Department of Gastroenterological Surgery, Dentistry and Pharmaceutical Sciences, Okayama University Graduate School of Medicine, Okayama, Japan.
Department of Clinical Oncology, Kawasaki Medical School, Kurashiki, Japan.
Oncology. 2023;101(2):105-116. doi: 10.1159/000527098. Epub 2023 Jan 2.
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.
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.
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.
GS subtype tumors have the potential to possess distinct signatures in DNA hypomethylation profiles in Wnt signaling pathway, especially in sig.
胃癌可根据与遗传改变相关的分子特征分为四个亚型，例如 EBV（Epstein-Barr 病毒）、微卫星不稳定高（MSI-high）、染色体不稳定（CIN）和基因组稳定（GS），称为 TCGA 分类。在这项研究中，我们试图根据 23 个位点的异常甲基化状态来阐明四种 GC 亚型的表观遗传特征。
本研究共纳入 98 例胃癌及其正常胃黏膜标本。我们根据 MSI-high、EBV、CIN 到 GS 的分子特征将胃癌分为 TCGA 亚型。使用 13 个多态性微卫星序列的位点来确定 CIN 的异质性缺失。MSI 状态通过三个单核苷酸重复标记物确定。通过从胃癌中收集的基因组 DNA 中恢复 EBV BNRF1 序列来确定 EBV 感染。通过联合亚硫酸氢盐限制性分析研究 23 个位点的甲基化状态。还确认了其他发现，例如 KRAS 突变、HER2 表达状态和幽门螺杆菌感染的状态。
胃癌分为 MSI（13%）、EBV（7%）、CIN（53%）和 GS（27%）。根据组织学分类，MSI-high 型肿瘤中分化差的腺癌更多，GS 和印戒细胞癌（sig）更多在 GS 中。在研究其甲基化状态的 23 个位点中，18 个位点在癌组织中明显呈高甲基化状态。无监督聚类将胃癌分为两个聚类，并显示大多数 GS 肿瘤聚集在一起，形成一个聚类，其甲基化水平较低，与其他亚型明显不同。变量间聚类显示，一个聚类包含三个属于 Wnt 信号级联（Wnt 相关基因座）的 SFRP2 区 1/2 和 APC 基因座。GS 肿瘤的 Wnt 相关基因座的平均甲基化评分最低（MSI-high：2.7[95%置信区间，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]）。相比之下，MSI-high 中其他 15 个基因座的平均甲基化评分显著较高，而 GS 中的平均甲基化评分与 EBV 或 CIN 相同（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 sig 肿瘤中观察到 Wnt 相关基因座的低甲基化评分。
GS 亚型肿瘤在 Wnt 信号通路的 DNA 低甲基化谱中具有独特的特征，特别是在 sig 肿瘤中。
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