Early detection of colorectal cancer: biomarker discovery

Colorectal cancer (CRC) is the third most common cancer worldwide, with about 1.2 million new cases diagnosed each year. CRC derived from the gradual accumulation of both genetic and epigenetic changes that transform the normal intestinal glandular epithelium into invasive cancer. While the genetic alterations are already used as prognostic and predictive markers, epigenetic alterations are currently the subject of intense research in the biomedical field because are considered as common and early molecular events in carcinogenesis that potentially could be used as molecular markers. The aims of this study were: to identify the alterations that characterize the CRC methylome; verify that these changes represent early events in the development of CRCs; explore the use of ultra-sensitive molecular techniques to track these alterations in biological matrices suitable for a non-invasive assessment (blood and stool); correlate the methylation alterations with the associated genes expression. The methylome analysis, conducted by Infinium HumanMethylation450 BeadChip on CRC and adenoma samples, has allowed us to delineate both the CRC methylation profile and that associated with precancerous stages. The gene-set/pathway enrichment analysis conducted by Toppgene and based on case/control differential methylation analysis results of CRCs and adenomas, allowed the identification of pathways involved in CRC carcinogenesis. The contribution of these pathways had never been widely emphasized and discussed in the literature. A very important result, emerged from the comparison of the genes belonging to the most altered significant pathways both in CRCs and adenomas, has been the identification of methylation alterations of regions, known as CpG islands, since the earliest stages of precancerous lesion suggesting that the alteration of specific pathways can lead the tumorigenic process. The selection of these regions has allowed us to identify a panel of biomarkers that can discriminate, with high specificity and sensitivity, CRCs and adenomas from peritumoral / normal counterpart. This panel has been extensively validated in silico in over 600 samples. We also evaluated the gene expression associated with these regions; more than 70% of hypermethylated CpG islands correlated with a downregulation in tumor tissue. To evaluate the usefulness of these biomarkers as a potential tool for non-invasive early diagnosis of CRC in clinical practice, we tried to trace through the use of ultra-sensitive techniques (methyl_BEAMING), the hypermethylation of three selected biomarkers in DNA extract from blood and stool. The hypermethylation of these regions, due to the presence of tumoral DNA, has been traced with great sensitivity and specificity in both matrices confirming the usefulness of these regions as possible biomarkers for the early diagnosis and traceability of residual disease of CRC.