Institut National de la Sante et de la Recherche Médicale, Université de Bourgogne, Dijon, France.
BMC Cancer. 2012 Jan 13;12:12. doi: 10.1186/1471-2407-12-12.
In colorectal carcinoma, extensive gene promoter hypermethylation is called the CpG island methylator phenotype (CIMP). Explaining why studies on CIMP and survival yield conflicting results is essential. Most experiments to measure DNA methylation rely on the sodium bisulfite conversion of unmethylated cytosines into uracils. No study has evaluated the performance of bisulfite conversion and methylation levels from matched cryo-preserved and Formalin-Fixed Paraffin Embedded (FFPE) samples using pyrosequencing.
Couples of matched cryo-preserved and FFPE samples from 40 colon adenocarcinomas were analyzed. Rates of bisulfite conversion and levels of methylation of LINE-1, MLH1 and MGMT markers were measured.
For the reproducibility of bisulfite conversion, the mean of bisulfite-to-bisulfite standard deviation (SD) was 1.3%. The mean of run-to-run SD of PCR/pyrosequencing was 0.9%. Of the 40 DNA couples, only 67.5%, 55.0%, and 57.5% of FFPE DNA were interpretable for LINE-1, MLH1, and MGMT markers, respectively, after the first analysis. On frozen samples the proportion of well converted samples was 95.0%, 97.4% and 87.2% respectively. For DNA showing a total bisulfite conversion, 8 couples (27.6%) for LINE-1, 4 couples (15.4%) for MLH1 and 8 couples (25.8%) for MGMT displayed significant differences in methylation levels.
Frozen samples gave reproducible results for bisulfite conversion and reliable methylation levels. FFPE samples gave unsatisfactory and non reproducible bisulfite conversions leading to random results for methylation levels. The use of FFPE collections to assess DNA methylation by bisulfite methods must not be recommended. This can partly explain the conflicting results on the prognosis of CIMP colon cancers.
在结直肠癌中，广泛的基因启动子过度甲基化被称为 CpG 岛甲基化表型（CIMP）。解释为什么 CIMP 和生存的研究结果存在冲突是至关重要的。大多数用于测量 DNA 甲基化的实验都依赖于亚硫酸氢盐将未甲基化的胞嘧啶转化为尿嘧啶。没有研究评估过焦磷酸测序中从匹配的冷冻保存和福尔马林固定石蜡包埋（FFPE）样本中获得的亚硫酸氢盐转化率和甲基化水平。
对 40 例结肠腺癌的冷冻保存和 FFPE 样本进行了配对分析。测量了 LINE-1、MLH1 和 MGMT 标记物的亚硫酸氢盐转化率和甲基化水平。
对于亚硫酸氢盐转化率的可重复性，亚硫酸氢盐-亚硫酸氢盐标准偏差（SD）的平均值为 1.3%。PCR/焦磷酸测序的运行间 SD 的平均值为 0.9%。在 40 对 DNA 中，只有 67.5%、55.0%和 57.5%的 FFPE DNA 分别在第一次分析后可用于 LINE-1、MLH1 和 MGMT 标记物。在冷冻样本中，完全转化的样本比例分别为 95.0%、97.4%和 87.2%。对于显示总亚硫酸氢盐转化率的 DNA，8 对（27.6%）的 LINE-1、4 对（15.4%）的 MLH1 和 8 对（25.8%）的 MGMT 标记物的甲基化水平存在显著差异。
冷冻样本的亚硫酸氢盐转化率和可靠的甲基化水平具有可重复性。FFPE 样本的亚硫酸氢盐转化率不理想且不可重复，导致甲基化水平的结果随机。不建议使用 FFPE 样本进行亚硫酸氢盐方法评估 DNA 甲基化。这可以部分解释 CIMP 结肠癌预后研究结果存在冲突的原因。
