Establishment of a DNA methylation marker to evaluate cancer cell fraction in gastric cancer

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 methylationspecific 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.

Detection of OSR2, VAV3, and PPFIA3 Methylation in the Serum of Patients with Gastric Cancer

“…In this study, we evaluated the methylation status of three genes together in both tissue and serum samples to improve the sensitivity. According to Zong et al [ 20 ] research, by using an Infinium HumanMethylation 450 BeadChip array, they identified that OSR2, VAV3, and PPFIA3 are barely methylated in normal cells but highly methylated in GC cells. To further assess the clinical value of methylated OSR2, VAV3, and PPFIA3, we first analyzed those genes' methylation status in GC tissue samples or PCHNTs samples.…”

“…Current microarray technology provides us with an opportunity for high-throughput unbiased methylation analysis of a large number of CpG sites [ 19 ]. By using an Infinium HumanMethylation 450 BeadChip array, Zong and colleagues [ 20 ] identified three genes (OSR2, VAV3, and PPFIA3) that were hypermethylated in GC tissue. Odd-skipped related 2 (OSR2), which contains DNA-binding C2H2-type zinc finger domains in the C-terminal half, plays an important role in cellular quiescence and proliferation under epigenetic regulation [ 21 , 22 ].…”

“…Odd-skipped related 2 (OSR2), which contains DNA-binding C2H2-type zinc finger domains in the C-terminal half, plays an important role in cellular quiescence and proliferation under epigenetic regulation [ 21 , 22 ]. Additionally, recent articles have reported that VAV3 (a member of VAV gene family which plays an important role in the process of tumor development and metastasis) and PTPRF-Interacting Protein Alpha-3 (PPFIA3) may associated with the tumorigenesis and development of GC [ 20 , 23 ]. In the present study, we sought to explore the feasibility of DNA methylation status of OSR2, VAV3, and PPFIA3 as a noninvasive screening tool for GC.…”

Hypermethylation of BEND5 contributes to cell proliferation and is a prognostic marker of colorectal cancer

“…The Illumina Infinium HumanMethylation450 BeadChip array has been used to detect genome-wide DNA methylation profile alterations in cancer [ 14 – 16 ]. Using this array, we analyzed 26 paired CRC and noncancerous colorectal tissues and found multiple hypermethylated loci in the promoter and exon 1 regions of BEND5 .…”

Pancreatic Cancer Cell Fraction Estimation in a DNA Sample

“…To estimate the cancer cell fraction in DNA samples, we previously established a unique method using cancer cell-specific DNA methylation in esophageal squamous cell carcinomas and gastric cancers [9,10]. The method utilizes a panel of a limited number of genes, typically three, specifically methylated in cancer cells and unmethylated in noncancer cells, such as normal epithelial cells, fibroblasts, and blood cells.…”