first, neuroblastomas have a much shorter history than colon cancers, and the accumulated number of methylated CGIs in neuroblastomas is expected to parallel the speed of occurrence of methylation. Second, methylation of the PCDHB family did not affect gene expression, and there should have been no selection of cells with the PCDHB methylation, in contrast to the case of promoter methylation of tumor suppressor genes. Investigation into the mechanism of the intrinsic tendency for methylation of multiple CGIs is necessary. Furthermore, alleviation of the intrinsic tendency could block progression of neuroblastomas and have potential therapeutic value.

Among the six CGI (groups) outside promoter regions analyzed here, CIMP in neuroblastomas preferentially affected four CGI (groups); those of the PCDHB family, the PCDHA family, HLP, and CYP26C1. Unexpectedly, three CGIs that are known to be frequently methylated in human colon cancers with CIMP, MINT1, MINT2, and MINT17 (16) were not methylated in neuroblastoma cell lines (data not shown). Among the nine CGIs in promoter regions analyzed, CIMP in neuroblastomas affected only three, those of RASSF1A, BLU, and DKFZp451I127. The nine CGIs were selected based upon previous reports as tumor suppressor genes (RASSF1A, BLU, p16, and hMLH1; refs. 21–23), the chromosomal location flanking the PCDHB family (PCDHB1 and TAF7), our previous report on the fidelity in inheriting methylation patterns (p41Arc and SIM2; ref. 19), and the findings here (DKFZp451I127). Because gene expression and possibly chromatin structures affect the frequency of de novo methylation (24, 25), the available data suggest that CGIs useful to sensitively detect CIMP might vary according to the tumor type.

The influence of CIMP on prognosis was here found to be comparable to that of the currently most reliable marker, N-myc amplification, and stronger than TrkA overexpression and DNA ploidy on univariate analysis. Subgroup analysis showed that the influence was independent of TrkA overexpression, DNA ploidy and age at diagnosis and CIMP had influence even in cases without N-myc amplification and in advanced stages. These points strongly indicated CIMP to be a promising new prognostic marker. However, the cutoff values adopted here are tentative, and the HRs obtained could have been overestimated. A validation study using independent samples is necessary for further evaluation. The fact that cases with CIMP contained almost all the cases with N-myc amplification suggested that a common molecular mechanism caused both alterations, or that CIMP may lead to N-myc amplification. Whatever the case, the findings might provide clues to molecular mechanisms of neuroblastoma development.

In summary, the present study showed that CIMP is present specifically in neuroblastomas with poor prognosis and that can be sensitively detected by focusing on PCDHB methylation. CIMP seems to be a promising new prognostic marker, and its evaluation and investigations into the mechanisms underlying CIMP in neuroblastomas seem warranted.

	Acknowledgments

 
Grant support: Grant-in-aid for the Third-term Cancer Control Strategy Program from the Ministry of Health, Labour, and Welfare, Japan and Research Resident Fellowship from the Foundation for Promotion of Cancer Research (M. Abe).

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 Drs. E. Okochi-Takada and G. S. Goldberg for critical reading of the rticle and the institutions for participation in the collection of clinical materials.

	Footnotes

 
Note: Supplementary data for this article are available at Cancer Research online (http://cancerres.aacrjournals.org/).

Received 7/27/04. Revised 11/14/04. Accepted 11/24/04.

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