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Novel epigenetic markers for gastric cancer risk stratification in individuals after Helicobacter pylori eradication

  • Masahiro Maeda
  • Satoshi Yamashita
  • Taichi Shimazu
  • Naoko Iida
  • Hideyuki Takeshima
  • Takeshi Nakajima
  • Ichiro Oda
  • Sohachi Nanjo
  • Chika Kusano
  • Akiko Mori
  • Hiroshi Moro
  • Harumi Yamada
  • Shoichiro Tsugane
  • Toshiro Sugiyama
  • Yoshiharu Sakai
  • Toshikazu Ushijima
Original Article

Abstract

Background

The risk stratification of healthy individuals after Helicobacter pylori eradication is an urgent issue. The assessment of aberrant DNA methylation accumulated in gastric tissues with normal appearance, which can reflect overall epigenomic damage, is a promising strategy. We aimed to establish novel epigenetic cancer risk markers for H. pylori-eradicated individuals.

Methods

Gastric mucosa was collected from eight healthy volunteers without H. pylori infection (G1), 75 healthy individuals with gastric atrophy (G2), and 94 gastric cancer patients (G3) after H. pylori eradication. Genome-wide analysis was conducted using Infinium 450 K and differentially methylated probes were screened using large difference and iEVORA-based methods. Bisulfite pyrosequencing was used for validation.

Results

Screening, using 8 G1, 12 G2, and 12 G3 samples, isolated 57 candidates unmethylated in G1 and differentially methylated in G3 compared with G2. Validation for nine candidate markers (FLT3, LINC00643, RPRM, JAM2, ELMO1, BHLHE22, RIMS1, GUSBP5, and ZNF3) in 63 G2 and 82 G3 samples showed that all of them had significantly higher methylation levels in G3 than in G2 (P < 0.0001). Their methylation levels were highly correlated, which indicated that they reflect overall epigenomic damage. The candidates had sufficient performance (AUC: 0.70–0. 80) and high odds ratios (5.43–23.41), some of which were superior to a previous marker, miR-124a-3. The methylation levels of our novel markers were not associated with gastric atrophy, gender, or age.

Conclusions

Novel epigenetic markers for gastric cancer risk optimized for H. pylori-eradicated individuals were established.

Keywords

Epigenetics Cancer risk marker DNA methylation Gastric cancer Helicobacter pylori 

Notes

Acknowledgements

The authors are grateful to Drs. Y. Kakugawa, Y. Otake and T. Gotoda for sample and data acquisition, and Dr. N. Hattori for her advice, and Drs. K. Ichimura, Y. Matsushita, and M. Kitahara of Division of Brain Tumor Translational Research in National Cancer Center Research Institute for their technical assistance with the experiments.

Funding

Practical Research for Innovative Cancer Control from Japan Agency for Medical Research and Development, AMED (15ck0106023h0002, 16ck0106023h0003, 17ck0106267h0001).

Compliance with ethical standards

Conflict of interest

MM and TU made a joint patent application with Sysmex Corporation for identified epigenetic markers.

Ethical approval

All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1964 and later versions. Informed consent or substitute for it was obtained from all patients for being included in the study.

Supplementary material

10120_2018_803_MOESM1_ESM.xlsx (48 kb)
Supplementary material 1 (XLSX 48 kb)
10120_2018_803_MOESM2_ESM.pptx (1.6 mb)
Supplementary Fig. 1 Effects of H. pylori eradication on methylation levels of the nine candidate methylation markers. Methylation levels (upper panels) and methylation changes (lower panels) before and after eradication G2 (n = 63) were analyzed. The horizontal line represents the mean methylation level in each group. Some markers showed significantly decreased methylation levels after eradication, in agreement with our previous report [6]. The other markers showed comparable methylation levels. Supplementary Fig. 2 Correlations among the methylation markers. Strong correlation coefficients were observed between novel markers in each method (Upper left, Large difference method, R = 0.97; Upper Right, iEVORA-based method, R = 0.88). ELMO1, a novel marker identified by the large difference method, showed a strong correlation with miR-124a-3, the previous marker (R = 0.93), compared to ZNF93, which was identified by the iEVORA-based method (R = 0.66). Supplementary Fig. 3 Methylation levels of the nine methylation markers and the previous marker according to (a) extent of gastric atrophy in the intermediate-risk group (G2, n = 63) and (b) the high-risk group (G3, n = 82) of the validation set. The horizontal line represents the mean methylation level in each group. There were no significant associations between methylation levels and the extent of gastric atrophy in all the methylation markers. Supplementary Fig. 4 Methylation levels of the nine methylation markers and the previous marker among individuals with moderate gastric atrophy in the validation set. The horizontal line represents the mean methylation level in each group. As in the entire population (Fig. 2), methylation levels of all the nine markers, as well as the previous marker, miR-124a-3, in the high-risk group (G3, n = 42), were significantly higher than those in the intermediate-risk group (G2, n = 26).Supplementary Fig. 5 Relationships between methylation levels and age in H. Pylori-eradicated individuals in the validation set (G2, n = 64; G3, n = 82). For some markers (RPRM, JAM2, ELMO1, RIMS1, and miR-124a-3), weak positive correlations were observed (R = 0.19–0.21)

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Copyright information

© The International Gastric Cancer Association and The Japanese Gastric Cancer Association 2018

Authors and Affiliations

  • Masahiro Maeda
    • 1
    • 2
  • Satoshi Yamashita
    • 1
  • Taichi Shimazu
    • 3
  • Naoko Iida
    • 1
  • Hideyuki Takeshima
    • 1
  • Takeshi Nakajima
    • 4
  • Ichiro Oda
    • 4
  • Sohachi Nanjo
    • 5
  • Chika Kusano
    • 6
  • Akiko Mori
    • 1
  • Hiroshi Moro
    • 1
  • Harumi Yamada
    • 1
    • 2
  • Shoichiro Tsugane
    • 3
  • Toshiro Sugiyama
    • 5
  • Yoshiharu Sakai
    • 2
  • Toshikazu Ushijima
    • 1
  1. 1.Division of EpigenomicsNational Cancer Center Research InstituteTokyoJapan
  2. 2.Department of Gastrointestinal SurgeryKyoto University Graduate School of MedicineKyotoJapan
  3. 3.Epidemiology and Prevention GroupCenter for Public Health Sciences, National Cancer CenterTokyoJapan
  4. 4.Endoscopy DivisionNational Cancer Center HospitalTokyoJapan
  5. 5.Third Department of Internal MedicineUniversity of ToyamaToyamaJapan
  6. 6.Division of Gastroenterology and Hepatology, Department of MedicineNihon University School of MedicineTokyoJapan

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