Introduction: Gastric cancer is divided into four subtypes by their molecular features linked with genetic alterations, e.g., Epstein-Barr virus (EBV), microsatellite instability-high (MSI-high), chromosomal instability (CIN), and genomically stable (GS), called as TCGA classification. In this study, we tried to clarify the epigenetic features of the four GC subtypes according to aberrant methylation status in 23 loci. Methods: A total of 98 gastric cancers and their normal gastric mucosa samples were included in this study. We divided gastric cancers into TCGA subtypes which were determined in line with MSI-high, EBV, CIN, to GS by their molecular features. The 13 loci of polymorphic microsatellite sequences were used to determine loss of heterogeneity for the detection of CIN. The MSI status was determined by three mononucleotide repeat markers. Infection of EBV was determined by recovering EBV BNRF1 sequence from genomic DNA collected from gastric cancers. Methylation status of 23 loci was investigated by the combined bisulfite restriction analysis. Status of other findings, e.g., KRAS mutations, HER2 expression status, and infection of helicobacter pylori were confirmed. Results: Gastric cancers were divided into MSI (13%), EBV (7%), CIN (53%), and GS (27%). By histological classification, poorly differentiated adenocarcinoma was more in tumors categorized in MSI-high, and GS and signet-ring cell carcinoma (sig) were more in GS. Among the 23 loci investigated their methylation status, 18 loci were significantly hypermethylated in caner tissues. An unsupervised clustering divided gastric cancers into two clusters and revealed that most GS tumors clustered together in a cluster that exhibited lower methylation levels, distinct from the other subtypes. The inter-variable clustering revealed that a cluster contained the three loci (SFRP2-region 1/2 and APC) belonging to the Wnt signal cascade (Wnt-associated loci). The mean methylation score of Wnt-associated loci was the lowest in GS tumors (MSI-high: 2.7 [95% confidence interval, 2.3–2.9]; EBV: 2.1 [1.2–3.1]; CIN: 2.4 [2.2–2.7]; GS: 1.3 [0.8–0.7]). In contrast, the mean methylation score of the other 15 loci was significantly higher in MSI-high, while that in GS was as same as that in EBV or CIN (MSI-high: 10.4 [8.3–12.4]; EBV: 5.7 [1.7–9.7]; CIN: 4.4 [3.6–5.1]; GS: 3.4 [2.2–4.6]). Additionally, the lower methylation score of Wnt-associated loci was observed only in sig tumors. Conclusions: GS subtype tumors have the potential to possess distinct signatures in DNA hypomethylation profiles in Wnt signaling pathway, especially in sig.

Keywords:
Gastric, Cancer, DNA methylation, Diffuse-type gastric cancers, Signet-ring cell carcinoma, Wnt signal pathway
1.
Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018 Nov;68(6):394–424.
2.
Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification. Acta Pathol Microbiol Scand. 1965;64:31–49.
3.
Mariette C, Carneiro F, Grabsch HI, van der Post RS, Allum W, de Manzoni G, et al. Consensus on the pathological definition and classification of poorly cohesive gastric carcinoma. Gastric Cancer. 2019 Jan;22(1):1–9.
4.
Cancer Genome Atlas Research Network. Comprehensive molecular characterization of gastric adenocarcinoma. Nature. 2014 Sep 11;513(7517):202–9.
5.
Hewitt LC, Inam IZ, Saito Y, Yoshikawa T, Quaas A, Hoelscher A, et al. Epstein-Barr virus and mismatch repair deficiency status differ between oesophageal and gastric cancer: a large multi-centre study. Eur J Cancer. 2018 May;94:104–14.
6.
Naseem M, Barzi A, Brezden-Masley C, Puccini A, Berger MD, Tokunaga R, et al. Outlooks on Epstein-Barr virus associated gastric cancer. Cancer Treat Rev. 2018 May;66:15–22.
7.
Kempers MJ, Kuiper RP, Ockeloen CW, Chappuis PO, Hutter P, Rahner N, et al. Risk of colorectal and endometrial cancers in EPCAM deletion-positive Lynch syndrome: a cohort study. Lancet Oncol. 2011 Jan;12(1):49–55.
8.
Peltomaki P. Deficient DNA mismatch repair: a common etiologic factor for colon cancer. Hum Mol Genet. 2001 Apr;10(7):735–40.
9.
Smyrk TC, Watson P, Kaul K, Lynch HT. Tumor-infiltrating lymphocytes are a marker for microsatellite instability in colorectal carcinoma. Cancer. 2001 Jun 15;91(12):2417–22.
10.
Morihiro T, Kuroda S, Kanaya N, Kakiuchi Y, Kubota T, Aoyama K, et al. PD-L1 expression combined with microsatellite instability/CD8+ tumor infiltrating lymphocytes as a useful prognostic biomarker in gastric cancer. Sci Rep. 2019 Mar 15;9(1):4633.
11.
Llosa NJ, Cruise M, Tam A, Wicks EC, Hechenbleikner EM, Taube JM, et al. The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. Cancer Discov. 2015 Jan;5(1):43–51.
12.
Nagasaka T, Koi M, Kloor M, Gebert J, Vilkin A, Nishida N, et al. Mutations in both KRAS and BRAF may contribute to the methylator phenotype in colon cancer. Gastroenterology. 2008;134(7):1950–60.e1.
13.
Deng N, Goh LK, Wang H, Das K, Tao J, Tan IB, et al. A comprehensive survey of genomic alterations in gastric cancer reveals systematic patterns of molecular exclusivity and co-occurrence among distinct therapeutic targets. Gut. 2012 May;61(5):673–84.
14.
Katona BW, Rustgi AK. Gastric cancer genomics: advances and future directions. Cell Mol Gastroenterol Hepatol. 2017 Mar;3(2):211–7.
15.
Fuchs CS, Tomasek J, Yong CJ, Dumitru F, Passalacqua R, Goswami C, et al. Ramucirumab monotherapy for previously treated advanced gastric or gastro-oesophageal junction adenocarcinoma (REGARD): an international, randomised, multicentre, placebo-controlled, phase 3 trial. Lancet. 2014 Jan 4;383(9911):31–9.
16.
Wilke H, Muro K, Van Cutsem E, Oh SC, Bodoky G, Shimada Y, et al. Ramucirumab plus paclitaxel versus placebo plus paclitaxel in patients with previously treated advanced gastric or gastro-oesophageal junction adenocarcinoma (RAINBOW): a double-blind, randomised phase 3 trial. Lancet Oncol. 2014 Oct;15(11):1224–35.
17.
Sohn BH, Hwang JE, Jang HJ, Lee HS, Oh SC, Shim JJ, et al. Clinical significance of four molecular subtypes of gastric cancer identified by the cancer genome Atlas project. Clin Cancer Res. 2017 Aug 1;23(15):4441–9.
18.
Toda K, Nagasaka T, Umeda Y, Tanaka T, Kawai T, Fuji T, et al. Genetic and epigenetic alterations of netrin-1 receptors in gastric cancer with chromosomal instability. Clin Epigenetics. 2015;7(1):73.
19.
Japanese Gastric Cancer Association. Japanese classification of gastric carcinoma: 3rd English edition. Gastric Cancer. 2011 Jun;14(2):101–12.
20.
Goel A, Nagasaka T, Hamelin R, Boland CR. An optimized pentaplex PCR for detecting DNA mismatch repair-deficient colorectal cancers. PLoS One. 2010 Feb 24;5(2):e9393.
21.
Hofmann M, Stoss O, Shi D, Buttner R, van de Vijver M, Kim W, et al. Assessment of a HER2 scoring system for gastric cancer: results from a validation study. Histopathology. 2008 Jun;52(7):797–805.
22.
Shigeyasu K, Nagasaka T, Mori Y, Yokomichi N, Kawai T, Fuji T, et al. Clinical significance of MLH1 methylation and CpG island methylator phenotype as prognostic markers in patients with gastric cancer. PLoS One. 2015;10(6):e0130409.
23.
Nagasaka T, Goel A, Notohara K, Takahata T, Sasamoto H, Uchida T, et al. Methylation pattern of the O6-methylguanine-DNA methyltransferase gene in colon during progressive colorectal tumorigenesis. Int J Cancer. 2008 Jun 1;122(11):2429–36.
24.
Deng G, Chen A, Hong J, Chae HS, Kim YS. Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res. 1999 May 1;59(9):2029–33.
25.
Nagasaka T, Tanaka N, Cullings HM, Sun DS, Sasamoto H, Uchida T, et al. Analysis of fecal DNA methylation to detect gastrointestinal neoplasia. J Natl Cancer Inst. 2009 Sep 16;101(18):1244–58.
26.
Flanagan DJ, Vincan E, Phesse TJ. Wnt signaling in cancer: not a binary ON:OFF switch. Cancer Res. 2019 Dec 1;79(23):5901–6.
27.
Phesse T, Flanagan D, Vincan E. Frizzled7: a promising achilles’ heel for targeting the Wnt receptor complex to treat cancer. Cancers. 2016 May 17;8(5):50.
28.
Flanagan DJ, Barker N, Costanzo NSD, Mason EA, Gurney A, Meniel VS, et al. Frizzled-7 is required for Wnt signaling in gastric tumors with and without apc mutations. Cancer Res. 2019 Mar 1;79(5):970–81.
29.
Nojima M, Suzuki H, Toyota M, Watanabe Y, Maruyama R, Sasaki S, et al. Frequent epigenetic inactivation of SFRP genes and constitutive activation of Wnt signaling in gastric cancer. Oncogene. 2007 Jul 12;26(32):4699–713.
30.
Miao J, Liu Y, Zhao G, Liu X, Ma Y, Li H, et al. Feasibility of plasma-methylated SFRP2 for early detection of gastric cancer. Cancer Control. 2020 Apr–Jun;27(2):1073274820922559.
31.
Heller RS, Dichmann DS, Jensen J, Miller C, Wong G, Madsen OD, et al. Expression patterns of Wnts, Frizzleds, sFRPs, and misexpression in transgenic mice suggesting a role for Wnts in pancreas and foregut pattern formation. Dev Dyn. 2002 Nov;225(3):260–70.
32.
Ilyas M. Wnt signalling and the mechanistic basis of tumour development. J Pathol. 2005 Jan;205(2):130–44.
33.
Kaur A, Webster MR, Marchbank K, Behera R, Ndoye A, Kugel CH 3rd, et al. sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance. Nature. 2016 Apr 14;532(7598):250–4.
34.
Cheng YY, Yu J, Wong YP, Man EPS, To KF, Jin VX, et al. Frequent epigenetic inactivation of secreted frizzled-related protein 2 (SFRP2) by promoter methylation in human gastric cancer. Br J Cancer. 2007 Oct 8;97(7):895–901.
35.
Suzuki H, Gabrielson E, Chen W, Anbazhagan R, van Engeland M, Weijenberg MP, et al. A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer. Nat Genet. 2002 Jun;31(2):141–9.
36.
Kang GH, Shim YH, Jung HY, Kim WH, Ro JY, Rhyu MG. CpG island methylation in premalignant stages of gastric carcinoma. Cancer Res. 2001 Apr 1;61(7):2847–51.
37.
Waki T, Tamura G, Tsuchiya T, Sato K, Nishizuka S, Motoyama T. Promoter methylation status of E-cadherin, hMLH1, and p16 genes in nonneoplastic gastric epithelia. Am J Pathol. 2002 Aug;161(2):399–403.
38.
Wang ZK, Liu J, Liu C, Wang FY, Chen CY, Zhang XH. Hypermethylation of adenomatous polyposis coli gene promoter is associated with novel Wnt signaling pathway in gastric adenomas. J Gastroenterol Hepatol. 2012 Oct;27(10):1629–34.
39.
Liu F, Lu X, Zhou X, Huang H. APC gene promoter methylation as a potential biomarker for lung cancer diagnosis: a meta-analysis. Thorac Cancer. 2021 Nov;12(21):2907–13.
40.
Hosoya K, Yamashita S, Ando T, Nakajima T, Itoh F, Ushijima T. Adenomatous polyposis coli 1A is likely to be methylated as a passenger in human gastric carcinogenesis. Cancer Lett. 2009 Nov 28;285(2):182–9.
41.
Clement G, Bosman FT, Fontolliet C, Benhattar J. Monoallelic methylation of the APC promoter is altered in normal gastric mucosa associated with neoplastic lesions. Cancer Res. 2004 Oct 1;64(19):6867–73.
42.
Togasaki K, Sugimoto S, Ohta Y, Nanki K, Matano M, Takahashi S, et al. Wnt signaling shapes the histologic variation in diffuse gastric cancer. Gastroenterology. 2021 Feb;160(3):823–30.
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