Methylation profiling of rectal cancer identifies novel markers of early-stage disease

Background: Radical surgery is the de facto treatment for early rectal cancer. Conservative surgery with transanal endoscopic microsurgery can achieve high rates of cure but the histopathological measures of outcome used to select local treatment lack precision. Biomarkers associated with disease progression, particularly mesorectal nodal metastasis, are urgently required. The aim was to compare patterns of gene-specific hypermethylation in radically excised rectal cancers with histopathological stage. Methods: Locus-specific hypermethylation of 24 tumour suppressor genes was measured in 105 rectal specimens (51 radically excised adenocarcinomas, 35 tissues adjacent to tumour and 19 normal controls) using the methylation-specific multiplex ligation-dependent probe assay (MS-MLPA). Methylation values were correlated with histopathological indices of disease progression and validated using bisulphite pyrosequencing. Results: Five sites (ESR1, CDH13, CHFR, APC and RARB) were significantly hypermethylated in cancer compared with adjacent tissue and normal controls (P < 0.050). Methylation at these sites was higher in Dukes' A than Dukes' 'D' cancers (P = 0.013). Methylation at two sites (GSTP1 and RARB) was individually associated with localized disease (N0 and M0 respectively; P = 0.006 and P = 0.008). Hypermethylation of at least two of APC, RARB, TIMP3, CASP8 and GSTP1 was associated with early (N0 M0) disease (N0, P = 0.002; M0, P = 0.044). Methylation levels detected by MS-MLPA and pyrosequencing were concordant. Conclusion: Locus-specific hypermethylation was more prevalent in early-than late-stage disease. Hypermethylation of two or more of a panel of five tumour suppressor genes was associated with localized disease.