DNA methylation as a molecular biomarker in gastric cancer.

Review For reprint orders, please contact: [email protected]

DNA methylation as a molecular biomarker in gastric cancer

DNA methylation plays a significant role in gastric carcinogenesis. The CpG island methylator phenotype (CIMP) characterizes distinct subtypes of gastric cancer (GC) and the relationship between specific methylation patterns and clinicopathological features has been evaluated. Altered DNA methylation is also observed in Helicobacter pylori-infected gastric mucosa, and its potential utility for GC risk estimation has been suggested. The ability to detect small amounts of methylated DNA among tissues allows us to use DNA methylation as a molecular biomarker in GC in a variety of samples, including serum, plasma and gastric washes. The DNA methylation status of nontargeted tissue, particularly blood, has been associated with predisposition to GC. We focus on the recent development of DNA methylation-based biomarkers in GC. Keywords: chromatin remodeling • CIMP • CpG island methylator phenotype • DNA methylation • epigenetics • gastric cancer • Helicobacter pylori • molecular biomarker

Gastric cancer (GC) is one of the most common malignancies worldwide, it accounts for approximately 70,000 new cases and 650,000 deaths per year [1,2] . Although improvements in early detection by screening have resulted in lower incidence rates in most parts of the world, many patients have advanced disease at diagnosis and treatment outcomes for such patients are not optimal [3] . In terms of risk assessment, although Helicobacter pylori infection is strongly associated with predisposition to GC [4] , the existence of H. pylori infection alone is not sufficient for predicting its risk in areas with high H. pylori infection rates, particularly in Asian countries. Therefore, identifying precise molecular markers of GC will improve our understanding of geastric carcinogenesis and will possibly allow clinicians to divide patients into relevant subgroups with the aim of developing novel molecular tailored therapies for the treatment of GC. Although the molecular mechanisms of gastric carcinogenesis remain unclear, as is the case with many other cancers, it has been shown that the accumulation of both genetic abnormalities and epigenetic abnormalities

10.2217/EPI.15.4 © 2015 Future Medicine Ltd

Tomomitsu Tahara*,1 & Tomiyasu Arisawa2 1 Department of Gastroenterology, Fujita Health University School of Medicine, 1–98 Dengakugakubo Kutsukake-cho, Toyoake, Aichi, 470–1192, Japan 2 Department of Gastroenterology, Kanazawa Medical University, Ishikawa, Japan *Author for correspondence: Tel.: +81 562 93 9240 Fax: +81 562 93 8300 [email protected]

plays an important role in carcinogenesis [5] . Epigenetic effects, including DNA methylation, histone modifications, microRNAs, noncoding RNAs and nucleosome positioning, are defined as processes that alter gene expression without changing the DNA sequence [6–10] . In the last few decades, it has become increasingly clear that altered epigenetic regulation plays a key role in many different diseases, particularly cancers [9] . To date, aberrant DNA methylation is the most extensively studied deregulated epigenetic mechanism in GC. For example, known tumor suppressor or tumor-related genes (p16, RUNX3, MLH1 and CDH1 etc.) are silenced by promoter methylation in GC and in its precancerous lesions [7] . Generally, aberrant DNA methylation in cancer is classified into two categories: ‘global DNA hypomethylation’ and ‘regional hypermethylation.’ Global DNA hypomethylation, which was discovered 30 years ago, is characterized as a decrease in 5-methylcytosine content throughout the genome [11] . Global DNA hypomethylation occurs at CpG

Epigenomics (2015) 7(3), 475–486

part of

ISSN 1750-1911

475

Review   Tahara & Arisawa dinucleotides, especially in repetitive sequences, which are typically methylated in normal tissues [12,13] . Hypomethylation in repetitive sequences is thought to contribute to carcinogenesis by inducing genomic instability [14,15] and leading to the formation of abnormal chromosomal structures [16,17] . Recent comprehensive studies using whole-genome bisulfite sequencing have also demonstrated that global DNA hypomethylation corresponds to nuclear-lamina–associated domains (LADs) [18,19] . Because genes in LADs are typically transcriptionally repressed [19] , global DNA hypomethylation in LADs are more likely to be associated with genes that exhibit increased expression in cancer. The latter type of DNA methylation, regional hypermethylation, occurs in CpG islands. Occurring preferentially at promoter CpG islands, regional hypermethylation plays a key role in carcinogenesis [20– 22] and leads to the inactivation of tumor suppressor genes in the absence of changes to the genetic sequence of these genes [23,24] . As previously mentioned, promoter methylation in tumor suppressor genes has been identified in many cancer types including GC [7] . The CpG island methylator phenotype (CIMP), defined by the occurrence of tumors with an increased accumulation of cancer-specific aberrant promoter hypermethylation, was first described in colorectal cancer [25] and has also been evaluated in GC. CIMP in GC is associated with several unique characteristics, including Epstein–Barr virus (EBV)-positive GC, microsatellite instability (MSI) and epigenetic silencing of the mismatch repair gene MLH1 [26] . Recent comprehensive molecular characterization of GC categorized two subtypes of CIMP in GC – EBV-CIMP and gastric-CIMP. EBV-CIMP was characterized as having CDKN2A (p16 ) methylation and a PIK3CA mutation, whereas gastric-CIMP was tightly linked to MSI and epigenetic silencing of MLH1 [27] . CpG island hypermethylation also occurs in H. pylori-infected nonneoplastic gastric mucosa [28,29] , which is at an increased risk of developing GC [4] . This phenomenon can be explained by the concept of an ‘epigenetic-field-defect,’ which constitutes the earliest steps toward neoplastic transformation by creating molecular diversity. To date, many studies have evaluated DNA methylation in the nonneoplastic gastric mucosa as a risk marker for GC [28,30,31] . On the other hand, the DNA methylation status of nontargeted tissue, particularly blood, is now increasingly being considered to be a marker for cancer predisposition [32,33] , and its use as a risk marker for GC has been evaluated [34,35] . Methylation profiling of GC may aid us in understanding the individual heterogeneity of tumors and may provide a basis for better understanding the

476

Epigenomics (2015) 7(3)

pathogenesis of individual cases. In addition, DNA methylation is commonly observed in H. pyloriinfected, nonneoplastic gastric mucosa, which exhibit a higher risk of GC. Moreover, it is possible to detect methylation from very small amounts of DNA, which makes it a useful tumor marker of GC when using many samples or samples of limited quantity [36] . In this review, we focus on the recent development of epigenetic biomarkers in GC with particular attention given to aberrant DNA methylation as this is the most extensively studied deregulated epigenetic mechanism in GC to date. CIMP in GC & the importance of methylation profiling in GC DNA hypermethylation of promoter CpG islands, which leads to transcriptional silencing of genes, is the most studied epigenetic alteration in human cancers including GC [7,20–22] . As shown in Table 1, many tumor suppressor and tumor-related genes (p16, RUNX3, CDH1, APC, CHFR, DAPK, GSTP1, etc.) are known to be silenced by hypermethylation in GC [7,37] . Aberrant promoter hypermethylation does not occur at only one gene but is usually accelerated across multiple gene promoters. This accumulation of a high degree of cancer-specific aberrant promoter hypermethylation is referred to as the CpG island methylator phenotype (CIMP). The concept of CIMP was first introduced in the context of the molecular pathways leading to certain colorectal cancers [25] . Colorectal cancers characterized by CIMP have distinctly different molecular and clinicopathological features compared with tumors derived from the traditional adenoma-carcinoma pathway. For example, CIMP-positive colorectal cancers are associated with increased age, female gender, relatively favorable survival and BRAF mutations [38–40] . As described above, CIMP-positive colorectal cancers display distinct molecular and clinical features. The discovery of CIMP in colorectal cancers opened many avenues for research in the methylation profiling of cancers in an effort to characterize the heterogeneity of tumors to better understand the pathogenesis of individual cancer cases. The presence of CIMP has been investigated in many types of cancers, including breast, endometrial, glioblastoma (gliomas), hepatocellular, lung, ovarian, prostate, leukemia and so on [41] . CIMP-positive GC was also discovered using the same panel of genes used to identify CIMP-positive colorectal cancers [42] . To date, many studies have evaluated the molecular and clinical features that are specific to CIMP-positive GC [26,43–48] . The results suggest that CIMP-positive GC also arises from a process that is different from CIMP-negative GCs. GCs with

future science group


Review

Table 1. Frequently methylated genes in primary gastric cancer tissue and its correlation with clinicopathological features. Gene

Correlation with methylation positive group

p16

GC tissue

[7,37,45]

Ref.

RUNX3

GC tissue, gastric dysplasia and Intestinal metaplasia

[7,37,69]

CDH1

GC tissue

[7,37]

APC

GC tissue

[7,37]

DAPK

GC tissue

[7,37]

GSTP1

GC tissue

[7,37]

MLH1

GC tissue, MSI positive

LOX

GC tissue

[45]

FLNc

GC tissue

[45]

HRASL

GC tissue

[45]

HAND

GC tissue

[45]

THBD

GC tissue

[45]

F2R

GC tissue

[45]

NT5E

GC tissue

[45]

GREM

GC tissue

[45]

ZNF177

GC tissue

[45]

CLDN3

GC tissue

[45]

PAX6

GC tissue

[45]

CTSL

GC tissue

[45]

ALX4

GC tissue

[47]

TMEFF2

GC tissue

[47]

CHCHD10

GC tissue

[47]

IGFBP3

GC tissue

[47]

NPR1

GC tissue

[47]

CHFR

GC tissue, sensitivity to microtubule inhibitors

ADAMTS9

GC tissue, worse overall survival

[58]

FOXD3


[59]

PAX5


[60]

[7,37,45]

[37,57]

GC: Gastric cancer; MSI: Microsatellite instability.

high level CIMP (CIMP-high) are Epstein–Barr Virus (EBV)-positive and are associated with upper stomach localization and diffuse-type histopathology, exhibiting less frequent TP53 or KRAS mutations, whereas EBV-negative CIMP-high GCs are closely associated with MLH1 methylation and microsatellite instability (MSI) [26] . Therefore, it is possible that EBV-positive and -negative cancers represent distinct subtypes of CIMP-high GCs. Recently, The Cancer Genome Atlas (TCGA) research network performed an integrated molecular characterization of GC in relation to its clinical characteristics [27] . The clustering analysis of DNA methylation at CpG sites for 295 GC samples identified two different subtypes of CIMP: EBV-CIMP and


Gastric-CIMP. EBV-CIMP was found in 9% of the cases (28/295). EBV-CIMP was present in the gastric fundus or body and mostly occurred in males, as previously suggested [26] . The molecular characteristics of EBV-CIMP were highlighted as being closely linked to CDKN2A (p16 ) methylation (in all cases) and PIK3CA mutations (in 80% of cases). Gastric-CIMP was found in 26% of the GC samples (77/295) and was closely linked to MSI and epigenetic silencing of MLH1 [27] . In embryonic stem (ES) cells, the Polycomb repressive complex (PRC) plays an important role in reversibly repressing gene expression. In ES cells, PRC target genes are more frequently methylated than non-PRC target genes in various cancers, including

www.futuremedicine.com

477

Review   Tahara & Arisawa CIMP-positive GCs [49,50] . An array-based genomewide methylation analysis of CIMP-positive GC showed significant enrichment of aberrant methylation in PRC target genes in CIMP-positive GC without EBV, whereas EBV-positive, CIMP-positive GC displayed extended methylation in both PRC target genes and non-PRC target genes. In vitro EBV infection experiments confirmed a causal role for EBV infection in inducing methylation, suggesting unique mechanisms in the accumulation of methylation in EBV-positive, CIMP-positive GC compared with EBV-negative cases [51] . Although the detailed molecular mechanisms leading to CIMP remain unclear, it is possible that promoter CpG island DNA methylation could be an alternative pathway for the inactivation of tumor suppressor genes. Indeed, DNA methylation of tumor suppressor genes has been observed in GC, and CIMP-positive GC exhibits less frequent mutations in the tumor suppressor gene TP53 [26] . However, genome-wide methylation analysis of GC revealed that the majority of cancer-specific epigenetic alterations are unrelated to gene expression, and the role of these genes in the development of CIMP-positive cases is not fully understood [48] . This supports the hypothesis that there may be other pathogenic origins leading to epigenetic disturbance in CIMP-positive GC. The field is still in the early stages of understanding the molecular basis for CIMP. Significant early contributions came from two studies, the first showing that glioblastomas with a hypermethylator phenotype are associated with somatic mutations in isocitrate dehydrogenase-1 (IDH1) [52] and the second showing that somatic mutations in IDH1 and IDH2 as well as loss-of-function mutations in ten-eleven translocation (TET)-methylcytosine dioxygenase-2 (TET2) establish a hypermethylation phenotype in leukemia [53] . In colorectal cancer, genes regulating

chromatin were frequently mutated in CIMP-high cases, most notably CHD7 and CHD8, which encode members of the chromodomain helicase/adenosine triphosphate-dependent chromatin remodeling family [54] . A recent study using exome sequencing of GC samples also found novel mutated genes and pathway alterations involved in chromatin modification, most notably in ARID1A, which encodes one of the subunits in the Switch/Sucrose Nonfermentable (SWI-SNF) chromatin remodeling complex. Importantly, the frequency of the ARID1A mutation distinctly differed across different molecular subtypes of GC such that it was 83% mutated in GCs with MSI, 73% mutated in GC with EBV and 11% mutated in GCs without MSI and EBV. ARID1A mutations were also negatively associated with TP53 mutations. Moreover, GCs with ARID1A alterations showed a trend toward prolonged, recurrence-free survival [55] . Another study also analyzed a spectrum of somatic alterations in GC via exome sequencing, and their results demonstrated that mutations in chromatin remodeling genes (ARID1A, MLL3, MLL, etc.) were found in nearly half of all GCs (47%). ARID1A mutations were detected in 8% of GCs (9/110) and were associated with MSI [56] . In a recent TCGA study, ARID1A mutations were detected in 14% of GCs and were even more frequent with the EB-CIMP phenotype (55%) [27] . These results suggest the importance of altered chromatin remodeling in the pathogenesis of GC. Although neither study evaluated a direct link between mutations in chromatin remodeling genes and CIMP in GC, a correlation between them would likely be positive based on the fact that the two major subtypes displaying ARID1A mutations (MSI and EBV-positive GCs) are closely linked to the CIMP phenotype. Table 2 lists somatic mutations in epigenetic modifier genes in GC. It is possible that mutation of genes encoding chromatin-remodeling

Table 2. Mutated epigenetic modifier genes identified in gastric cancer. Gene

Frequency, feature

Ref.

ARID1A

78% (18/23) in MSI GC, 47% (7/15) in EBV-positive GC

[55]

ARID1A

8% (9/110) in over all GC

[56]

ARID1A

14% in over all GC, 47% in EBV-CIMP

[27]

MLL3

13% (2/15)

[56]

MLL

6.7% (1/15)

[56]

DNMT3A

6.7% (1/15)

[56]

SETD1A

6.7% (1/15)

[56]

KDM2B

6.7% (1/15)

[56]

BAZ1B

6.7% (1/15)

[56]

CHD4

6.7% (1/15)

[56]

EBV: Epstein–Barr virus; EBV-CIMP: Epstein–Barr virus-CpG island methylator phenotype; GC: Gastric cancer; MSI: Microsatellite instability.

478




enzymes may activate an alternative pathway of carcinogenesis in CIMP-positive GC that drives cancer progression through epigenetic disorder. Several researchers have indicated that the DNA methylation status of a single marker gene can be associated with clinical outcome in GC (Table 1) . DNA methylation of the checkpoint with forkhead-associated and ring finger (CHFR) gene, a mitotic checkpoint gene, is associated with sensitivity to microtubule inhibitors in GC [57] . Methylation of ADAMTS9 [58] , FOXD3 [59] and PAX5 [60] are all associated with worse overall survival in patients with GC. These findings suggest the potential utility of methylation profiling in GC for better clinical implementation reflecting individual tumor heterogeneity. Regarding the CIMP phenotype, however, the data are controversial and the prognostic value of CIMP for GC has not been confirmed. Some CIMP-positive GCs are associated with better overall survival [26,44,45] , whereas other CIMP-positive GCs are associated with worse overall survival [46–48] (Table 3) . This may be attributed to differences in sample size or the occurrence of confounding variables, such as differences in the criteria used to define CIMP. In reference to the panel of genes used to define CIMP in colorectal cancer, Weisenberger et al. identified a robust 5-gene panel that recognized CIMP-positive colorectal cancer cases [61] . This panel was used to validate the phenotype in colorectal cancer and has been further validated in a large, population-based sample [62] . It is considered the ‘best panel’ for defining CIMP in colorectal cancer. Table 3 lists a series of studies of CIMP in GC and their criteria for defining CIMP. In some cases of GC, however, the observation of CIMP in a tumor is determined according to methylation status using different panels or genes, where a subgroup of tumors with a higher degree of DNA methylation than the remaining tumors constitutes CIMP. Although conflicting results have been generated in relation to patient prognosis, it seems a close link exists between CIMP-high and MSI or EBV-positive GC cases irrespective of the definition of CIMP that is used. A recent meta-analysis did not confirm any clinical-pathological association with CIMP-positive GC other than H. pylori, EBV and MSI [63] . Still, the findings regarding the prognostic value of CIMP for GC are limited, especially for patients being treated with chemotherapy. Importantly, CIMP-positive colorectal cancers are usually associated with a better prognosis [40] ; however, patients with CIMP-positive colorectal cancers do not benefit from 5-FU-based adjuvant chemotherapy [64] . Additional well-designed studies will be needed for the establishment of CIMP as the prognostic indicator in GC patients.


Review

Another point that needs to be addressed is the question of how universal CIMP is across tumor types. The close link between MSI and MLH1 epigenetic silencing seems to be a common molecular feature of CIMP across gastric and colorectal cancers. However, a different spectrum of mutated genes in CIMP-positive GC (PIK3CA) compared with other CIMP tumors (BRAF in colorectal cancers, IDH1 in glioblastomas) also suggests that genetic and epigenetic changes in cancers occur via unique processes that differ greatly among tumors arising in different organs. Aberrant DNA methylation in nonneoplastic gastric mucosa induced by H. pylori infection & its role in gastric carcinogenesis H. pylori infection has been associated with predisposition to the development of GC through inducing gastric mucosal inflammation and atrophy [4] . H. pylorirelated chronic active gastritis is characterized by enhanced infiltration of neutrophils and mononuclear cells into the gastric mucosa. These activated neutrophils and mononuclear cells generate proinflammatory cytokines, including IL-1β, IL-8 and TNF-α [65,66] . These inflammatory states are terminated by the development of gastric atrophy and intestinal metaplasia. Patients with active gastritis, severe gastric mucosal atrophy and intestinal metaplasia are considered to be in a premalignant state of GC because these patients have a much greater risk for developing GC [4] . A number of studies have provided evidence that epigenetic alterations occur frequently in GC, whereas changes are also frequently observed at the premalignant stages [67] , suggesting that aberrant methylation occurs early during the multistep process of gastric carcinogenesis. Moreover, consequent studies have demonstrated that aberrant DNA methylation is not only observed in neoplastic gastric tissue but also in the nonneoplastic gastric mucosa in relation to age, gender, intestinal metaplasia and chronic inflammation [68] . The induction of methylation in gastric mucosa resulting from H. pylori infection was clearly described by Maekita et al. H. pylori infection strongly induced methylation of multiple genes (p16, LOX, THBD, FLNc, HAND1, etc.) at 5.4–303-fold higher levels than in H. pylori-negative subjects [28] . The DNA methylation status of gastric mucosa is associated with the severity of H. pylori-related gastric mucosal inflammation and atrophy [29] , which is a risk factor for GC [4] . Additionally, accelerated DNA methylation is shown in premalignant stages of GC, such as intestinal metaplasia, indefinite dysplasia and dysplasia [69] . Therefore, the accumulation of aberrant methylation in gastric mucosa can be thought of as an ‘epigeneticfield-defect,’ which constitutes the earliest steps toward


479

Review   Tahara & Arisawa

Table 3. Studies of CpG island methylator phenotype in gastric cancer and its correlation with clinicopathological and molecular features. Panels of marker or gene

Method

Criteria for defining CIMP

Correlation with CIMP

Ref.

MINT1, MINT2, MINT12, MINT25, MINT31

MSP

CIMP-high (3-5), CIMPintermediate (2),

CIMP-high with MSI-H

[43]

MINT1, MINT2, MINT25, MINT31, MSP MLH1

CIMP-negative (1 or less)

CIMP-high (3 or more), CIMPlow (2, 1),

CIMP-high with MSI,

CIMP-negative (0)

better overall survival

MINT1, MINT2, MINT12, MINT25, MINT31

COBRA

CIMP-high (4-5), CIMPintermediate (1-3),

CIMP-high with EBVpositive,

CIMP-negative (0)


p16, hMLH, LOX, FLNc, HRASL, HAND, THBD, F2R, NT5E, GREM,

Real-time MSP

CIMP-high (5 or more), CIMPlow (1-4),

CIMP-high with EBVpositive,

ZNF177, CLDN3, PAX6, CTSL

CIMP-negative (0)


ALX4, TMEFF2, CHCHD10, IGFBP3, NPR1

MethyLight

CIMP-high (4 or more), CIMPlow (1-3),

CIMP-high with worse overall survival,

CIMP-negative (0)

distant lymph node metastasis

BCL2, BDNF, CACNA1G, CALCA, CHFR, CYP1B1, DLEC1, GRIN2B,

MethyLight

CIMP-high (14-16), CIMP-low (1-3),

CIMP-high with worse overall survival

RUNX3, SEZ6L, SFRP4, TERT, THBS1, TIMP3, TP73, TWIST1

CIMP-negative (0)

27,578 CpG sites

Infinium 27K methylation arrays

Custer analysis of 1,653 expression-

CIMP with young patient age,

associated CpG sites

worse overall survival

27,578 CpG sites

Infinium 27K methylation arrays

Custer analysis of 1,315 CpG sites

CIMP with EBV-positive, MSI

[44]

[26]

[45]

[47]

[46]

[48]

[27]

CIMP: CpG island methylator phenotype; EBV: Epstein Barr virus; MSI: Microsatellite instability; MSP: Methylation-specific PCR.

neoplastic transformation by creating molecular diversity and reflects individual GC risk. To date, many researchers have provided evidence that epigenetic alterations in nonneoplastic gastric mucosa are linked to the risk of GC (Table 4) . For example, the methylation levels of tumor suppressor and tumor-related genes, such as p16, CDH1 and DAPK, were significantly higher in noncancerous gastric mucosa from GC patients [30,70] . The methylation levels of IRF4 [71] and FLNc [31] are associated with increased incidence of multiple GC. It has also been reported that patients with enlarged-fold gastritis, which is a form of gastritis with an increased risk of GC, exhibit global DNA hypomethylation, which is correlated with promoter CpG island hypermethylation of the CDH1, CDH13 and PGP9.5 genes [72] . In addition to coding genes, DNA methylation of small noncoding RNAs (miRNAs), such as miR-34b/c, is also associated with the increased incidence of multiple GCs [73] . In areas with high H. pylori infection rates, especially in Asian countries, the existence of H. pylori

480


infection alone is not adequate for the assessment of GC risk; therefore, the methylation signature in the nonneoplastic gastric mucosa has become a promising biomarker for GC risk estimation. Although the induction of methylation in the gastric mucosa by H. pylori involves complex biological processes that are still not completely understood, chronic inflammation may play a crucial role in methylation induction. H. pylori infection induces chronic inflammation and enhances the expression of proinflammatory cytokines, such as IL-1β, IL-8 and TNF-α [65,66] , causing oxidative stress to the gastric epithelium [74] . Studies in patients with ulcerative colitis and chronic hepatitis also provide evidence for a role of chronic inflammation in the induction of methylation [75,76] . Suppression of inflammation by an immunosuppressant, cyclosporine A, reduced the induction of methy lation in H. pylori-infected Mongolian gerbils, suggesting that inflammation induced by H. pylori infection but not by H. pylori itself is involved in the induction of methylation in the gastric mucosa [77] .



Review

Table 4. Suggested methylation markers in nonneoplastic gastric mucosa and its correlation with clinicopathological features. Suggested methylation markers


Ref.

LOX

Helicobacter pylori infection, GC occurrence

[28]

THBD


[28]

FLNC


[28]

HAND1


[28]

p16

Helicobacter pylori infection, severity of gastritis, GC occurrence

[29,30,70]

DAPK


[29,30,70]

CDH1


[29,30,70]

RUNX3

Intestinal metaplasia, dysplasia

[69]

IRF4

Incidence of multiple GC

[71]

FLNc


[31]

LINE1

Enlarged-fold gastritis

[72]

CDH1


[72]

CDH13


[72]

PGP9.5


[72]

miR-34b/c


[73]

GC: Gastric cancer.

It has been suggested that eradication of H. pylori reduces the risk of GC [78] , and this is supported by evidence of its effect on DNA methylation. After H. pylori eradication, methylation of FLNc and THBD significantly decrease; however, methylation levels are still higher than in healthy individuals without H. pylori infection [79] . Methylation of MOS decreases after H. pylori eradication among cases without intestinal metaplasia, and MOS methylation level is persistently increased in patients with dysplasia or GC [80] . This indicates that decreased but measurable levels of methylation after H. pylori eradication provide profiles that are unique to individuals and reflect past exposure to chronic inflammation. Most patients with intestinaltype gastric cancer are thought to have a history of H. pylori infection, even if the clinical test for H. pylori is negative. Furthermore, H. pylori eradication does not completely restore DNA methylation to normal levels [79,80] . Thus, individuals with sustained alterations in DNA methylation would be at higher risk even after H. pylori eradication. From this view point, evaluation of DNA methylation among H. pylori-negative subjects, including those for whom H. pylori has been eradicated, may also serve as a useful diagnostic marker for estimation of GC risk. The relationship between residual methylation and GC risk after H. pylori eradication needs to be further clarified.


Potential usefulness of DNA methylation as a molecular biomarker of GC in samples from a variety of sources DNA methylation is frequently observed in GC at the precancerous stage. Additionally, it is possible to detect very small amounts of methylated DNA among tissues [36] . Therefore, aberrant methylation can be used as a molecular biomarker of GC in samples from a variety of sources (Table 5) . It has been proposed that tumor cells can release DNA into the peripheral blood and that an enriched circulating DNA level can be found in the serum of cancer patients, which can be several times higher than in cancer-free subjects. A number of studies have investigated the utility of measuring serum or plasma DNA methylation to detect DNA methylation from tumor-derived circulating DNA as a potential molecular diagnostic marker for GC. Methylation of p16, CDH1, MGMT, RARB and RNF180 are significantly higher in DNA from serum or plasma from GC subjects than in that from control subjects [81–84] . Methylation of the CDH1 promoter in preoperative peritoneal washes is also significantly associated with more aggressive clinicopathological subtypes of GC, including larger tumors, infiltration type, lymphatic and venal invasion, higher T stage, lymph node and distant metastasis and worse disease-free survival [85] . Methylation of MINT25,


481

Review   Tahara & Arisawa

Table 5. Suggested methylation markers in gastric cancer in a variety of sources of samples. Sample type

Suggested methylation markers


Ref.

Serum

p16

GC occurrence

Serum

CDH1

GC occurrence

[81]

Serum

RARB

GC occurrence

[82]

Plasma

MGMT

GC occurrence

[83]

Plasma

RNF180

GC occurrence

[84]

Preoperative peritoneal washes

CDH1

Worse disease-free survival

[85]

[81]

Gastric wash fluid

MINT25

Detection of GC occurrence

[86]

Gastric wash fluid

RORA


[86]

Gastric wash fluid

GDNF


[86]

Gastric wash fluid

ADAM23


[86]

Gastric wash fluid

PRDM5


[86]

Gastric wash fluid

MLF1


[86]

Gastric wash fluid

Sox17

Prediction of residual GC after endoscopic resection

[87]

Whole blood

SFRP1

GC occurrence

[34]

GC: Gastric cancer.

RORA, GDNF, ADAM23, PRDM5, MLF1 from gastric washes are significantly associated with the occurrence of GC [86] . The usefulness of measurement of Sox17 methylation in gastric washes for the prediction of residual GC after endoscopic resection has also been reported [87] . All of these studies are based on the concept that DNA methylation from targeted tissues can also be detected in samples from a variety of sources. On the other hand, it is suggested that the DNA methylation status of nontargeted tissue, particularly in blood, is associated with aging and exposures throughout life [88,89] and therefore is linked to a mechanism for cancer predisposition [32,33] . Evaluating whole blood DNA methylation as a risk marker for GC is of particular interest because peripheral blood DNA is a convenient tissue to assay for constitutional methylation as its collection is considered noninvasive. A recent study evaluated this concept using 14 candidate CpG island promoters [34] . The results suggest that the SFRP1 promoter is one of the regions where methylation in blood could reflect GC predisposition, but the differences between cases and controls were small. The methylation levels of the IGF2 differentially methylated region 0 (DMR0) and the Alu and LINE1 repetitive elements tended to be lower in blood from patients with GC than from control patients, but the association in subjects overall was not significant (p > 0.05 for all) [35,90] . Another study explored the association between methylation in prediagnostic blood leukocyte DNA and GC risk in the prospec-

482


tive cohort [91] . The results showed that Alu methylation was inversely associated with GC risk, mainly among cases diagnosed one or more years later but not across all subjects [91] . Taken together, it is suggested that methylation in whole blood might reflect cancer predisposition. However, these changes are always minimal and there is often a large overlap between cases and controls; therefore, the potential usefulness of blood DNA methylation as a screening/diagnostic biomarker for cancer may be limited. Conclusion & future perspective We have discussed the recent studies on DNA methy lation as a molecular biomarker for GC. DNA methy lation status can be associated with the clinicopathological features and outcome of GC, suggesting that DNA methylation can be used as a marker to classify GC cases more accurately in a way that is relevant to diagnosis and therapy. DNA methylation is also commonly observed in H. pylori-infected nonneoplastic gastric mucosa, which exhibit a higher risk of GC. Therefore, DNA methylation can also serve as a useful diagnostic tool for GC risk estimation. Moreover, the possibility to detect very small amounts of methylated DNA among tissues will allow us to explore the novel molecular biomarker in GC using samples from a variety of sources. Recent advances in genome-wide DNA methylation analysis have shifted the focus to characterizing the methylation signatures on a genome-wide scale, which provides



a better understanding of individual prognosis and risk and allows for improved clinical implementation [27,48,50,51] . Because epigenetic dysregulation of gene expression is reversible, one goal of the discovery of specific methylation changes would be the possibility of targeting epigenetic abnormalities for the treatment and prevention of GC.

Review

Financial & competing interests disclosure The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Executive summary • DNA methylation plays a significant role in the development and progression of gastric cancer (GC) and specific methylation patterns provide distinct clinicopathological subgroups of GC. • The CpG island methylator phenotype (CIMP) is associated with specific subtypes of GC, such as Epstein–Barr virus-positive and microsatellite instability (MSI). Somatic mutations in chromatin modifiers were identified in GC, and these might aid in our understanding of the molecular origin of this phenotype. • DNA methylation observed in Helicobacter pylori-infected gastric mucosa reflects an epigenetic-field-defect, and this might be useful for risk estimation of GC. • DNA methylation can serve as a molecular biomarker in GC in a variety of samples, including serum, plasma and gastric washes. • The DNA methylation status of nontargeted tissue, particularly blood, is thought to be associated with GC predisposition, but the clinical usefulness of GC risk estimation should be carefully evaluated.

References Papers of special note have been highlighted as: • of interest 1

2

3

Ferlay J, Bray F, Parkin DM, Pisani P (Eds). Gobocan 2000: Cancer Incidence and Mortality Worldwide (IARC Cancer Bases No. 5). Lyon: IARC Press (2001). Lau M, Le A, El-Serag HB. Noncardia gastric adenocarcinoma remains an important and deadly cancer in the United States: secular trends in incidence and survival. Am. J. Gastroenterol. 101(11), 2485–2492 (2006). Nashimoto A, Akazawa K, Isobe Y et al. Gastric cancer treated in 2002 in Japan: 2009 annual report of the JGCA nationwide registry. Gastric Cancer 16(1), 1–27 (2013).

4

Uemura N, Okamoto S, Yamamoto S et al. Helicobacter pylori infection and the development of gastric cancer. N. Engl. J. Med. 345(11), 784–789 (2001).

5

Vogelstein B, Papadopoulos N, Velculescu VE et al. Cancer genome landscapes. Science 339, 1546–1558 (2013).

6

Ushijima T, Asada K. Aberrant DNA methylation in contrast with mutations. Cancer Sci. 101, 300–305 (2010).

7

Calcagno DQ, Gigek CO, Chen ES, Burbano RR, Smith MA. DNA and histone methylation in gastric carcinogenesis. World J. Gastroenterol. 19, 1182–1192 (2013).

8

Tsai KW, Wu CW, Hu LY et al. Epigenetic regulation of miR-34b and miR-129 expression in gastric cancer. Int. J. Cancer 129, 2600–2610 (2011).

9

Ziogas D, Roukos D. Epigenetics in gastric cancer: challenges for clinical implications. Ann. Surg. Oncol. 16, 2077–2078 (2009).

10

Wolffe AP, Matzke MA. Epigenetics: regulation through repression. Science 286, 481–486 (1999).

11

Gama-Sosa MA, Slagel VA, Trewyn RW et al. The 5-methylcytosine contentof DNA from human tumors. Nucleic Acids Res. 11, 6883–6894 (1983).


12

Dunn BK. Hypomethylation: one side of a larger picture. Ann. NY Acad. Sci. 983, 28–42 (2003).

13

Esteller M, Herman JG. Cancer as an epigenetic disease: DNA methylation and chromatin alterations in human tumours. J. Pathol. 196, 1–7 (2002).

14

Eden A, Gaudet F, Waghmare A, Jaenisch R. Chromosomal instability and tumors promoted by DNA hypomethylation. Science 300, 455 (2003).

15

Gaudet F, Hodgson JG, Eden A et al. Induction of tumors in mice by genomic hypomethylation. Science 300, 489–492 (2003).

16

Ehrlich M. DNA methylation in cancer: too much, but also too little. Oncogene 21, 5400–5413 (2002).

17

Ehrlich M. DNA hypomethylation, cancer, the immunodeficiency, centromeric region instability, facial anomalies syndrome and chromosomal rearrangements. J. Nutr. 132, 2424S–2429S (2002).

18

Berman BP, Weisenberger DJ, Aman JF et al. Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nat. Genet. 44(1), 40–46 (2011).

19

Timp W, Feinberg AP. Cancer as a dysregulated epigenome allowing cellular growth advantage at the expense of the host. Nat. Rev. Cancer 13(7), 497–510 (2013).

20

Jones PA, Baylin SB. The epigenomics of cancer. Cell 128(4), 683–692 (2007).

21

Toyota M, Issa JP. Epigenetic changes in solid and hematopoietic tumors. Semin. Oncol. 32(5), 521–530 (2005).

22

Ushijima T. Detection and interpretation of altered methylation patterns in cancer cells. Nat. Rev. Cancer 5(3), 223–231 (2005).

23

Bird AP, Wolffe AP. Methylation-induced repression–belts, braces, and chromatin. Cell 99(5), 451–454 (1999).


483

Review   Tahara & Arisawa 24

Jones PA, Laird PW. Cancer epigenetics comes of age. Nat. Genet. 21(2), 163–7 (1999).

25

Toyota M, Ahuja N, Ohe-Toyota M, Herman JG, Baylin SB, Issa JP. CpG island methylator phenotype in colorectal cancer. Proc. Natl Acad. Sci. USA 96(15), 8681–8686 (1999).

26

Kusano M, Toyota M, Suzuki H et al. Genetic, epigenetic, and clinicopathologic features of gastric carcinomas with the CpG island methylator phenotype and an association with Epstein-Barr virus. Cancer 106(7), 1467–1479 (2006).

••

27

••

Shen L, Toyota M, Kondo Y et al. Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer. Proc. Natl Acad. Sci. USA 104(47), 18654–18659 (2007).

39

Ahn JB, Chung WB, Maeda O et al. DNA methylation predicts recurrence from resected stage III proximal colon cancer. Cancer 117(9), 1847–1854 (2011).

40

This study comprehensively investigated CIMP in gastric cancer (GC) in relation to clinicopathological and molecular features.

Ogino S, Nosho K, Kirkner GJ et al. CpG island methylator phenotype, microsatellite instability, BRAF mutation and clinical outcome in colon cancer. Gut 58(1), 90–96 (2009).

41

Cancer Genome Atlas Network. Comprehensive molecular characterization of gastric adenocarcinoma. Nature 513(7517), 202–209 (2014).

Hughes LA, Melotte V, de Schrijver J et al. The CpG island methylator phenotype: what’s in a name? Cancer Res. 73(19), 5858–5868 (2013).

42

This comprehensive study provided detailed molecular landscape in GC.

Toyota M, Ahuja N, Suzuki H et al. Aberrant methylation in gastric cancer associated with the CpG island methylator phenotype. Cancer Res. 59(21), 5438–5442 (1999).

••

This study first identified CIMP phenotype in GC. Kim H, Kim YH, Kim SE, Kim NG, Noh SH, Kim H. Concerted promoter hypermethylation of hMLH1, p16INK4A, and E-cadherin in gastric carcinomas with microsatellite instability. J. Pathol. 200(1), 23–31 (2003).

28

Maekita T, Nakazawa K, Mihara M et al. High levels of aberrant DNA methylation in Helicobacter pylori-infected gastric mucosae and its possible association with gastric cancer risk. Clin. Cancer Res. 12(3 Pt 1), 989–995 (2006).

43

••

This study confirmed positive role of Helicobacter pylori infection in methylation induction.

44

29

Tahara T, Arisawa T, Shibata T et al. Increased number of methylated CpG islands correlates with Helicobacter pylori infection, histological and serological severity of chronic gastritis. Eur. J. Gastroenterol. Hepatol. 21(6), 613–619 (2009).

An C, Choi IS, Yao JC et al. Prognostic significance of CpG island methylator phenotype and microsatellite instability in gastric carcinoma. Clin. Cancer Res. 11(2 Pt 1), 656–663 (2005).

45

Tahara T, Shibata T, Nakamura M et al. Increased number of CpG island hypermethylation in tumor suppressor genes of non-neoplastic gastric mucosa correlates with higher risk of gastric cancer. Digestion 82(1), 27–36 (2010).

Enomoto S, Maekita T, Tsukamoto T et al. Lack of association between CpG island methylator phenotype in human gastric cancers and methylation in their background non-cancerous gastric mucosae. Cancer Sci. 98(12), 1853–1861 (2007).

46

Nakajima T, Maekita T, Oda I et al. Higher methylation levels in gastric mucosae significantly correlate with higher risk of gastric cancers. Cancer Epidemiol. Biomarkers Prev. 15(11), 2317–2321 (2006).

Park SY, Kook MC, Kim YW et al. CpG island hypermethylator phenotype in gastric carcinoma and its clinicopathological features. Virchows Arch. 457(4), 415–422 (2010).

47

Marsit CJ, Koestler DC, Christensen BC, Karagas MR, Houseman EA, Kelsey KT. DNA methylation array analysis identifies profiles of blood-derived DNA methylation associated with bladder cancer. J. Clin. Oncol. 29(9), 1133–1139 (2011).

Chen HY, Zhu BH, Zhang CH et al. High CpG island methylator phenotype is associated with lymph node metastasis and prognosis in gastric cancer. Cancer Sci. 103(1), 73–79 (2012).

48

Zouridis H, Deng N, Ivanova T et al. Methylation subtypes and large-scale epigenetic alterations in gastric cancer. Sci. Transl. Med. 4(156), 156ra140 (2012).

49

Widschwendter M, Fiegl H, Egle D et al. Epigenetic stem cell signature in cancer. Nat. Genet. 39, 157–158 (2007).

50

Loh M, Liem N, Vaithilingam A et al. DNA methylation subgroups and the CpG island methylator phenotype in gastric cancer: a comprehensive profiling approach. BMC Gastroenterol. 14, 55 (2014).

51

Matsusaka K, Kaneda A, Nagae G et al. Classification of Epstein-Barr virus-positive gastric cancers by definition of DNA methylation epigenotypes. Cancer Res. 71(23), 7187–7197 (2011).

52

Noushmehr H, Weisenberger DJ, Diefes K et al. Identification of a CpG island methylator phenotype that defines a distinct subgroup of glioma. Cancer Cell 17(5), 510–522 (2010).

53

Figueroa ME, Abdel-Wahab O, Lu C et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype,

30

31

32

33

484

38

Wong EM, Southey MC, Fox SB et al. Constitutional methylation of the BRCA1 promoter is specifically associated with BRCA1 mutation-associated pathology in early-onset breast cancer. Cancer Prev. Res. (Phila). 4(1), 23–33 (2011).

34

Tahara T, Maegawa S, Chung W et al. Examination of whole blood DNA methylation as a potential risk marker for gastric cancer. Cancer. Prev. Res. (Phila). 6(10), 1093–1100 (2013).

35

Yuasa Y, Nagasaki H, Oze I et al. Insulin-like growth factor 2 hypomethylation of blood leukocyte DNA is associated with gastric cancer risk. Int. J. Cancer 131(11), 2596–2603 (2012).

36

Laird PW. The power and the promise of DNA methylation markers. Nat. Rev. Cancer 3(4), 253–266 (2003).

37

Suzuki H, Tokino T, Shinomura Y, Imai K, Toyota M. DNA methylation and cancer pathways in gastrointestinal tumors. Pharmacogenomics 9(12), 1917–1928 (2008).




disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell 18(6), 553–567 (2010). 54

55

Tahara T, Yamamoto E, Madireddi P et al. Colorectal carcinomas with CpG island methylator phenotype 1 frequently contain mutations in chromatin regulators. Gastroenterology 146(2), 530–538.e5 (2014). Wang K, Kan J, Yuen ST et al. Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of gastric cancer. Nat. Genet. 43(12), 1219–1223 (2011).

••

This study identified somatic mutation in chromatin remodeling gene ARID1A in specific subtypes of GC.

56

Zang ZJ, Cutcutache I, Poon SL et al. Exome sequencing of gastric adenocarcinoma identifies recurrent somatic mutations in cell adhesion and chromatin remodeling genes. Nat. Genet. 44(5), 570–574 (2012).

••

This study identified chromatin remodeling as an altered pathways in GC.

57

Satoh A, Toyota M, Itoh F et al. Epigenetic inactivation of CHFR and sensitivity to microtubule inhibitors in gastric cancer. Cancer Res. 63(24), 8606–8613 (2003).

58

Du W, Wang S, Zhou Q et al. ADAMTS9 is a functional tumor suppressor through inhibiting AKT/mTOR pathway and associated with poor survival in gastric cancer. Oncogene 32(28), 3319–3328 (2013).

59

Cheng AS, Li MS, Kang W et al. Helicobacter pylori causes epigenetic dysregulation of FOXD3 to promote gastric carcinogenesis. Gastroenterology 144(1), 122–133.e9 (2013).

60

Li X, Cheung KF, Ma X et al. Epigenetic inactivation of paired box gene 5, a novel tumor suppressor gene, through direct upregulation of p53 is associated with prognosis in gastric cancer patients. Oncogene 31(29), 3419– 3430 (2012).

61

62

63

Weisenberger DJ, Siegmund KD, Campan M et al. CpG island methylator phenotype underlies sporadic microsatellite instability and is tightly associated with BRAF mutation in colorectal cancer. Nat. Genet. 38, 787–793 (2006). Ogino S, Kawasaki T, Kirkner GJ, Kraft P, Loda M, Fuchs CS. Evaluation of markers for CpG island methylator phenotype (CIMP) in colorectal cancer by a large population-based sample. J. Mol. Diagn. 9, 305–314 (2007). Zong L, Seto Y. CpG island methylator phenotype, Helicobacter pylori, Epstein-Barr virus, and microsatellite instability and prognosis in gastric cancer: a systematic review and meta-analysis. PLoS ONE 9(1), e86097 (2014).

64

Jover R, Nguyen TP, Perez-Carbonell L et al. 5-fluorouracil adjuvant chemotherapy does not increase survival in patients with CpG island methylator phenotype colorectal cancer. Gastroenterology 140(4), 1174–1181 (2011).

65

Wang M, Furuta T, Takashima M et al. Relation between interleukin-1beta messenger RNA in gastric fundic mucosa and gastric juice pH in patients infected with Helicobacter pylori. J. Gastroenterol. 34(Suppl. 11), 10–17 (1999).

66

Yamaoka Y, Kita M, Kodama T, Sawai N, Kashima K, Imanishi J. Induction of various cytokines and development of severe mucosal inflammation by cagA gene positive Helicobacter pylori strains. Gut 41(4), 442–451 (1997).


67

Kang GH, Shim YH, Jung HY, Kim WH, Ro JY, Rhyu MG. CpG island methylation in premalignant stages of gastric carcinoma. Cancer Res. 61(7), 2847–2851 (2001).

68

Kang GH, Lee HJ et al. Aberrant CpG island hypermethylation of chronic gastritis, in relation to aging, gender, intestinal metaplasia, and chronic inflammation. Am. J. Pathol. 163(4), 1551–1556 (2003).

69

Li WQ, Pan KF, Zhang Y et al. RUNX3 methylation and expression associated with advanced precancerous gastric lesions in a Chinese population. Carcinogenesis 32(3), 406–410 (2011).

70

Kaise M, Yamasaki T, Yonezawa J, Miwa J, Ohta Y, Tajiri H. CpG island hypermethylation of tumor-suppressor genes in H. pylori-infected nonneoplastic gastric mucosa is linked with gastric cancer risk. Helicobacter 13(1), 35–41 (2008).

71

Yamashita M, Toyota M, Suzuki H et al. DNA methylation of interferon regulatory factors in gastric cancer and noncancerous gastric mucosae. Cancer Sci. 101(7), 1708–1716 (2010).

72

Yamamoto E, Toyota M, Suzuki H et al. LINE-1 hypomethylation is associated with increased CpG island methylation in Helicobacter pylori-related enlarged-fold gastritis. Cancer Epidemiol. Biomarkers Prev. 17(10), 2555– 2564 (2008).

73

Suzuki H, Yamamoto E, Nojima M et al. Methylationassociated silencing of microRNA-34b/c in gastric cancer and its involvement in an epigenetic field defect. Carcinogenesis 31(12), 2066–2073 (2010).

74

Meyer-ter-Vehn T, Covacci A, Kist M, Pahl HL. Helicobacter pylori activates mitogen-activated protein kinase cascades and induces expression of the proto-oncogenes c-fos and c-jun. J. Biol. Chem. 275(21), 16064–16072 (2000).

75

Issa JP, Ahuja N, Toyota M, Bronner MP, Brentnall TA. Accelerated age-related CpG island methylation in ulcerative colitis. Cancer Res. 61(9), 3573–357 (2001).

76

Nishida N, Nagasaka T, Nishimura T, Ikai I, Boland CR, Goel A. Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma. Hepatology 47(3), 908–918 (2008).

77

Niwa T, Tsukamoto T, Toyoda T et al. Inflammatory processes triggered by Helicobacter pylori infection cause aberrant DNA methylation in gastric epithelial cells. Cancer Res. 70(4), 1430–1440 (2010).

78

Fukase K, Kato M, Kikuchi S et al. Effect of eradication of Helicobacter pylori on incidence of metachronous gastric carcinoma after endoscopic resection of early gastric cancer: an open-label, randomised controlled trial. Lancet 372(9636), 392–397 (2008).

79

Nakajima T, Enomoto S, Yamashita S et al. Persistence of a component of DNA methylation in gastric mucosae after Helicobacter pylori eradication. J. Gastroenterol. 45(1), 37–44 (2010).

80

Shin CM, Kim N, Park JH et al. Prediction of the risk for gastric cancer using candidate methylation markers in the non-neoplastic gastric mucosae. J. Pathol. 226(4), 654–665 (2012).


Review

485

Review   Tahara & Arisawa 81

Ichikawa D, Koike H, Ikoma H et al. Detection of aberrant methylation as a tumor marker in serum of patients with gastric cancer. Anticancer Res. 24(4), 2477–2481 (2004).

••

Demonstrated the clinical utility of DNA methylation gastric washes as promising diagnostic markers in GC.

87

82

Kolesnikova EV, Tamkovich SN, Bryzgunova OE et al. Circulating DNA in the blood of gastric cancer patients. Ann. NY Acad. Sci. 1137, 226–231 (2008).

Oishi Y, Watanabe Y, Yoshida Y et al. Hypermethylation of Sox17 gene is useful as a molecular diagnostic application in early gastric cancer. Tumour Biol. 33(2), 383–393 (2012).

88

83

Ikoma H, Ichikawa D, Daito I et al. Clinical application of methylation specific-polymerase chain reaction in serum of patients with gastric cancer. Hepatogastroenterology 54(75), 946–950 (2007).

Christensen BC, Houseman EA, Marsit CJ et al. Aging and environmental exposures alter tissuespecific DNA methylation dependent upon CpG island context. PLoS Genet. 5(8), e1000602 (2009).

89

84

Cheung KF, Lam CN, Wu K et al. Characterization of the gene structure, functional significance, and clinical application of RNF180, a novel gene in gastric cancer. Cancer 118(4), 947–959 (2012).

Teschendorff AE, Menon U, Gentry-Maharaj A et al. Agedependent DNA methylation of genes that are suppressed in stem cells is a hallmark of cancer. Genome Res. 20(4), 440–446 (2012).

90

85

Yu QM, Wang XB, Luo J et al. CDH1 methylation in preoperative peritoneal washes is an independent prognostic factor for gastric cancer. J. Surg. Oncol. 106(6), 765–771 (2012).

Hou L, Wang H, Sartori S et al. Blood leukocyte DNA hypomethylation and gastric cancer risk in a high-risk Polish population. Int. J. Cancer 127(8), 1866–1874 (2010).

91

Gao Y, Baccarelli A, Shu XO et al. Blood leukocyte Alu and LINE-1 methylation and gastric cancer risk in the Shanghai Women’s Health Study. Br. J. Cancer 106(3), 585–591 (2012).

86

486

Watanabe Y, Kim HS, Castoro RJ et al. Sensitive and specific detection of early gastric cancer with DNA methylation analysis of gastric washes. Gastroenterology 136(7), 2149–2158 (2009).



DNA methylation as an epigenetic biomarker in colorectal cancer.

DNA methylation as a biomarker for preeclampsia.

DNA Methylation as Surrogate Marker For Gastric Cancer.

DNA methylation downregulated mir-10b acts as a tumor suppressor in gastric cancer.

PAX1 methylation as a potential biomarker for cervical cancer screening.

Methylation as a biomarker for head and neck cancer.

Circulating DNA as biomarker in breast cancer.

DNA methylation: potential biomarker in Hepatocellular Carcinoma.

DNA methylation pattern of CALCA in preterm neonates with bacterial sepsis as a putative epigenetic biomarker.

FOXP3 DNA methylation levels as a potential biomarker in the development of periapical lesions.

DNA Methylation Score as a Biomarker in Newborns for Sustained Maternal Smoking during Pregnancy.

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

Aberrant DNA Methylation as a Biomarker and a Therapeutic Target of Cholangiocarcinoma.

Free circulating DNA as a biomarker of colorectal cancer.

Circulating Tumor DNA as a Cancer Biomarker: Fact or Fiction?

DNA methylation status as a biomarker of anti-epidermal growth factor receptor treatment for metastatic colorectal cancer.

DNA methylation of BDNF as a biomarker of early-life adversity.

Protein expression and promoter methylation of the candidate biomarker TCF21 in gastric cancer.

MicroRNA-10a is down-regulated by DNA methylation and functions as a tumor suppressor in gastric cancer cells.

Promoter DNA methylation analysis reveals a combined diagnosis of CpG-based biomarker for prostate cancer.

DNA methylation pattern as important epigenetic criterion in cancer.

Detection of SNCA and FBN1 methylation in the stool as a biomarker for colorectal cancer.

Zic1 Promoter Hypermethylation in Plasma DNA Is a Potential Biomarker for Gastric Cancer and Intraepithelial Neoplasia.

Clinical significance of CDH13 promoter methylation as a biomarker for bladder cancer: a meta-analysis.