DNA Methylation in Rectal Cancer: Validating Findings of an Epigenome-Wide Association Study

Abstract

Background: Preliminary studies conducted by our group utilised the Illumina Infinium Human Methylation 450k Beadchip array to perform an epigenome-wide association study (EWAS) of 15 matched rectal tumour (RT) and adjacent mucosa (AM) samples. 176 differentially methylated probes (DMPs) were identified (P<0.00001). RT was also characterised by significantly reduced global methylation in comparison to AM.

Aims: This study aimed to validate specific and global DNA methylation differences identified by our preliminary work. We then sought to replicate the findings in additional samples. Finally, we attempted to identify correlations between DNA methylation differences and clinicopathological tumour features.

Materials and Methods: Polymerase chain reaction (PCR) and bisulphite pyrosequencing assays were designed and optimised to quantify DNA methylation at nine DMPs nominated by our EWAS. Pearson’s test was used to calculate the correlation between 450k and pyrosequencing methylation values. Replication was performed in an additional cohort of 68 matched colorectal tumour and AM pairs. Global DNA methylation of the discovery cohort was quantified using the luminometric methylation assay (LUMA). Potential relationships between tumour features and differential methylation were investigated using univariate (t-tests or ANOVA) and multivariate analyses (logistic regression).

Results: All DMPs selected for validation showed strong correlations between bisulphite pyrosequencing and Illumina 450k methylation values (r= 0.87-0.97). Global hypomethylation was observed in RT (54.6%) when compared to AM (63.5%, P = 0.021). All probes displayed significant levels of differential methylation in the replication cohort (P = <2.2e-16). No significant associations were observed between DNA methylation and clinicopathological tumour features, however this may reflect the relatively small number of samples assessed.

Conclusions: Our studies have identified and validated a novel methylomic signature of rectal cancer. Although no clinicopathological correlations were observed with the DMPs investigated, others may represent potential targets in the diagnosis, monitoring and risk stratification of rectal cancer.