Sakai Eiji, Fukuyo Masaki, Ohata Ken, Matsusaka Keisuke, Doi Noriteru, Mano Yasunobu, Takane Kiyoko, Abe Hiroyuki, Yagi Koichi, Matsuhashi Nobuyuki, Fukushima Junichi, Fukayama Masashi, Akagi Kiwamu, Aburatani Hiroyuki, Nakajima Atsushi, Kaneda Atsushi
Department of Gastroenterology, Yokohama City University School of Medicine, Yokohama, Japan.
Department of Molecular Oncology, Graduate School of Medicine, Chiba University, Chiba, Japan.
Int J Cancer. 2016 Apr 1;138(7):1634-44. doi: 10.1002/ijc.29903. Epub 2015 Nov 12.
To clarify molecular alterations in serrated pathway of colorectal cancer (CRC), we performed epigenetic and genetic analyses in sessile serrated adenoma/polyps (SSA/P), traditional serrated adenomas (TSAs) and high-methylation CRC. The methylation levels of six Group-1 and 14 Group-2 markers, established in our previous studies, were analyzed quantitatively using pyrosequencing. Subsequently, we performed targeted exon sequencing analyses of 126 candidate driver genes and examined molecular alterations that are associated with cancer development. SSA/P showed high methylation levels of both Group-1 and Group-2 markers, frequent BRAF mutation and occurrence in proximal colon, which were features of high-methylation CRC. But TSA showed low-methylation levels of Group-1 markers, less frequent BRAF mutation and occurrence at distal colon. SSA/P, but not TSA, is thus considered to be precursor of high-methylation CRC. High-methylation CRC had even higher methylation levels of some genes, e.g., MLH1, than SSA/P, and significant frequency of somatic mutations in nonsynonymous mutations (p < 0.0001) and insertion/deletions (p = 0.002). MLH1-methylated SSA/P showed lower methylation level of MLH1 compared with high-methylation CRC, and rarely accompanied silencing of MLH1 expression. The mutation frequencies were not different between MLH1-methylated and MLH1-unmethylated SSA/P, suggesting that MLH1 methylation might be insufficient in SSA/P to acquire a hypermutation phenotype. Mutations of mismatch repair genes, e.g., MSH3 and MSH6, and genes in PI3K, WNT, TGF-β and BMP signaling (but not in TP53 signaling) were significantly involved in high-methylation CRC compared with adenoma, suggesting importance of abrogation of these genes in serrated pathway.
为了阐明结直肠癌（CRC）锯齿状途径中的分子改变，我们对无蒂锯齿状腺瘤/息肉（SSA/P）、传统锯齿状腺瘤（TSA）和高甲基化CRC进行了表观遗传学和遗传学分析。使用焦磷酸测序法定量分析了我们之前研究中确定的6个第1组和14个第2组标志物的甲基化水平。随后，我们对126个候选驱动基因进行了靶向外显子测序分析，并检查了与癌症发生相关的分子改变。SSA/P表现出第1组和第2组标志物的高甲基化水平、频繁的BRAF突变以及发生在近端结肠，这些都是高甲基化CRC的特征。但TSA表现出第1组标志物的低甲基化水平、较少的BRAF突变以及发生在远端结肠。因此，SSA/P而非TSA被认为是高甲基化CRC的前体。高甲基化CRC某些基因（如MLH1）的甲基化水平甚至高于SSA/P，并且非同义突变（p < 0.0001）和插入/缺失（p = 0.002）的体细胞突变频率显著。与高甲基化CRC相比，MLH1甲基化的SSA/P显示出较低的MLH1甲基化水平，并且很少伴有MLH1表达的沉默。MLH1甲基化和未甲基化的SSA/P之间的突变频率没有差异，这表明在SSA/P中MLH1甲基化可能不足以获得高突变表型。与腺瘤相比，错配修复基因（如MSH3和MSH6）以及PI3K、WNT、TGF-β和BMP信号通路（但不包括TP53信号通路）中的基因突变在高甲基化CRC中显著参与，这表明在锯齿状途径中消除这些基因很重要。
