LINE-1 hypomethylation is not a common event in preneoplastic stages of gastric carcinogenesis

Abstract

LINE-1 hypomethylation is widely accepted as marker for global genomic DNA hypomethylation, which is a frequent event in cancer. The aim of the study was to evaluate LINE-1 methylation status at different stages of gastric carcinogenesis and evaluate its prognostic potential in clinical settings. LINE-1 methylation was analyzed in 267 tissue samples by bisulfite pyrosequencing including primary colorectal cancer tissues (T-CRC) with corresponding adjacent colon mucosa (N-CRC), gastric cancer tissues (T-GC) with corresponding gastric mucosa (N-GC), normal gastric tissues (N), chronic non-atrophic and atrophic gastritis (CG). LINE-1 methylation level was lower in both T-GC and T-CRC when compared to paired adjacent tissues. No difference was observed for LINE-1 methylation status between patients with normal gastric mucosa, CG and N-GC. LINE-1 methylation in T-GC but not N-GC tended to correlate with age. Subgroup stratification analysis did not reveal significant differences in LINE-1 methylation status according to tumor stage, anatomical location, histological subtype, differentiation grade. We observed similar overall survival data between patients with high or low LINE-1 levels. In summary, LINE-1 hypomethylation is a characteristic feature in GC but not very common in early preneoplastic stages of gastric carcinogenesis. Prognostic role of LINE-1 hypomethylation in GC patients could not be confirmed in this cohort.

Figure 1

Quantitative LINE-1 methylation analyses in paired colorectal (CRC) and gastric cancer (GC) tissues. (A) LINE-1 methylation in paired T-CRC and adjacent N-CRC tissues (n = 24) (p = 0.0005). (B) LINE-1 methylation level in T-GC and adjacent N-GC tissues (n = 80) (p = 0.002). (C and D) Absolute difference between LINE-1 methylation in matching (C) T-CRC and N-CRC, and (D) T-GC and N-GC tissues. Wilcoxon test has been used for paired analyses **P < 0.005.

Figure 2

Quantitative LINE-1 methylation analyses in gastric and colon tissues. (A) LINE-1 methylation values were obtained using bisulfite pyrosequencing in gastric tissues from controls (N), patients with chronic gastritis (CG), adjacent non-tumor tissues (N-GC) and gastric cancer tumor tissues (T-GC) (p > 0.05). (B) LINE-1 methylation level comparison between normal gastric (N), adjacent non-tumor gastric tissues (N-GC) and non-tumoral adjacent colon tissue (N-CRC) (p = 0.2). Statistical analyses where performed using Mann-Whitney test *P < 0.05.

Figure 3

Correlation between LINE-1 methylation status in tumorous and adjacent non-tumorous tissue. LINE-1 methylation obtained using bisulfite pyrosequencing did not correlate between (A) N-GC and T-GC and (B) N-CRC and T-CRC tissues. (C and D) Correlation analysis between patient’s age and (C) absolute LINE-1 methylation in T-GC; and (D) difference in LINE-1 methylation between T-GC and N-GC tissues. Analyses were performed using Spearman’s test.

Figure 4

Subgroup analyses of LINE-1 methylation in gastric cancer patients according to clinicopathological data. LINE-1 methylation analyses based on (A) anatomical tumour localization (p = 0.41), (B) T- (p = 0.20), (C) N-, (p = 0.11) and (D) M-tumor staging (p = 0.17). (E) LINE-1 methylation differences in patients with low and high-grade tumors (p = 0.26). LINE-1 methylation differences in GC patients according to (F) Lauren’s classification of GC type (p = 0.39), (G) gender (p = 0.83) and (H) H. pylori status (p = 0.70). Statistical analyses were performed using Mann-Whitney for two and Kruskal-Wallis test with Dunn’s posttest for multiple comparison analyses.

Figure 5

Overall survival analyses of patients with GC based on LINE-1 methylation. (A) Patients with GC with high and low LINE-1 methylation status defined by cut-off 60% based on LINE-1 methylation in T-GC sample (low LINE-1 methylation 21.5%). (B) Kaplan-Meier analyses based on UICC tumor stage with significant surivival difference among the groups. (D) Kaplan-Meier analyses based on high (n = 63) and low (n = 17) LINE-1 methylation status (cut-off methylation 60%) (p = 0.59). (D) Kaplan-Meier analyses based on high ≥ 65% (n = 45), middle > 55% and <65% (n = 25) and low ≤55% (n = 10) LINE-1 methylation status (p = 0.51). (E) Kaplan-Meier analyses of survival difference based on LINE-1 methylation status in intestinal/mixed type GC (p = 0.36) and in (F) diffuse type GC (p = 0.63). Statistical comparison between curves was performed with Log-rank (Mantel-Cox) test.