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1467
Genetic, Epigenetic, and Clinicopathologic Features of
Gastric Carcinomas with the CpG Island Methylator
Phenotype and an Association with Epstein–Barr Virus
Masanobu Kusano, M.D.1
Minoru Toyota, M.D., Ph.D.1–3
Hiromu Suzuki, M.D., Ph.D.1,4
Kimishige Akino, M.D.1,2
Fumio Aoki, Ph.D.5
Masahiro Fujita, M.D., Ph.D.6
Masao Hosokawa, M.D.7
Yasuhisa Shinomura, M.D., Ph.D.1
Kohzoh Imai, M.D., Ph.D.1
Takashi Tokino, M.D., Ph.D.2
BACKGROUND. The CpG island methylator phenotype (CIMP), which is character-
1
RESULTS. The methylation profiles of 12 genes showed nonrandom methylation,
supporting the presence of CIMP in gastric carcinoma. No p53 mutations were
detected among CIMP-H tumors, and no EBV association was detected in tumors
that showed mutation of p53 and K-ras. In a multiple logistic regression model with
CIMP-H as the dependent variable, proximal location (P ⫽ .011), diffuse type
(P ⫽ .019), and less advanced pathologic TNM status (P ⫽ .043) contributed significantly to CIMP-H. Patients who had CIMP-N gastric tumors had a significantly
worse survival than patients who had CIMP-H tumors (P ⫽ .004) or CIMP-L tumors
(P ⫽ .012). EBV-associated tumors were associated strongly with CIMP-H, hypermethylation of tumor-related genes, and no p53 or K-ras mutation.
CONCLUSIONS. CIMP status appeared to be associated with distinct genetic, epigenetic, and clinicopathologic features in gastric carcinomas. The finding that
gastric carcinomas arose through different molecular pathways may affect not only
tumor characteristics but also patient prognosis. Cancer 2006;106:1467–79.
© 2006 American Cancer Society.
First Department of Internal Medicine, Cancer
Research Institute, Sapporo Medical University,
Sapporo, Japan.
2
Department of Molecular Biology, Cancer Research Institute, Sapporo Medical University, Sapporo, Japan.
3
Precursory Research for Embryonic Science and
Technology (PRESTO), Japan Science and Technology Agency, Kawaguchi, Japan.
4
Department of Public Health, Sapporo Medical
University, Sapporo, Japan.
5
Information Center for Computer Communication,
Sapporo Medical University, Sapporo, Japan.
6
Keiyukai Institute of Clinical Pathology, Keiyukai
Sapporo Hospital, Sapporo, Japan.
7
Department of Surgery, Keiyukai Sapporo Hospital, Sapporo, Japan.
Supported in part by Grants-in-Aid for Scientific
Research on Priority Areas (C) from the Ministry of
Education, Culture, Sports, Science, and Technology (M.T., K.I., and T.T.).
The authors thank Dr. Tomoko Sonoda, Dr. William
F. Goldman, and Mr. Robert Holmes for statistical
analysis and editing the article.
Address for reprints: Minoru Toyota, M.D., Ph.D.,
First Department of Internal Medicine, Sapporo
Medical University, South 1,West 16, Chuo-ku,
Sapporo 060-8543, Japan; Fax: (011) 81-11-6183313; E-mail: [email protected]
Received April 26, 2005; revision received September 21, 2005; accepted October 19, 2005
ized by simultaneous methylation of the CpG islands of multiple genes, has been
recognized as one of the important mechanisms in gastrointestinal carcinogenesis.
METHODS. Methylation of the 5 methylated-in-tumors (MINT) loci and 12 tumorrelated genes in 78 primary gastric carcinomas was examined using combined
bisulfite-restriction analysis. Epstein–Barr virus (EBV)-associated gastric tumors
were detected using real-time polymerase chain reaction analysis followed by an
evaluation of the correlations between CIMP status, EBV-association, and genetic
alteration of p53 and K-ras. The authors compared the clinicopathologic features
of gastric carcinomas that had high CIMP methylation (CIMP-H) with tumors that
had low CIMP methylation (CIMP-L) or negative CIMP methylation (CIMP-N).
KEYWORDS: gastric carcinoma, DNA methylation, molecular profiling, clinicopathologic features, Epstein–Barr virus.
G
astric carcinoma is one of the most common human neoplasms
and is the second leading cause of cancer-related death in the
world.1 Promoter hypermethylation that leads to epigenetic silencing
of multiple genes has been recognized as an important mechanism in
gastrointestinal carcinogenesis. In that regard, promoter methylation
of the so called CpG islands, which are CpG dinucleotide-rich areas
located within the promoters of approximately 60% of human genes,2
usually is associated with long-term, irreversible epigenetic silencing
of X-linked and imprinted genes, but it also is known that CPG
promoter methylation silences tumor-related genes.3 Furthermore,
concordant promoter hypermethylation of multiple genes, which is
© 2006 American Cancer Society
DOI 10.1002/cncr.21789
Published online 3 March 2006 in Wiley InterScience (www.interscience.wiley.com).
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CANCER April 1, 2006 / Volume 106 / Number 7
termed descriptively as the “CpG island methylator
phenotype” (CIMP), was described recently in both
gastric and colorectal carcinomas.4 –7
Helicobacter pylori (HP) infection of the stomach
is associated with an increased risk of gastric carcinoma.8 Although HP infection is extraordinarily common, gastric adenocarcinoma occurs in only a minority of infected individuals. In addition, Epstein–Barr
virus (EBV) is a ubiquitous herpes virus that infects
most children during early childhood and causes few
if any symptoms. However, EBV also is involved in a
subset of gastric carcinomas, although its specific role
in carcinogenesis remains unclear. It has been shown
that p16 cyclin dependent kinase 4A inhibitor
(p16INK4A) expression is absent significantly more often with EBV-associated gastric carcinoma than with
EBV-negative gastric carcinoma9,10 and that this loss is
associated with not only p16INK4A methylation but also
with methylation of other tumor-suppressor genes.11
In addition, evidence suggests a close association between aberrant methylation and the entry of foreign
viral DNA into host cells.12,13
The most frequently observed genetic alteration
in gastric carcinoma is mutation of the p53 gene,
which is believed to play a central role in carcinogenesis of the stomach.14 By contrast, there is wide variation in the frequency of K-ras mutations in upper and
lower gastrointestinal tract tumors, with several studies showing the frequency in gastric carcinomas to be
quite low.15 The frequency of the occurrence of p53
and/or K-ras mutations in gastric tumors with multiple concordant methylation events and in EBV-associated gastric tumors remains unknown.
In the current study, we evaluated the methylation status of 12 tumor-related genes along with
the 5 methylated-in-tumors (MINT) loci. In addition, to clarify the characteristics of gastric tumors
with hypermethylation of MINT loci and to shed
light on their underlying mechanisms, we initially
assessed their clinicopathologic features, including
EBV and HP status and then analyzed the genetic
alterations of p53 and K-ras. Finally, we evaluated
the prognostic significance of CIMP status in gastric
carcinomas.
MATERIALS AND METHODS
Cell Lines and Specimens
The gastric carcinoma cell lines that were used in this
study were obtained from the Japanese Collection of
Research Bioresources (Tokyo, Japan) or the American
Tissue Type Collection (Manassas, VA) and then were
cultured in the appropriate medium. The 78 gastric
tumor specimens and their paired normal tissue specimens were from 78 randomly selected Japanese pa-
tients; these specimens were removed surgically, immediately frozen, and stored at ⫺ 80 °C until they were
used. Informed consent was obtained from all patients
before the samples were collected. DNA was extracted
using the standard phenol/chloroform method. Total
RNA was extracted using Trizol (Invitrogen, Carlsbad,
CA).
Reverse Transcription-Polymerase Chain Reaction
Five micrograms of total RNA were reverse-transcribed using SuperScript III Reverse Transcriptase
(Invitrogen); then, polymerase chain reaction (PCR)
analysis was carried out as described previously.16,17
The integrity of the cDNA was confirmed by amplifying glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), as described previously.18 To analyze restoration of chondroitin sulfate proteoglycan 2 (CSPG2),
MKN74, and KATOIII, cells were incubated for 72
hours with 0.2 ␮M 5-aza-2⬘-deoxycytidine (5-aza-dC)
(Sigma, St. Louis, MO), which is a methyltransferase
inhibitor.
Combined Bisulfite Restriction Analysis
Combined bisulfite restriction analysis (COBRA) consists of a standard sodium bisulfite treatment and PCR
amplification followed by restriction enzyme digestion
and a quantification step. We applied COBRA to assess
gene methylation using primers that were designed to
amplify the regions around the transcription start sites
of the target genes.19 Bisulfite modification was carried out as described previously.20 Briefly, 2 ␮g of
genomic DNA were incubated with 3 M sodium bisulfite (Sigma) for 16 hours; then, the DNA was purified
using a DNA Purification System (Promega, Madison,
WI), and it was stored at ⫺ 20 °C until it was used.
Selected for study were the 5 MINT loci (MINT1,
MINT2, MINT12, MINT25, and MINT31), which were
examined as described previously,4,5 and 12 tumorrelated genes. The primer sequences, annealing temperatures, and restriction enzymes that were used are
listed in Table 1. Initially, PCR was carried out in a
reaction medium that contained 1 ⫻ methylationspecific PCR (MSP) buffer (67 mM Tris-HCl, pH 8.8;
16.6 mM [NH4]2SO4; 6.7 mM MgCl2; and 10 mM
2-mercaptoethanol), 0.25 mM deoxyribonucleoside
triphosphate mixture, 0.5 ␮M primer, and 1.0 U of Ex
Taq (Hot Start Version; TaKaRa, Tokyo, Japan) using
primers that amplified both the methylated and unmethylated alleles. The products were then digested
using restriction enzymes that cleave only methylated
CpG sites.19 All of the restriction enzymes were purchased from TaKaRa, except BstU I (New England
BioLabs, Beverly, MA) and Tai I (Fermentas Inc., MD).
After digestion, the products were electrophoresed on
Gastric CA with CIMP and Association to EBV/Kusano et al.
1469
TABLE 1
Primer Sequences Used in the Current Study
Primer Sequence*
MINT1
F: 5⬘-GGGTTGGAGAGTAGGGGAGTT-3⬘
R: 5⬘-CCATCTAAAATTACCTCRATAACTTA-3⬘
MINT2
F: 5⬘-YGTTATGATTTTTTTGTTTAGTTAAT3⬘
R: 5⬘-TACACCAACTACCCAACTACCTC-3⬘
MINT12
F: 5⬘-YGGGTTATGTTTTATTTTTTGTGTTT-3⬘
R: 5⬘-CTCAAAAAAATCAAACAACCAACCAA-3⬘
MINT25
F: 5⬘-GGYGTATTAGGYGTAGTAGGAA-3⬘
R: 5⬘-CRACTTAACCRCCCACCTAAC-3⬘
MINT31
F: 5⬘-GAYGGYGTAGTAGTTATTTTGTT-3⬘
R: 5⬘-CATCACCACCCCTCACTTTAC-3⬘
CSPG2
F: 5⬘-TATGTTATTGAGTGAGTTTTTGAATG-3⬘
R: 5⬘-TTCAACCACTCCTAAAAATCCA-3⬘
BNIP3
F: 5⬘-TTYGGTYGGAGGAATTTATAGGGTAG-3⬘
R: 5⬘-CCCTCRCCCACCRCAAAAC-3⬘
CHFR
F: 5⬘-YGTTTATTAAGAGYGGTAGTTAAAG-3⬘
R: 5⬘-AAAATCCTTAAAACTTCCAATCC-3⬘
p16INK4A
F: 5⬘-GGTTTTGGYGAGGGTTGTTT-3⬘
R: 5⬘-ACCCTATCCCTCAAATCCTCTAAAA-3⬘
HLTF
F: 5⬘-GTTTTTTTGGATYGTTTTYGAGT-3⬘
R: 5⬘-CRACRCTAATCTCCCAAATTATTAC-3⬘
PAX5␤
F: 5⬘-TTTTTYGTTTTTTGAGTGAAGTTAAG-3⬘
R: 5⬘-CCTCCCTAACTAACTCAAACAACA-3⬘
HRK
F: 5⬘ AAAYGTATAATATAAGGAGAAATTTGG-3⬘
R: 5⬘-RATACAAAAAACACRAACACATAAC-3⬘
SLC5A8
F: 5⬘-TAAAATTTGTTTAGAGYGTTTTTTGT-3⬘
R: 5⬘-CCCAAATATAAAACCTCRAAAAATC-3⬘
TIG1
F: 5⬘-GAGAGAATTTAGGGGTTG-3⬘
R: 5⬘-AACCAAAAAACAAACAACC-3⬘
P57KIP2
F: 5⬘-GGTTGGGYGTTTTATAGGTTA-3⬘
R: 5⬘-ACCTAACTATCCGATAATAAACTCTTC-3⬘
HMLH1
F: 5⬘-TAGTAGTYGTTTTAGGGAGGGA-3⬘
R: 5⬘-TCTAAATACTCAACRAAAATACCTT-3⬘
SOCS-3
F: 5⬘-TATYGTATTTYGGGGGGTTG-3⬘
R: 5⬘-AACTCCRTAAAACRCCTAAATC-3⬘
MINT: methylated in tumor loci; F: forward primer; R: reverse primer.
* Y: C or T; R: A or G.
Annealing Temperature in °C (No. of Cycles)
Restriction Enzyme
55 (35)
Taq I
60 (3), 58 (4), 56 (5), 54 (26)
BstU I
64 (3), 61 (4), 58 (5), 55 (26)
Tai I
58 (3), 56 (4), 54 (5), 52 (26)
BstU I
58 (3), 56 (4), 54 (5), 52 (26)
BstU I
58 (3), 56 (4), 54 (5), 52 (28)
Taq I
58 (3), 56 (4), 54 (5), 52 (26)
Afa I
55 (3), 53 (4), 51 (5), 49 (26)
Nru I
58 (3), 56 (4), 54 (5), 52 (26)
Taq I
58 (3), 56 (4), 54 (5), 52 (26)
Nru I
58 (3), 56 (4), 54 (5), 52 (26)
BspT104 I
58 (3), 56 (4), 54 (5), 52 (26)
Taq I
58 (3), 56 (4), 54 (5), 52 (26)
EcoR I
58 (3), 56 (4), 54 (5), 52 (26)
Hinf I
58 (3), 56 (4), 54 (5), 52 (26)
EcoR I
53 (35)
Afa I
58 (3), 56 (4), 54 (5), 52 (26)
Taq I
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CANCER April 1, 2006 / Volume 106 / Number 7
2.5% agarose gels, stained with ethidium bromide, and
examined for methylation density using Lane & Spot
Analyzer (version 6.0 for Windows; ATTO, Tokyo, Japan).
Detection of the EBV Genome and HP
To detect the EBV genome in gastric tumors, we performed real-time PCR using 2 sets of primers that
detect BamHI-W and the EBV-encoded protein EBNA,
as described previously.21 Both fluorescent probes
contained a 3⬘-blocking phosphate group to prevent
their extension during PCR. A calibration curve was
run in parallel using DNA extracted from the EBVpositive Raji cell line (American Type Culture Collection) as a standard. Consistent results were obtained
with both systems. HP infection was identified by conducting histologic review of hematoxylin and eosinstained tissue specimens and PCR assays. A patient
was classified as histopathologically HP-positive on
the basis of the presence of curved, rod-shaped bacteria on the tumor neighboring and/or antral gastric
mucosa. Genomic DNA from normal mucosa was analyzed using PCR with primers that were derived from
the internal 411-base-pair fragment of the urease A
gene, as described by Clayton et al.22
indicate moderate correspondence, values from 0.61
to 0.8 indicate substantial correspondence, and values
⬎ 0.8 indicate near perfect correspondence. The hypothesis that ␬ ⫽ 0.0 was tested using the exact test.
Each tumor was classified using tumor location, macroscopic type; lymphatic invasion; venous invasion
(Japanese Gastric Cancer Association, 1998)24; the
pathologic tumor, lymph node, metastasis (pTNM)
classification (5th edition, 1997)25; and the Lauren
classification.26 CIMP status was compared using the
Student t test for age; the Mann–Whitney U test for
tumor size, pT status, pN status, and disease stage;
and the Fisher exact test for gender, tumor location,
macroscopic type, histology, lymphatic invasion, venous invasion, pM status, EBV association, lymphoepithelioma-like carcinoma, HP status, and p53 and Kras mutation. Survival was assessed using the Kaplan–
Meier method; survival curves were compared using
the log-rank test. The Fisher exact test was carried out
using SAS (SAS Institute Inc., Cary, NC), and all other
statistical analyses (indicated in the text) were carried
out using SPSS software (version 11.0; SPSS Inc., Chicago, IL). All tests were 2-tailed, and values of P ⬍ .05
were considered significant.
Mutational Analysis
RESULTS
Mutations in codon 12 or 13 of K-ras were detected by
direct sequencing of PCR products after the amplification of K-ras exon 2. Mutations of p53 were detected
by single-strand conformation polymorphism followed by direct sequencing. Genomic DNA was amplified using exon-specific primers for p53 exons 2
through 11; the primer sequences were obtained from
the literature23 and were used with only minor modification. The mutations that were identified were examined using a Genephor Electrophoresis System
(Amersham Biosciences, Uppsala, Sweden) and a DNA
Silver Staining Kit (Amersham Biosciences). Shifted
bands were excised from gels and reamplified using
the same sets of primers. The resultant PCR products
were purified using a DNA Purification System (Promega); then, the mutated sequences were determined
by direct sequencing using a Big Dye Terminator v3.1
Cycle Sequencing Kit with an ABI PRISM 3100 Genetic
Analyzer (Applied Biosystems, Foster City, CA).
Gene Methylation and CIMP Status in Gastric Carcinoma
Statistical Analysis
The ␬ statistic was used to describe the correspondence between tumors that had high CIMP methylation (CIMP-H) or EBV-association and methylation
of each of the 12 tumor-related genes studied. It is
accepted generally that ␬ values from 0.0 to 0.4 indicate poor correspondence, values from 0.41 to 0.6
Of the 12 tumor-related genes that were studied, 8
genes were selected for the current analysis based on
the finding that their methylation occurs exclusively in
cancerous tissues and not in adjacent normal gastric
mucosa. These included p16INK4A,4 the human MutL
homologue 1 gene (hMLH1),4 the p57 cyclin-dependent kinase 2 inhibitor gene (p57KIP2),27 the helicaselike transcription factor gene (HLTF),28 the mitotic
checkpoint gene CHFR,29 the human harakiri gene
(HRK),30 the solute carrier family 5 (iodine transporter) member 8 gene (SLC5A8),31 and the BCL2/
adenovirus E1B 19-kDa interacting protein 3 gene
(BNIP3),32 all of which are inactivated by methylation
in gastric carcinoma. The other 4 genes that were
studied, CSPG2,16 paired box gene 5␤ (PAX5␤),33 suppressor of cytokine signaling 3 gene (SOCS-3),34 and
tazarotene-induced gene 1 (TIG1),17 all have been
shown to be methylated in various human malignancies other than gastric carcinoma. We deemed that a
gene was methylated if it showed a methylation density ⱖ 10%. No methylation was detected in normal
mucosa adjacent to tumors. Confirmation of methylation-induced silencing of affected genes in gastric
carcinoma cell lines was followed by methylation
analysis of the primary tumors. Representative results
that show the methylation and expression of CSPG2
Gastric CA with CIMP and Association to EBV/Kusano et al.
1471
ation profiles and the EBV and HP status of the 78
gastric tumors studied are summarized in Figure 2.
CIMP Status and the Frequency of Methylation of TumorRelated Genes
FIGURE 1. Methylation and expression of the chondroitin sulfate proteoglycan 2 gene (CSPG2) and tazarotene-induced gene 1 (TIG1) are shown in gastric
carcinoma cell lines and primary gastric carcinomas. (A) For reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CSPG2 and TIG1,
PCR was carried out using samples that were prepared with (RT ⫹) or without
(RT ⫺) reverse transcriptase. Glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) was amplified to confirm the quality of the cyclic DNA. (B) Reexpression of CSPG2 and TIG1 is shown after treatment with the methyltransferase
inhibitor 5-aza-2⬘-deoxycytidine (5-aza-dC). Expression was restored in methylated cells after treatment with 2 ␮M 5-aza-dC for 72 hours. (C) Representative results are shown from an analysis of CSPG2 and TIG1 methylation. The
calculated percentage of DNA methylation is indicated below each lane.
Primary gastric carcinomas were considered methylation-positive when the
percentage of digested DNA was ⱖ 10%. Cell lines and tumors are indicated
above the data. The genes that were analyzed are shown on the left. M:
methylated alleles; N: gastric normal mucosa adjacent to tumors; T: gastric
tumors.
and TIG1 in various gastric carcinoma cell lines and
primary gastric carcinomas are depicted in Figure 1.
To evaluate CIMP status, we classified gastric tumors, first, based on the presence or absence of methylation within each of the five MINT loci and, second,
based on the numbers of methylated loci—i.e., tumors
were classified as CIMP-H (4 or 5 MINT loci showed
methylation), CIMP-low (CIMP-L) (from 1 to 3 MINT
loci showed methylation), or CIMP-negative (CIMP-N)
(no MINT loci showed methylation). Of the 78 primary
gastric tumors studied, 19 tumors were classified as
CIMP-H, 39 tumors were classified as CIMP-L, 20 tumors were classified as CIMP-N, and the frequency of
MINT locus methylation ranged from 23.1% (MINT12)
to 64.1% (MINT25). In addition, we determined the
methylation status of 12 tumor-related genes and
found that the frequency of their methylation ranged
from 6.4% (hMLH1) to 48.7% (CSPG2). The methyl-
Figure 3 shows that the frequency of methylation in 11
of 12 tumor-related genes studied was much greater in
CIMP-H tumors compared with the frequency of
methylation in CIMP-L or CIMP-N tumors. The exception was SOCS-3, which was methylated slightly more
frequently in CIMP-L tumors than in CIMP-H tumors.
To calculate the expected number of tumors that
would arise for a given number of methylated loci
(from 0 to 12 loci), we created a web-based program,
“Tool for Gene Methyl Possibility” (TGMP; available at
URL: http://info.bio.sunysb.edu/methyl.html) [accessed
February 2006]), which worked on the assumption
that methylation of individual genes occurs independent of the methylation of other genes. We then compared the observed distribution of tumors that had
each number of methylated loci with the expected
distribution among our 78 tumor specimens; the expected distribution was calculated using the TGMP
program based on the methylation frequency of the
aforementioned 12 genes (Fig. 4). In this comparison,
tumors with five or more methylated loci were pooled
into one group, because the number of these tumors
in the expected distribution was less than five. There
were no tumors with 7, 11, or 12 methylated loci in the
observed distribution. The expected distribution was
unimodal, with a peak at 2 loci per tumor occurring in
22.5 tumors. By contrast, the observed distribution
was not unimodal and differed significantly from the
expected distribution (goodness-of-fit test: chi-square
statistic, 92.576; 5 degrees of freedom; P ⬍ .0005).
To evaluate the correlation between the number
of methylated MINT loci (CIMP status) and the number of methylated loci among the 12 tumor-related
genes in more detail, the 78 tumor specimens first
were divided into 6 groups based on the number of
methylated MINT loci (from 0 to 5 loci) in each tumor.
Then, which the average numbers of methylated tumor-related genes were determined (Fig. 5). One-way
analysis of variance (ANOVA) showed that the numbers of methylated tumor-related genes were affected
significantly by the numbers of methylated MINT loci
(F[5, 72] ⫽ 16.376; P ⬍ .0005), and a post-hoc Tukey
test showed that, in tumors that had 4 or 5 methylated
MINT loci, there also were significantly greater numbers of methylated loci among the 12 tumor-related
genes than among tumors that had fewer methylated
MINT loci. The ␬ statistic, which indicated the degree
of correspondence between CIMP-H and methylation
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CANCER April 1, 2006 / Volume 106 / Number 7
FIGURE 2. These profiles characterize methylation of the 5 methylated-in-tumors (MINT) loci and 12 tumor-related genes as well as the Epstein–Barr virus (EBV),
Helicobacter pylori (H. pylori), and p53 and K-ras mutation status in 78 gastric carcinomas. Methylated MINT loci and loci from the 12 tumor-related genes
(chondroitin sulfate proteoglycan-2 gene [CSPG2], BCL2/adenovirus E1B 19-kDa interacting protein 3 gene; [BNIP3], mitotic checkpoint gene [CHFR], p16 cyclin
dependent kinase 4A inhibitor gene [p16INK4A], helicase-like transcription factor gene [HLTF], paired box gene 5␤ [PAX5␤], human harakiri gene [HRK], solute carrier
family 5 [iodine transporter] member 8 gene [SLC5A8], tazarotene-induced gene 1 [TIG1], p57 cyclin-dependent kinase 2 inhibitor gene [p57KIP2], human MutL
homologue 1 [hMLH1], and suppressor of cytokine signaling 3 gene [SOCS]) are shown in blue and green, respectively; the presence of EBV and lymphoepitheliomalike carcinoma (LELC) are indicated by charcoal gray and light gray, respectively; mutations of K-ras and p53 are indicated by purple, and the presence of H. pylori
detected by polymerase chain reaction (PCR) analysis and hematoxylin and eosin (H&E) staining is indicated by light brown. CIMP-H: high CpG island methylator
phenotype (CIMP) methylation; CIMP-L: low CIMP methylation; CIMP-N: negative CIMP methylation.
of the 12 genes, varied from 0.006 to 0.544 (Table 2).
Correspondence was moderate for the HRK, HLTF,
BNIP3, p16INK4A, TIG1, and PAX5␤ genes but was poor
for the other 6 genes. In addition, we evaluated the
correspondence between EBV association and gene
methylation (Table 2) and found that it was substantial for p16INK4A and TIG1 and moderate for p57KIP2,
HLTF, PAX5␤, and HRK.
CIMP Status and the Frequency of EBV Association and
Genetic Alteration of p53 and K-ras
EBV was detected in 10 of 78 tumors (12.8%) using
real-time quantitative PCR. Mutations in p53 were
found in 19 of 78 tumors (24.4%), including 14 tumors
that contained single point mutations between exons
4 and 10 and 4 tumors that contained from 1 to 21
base pair deletions between exons 4 and 7. K-ras mutations were detected at codon 12 in 4 of 78 gastric
tumors, a GGT 3 GAT substitution was found in 3
tumors, and a GGT 3 GTT substitution was found in 1
tumor. No K-ras mutations were detected at codon 13.
The frequencies of EBV association, p53 mutation, and
K-ras mutation in the 78 gastric tumors are summarized in Figure 2. The median number of methylated
loci of the 12 tumor-related genes was significantly
greater in EBV-associated gastric tumors than in EBVnegative gastric tumors (7 loci vs. 1 locus; P ⬍.0005;
Mann–Whitney U test). All EBV-associated tumors be-
Gastric CA with CIMP and Association to EBV/Kusano et al.
1473
FIGURE 3. Methylation frequency among 12 tumor-related genes in gastric
carcinomas (chondroitin sulfate proteoglycan-2 gene [CSPG2], BCL2/adenovirus E1B 19-kDa interacting protein 3 gene; [BNIP3], mitotic checkpoint gene
[CHFR], p16 cyclin dependent kinase 4A inhibitor gene [p16INK4A], helicase-like
transcription factor gene [HLTF], paired box gene 5␤ [PAX5␤], human harakiri
gene [HRK], solute carrier family 5 [iodine transporter] member 8 gene
[SLC5A8], tazarotene-induced gene 1 [TIG1], p57 cyclin-dependent kinase 2
inhibitor gene [p57KIP2], human MutL homologue 1 [hMLH1], and suppressor of
cytokine signaling 3 gene [SOCS]). Gastric carcinomas were classified as high
CpG island methylator phenotype (CIMP) methylation (CIMP-H) (yellow column;
methylation of 4 or 5 methylated-in-tumors [MINT] loci), low CIMP methylation
(CIMP-L) (red column; methylation of 1, 2, or 3 MINT loci), or negative CIMP
methylation (CIMP-N) (blue column; no MINT methylation). The corresponding
gene is indicated below each row of bars.
FIGURE 5. The average numbers of methylated loci are shown among 12
tumor-related genes in 78 gastric carcinomas with the indicated numbers (0-5)
of methylated-in-tumors (MINT) loci. A 1-way analysis of variance followed by
a Tukey test showed that tumors with 4 or 5 methylated MINT loci differed
significantly from tumors with 0 (*P ⬍ .0005; **P ⬍ .0005), 1 (*P ⫽ .001; **P
⬍ .0005), 2 (*P ⬍ .005; **P ⬍ .0005) and 3 (*P ⬍ .05; **P ⬍ .0005)
methylated MINT loci.
longed to the CIMP-H group, with 10 of 19 CIMP-H
tumors (52.6%) showing an EBV-association. Tumors
with p53 and/or K-ras mutations showed neither
hMLH1 methylation nor EBV association, with the
exception of 1 tumor (KG12): In other words, these
genetic alterations almost never occurred together
with hMLH1 methylation or EBV association. The correlation between CIMP status and genetic alteration of
the p53 and K-ras genes is summarized in Table 3. No
p53 mutations were detected among CIMP-H tumors,
whereas 19 of 59 CIMP-L/CIMP-N tumors (32.2%)
showed p53 mutation (P ⫽ .004). Conversely, no significant difference was noted in the frequency of K-ras
mutation between CIMP-H tumors and CIMP-L/
CIMP-N tumors (P ⫽ .248).
CIMP Status and Clinicopathologic Characteristics
FIGURE 4. This chart illustrates a comparison of the observed and expected
numbers of tumors containing the indicated number of methylated loci among
12 tumor-related genes in 78 gastric carcinomas. Vertical bars indicate the
observed numbers of tumors that contained the indicated number of methylated loci. Solid lines indicate the expected numbers of tumors that were
calculated using the “Tool for Gene Methyl Possibility” program (available at
URL: http://info.bio.sunysb.edu/methyl.html) based on the individual methylation frequencies of the 12 genes. The observed distribution differed significantly from the expected distribution (chi-square statistic, 92.576; 5 degrees of
freedom; P ⬍ .0005).
Bearing in mind the results described above, our objective was to gain a better understanding of the clinicopathologic characteristics of CIMP by comparing
the characteristics of patients in the CIMP-H group
with the characteristics of patients in a combined
CIMP-L/CIMP-N group. Univariate analysis revealed
no differences between the CIMP-H and CIMP-L/
CIMP-N groups with respect to age, gender, tumor
size, macroscopic type, or pT status, pN status, or pM
status. There were significant differences between patients sin the CIMP-H and CIMP-L/CIMP-N groups
with respect to tumor location (P ⫽ .005), histology
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CANCER April 1, 2006 / Volume 106 / Number 7
TABLE 2
The ␬ Statistic for Agreement of Gene Methylation with High CpG
Island Methylator Phenotype Expression and with
Epstein–Barr Virus Association
Agreement with CIMP-H
Agreement with EBV
Gene
␬
P
␬
P
HRK
HLTF
BNIP3
p16INK4A
TIG1
PAX5␤
CHFR
CSPG2
P57KIP2
hMLH1
SLC5A8
SOCS-3
0.544
0.532
0.528
0.503
0.469
0.423
0.351
0.350
0.321
0.165
0.155
0.006
⬍ .0005
⬍ .0005
⬍ .0005
⬍ .0005
⬍ .0005
⬍ .0005
.002
⬍ .0005
⬍ .0005
.055
.170
.955
0.426
0.542
0.271
0.706
0.671
0.426
0.250
0.164
0.585
⫺ 0.093
0.269
⫺ 0.163
⬍ .0005
⬍ .0005
.003
⬍ .0005
⬍ .0005
⬍ .0005
.008
.034
⬍ .0005
.375
.013
.149
CIMP-H high CpG island methylator phenotype methylation; EBV: Epstein–Barr virus; HRK: human
harakiri gene; HLTF: helicase-like transcription factor gene; BNIP3: BCL2/adenovirus E1B 19-kDa
interacting protein 3 gene; p16INK4A: p16 cyclin dependent kinase 4A inhibitor gene; TIG1: tazaroteneinduced gene 1; PAX5␤: paired box gene 5␤; CHFR: mitotic checkpoint gene; CSPG2: chondroitin sulfate
proteoglycan-2 gene; p57KIP2: p57 cyclin-dependent kinase 2 inhibitor gene; hMLH1: human MutL
homologue 1 gene; SLC5A8: solute carrier family 5 (iodine transporter) member 8 gene; SOCS-3:
suppressor of cytokine signaling 3 gene.
(P ⫽ .030), venous invasion (P ⫽ .009), pTNM stage
(P ⫽ .034), EBV status (P ⬍ .0005), and p53 mutation
status (P ⫽ .004) (Table 3). In a multiple logistic regression model in which CIMP-H was the dependent
variable, the clinicopathologic features, including tumor location, tumor histology, venous invasion, and
pTNM stage, were investigated as independent variables. Proximal location (P ⫽ .011), diffuse type tumors (P ⫽ .018), and less advanced pTNM stages
(P ⫽ .037) were selected as significant variables (Table
4). Because there were no CIMP-H tumors with p53
mutations and no EBV-positive CIMP-L/CIMP-N tumors, it was impossible to include p53 mutation and
EBV association as variables in the current analysis,
although both were significant in the univariate analysis. The comparison of EBV-positive and EBV-negative tumors in the CIMP-H group is summarized in
Table 5. There were five tumors that showed lymphoepithelioma-like carcinoma, and all were EBV-associated tumors (Fig. 6).
Survival among patients with CIMP-H, CIMP-L,
and CIMP-N tumors was characterized using the
Kaplan–Meier method and was compared using the
log-rank test (Fig. 7A). Values of P ⬍ .016 were considered significant based on the Bonferroni correction
for comparison between 2 different groups. Patients
with CIMP-H gastric tumors tended to survive longer
than patients with CIMP-L gastric tumors, but the
difference was not significant (P ⫽ .350). Patients who
had CIMP-N gastric tumors showed significantly
worse survival than patients with CIMP-H tumors
(P ⫽ .004) or patients with CIMP-L tumors (P ⫽ .012).
There was no difference in survival between patients
with CIMP-H/EBV-positive tumors and CIMP-H/EBVnegative tumors (Fig. 7B).
DISCUSSION
It is noteworthy that the term CIMP has had a variety
of usages in the context of gastric carcinoma. Its original definition was based on quantitative techniques
that were relatively insensitive to methylation levels in
normal mucosa. However, careful quantitative evaluation showed that many genes that are highly methylated in carcinoma also show a low but measurable
degree of methylation in normal mucosa.35 Conversely, in studies using MSP, which is a nonquantitative and highly sensitive method, the incidence of
methylation was substantially higher. For instance,
the frequency of p16INK4A methylation in gastric carcinoma detected using MSP was reportedly 27%,36
33%,37 or 42%,38 rates much higher than what we
obtained in the current study by using COBRA.
We compared the observed distribution of tumors
that carried each number of methylated tumor-related
genes with the expected distribution, which was calculated using our TGMP program. If methylation of
these genes occurred independently of one another,
then the observed distribution should have resembled
the expected distribution. Our finding of a significant
difference between the observed and expected distributions suggests that promoter methylation of tumorrelated genes in gastric tumors is not a random occurrence. In that regard, we defined CIMP status on the
basis of the degree of methylation in the five MINT
loci. One-way ANOVAs showed that tumors with 4 or 5
methylated MINT loci (CIMP-H) had significantly
greater numbers of methylated loci among the 12 tumor-related genes that among tumors that had fewer
methylated MINT loci (CIMP-L or CIMP-N), a finding
that validated our operational definition of the threshold between CIMP-H tumors and CIMP-L tumors (between 3 and 4 methylated MINT loci).
We observed no significant difference in the age
or gender of patients with CIMP-H tumors and patients with CIMP-L/CIMP-N tumors. The strong association between CIMP-H and EBV-associated gastric
carcinoma appears to have influenced the result of the
multiple logistic regression model, because diffuse
type tumors and a proximal location were selected as
significant variables for CIMP-H tumors. Evaluation of
the correspondence between EBV association and
methylation of the 12 genes studied showed the high-
TABLE 3
Clinicopathologic Features of Gastric Carcinomas with High CpG Island Methylator Phenotype Expression and Low or Negative CpG Island
Methylator Phenotype Expression: Univariate Analysis
No. of Patients (%)
Characteristic
Total
CIMP-H
CIMP-L/CIMP-N
P
No. of patients
Mean age ⫾ SD (yrs)
Gender
Male
Female
Tumor size (cm)
Range
Median
Gastric tumor location
Upper one-third
Middle one-third
Lower one-third
Macroscopic type
Type 0
Type 1
Type 2
Type 3
Type 4
Histology (Lauren)
Intestinal
Diffuse
Lymphatic invasion
Negative
Positive
Venous invasion
Negative
Positive
Pathologic tumor classification
pT1
pT2
pT3
pT4
Pathologic lymph node status
pN0
pN1
pN2
pN3
Pathologic metastasis status
pM0
pM1
Stage (pTNM)
Stage IA
Stage IB
Stage II
Stage IIIA
Stage IIIB
Stage IV
Epstein–Barr virus
Positive
Negative
p53 mutation
Positive
Negative
K-ras mutation
Positive
Negative
Helicobacter pylori
Positive
Negative
78
64.5 ⫾ 12.0
19
65.9 ⫾ 13.3
59
64.0 ⫾ 11.6
52 (66.7)
26 (33.3)
12 (63.2)
7 (36.8)
40 (67.8)
19 (32.2)
.782
2.3-21.0
7.0
2.3-20.5
7.0
2.3-21.0
7.5
.709
22 (28.2)
23 (29.5)
33 (42.3)
8 (42.1)
7 (36.8)
4 (21.1)
14 (23.7)
16 (27.1)
29 (49.2)
.005
4 (5.1)
6 (7.7)
30 (38.5)
30 (38.5)
8 (10.2)
3 (15.8)
1 (5.3)
9 (47.3)
5 (26.3)
1 (5.3)
1 (1.7)
5 (8.5)
21 (35.6)
25 (42.3)
7 (11.9)
.122
36 (46.2)
42 (53.8)
5 (26.3)
14 (73.7)
31 (52.5)
28 (47.5)
.030
20 (25.6)
58 (74.4)
7 (36.8)
12 (63.2)
13 (22.0)
46 (78.0)
.233
37 (47.4)
41 (52.6)
14 (73.7)
5 (26.3)
23 (39.0)
36 (61.0)
.009
5 (6.4)
43 (55.1)
28 (35.9)
2 (2.6)
3 (15.8)
10 (52.6)
6 (31.6)
0 (0.0)
2 (3.4)
33 (55.9)
22 (37.3)
2 (3.4)
.205
22 (28.2)
28 (35.9)
16 (20.5)
12 (15.4)
7 (36.8)
8 (42.1)
3 (15.8)
1 (5.3)
15 (25.4)
20 (33.9)
13 (22.0)
11 (18.7)
.114
66 (84.6)
12 (15.4)
18 (94.7)
1 (5.3)
48 (81.4)
11 (18.6)
.274
3 (3.8)
15 (19.2)
16 (20.5)
13 (16.7)
8 (10.3)
23 (29.5)
3 (15.8)
3 (15.8)
5 (26.3)
4 (21.1)
2 (10.5)
2 (10.5)
0 (0.0)
12 (20.3)
11 (18.6)
9 (15.3)
6 (10.2)
21 (35.6)
.034
10 (12.8)
68 (87.2)
10 (52.6)
9 (47.4)
0 (0.0)
59(100.0)
⬍ .0005
19 (24.4)
59 (75.6)
0 (0.0)
19(100.0)
19 (32.2)
40 (67.8)
.004
4 (5.1)
74 (94.9)
2 (10.5)
17 (89.5)
2 (3.4)
57 (96.6)
.248
65 (83.3)
13 (16.7)
17 (89.5)
2 (10.5)
48 (81.4)
11 (18.6)
.778
.580
CIMP-H: high CpG island methylator phenotype methylation; CIMP-L/CIMP-N: low/negative CpG island methylator phenotype methylation; SD: standard deviation; pTNM: pathologic tumor, lymph node,
metastasis status according to the International Union Against Cancer classification system.
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CANCER April 1, 2006 / Volume 106 / Number 7
TABLE 4
Multiple Logistic Regression Model of High CpG Island Methylator Phenotype Using Clinicopathologic
Features in Gastric Carcinoma
Variable
Category
␤
P
Gastric tumor location
Histology (Lauren)
Venous invasion
Stage (pTNM)
Upper one-third/middle one-third/lower one-third
Intestinal/diffuse
Negative/positive
Stage IA/Stage IB/Stage II/Stage IIIA/Stage IIIB/Stage IV
⫺ 1.035
1.635
⫺ 1.242
⫺ 0.529
.011
.018
.098
.037
␤: logistic regression coefficient; pTNM: pathologic Tumor, Lymph Node, Metastasis status according to the International Union Against Cancer classification
system.
TABLE 5
Comparison between High CpG Island Methylator Phenotype
Expressing/Epstein–Barr Virus-Positive Tumors and High CpG Island
Methylator Phenotype Expressing/Epstein–Barr Virus-Negative
Tumors
CIMP-H
Characteristic
EBV-Positive
EBV-Negative
Total no. of patients
Mean age ⫾ SD, y
Gender
Male
Female
Gastric tumor location
Upper third
Middle third
Lower third
Histology (Lauren)
Intestinal
Diffuse
Stage (pTNM)
Stage IA
Stage IB
Stage II
Stage IIIA
Stage IIIB
Stage IV
LELC
Positive
Negative
Helicobacter pylori
Positive
Negative
10
65.9 ⫾ 13.3
9
64.0 ⫾ 11.6
7
3
5
4
6
4
0
2
3
4
1
9
5
4
1
2
3
2
1
1
2
1
2
2
1
1
5
5
0
9
9
1
8
1
P
.497
.650
.061
.057
.934
.033
.999
CIMP-H: high CpG island methylator phenotype methylation; EBV: Epstein–Barr virus; SD: standard
deviation; pTNM: pathologic Tumor: Lymph Node, Metastasis status according to the International
Union Against Cancer classification system; LELC: lymphoepithelioma-like carcinoma.
est correspondence with the p16INK4A gene (␬ ⫽ 0.706):
The product of that gene is an inhibitor of G1/S phase
transition, the loss of which promotes uncontrolled
cell growth. The p16INK4A gene is a common target of
inactivation by epigenetic mechanisms in gastric carcinoma.4,39-41 In our current results, p16INK4A was
methylated frequently in CIMP-H tumors (52.6%) but
rarely was methylated in CIMP-L/CIMP-N tumors
(6.8%). It is worth noting that the frequency of
p16INK4A methylation in EBV-associated tumors was
remarkably high (90.0%). Thus, it appears that epigenetic silencing of this gene is associated strongly with
the development of EBV-associated gastric carcinoma.
Approximately 50% of CIMP-H gastric carcinomas
are EBV-negative; presumably, CIMP-H tumors without EBV association are mediated by different, as yet
unknown mechanisms. Compared with EBV-positive
CIMP-H tumors, it appeared that EBV-negative
CIMP-H tumors were less likely to be diffuse carcinomas or to be located in the upper one-third of the
stomach, although there was no meaningful prognostic difference between the two groups. No lymphoepithelioma-like carcinomas were detected among the
EBV-negative tumors. A subset of CIMP gastric tumors
are microsatellite instability-positive tumors because
of hMLH1 methylation.4,5 Kang et al. reported that
EBV-positive gastric tumors are subset of CIMP-positive tumors, although only 2 of 21 EBV-positive tumors
(9.5%) showed hMLH1 methylation.11 This is consistent with our finding that the carcinogenesis of EBVassociated gastric tumors commonly involves hypermethylation of multiple genes without involvement of
hMLH1 methylation.
It is plausible that EBV may activate a methylation
pathway that affects multiple genes during gastric carcinogenesis; however, the molecular mechanism underlying EBV-related aberrant methylation currently is
unknown. Consistent with the idea that oncogenic
viruses induce aberrant methylation of tumor suppressor genes, Shivapurkar et al. reported that methylation was completely absent in Simian virus 40
(SV40)-uninfected and EBV-infected peripheral blood
mononuclear lymphocytes but that the presence of
SV40 in hematologic malignancies was associated with
promoter methylation of tumor suppressor genes.42
Moreover, Soejima et al. reported that malignant
transformation of normal human bronchial epithelial
cells that expressed telomerase, SV40 large-T antigen,
Gastric CA with CIMP and Association to EBV/Kusano et al.
1477
FIGURE 6.
This photomicrograph shows a representative lymphoepithelioma-like carcinoma (Patient KG223). Diffuse infiltrating nests of undifferentiated
carcinoma cells are surrounded by a uniformly dense and diffuse lymphoid cell
infiltration (hematoxylin and eosin stain; original magnification, ⫻ 50).
and activated Ras led to DNA methyltransferase 3b
expression, which was correlated with the methylation
and down-regulation of tumor suppressor genes.43
Determining precisely how EBV infection leads to
methylation in gastric carcinogenesis seems to be a
key question to be addressed in future studies.
Previous studies identified differences in the frequency of p53 mutation in EBV-associated malignancies, including Burkitt lymphoma, posttransplantation
lymphoma, and nasopharyngeal carcinoma.44-47 Consistent with the finding that p53 is overexpressed infrequently in EBV-associated gastric carcinomas,48,49
no p53 mutations were detected among the 10 EBVassociated gastric tumors we studied, all of which were
classified as CIMP-H, suggesting that an EBV viral
gene product may interfere with some functions of
p53 by eliminating a section for mutational inactivation. Szekely et al. reported that, on binding in vitro,
the EBV-encoded protein EBNA-5 (alternatively designated EBNA-LP) forms a molecular complex with both
p53 and retinoblastoma (Rb) proteins, thereby inactivating the tumor suppressor pathway.50 This means
that, in EBV-associated gastric carcinoma, the p53/Rb
pathway can be inactivated without mutation of the
p53 gene. Still, 42 of 78 tumor specimens (53.8%) in
the current study showed no EBV association, no
methylation of hMLH1, and no genetic alterations of
the p53 or K-ras genes. Thus, these traits do not explain the mechanism of carcinogenesis in approximately 50% of our 78 tumors.
Finally, our clinicopathologic study using multivariate analysis revealed that less advanced pTNM
stages contributed significantly to CIMP-H. Although
FIGURE 7. (A) Survival among patients who had tumors with high CpG island
methylator phenotype (CIMP) methylation (CIMP-H), low CIMP methylation
(CIMP-L), and negative CIMP methylation (CIMP-N) was assessed using the
Kaplan–Meier method. Survival curves were compared using the log-rank test
(CIMP-H vs. CIMP-L, P ⫽ .350; CIMP-L vs. CIMP-N, P ⫽ .012; CIMP-H vs.
CIMP-N, P ⫽ .004). (B) There was no meaningful difference in survival between
patients with Epstein–Barr virus (EBV)-positive tumors and patients with EBVnegative CIMP-H tumors.
venous invasion differed significantly between CIMPH and CIMP-L/CIMP-N in univariate analysis, it was
not selected as a significant variable in the multiple
logistic regression model of CIMP-H, because positive
venous invasion was linked strongly to advanced
pTNM stages. Furthermore, among the 20 tumors that
were classified as CIMP-N, 6 tumors (30.0%) showed
distant metastasis to either the liver or peritoneum at
the time of resection, whereas 5 of 39 CIMP-L tumors
(12.8%) and only 1 of 19 CIMP-H tumors (5.3%)
showed such distant metastasis. Thus, the clinical
characteristics of CIMP-H, CIMP-L, and CIMP-N gastric carcinomas appear to differ. An interesting ques-
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CANCER April 1, 2006 / Volume 106 / Number 7
tion in this regard is whether patients who have hypomethylated tumors tend to present at more
advanced stages and, thus, have poorer prognoses.
Because the answer to that question is affirmative, the
five MINT loci may be useful not only as hypermethylation markers but also as hypomethylation markers
and may serve as valuable prognostic markers that
facilitate decisions about whether chemotherapy is
indicated for an individual patient.
17.
18.
19.
REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Hohenberger P, Gretschel S. Gastric cancer. Lancet. 2003;
362:305-315.
Feltus FA, Lee EK, Costello JF, Plass C, Vertino PM. Predicting aberrant CpG island methylation. Proc Natl Acad Sci
USA. 2003;100:12253-12258.
Jones PA, Baylin SB. The fundamental role of epigenetic
events in cancer. Nat Rev Genet. 2002;3:415-428.
Toyota M, Ahuja N, Suzuki H, et al. Aberrant methylation in
gastric cancer associated with the CpG island methylator
phenotype. Cancer Res. 1999;59:5438-5442.
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. 1999;96:8681-8686.
Toyota M, Ohe-Toyota M, Ahuja N, Issa JP. Distinct genetic
profiles in colorectal tumors with or without the CpG island
methylator phenotype. Proc Natl Acad Sci USA. 2000;97:710715.
An C, Choi IS, Yao JC, et al. Prognostic significance of CpG
island methylator phenotype and microsatellite instability
in gastric carcinoma. Clin Cancer Res. 2005;11:656-663.
Parsonnet J, Friedman GD, Vandersteen DP, et al. Helicobacter pylori infection and the risk of gastric carcinoma.
N Engl J Med. 1991;325:1127-1131.
Schneider BG, Gulley ML, Eagan P, Bravo JC, Mera R,
Geradts J. Loss of p16/CDKN2A tumor suppressor protein in
gastric adenocarcinoma is associated with Epstein-Barr virus and anatomic location in the body of the stomach. Hum
Pathol. 2000;31:45-50.
Lee HS, Chang MS, Yang HK, Lee BL, Kim WH. Epstein-Barr
virus-positive gastric carcinoma has a distinct protein expression profile in comparison with Epstein-Barr virus-negative carcinoma. Clin Cancer Res. 2004;10:1698-1705.
Kang GH, Lee S, Kim WH, et al. Epstein-Barr virus-positive
gastric carcinoma demonstrates frequent aberrant methylation of multiple genes and constitutes CpG island methylator phenotype-positive gastric carcinoma. Am J Pathol.
2002;160:787-794.
Remus R, Kammer C, Heller H, Schmitz B, Schell G, Doerfler
W. Insertion of foreign DNA into an established mammalian
genome can alter the methylation of cellular DNA sequences. J Virol. 1999;73:1010-1022.
Toyooka S, Pass HI, Shivapurkar N, et al. Aberrant methylation and simian virus 40 tag sequences in malignant mesothelioma. Cancer Res. 2001;61:5727-5730.
Maehara Y, Tomoda M, Hasuda S, et al. Prognostic value of
p53 protein expression for patients with gastric cancer—a
multivariate analysis. Br J Cancer. 1999;79:1255-1261.
Nanus DM, Kelsen DP, Mentle IR, Altorki N, Albino AP.
Infrequent point mutations of ras oncogenes in gastric cancers. Gastroenterology. 1990;98:955-960.
Toyota M, Ho C, Ahuja N, et al. Identification of differen-
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
tially methylated sequences in colorectal cancer by methylated CpG island amplification. Cancer Res. 1999;59:23072312.
Youssef EM, Chen XQ, Higuchi E, et al. Hypermethylation
and silencing of the putative tumor suppressor Tazaroteneinduced gene 1 in human cancers. Cancer Res. 2004;64:24112417.
Suzuki H, Itoh F, Toyota M, Kikuchi T, Kakiuchi H, Imai K.
Inactivation of the 14-3-3 ␴ gene is associated with 5⬘ CpG
island hypermethylation in human cancers. Cancer Res.
2000;60:4353-4357.
Xiong Z, Laird PW. COBRA: a sensitive and quantitative DNA
methylation assay. Nucleic Acids Res. 1997;25:2532-2534.
Clark SJ, Harrison J, Paul CL, Frommer M. High sensitivity
mapping of methylated cytosines. Nucleic Acids Res. 1994;
22:2990-2997.
Lo YM, Chan LY, Lo KW, et al. Quantitative analysis of
cell-free Epstein-Barr virus DNA in plasma of patients with
nasopharyngeal carcinoma. Cancer Res. 1999;59:1188-1191.
Clayton CL, Kleanthous H, Coates PJ, Morgan DD,
Tabaqchali S. Sensitive detection of Helicobacter pylori by
using polymerase chain reaction. J Clin Microbiol. 1992;30:
192-200.
Rhei E, Bogomolniy F, Federici MG, et al. Molecular genetic
characterization of BRCA1- and BRCA2-linked hereditary
ovarian cancers. Cancer Res. 1998;58:3193-3196.
Japanese Gastric Cancer Association. Japanese classification
of gastric carcinoma—2nd English edition. Gastric Cancer.
1998;1:10-24.
Sobin LH, Wittekind C, editors. TNM Classification of Malignant Tumors, 5th Edition. New York: John Wiley & Sons,
1997.
Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An
attempt at a histo-clinical classification. Acta Pathol Microbiol Scand. 1965;64:31-49.
Kikuchi T, Toyota M, Itoh F, et al. Inactivation of p57KIP2 by
regional promoter hypermethylation and histone deacetylation in human tumors. Oncogene. 2002;21:2741-2749.
Hamai Y, Oue N, Mitani Y, et al. DNA hypermethylation and
histone hypoacetylation of the HLTF gene are associated
with reduced expression in gastric carcinoma. Cancer Sci.
2003;94:692-698.
Satoh A, Toyota M, Itoh F, et al. Epigenetic inactivation of
CHFR and sensitivity to microtubule inhibitors in gastric
cancer. Cancer Res. 2003;63:8606-8613.
Obata T, Toyota M, Satoh A, et al. Identification of HRK as a
target of epigenetic inactivation in colorectal and gastric
cancer. Clin Cancer Res. 2003;9:6410-6418.
Ueno M, Toyota M, Akino K, et al. Aberrant methylation and
histone deacetylation associated with silencing of SLC5A8 in
gastric cancer. Tumor Biol. 2004;25:134-140.
Murai M, Toyota M, Suzuki H, et al. Aberrant methylation
and silencing of the BNIP3 gene in colorectal and gastric
cancer. Clin Cancer Res. 2005;11:1021-1027.
Palmisano WA, Crume KP, Grimes MJ, et al. Aberrant promoter methylation of the transcription factor genes PAX5 ␣
and ␤ in human cancers. Cancer Res. 2003;63:4620-4625.
He B, You L, Uematsu K, et al. SOCS-3 is frequently silenced
by hypermethylation and suppresses cell growth in human
lung cancer. Proc Natl Acad Sci USA. 2003;100:14133-14138.
Rashid A, Issa JP. CpG island methylation in gastroenterologic neoplasia: a maturing field. Gastroenterology. 2004;
127:1578-1588.
Gastric CA with CIMP and Association to EBV/Kusano et al.
36. Oue N, Motoshita J, Yokozaki H, et al. Distinct promoter
hypermethylation of p16INK4a, CDH1, and RAR-beta in intestinal, diffuse-adherent, and diffuse-scattered type gastric
carcinomas. J Pathol. 2002;198:55-59.
37. Kim H, Kim YH, Kim SE, Kim NG, Noh SH. Concerted
promoter hypermethylation of hMLH1, p16INK4A, and Ecadherin in gastric carcinomas with microsatellite instability. J Pathol. 2003;200:23-31.
38. Shim YH, Kang GH, Ro JY. Correlation of p16 hypermethylation with p16 protein loss in sporadic gastric carcinomas.
Lab Invest. 2000;80:689-695.
39. Suzuki H, Itoh F, Toyota M, et al. Distinct methylation
pattern and microsatellite instability in sporadic gastric cancer. Int J Cancer. 1999;83:309-313.
40. Vo QN, Geradts J, Gulley ML, Boudreau DA, Bravo JC,
Schneider BG. Epstein-Barr virus in gastric adenocarcinomas: association with ethnicity and CDKN2A promoter
methylation. J Clin Pathol. 2002;55:669-675.
41. Osawa T, Chong JM, Sudo M, et al. Reduced expression and
promoter methylation of p16 gene in Epstein-Barr virusassociated gastric carcinoma. Jpn J Cancer Res. 2002;93:
1195-1200.
42. Shivapurkar N, Takahashi T, Reddy J, et al. Presence of
simian virus 40 DNA sequences in human lymphoid and
hematopoietic malignancies and their relationship to aberrant promoter methylation of multiple genes. Cancer Res.
2004;64:3757-3760.
1479
43. Soejima K, Fang W, Rollins BJ. DNA methyltransferase 3b
contributes to oncogenic transformation induced by SV40T
antigen and activated Ras. Oncogene. 2003;22:4723-4733.
44. Farrell PJ, Allan GJ, Shanahan F, Vousden KH, Crook T. p53
is frequently mutated in Burkitt’s lymphoma cell lines.
EMBO J. 1991;10:2879-2887.
45. Edwards RH, Raab-Traub N. Alterations of the p53 gene in
Epstein-Barr virus-associated immunodeficiency-related
lymphomas. J Virol. 1994;68:1309-1315.
46. Effert P, McCoy R, Abdel-Hamid M, et al. Alterations of the
p53 gene in nasopharyngeal carcinoma. J Virol. 1992;66:
3768-3775.
47. Spruck CH 3rd, Tsai YC, Huang DP, et al. Absence of p53
gene mutations in primary nasopharyngeal carcinomas.
Cancer Res. 1992;52:4787-4790.
48. Ojima H, Fukuda T, Nakajima T, Nagamachi Y. Infrequent
overexpression of p53 protein in Epstein-Barr virus-associated gastric carcinomas. Jpn J Cancer Res. 1997;88:262-266.
49. van Rees BP, Caspers E, zur Hausen A, et al. Different
pattern of allelic loss in Epstein-Barr virus-positive gastric
cancer with emphasis on the p53 tumor suppressor pathway. Am J Pathol. 2002;161:1207-1213.
50. Szekely L, Selivanova G, Magnusson KP, Klein G, Wiman KG.
EBNA-5, an Epstein-Barr virus-encoded nuclear antigen,
binds to the retinoblastoma and p53 proteins. Proc Natl
Acad Sci USA. 1993;90:5455-5459.