Estimation of the fraction of cancer cells in a tumor DNA sample using DNA methylation.
Estimation of the fraction of cancer cells in a tumor DNA sample using DNA methylation.
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DOI：
10.1371/journal.pone.0082302
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Ushijima T
中科院分区：
文献类型：
--
作者：
Takahashi T;Matsuda Y;Yamashita S;Hattori N;Kushima R;Lee YC;Igaki H;Tachimori Y;Nagino M;Ushijima T
正常细胞的污染几乎总是存在于肿瘤样本中，并影响其分子分析。DNA甲基化是一种稳定的表观遗传修饰，与细胞类型有关，在癌症细胞和正常细胞之间有所不同。在这里，我们的目的是证明DNA甲基化可以用来估计肿瘤DNA样本中癌细胞的比例，以食管鳞状细胞癌（ESCC）为例。首先，通过Infinium HumanMethylation450 BeadChip阵列，我们分离了3个基因组区域（TFAP2B、ARHGEF4和RAPGEFL1）： 1)在4个ESCC细胞系中高度甲基化，2)在非癌性粘膜、正常食管粘膜和外周白细胞的混合样本中几乎不甲基化，3)在28个ESCC中频繁甲基化（TFAP2B, 24/28; ARHGEF4, 20/28; RAPGEFL1, 19/28）。其次，利用激光捕获显微解剖制备的8对癌细胞和非癌细胞样品，我们证实了ESCC细胞中三个区域中至少有一个几乎完全甲基化，而非癌细胞中三个区域几乎完全未甲基化。我们还证实，在15份ESCC样本中，这三个区域的DNA拷贝数改变是罕见的，并且不影响癌细胞比例的估计。然后，肿瘤DNA样本中的癌细胞部分被定义为三个区域中甲基化水平最高的部分，我们证实了DNA甲基化分数标记评估的分数与病理学家评估的分数之间的高度相关性（r=0.85; p<0.001）。最后，我们观察到，通过校正癌细胞含量，肿瘤抑制基因启动子区域的CpG岛几乎完全甲基化。这些结果表明，DNA甲基化可以用来估计肿瘤DNA样本中癌细胞的比例。
Contamination of normal cells is almost always present in tumor samples and affects their molecular analyses. DNA methylation, a stable epigenetic modification, is cell type-dependent, and different between cancer and normal cells. Here, we aimed to demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample, using esophageal squamous cell carcinoma (ESCC) as an example. First, by an Infinium HumanMethylation450 BeadChip array, we isolated three genomic regions (TFAP2B, ARHGEF4, and RAPGEFL1) i) highly methylated in four ESCC cell lines, ii) hardly methylated in a pooled sample of non-cancerous mucosae, a pooled sample of normal esophageal mucosae, and peripheral leukocytes, and iii) frequently methylated in 28 ESCCs (TFAP2B, 24/28; ARHGEF4, 20/28; and RAPGEFL1, 19/28). Second, using eight pairs of cancer and non-cancer cell samples prepared by laser capture microdissection, we confirmed that at least one of the three regions was almost completely methylated in ESCC cells, and all the three regions were almost completely unmethylated in non-cancer cells. We also confirmed that DNA copy number alterations of the three regions in 15 ESCC samples were rare, and did not affect the estimation of the fraction of cancer cells. Then, the fraction of cancer cells in a tumor DNA sample was defined as the highest methylation level of the three regions, and we confirmed a high correlation between the fraction assessed by the DNA methylation fraction marker and the fraction assessed by a pathologist (r=0.85; p<0.001). Finally, we observed that, by correction of the cancer cell content, CpG islands in promoter regions of tumor-suppressor genes were almost completely methylated. These results demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample.
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