Aberrant methylation of microRNA-34b/c is a predictive marker of metachronous gastric cancer risk

The profiles of the study participants are shown in Fig. 1. After excluding patients who were lost to follow-up or died of extragastric disease, 129 GC patients were enrolled in the study. The median follow-up period for the 129 GC patients was 1.5 (range 0.1–6.1) years. A total of 86 patients exhibited H. pylori infection; of those, 51 underwent eradication therapy after endoscopic resection of their GC, and the H. pylori was successfully eradicated in 49 (96 %). Twenty-six patients were H. pylori-negative, although the majority of those were presumed to have been infected in the past, as they exhibited intestinal metaplasia or gastric mucosal atrophy (data not shown). H. pylori status was not available for 17 patients. During the follow-up period, 17 patients (13 %) developed metachronous GCs. This included 12 H. pylori-positive patients, among whom five were successfully treated for H. pylori after endoscopic resection of their GC (Table 1).

The primary endpoint of this study was the occurrence of metachronous GC during the follow-up period. At the time each patient entered the study, biopsy specimens of noncancerous gastric mucosa were collected from the antrum and body of the stomach, after which methylation levels of miR-34b/c, SFRP1, SFRP2, SFRP5, DKK2 and DKK3 were determined using quantitative bisulfite pyrosequencing. These six genes are known to be frequently methylated in H. pylori-related gastritis as well as in GC [17, 20, 21]. Based on the results, patients were categorized into a high-methylation or low-methylation group, relative to the median baseline methylation levels in the gastric body and antrum among the 129 GC patients. Thereafter, we assessed the correlations between methylation of the respective genes and the incidence of metachronous GC. Using a univariate Cox proportional hazards model, the greatest hazard ratio (HR) was obtained for patients showing higher levels of miR-34b/c methylation in the gastric body when a cutoff of 18.6 % was employed (HR, 10.01; 95 % CI, 2.26–44.23, p = 0.002). Similarly, we found an elevated risk of metachronous GC in patients with higher levels of SFRP2 (cutoff, 43.3 %; HR, 3.33; 95 % CI, 1.08–10.25, p = 0.02) and DKK2 (cutoff, 16.9 %; HR, 3.66; 95 % CI, 1.19–11.25, p = 0.01) methylation in the gastric body (Fig. 2a). In addition, Kaplan–Meier analysis revealed that greater methylation of these three genes in the gastric body was significantly associated with shorter metachronous GC-free survival (miR-34b/c, p ≤ 0.001; SFRP2, p = 0.025; DKK2, p = 0.015 by log-lank test) (Fig. 2b, Supplementary Figure 1). By contrast, we found no significant correlation between the incidence of metachronous GC and methylation in the antral mucosa (Supplementary Figure 2).

The results summarized above prompted us to select miR-34b/c methylation in the gastric body for further analysis (Supplementary Figure 3). The 2-year cumulative incidence of metachronous GC in the high-methylation group (≥18.6 %) was 30.4 % (95 % CI, 15.9–44.9), while that for the low-methylation group (≤18.6 %) was only 3.8 % (95 % CI, 0–12.6) (Table 2). When we then divided the 129 GC patients into four quartiles based on the level of miR-34b/c methylation in the gastric body, we found that the cumulative incidence of metachronous GC significantly differed among the four groups (p ≤ 0.001 by log-lank test) (Supplementary Figure 4). These results suggest that levels of miR-34b/c methylation in the gastric body mucosa could be a predictive marker of the risk of metachronous GC.

The associations between the clinicopathological features and miR-34b/c methylation are summarized in Table 3. Among the GC patients, miR-34b/c methylation was associated with older age and a greater degree of inflammation, activity, atrophy and metaplasia in the gastric body (Table 3). By contrast, no significant differences were found with respect to sex, H. pylori infection or pathological findings in the antral mucosa (Table 3).

We next used univariate analysis and multivariate Cox proportional-hazards analysis to evaluate the utility of the miR-34b/c methylation level in the mucosa of the noncancerous gastric body as a predictive marker of metachronous GC risk. In the univariate analysis, miR-34b/c methylation and mucosal atrophy in the gastric body were significantly associated with the occurrence of metachronous GC (Table 4). On the other hand, inflammation, activity and metaplasia were not significant predictors of the occurrence of metachronous GC. In a multivariate analysis adjusted for miR-34b/c methylation, age, sex, H. pylori status and pathological findings (inflammation, activity, atrophy and metaplasia), miR-34b/c methylation and inflammation were independently associated with metachronous GC (Table 4).

Recent studies demonstrated that eradicating H. pylori after endoscopic resection of early GC reduces the risk of metachronous GC [9]. To assess the relationship between miR-34b/c methylation and H. pylori status, we compared GC patients who had undergone successful eradication therapy after enrolling in this study (n = 49) with those who were H. pylori-negative at the start of the study but were presumed to have had a past infection (n = 26). Kaplan–Meier analysis indicated that miR-34b/c methylation in the noncancerous gastric body was significantly associated with a risk of metachronous GC in patients successfully treated for H. pylori (p ≤ 0.001) (Fig. 3a). Moreover, we also found a significant correlation between methylation and metachronous GC in H. pylori-negative patients (p = 0.026) (Fig. 3b). These results suggest that miR-34b/c methylation in the mucosa of the gastric body may be a useful marker for predicting metachronous GC risk, irrespective of the presence of H. pylori.