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. 2012 Aug;4(2):268-274.
doi: 10.3892/ol.2012.708. Epub 2012 May 9.

Identification of a DNA methylation marker that detects the presence of lymph node metastases of gastric cancers

Yasuyuki Shigematsu  1 , Tohru Niwa, Satoshi Yamashita, Hirokazu Taniguchi, Ryoji Kushima, Hitoshi Katai, Seiji Ito, Tetsuya Tsukamoto, Masao Ichinose, Toshikazu Ushijima
Affiliations

Identification of a DNA methylation marker that detects the presence of lymph node metastases of gastric cancers

Yasuyuki Shigematsu et al. Oncol Lett. 2012 Aug.

Abstract

The accurate detection of the presence of lymph node metastases (LNM) of gastric cancers (GCs) is useful for the implementation of necessary and sufficient treatment, but current methods of detection are unsatisfactory. In the present study, we focused on DNA methylation markers since they have several advantages, including biological and chemical stability and informativeness even in the presence of contaminating cells. Using three metastatic lymph nodes and three primary GCs without LNM, methylation bead array analyses were performed, which enabled the interrogation of 485,577 CpG sites. A total of 31 CpG sites that were hypermethylated in the metastatic lymph nodes, compared with the GCs without LNM, were isolated. Using primary GCs with and without LNM (28 GCs with LNM and 10 without), their methylation levels were measured using quantitative PCR following treatment with sodium bisulfite or a methylation-sensitive restriction enzyme. Of the genomic regions around the 31 CpG sites, 10 regions demonstrated higher methylation levels in the GCs with LNM compared with the GCs without LNM (P<0.05). Finally, the hypermethylation of the 10 regions was validated using another set of samples (129 GCs with LNM and 20 without). Hypermethylation of the region around the cg06436185 CpG site predicted the presence of LNM at a sensitivity of 43% and specificity of 85%. Additionally, the hypermethylation of the region was associated with a poor survival rate among GC patients with LNM. The results of the present study indicated that the methylation status of the region was a promising candidate marker to detect the presence of LNM of GCs and may reflect the malignant potential of GCs.

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Figures

Figure 1
Figure 1
Representative genomic regions around the CpG sites differentially methylated between metastatic lymph nodes and GCs without LNM and primer design in the regions. Below the genomic structure of a region, β values (methylation levels) of the CpG sites carried by Infinium bead array are shown. The differentially methylated CpG site is marked by a rectangle with dotted line. A CpG map is drawn at the bottom, vertical lines (solid and broken lines) indicate CpG sites and broken lines indicate CpG sites whose β values were measured. (A) A region whose methylation level was assessed by qMSP. Primers specific to the methylated sequence (closed arrows) were designed on CpG sites around the differentially methylated sites based on the bisulfite-modified sequence. (B) A region whose methylation level was assessed by qPTMR. Primers (open arrows) were designed to amplify the region encompassing the MspJI-cleaved site (thin vertical arrow) based on the unmodified sequence. GC, gastric cancer; MLN, metastatic lymph nodes; LNM, lymph node metastases; qMSP, quantitative methylation-specific PCR; qPTMR, quantitative PCR following treatment with a methylation-dependent restriction enzyme.
Figure 2
Figure 2
Methylation levels of the candidate genomic regions in primary GCs with and without LNM. Methylation levels were measured by (A) qMSP and (B) qPTMR in the screening sets. The screening set consisted of 10 GCs without LNM and 28 with LNM. (C) Methylation level of the region around cg06436185 in the validation sets was measured by qMSP. The validation set consisted of 20 GCs without LNM and 129 with LNM. Meta (−), GCs without LNM; Meta (+), those with LNM. Horizontal dotted lines are the cut-off methylation levels and the number on the line indicates the value of the level. GC, gastric cancer; LNM, lymph node metastases; qMSP, quantitative methylation-specific PCR; qPTMR, quantitative PCR following treatment with a methylation-dependent restriction enzyme; PUR, percentage of the value of unmethylated reference; PMR, percentage of the value of methylated DNA reference.
Figure 3
Figure 3
Kaplan-Meier analysis of 55 GC patients with LNM. (A) Overall survival in all the patients with LNM. (B and C) The survival curves of patients categorized into T3 and T4. Methylation (+), GCs with PMR of the region around cg06436185>28.8; Methylation (−), GCs with the PMR<28.8. GC, gastric cancer; LNM, lymph node metastases; PMR, percentage of the value of methylated DNA reference.
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