INTRODUCTION

The incidence of gastric cancer has declined; however, it continues to be the second most common malignant neoplasms across the world and the second leading cause of cancer death 1. Epigenetics has an important role in biological research and affects many different areas of study including cancer biology 2, 3, viral latency 4, 5, 6, 7, activity of mobile elements 8, somatic gene therapy 9, 10, 11, 12, 13, 14, cloning and transgenic technologies, genomic imprinting 15, 16, and developmental abnormalities 15, 16. Epigenetic silencing of tumor-related genes due to CpG island methylation has been recently reported in gastric carcinoma 17. CpG islands are 0.5 to 2 kb regions rich in cytosine-guanine dinucleotides and are present in the 5′ promoter region of approximately 40-50% of human genes 18. Methylation of cytosines within CpG islands is associated with loss of gene expression by repression of transcription and is observed in tumorigenesis, as well as in physiological conditions such as X chromosome inactivation and aging 19, 20, 21, 22, 23, 24.

Gastric carcinoma can arise either from precursor lesions or de novo. It has been demonstrated that some gastric carcinoma may arise from gastric adenomas or flat dysplasias, similar to the consequence of colorectal adenoma derived from adenocarcinoma 25, 26, 27, 28. The cumulative prevalence of malignant transformation of gastric dysplasia/adenoma has been reported to be greater than 10% in long-term follow-up studies 29, 30. Epigenetic alterations have been acknowledged as an important mechanism contributing to early gastric carcinogenesis. Previous studies have characterized epigenetic abnormalities in intestinal metaplasia (IM) and adenoma, which are precursors of invasive adenocarcinoma 31, 32.

DNA METHYLATION IN CANCER

DNA methylation has become the topic of intense investigation in cancer cells. As compared with normal cells, the malignant cells show major disruptions in their DNA methylation patterns 32. The tumor cells are characterized by significant modifications of DNA methylation system including general genome demethylation, increase in DNA methyltransferase activity, and local hypermethylation 34, 35. These modifications seem to be contradictory, since it seems difficult to explain the increase in DNA methyltransferase activity with local hypermethylation of the genome and overall demethylation on the remaining genome. This contradiction is further clarified by better understanding of the DNA methylation system. The demethylation has been estimated to involve almost exclusively the “dispersed” CpG (∼80% of total content, and they are methylated in normal cells). In contrast, local hypermethylation occurs on the CpG islands (about 20% of total content, and are not methylated in normal cells excluding certain exceptions) does not compensate the demethylation of “dispersed” CpG to give general genome demethylation. In tumor cells the reciprocal relations between the methylation levels of the CpG islands and of “dispersed” CpG, are also preserved. The increased activity of DNA methyltransferase frequently seen in tumor cells can be one of the factors responsible for the aberrant methylation of CpG islands.

Methylation of tumor suppressor genes

Inactivation of tumor suppressor genes by DNA methylation in promoter region plays an important role in carcinogenesis. The tumor suppressor genes that undergo aberrant CpG island methylation in human cancer can affect important cellular pathways including cell cycle regulation and proliferation. For example, in p16INK4a/Rb/cdk4 pathway, the cell-cycle inhibitor p16INK4a is methylated in various human primary tumors and cell lines 36, 37, allowing the cancer cells to escape senescence and to proliferate Rb itself can also be occasionally inactivated by aberrant methylation 38, 39. APC/β-catenin/E-cadherin pathway has been altered in a variety of human cancers. APC gene is commonly mutated in sporadic colon cancer but the role of the APC mutation in other tumor systems is not clear. Recently, it is been shown that aberrant methylation of APC is a common phenomenon in other aerodigestive tract neoplasms 40 and that E-cadherin promoter hypermethylation is important for the cancer biology of breast and other tumor types 41, 42.

Methylation in DNA mismatch repair gene

DNA mismatch is in the crossroad of all other cellular pathways. DNA methylation is one of the major players in causing alterations of DNA mismatch repair genes. Methylation-mediated silencing of the mismatch DNA repair gene hMLH1 in sporadic cases of colorectal, endometrial, and gastric cancers are responsible for the high level of microsatellite instability in tumors 43, 44, 45, 46, 47. The promoter hypermethylation of MGMT 48 that prevents the removal of groups at the O6 position of the guanine is associated with particular type of K-ras and p53 mutations 49, 50; and the somatic inactivation of BRCA1 by aberrant methylation altering its role in the repair of DNA double-strand breaks in breast and ovarian tumors 51.

Two types of DNA methylation

Not all of the DNA methylation are tumor specific. In colon tissues, studies have shown DNA methylation in certain genes such as ERα, N33 and MYOD are age-related, and this type of methylation is classified as Type A methylation. In contrast, tumor-specific methylation such as p16 and hMLH1 is classified as Type C methylation 17. A similar distinction between age-related and tumor-specific methylation has also been shown in gastric cancer 52, 53. In stomach, age-related Type A methylation genes include E-cadherin and APC 32, 53. In addition, Waki et al found that methylation of DAP-kinase, like that of E-cadherin, was also age-related. The methylation of DAP-kinase in non-neoplastic epithelia frequently appears at around age 45 54.

The significance of the detection of the methylated gene can depend on the position of the CpG sites examined. Methylation of hMLH1 gene, which was thought to be cancer-specific 17, was found to be a common age-related event in normal colonic mucosa, when the entire hMLH1 promoter ∼700 bp region was analyzed 55. Partial methylation on hMLH1 promoter region is frequently present in normal colonic mucosa, especially in older patients. The hMLH1 gene will ultimately shut down when the methylation spreads to reach a threshold 55. Therefore, these contradictory results might have been due to analysis of different CpG sites 17, 55. In addition, it has recently been reported that DAP-kinase methylation was present in virtually every tumor and normal gastric and colorectal sample when the edge of CpG islands was examined, although it turned out to be a rather infrequent, cancer-specific phenomenon when the central region of the CpG islands was analyzed 56.

CpG island methylator phenotype

Tumors with concurrent methylation in multiple genes or loci have been defined as CpG island methylation phenotype-high (CIMP-H) in colorectal and gastric cancers 17, 52. The mechanism of concurrent hypermethylation of multiple genes remains obscure. Apparently, the increase in DNA methyltransferase activity plays an important role. Transfection of a cloned human DNMT1 gene into immortalized human fibroblasts leads to the aberrant methylation of CpG islands in promoter regions of several genes, including E-cadherin and HIC1, but CpG islands associated with other genes (for example, p16INK4a) are not altered in methylation state, although DNMT1 is expressed constantly 57. Therefore, it is evident that the increase in DNMT1 activity can play a significant role in the selective aberrant methylation of CpG islands, but not in total CpG island methylation.

CIMP-H affects only a subset of tumors and a limited number of genes. The defect that leads to CIMP-H could be either aberrant de novo methylation (through a mutation in DNA-methyltransferase for example) or loss of protection against de novo methylation through the loss of a trans-activating factor 58, 59, 60. These CIMP-H tumors may then develop through a pathway that heavily relies on this methylation defect, whereas others rarely show tumor suppressor gene methylation. Aberrant methylation often occurs in CpG islands outside of promoter regions in which it may not affect gene transcription 61. An important question is whether the concordant methylation described here provides a growth advantage to affect cells or whether it just accompanies tumor development. Additional studies are necessary to clarify whether the genes methylated in cancer simply reflect the genome wide methylation defect or whether stochastic methylation of each CpG island results from selective pressures.

EPIGENETIC ALTERATIONS IN EARLY GASTRIC TUMORIGENESIS

There are two major histological types of gastric adenocarcinoma (intestinal and diffuse) according to the Lauren's classification 62. The pathogenesis and genetic alterations for these two distinct types of adenocarcinoma are also different 63, 64, 65. The most frequent gastric malignancy is the intestinal type, which is often preceded by sequential steps of precancerous changes, including atrophic gastritis, intestinal metaplasia, and either dysplasia or adenoma. In contrast, the diffuse type of gastric carcinoma tends to arise de novo and is infrequently associated with dysplasia or adenoma 66, 67, 68, 69. The sequential accumulation of alternations of APC and K-ras genes, characteristic of the colorectal adenoma-carcinoma sequence, however, does not occur frequently between adenoma and intestinal type adenocarcinoma of the stomach 27, 70, 71, 72, 73, 74, 75, 76, 77. There are two lines of evidence indicating that not all gastric dysplastic lesions are precursor lesions for gastric carcinoma. First, gastric dysplasia can undergo spontaneous regression clinically, especially low-grade dysplastic lesions, and only 11–40% of adenoma/dysplasia progress to carcinoma 29, 78, 79, 80, 81. Secondly, APC mutations have been reported to occur more frequently in gastric adenomas than in gastric adenocarcinomas 75, 76, 77, 81.

The status of methylation in multiple genes or loci has been studied extensively in early gastric tumorigenesis following the stepwise morphologic changes as summarized in Fig. 1 and Tab. 1. Methylation of tumor suppressor genes are frequently present in the non-neoplastic gastric mucosa including chronic gastritis and intestinal metaplasia (IM). Kang et al 32 determined the methylation frequency of 12 genes, including APC, COX-2, DAP-kinase, E-cadherin, GSTP1, hMLH1, MGMT, p16, p14, RASSF1A, THBS1, and TIMP3, by methylation-specific PCR in this progression sequence. In this study five different classes of methylation behaviors were found: (1) genes only methylated in carcinoma such as GSTP1 and RASSF1A; (2) genes showing low methylation frequency in chronic gastritis, IM, and gastric adenoma but significantly higher methylation frequency in carcinoma such as COX-2, hMLH1, and p16; (3) a gene with low and similar methylation frequency in four-step lesions such as MGMT, (4) genes with high and similar methylation frequency in four-step lesions such as APC and E-cadherin, and (5) genes showing an increasing tendency of the methylation frequency along the progression such as DAP-kinase, p14, THBS1, and TIMP-3. A similar result has also been shown by other studies. To et al showed three differential methylation patterns: methylation was more frequent in cancer than in IM (DAP-kinase, p14, p15 and p16); comparable frequencies of methylation in cancer and IM (E-cadherin and hMLH1); and no methylation (GSTP1) 82. Among these genes evaluated, it appears that at least methylation of APC and E-cadherin are not tumor specific but rather an age-related phenomenon (Type A methylation gene for stomach). Therefore, it is crucial to include control non-neoplastic gastric mucosa; in methylation study of gastric precursor lesions and carcinomas. It is however, difficult to classify genes such as DAP-kinase, THBS1 and TIMP-3 in which methylation is frequently detected in non-neoplastic mucosa and precursor lesions but a higher frequency of methlyation is present in carcinoma stage based on Type A or Type C methlyation gene classification (Fig. 1).

Figure 1
figure 1

Epigenetic alterations in the multistep gastric carcinogenesis pathway following the chronic gastritis-intestinal metaplasia-adenoma/dysplasia-adenocarcinoma sequence. Methylation of APC and E-cadherin genes occur frequently in normal gastric mucosa and chronic gastritis, and uniformly through the sequence is classified as Type A (age-related) methylation genes. The remaining genes are classified as Type C (tumor-specific) methylation genes. Genes such as DAP-kinase, MGMT, TIMP3 and THBS1 are frequently methylated in intestinal metaplasia stage, but the methylation frequency is higher in precursor lesions or adenocarcinoma, therefore also classified as Type C methylation genes. Gastric carcinoma can be further subclassified based on the extent of methylation; CIMP-high (CIMP-H) with concurrent methylation of multiple genes in ≥50% of assessed genes, CIMP-low (CIMP-L) with methylation in <50% of assessed genes, or CIMP-negative (CIMP-N) with no methylated genes. In tumor with CIMP-H, methylation of hMLH1 gene is also associated with microsatellite instability-high (MSI-H) phenotype.

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Table 1 Genes and frequency of methylation occur in the multistep gastric carcinogenesis pathway
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Concurrent methylation of multiple tumor-related genes has been detected in 20% of normal tissues adjacent to gastric carcinoma by Leung et al 83. We have also shown concurrent methylation (CIMP-H) in 15% of IM but not in chronic gastritis or normal gastric mucosa using a panel of six genes/loci including p16, hMLH1 and four CpG islands (MINT1, MINT2, MINT25 and MINT31) 81. MINT loci were methylated in 6–21% of normal mucosa, and in 10–41% of IM. In particular, methylation of MINT25 was more frequent in normal/chronic gastritis mucosa contiguous with neoplasms and in IM than in noncontiguous normal/chronic gastritis mucosa. The mean methylation index and frequency of CIMP-H increased following the normal/chronic gastritis, IM, adenoma/dysplasia and early adenocarcinoma sequence indicating accumulation of methylation events may play an important role in early gastric tumorigenesis (Fig. 2) 81.

Figure 2
figure 2

The frequency of concurrent methylation in multiple genes/loci (CIMP-H) increased with the histological progression from normal (NM)/chronic gastritis (CG), to Intestinal metaplasia (IM), adenomas or dysplasias, and early adenocarcinomas. The majorities of the non-neoplastic mucosa (NM, CG and IM) are CIMP-L or CIMP-N.

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There are two morphologically distinct gastric precancerous lesions: adenoma (polypoid dysplastic mucosal lesion) and flat dysplasia. Genetic and epigenetic alterations separating these two distinct morphological precancerous lesions remain unclear. We have shown that there was no difference in the frequency of individual gene/loci or in frequency of CIMP-H between flat dysplasias (50%) and polypoid adenomas (51%) 81. Interestingly, we have found that methylation of p16 gene was more frequent in adenocarcinoma-associated dysplasia/adenoma and adenocarcinomas as compared to adenoma/dysplasia unassociated with adenocarcinoma, indicating methylation of p16 gene may contribute to the malignant transformation of gastric precursor lesions 81.

EPIGENETIC ALTERATIONS IN GASTRIC CARCINOMA

The roles of epigenetic alterations in the pathogenesis of gastric carcinomas have been recently elucidated. Toyota et al first demonstrated that about one-half of all gastric cancers had frequent methylation in multiple genes/loci including p16, hMLH1 and multiple tumor specific CpG islands (MINT1, MINT2, MINT12, MINT25, and MINT31) that seemed to be methylated de novo during cancer progression and play an important role in gastric carcinogenesis 52. Subsequently, numerous other genes with methylation have been found in gastric carcinomas as shown in Fig. 1 and Tab. 1.

Similar to colorectal cancer, the presence of methylation of hMLH1 gene is strongly associated with loss of hMLH1 protein expression and MSI-H phenotype in gastric carcinomas 52, 81, 83. In contrast to gastric carcinoma, methylation of hMLH1 genes in gastric precursor lesions appears to be less frequently associated with MSI-H phenotype and loss of hMLH1 protein expression 81. The reason for this discrepancy is not clear, but it is possible that hMLH1 methylation precede the loss of protein expression.

Genetic alteration of E-cadherin gene has been frequently detected in gastric carcinomas, especially for diffuse type adenocarcinoma. Tamura et al reported that E-cadherin promoter hypermethylation was seen in 27 (51%) of 53 primary gastric carcinomas, including 15 (83%) of 18 undifferentiated (diffuse) type, and E-cadherin promoter hypermethylation was seen at similar frequencies in both early and advanced cases 84. However, the presence of E-cadherin gene methylation in non-neoplastic gastric mucosa makes difficult to determine its role in gastric carcinogenesis.

In gastric cancer, CIMP-H has been described in 41% of the tumors by Toyota et al 52. We have also detected CIMP-H in 31% of the gastric carcinomas using a similar panel of genes/loci 85. Etoh et al have shown that increased DNMT1 protein expression correlated significantly with DNA methylation of multiple CpG islands in poorly differentiated gastric cancers, suggested that DNMT1 may play a significant role in the development of poorly differentiated gastric cancers by inducing frequent DNA methylation of multiple CpG islands 86.

CLINICAL IMPLICATIONS

The clinical significance of presence of epigenetic alterations in gastric cancer remains unclear. It would be important to know whether the presence or absence of certain epigenetic changes affects the prognosis. This would also help in modifying initial patient treatment options and monitoring response to therapy. It is particularly important to identify any specific epigenetic alterations in gastric precursor lesions for predicting malignant transformation since not all the gastric precursor lesions carry the same malignant transforming potential. Epigenetic silencing of a tumor suppressor gene could be the rate-limiting step that initiates the series of events leading to an invasive malignant tumor. Methylation of p16 gene appears to be a promising candidate to serve this purpose as shown in our study 81 and supported in a population-based study by Sun et al that aberrant methylation of p16 promoter CpG islands might be useful to predict the malignant potential of dysplasia identified specifically in gastric biopsies 87.

The prognosis of methylation of single genes/loci is unclear in gastric cancer, but it has been reported that methylation of MGMT gene was associated with advance stage and poor prognosis 88. We have recently shown that concordant methylation of multiple gene/loci (CIMP-H) is associated with better survival but is not an independent predictor of prognosis in resected gastric cancer 85. The sensitivity and specificity of DNA methylation markers in cancer diagnosis depends on several factors, including the type of cancer and the gene to be studied, the type of body fluid to be used, and the techniques involved. The assay needs to be standardized and shown to be useful in a prospective fashion before it can become clinically useful. A more comprehensive methylation profiling for gastric cancer will be needed to achieve this goal.