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Introduction: Risk prediction of gastric cancers is important to implement appropriate screening procedures. Although aberrant DNA methylation is deeply involved in gastric carcinogenesis, its induction by Helicobacter pylori, a strong gastric carcinogen, is unclear. Here, we analyzed the effect of H. pylori infection on the quantity of methylated DNA molecules in noncancerous gastric mucosae and examined its association with gastric cancer risk.Experimental Design: Gastric mucosae were collected from 154 healthy volunteers (56 H. pylori negative and 98 H. pylori positive) and 72 cases with differentiated-type gastric cancers (29 H. pylori negative and 43 H. pylori positive) by endoscopy. The numbers of DNA molecules methylated and unmethylated for eight regions of seven CpG islands (CGI) were quantified by quantitative PCR after bisulfite modification, and fractions of methylated molecules (methylation levels) were calculated.Results: Among healthy volunteers, methylation levels of all the eight regions were 5.4- to 303-fold higher in H. pylori positives than in H. pylori negatives (P < 0.0001). Methylation levels of the LOX, HAND1, and THBD promoter CGIs and p41ARC exonic CGI were as high as 7.4% or more in H. pylori–positive individuals. Among H. pylori–negative individuals, methylation levels of all the eight regions were 2.2- to 32-fold higher in gastric cancer cases than in age-matched healthy volunteers (P ≤ 0.01). Among H. pylori–positive individuals, methylation levels were highly variable, and that of only HAND1 was significantly increased in gastric cancer cases (1.4-fold, P = 0.02).Conclusions: It was indicated that H. pylori infection potently induces methylation of CGIs to various degrees. Methylation levels of specific CGIs seemed to reflect gastric cancer risk in H. pylori–negative individuals.

Supplementary Table S1 from High Levels of Aberrant DNA Methylation in Helicobacter pylori–Infected Gastric Mucosae and its Possible Association with Gastric Cancer Risk

Supplementary Figure 4 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Figure 2 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Figure Legends 1-8 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Figure 3 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Tables1-4 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Figure 5 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Figure 1 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Supplementary Figures 6-8 from Inflammatory Processes Triggered by Helicobacter pylori Infection Cause Aberrant DNA Methylation in Gastric Epithelial Cells

Aberrant DNA methylation, induced by aging and accelerated by chronic inflammation, accumulates in normal tissue. Accumulation of DNA methylation in multiple genes, namely epimutation burden, is closely associated with cancer risk. Indeed, we demonstrated that the measurement of the methylation level of a marker gene in normal tissue is useful for cancer risk prediction of metachronous cancers, by a multicenter prospective study with gastric cancer patients (Asada, Gut, 64:388, 2015: Maeda, Gut, 66:1721, 2017). In this study, we attempted to predict the risk of primary gastric cancer by performing a new multicenter prospective study. We recruited 1,880 healthy people, after Helicobacter pylori eradication, with a clinically high risk of gastric cancer. Biopsy specimens were obtained from the antrum and body, and the methylation level of RIMS1, which is a preselected epigenomic risk marker for this study, was measured in 1,757 participants. The participants are annually being followed-up to detect primary gastric cancer, for 5-8 years. Currently, the median follow-up is 4.05 years, and 27 people have developed primary gastric cancers (as of September 2022). The results of statistical analysis are being determined. This study will establish the clinical utility of a cancer risk diagnosis by epimutation burden in normal tissue, and is promising for application in various inflammation-associated cancers. Citation Format: Harumi Yamada, Masahiro Maeda, Seiichiro Abe, Taichi Shimazu, Takayuki Ando, Kazunari Murakami, Shinji Tanaka, Takao Maekita, Osamu Inatomi, Ken Sugimoto, Tetsuro Kawagoe, Nobutake Yamamichi, Toshikazu Ushijima. Cancer risk diagnosis by epimutation burden: a multicenter prospective study. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6014.

Aberrant DNA methylation is involved in many types of human cancers, and is induced by aging. In addition, it is known to be induced by chronic inflammation, and this inflammation-induced DNA methylation is considered as acceleration of age-related methylation. However, few studies addressed target genomic regions of age-related and inflammation-induced DNA methylation in a genome-wide manner. We previously demonstrated that Helicobacter pylori(HP) infection induces aberrant DNA methylation potently in human and gerbil gastric mucosa [Niwa, Cancer Res, 70:1430, 2010; Ushijima, Clin Cancer Res, 18:923, 2012]. Here, taking advantage of the potent methylation induction, we aimed to clarify the targets of methylation induction by aging and inflammation. We collected gastric mucosae from i) HP-never infected young individuals (under 40 years; n=4; G1), ii) HP-currently infected young individuals (n=4; G2), iii) HP-never-infected old individuals (above 60 years; n=4; G3), and vi) HP-currently-infected old individuals (n=4; G4). DNA methylation of 482,421 CpG probes was analyzed by Infinium Human Methylation 450K, and the probes were grouped into 270,249 genomic blocks. It was revealed that high levels of methylation were induced in 44,461 (16.5%) genomic blocks by current inflammation (G2/G1), even after correction of the influence of leucocyte infiltration. 61.8 % of the hypermethylation was acceleration of age-related methylation (G3/G1) while 21.6 % was specific to inflammation. Regions with H3K27me3 were frequently hypermethylated both by aging and inflammation. Basal methylation levels were essential for age-related hypermethylation while even regions with little basal methylation were hypermethylated by inflammation. When limited to promoter CpG islands, being a microRNA gene and high basal methylation levels strongly enhanced hypermethylation while H3K27me3 strongly enhanced inflammation-induced hypermethylation. Inflammation was capable of overriding active transcription. In young gastric mucosae (G2/G1), genes with high expression and frequent mutations in gastric cancers were more frequently methylated than in old ones (G4/G3).Methylation by inflammation was not simple acceleration of age-related methylation. Targets of aberrant DNA methylation were different between young and old gastric mucosae, and driver genes were preferentially methylated in young gastric mucosa [Yamashita, Clin Epigenet, in press]. Citation Format: Satoshi Yamashita, Sohachi Nanjo, Emil Rehnberg, Naoko Iida, Hideyuki Takeshima, Takayuki Ando, Takao Maekita, Toshiro Sugiyama, Toshikazu Ushijima. Distinct DNA methylation targets by aging and chronic inflammation [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1079.

Altered patterns of DNA methylation associated with Helicobacter pylori (HP) infection of gastric epithelial cells are thought to contribute to gastric cancer risk. However, it is unclear whether this increased risk reflects an infection-associated inflammatory response or the infection itself. In this study, we sought to clarify mechanisms in a gerbil model of gastric cancer where we showed that HP infection is causally involved in induction of aberrant DNA methylation. By genome-wide screening, CpG islands that were aberrantly methylated in gerbil gastric cancer cell lines were isolated, and 10 islands were shown to be specifically methylated only in gastric mucosae infected with HP. By temporal analysis, methylation levels in gastric epithelial cells started to increase at 5 to 10 weeks after infection and reached high levels by 50 weeks. When HP was eradicated, methylation levels markedly decreased 10 and 20 weeks later, but they remained higher than those in gerbils that were not infected by HP. Expression levels of several inflammation-related genes (CXCL2, IL-1β, NOS2, and TNF-α) paralleled the temporal changes of methylation levels. Significantly suppressing inflammation with the immunosuppressive drug cyclosporin A did not affect colonization by HP but blocked the induction of altered DNA methylation. Our findings argue that DNA methylation alterations that occur in gastric mucosae after HP infection are composed of transient components and permanent components, and that it is the infection-associated inflammatory response, rather than HP itself, which is responsible for inducing the altered DNA methylation. Cancer Res; 70(4); 1430–40

Aberrant DNA methylation can be accumulated in normal-appearing tissues, and its degree can be correlated with cancer risk [Hattori, Genome Med, 8:10, 2016]. Such accumulation is especially prominent in inflammation-associated cancers, including gastric cancer. To bring the findings into cancer risk diagnosis, we conducted a multicenter prospective cohort study with 826 patients [Asada, Gut, 64:388, 2015]. Between 2008 and 2010, 826 patients after endoscopic treatment of gastric cancer and without current H. pylori infection were enrolled, and a gastric biopsy was collected. Methylation levels of pre-defined three marker genes (miR-124a-3, EMX1 and NKX6-1) were measured by quantitative methylation-specific PCR. Patients were classified into quartiles according to methylation levels of individual genes (Q1, the lowest; Q4, the highest). Among the 826 enrolled patients, 795 patients received annual follow-ups by endoscopy with a median period of 5.46 years (interquartile range: 3.95 - 6.09). By the end, 133 patients developed a metachronous gastric cancer, and 116 of them developed a cancer after 1 year of the enrollment (authentic metachronous cancer). Kaplan-Meier analysis showed that Q4 had a higher cumulative incidence of metachronous gastric cancer than Q1 with p-values of The results clearly showed that accumulation levels of aberrant DNA methylation can be used for cancer risk diagnosis. Even among the patients who carried a high risk of metachronous gastric cancer, cancer risk stratification was achieved. The strong influence of methylation accumulation on gastric cancer risk indicated a large contribution of aberrant DNA methylation to H. pylori infection-induced gastric carcinogenesis. Epigenetic cancer risk diagnosis is now becoming real. Citation Format: Masahiro Maeda, Takeshi Nakajima, Ichiro Oda, Taichi Shimazu, Nobutake Yamamichi, Takao Maekita, Kiyoshi Asada, Chizu Yokoi, Takayuki Ando, Takeichi Yoshida, Sohachi Nanjo, Mitsuhiro Fujishiro, Takuji Gotoda, Masao Ichinose, Toshikazu Ushijima. High impact of methylation accumulation on cancer risk: 5-year follow-up of a multicenter prospective cohort study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-092. doi:10.1158/1538-7445.AM2017-LB-092