Implementation of a MSRE ddPCR method for the detection of methylated WIF1 and NPY genes in colorectal cancer patients

Background: Colorectal cancer is a worldwide leading cause of death accounting for high-rate mortality. Mutations in the epidermal growth factor receptor and RAS/MAPK pathways, as well as altered methylation genes profiles, have been described as molecular mechanisms promoting and sustaining tumour development and progression. Aberrant methylation is a well-known epigenetic mechanism involved in gene regulation; particularly several genes were reported as hypermethylated in CRC. Recently, it was shown that epigenetic alterations in genes such as neuropeptide y, proenkephalin and Wnt inhibitory factor 1 can be used as promising disease biomarkers. Almost all methods developed for the DNA methylation analysis combined next generation sequencing, conventional qRT-PCR or ddPCR with the prior DNA modification with sodium bisulfite. Methods and results: We implemented a ddPCR method to assess the methylation status of Wnt inhibitory factor 1 and neuropeptide y using the methylation sensitive restriction enzyme approach that does not impact on DNA quality and guarantees the discrimination of DNA methylation independent of bisulfite conversion. Conclusions: We showed that this method is robust and sensitive also allowing the monitoring of CRC disease progression when applied to circulating free DNA samples from liquid biopsies, proving to be a fast and easy to implement assay to be used for the monitoring of the methylation pattern of clinically relevant target genes.