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Clinical Cancer Research Vol. 12, 2995-3002, May 15, 2006
© 2006 American Association for Cancer Research

Human Cancer Biology

CpG Island M`ethylation Status in Gastric Carcinoma with and without Infection of Epstein-Barr Virus

Moon-Sung Chang1,3, Hiroshi Uozaki1, Ja-Mun Chong1, Tetsuo Ushiku1, Kazuya Sakuma4, Shunpei Ishikawa2, Rumi Hino1, Rita Rani Barua1, Yoshiaki Iwasaki5, Kuniyoshi Arai5, Hideki Fujii3, Hideo Nagai4 and Masashi Fukayama1

Authors' Affiliations: 1 Department of Pathology, Graduate School of Medicine and 2 Genome Science Division, Research Center for Advanced Science and Technology, University of Tokyo, Tokyo; 3 Department of Surgery, University of Yamanashi, Yamanashi; 4 Department of Surgery, Jichi Medical School, Tochigi and 5 Department of Surgery, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan

Requests for reprints: Masashi Fukayama, Department of Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo, 113-0033, Japan. Phone: 81-3-5841-3341; Fax: 81-3-3815-8379; E-mail: mfukayama-tky{at}umin.ac.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: EBV-associated gastric carcinoma shows global CpG island methylation of the promoter region of various cancer-related genes. To further clarify the significance of CpG island methylator phenotype (CIMP) status in gastric carcinoma, we investigated methylation profile and clinicopathologic features including overall survival in four subgroups defined by EBV infection and CIMP status: EBV-associated gastric carcinoma and EBV-negative/CIMP-high (H), EBV-intermediate (I), and EBV-negative (N) gastric carcinoma.

Experimental Design: Methylation-specific PCR was applied to 106 gastric carcinoma cases. CIMP-N, CIMP-I, and CIMP-H status was determined by the number (0, 1-3, and 4-5, respectively) of methylated marker genes (LOX, HRASLS, FLNc, HAND1, and TM), that were newly identified as highly methylated in gastric cancer cell lines. The methylation status of 10 other cancer-related genes (p14, p15, p16, p73, TIMP-3, E-cadherin, DAPK, GSTP1, hMLH1, and MGMT) was also evaluated.

Results: Nearly all (14 of 15) of EBV-associated gastric carcinoma exhibited CIMP-H, constituting a homogenous group (14%). EBV-negative gastric carcinoma consisted of CIMP-H (24%), CIMP-I (38%), and CIMP-N (24%). EBV-associated gastric carcinoma showed significantly higher frequencies of methylation of cancer-related genes (mean number ± SD = 6.9 ± 1.5) even if compared with EBV-negative/CIMP-H gastric carcinoma (3.5 ± 1.8). Among EBV-negative gastric carcinoma subgroups, CIMP-H gastric carcinoma showed comparatively higher frequency of methylation than CIMP-I or CIMP-N, especially of p16 and hMLH1. CIMP-N gastric carcinoma predominantly consisted of advanced carcinoma with significantly higher frequency of lymph node metastasis. The prognosis of the patients of CIMP-N was significantly worse compared with other groups overall by univariate analysis (P = 0.0313).

Conclusion: The methylation profile of five representative genes is useful to stratify gastric carcinomas into biologically different subgroups. EBV-associated gastric carcinoma showed global CpG island methylation, comprising a pathogenetically distinct subgroup in CIMP-H gastric carcinoma.

Epigenetic alterations, such as CpG island methylation of the promoter region of various cancer-associated genes, are now regarded as one of major mechanisms of the development and progression of gastric carcinoma (1, 2). In the investigation of EBV-associated gastric carcinoma, we found that global DNA methylation and subsequent gene silencing occurs typically in this specific type of gastric carcinoma (3–6). Both p16 and E-cadherin expression were decreased or abnormal in association with the promoter methylation of each corresponding gene in EBV-associated gastric carcinoma, but such correlation was equivocal in EBV-negative gastric carcinoma (3, 5, 7). Because EBV-associated gastric carcinoma shows distinct clinicopathologic characteristics, such as male predominance, preferred occurrence of gastric corpus, and a diffuse type of histology (8), it is reasonable to assume that the CpG island methylator phenotype (CIMP) status, high or low, is also closely associated with some remarkable features in EBV-negative gastric carcinoma.

Toyota et al. first attempted to define CIMP-high (H) gastric carcinoma based on the methylation of MINT clones (9), and thereafter, CIMP-H gastric carcinoma has been reported as 24% to 41% of total gastric carcinoma (10–13). However, the significance of CIMP status in gastric carcinoma has not been fully clarified, although CIMP-H gastric carcinoma is likely to show p16 and hMLH1 methylation (9, 11, 13, 14). In this study, we adopted the following strategies to clarify the significance of CIMP in gastric carcinoma. We used five genes as marker genes for the analysis [lysyl oxidase (LOX), HRAS-like suppressor (HRASLS), filamin C (FLNc), HAND1, and thrombomodulin (TM)], which were recently identified from gastric cancer cell lines (15). CIMP status in carcinomas of various organs has been defined using MINT clones. However, these clones were originally identified from a colorectal cancer cell line (16). The methylation frequency of each MINT clone varies in carcinomas of various organs (2, 17), and some CpG islands might be specifically methylated in the tumor in tumor type–specific or organ-specific manner. Second, we defined CIMP-negative (CIMP-N) and CIMP-H gastric carcinoma more strictly by the methylation number of none and 4-5 of five of these genes to underscore both subgroups. Third, as emphasized above, we classified EBV-associated gastric carcinoma as a separated group to exclude its influence in the analysis of CIMP-H gastric carcinoma (3–6). Thus, in this study using these strategies, the methylation status of various cancer-related genes, the clinicopathologic features, including the prognosis of the patients, were comparatively evaluated in four subgroups: EBV-associated gastric carcinoma, EBV-negative/CIMP-H, CIMP-intermediate (CIMP-I), and CIMP-N gastric carcinoma.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Patients and samples. One hundred six gastric cancer samples from 106 patients were studied (21-85 years old; 81 males and 25 females). Tumor samples were surgically resected at Tokyo Metropolitan Komagome Hospital and Jichi Medical School from 1996 to 1998. Cancerous tissues were frozen immediately after surgical resection and stored at –80°C until genomic DNA was extracted. Only patients with primary gastric carcinoma without preoperative neoadjuvant therapy were included. All cases were histologically diagnosed according to the Japanese Classification of Gastric Carcinoma (18). Histologic classification was also made according to Lauren's classification system (intestinal type and diffuse type; ref. 19).

To determine the presence of EBV, EBER1 in situ hybridization was applied to formalin-fixed and paraffin-embedded specimens in all cases. The method of EBER1 in situ hybridization has been reported previously (20). Clinical follow-up data were available from all the patients with gastric carcinoma analyzed in the present study. Median follow-up duration of the study population was 32 months (range, 1-116 months).

Bisulfite modification and methylation-specific PCR. Bisulfite conversion was carried out using 1 µg of genomic DNA (21), using the CpGenome DNA Modification kit (Intergen, Purchase, NY). This process converts unmethylated cytosine residues to uracil, and methylated cytosine residues remain unchanged. Methylation-specific PCR was then done to examine the methylation status of LOX, HRASLS, FLNc, HAND1, and TM and 10 other cancer-related genes: cell cycle regulation (p14, p15, p16, and p73), repair and protection of DNA (GSTP1, MGMT, and hMLH1), cell adherence and metastasis (TIMP-3 and E-cadherin), and apoptosis (DAPK). One-microliter aliquots of bisulfite-modified DNA were used as templates for PCR reactions with primers specific for methylated or unmethylated sequences. The primer sequences of each CpG island and PCR condition have been described in previous reports (4, 15). A pair of positive (universal methylated and unmethylated DNA; Intergen) and negative controls (distilled water) accompanied every amplification reaction. The PCR products were electrophoresed on 2% agarose gel, stained with ethidium bromide, and visualized under a UV illuminator. Distinct visible band of the amplicon with methylation-specific primers was considered positive. All experiments were done in duplicate.

Statistical analysis. Statistical analysis was done using the Student's t test, the {chi}2 test, or two-tailed Fisher's exact test. As for the analysis of the prognosis of the patients, survival was calculated from the date of surgery until death or the date of last follow-up. Survival was analyzed by the Kaplan-Meier method, and differences in the distribution were evaluated using log-rank test. Multivariate survival analysis was carried out on all variables that were found to be significant on univariate analysis, using the Cox proportional hazards models. Results from this model are reported as relative risks with 95% confidence intervals. For all of the analyses, differences were considered significant when P < 0.05. All data were analyzed with the use of StatView (Abacus Concepts, Inc., Berkeley, CA).


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
The methylation-specific PCR analysis results of representative cases are presented in Fig. 1 . The methylation frequency of the five genes by methylation-specific PCR (Table 1 ) was 44% in LOX, 63% in HRASLS, 53% in FLNc, 62% in HAND1, and 40% in TM. For other cancer-related genes, the frequency varied: 44% in p14, 41% in p15, 38% in p16, 19% in p73, 21% in TIMP-3, 55% in E-cadherin, 32% in DAPK, 7% in GSTP1, 19% in hMLH1, and 25% in MGMT.


Figure 1
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  		Fig. 1. Representative examples of methylation-specific PCR analysis of marker genes and cancer-related genes. Methylation status of five marker genes (LOX, HRASLS, FLNc, HAND1, and TM) and 10 cancer-related genes (p14, p15, p16, p73, TIMP-3, E-cadherin, DAPK, GSTP1, hMLH1, and MGMT) were analyzed in EBV-associated gastric carcinoma (J355) and EBV-negative gastric carcinoma (J570, J368, and J526). According to the definition, J355 and J570 were CIMP-H, J368 was CIMP-I, and J526 was CIMP-N. Note that methylation in 10 cancer-related genes was much more frequent in EBV-associated gastric carcinoma and EBV-negative/CIMP-H gastric carcinoma compared with in CIMP-I and CIMP-N gastric carcinoma. M, methylated alleles; U, unmethylated alleles.

 

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  		Table 1. CpG island methylation of marker genes and cancer-related genes in gastric carcinomas

 
The gastric carcinoma cases in this study consisted of 15 EBV-associated and 91 EBV-negative gastric carcinoma, disclosed by EBER1 in situ hybridization. When the overall frequency of promoter methylation was correlated with the clinicopathologic data (Table 1), the most striking was the global methylation in EBV-associated gastric carcinoma. All five marker genes and 8 of 10 cancer-related genes showed significantly higher methylation frequency in EBV-associated gastric carcinoma compared with EBV-negative gastric carcinoma. Four genes (p73, TIMP-3, FLNc, and TM) showed significant differences regarding the anatomic location of all samples in gastric carcinoma. These differences seemed primarily due to EBV-associated gastric carcinoma predominantly occurring in the gastric corpus, as evidenced by negative results in EBV-negative gastric carcinoma (Table 2 ). Other findings to be noted were correlation between methylation of marker genes and "negative" lymph node metastasis. These correlations were primarily observed in EBV-negative gastric carcinoma (Table 2).


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  		Table 2. Statistically significant correlations between pathologic factors and CpG island methylation in EBV-negative gastric carcinomas

 
Four subgroups of gastric carcinoma: EBV-associated gastric carcinoma and EBV-negative/CIMP-H, CIMP-I, and CIMP-N gastric carcinoma. For gastric carcinoma subgrouping, we first applied two-directional clustering to the whole data. The data showed that the major cluster of genes defining the highly methylated cluster were these five genes (LOX, HRASLS, FLNc, HAND1, and TM), and that all cases of EBV-associated gastric carcinoma belonged to the cluster of the highly methylated group (data not shown). To simplify the definition, we thus defined CIMP-N, CIMP-I, and CIMP-H by the methylation number of 0, 1-3, and 4-5 of five of these genes.

When the criteria were applied to EBV-associated gastric carcinoma, nearly all of EBV-associated gastric carcinoma (14 of 15) constituted CIMP-H. On the other hand, for the proportion of subgroups within EBV-negative gastric carcinoma, both CIMP-H (25 of 91, 27%) and CINP-N (25 of 91, 27%) included much greater numbers of cases compared with the expected ones (12 of 91, 13% and 4 of 91, 4%, respectively), when methylation was assumed to occur randomly with the observed frequency for each gene (e.g., multiplication of the observed ratios of unmethylation of the five genes, 0.63 x 0.43 x 0.42 x 0.55 x 0.69 gives an expected ratio, 0.04, 4% for CIMP-N gastric carcinoma). Such clustering suggests that high or low methylation numbers of indicator genes define specific groups in EBV-negative gastric carcinoma. Thus, we classified tumors in gastric cancer under investigation (n = 106) into four subgroups: EBV-associated gastric carcinoma (14%, n = 15), EBV-negative/CIMP-H (24%, n = 25), CIMP-I (38%, n = 41), and CIMP-N (24%, n = 25).

Evaluation of the number of methylated genes. To confirm that this classification was reasonable, the number of methylated genes of 10 other cancer-related genes (p14, p15, p16, p73, TIMP-3, E-cadherin, DAPK, GSTP1, hMLH1, and MGMT) was plotted against that of five marker genes (Fig. 2 ; Table 3 ). EBV-associated gastric carcinoma showed a significantly higher number of methylated genes (6.9 ± 1.5) than EBV-negative/CIMP-H (3.5 ± 1.8), CIMP-I (2.0 ± 1.4), and CIMP-N gastric carcinoma (1.8 ± 1.6; P < 0.001). In addition, the number of methylated genes in EBV-negative/CIMP-H gastric carcinoma was significantly higher than in CIMP-I and CIMP-N gastric carcinoma, respectively.


Figure 2
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  		Fig. 2. Scatter plot of the number of methylated genes of cancer-related genes against that of marker genes. Vertical axis indicates the number of methylated genes of 10 other cancer-related genes (p14, p15, p16, p73, TIMP-3, E-cadherin, DAPK, GSTP1, hMLH1, and MGMT), and the horizontal axis indicates that of five marker genes (LOX, HRASLS, FLNc, HAND1, and TM). Four subgroups are plotted using a closed circle for EBV-associated gastric carcinoma, open circle for EBV-negative/CIMP-H gastric carcinoma (the number of methylated marker genes, n = 4-5), hatched triangle for CIMP-I (n = 1-3), and closed star for CIMP-N gastric carcinoma (n = 0). Note that the plots of EBV-associated gastric carcinoma accumulate in the top right corner of the scattergram. The plots of EBV-negative/CIMP-H gastric carcinoma distribute widely but relatively in higher numbers of cancer-related genes compared with those of CIMP-N and CIMP-I.

 

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  		Table 3. Clinicopathologic features of EBV-associated gastric carcinoma and EBV-negative CIMP-H, CIMP-I, and CIMP-N gastric carcinoma

 
Evaluation of the profile of methylated genes. The profile of methylated genes was also different among the four groups (Fig. 3 ). In EBV-associated gastric carcinoma, even when compared with EBV-negative/CIMP-H gastric carcinoma, the methylation frequency was significantly high in p14, p15, p16, p73, E-cadherin, TIMP-3, DAPK, and GSTP1.


Figure 3
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  		Fig. 3. Methylation profile of cancer-related genes in EBV-associated gastric carcinoma and EBV-negative/CIMP-H, CIMP-I, and CIMP-N gastric carcinoma. The percentage of cases showing methylation is presented as a bar for each cancer-related gene (p14, p15, p16, p73, TIMP-3, E-cadherin, DAPK, GSTP1, hMLH1, and MGMT) in each subgroup of gastric carcinoma. EBV-associated gastric carcinoma showed significantly higher frequencies of aberrant methylation of cancer-related genes even if compared with EBV-negative/CIMP-H gastric carcinoma. *, statistically significant differences are observed between EBV-associated gastric carcinoma and EBV-negative/CIMP-H gastric carcinoma in 8 of 10 genes. Among EBV-negative gastric carcinoma subgroups, CIMP-H gastric carcinoma shows significantly higher frequency in methylation of p16 (#1) compared with CIMP-I or CIMP-N gastric carcinoma. A statistically significant difference (#2) is also noted in the frequency of methylation of hMLH1 between CIMP-H gastric carcinoma and CIMP-N gastric carcinoma.

 
On the other hand, among 10 cancer-related genes, both p16 and hMLH1 showed higher frequencies of methylation in EBV-negative/CIMP-H gastric carcinoma: the frequency in p16 was 56% in CIMP-H, significantly higher than 24% in CIMP-I and 12% in CIMP-N gastric carcinoma. The methylation frequency in hMLH1 was 36%, 15%, and 4% in CIMP-H, CIMP-I, and CIMP-N gastric carcinoma, respectively, and statistical difference was noted between CIMP-H and CIMP-N.

Clinicopathologic features of four groups. The clinicopathologic features were then compared among four groups (Table 3). When compared with EBV-negative/CIMP-H gastric carcinoma, EBV-associated gastric carcinoma showed predominant localization of the gastric corpus (P = 0.006) and relatively high frequency of lymphatic invasion (P = 0.060; data not shown). In EBV-negative/CIMP-H, the diffuse type of histology was relatively frequent, and the difference was significant when compared with CIMP-I gastric carcinoma (P = 0.031). The CIMP-I subgroup showed relatively high frequency of distant metastasis (9 of 41, 22%) compared with CIMP-N (2 of 25, 8%), but the difference was not statistically significant (P = 0.1846). On the other hand, CIMP-N gastric carcinoma showed high frequency of advanced carcinoma (22 of 25, 88%) and lymph node metastasis (24 of 25, 96%). The latter frequency was significantly higher than those in CIMP-H and CIMP-I gastric carcinoma.

Prognostic significance of CIMP status. The 5-year survival rate of the patients was 65% in EBV-associated gastric carcinoma and 71%, 64%, and 35% in EBV-negative/CIMP-H, CIMP-I, and CIMP-N gastric carcinoma, respectively (Table 3). By Kaplan-Meier analysis among four subgroups, significant differences were observed between CIMP-N and CIMP-H subgroups (P = 0.0311) and between CIMP-N and a combined group of EBV-associated gastric carcinoma and EBV-negative/CIMP-H (P = 0.0254; Fig. 4A ). Thus, CIMP-N subgroup was the only one that showed significantly shorter survival compared with other groups in all tumors under investigation (P = 0.0313; Fig. 4B; Table 4 ).


Figure 4
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  		Fig. 4. Kaplan-Meier survival analysis of patients with gastric cancer, stratified by EBV-infection and CIMP status of the carcinoma. A, survival curves of four subgroups: EBV-associated gastric carcinoma and EBV-negative/CIMP-H, CIMP-I, and CIMP-N gastric carcinoma. B, survival curves of CIMP-N gastric carcinoma and others. Significant difference is observed (P = 0.0313).

 

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  		Table 4. Univariate and multivariate analyses of overall survival (n = 106)

 
To further clarify the prognostic significance of CIMP status in gastric carcinoma, univariate analysis was also done using clinical factors (age and gender of patients), and pathologic factors (histologic type, invasion depth, lymph node metastasis, and hematogenous metastasis of gastric carcinoma) as variables (Table 4). Diffuse type of histology, invasion beyond muscularis propria, and positive lymph node and hematogenous metastasis were significantly correlated with worse prognosis in addition to CIMP-N status. Then, the multivariate Cox regression analysis showed that lymph node metastasis (P = 0.0115) and distant metastasis (P = 0.0016) were only statistically significant as the prognostic determinant of gastric carcinoma patients (Table 4).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Epigenetic alterations have been observed with gastric carcinoma development, such as global hypomethylation (22) or CpG island methylation of the promoter region of various cancer-associated genes (9–14). In this study, we focused on five unique genes (LOX, HRASLS, FLNc, HAND1, and TM), whose promoter CpG islands are densely methylated in gastric cancer cell lines (15). We confirmed and extended our previous observation that EBV-associated gastric carcinoma is a specific subset of gastric carcinoma with global methylation (3–6). When concordant CIMP in multiple genes (CIMP-H) was defined as the methylation of 4-5 of five indicator genes, nearly all of EBV-associated gastric carcinoma belonged to this group, and the number of methylated genes in 10 other cancer-related genes in EBV-associated gastric carcinoma was much higher (6.9 ± 1.5) compared with that in EBV-negative/CIMP-high gastric carcinoma (3.5 ± 1.8). Because the methylation-mediated suppression of cellular genes has been observed in HTLV-1 without the association of chronic inflammatory process (23), an EBV-specific de novo or maintenance methylation mechanism (6) may have direct action in the development of this specific type of gastric carcinoma.

LOX, HRASLS, FLNc, HAND1, and TM, used as CIMP status markers in this study, were recently identified by methylation-sensitive representational difference analysis as being completely methylated in two gastric cancer cell lines, MKN28 and MKN74 (15). On the other hand, CIMP in various types of carcinomas has been defined by the methylation status of MINT clones, which were originally identified from a colon cancer cell line (16). It has not been clarified yet what sets of marker genes effectively define CIMP status, and whether there is a universal set for various organs. We comparatively analyzed methylation status of four MINT clones (MINT1, MINT2, MINT25, and MINT31) in 31 EBV-negative gastric carcinoma, and the number of methylated MINT clones was 2.0 ± 1.6, 1.9 ± 1.3, and 2.1 ± 1.1 in CIMP-H (n = 9), CIMP-I (n = 13), and CIMP-N gastric carcinoma (n = 9), respectively.6 The fact indicates that methylation status of five marker genes is independent of MINT clones in the carcinoma of the stomach and suggests that CIMP marker genes should be cautiously selected according to organs and tumor types.

Kaneda et al. originally analyzed the methylation status of these five genes in 41 cases of gastric carcinoma and found that all cases with a high prevalence of aberrant methylation (CIMP-H) were the diffuse type in histology (15). In this study, no such vital relationship was observed in EBV-negative/CIMP-H gastric carcinoma, but the proportion of the diffuse type was statistically higher than that in CIMP-I gastric carcinoma. Furthermore, several features were observed in this subgroup compared with other EBV-negative gastric carcinoma. First, CpG island methylation was augmented in this subgroup, as shown by increased number of methylated genes of other cancer-related genes and higher frequency of both p16 and hMLH1 methylation. Second, the frequency of lymph node metastasis was lower, and the prognosis of the patients was relatively better in this subgroup. It is of interest to note the correlation of LOX, HRASLS, HAND1, and p14 methylation with "negative" lymph node metastasis. Because a tumor suppressor function in gastric carcinoma has been shown in LOX (24) and p14, this correlation seems to be contradictory to that might be expected. However, the facts rather indicate that accumulation of CpG island methylation simply occurs in the early stage of development CIMP-H gastric carcinoma, without contribution to its further progression.

We strictly defined the CIMP-N gastric carcinoma in the present study, and such classification showed that CIMP-N gastric carcinoma subtypes showed characteristic clinical features. Although hematogenous metastasis was relatively frequent in CIMP-I gastric carcinoma, lymph node metastasis was significantly higher in CIMP-N subgroup. Furthermore, most cases of CIMP-N gastric carcinoma were advanced carcinoma, showing the worst prognosis among the subgroups. Because global DNA hypomethylation and satellite DNA hypomethylation is independent of the CIMP-positive or CIMP-negative status (22, 25, 26), the underlying mechanism in the aggressive potential in CIMP-N gastric carcinoma is totally unknown at present. However, it is possible that classic loss of heterozygosity mechanisms may act in this subgroup, thus enabling carcinoma cells to retain their capability of blocking methylation pressure in CpG island DNA.

In conclusion, using the methylation profile of five unique genes (LOX, HRASLS, FLNc, HAND1, and TM) as marker genes, we confirmed that EBV-associated gastric carcinoma is a specific subset of gastric carcinoma showing global methylation, the mechanism of which should be intensively investigated. EBV-negative/CIMP-H gastric carcinoma also represents a subgroup, in which promoter methylation plays an important role in the developmental process. CIMP-N gastric carcinoma is an aggressive group, which is associated with worse patient prognosis through lymph node metastasis. The methylation profile of five representative genes is useful to stratify gastric carcinomas into biologically different subgroups.


   Footnotes
 
Grant support: Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

6 Unpublished data. Back

Received 8/ 4/05; revised 1/30/06; accepted 3/ 9/06.


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Y.-F. Zhao, Y.-G. Zhang, X.-X. Tian, Juan Du, and Jie Zheng
Aberrant Methylation of Multiple Genes in Gastric Carcinomas
International Journal of Surgical Pathology, July 1, 2007; 15(3): 242 - 251.
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