Zou Hongzhi, Harrington Jonathan J, Shire Abdirashid M, Rego Rafaela L, Wang Liang, Campbell Megan E, Oberg Ann L, Ahlquist David A
Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55905, USA.
Cancer Epidemiol Biomarkers Prev. 2007 Dec;16(12):2686-96. doi: 10.1158/1055-9965.EPI-07-0518.
Discriminant markers are required for accurate cancer screening. We evaluated genes frequently methylated in colorectal neoplasia to identify the most discriminant ones. Four genes specifically methylated in colorectal cancer [bone morphogenetic protein 3 (BMP3), EYA2, aristaless-like homeobox-4 (ALX4), and vimentin] were selected from 41 candidate genes and evaluated on 74 cancers, 62 adenomas, and 70 normal epithelia. Methylation status was analyzed qualitatively and quantitatively and confirmed by bisulfite genomic sequencing. Effect of methylation on gene expression was evaluated in five colon cancer cell lines. K-ras and BRAF mutations were detected by sequencing. Methylation of BMP3, EYA2, ALX4, or vimentin was detected respectively in 66%, 66%, 68%, and 72% of cancers; 74%, 48%, 89%, and 84% of adenomas; and 7%, 5%, 11%, and 11% of normal epithelia (P < 0.01, cancer or adenoma versus normal). Based on area under the curve analyses, discrimination was not significantly improved by combining markers. Comethylation was frequent (two genes or more in 72% of cancers and 84% of adenomas), associated with proximal neoplasm site (P < 0.001), and linked with both BRAF and K-ras mutations (P < 0.01). Cell line experiments supported silencing of expression by methylation in all four study genes. This study shows BMP3, EYA2, ALX4, and vimentin genes are methylated in most colorectal neoplasms but rarely in normal epithelia. Comethylation of these genes is common, and pursuit of complementary markers for methylation-negative neoplasms is a rational strategy to optimize screening sensitivity.
准确的癌症筛查需要鉴别标志物。我们评估了在结直肠肿瘤中频繁发生甲基化的基因，以确定最具鉴别性的基因。从41个候选基因中选择了4个在结直肠癌中特异性甲基化的基因[骨形态发生蛋白3（BMP3）、EYA2、无翅型同源盒基因4（ALX4）和波形蛋白]，并在74例癌症、62例腺瘤和70例正常上皮组织中进行评估。对甲基化状态进行了定性和定量分析，并通过亚硫酸氢盐基因组测序进行了确认。在5种结肠癌细胞系中评估了甲基化对基因表达的影响。通过测序检测K-ras和BRAF突变。BMP3、EYA2、ALX4或波形蛋白的甲基化分别在66%、66%、68%和72%的癌症中被检测到；在74%、48%、89%和84%的腺瘤中被检测到；在7%、5%、11%和11%的正常上皮组织中被检测到（P<0.01，癌症或腺瘤与正常组织相比）。基于曲线下面积分析，联合使用标志物并没有显著提高鉴别能力。共甲基化很常见（72%的癌症和84%的腺瘤中有两个或更多基因发生共甲基化），与肿瘤近端部位相关（P<0.001），并且与BRAF和K-ras突变均有关联（P<0.01）。细胞系实验支持所有4个研究基因的甲基化导致表达沉默。本研究表明，BMP3、EYA2、ALX4和波形蛋白基因在大多数结直肠肿瘤中发生甲基化，但在正常上皮组织中很少发生。这些基因的共甲基化很常见，寻找甲基化阴性肿瘤的互补标志物是优化筛查敏感性的合理策略。
