Another pathway contributing to CRC is DNA mismatch repair, leading to microsatellite instability (MSI) when dysregulated. MSI is frequently caused by promoter hypermethylation of the mismatch repair gene mutL homolog 1 (MLH1), but may also be caused by mutations to MLH1 or other mismatch repair genes.(4) A number of epigenetic events distinguishing the MSI subtype from microsatellite stable (MSS) CRCs have been demonstrated. We have previously shown that methylation of secreted frizzled-related protein 1 (SFRP1), an extracellular Wnt antagonist, is associated with MSS CRC.5 Conversely, methylation of a different Wnt antagonist, dickkopf Wnt signaling pathway inhibitor 1 (DKK1), is associated with MSI-high (MSI-H) CRC.5 Methylation of Wnt5a, which activates non-canonical Wnt signaling, is also associated with MSI. (6)
In order to explore CRC subtype-specific methylation events occurring at Wnt signaling genes, we selected two genes for further analysis, APC and immunoglobulin transcription factor 2 (ITF2). DNA methylation of the tumour suppressor APC has been observed in CRC. However, to what extent APC methylation plays a role in colorectal carcinogenesis is unclear, as a wide range of methylation levels has been reported. (7-10) Conflicting evidence exists regarding APC methylation in MSI CRCs. Some small-scale studies (MSI tumours N ≤ 29) have suggested that APC methylation may be associated with the MSI subtype but others show no significant difference (MSI tumours N ≤ 44).8,9,11-15 The transcription factor ITF2, a Wnt signaling target gene that is overexpressed in some adenomas and CRCs, is shown to incur methylation in several cancer types.(16-19)
In this study we quantified promoter methylation of ITF2 and APC using the semi-quantitative real-time PCR-based MethyLight technique in two case-case studies nested in population-based CRC cohorts from the Ontario Familial Colorectal Cancer Registry (n = 330) and the Newfoundland Familial Colorectal Cancer Registry (n = 102) comparing tumour subtypes stratified by MSI status. We also compared methylation of both genes in matched non-neoplastic colorectal mucosa and tumour (n = 47). We established a methylation cutoff of 10% in order to dichotomize values into two groups: unmethylated or methylated. Using this cutoff we determined that ITF2 and APC methylation are significantly associated with tumor versus normal state (both P < 1.0×10-). (6)
In our population from Ontario 26.1% of MSS cases incurred ITF2 methylation compared to 42.4% of MSI-H cases (P = 0.002). Similarly in Newfoundland 29.4% of MSS cases and 56.9% of MSI-H cases were methylated (P = 0.005). Ontario and Newfoundland data was then pooled for further analysis. Among MSI-H cases, ITF2 methylation was associated with MLH1 promoter hypermethylation (P = 6.72×10-4). ITF2 methylation was not found to be associated with age, sex, stage, grade, tumour location, histological type, CIMP status, or survival. APC methylation, although tumor-specific, did not show a significant association with MSI status, age, sex, stage, or other clinicopathological variables, indicating that it is a general tumour-specific CRC biomarker.
Based on its high specificity for CRC, APC methylation may offer usefulness as a marker within a panel of other genes for CRC detection and ITF2 may be useful for detection of MSI-H tumours. Future studies to independently validate these findings are warranted, especially in non-invasive biological sources such as serum or stool. Overall, this study has investigated methylation of the Wnt genes APC and ITF2 in two large cohorts of MSI-H and matched MSS CRC tumours and determined that ITF2 methylation is significantly associated with MSI-H tumours while APC methylation is a tumour- specific event in CRC, which does not differ significantly between MSI-H and MSS subtypes or other clinicopathological variables.
Written by Andrea J. Savio1,2 and Bharati Bapat1,2,3
- Lunenfeld-Tanenbaum Research Institute, Sinai Health System
- Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto
- Department of Pathology, University Health Network
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