Selected Alu methylation levels in the gastric carcinogenesis cascade

Abstract

Background: Genome-wide hypomethylation, a common epigenetic change that occurs during cancer development, primarily affects repetitive elements, such as Alu repeats. Consequently, Alu repeats can be used as a surrogate marker of genomic hypomethylation.

Methods: In this study, we aimed to investigate the correlation between Alu methylation levels and the multistage course of gastric carcinogenesis.

Results: We found that the Alu methylation levels in gastric cancer tissue decreased compared with those in normal gastric tissue, with the change in methylation levels and pattern being most significant between chronic gastritis and intestinal metaplasia. Moreover, Alu methylation levels were not associated with Helicobacter pylori or Epstein-Barr virus infection.

Conclusions: Finally, our sensitivity and specificity analyses suggested that Alu methylation level can be used to distinguish gastric cancer tissue from normal tissue. Thus, Alu methylation level shows promise as biomarker for gastric cancer diagnosis.

Keywords: Epstein–Barr virus; Gastric cancer; Helicobacter pylori; Hypomethylation.

Figure 1. Alu methylation patterns across sample groups.

(A) The overall Alu methylation (mC) decreased significantly from NS and CG to IM and GA (p < 0.0001) for these comparisons. (B) Significant differences in mCmC Alu methylation were observed between CG and IM (p < 0.0001), NS and IM (p = 0.0002), CG and GA (p = 0.0058), and IM and GD (p = 0.0216). (C) The mCuC methylation pattern also significantly differed between CG and IM (p < 0.0001), NS and GA (p = 0.0348), CG and GA (p = 0.0008), and IM and GA (p = 0.0258). (D) A notable increase in uCmC Alu methylation was observed in IM compared with that in CG (p < 0.0001), GD (p = 0.0100), and GA (p = 0.0029). (E) In contrast to other patterns, the frequency of uCuC Alu methylation progressively increased from NS to CG, GD, and GA, with differences found between NS and GA (p < 0.0001), CG and GA (p = 0.0002), IM and GA (p = 0.0134), and GD and GA (p = 0.0085). NS, normal gastric tissue; CG, chronic gastritis; IM, intestinal metaplasia; GD, gastric dysplasia; GA, gastric adenocarcinoma. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.

Figure 2. Alu methylation levels in relation to Helicobacter pylori and Epstein–Barr virus (EBV) infections.

(A) Alu methylation in CG samples was similar between H. pylori-negative and -positive cases (44.73 ± 2.88% vs. 44.36 ± 1.87%, respectively), whereas, in IM samples, H. pylori-negative cases showed higher methylation levels than positive cases, though not statistically significant (39.31 ± 2.04% vs. 38.51 ± 1.79%). (B) In GA samples, Alu methylation between H. pylori-negative (38.76 ± 1.88%) and -positive (38.88 ± 1.58%) cases did not significantly differ. (C) EBV-encoded small RNA (EBER)-positive GA cases exhibited slightly higher Alu methylation levels than EBER-negative cases (39.24 ± 1.49% and 38.72 ± 1.88%, respectively), but the difference was not significant. NS, normal gastric tissue; CG, chronic gastritis; IM, intestinal metaplasia; GD, gastric dysplasia; GA, gastric adenocarcinoma.

Figure 3. Diagnostic efficacy of Alu methylation levels in detecting GA from biopsy lesions.

(A) Alu methylation levels were significantly reduced in gastric adenocarcinoma (GA; n = 29) compared to non-cancerous tissues, including normal gastric tissue and chronic gastritis (NS + CG; n = 46), with a highly significant difference (p < 0.0001). These data suggest that Alu hypomethylation is associated with malignant transformation in gastric tissue. (B) Receiver operating characteristic (ROC) analysis demonstrated that Alu methylation has strong diagnostic potential for gastric adenocarcinoma (GA), with an AUC of 0.9344. Using a cut-off value of 40.98%, the assay achieved 86.21% sensitivity and 86.96% specificity. These findings highlight Alu methylation as a promising biomarker for the accurate detection of GA, even in biopsy samples with limited tumor content. ****p < 0.0001.