Inoue A, Okamoto K, Fujino Y, Nakagawa T, Muguruma N, Sannomiya K, Mitsui Y, Takaoka T, Kitamura S, Miyamoto H, Okahisa T, Fujimori T, Imoto I, Takayama T
Department of Gastroenterology and Oncology, Institute of Health Biosciences, The University of Tokushima Graduate School, Tokushima 770-8503, Japan.
Department of Surgical and Molecular Pathology, Dokkyo University School of Medicine, 880 Kitakobayashi, Mibu, Tochigi 321-0293, Japan.
Br J Cancer. 2015 Jan 20;112(2):403-12. doi: 10.1038/bjc.2014.545. Epub 2014 Oct 14.
Sessile serrated adenomas/polyps (SSA/Ps) are a putative precursor of colon cancer with microsatellite instability (MSI). However, the developmental mechanism of SSA/P remains unknown. We performed genetic analysis and genome-wide DNA methylation analysis in aberrant crypt foci (ACF), SSA/P, and cancer in SSA/P specimens to show a close association between ACF and the SSA/P-cancer sequence. We also evaluated the prevalence and number of ACF in SSA/P patients.
ACF in the right-side colon were observed in 36 patients with SSA/Ps alone, 2 with cancers in SSA/P, and 20 normal subjects and biopsied under magnifying endoscopy. B-RAF mutation and MSI were analysed by PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP, respectively, in 15 ACF, 20 SSA/P, and 2 cancer specimens. DNA methylation array analysis of seven ACF, seven SSA/P, and two cancer in SSA/P specimens was performed using the microarray-based integrated analysis of methylation by isochizomers (MIAMI) method.
B-RAF mutations were frequently detected in ACF, SSA/P, and cancer in SSA/P tissues. The number of methylated genes increased significantly in the order of ACF<SSA/P<cancer. The most commonly methylated genes in SSA/P were PQLC1, HDHD3, RASL10B, FLI1, GJA3, and SLC26A2. Some of these genes were methylated in ACF, whereas all genes were methylated in cancers. Immunohistochemistry revealed their silenced expression. Microsatellite instability and MLH1 methylation were observed only in cancer. The prevalence and number of ACF were significantly higher in SSA/P patients than in normal subjects. A significant correlation was seen between the numbers of SSA/P and ACF in SSA/P patients.
Our results suggest that ACF are precursor lesions of the SSA/P-cancer sequence in patients with SSA/P, where ACF arise by B-RAF mutation and methylation of some of the six identified genes and develop into SSA/Ps through accumulated methylation of these genes.
无蒂锯齿状腺瘤/息肉（SSA/P）是具有微卫星不稳定性（MSI）的结肠癌的一种假定前体。然而，SSA/P的发育机制仍不清楚。我们对SSA/P标本中的异常隐窝灶（ACF）、SSA/P和癌进行了基因分析及全基因组DNA甲基化分析，以显示ACF与SSA/P - 癌序列之间的密切关联。我们还评估了SSA/P患者中ACF的患病率和数量。
在36例单纯SSA/P患者、2例SSA/P合并癌患者及20例正常受试者的右侧结肠中观察ACF，并在放大内镜下进行活检。分别采用聚合酶链反应 - 限制性片段长度多态性（PCR - RFLP）和聚合酶链反应 - 单链构象多态性（PCR - SSCP）分析15个ACF、20个SSA/P和2个癌标本中的B - RAF突变和MSI。使用基于同裂酶的甲基化微阵列综合分析（MIAMI）方法对7个ACF、7个SSA/P和2个SSA/P合并癌标本进行DNA甲基化阵列分析。
在SSA/P组织中的ACF、SSA/P和癌中经常检测到B - RAF突变。甲基化基因的数量按ACF＜SSA/P＜癌的顺序显著增加。SSA/P中最常甲基化的基因是PQLC1、HDHD3、RASL10B、FLI1、GJA3和SLC26A2。其中一些基因在ACF中甲基化，而在癌中所有基因均甲基化。免疫组织化学显示它们的表达沉默。仅在癌中观察到微卫星不稳定性和MLH1甲基化。SSA/P患者中ACF的患病率和数量显著高于正常受试者。SSA/P患者中SSA/P的数量与ACF的数量之间存在显著相关性。
我们的结果表明，ACF是SSA/P患者中SSA/P - 癌序列的前体病变，其中ACF通过B - RAF突变和六个已鉴定基因中的一些基因的甲基化产生，并通过这些基因的累积甲基化发展为SSA/P。
