RASSF1A promoter region CpG island hypermethylation in phaeochromocytomas and neuroblastoma tumours

Deletions of chromosome 3p are frequent in many types of neoplasia includin g neural crest tumours such as neuroblastoma (NB) and phaeochromocytoma. Re cently we isolated several candidate tumour suppressor genes (TSGs) from a 120 kb critical interval at 3p21.3 defined by overlapping homozygous deleti ons in lung and breast tumour lines. Although mutation analysis of candidat e TSGs in lung and breast cancers revealed only rare mutations, expression of one of the genes (RASSF1A) was absent in the majority of lung tumour cel l lines analysed. Subsequently methylation of a CpG island in the promoter region of RASSF1A was demonstrated in a majority of small cell lung carcino mas and to a lesser extent in non-small cell lung carcinomas. To investigat e the role of 3p TSGs in neural crest tumours, we (a) analysed phaeochromoc ytomas for 3p allele loss (n = 41) and RASSF1A methylation (n = 23) and (b) investigated 67 neuroblastomas for RASSF1A inactivation. 46% of phaeochrom ocytomas showed 3p allele loss (38.5% at 3p21.3). RASSF1A promoter region h ypermethylation was found in 22% (5/23) of sporadic phaeochromocytomas and in 55% (37/67) of neuroblastomas analysed but RASSF1A mutations were not id entified. In two neuroblastoma cell lines, methylation of RASSF1A correlate d with loss of RASSF1A expression and RASSF1A expression was restored after treatment with the demethylating agent 5-azacytidine. As frequent methylat ion of the CASP8 gene has also been reported in neuroblastoma, we investiga ted whether RASSF1A and CASP8 methylation were independent or related event s. CASP8 methylation was detected in 56% of neuroblastomas with RASSF1A met hylation and 17% without RASSF1A methylation (P = 0.0031). These results in dicate that (a) RASSF1A inactivation by hypermethylation is a frequent even t in neural crest tumorigenesis, particularly neuroblastoma, and that RASSF 1A is a candidate 3p21.3 neuroblastoma TSG and (b) a subset of neuroblastom as may be characterized by a CpG island methylator phenotype.