Prognostic Relevance of Aberrant DNA Methylation in G1 and G2 Pancreatic Neuroendocrine Tumors

Background/Aims: The occurrence and clinical relevance of DNA hypermethylation and global hypomethylation in pancreatic neuroendocrine tumours (PanNETs) are still unknown. We evaluated the frequency of both epigenetic alterations in PanNETs to assess the relationship between methylation profiles and chromosomal instability, tumour phenotypes and prognosis. Methods: In a well-characterized series of 56 sporadic G1 and G2 PanNETs, Methylation-Sensitive Multiple Ligation-dependent Probe Amplification was performed to assess hypermethylayion of 33 genes and copy number alterations (CNA) of 53 chromosomal regions. Long Interspersed Nucleotide Element-1 (LINE-1) hypomethylation was quantified by pyrosequencing. Results: Unsupervised hierarchical clustering allowed to identify a subset of 22 PanNETs (39%) exhibiting high frequency of gene specific methylation and low CNA percentages. This tumour cluster was significantly associated with stage IV (p = 0.04) and with poor prognosis in univariable analysis (p = 0.004). LINE-1 methylation levels in PanNETs were significantly lower than in normal samples (p < 0.01) and were approximately normally distributed. Twelve tumours (21%) were highly hypomethylated, showing variable levels of CNA. Interestingly, only five PanNETs (9%) were observed to show simultaneously LINE-1 hypomethylation and high frequency of gene specific methylation. LINE-1 hypomethylation was strongly correlated with advanced stage (p = 0.002) and with poor prognosis (p < 0.0001). In the multivariable analysis low LINE-1 methylation status and methylation clusters were the only independent significant predictors of outcome (p = 0.034 and p = 0.029, respectively). Conclusion: The combination of global DNA hypomethylation and gene hypermethylation analyses may be useful to define distinct subsets of PanNETs. Both alterations are common in PanNETs and could be directly correlated with tumour progression