Supplementary Material for: Genomically stable gastric cancer characterized by hypomethylation in Wnt signal cascade

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 (LOH) 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 (COBRA). 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 (por) was more in tumors categorized in MSI-high, and GS and signet-ring cell carcinoma (sig) was more in GS. Among the 23 loci investigated their methylation status, 18 loci were significantly hypermethylated in caner tissues. A 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 (CI), 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 signet-ring cell carcinoma.

引言 胃癌可根据与遗传改变相关的分子特征分为四个亚型，即EB病毒（Epstein-Barr virus, EBV）、微卫星不稳定高型（microsatellite instability-high, MSI-high）、染色体不稳定型（chromosomal instability, CIN）和基因组稳定型（genomically stable, GS），该分型被称为TCGA分类。本研究旨在通过23个基因位点的异常甲基化状态，阐明四种胃癌亚型的表观遗传特征。材料与方法 本研究共纳入98例胃癌组织及其配对的正常胃黏膜样本。研究人员依据MSI-high、EBV、CIN及GS的分子特征，将胃癌划分为TCGA四大亚型。通过13个多态性微卫星序列位点检测杂合性缺失（loss of heterogeneity, LOH），以此判定CIN状态。MSI状态通过3个单核苷酸重复标记进行确认。EB病毒感染情况通过从胃癌组织基因组DNA中扩增EBV BNRF1序列予以验证。采用联合亚硫酸氢盐限制性分析（combined bisulfite restriction analysis, COBRA）检测23个基因位点的甲基化水平。此外，本研究还对KRAS突变、HER2表达状态及幽门螺杆菌（helicobacter pylori）感染情况进行了确认。结果 本研究纳入的胃癌病例中，MSI型占13%、EBV型占7%、CIN型占53%、GS型占27%。组织学分型结果显示，MSI-high型与GS型胃癌中低分化腺癌（poorly differentiated adenocarcinoma, por）的占比更高，而GS型胃癌中印戒细胞癌（signet-ring cell carcinoma, sig）的占比更高。在本次检测的23个基因位点中，有18个位点在癌组织中呈现显著高甲基化。无监督聚类将胃癌分为两个聚类簇，结果显示绝大多数GS型肿瘤聚集于甲基化水平更低的聚类簇中，与其余亚型界限清晰。变量间聚类分析显示，其中一个聚类簇包含属于Wnt信号级联反应（Wnt signal cascade）的3个基因位点（SFRP2区域1/2及APC），即Wnt相关位点。Wnt相关位点的平均甲基化评分在GS型肿瘤中最低（MSI-high: 2.7 [95%置信区间（confidence interval, CI）, 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]）。与之相反，其余15个位点的平均甲基化评分在MSI-high型中显著升高，而GS型的评分与EBV型或CIN型相当（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]）。此外，仅在印戒细胞癌肿瘤中观察到Wnt相关位点的低甲基化评分。结论 GS型胃癌具有独特的DNA低甲基化特征谱，尤其在Wnt信号通路中，且该特征在印戒细胞癌亚型中尤为显著。