Long-term Changes In Aberrant DNA Methylation And Gastritis After Helicobacter Pylori Eradication Focused On Metachronous Gastric Cancer

DOI: https://doi.org/10.21203/rs.3.rs-1187733/v1

Abstract

Background

A persistently high methylation level in gastric mucosa after Helicobacter pylori (H. pylori) eradication is presumed to be a risk for metachronous gastric cancer (MGC); however, long-term changes in aberrant DNA methylation and histological gastritis have been unclear. Our aim was to examine changes in DNA methylation and histological gastritis according to the occurrence of MGC.

Methods

Subjects were classified into 3 groups: 25 patients in whom metachronous gastric cancers occurred after the initial endoscopic resection (ER) for early gastric cancer and H. pylori eradication (MGC group), 17 patients in whom MGC did not occur for more than 5 years after initial ER and H. pylori eradication (non-MGC group) and 29 patients without a history of gastric cancer who succeeded in eradication more than 5 years ago (HP group). Aberrance of DNA methylation in 3 genes (miR-124a-3, EMX1, NKX6-1) and histological score of atrophy and intestinal metaplasia (IM) were evaluated using biopsy samples before and more than a mean of 5 years after H. pylori eradication.

Results

The methylation level of miR-124a-3 in the HP group and non-MGC group and that of EMX1 in the HP group significantly decreased in the long term after eradication. In the MGC group, H. pylori eradication did not improve aberrant methylation, and the Z-score significantly increased. There were significant positive correlations between methylation levels in miR-124a-3 and EMX1 and histological findings after eradication.

Conclusions

A persistently high methylation level after H. pylori eradication reflected precancerous mucosal conditions and led to long-term MGC.

Introduction

According to the GLOBOCAN 2018 report, gastric cancer is the third deadliest cancer, with an estimated 783,000 deaths and 1,000,000 new cases worldwide in 2018 (2018). Helicobacter pylori (H. pylori) infection induces histological inflammatory changes in the gastric mucosa, such as atrophic gastritis and intestinal metaplasia (IM), which is a risk factor for gastric cancer (Kuipers et al. 1995; Uemura et al. 2001). The International Agency for Research on Cancer working group declared in 1994 that H. pylori was a human carcinogen and that screening and treatment programs for H. pylori were recommended in all countries (Schistosomes 1994; IARC Working Group Reports 2014). The Kyoto global consensus report in 2015 stated that all H. pylori-positive individuals worldwide should receive eradication therapy (Sugano et al. 2015). Successful eradication of H. pylori prevents gastric cancer development (Fukase et al. 2008). Several randomized controlled trials and meta-analyses showed the cancer-preventive effects of H. pylori eradication, but gastric cancer also occurred to some degree after H. pylori eradication (1.0-1.6% in primary gastric cancer, 2.7-6.1% in metachronous gastric cancer (MGC)) (Ford et al. 2014; Yoon et al. 2014; Chen et al. 2016; Lee et al. 2016; Terasawa et al. 2019; Fan et al. 2020). Risk factors for MGC after successful H. pylori eradication have been reported to be age, male sex, severe atrophy and IM. Atrophy and IM are considered to be the most important factors in gastric carcinogenesis, and H. pylori eradication improves atrophy and IM, which leads to the prevention of gastric cancer (Correa 1992; Ito et al. 2002; Kamada et al. 2005; Toyokawa et al. 2010; Park and Kim 2015). On the other hand, there have been some reports on conflicting results, and whether H. pylori eradication improves atrophy and IM is controversial (Lahner et al. 2005; Wang et al. 2011; Kang et al. 2012).

DNA methylation, which accumulates in both gastric cancers and noncancerous mucosa, is an important epigenetic change associated with gastric carcinogenesis (Ushijima and Sasako 2004; Oka et al. 2006; Yamashita et al. 2011; Yousefi et al. 2019). H. pylori infection accumulates DNA methylation in the gastric mucosa (Maekita et al. 2006), and H. pylori eradication decreases aberrant methylation in some gene promoters, which also decreases the risk of gastric cancers (Chan et al. 2006; Leung et al. 2006; Nakajima et al. 2010). Asada et al. reported that the occurrence of MGC after endoscopic resection (ER) of early gastric cancers in patients with H. pylori-negative or H. pylori-eradicated gastric cancer was associated with a high methylation level of miR-124a-3 at a median 3-year follow-up (Asada et al. 2015). A persistently high methylation level in gastric mucosa after H. pylori eradication is presumed to be a risk factor for MGC (Ushijima et al. 2006; Maeda et al. 2017a). Although a positive correlation between histological IM and methylation levels in gastric mucosa has been reported (Shin et al. 2013), there have been no reports on both of these changes for long-term surveillance of MGC. Based on the fact that recovery of atrophy and IM takes for at least several years after H. pylori eradication, herein, we examined the relationship between histological gastritis and methylation level focused on the long-term occurrence of MGC (Kodama et al. 2012).

Methods

Patients

Between January 2004 and December 2018, 184 patients were positive for H. pylori at the initial ER for early gastric cancer and were successfully eradicated after ER at our institution. Among these patients, patients who received annual endoscopy for more than 5 years and in whom biopsy specimens were properly obtained were selected, and they were divided into groups with and without MGC. MGC was defined as newly discovered cancer more than 1 year after eradication. The criteria of MGC were as follows: 1) each lesion was histopathologically malignant, 2) each lesion was separated from another and 3) each lesion was not the result of a local extension or metastasis of other lesions (Moertel et al. 1957). The following patients were excluded: 1) those who were H. pylori-negative at the initial ER (including those who had spontaneous or previous eradication), 2) those who failed or refused H. pylori eradication, 3) those whose biopsy specimens were not properly collected or too small, 4) those whose follow-up was less than 5 years after eradication, and 5) those in whom local recurrence was found, 6) those who received surgical gastrectomy before and after eradication.

Finally, 25 patients in whom MGCs occurred were included in the MGC group, and 17 patients in whom MGC did not occur were included in the non-MGC group. As a control (HP group), 29 patients without gastric cancer for more than 5 years in annual endoscopy follow-up after successful eradication were analyzed (Fig. 1, Online Resource 1).

This study was performed in accordance with the ethical standards detailed in the Declaration of Helsinki. The authors’ institutional ethics committee approved this study, and all patients provided written informed consent (Hokkaido University Hospital Review Board 019-015).

Determination of H. pylori infection status

H. pylori infection status was investigated by using the urea breath test (cutoff value < 2.5‰, Ubit®, Otsuka Pharmaceutical, Tokyo, Japan), and serum anti-H. pylori antibody test (cutoff value < 10 U/ml, E-Plate Eiken H. pylori antibody, Eiken Chemical Co., Ltd., Tokyo, Japan) and histopathology from the greater curvatures in the antrum and the corpus. H. pylori-positive status was determined when at least one test was positive. For judgment, at least two tests were performed more than 8 weeks after completion of eradication therapy.

Biopsy samples

Endoscopic biopsy specimens obtained from noncancerous gastric mucosa of the antrum (2 cm from the pylorus at the greater curvature) were used for measurement of methylation and histological assessment. For the MGC group, biopsy samples before the first ER (before eradication) and when MGCs were detected after eradication were used. In the HP group and non-MGC group, biopsy samples before and at 5 years after eradication were prepared.

Histological assessment of atrophy and IM

All biopsy specimens were stained with hematoxylin and eosin and evaluated according to the updated Sydney system by pathologists who had no information about the results of endoscopy and H. pylori tests (Dixon et al. 1996). A visual analog scale according to the updated Sydney system was converted to a score in which 0 = absent, 1 = mild, 2 = moderate, and 3 = severe (Kodama et al. 2012).

Quantitative methylation analysis

The DNA methylation levels of three gene promoters (miR-124a-3, EMX1 and NKX6-1) related to gastric cancer after H. pylori eradication were analyzed (Asada et al. 2015; Maeda et al. 2017).

From formalin-fixed paraffin-embedded specimens, twenty 10-µm-thick tissue sections were cut for DNA extraction. DNA was extracted using the QIAamp DNA FFPE Tissue Kit (QIAGEN, Valencia, CA). Bisulfite treatment of DNA was performed using an EpiTect bisulfite kit (QIAGEN, Valencia, CA). Biotinate polymerase chain reaction was performed using primers that specifically amplify the sequences of the three genes (Online Resource 2). All PCR assays included a denaturation step at 95°C for 30 seconds, followed by an annealing step at various temperatures for 30 seconds and an extension step at 72°C for 30 seconds. Pyrosequencing was performed using PSQ HS 96 Gold single-nucleotide polymorphism reagents on a Pyromark Q24 pyrosequencing machine (Biotage, Uppsala, Sweden). The protocol for pyrosequencing has been described in detail previously (Colella et al. 2003; Watanabe et al. 2009; Yamamoto et al. 2016). Pyrosequencing quantitatively measures the methylation status of several CpG sites in a given promoter. These adjacent sites usually show highly concordant methylation. Therefore, the mean percentage of methylation of detected sites was used as a representative value for each gene promoter.

Statistical analysis

Continuous variables such as methylation level, age and period were compared using Student’s t-test or paired t-test. Histological atrophy and IM scores were compared using the Mann–Whitney U test or Wilcoxon’s rank-sum test. Correlations between methylation level and histological atrophy/IM score were analyzed using Spearman’s rank correlation coefficient. Z-score analysis was used to normalize the methylation level of the three genes in each sample. The Z-score for each gene was calculated as follows: Z-score = (methylation level of each sample – mean value of methylation level)/standard deviation of methylation level. A P value of < .05 in each analysis was considered statistically significant.

Results

Characteristics of patients and histological score

The characteristics of the patients and samples used are shown in Table 1. Table 2 shows histological score of atrophy and IM. The atrophy score before eradication was significantly lower in the HP group than in the MGC group. For the IM score, there were significant differences between the HP group and MGC group before and long term after eradication therapy. However, there was no significant improvement in the histological score after eradication in each group.

Table1. Characteristics of patients and used samples.

HP group

N = 29

Non-MGC group

N = 17

MGC group

N = 25

Sex, M: F

22: 7

14: 3

21: 4

Age at the initial ER†, mean ± SD‡, y

−

66.2 ± 8.8

69.3 ± 6.9

Age at eradication, mean ± SD, y

67.5 ± 5.5

66.8 ± 8.9

70.8 ± 7.0

Follow-up periods after eradication, mean ± SD, m 

83.2 ± 18

102.5 ± 29

97.4 ± 37

Periods from the initial ER to the sampling after eradication, mean ± SD, m

−

67.9 ± 10.2

83.8 ± 44.3

Periods from eradication to the sampling after eradication, mean ± SD, m

61.1 ± 2.7

62.2 ± 3.6

67.0 ± 37.2

†ER: endoscopic resection, ‡SD: standard deviation.

In all the items, there were no significant differences among the three groups.

Table 2

Changes in histological atrophy and intestinal metaplasia.
 

HP group

N = 29

Non-MGC group

N = 17

MGC-group

N = 25

Atrophy score before eradication

0.95 ± 0.67

1.45 ± 0.82

1.66 ± 0.98*

Atrophy score after eradication

0.89 ± 0.70

1.00 ± 1.00

1.50 ± 1.17

Changes in atrophy score

-0.07 ± 1.21

-0.83 ± 0.98

-0.71 ± 1.38

IM† score before eradication

0.54 ± 0.88

0.94 ± 0.97

1.21 ± 1.18**

IM score after eradication

0.45 ± 0.78

0.82 ± 1.13

1.42 ± 1.21***

Changes in IM score

-0.07 ± 1.12

-0.11 ± 0.86

0.22 ± 1.28
†IM: intestinal metaplasia, *HP group vs MGC group, P = 0.01, **HP group vs MGC group, P = 0.03, ***HP group vs MGC group, P = 0.002

Aberrant DNA methylation

Figure 2 shows changes in methylation levels before and after eradication in each group. There were no significant differences in the methylation level, including 3 genes and Z-score before eradication, among the 3 groups. In the HP group, the mean methylation levels of miR-124a-3 and EMX1 significantly decreased after eradication (miR-124a-3: P < 0.01, EMX1: P = 0.03). In the non-MGC group, only the mean methylation level in miR-124a-3 significantly decreased (P < 0.01). In the MGC group, the mean methylation level in NKX6-1 was increased after eradication (P = 0.04), and the Z-score was significantly increased (P = 0.03). The mean Z-score decreased after eradication in the HP group (before: 0.13 ± 0.59, after: -0.31 ± 0.56, P < 0.01) but increased in the MGC group (before: -0.12 ± 0.71, after: 0.30 ± 0.78, P = 0.03).

There were significant differences in the mean methylation levels of miR-124a-3 and EMX1 after H. pylori eradication between the MGC group and HP group (P < 0.01). Individual data is shown in Online Resource 3.

In the MGC group, 6 patients had repeated metachronous cancers more than twice after eradication, and methylation was measured using samples at the occurrence of MGC. In 4 patients who repeated MGC for more than 5 years after eradication, the methylation level showed an increase during follow-up (Fig. 3).

Correlations between histological atrophy/IM and DNA methylation

Correlations between histological atrophy/IM and DNA methylation are shown in Fig. 4. There was a good positive correlation between the methylation level of miR-124a-3 and the IM score after eradication (P < 0.01, r = 0.82). Additionally, the EMX1 gene promoter methylation level also had a moderate correlation with atrophy and IM after eradication (P <0.001, r = 0.59: P < 0.001, r = 0.64).

Discussion

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.

Declarations

Funding

There is no funding for the study.

Conflict of Interest

The authors declare that they have no conflict of interest.

Author contributions

Conceptualization: Shoko Ono and Naoya Sakamoto, Methodology: Ikko Tanaka, Shoko Ono and Yoshiyuki Watanabe, Formal analysis and investigation: Ikko Tanaka, Yoshoyuki Watanabe, Hiroyuki Yamamoto, Ritsuko Oikawa, Shogo Matsumoto, Marina Kubo, Yusuke Nishimura, Yoshihiko Shimoda, Masayoshi Ono, and Keiko Yamamoto, Writing - original draft preparation: Ikko Tanaka, Writing - review and editing: Shoko Ono and Yoshiyuki Watanabe, Supervision: Hiroyuki Yamamoto and Naoya Sakamoto.

Ethics approval

This study was performed in accordance with the ethical standards detailed in the Declaration of Helsinki. The study was reviewed and approved by the Hokkaido University Hospital Review Board 019-015.

Consent to participate

All patients provided written informed consent.

Consent for publication

Not applicable.

References

  1. Asada K, Nakajima T, Shimazu T et al (2015) Demonstration of the usefulness of epigenetic cancer risk prediction by a multicentre prospective cohort study. Gut 64:388–396. https://doi.org/10.1136/gutjnl-2014-307094
  2. Chan AOO, Peng JZ, Lam SK et al (2006) Eradication of Helicobacter pylori infection reverses E-cadherin promoter hypermethylation. Gut 55. https://doi.org/10.1136/gut.2005.077776
  3. Chen HN, Wang Z, Li X, Zhou ZG (2016) Helicobacter pylori eradication cannot reduce the risk of gastric cancer in patients with intestinal metaplasia and dysplasia: evidence from a meta-analysis. Gastric Cancer 19:166–175. https://doi.org/10.1007/s10120-015-0462-7
  4. Choi IJ, Kook M-C, Kim Y-I et al (2018) Helicobacter pylori Therapy for the Prevention of Metachronous Gastric Cancer. N Engl J Med 378:1085–1095. https://doi.org/10.1056/nejmoa1708423
  5. Colella S, Shen L, Baggerly KA et al (2003) Sensitive and quantitative universal PyrosequencingTM methylation analysis of CpG sites. Biotechniques 35:146–150. https://doi.org/10.2144/03351md01
  6. Correa P (1992) Human gastric carcinogenesis: a multistep and multifactorial process--First American Cancer Society Award Lecture on Cancer Epidemiology and Prevention. Cancer Res 52:6735–6740
  7. Dixon MF, Genta RM, Yardley JH et al (1996) Classification and grading of Gastritis: The updated Sydney system. Am J Surg Pathol 20:1161–1181. https://doi.org/10.1097/00000478-199610000-00001
  8. Fan F, Wang Z, Li B, Zhang H (2020) Effects of eradicating Helicobacter pylori on metachronous gastric cancer prevention: A systematic review and meta-analysis. J Eval Clin Pr 26:308–315. https://doi.org/10.1111/jep.13179
  9. Ford AC, Forman D, Hunt RH et al (2014) Helicobacter pylori eradication therapy to prevent gastric cancer in healthy asymptomatic infected individuals: systematic review and meta-analysis of randomised controlled trials. BMJ 348:g3174. https://doi.org/10.1136/bmj.g3174
  10. Fukase K, Kato M, Kikuchi S et al (2008) Effect of eradication of Helicobacter pylori on incidence of metachronous gastric carcinoma after endoscopic resection of early gastric cancer: an open-label, randomised controlled trial. Lancet 372:392–397. https://doi.org/10.1016/s0140-6736(08)61159-9
  11. IARC Helicobacter pylori Working Group (2014) Helicobacter pylori eradication as a strategy for preventing gastric cancer. Lyon, France: International Agency for Research on Cancer.IARC Working Group ReportsNo.8
  12. Ito M, Haruma K, Kamada T et al (2002) Helicobacter pylori eradication therapy improves atrophic gastritis and intestinal metaplasia: a 5-year prospective study of patients with atrophic gastritis. Aliment Pharmacol Ther 16:1449–1456. https://doi.org/10.1046/j.1365-2036.2002.01311.x
  13. Kamada T, Hata J, Sugiu K et al (2005) Clinical features of gastric cancer discovered after successful eradication of Helicobacter pylori: results from a 9-year prospective follow-up study in Japan. Aliment Pharmacol Ther 21:1121–1126. https://doi.org/10.1111/j.1365-2036.2005.02459.x
  14. Kang JM, Kim N, Shin CM et al (2012) Predictive factors for improvement of atrophic gastritis and intestinal metaplasia after Helicobacter pylori eradication: a three-year follow-up study in Korea. Helicobacter 17:86–95. https://doi.org/10.1111/j.1523-5378.2011.00918.x
  15. Kim HJ, Kim N, Kim HW et al (2021) Promising aberrant DNA methylation marker to predict gastric cancer development in individuals with family history and long-term effects of H. pylori eradication on DNA methylation. Gastric Cancer 24:302–313. https://doi.org/10.1007/s10120-020-01117-w
  16. Kodama M, Murakami K, Okimoto T et al (2012) Ten-year prospective follow-up of histological changes at five points on the gastric mucosa as recommended by the updated Sydney system after Helicobacter pylori eradication. J Gastroenterol 47:394–403. https://doi.org/10.1007/s00535-011-0504-9
  17. Kuipers EJ, Uyterlinde AM, Peña AS et al (1995) Long-term sequelae of Helicobacter pylori gastritis. Lancet 345:1525–1528. https://doi.org/10.1016/s0140-6736(95)91084-0
  18. Lahner E, Bordi C, Cattaruzza MS et al (2005) Long-term follow-up in atrophic body gastritis patients: atrophy and intestinal metaplasia are persistent lesions irrespective of Helicobacter pylori infection. Aliment Pharmacol Ther 22:471–481. https://doi.org/10.1111/j.1365-2036.2005.02582.x
  19. Lee YC, Chiang TH, Chou CK et al (2016) Association Between Helicobacter pylori Eradication and Gastric Cancer Incidence: A Systematic Review and Meta-analysis. Gastroenterology 150:1113–1124e5. https://doi.org/10.1053/j.gastro.2016.01.028
  20. Leung WK, Man EP, Yu J et al (2006) Effects of Helicobacter pylori eradication on methylation status of E-cadherin gene in noncancerous stomach. Clin Cancer Res 12:3216–3221. https://doi.org/10.1158/1078-0432.ccr-05-2442
  21. Maeda M, Moro H, Ushijima T (2017a) Mechanisms for the induction of gastric cancer by Helicobacter pylori infection: aberrant DNA methylation pathway. Gastric Cancer 20:8–15. https://doi.org/10.1007/s10120-016-0650-0
  22. Maeda M, Nakajima T, Oda I et al (2017b) High impact of methylation accumulation on metachronous gastric cancer: 5-year follow-up of a multicentre prospective cohort study. Gut. Division of Epigenomics, National Cancer Center Research Institute, Tokyo, Japan. Department of Gastrointestinal Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan. Endoscopy Division, National Cancer Center Hospital, Tokyo, Japan. Preve, pp 1721–1723
  23. Maekita T, Nakazawa K, Mihara M et al (2006) High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk. Clin Cancer Res 12:989–995. https://doi.org/10.1158/1078-0432.ccr-05-2096
  24. Michigami Y, Watari J, Ito C et al (2018) Long-term effects of H. pylori eradication on epigenetic alterations related to gastric carcinogenesis. Sci Rep 8:1–13. https://doi.org/10.1038/s41598-018-32717-3
  25. Moertel CG, Bargen JA, Soule EH (1957) Multiple gastric cancers; review of the literature and study of 42 cases. Gastroenterology 32:1095–1103
  26. Nakajima T, Enomoto S, Yamashita S et al (2010) Persistence of a component of DNA methylation in gastric mucosae after Helicobacter pylori eradication. J Gastroenterol 45:37–44. https://doi.org/10.1007/s00535-009-0142-7
  27. Oka S, Tanaka S, Kaneko I et al (2006) Advantage of endoscopic submucosal dissection compared with EMR for early gastric cancer. Gastrointest Endosc 64:877–883. https://doi.org/10.1016/j.gie.2006.03.932
  28. Park YH, Kim N (2015) Review of atrophic gastritis and intestinal metaplasia as a premalignant lesion of gastric cancer. J Cancer Prev 20:25–40. https://doi.org/10.15430/jcp.2015.20.1.25
  29. Shimazu T, Asada K, Charvat H et al (2015) Association of gastric cancer risk factors with DNA methylation levels in gastric mucosa of healthy Japanese: a cross-sectional study. Carcinogenesis 36:1291–1298. https://doi.org/10.1093/carcin/bgv125
  30. Shin CM, Kim N, Lee HS et al (2013) Changes in aberrant DNA methylation after Helicobacter pylori eradication: a long-term follow-up study. Int J Cancer 133:2034–2042. https://doi.org/10.1002/ijc.28219
  31. Sugano K, Tack J, Kuipers EJ et al (2015) Kyoto global consensus report on Helicobacter pylori gastritis. Gut 64:1353–1367. https://doi.org/10.1136/gutjnl-2015-309252
  32. Suzuki R, Yamamoto E, Nojima M et al (2014) Aberrant methylation of microRNA-34b/c is a predictive marker of metachronous gastric cancer risk. J Gastroenterol 49:1135–1144. https://doi.org/10.1007/s00535-013-0861-7
  33. Take S, Mizuno M, Ishiki K et al (2011) The long-term risk of gastric cancer after the successful eradication of Helicobacter pylori. J Gastroenterol 46:318–324. https://doi.org/10.1007/s00535-010-0347-9
  34. Terasawa T, Hamashima C, Kato K et al (2019) Helicobacter pylori eradication treatment for gastric carcinoma prevention in asymptomatic or dyspeptic adults: systematic review and Bayesian meta-analysis of randomised controlled trials. BMJ Open 9:e026002. https://doi.org/10.1136/bmjopen-2018-026002
  35. The international agency for research on cancer. cancer today (2018) Available from: https://gco.iarc.fr/today/home
  36. Toyokawa T, Suwaki K, Miyake Y et al (2010) Eradication of Helicobacter pylori infection improved gastric mucosal atrophy and prevented progression of intestinal metaplasia, especially in the elderly population: a long-term prospective cohort study. J Gastroenterol Hepatol 25:544–547. https://doi.org/10.1111/j.1440-1746.2009.05995.x
  37. Uemura N, Okamoto S, Yamamoto S et al (2001) Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 345:784–789. https://doi.org/10.1056/NEJMoa001999
  38. Ushijima T, Nakajima T, Maekita T (2006) DNA methylation as a marker for the past and future. J Gastroenterol 41:401–407. https://doi.org/10.1007/s00535-006-1846-6
  39. Ushijima T, Sasako M (2004) Focus on gastric cancer. Cancer Cell 5:121–125. https://doi.org/10.1016/s1535-6108(04)00033-9
  40. Wang J, Xu L, Shi R et al (2011) Gastric atrophy and intestinal metaplasia before and after Helicobacter pylori eradication: a meta-analysis. Digestion 83:253–260. https://doi.org/10.1159/000280318
  41. Watanabe Y, Kim HS, Castoro RJ et al (2009) Sensitive and Specific Detection of Early Gastric Cancer with DNA Methylation Analysis of Gastric Washes. Gastroenterology 136:2149–2158. https://doi.org/10.1053/j.gastro.2009.02.085
  42. Yamamoto H, Watanabe Y, Oikawa R et al (2016) BARHL2 Methylation Using Gastric Wash DNA or Gastric Juice Exosomal DNA is a Useful Marker For Early Detection of Gastric Cancer in an H. pylori -Independent Manner. Clin Transl Gastroenterol 7:e184. https://doi.org/10.1038/ctg.2016.40
  43. Yamashita K, Sakuramoto S, Watanabe M (2011) Genomic and epigenetic profiles of gastric cancer: Potential diagnostic and therapeutic applications. Surg Today 41:24–38. https://doi.org/10.1007/s00595-010-4370-5
  44. Yamashita S, Nanjo S, Rehnberg E et al (2019) Distinct DNA methylation targets by aging and chronic inflammation: a pilot study using gastric mucosa infected with Helicobacter pylori. Clin Epigenetics 11:191. https://doi.org/10.1186/s13148-019-0789-8
  45. Yoon SB, Park JM, Lim CH et al (2014) Effect of Helicobacter pylori eradication on metachronous gastric cancer after endoscopic resection of gastric tumors: a meta-analysis. Helicobacter 19:243–248. https://doi.org/10.1111/hel.12146
  46. Yousefi B, Mohammadlou M, Abdollahi M et al (2019) Epigenetic changes in gastric cancer induction by Helicobacter pylori. J Cell Physiol 234:21770–21784. https://doi.org/10.1002/jcp.28925
  47. Schistosomes liver flukes and Helicobacter pylori. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans.Lyon, 7-14 June 1994