Endocrine Abstracts

Pancreatic neuroendocrine tumours (PNETs) have a lower mutational burden than other tumours, indicating that other mechanisms contribute to tumourigenesis. One such reported mechanism is DNA methylome dysregulation, however, inconsistencies have been observed between gene methylation and protein expression, potentially stemming from the use of standard methylation assessment methods which do not distinguish methylation (5’methylcytosine (5’mC), repressive mark) from hydroxymethylation (5’hydroxymethylcytosine (5’hmC)), at CpG (Cytosine cis-Guanine) sites, the latter protecting against promoter methylation. The aim of our study was therefore to investigate the hydroxymethylome in PNETs. Nine PNETs (5 non-functioning and 4 insulinomas), 5 with adjacent normal tissue, and 2 additional normal pancreata were investigated for 5’hmC by immunohistochemistry. Using paired DNA samples in which one had undergone oxidation prior to bisulfite conversion, specific 5’hmc sites and enrichment of pathways were also interrogated using the Illumina MethylationEPIC array (consisting of >850,000 CpG sites, annotated to 28,637 genes). Normal islets were significantly enriched in 5’hmC, compared to exocrine tissue and PNETs, P<0.0001. Moreover, MethylationEPIC array analysis revealed that PNETs had significantly (P<0.001) lower 5’hmC (0.01% (57/723,389)) compared to normal tissue (8.9% (64,478/727,322), and that 4.1% (29,761/724,156) of CpGs were differentially methylated, of which 50.8% (15,104/29,761) were hypermethylated and 24.9% (3616/15,104) of these were hydroxymethylated in normal tissue (P<0.0001, vs 5’hmC in normal tissue (8.9%)). In normal tissue, 5’hmC were enriched in 36 Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathways (P<0.01, false discovery rate (FDR) <0.05), that included insulin and glucagon signalling. In PNETs, 5’hmC loss and 5’mC gain at the same CpG site were present in >300 genes including DAXX, (P<0.01). Overall, our results indicate that 5’hmC is lost in PNETs, compared to normal islets, and thus important cell specific CpG sites are no longer protected from methylation, and this may therefore lead to the downregulation of anti-tumourigenic pathways.