de Maat Michiel F G, Umetani Naoyuki, Sunami Eiji, Turner Roderick R, Hoon Dave S B
Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Boulevard, Santa Monica, CA 90404, USA.
Mol Cancer Res. 2007 May;5(5):461-71. doi: 10.1158/1541-7786.MCR-06-0358.
To date, the epigenetic events involved in the progression of colorectal cancer are not well described. To study, in detail, methylation during colorectal cancer development in high-risk adenomas, we developed an assay combining in situ (on-slide) sodium bisulfite modification (SBM) of paraffin-embedded archival tissue sections with absolute quantitative assessment of methylated alleles (AQAMA). We tested the performance of the assay to detect methylation level differences between paired pre-malignant and malignant colorectal cancer stages. AQAMA assays were used to measure methylation levels at MINT (methylated in tumor) loci MINT1, MINT2, MINT12, and MINT31. Assay performance was verified on cell line DNA and standard cDNA. On-slide SBM, allowing DNA methylation assessment of 1 to 2 mm(2) of paraffin-embedded archival tissue, was employed. Methylation levels of adenomatous and cancerous components within a single tissue section in 72 colorectal cancer patients were analyzed. AQAMA was verified as accurately assessing CpG island methylation status in cell lines. The correlation between expected and measured cDNA methylation levels was high for all four MINT AQAMA assays (R >or= 0.966, P<0.001). Methylation levels at the four loci increased in 11% and decreased in 36% of specimens comparing paired adenoma and cancer tissues (P<0.0001 by Kolmogorov-Smirnov test). Single-PCR AQAMA provided accurate methylation level measurement. Variable MINT locus methylation level changes occur during malignant progression of colorectal adenoma. Combining AQAMA with on-slide SBM provides a sensitive assay that allows detailed histology-oriented analysis of DNA methylation levels and may give new, accurate insights into understanding development of epigenetic aberrancies in colorectal cancer progression.
迄今为止，结直肠癌进展过程中涉及的表观遗传事件尚未得到充分描述。为了详细研究高危腺瘤性结直肠癌发生过程中的甲基化情况，我们开发了一种检测方法，将石蜡包埋存档组织切片的原位（载玻片上）亚硫酸氢钠修饰（SBM）与甲基化等位基因的绝对定量评估（AQAMA）相结合。我们测试了该检测方法在检测配对的癌前和恶性结直肠癌阶段之间甲基化水平差异方面的性能。使用AQAMA检测方法测量MINT（肿瘤中甲基化）位点MINT1、MINT2、MINT12和MINT31的甲基化水平。在细胞系DNA和标准cDNA上验证了检测方法的性能。采用载玻片上的SBM，可对1至2平方毫米的石蜡包埋存档组织进行DNA甲基化评估。分析了72例结直肠癌患者单个组织切片中腺瘤和癌性成分的甲基化水平。已证实AQAMA能准确评估细胞系中的CpG岛甲基化状态。对于所有四种MINT AQAMA检测方法，预期和测量的cDNA甲基化水平之间的相关性都很高（R≥0.966，P<0.001）。比较配对的腺瘤和癌组织时，11%的标本中四个位点的甲基化水平升高，36%的标本中甲基化水平降低（通过柯尔莫哥洛夫-斯米尔诺夫检验，P<0.0001）。单重PCR AQAMA提供了准确的甲基化水平测量。在结直肠腺瘤的恶性进展过程中，MINT位点的甲基化水平变化各异。将AQAMA与载玻片上的SBM相结合，提供了一种灵敏的检测方法，可对DNA甲基化水平进行详细的组织学导向分析，并可能为理解结直肠癌进展过程中表观遗传异常的发生提供新的、准确的见解。
