Quantitative global and gene-specific promoter methylation in relation to biological properties of neuroblastomas

<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>In this study we aimed to quantify tumor suppressor gene (TSG) promoter methylation densities levels in primary neuroblastoma tumors and cell lines. A subset of these TSGs is associated with a CpG island methylator phenotype (CIMP) in other tumor types.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>The study panel consisted of 38 primary tumors, 7 established cell lines and 4 healthy references. Promoter methylation was determined by bisulphate Pyrosequencing for 14 TSGs; and<jats:italic>LINE-1</jats:italic>repeat element methylation was used as an indicator of global methylation levels.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Overall mean TSG Z-scores were significantly increased in cases with adverse outcome, but were unrelated to global<jats:italic>LINE-1</jats:italic>methylation. CIMP with hypermethylation of three or more gene promoters was observed in 6/38 tumors and 7/7 cell lines. Hypermethylation of one or more TSG (comprising TSGs<jats:italic>BLU</jats:italic>,<jats:italic>CASP8</jats:italic>,<jats:italic>DCR2</jats:italic>,<jats:italic>CDH1</jats:italic>,<jats:italic>RASSF1A</jats:italic>and RASSF2) was evident in 30/38 tumors. By contrast only very low levels of promoter methylation were recorded for<jats:italic>APC</jats:italic>,<jats:italic>DAPK1</jats:italic>,<jats:italic>NORE1A</jats:italic>,<jats:italic>P14</jats:italic>,<jats:italic>P16</jats:italic>,<jats:italic>TP73</jats:italic>,<jats:italic>PTEN</jats:italic>and<jats:italic>RARB</jats:italic>. Similar involvements of methylation instability were revealed between cell line models and neuroblastoma tumors. Separate analysis of two proposed<jats:italic>CASP8</jats:italic>regulatory regions revealed frequent and significant involvement of CpG sites between exon 4 and 5, but modest involvement of the exon 1 region.</jats:p></jats:sec><jats:sec><jats:title>Conclusions/significance</jats:title><jats:p>The results highlight the involvement of TSG methylation instability in neuroblastoma tumors and cell lines using quantitative methods, support the use of DNA methylation analyses as a prognostic tool for this tumor type, and underscore the relevance of developing demethylating therapies for its treatment.</jats:p></jats:sec>