Our data showed that methylation in gastric mucosa did not improve long term after H. pylori eradication in the MGC group, and a persistently high methylation level that was correlated with severe atrophy and IM after successful eradication would be a risk factor for MGC.
It has been reported that methylation before eradication reflects both temporary factors such as inflammation and permanent factors such as chronic gastritis, and methylation levels before eradication are not necessarily associated with gastric cancer risk (Ushijima et al. 2006; Asada et al. 2015). High methylation levels after H. pylori eradication are one of the risk factors for MGC (Maekita et al. 2006; Shin et al. 2013; Suzuki et al. 2014; Asada et al. 2015; Maeda et al. 2017). H. pylori eradication reduces methylation and prevents carcinogenesis (Nakajima et al. 2010; Leung et al. 2006; Chan et al. 2006). However, persistence of high methylation caused MGC, and patients with repeated MGC tended to have increased methylation. Asada et al reported that high methylation in gastric mucosa after ESD for early gastric cancer related with high incidence of MGC, but in many cases the timing of eradication is unclear and long-term changes in methylation levels after eradication were not studied (Asada et al. 2015; Maeda et al. 2017). In other previous studies, individual methylation data had not been followed-up, and the observation periods were short (Shin et al. 2013; Michigami et al. 2018). The strength of our study was that we were able to measure methylation levels and to examine changes in histology in the long term after successful eradication.
The mechanism by which methylation improves after eradication is postulated to be as follows (Ushijima et al. 2006; Maeda et al. 2017). First, H. pylori infection induced methylation of differentiated cells. After H. pylori eradication, differentiated cells with methylation disappear over time and are replaced by normal differentiated cells derived from a stem cell without methylation. In contrast, if a stem cell is methylated, the methylation of differentiated cells in that gland duct will not improve after eradication because an entire gland reflects the methylation status of its stem cell and methylation status in stem cells is preserved and replicated. In cases with MGC after eradication, many stem cells had been methylated before eradication. Even if many methylated cells are shed after H. pylori eradication, because cell differentiation from methylated stem cells continues, it is possible that the methylation level does not improve after eradication. Additionally, there may be other factors besides H. pylori that increase methylation levels in gastric mucosa, such as age, familial history and smoking (Shimazu et al. 2015; Yamashita et al. 2019; Kim et al. 2021).
Wong et al. suggested a “point of no return”, in which the benefit of H. pylori eradication diminished when H. pylori-associated gastritis reached a certain degree (Wong et al. 2004). Kiriyama et al. also stated that mucosal damage with IM may not recover to gastric-type mucosa for a “histological point of no return” with eradication of H. pylori (Kirishima et al. 2016). Methylation levels, especially miR-124a-3 in gastric mucosa, were positively associated with IM after eradication. These results support that atrophy and IM after eradication are associated with carcinogenesis depending on epigenetic changes. Our results also show that mucosal damage in cases with MGC has accumulated to the point of no histological and genetic improvement.
This study has some limitations. First, the study was a single-center retrospective study with a small sample size. Second, methylation after eradication in the other group except for the repeated MGC group was evaluated at only one point. Because it takes a long time to change chronic gastritis after successful eradication, we didn’t evaluate short-term changes in this study (Kamada et al. 2005; Toyokawa et al. 2010; Ito et al. 2002). Third, we defined the control group as those patients who did not develop cancer for more than 5 years after eradication. Although there have been some reports of cases with gastric cancer detected more than 5 years after eradication (Take et al. 2011; Choi et al. 2018), our subjects continued annual endoscopy after successful eradication and were retroactively confirmed to have no occurrences of gastric cancer to date. Finally, the DNA methylation was measured using FFPE samples in this study. Generally, fresh frozen samples are more suitable for methylation analysis because of good DNA quality (Ohomomo et al. 2021; Ueda et al. 2021). Actually, the EMX1 and NKX6-1 genes were not amplified by PCR in some samples. We have to perform a further study using a large sample size from multiple centers.
In conclusion, the persistence of a high level of aberrant DNA methylation in gastric mucosa after H. pylori eradication reflected severe atrophy and IM and was associated with the occurrence of MGC in the long term.