Abstract
Aberrant methylation of DNA is supposed to be a major and early driver of colonic adenoma development, which may result in colorectal cancer (CRC). Although gene methylation assays are used already for CRC screening, differential epigenetic alterations of recurring and nonrecurring colorectal adenomas have yet not been systematically investigated. Here, we collected a sample set of formalin-fixed paraffin-embedded colorectal low-grade adenomas (n = 72) consisting of primary adenomas without and with recurrence (n = 59), recurrent adenomas (n = 10), and normal mucosa specimens (n = 3). We aimed to unveil differentially methylated CpG positions (DMPs) across the methylome comparing not only primary adenomas without recurrence vs primary adenomas with recurrence but also primary adenomas vs recurrent adenomas using the Illumina Human Methylation 450K BeadChip array. Unsupervised hierarchical clustering exhibited a significant association of methylation patterns with histological adenoma subtypes. No significant DMPs were identified comparing primary adenomas with and without recurrence. Despite that, a total of 5094 DMPs (false discovery rate <0.05; fold change >10%) were identified in the comparisons of recurrent adenomas vs primary adenomas with recurrence (674; 98% hypermethylated), recurrent adenomas vs primary adenomas with and without recurrence (241; 99% hypermethylated) and colorectal adenomas vs normal mucosa (4179; 46% hypermethylated). DMPs in cytosine-phosphate-guanine (CpG) islands were frequently hypermethylated, whereas open sea- and shelf-regions exhibited hypomethylation. Gene ontology analysis revealed enrichment of genes associated with the immune system, inflammatory processes, and cancer pathways. In conclusion, our methylation data could assist in establishing a more robust and reproducible histological adenoma classification, which is a prerequisite for improving surveillance guidelines.
Highlights
Colorectal cancer (CRC) has the third highest cancer incidence worldwide with approximately 1.8 million new cases in 2018.1 As colorectal cancer (CRC) develops mainly via the adenoma-carcinoma sequence, endoscopic resection of adenomas reduces CRC risk significantly.[2]
Several distinct molecular pathways have been described: (a) the chromosomal instability (CIN) pathway that is associated with aneuploidy, copy number alterations (CNAs), and mutations most frequently affecting APC, TP53, and KRAS7,8; (b) the microsatellite instability pathway that is caused by DNA hypermutation[9]; (c) the CpG island mismatch repair phenotype (CIMP), which is defined by extensive methylation of CpG islands.[10]
The FDA approved a SEPT9 gene methylation assay in 2016 as an additional CRC screening option to be considered for patients who have a history of an intermittent completion of colonoscopy and fecal occult blood tests.[35]
Summary
Colorectal cancer (CRC) has the third highest cancer incidence worldwide with approximately 1.8 million new cases in 2018.1 As CRC develops mainly via the adenoma-carcinoma sequence, endoscopic resection of adenomas reduces CRC risk significantly.[2]. Several distinct molecular pathways have been described: (a) the chromosomal instability (CIN) pathway that is associated with aneuploidy, copy number alterations (CNAs), and mutations most frequently affecting APC, TP53, and KRAS7,8; (b) the microsatellite instability pathway that is caused by DNA hypermutation[9]; (c) the CpG island mismatch repair phenotype (CIMP), which is defined by extensive methylation of CpG islands.[10] Abnormal DNA methylation is supposed to happen very early and affects up to 85% of adenomas and CRCs.[11] These tumors exhibit pathological methylation patterns predominantly affecting canonical and noncanonical WNT-pathway genes (eg, AXIN2, CTNNB1, SFRP1, and SFRP2) and Polycomb group genes.[12,13] it is reasonable to assume that adenoma recurrence after polypectomy might at least partially be triggered by methylation pattern changes. Selected markers were subsequently quantitatively analyzed by pyrosequencing to validate the array results
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