Epigenetic modifications such as methylation of CpG islands in tumor-suppressor gene promoter regions have been associated with tumor development in many human cancers. Using methylation specific multiplex ligation-dependent probe amplification method, we analyzed the methylation status of 35 different genes in 16 neuroblastoma (NB) cell lines and 50 NB tumor samples (NBs), and investigated whether specific hypermethylation was associated with biological and/or clinical parameters. Among the genes found hypermethylated, the effect of GSTP1 hypermethylation on mRNA and protein expression was also explored. The median number of hypermethylated genes was higher in cell lines compared to NBs (5.5 vs. 2). For eight genes, aberrant methylation of CpG-islands in NB was not (ESR1, PAX5, WT1, CADM1, MSH6, and CDKN2B) or very rarely (CDH13 and GSTP1) reported in literature. GSTP1 was found hypermethylated in 44% of the NB cell lines and in 33% of the stage 4-11qLOH -non MYCN-amplified high risk NBs. Hypermethylation was correlated with reduced mRNA and protein expression. In the whole NBs cohort, GSTP1 hypermethylation was less frequently detected (8%), but found to be associated with lower event-free (EFS) and overall survival. Hypermethylation of GSTP1 showed also association with lower EFS in high risk subgroups as stage 4 and older patients (≥547 days). Our results suggest that, as in several adult cancers, aberrant methylation of GSTP1 may contribute to the carcinogenetic process in NB and could be potentially used as a new marker leading to define an ultra-high risk subgroup.

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10058 Background: Neuroblastoma (NB) is the most common malignant disease of infancy and accounts for approximately 8% of all childhood cancers. The clinical hallmark of this tumor is the marked variability in prognosis depending of the age, stage, and biological characteristics. There is evidence to suggest that the long arm of chromosome 11 (11q) plays a role in NB biology. The ATM gene is located at 11q22–23 and hereditary mutations of this gene cause ataxia-telangiectasia, a recessive disorder associated with a high incidence of neoplasia. The aim of this project was to determine the prevalence of ATM gene mutation and ATM methylation status in 50 NB samples. Methods: Following DNA extraction, PCR products of the 65 exons of the ATM gene and its promoter were screened by DHPLC. This screening was also performed on DNA from 60 blood donors. Alterations detected were analyzed by direct sequencing. Direct and indirect criteria were used to classify the observed nucleotide alterations as mutation (if pathogenic), rare variant (if the allelic frequency in controls was < or = 1%), variant (1.1–2.4%) or polymorphism (> or = 2.5%). The ATM methylation status was analyzed by MS-MLPA (Methylation-Specific Multiplex Ligation-dependent Probe Amplification). Results: Except polymorphisms, 17 different sequence alterations were found in 17 NB samples (34%). Ten of these 17 alterations, found in 11 NB (22%), were rare variants (RV). In 5 NB (10%), RV were found homozygous. At the same time, we found 20 different sequence alterations in 19 controls (32%). Sixteen of these 20 alterations were RV and one was a heterozygous pathogenic mutation. These 17 alterations concern 15 controls (25%). No homozygous RV was found in controls. We found no evidence of ATM promoter hypermethylation in the 48 NB samples analyzed. Conclusions: We found no difference in ATMvariant and RV frequency between NB and control samples. However, as ATM deletion is a frequent event in NB, we found a high frequency of homozygous RV (10%). At present, we are completing this study by screening ATMlarge genomic deletion/duplication using MLPA. Finally, our observations indicate that epigenetic ATM silencing by methylation is uncommon in neuroblastoma. No significant financial relationships to disclose.