Evaluation of global and intragenic hypomethylation in colorectal adenomas improves patient stratification and colorectal cancer risk prediction

Abstract

Background: Aberrant DNA hypomethylation of the long interspersed nuclear elements (LINE-1 or L1) has been recognized as an early event of colorectal transformation. Simultaneous genetic and epigenetic analysis of colorectal adenomas may be an effective and rapid strategy to identify key biological features leading to accelerated colorectal tumorigenesis. In particular, global and/or intragenic LINE-1 hypomethylation of adenomas may represent a helpful tool for improving colorectal cancer (CRC) risk stratification of patients after surgical removal of polyps. To verify this hypothesis, we analyzed a cohort of 102 adenomas derived from 40 high-risk patients (who developed CRC in a post-polypectomy of at least one year) and 43 low-risk patients (who did not develop CRC in a post-polypectomy of at least 5 years) for their main pathological features, the presence of hotspot variants in driver oncogenes (KRAS, NRAS, BRAF and PIK3CA), global (LINE-1) and intragenic (L1-MET) methylation status.

Results: In addition to a significantly higher adenoma size and an older patients' age, adenomas from high-risk patients were more hypomethylated than those from low-risk patients for both global and intragenic LINE-1 assays. DNA hypomethylation, measured by pyrosequencing, was independent from other parameters, including the presence of oncogenic hotspot variants detected by mass spectrometry. Combining LINE-1 and L1-MET analyses and profiling the samples according to the presence of at least one hypomethylated assay improved the discrimination between high and low risk lesions (p = 0.005). Remarkably, adenomas with at least one hypomethylated assay identified the patients with a significantly (p < 0.001) higher risk of developing CRC. Multivariable analysis and logistic regression evaluated by the ROC curves proved that methylation status was an independent variable improving cancer risk prediction (p = 0.02).

Conclusions: LINE-1 and L1-MET hypomethylation in colorectal adenomas are associated with a higher risk of developing CRC. DNA global and intragenic hypomethylation are independent markers that could be used in combination to successfully improve the stratification of patients who enter a colonoscopy surveillance program.

Keywords: Bisulfite pyrosequencing; CRC risk; Colorectal adenomas; L1-MET; LINE-1 hypomethylation.

Fig. 1

Adenoma clinical and pathological features. a Adenoma histology: tubular adenomas (TB) versus tubulovillous/villous adenomas (TBV/V). b Adenoma dysplasia: low dysplasia grade versus high grade. c Adenoma diameter (mm): the adenomas were classified in two groups, using as a threshold 10 mm, a parameter currently adopted to identify higher CRC risk patients. d Number of polyps per patient: patients were divided using as a threshold 3 adenomas removed at the same time, a parameter currently used to identify higher CRC risk patients. e Adenoma mutational status: wild-type (WT) versus mutated adenomas. f Mutations identified analyzing adenomas were classified in transversions or transitions. High-risk group (dark gray) and Low-risk group (light gray)

Fig. 2

Methylation assays in high-risk and low-risk patients. Adenomas were considered non-hypomethylated (in dark gray) when assays showed methylation levels above 60%. Adenomas were considered hypomethylated (in white) when assays displayed methylation level below 60%. a Global L1 methylation assay. b Intragenic L1-MET methylation assay. c Combination of the two methylation assays: we consider hypomethylated patients with at least one assay below 60%

Fig. 3

Methylation level and time free from CRC. Percentage of patients who had not developed CRC is reported on Y-axis, whereas on X-axis time is represented in months. Gray curve shows patients with hypomethylated adenomas, and black curve displays patients with non-hypomethylated adenomas. a Hypomethylation was assessed when at least one of the two assays was below 60%. b Hypomethylation according to only the LINE-1 assay below 60%. c Hypomethylation according to only the L1-MET assay below 60%

Fig. 4

ROC curves representing the logistic models to identify higher CRC risk patients. MOD1 is the logistic model without the methylation parameter, while MOD2 comprises the methylation value. Black curves show the efficiency of the models built using the current parameters, while gray curves display the efficiency of the new models comprising the methylation level. a All adenomas of the study were evaluated. b The subset consisting only of tubular adenomas was considered. c The subset including only the controls with at least 5 years of follow-up and the cases that had developed CRC within 10 years was assessed