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 胃癌可依据与遗传改变相关的分子特征分为四种亚型，即爱泼斯坦-巴尔病毒（Epstein-Barr virus, EBV）阳性型、微卫星高度不稳定（microsatellite instability-high, MSI-high）型、染色体不稳定（chromosomal instability, CIN）型以及基因组稳定（genomically stable, GS）型，该分类方法被称为TCGA分类系统。本研究旨在基于23个基因座的异常甲基化状态，阐明四种胃癌（GC）亚型的表观遗传学特征。
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。研究人员根据分子特征，将胃癌组织划分为符合TCGA分类标准的四种亚型：MSI-high型、EBV阳性型、CIN型及GS型。采用13个多态性微卫星序列基因座检测杂合性缺失（loss of heterogeneity, LOH）以判断CIN状态；通过3个单核苷酸重复标记物确定MSI状态；从胃癌组织提取的基因组DNA中扩增EBV BNRF1序列，以此确定EBV感染情况；采用联合亚硫酸氢盐限制性分析（combined bisulfite restriction analysis, COBRA）检测23个基因座的甲基化状态。同时确认了其他相关指标的状态，包括KRAS突变、HER2表达情况以及幽门螺杆菌（Helicobacter pylori）感染情况。
结果 本研究中的胃癌组织被划分为四类：MSI-high型（13%）、EBV阳性型（7%）、CIN型（53%）及GS型（27%）。组织学分类结果显示，MSI-high型及GS型胃癌中低分化腺癌（poorly differentiated adenocarcinoma, por）占比更高，而GS型胃癌中印戒细胞癌（signet-ring cell carcinoma, sig）占比更高。在本次检测的23个基因座中，有18个基因座在癌组织中呈现显著高甲基化状态。无监督聚类分析将胃癌组织分为两个聚类簇，结果显示绝大多数GS型肿瘤聚集于甲基化水平较低的聚类簇中，与其他亚型肿瘤明显区分开来。变量间聚类分析显示，其中一个聚类簇包含3个属于Wnt信号级联反应的基因座（SFRP2区域1/2及APC），即Wnt相关基因座。Wnt相关基因座的平均甲基化评分在GS型肿瘤中最低（MSI-high型：2.7 [95%置信区间（confidence interval, CI）：2.3~2.9]；EBV阳性型：2.1 [1.2~3.1]；CIN型：2.4 [2.2~2.7]；GS型：1.3 [0.8~0.7]）。与之相反，其余15个基因座的平均甲基化评分在MSI-high型肿瘤中显著升高，而GS型肿瘤的该评分与EBV阳性型或CIN型肿瘤无显著差异（MSI-high型：10.4 [8.3~12.4]；EBV阳性型：5.7 [1.7~9.7]；CIN型：4.4 [3.6~5.1]；GS型：3.4 [2.2~4.6]）。此外，仅在印戒细胞癌（sig）肿瘤中观察到Wnt相关基因座的甲基化评分较低。
结论 GS型胃癌具有独特的Wnt信号通路DNA低甲基化特征谱，这一特征在印戒细胞癌亚型中尤为显著。