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. 2014 Sep 1;20(17):4598-612.
doi: 10.1158/1078-0432.CCR-13-3380. Epub 2014 Jul 9.

Large-scale characterization of DNA methylation changes in human gastric carcinomas with and without metastasis

Zhaojun Liu  1 , Jun Zhang  2 , Yanhong Gao  1 , Lirong Pei  3 , Jing Zhou  1 , Liankun Gu  1 , Lianhai Zhang  4 , Budong Zhu  5 , Naoko Hattori  6 , Jiafu Ji  4 , Yasuhito Yuasa  7 , Wooho Kim  8 , Toshikazu Ushijima  6 , Huidong Shi  9 , Dajun Deng  10
Affiliations

Large-scale characterization of DNA methylation changes in human gastric carcinomas with and without metastasis

Zhaojun Liu et al. Clin Cancer Res. .

Abstract

Purpose: Metastasis is the leading cause of death for gastric carcinoma. An epigenetic biomarker panel for predicting gastric carcinoma metastasis could have significant clinical impact on the care of patients with gastric carcinoma. The main purpose of this study is to characterize the methylation differences between gastric carcinomas with and without metastasis.

Experimental design: Genome-wide DNA methylation profiles between 4 metastatic and 4 nonmetastatic gastric carcinomas and their surgical margins (SM) were analyzed using methylated-CpG island amplification with microarray. The methylation states of 73 candidate genes were further analyzed in patients with gastric carcinoma in a discovery cohort (n=108) using denatured high performance liquid chromatography, bisulfite-sequencing, and MethyLight. The predictive values of potential metastasis-methylation biomarkers were validated in cohorts of patients with gastric carcinoma in China (n=330), Japan (n=129), and Korea (n=153).

Results: The gastric carcinoma genome showed significantly higher proportions of hypomethylation in the promoter and exon-1 regions, as well as increased hypermethylation of intragenic fragments when compared with SMs. Significant differential methylation was validated in the CpG islands of 15 genes (P<0.05) and confirmed using bisulfite sequencing. These genes included BMP3, BNIP3, CDKN2A, ECEL1, ELK1, GFRA1, HOXD10, KCNH1, PSMD10, PTPRT, SIGIRR, SRF, TBX5, TFPI2, and ZNF382. Methylation changes of GFRA1, SRF, and ZNF382 resulted in up- or downregulation of their transcription. Most importantly, the prevalence of GFRA1, SRF, and ZNF382 methylation alterations was consistently and coordinately associated with gastric carcinoma metastasis and the patients' overall survival throughout discovery and validation cohorts in China, Japan, and Korea.

Conclusion: Methylation changes of GFRA1, SRF, and ZNF382 may be a potential biomarker set for prediction of gastric carcinoma metastasis.

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Conflict of interest statement

Disclosure of potential conflicts of interest: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Distribution of probes with significant GC-related differential methylation changes in the human GC genome
(A) More hypomethylation was observed in the promoter and exon-1 regions, whereas more hypermethylation was observed in the gene body region. (B) Heatmap of the top-100 probes with differential methylation changes between GC and SM samples in supervised analysis. (C). Patterns of detailed regional methylation trends for each chromosome arm in GCs are displayed. The regional methylation value represents the average value of normalized methylation signal ratios between 8 GCs and 8 paired SMs for each sliding window (sequence or region) covering 51 probe-matched fragments. The long-range hypermethylated and hypomethylated regions are indicated with deep-red and blue color, respectively. Double-triangle indicates centromere. (D) Heatmap of the top-100 probes with differential methylation changes between metastatic samples (marked with “+”) and non-metastatic GC samples (marked with “−”) in supervised analysis.
Figure 2
Figure 2. DNA methylation of GFRA1, SRF, and ZNF382 in GC samples
(A) Representative DHPLC chromatograms of bisulfite PCR amplicons of GFRA1, SRF, and ZNF382 CGIs, respectively. The hypermethylated (M) and hypomethylated (U) PCR products of each gene in the 8 pairs of GC and SM samples were separated with the DNASep analytical column at partial denaturing temperature as described in the method section. The peak areas corresponding to the methylated and unmethylated PCR products were used to calculate the percentage of methylated copies [proportion of hypermethylated copies = methylation-peak area/ total peak area] for each gene analyzed. (B) Representative bisulfite clone sequencing results of GFRA1, SRF, and ZNF382 in the representative GC and paired SM samples. The dark red dots indicate methylated CpG sites. Locations of the primer sets and probes used in the MethyLight assays are also illustrated.
Figure 3
Figure 3. Kaplan-Meier survival curves of GC patients with different GFRA1, SRF, and ZNF382 methylation states
(A, B, C) GFRA1 and ZNF382 methylation-high and SRF methylation-positive in GC or SM tissues were good survival factors with statistical significance for GC patients in the Chinese-discovery cohorts, Chinese, Japanese, and Korean validation cohorts. (D) Synergistic analysis of three methylation markers. Distribution of the number of patients with methylation changes in one to three genes (GFRA1, SRF, and ZNF382) in metastatic and non-metastatic GC groups. The number of patients with one more differentially methylated genes in non-metastatic GCs was significantly higher than metastatic GCs (left chart). The more number of genes associates with differential methylation, the longer of overall survival of GC patients will be (right chart).
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