Aberrant methylation of multiple genes in neuroblastic tumours: relationship with MYCN amplification and allelic status at 1p

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Abstract

Aberrant hypermethylation occurs in tumour cell CpG islands and is an important pathway for the repression of gene transcription in cancers. We investigated aberrant hypermethylation of 11 genes by methylation-specific polymerase chain reaction (PCR), after treatment of the DNA with bisulphite, and correlated the findings with MYCN amplification and allelic status at 1p in a series of 44 neuroblastic tumours. This tumour series includes five ganglioneuromas (G), one ganglioneuroblastoma (GN) and 38 neuroblastomas (six stage 1 tumours; five stage 2 tumours; six stage 3 cases; 19 stage 4 tumours, and two stage 4S cases). Aberrant methylation of at least one of the 11 genes studied was detected in 95% (42 of 44) of the cases. The frequencies of aberrant methylation were: 64% for thrombospondin-1 (THBS1); 30% for tissue inhibitor of metalloproteinase 3 (TIMP-3); 27% for O6-methylguanine-DNA methyltransferase (MGMT); 25% for p73; 18% for RB1; 14% for death-associated protein kinase (DAPK), p14ARF, p16INK4a and caspase 8, and 0% for TP53 and glutathione S-transferase P1 (GSTP1). No aberrant methylation was observed in four control normal tissue samples (brain and adrenal medulla). MYCN amplification was found in 11 cases (all stage 4 neuroblastomas), whereas allelic loss at 1p was identified in 16 samples (13 stage 4 and two stage 3 neuroblastomas, and one ganglioneuroma). All but one case with caspase 8 methylation also displayed MYCN amplification. Our results suggest that promoter hypermethylation is a frequent epigenetic event in the tumorigenesis of neuroblastic tumours, but no specific pattern of hypermethylated genes could be demonstrated.

Introduction

According to Schwab and colleagues [1], neuroblastic tumours are “childhood embryonal neoplasms composed of migrating neuroectodermal cells derived from the neural crest and destined for the adrenal medulla and sympathetic nervous system”. Four types of neuroblastic tumours are characterised by a distinct schwannian stroma component and they are classified as neuroblastoma, nodular or intermixed ganglioneuroblastoma, and ganglioneuroma [1]. Neuroblastoma is one of the most common paediatric solid tumours and is clinically variable since some tumours undergo spontaneous regression, while others have metastasised at presentation. Accordingly, the tumour is commonly classified by the stage of tumour development [2]. Patients with early stage (1, 2 and 3) tumours have good/intermediate overall survival chances; however, stage 4 tumours are generally characterised by an unfavourable prognosis. The disseminated form of the disease (stage 4S) involves multiple organs/tissues and has a better prognosis than regular stage 4 tumours.

The molecular pathology of neuroblastoma shows several genomic alterations including amplification of the MYCN oncogene, loss of heterozygosity (LOH) at 1p, 2q, 9p, 11q, 14q and 18q, and gain of genetic material at 17q (for a review see Ref. [1]). The p16INK4a gene on 9p21 and the dcc gene on 18q21 have been proposed as candidate tumour suppressor genes that are inactivated in neuroblastoma 3, 4, and up to three distinct genes of this category involved in neuroblastoma development appear to be located on 1p35.36 [5]. MYCN amplification and 1p deletion are indicators of poor prognosis; since both anomalies frequently present in association (90% of cases with MYCN amplifcation also display 1p loss), they characterise a genetically distinct subgroup of aggressive neuroblastomas [6].

CpG islands are 0.5 to 2.0 Kb DNA regions rich in cytosine-guanine dinucleotides, present in the 5′ region of approximately half of human genes [7]. Aberrant methylation of cytosines within CpG islands is associated with loss of gene expression by repression of transcription of tumour-related genes, and this loss contributes to the pathogenesis and the progression of malignant neoplasms [7]. Examples of this process have been reported in a wide range of tumour types and cancer-related genes [8], but little information is available for neuroblastic tumours. There is a report suggesting that p73 gene silencing is independent of promoter methylation in neuroblastomas [9] and reduced expression of caspase 8 in primary tumours and neuroblastoma-derived cell lines has been correlated with methylation of the corresponding CpG island [10].

We, therefore, determined the methylation profile for 44 neuroblastic tumours, studying eleven genes that frequently show promoter region methylation in other neoplasms (lung, breast, gastric, colon cancer, brain tumours, etc). The genes were: the detoxifying gene glutathione S-transferase P1 (GSTP1), O6-methylguanine-DNA methyltransferase (MGMT), death-associated protein kinase (DAPK), p14ARF, thrombospondin-1 (THBS1), tissue inhibitor of metalloproteinase 3 (TIMP-3), p73, p16INK4a, RB1, TP53, and caspase 8. These genes were selected for analysis on the basis of either, their localisation at genomic regions involved in chromosome deletions in neuroblastomas, or their key tumour development related function. As controls, two normal brain tissue samples and two adrenal medulla samples were also studied using polymerase chain reaction (PCR)-based techniques involving sodium bisulphite modification of DNA (MSP). We also determined the MYCN amplification and allelic status at 1p in the same series of tumours.

Section snippets

Tissue samples and DNA purification

Unfixed frozen tumours and corresponding normal tissues (peripheral blood lymphocytes) from 44 children with neuroblastic tumours were studied. Histological diagnosis was performed according to the World Health Organization (WHO) guidelines [1] as: ganglioneuromas (G; five cases); ganglioneuroblastoma (GN; one sample) and neuroblastoma (N; 38 cases). Staging of the neuroblastomas was done according to the International Neuroblastoma Staging System (INSS) [2], and identified six stage 1 tumours;

Results

As shown in Table 2, MYCN amplification (10-50-fold amplification) was detected in 11 samples. All of them corresponded to the stage 4 neuroblastomas. Allelic loss at 1p was found in 16 tumours: 13 N4, two N3, and one G. Both anomalies were present in nine N4 tumours. Examples of MYCN amplification and 1p allelic loss are shown in Fig. 1.

The results of the MSP for the 11 loci are shown in Table 2, Table 3. A total of 42 of the 44 samples (95%) showed methylation in at least one of these loci.

Discussion

This study of a series of 44 neuroblastic tumours analysed the methylation status of a panel of loci, which have been identified as frequently methylated in other cancers or cell lines. Moreover, the chosen loci are located at genomic regions involved in chromosomal deletions in neuroblastomas (p73 at 1p; caspase 8 at 2q; p14ARF and p16INK4a at 9p; GSTP1 at 11q) or they play key tumour-related functions (RB1, TP53, MGMT, etc.). Our findings also evaluated the analysis of MYCN amplification and

Acknowledgements

Support for this work was provided by grants 00/0331 and 01/0279 from FIS, Ministerio de Sanidad. This work is part of a research partially supported by Programa de Cooperación Cientı́fica con Iberoamérica (Brasil), from Ministerio de Educación (Spain) and Agencia Española de Cooperación Cientı́fica con Iberoamérica. C. Casartelli is supported by Fundaçao de Amparo a Pesquisa do Estado de Sao Paolo (FAPESP) and Coordenaçcao de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES). M. Eva Alonso

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