DNA methylation signatures of sporadic colorectal cancer with microsatellite instability

Colorectal cancer (CRC) is a heterogeneous disease shaped by genetic and epigenetic alterations. Approximately 20% of CRCs exhibit widespread CpG island hypermethylation, termed the CpG Island Methylator Phenotype (CIMP), frequently accompanied by MLH1 promoter hypermethylation, deficient mismatch repair (dMMR) and microsatellite instability (MSI). However, methylation patterns associated with MSI, independent of CIMP and MLH1 silencing, and the influence of anatomical location and patient age on the CRC methylome remain incompletely defined. We performed epigenome-wide DNA methylation profiling of 259 sporadic CRCs using the Illumina EPICv2 array. Differential methylation between MSI and microsatellite stable (MSS) CRCs was assessed after adjustment for tumour purity and anatomical location, then MLH1 promoter methylation and CIMP status, to delineate MSI-associated methylation changes. Additionally, we evaluated the effects of anatomical location and age on methylation patterns. While differential methylation between MSS and MSI CRCs was dominated by MLH1 promoter hypermethylation, additional adjustment for MLH1 hypermethylation and CIMP identified 656 CpG sites associated with MSI, beyond the global methylator phenotype. These included hypermethylation at LRP6, GSK3β, and CDK12, identifying differential methylation of genes involved in WNT signalling and transcriptional regulation. Within MSI CRCs, we observed the co-occurrence of MLH1 hypermethylation with promoter hypermethylation at TXNRD1. Anatomical location was strongly associated with methylation, whereas age had more modest effects. These results identify methylation changes associated with sporadic MSI beyond CIMP status and MLH1 hypermethylation, reveal heterogeneity within MSI CRCs, and indicate that anatomical location is a major determinant of the CRC methylome, advancing molecular stratification of CRC.