Nosho Katsuhiko, Irahara Natsumi, Shima Kaori, Kure Shoko, Kirkner Gregory J, Schernhammer Eva S, Hazra Aditi, Hunter David J, Quackenbush John, Spiegelman Donna, Giovannucci Edward L, Fuchs Charles S, Ogino Shuji
Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA.
PLoS One. 2008;3(11):e3698. doi: 10.1371/journal.pone.0003698. Epub 2008 Nov 12.
The CpG island methylator phenotype (CIMP) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. Recent investigations have selected 5 promoters (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1) as surrogate markers for CIMP-high. However, no study has comprehensively evaluated an expanded set of methylation markers (including these 5 markers) using a large number of tumors, or deciphered the complex clinical and molecular associations with CIMP-high determined by the validated marker panel. METHOLODOLOGY/PRINCIPAL FINDINGS: DNA methylation at 16 CpG islands [the above 5 plus CDKN2A (p16), CHFR, CRABP1, HIC1, IGFBP3, MGMT, MINT1, MINT31, MLH1, p14 (CDKN2A/ARF) and WRN] was quantified in 904 colorectal cancers by real-time PCR (MethyLight). In unsupervised hierarchical clustering analysis, the 5 markers (CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1), CDKN2A, CRABP1, MINT31, MLH1, p14 and WRN were generally clustered with each other and with MSI and BRAF mutation. KRAS mutation was not clustered with any methylation marker, suggesting its association with a random methylation pattern in CIMP-low tumors. Utilizing the validated CIMP marker panel (including the 5 markers), multivariate logistic regression demonstrated that CIMP-high was independently associated with older age, proximal location, poor differentiation, MSI-high, BRAF mutation, and inversely with LINE-1 hypomethylation and beta-catenin (CTNNB1) activation. Mucinous feature, signet ring cells, and p53-negativity were associated with CIMP-high in only univariate analysis. In stratified analyses, the relations of CIMP-high with poor differentiation, KRAS mutation and LINE-1 hypomethylation significantly differed according to MSI status.
Our study provides valuable data for standardization of the use of CIMP-high-specific methylation markers. CIMP-high is independently associated with clinical and key molecular features in colorectal cancer. Our data also suggest that KRAS mutation is related with a random CpG island methylation pattern which may lead to CIMP-low tumors.
CpG岛甲基化表型（CIMP）是一种与结肠癌微卫星不稳定性（MSI）和BRAF突变相关的独特表型。最近的研究选择了5个启动子（CACNA1G、IGF2、NEUROG1、RUNX3和SOCS1）作为CIMP高的替代标志物。然而，尚无研究使用大量肿瘤全面评估一组扩展的甲基化标志物（包括这5个标志物），或解读经验证的标志物组所确定的与CIMP高相关的复杂临床和分子关联。
方法/主要发现：通过实时PCR（MethyLight）对904例结直肠癌中16个CpG岛（上述5个加上CDKN2A（p16）、CHFR、CRABP1、HIC1、IGFBP3、MGMT、MINT1、MINT31、MLH1、p14（CDKN2A/ARF）和WRN）的DNA甲基化进行定量。在无监督层次聚类分析中，5个标志物（CACNA1G、IGF2、NEUROG1、RUNX3和SOCS1）、CDKN2A、CRABP1、MINT31、MLH1、p14和WRN通常相互聚类，并与MSI和BRAF突变聚类。KRAS突变未与任何甲基化标志物聚类，表明其与CIMP低的肿瘤中的随机甲基化模式相关。利用经验证的CIMP标志物组（包括这5个标志物），多因素逻辑回归表明，CIMP高与年龄较大、近端位置、低分化、MSI高、BRAF突变独立相关，与LINE-1低甲基化和β-连环蛋白（CTNNB1）激活呈负相关。仅在单因素分析中，黏液特征、印戒细胞和p53阴性与CIMP高相关。在分层分析中，CIMP高与低分化、KRAS突变和LINE-1低甲基化的关系根据MSI状态有显著差异。
我们的研究为CIMP高特异性甲基化标志物的标准化使用提供了有价值的数据。CIMP高与结直肠癌的临床和关键分子特征独立相关。我们的数据还表明，KRAS突变与可能导致CIMP低肿瘤的随机CpG岛甲基化模式相关。
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