Liang Zong1,2, Naoko Hattori1, Yukie Yoda1, Satoshi Yamashita1, Hideyuki Takeshima1, Takamasa Takahashi1, Masahiro Maeda1, Hitoshi Katai3, Sohachi Nanjo4, Takayuki Ando4, Yasuyuki Seto2, Toshikazu Ushijima5. 1. Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan. 2. Department of Gastrointestinal Surgery, Graduate School of Medicine, The University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. 3. Gastric Surgery Division, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan. 4. Third Department of Internal Medicine, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan. 5. Division of Epigenomics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan. tushijim@ncc.go.jp.
Abstract
BACKGROUND: Tumor samples are unavoidably contaminated with coexisting normal cells. Here, we aimed to establish a DNA methylation marker to estimate the fraction of gastric cancer (GC) cells in any DNA sample by isolating genomic regions specifically methylated in GC cells. METHODS: Genome-wide and gene-specific methylation analyses were conducted with an Infinium HumanMethylation450 BeadChip array and by quantitative methylation-specific PCR, respectively. Purified cancer and noncancer cells were prepared by laser-capture microdissection. TP53 mutation data were obtained from our previous study using next-generation target sequencing. RESULTS: Genome-wide DNA methylation analysis of 12 GC cell lines, 30 GCs, six normal gastric mucosae, one sample of peripheral leukocytes, and four noncancerous gastric mucosae identified OSR2, PPFIA3, and VAV3 as barely methylated in normal cells and highly methylated in cancer cells. Quantitative methylation-specific PCR using 26 independent GCs validated that one or more of them was highly methylated in all of the GCs. Using four pairs of purified cells, we confirmed the three genes were highly methylated (85 % or more) in cancer cells and barely methylated (5 % or less) in noncancer cells. The cancer cell fraction assessed by the panel of the three genes showed good correlation with that assessed by the TP53 mutant allele frequency in 13 GCs (r = 0.77). After correction of the GC cell fraction, unsupervised clustering analysis of the genome-wide DNA methylation profiles yielded clearer clustering. CONCLUSIONS: A DNA methylation marker-namely, the panel of the three genes-is useful to estimate the cancer cell fraction in GCs.
BACKGROUND: Tumor samples are unavoidably contaminated with coexisting normal cells. Here, we aimed to establish a DNA methylation marker to estimate the fraction of gastric cancer (GC) cells in any DNA sample by isolating genomic regions specifically methylated in GC cells. METHODS: Genome-wide and gene-specific methylation analyses were conducted with an Infinium HumanMethylation450 BeadChip array and by quantitative methylation-specific PCR, respectively. Purified cancer and noncancer cells were prepared by laser-capture microdissection. TP53 mutation data were obtained from our previous study using next-generation target sequencing. RESULTS: Genome-wide DNA methylation analysis of 12 GC cell lines, 30 GCs, six normal gastric mucosae, one sample of peripheral leukocytes, and four noncancerous gastric mucosae identified OSR2, PPFIA3, and VAV3 as barely methylated in normal cells and highly methylated in cancer cells. Quantitative methylation-specific PCR using 26 independent GCs validated that one or more of them was highly methylated in all of the GCs. Using four pairs of purified cells, we confirmed the three genes were highly methylated (85 % or more) in cancer cells and barely methylated (5 % or less) in noncancer cells. The cancer cell fraction assessed by the panel of the three genes showed good correlation with that assessed by the TP53 mutant allele frequency in 13 GCs (r = 0.77). After correction of the GC cell fraction, unsupervised clustering analysis of the genome-wide DNA methylation profiles yielded clearer clustering. CONCLUSIONS: A DNA methylation marker-namely, the panel of the three genes-is useful to estimate the cancer cell fraction in GCs.
Entities:
Keywords:
Cancer cell fraction; DNA methylation; Epigenetics; Gastric cancer
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