Epigenetic investigation of multifocal small intestinal neuroendocrine tumours reveals accelerated ageing of tumours and epigenetic alteration of metabolic genes

BackgroundSmall intestinal neuroendocrine tumours (SI-NETs) are the most common malignancy of the small intestine and around 50% of patients present in clinic with multifocal disease. Recent investigations into the genomic architecture of multifocal SI-NETs have found evidence that these synchronous primary tumours evolve independently of each other. They also have extremely low mutational burden and few known driver genes, suggesting that epigenetic dysregulation may be driving tumorigenesis. Very little is known about epigenetic gene regulation, metabolism and ageing in these tumours, and how these traits differ across multiple tumours within individual patients. MethodsIn this study, we performed the first investigation of genome-wide DNA methylation in multifocal SI-NETs, assessing multiple primary tumours within each patient (n=79 primary tumours from 14 patients) alongside matched metastatic tumours (n=12) and normal intestinal epithelial tissue (n=9). We assessed multifocal SI-NET differential methylation using a novel method, comparing primary tumours with matched normal epithelial tissue and an enterochromaffin-enriched cell line to enrich for tumour-specific effects. This method reduced the identification of false positive methylation differences driven by cell composition differences between tumour and normal epithelial tissue. We also assessed tumour ageing using epigenetic clocks and applied metabolic predictors in the dataset to assess methylation variation across key metabolic genes. ResultsWe have identified 12,392 tumour-specific differentially methylated positions (Bonferroni corrected p<0.05) which were enriched for neural pathways. The expression levels of the genes associated with top sites were also found to be significantly altered in SI-NETs. Age acceleration was observed across SI-NETs and a variability in epigenetic age of tumours within each patient, which we believe is reflecting the order in which tumours have developed. This is supported by the correlation of age acceleration with somatic mutational count in the tumours. We have identified SI-NET associated alterations to the methylation patterns in key metabolic genes compared to matched normal tissue, which is more pronounced in metastatic tumours and tumours harbouring chromosome 18 loss of heterozygosity, indicating metabolic differences in these tumour subtypes. ConclusionsWe have identified accelerated ageing and changes to regulation of metabolic genes, alongside an epigenetic signature of multifocal SI-NETs. These findings add to our understanding of multifocal SI-NET biology and their molecular differences which may be instrumental in the development of these elusive tumours.