CLINICAL REVIEW

Epstein-Barr Virus-associated Gastric Carcinoma

A Newly Defined Entity
Jian-Ning Chen, MD, Dan He, PhD, Fang Tang, MD, and Chun-kui Shao, MD, PhD

such as the Hodgkin lymphoma and B-cell lymphomas

Abstract: Epstein-Barr virus (EBV)-associated gastric carcinoma among immunosuppressed patients, nasal NK/T-cell lym-
(EBVaGC) is a recently recognized entity, which is defined by the phoma, and so on.2 EBV has also been suspected to cause
presence of EBV in the gastric carcinoma cells. EBVaGC represents some epithelial malignancies, such as nasopharyngeal car-
about 10% of gastric carcinoma worldwide, and > 80,000 patients
are estimated to develop EBVaGC annually. EBVaGC shows some
cinoma (NPC) and a subset of gastric carcinoma.2 In 1990,
distinct clinicopathologic characteristics, such as male predom- Burke3 first reported the presence of EBV genome in lym-
inance, predisposition to the proximal stomach, and a high pro- phoepithelioma-like carcinomas (LELCs) of the stomach,
portion in diffuse-type gastric carcinomas. Besides, EBVaGC also using polymerase chain reaction (PCR) technique. Sub-
shows characteristic molecular abnormality, that is, global and sequently, in 1991 and 1992, using in situ hybridization
nonrandom CpG-island methylation of the promoter region of (ISH) technique to detect EBV-encoded small RNAs
many cancer-related genes, which causes downregulation of their (EBERs) in gastric tissue, Shibata reported the presence of
expression. Moreover, EBVaGC has a relative favorable prognosis. EBV genome in 75% (6/8) of LELCs 4 and 16% (22/138) of
The uniform presence of EBV-encoded small RNA in tumor cells conventional gastric adenocarcinomas5 in North America,
but not in the surrounding normal epithelial cells, and the detection
of monoclonal EBV episomes in EBVaGC, strongly suggests that
respectively. A large-scale study in Japan, published in
EBV play an etiological role in gastric carcinogenesis. Therefore, 1993, also showed the presence of EBERs in 6.9% of gas-
EBVaGC should be regarded as a distinct entity of gastric carcinoma, tric carcinomas.6 Subsequent studies revealed that the
although it only accounts for a relatively small fraction of total frequency of EBV in gastric carcinoma differed from country
gastric carcinomas. In this review, the epidemiological and clin- to country, and ranged from 1.3% to 20.1%, with an average
icopathologic features of EBVaGC and the genetic abnormalities of of about 10% worldwide.7–9 This subset of gastric carcinoma
EBVaGC cell including chromosomal and epigenetic abnormalities was defined as EBV-associated gastric carcinoma (EBVaGC)
are described. The roles of EBV in gastric carcinogenesis are dis- by the presence of EBV in neoplastic cells. PCR to amplify
cussed. We make an emphasis on the EBV latency pattern and ge- EBV DNA fragments and EBER-ISH techniques could be
nome polymorphisms as well as local immunity in EBVaGC. In
used to detect EBV in tumor tissues; however, as PCR is very
addition, the treatment of EBVaGC is also briefly discussed. Taken
together, this review aims to give the reader a full understanding of a sensitive and may cause false-positive results, and EBERs
newly defined entity of gastric carcinoma, EBVaGC. (EBER1 and EBER2) are always abundantly expressed in
nearly all the neoplastic cells of the tumor tissues (˜107 copies
Key Words: Epstein-Barr virus, gastric carcinoma, clinicopathologic per infected cell), EBER-ISH technique is served as gold
characteristics, prognosis standard to defined EBVaGC (Fig. 1).
(J Clin Gastroenterol 2012;46:262–271) EBVaGC is a nonendemic disease as it is distributed
throughout the world. This is different from other EBV-
associated malignancies, such as Burkitt lymphoma and
NPC, which is endemic in equatorial Africa and southeast
E pstein-Barr virus (EBV), also called human herpesvirus 4,
is a herpes virus, which is grouped as a member of the
Herpesviridae family, subfamily gammmaherpesvirinae,
China, respectively. EBVaGC represents about 10% of
gastric carcinoma worldwide; thus, > 80,000 patients in the
genus lymphocryptovirus. It is a human herpes virus that world are estimated to develop EBVaGC annually.
infects most humans by adulthood and is associated with a EBVaGC shows some distinct clinicopathologic charac-
number of different cancers. teristics, such as male predominance, predisposition to the
Burkitt lymphoma is the first neoplasm that is found to proximal stomach, and a high proportion in diffuse-type
be associated with EBV infection in 1964.1 After that, EBV gastric carcinomas. Besides, the frequency of EBVaGC in
is demonstrated to cause many other lymphoid neoplasms, gastric remnant carcinoma (GRC) is significantly higher than
that in conventional gastric carcinoma (CGC) which occurs in
the intact stomach. EBVaGC also shows some characteristic
From the Department of Pathology, The Third Affiliated Hospital,
molecular abnormality, that is, global and nonrandom CpG-
Sun Yat-sen University, Guangzhou, China. island methylation of the promoter region of many cancer-
Supported by the National Natural Science Foundation of China (nos. related genes, such as p16,10,11 p73,12 and E-cadherin,13,14 that
30672409 & 81071893), the Guangdong Natural Science Foundation causes downregulation of their expression. Furthermore,
(nos. 8151008901000132 & 05001748), the Guangdong Science and
Technology Project (no. 2009B060700034), and the Guangzhou
EBVaGC shows a relative favorable prognosis.15
Science and Technology Project (no. 2011J4100106), Guangdong Conversely, as mentioned above, EBERs are abun-
Province, China. dantly expressed in almost every tumor cell but not in the
The authors declare that they have nothing to disclose. surrounding normal epithelial cells and dysplastic cells.
Reprints: Chun-kui Shao, MD, PhD, Department of Pathology, The Third
Affiliated Hospital, Sun Yat-sen University, No. 600 Tianhe Road,
Besides, elevation of IgG and IgA antibodies against viral
Guangzhou 510630, China (e-mail: [email protected]). capsid antigen was found several months before the diag-
Copyright r 2012 by Lippincott Williams & Wilkins nosis of EBVaGC.16 Moreover, clonal analysis of EBV in

262 | www.jcge.com J Clin Gastroenterol  Volume 46, Number 4, April 2012

J Clin Gastroenterol  Volume 46, Number 4, April 2012 EBV-associated Gastric Carcinoma

FIGURE 1. Histology of an EBVaGC. A, H&E staining of a diffuse-type gastric carcinoma case. B, EBER1 in situ hybridization of the same
case. Note the even distribution of EBER1-positive signals labeled in all tumor nuclei but not in surrounding nontumor cells (original
magnification  200). EBER indicates Epstein-Barr virus-encoded small RNA; EBVaGC, Epstein-Barr virus-associated gastric carcinoma.

tumor tissues demonstrated that monoclonal EBV episomes Age and Sex Distribution
are present in EBVaGC.17 These findings strongly suggest Most studies showed no evident age dependence of
that EBV play an etiological role in gastric carcinogenesis. EBVaGC frequency. However, a study in Mexico showed
Therefore, the role of EBV in EBVaGC is being inves- an evident age-dependent increase of its frequency.39 In
tigated for the past 2 decades. The EBV latency pattern contrast, several studies showed an age-dependent decrease of
and genome polymorphisms, as well as the interactions EBVaGC.15,36,40 Whether the age dependence of EBVaGC
between EBV and host immunity in EBVaGC are the hot frequency exists and the mechanism involved in the age de-
topics. They have made some progress; however, the precise pendence of EBVaGC frequency needs to be elucidated.
role of EBV in gastric carcinogenesis still needs to be fur- The association of EBVaGC and age is uncertain,
ther elucidated. however, there was a high association of EBVaGC with the
Taken together, due to its distinct clinicopathologic male sex. This is an interesting feature of EBVaGC. To our
characteristics, distinct mechanisms of gastric carcino- knowledge, no study reported an evident female predom-
genesis, and a relative favorable prognosis, EBVaGC inance. Almost all of the studies so far showed a male
should be regarded as a distinct entity of gastric carcinoma, predominance of EBVaGC.5,6,18,19,22,23,25,26,28–32,34,36–38 In
although it only accounts for a relative small fraction of the recent meta-analysis, the prevalence of EBVaGC in
total gastric carcinomas. males is 2-fold higher than that in females.42 The male
In this review, the epidemiological and clinicopatho- predominance suggests that lifestyles and occupational
logic features of EBVaGC and the genetic abnormalities factors that are common among males may contribute to
of EBVaGC cell including chromosomal and epigenetic the development of EBVaGC.44 In addition, sex-related
abnormalities are reviewed. The roles of EBV in gastric hormonal and/or immune factors might also influence the
carcinogenesis are discussed. We make an emphasis on the development of EBVaGC.
EBV latency pattern and genome polymorphisms as well as
local immunity in EBVaGC. In addition, the treatment of Epidemiological Features of EBVaGC in GRC
EBVaGC is also briefly discussed. Another interesting finding is that the proportion of
EBVaGC in GRC is significantly higher than that in CGC
which occurs in the intact stomach. GRC is defined as a
carcinoma occurring in the gastric stump at least 5 years
EPIDEMIOLOGICAL FEATURES OF EBVaGC after surgery for benign diseases such as gastric ulcer and
duodenal ulcer.45,46 The proportion of EBVaGC in GRC is
Geographical Distribution 27% to 42%,47–50 which was 3 to 4 times higher than that in
Since the first discovery of EBVaGC in 1990s, EBVaGC CGC. In our preliminary study, we found that the pro-
was reported in many areas of the world.15,18–40 The portion of EBVaGC in GRC and CGC in Guangzhou was
frequency of EBVaGC in gastric carcinoma varies from 1.3% 30.8% versus 6.7%.51 Similar findings were also reported
to 20.1% in different areas, with an average of 10% world- by other groups in Japan (27.1% vs. 6.4%),47 Korea (29%
wide.7–9 The highest reported is 20.1% in Japan,41 followed vs. 6%),48 and The Netherlands (35% vs. 8%).49 This raises
by 18% in Germany,33 16.8% in Chile,24 and 16% in the a question: do more aggressive EBV variants exist in
United States5; the lowest is 1.3% in Papua New Guinea32 EBVaGC in GRC? To answer this question, we inves-
and 1.9% in Pakistan.35 According to a recent meta-analysis, tigated the EBV genome polymorphisms and clin-
the pooled estimate of frequency of EBVaGC in gastric car- icopathologic features of EBVaGC in GRC and CGC in
cinoma in America, Europe, and Asia was 9.9%, 9.2%, and Guangzhou. We found that the EBV genome poly-
8.3%, respectively.42 Thus, nearly 80,000 patients worldwide morphisms and clinicopathologic features of EBVaGC in
are estimated to develop EBVaGC annually, and it is the most GRC were similar to those in CGC, whereas the proportion
prevalent among EBV-associated malignancies.43 of EBVaGC in GRC was significantly higher than that in

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Chen et al J Clin Gastroenterol  Volume 46, Number 4, April 2012

CGC, suggesting that the injuries of gastric mucosa and/or In 1 meta-analysis, it is demonstrated that EBVaGC gen-
changes of the microenvironment within the remnant erally showed the diffuse-type histology. The overall odd
stomach may be involved in the development of EBVaGC ratio for EBVaGC in the diffuse-type carcinoma was
in GRC.51 Besides, gastritis cystica polyposa, which is fre- 1.745.8 However, another meta-analysis, conversely,
quently observed in the remnant stomach, especially with showed that prevalence did not significantly differ between
Billroth II anastomosis, might be a prerequisite for the the diffuse-type and intestinal-type gastric carcinoma.42
development of EBVaGC.52 Another interesting finding is that over 80% of LELCs
were EBV positive.54,55 LELC is a special kind of poorly
Risk Factors differentiated carcinoma with dense infiltration of lym-
Salt intake, which strongly associates with the risk of phocytes. The high prevalence of EBV in this particular
gastric carcinoma and its precursor lesions, is also a risk subtype, to some extent, suggests that EBV might influence
factor for EBVaGC.44 Other possible dietary factors asso- the histology of the tumor (ie, causes dense infiltration by
ciated with high risk of EBVaGC include frequent drinking lymphocytes), and play a role in the development of this
of coffee and high-temperature drinks, as well as frequent kind of gastric carcinoma.
intake of spicy foods.44 In addition, wood dust and/or iron On macroscopic examination, EBVaGC often takes
filings exposure are also risk factors related to EBVaGC.44 the form of an ulcerated or saucer-like tumor accompanied
Bile reflux, which causes damage to the gastric by marked thickening of the gastric wall.56 These features
mucosa, is also a risk of EBVaGC.51 This accounts for the are well discernible on endoscopic ultrasonograpy and
high proportion of EBVaGC in GRC, especially when the computed tomography scans of the stomach.57
Bilroth II anastomosis, which increases bile reflux, was Besides, the frequency of EBVaGC is relatively lower
performed. in early gastric carcinoma compared with that in advanced
It is of note that Helicobacter pylori infection is a gastric carcinoma.6 Moreover, early-stage EBVaGCs show
strong risk factor for gastric carcinoma, especially for the a unique morphologic feature, “lace pattern” that consists
carcinoma arising in the antrum of the stomach. However, of connection and fusion of neoplastic glands at the inter-
H. pylori infection is not a risk factor for EBVaGC. mediate zone of proper mucosa.58,59
According to a meta-analysis,8 H. pylori infection rate was
similar in EBVaGC and in EBV-negative gastric carcinoma Prognosis
(EBVnGC); thus, EBVaGC was not associated with EBVaGC shows a lower rate of lymph node involve-
H. pylori infection. Because H. pylori-positive gastric car- ment, especially in its early stage in the submucosa,15,60 and
cinoma is predominant in antrum and predisposes toward has a relatively favorable prognosis compared with
intestinal-type gastric carcinoma, whereas EBVaGC has a EBVnGC.15 In addition, LELCs, which are closely asso-
tendency of predominance in cardia/body and is usually ciated with EBV, also have a better prognosis.55
found to be diffuse-type gastric carcinoma, it is possible
that H. pylori-positive gastric carcinoma and EBVaGC EBV LATENCY PATTERN IN EBVaGC
follow 2 distinct pathogenetic pathways. Like other herpes viruses, EBV persists in its hosts
through its ability to establish a latent infection that peri-
CLINICOPATHOLOGIC CHARACTERISTICS odically reactivates. EBV establishes latent infection as an
OF EBVaGC episome, which is a self-replicating extrachromosomal
nucleic acid.61 Latent EBV infection takes 3 transcription-
Clinicopathologic Characteristics ally distinct forms of latency in terms of expression of EBV
EBVaGC shows some distinct clinicopathologic char- latent genes: latency I, II, and III (Table 1).62,63 Latency I is
acteristics. In addition to the male predominance discussed limited to only EBERs and EBV-determined nuclear anti-
in the above section, the proportion of EBVaGCs appears gen (EBNA) 1 expression; latency II includes latent mem-
to vary by tumor location and histology. The EBVaGCs are brane protein (LMP) 1 and 2 in addition; and latency III is
not evenly distributed topographically in the stomach. The defined by expression of EBERs, all 6 EBNA proteins
proportion of EBVaGC in carcinomas of the cardia and the (EBNA1, 2, 3A, 3B, 3C, and LP) and 3 LMP proteins
middle stomach is varying 2- to 4-fold or more than that in (LMP1, 2A, and 2B).
the antrum.15,53 EBVaGCs seem to be more common in EBV shows different latency pattern in different EBV-
diffuse-type gastric carcinomas than in intestinal-type ones. associated malignancies (Table 1).64 These different latency

TABLE 1. EBV Latency Patterns in EBV-associated Malignancies

EBNA2, LMP2A,
Latency EBNA1 3s, LP LMP1 2B EBERs BARTs miRs Neoplasms
I + +/ + + + Burkitt lymphoma
EBVaGC
II + + + + + + Hodgkin lymphoma
NPC
NK/T-cell lymphoma
III + + + + + + + AIDS-associated B-cell
lymphomas
Pyothorax-associated lymphoma
BARTs indicates BamHI A region rightward transcripts; EBERs, EBV-encoded small RNAs; EBNA, EBV-determined nuclear antigen; EBV, Epstein-Barr
virus; EBVaGC, EBV-associated gastric carcinoma; LMP, latent membrane protein; miRs, microRNAs; NPC, nasopharyngeal carcinoma.

264 | www.jcge.com r 2012 Lippincott Williams & Wilkins

J Clin Gastroenterol  Volume 46, Number 4, April 2012 EBV-associated Gastric Carcinoma

patterns reflect the complex interplay between EBV and the Interestingly, EBVaGC expressed only miR-BARTs but not
host cell environment; therefore, studies on the functions of miR-BHRF1s.82 MiR-BART2 is antisense to the BALF5
these key latent genes will be of help in understanding the open reading frame and has been proposed to have a role in
contribution of these EBV genes to the pathogenesis of the the regulation of viral DNA polymerase expression via the
EBV-associated malignancies so as to clarify the pathogenic degradation mechanism.79 In addition, several miR-BARTs
roles of EBV in these malignancies. EBVaGC belongs to were also found to regulate the expression of LMP1 viral
latency I, in which the expression of viral latent genes includes oncoprotein.83 Recently, miR-BART5 was reported to target
EBNA1, EBERs, and transcripts from the BamHI A region a cellular protein named p53 upregulated modulator of
(BARF0 and BARF1) rather than LMP1 and EBNA2.36,65 apoptosis thus facilitate the establishment of latent infection
In addition, LMP2A was also expressed in about half of the by promoting host cell survival in EBVaGC.84 However, the
EBVaGC cases.66,67 The functions of each of the latent genes functions of the EBV-encoded miRs are still largely unknown
in EBVaGC will be described in detail as follows. and are worthy of being deeply investigated.

EBNA1
EBNA1 is the only viral protein consistently expressed EBV GENOME POLYMORPHISMS IN EBVaGC
in all EBV-associated malignant tissues.64 EBNA1 is essential The first complete genomic sequence of EBV strain was
for the persistence and replication of the EBV genome in B95.8, which was published by Baer et al.85 After that, many
latently infected cells.68–70 In addition, EBNA1 contains a investigators turned to the studies on the EBV genome
glycine-alanine repeat sequence, which is an inhibitor of an- polymorphisms in EBV-associated malignancies and EBV
tigen presentation and processing.71 healthy carriers. They intended to find some correlations
between EBV genome polymorphisms and particular EBV-
EBERs associated disease. By now, 3 complete EBV genome se-
EBER1 upregulates insulin-like growth factor 1 ex- quences have been published, that is B95.8 (GenBank
pression, and secreted insulin-like growth factor 1 acts as an Accession No.: V01555), AG876 (GenBank Accession No.:
autocrine growth factor in NU-GC-3 gastric cancer cell line.72 DQ279927), and GD1 (GenBank Accession No.: AY961628)
that are derived from infectious mononucleosis, Burkitt
BARF1 lymphoma, and NPC, respectively.86 No complete EBV ge-
BARF1 was detected in nearly 100% of EBVaGCs; it nome sequence derived from gastric carcinoma is available
may be acting as the viral oncogene in the absence of LMP1 in yet. Although, progresses have been made in EBV genome
EBVaGC.65 BARF1 has an antiapoptotic role in EBVaGC, polymorphisms in EBVaGC as many studies investigated the
through an increased Bcl-2-to-Bax ratio, thus promoting polymorphisms of specific fragments of EBV genome.
cancer cell survival.73 This indicates that BARF1 may play a EBV shows some differences in the DNA sequence;
positive role in the development of EBVaGC. based on these differences, 2 subtypes and several variants of
EBV are identified. EBV has 2 major subtypes, subtype A
LMP2A and subtype B (also referred as subtype 1 and subtype 2)
LMP2A is reported to inhibit transforming growth which differ in the sequence of EBNA2, 3A, 3B, 3C, and LP
factor-b1–induced apoptosis in a gastric carcinoma cell genes and in their capacity to transform B lymphocytes into a
line.74 Recently, it is demonstrated that LMP2A upregu- state of continuous proliferation.64 Subtype A, which is much
lated the cellular survivin gene expression through the more efficient at immortalizing B cells than subtype B, is the
nuclear factor-kB pathway in gastric carcinoma cell lines with predominant strain in western and Asian countries, whereas
EBV infection.75 In addition, LMP2A upregulates cellular subtype B is frequently found in equatorial Africa and is seen
DNA methyltransferase 1 (DNMT1) in EBVaGC through more commonly in immunosuppressed patients.87,88 In ad-
the phosphorylation of STAT3, causing promoter hyper- dition, several variants of EBV have been identified based on
methylation of a tumor suppressor gene, PTEN.76 alignment to the prototype B95.8 genome.
In summary, the EBV latency pattern in EBVaGC is In 1994, Fukayama et al89 firstly studied the genotypes
latency I but somewhat different from the conventional of EBV in EBVaGC in Japan. They found that the domi-
latency I represented by Burkitt lymphoma.77 As LMP2A nant genotype of EBV in EBVaGC was subtype A (7/8),
has been demonstrated to play important roles in the on- type C (6/8), and type F (8/8) that were similar to the
cogenic processes in EBVaGC,75,76 and about half of the predominant type of EBV found in throat washing of the
EBVaGCs express LMP2A, we suggest designating the general population in Japan. Type F is defined as lack of a
EBV latency pattern in EBVaGC as latency Ia and Ib, BamHI enzyme site at BamHI-F region; in the contrary,
based on the absence and presence of LMP2A expression, type “f ” variant is featured by the presence of an extra
respectively. BamHI site at BamHI-F region, which is predominant in
Apart from these latent products, microRNAs (miRs) southern China and associated with NPC.90,91 Type C is
have also been concerned recently. miRs are small, non- contrasted to type D (also called type I and type “i,” re-
coding RNA molecules of about 22 nucleotides in length spectively); they are defined by the BamHI restriction site
and have been shown to play a role in the posttranscrip- polymorphism at BamHI W1/I1 boundary region. Type C
tional downregulation of target mRNAs.78 EBV was the lacks the BamHI site,92,93 whereas type D keeps the
first human virus found to encode miRs.79 By far, 25 species BamHI site.94 After that, EBV genotypes in EBVaGC in
of EBV-miRs have been identified.80 They are arranged in 2 other areas of the world was also been intensively inves-
clusters within the viral genome: 3 miRs adjacent to the tigated.21,29,36,38,93,95 In different areas, different results
BHRF1 gene (the BHRF1 cluster, miR-BHRF1-1 to miR- were found. Type F is almost exclusively found in EBVaGC
BHRF1-3) and the remaining 22 miRs located in the irrespective of the origin.21,29,36,38,93,95 However, the EBV
introns of the BamHI A region rightward transcripts (BART) variants in EBVaGC in Latin American95 and North
(the BART cluster, miR-BART1 to miR-BART22).79–81 Africa38 are predominantly type D and “XhoI+,” whereas

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Chen et al J Clin Gastroenterol  Volume 46, Number 4, April 2012

those in Asia are predominantly type C and “XhoI .”36,89,93,96 development and proliferation of hematopoietic cell line-
This may suggest that EBV variants be geographically dis- ages.107 However, whether GATA-2 involves in the devel-
tributed. Types “XhoI+” and “XhoI ” were defined by the opment of the EBVaGC is still uncertain. Therefore, the
presence and absence of XhoI restriction site in exon 1 of the function of the mutation in EBVaGC needs to be further
LMP1 gene. investigated.
Apart from the restriction enzyme polymorphisms In summary, EBV variants not only represent geo-
mentioned above, polymorphisms in EBV latent products graphical polymorphism but also show disease-related as-
were also been concerned. LMP1 and EBNA1 are the relative sociation. Although the function of the mut-W1/I1 EBV
intensively investigated proteins. Analysis of exon 3 of the variant in EBVaGC needs to be further clarified, its sig-
LMP1 gene leads to 2 variants, wt-LMP1 and del-LMP1. nificance may be equal to that of the well known “f ” var-
Del-LMP1 variant, which has a 30-bp sequence deletion when iant of NPC. More evidence should be accumulated to shed
compared with the wt-LMP1 variant, predominates in Chi- light on the correlations and roles of EBV variants in EBV-
nese and Taiwanese population.97 In EBNA1 variation associated malignancies.
analysis, 5 EBNA1 subtypes were determined based on the
signature amino acid residue 487 as well as particular amino
GENETIC AND EPIGENETIC ABNORMALITIES OF
acid alterations in other sites, proposed by Bhatia et al98 and
Gutiérrez et al.99 To date, there were only 2 papers concerning EBVaGC
about the EBNA1 variations in EBVaGC. Chen et al100 for Genetic Abnormalities of EBVaGC
the first time examined EBNA1 variations in EBVaGC cases The development of gastric carcinoma involves accu-
from Japan and the United States. They found that V-val mulation of multiple genetic alterations, including activation
EBNA1 variant was the only variant in all 25 EBVaGC cases of oncogenes and inactivation of tumor suppressor genes
from Japan; whereas in American EBVaGC cases, there were resulting from the abnormal structure of the chromosomes.
3 P-ala or P-ala variants, 9 P-thr, 4 V-leu or V-leu variants, Over the years, numerous studies revealed a variety of genetic
and 1 V-val. Recently, Wang et al101 investigated the EBNA1 changes, including changes of DNA copy numbers and
variations in 41 EBVaGC from northern China and found microsatellite instability (MSI), could be observed in gastric
that the V-val variant was predominant (78.1%) in EBVaGC. carcinoma. For EBVaGC, the studies of genetic instability at
Both of these 2 studies found that the EBNA1 variants in the chromosomal level are limited and sparse.
EBVaGCs were similar to those in normal background Comparative genome hybridization (CGH) provides a
populations, suggesting that the differences in EBNA1 gross and complete investigation of each chromosome of
sequences do not contribute to tissue tropism or tumor tumor cells for genetic amplification and deletions. There
formation and only reflect geographical polymorphism. Se- are few studies of EBVaGC with CGH and the results are
quence variations of LMP2A was also been investigated inconsistent. In 2001, zur Hausen et al108 first reported the
by 2 groups.102,103 The results indicated that LMP2A of CGH study on 10 EBVaGC and showed loss of 4p, 11p,
EBVaGC was very similar to B95.8, but contained some 18q was significantly more frequent in EBVaGC. Sub-
significant nucleotide variations. Recently, the variations sequently, Chan and colleagues performed CGH analysis of
of EBERs was also been studied by Wang et al.104 They 6 EBVaGC by removing the tumor infiltrative lymphocytes
identified 3 main distinct variants of EBERs, designated as in tumor specimens to eliminate the interference of these
EB-6m, EB-8m, and EB-10m. EB-6m was the predominant cells. They showed the common chromosomal alterations in
variants found in EBVaGCs, accounting for 96% (48/50). EBVaGC included gain of chromosome 11 as well as loss of
Finally, 2 recently identified variants, mut-W1/I1 and chromosome 15q15.109 Although CGH is a powerful mo-
wt-W1/I1, were designated by our group.36 Mut-W1/I1 lecular genetic method, it does not detect small regions of
variant, which shows a T to C mutation at position 148,972 genomic alterations. To overcome the methodological lim-
(wild-type EBV coordinates), was found in the majority of itations of CGH, another more sensitive method, loss of
the EBVaGCs in southern China.36,51 Wt-W1/I1variant, heterozygosity, was used to detect allelic deletion. In the
which keeps the original T at position 148,972, however, previous loss of heterozygosity studies, no association was
was found predominant in throat washing samples of found between allelic loss and EBV status. Van Rees et al110
healthy EBV carriers in the same area.51 In addition, the and Chong et al111 both demonstrated that chromosomal
representative NPC-derived EBV strain GD1 is also a losses were extremely rare in EBVaGC in contrast to the
wt-W1/I1 variant.105 These findings suggested that EBV high frequency in EBVnGC.
variants are not only geographically distributed but also MSI is a common feature of gastric carcinoma due to
disease restricted. Given that the mut-W1/I1 variant may be the defects in mismatch repair genes. The studies regarding
associated with EBVaGC, using bioinformatics analysis, we the relationships between EBVaGC and MSI are con-
found that the mutation is not in the coding region but in troversial and limited. Leung et al112 and Wu et al13 reported
the intron between exon 1 and exon 2 of the RPMS1. that high-level MSI was observed in gastric carcinomas re-
RPMS1 is the only full-length cDNA confirmed so far and gardless of the EBV status. In contrast, Chong et al111 and
one of the most abundant spliced forms in the BARTs Chang et al40 found no MSI in EBVaGCs.
family; and it is considered to play an important role in the In conclusion, compared with EBVnGC, the distinct
development of NPC.106 However, by now, no study con- chromosomal changes of EBVaGC strongly suggest that
cerning RPMS1 in EBVaGC is available. Besides, prediction EBVaGC follow a distinct pathogenetic pathway due to the
for transcription factor binding site using the tool at the existence of EBV.
website (http://www.cbrc.jp/research/db/TFSEARCH.html)
showed that the mutation located in the binding site
(GCGGATCCCG) of the GATA-2 transcription factor and EPIGENETIC ABNORMALITIES OF EBVaGC
it might affect GATA-2 binding. GATA-2 plays an essen- For the past few years, promoter hypermethylation
tial role in regulating transcription of genes involved in the has been recognized as an alternative way to inactivation

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J Clin Gastroenterol  Volume 46, Number 4, April 2012 EBV-associated Gastric Carcinoma

tumor suppressor genes in cancer. In contrast to the con- Although both immune responses are important for host to
sistently negative findings in genetic alterations, epigenetic resist the tumor invasion, the local immunity of tumor sites is
inactivation of the multiple genes plays an important role in the frontier of host immune responses and is recognized as
the tumourigenesis of EBVaGC. the most direct and specific response to malignant cells.
In 1999, Toyota et al113 first defined CpG-island EBVaGCs are known to be accompanied by massive
methylator phenotype (CIMP) in the gastric cancer using lymphocytes and mature dendritic cells infiltration,5,6,54,124
methylation of 5 MINT (methylation in tumor). By the suggesting that local triggering of cellular immune re-
numbers of methylation markers, 0, 1 to 3, 4 to 5, tumor sponses in EBVaGC has unique characteristics. Saiki
defined as CIMP none, low, high (CIMP-N, CIMP-L, et al125 firstly reported that lymphocytes infiltrating in
CIMP-H). According to the classification, Kusano et al114 EBVaGC were predominantly CD8+ T cells with high
showed EBVaGC were associated strongly with CIMP-H proliferation and cytotoxicity, many of which expressed
by using the methylation-specific polymerase chain method. perforin and granzyme B. This result was also confirmed by
Subsequently, another group adopted other 5 genes (LOX, other authors.23,126,127 In vitro, these CD8+ cytotoxic
HRASLS, FLNC, HAND1, THBD), which were densely T lymphocytes (CTLs) separated from EBVaGC could spe-
methylated in gastric carcinoma, as marker for the analysis, cifically kill autologous EBV-transformed lymphoblastoid
and they also demonstrated that EBVaGC exhibited cells.128 Apart from CD8+ T cells, CD4+ T cells, B cells,
CIMP-H phenotype.115 Meanwhile, a series of studies re- and NK cells were also existed in EBVaGC. However, the
vealed that EBVaGC showed simultaneous methylation of numbers of these cells infiltrated in EBVaGC were similar
multiple genes, including cell cycle regulation (p14, p15, to those in EBVnGC (our unpublished data). Thus, it seems
p16, cox2), DNA repair and protection (HMLH1, MGMT, that CD8+ CTLs play an important role in local immune
GSTP1), cell adherence and metastasis (E-cadherin, TIMP- response. Besides, van Beek et al126 demonstrated that
3), angiogenesis (THBS1), apoptosis (DAP-kinase, bcl-2, EBVaGC harbored more CD83+ mature dendritic cells
p73), and signal transduction (APC, PTEN, RASSF1A).116,117 than EBVnGC. These local immune responses may be as-
In comparison with EBVnGC, obvious higher frequencies of sociated with the generally favorable prognosis and lower
methylation of cancer-related genes were found in EBVaGC. frequency of lymph node metastases of EBVaGC.126
These findings suggested a relationship of EBV and aberrant Despite of potent local cellular immune responses, the
methylation in this tumor. ability of EBV to persist in EBVaGC cells indicated that the
It is of interest to note that p7312 and Hoxa10,118 are virus has evolved strategies to elude the immune system.
overwhelmingly methylated in EBVaGC but not in Firstly, limited expression of EBV latent genes was prob-
EBVnGC. Thus, these facts suggest that the pattern of host ably one of the immune escape strategies. EBVaGC belongs
genome methylation in EBVaGC is different from the to latency I, it is free from expression of the EBV latent
pattern of gene methylation seen in EBVnGC. Another proteins such as EBNA2, EBNA3s, and LMP1 which are
special finding regarding methylation of EBVaGC is that the target proteins of EBV-specific CTLs.129 Secondly, the
the association between promoter methylation and gene glycine-alanine repeat domain within EBNA1 inhibited
silencing is usually consistent in EBVaGC, such as p16,11 proteasomal processing for presentation of EBNA1, which
E-cadherin,14 and p73.12 Moreover, the methylation of p14 avoided triggering the activation of CTLs.71,130 Thirdly,
and p16 promoter regions was distributed uniformly in all EBVaGC cells also secreted some immune suppressive cy-
CpG sites in EBVaGC, whereas it was sporadic or variable tokines which enabled the tumor cells to evade the immune
in EBVnGC.119 response. Recently, we found that the expression of inter-
A few studies have demonstrated that viral proteins leukin (IL)-1b, IL-10 was upregulated in EBVaGC tumor
were involved either directly or indirectly in activation of cells (unpublished data). The perturbation in the level and
DNMTs. DNMT1 is responsible for the maintenance of activity of IL-1b secreted by the tumor cells was reported as
cytosine methylation. Tsai et al120 demonstrated that re- the key immune suppressive factor in EBVaGC.131,132
duction of E-cadherin is the result of LMP1-induced hy- Finally, the CD4+CD25+FoxP3+ regulatory T cells were
permethylation of E-cadherin gene promoter through predominant in EBVaGC than in EBVnGC (our un-
activation of DNMT1. The recombinant EBV infection in published data). The accumulation of regulatory T cells
gastric carcinoma cell lines demonstrates that viral latent impaired the cytotoxicity of infiltration lymphocytes in
membrane protein 2A (LMP2A) upregulates DNMT1, EBVaGC and suppressed EBV-specific immune responses.
leading to an increase in methylation of the PTEN pro- This is another mechanism of EBVaGC cells escaping the
moter.76 host immune responses.
Other than silencing of host genes, methylation of viral
genes may be involved in the maintenance of specific EBV
latency in cancer cells. DNA methylation contributes to TREATMENT OF EBVaGC
silencing of Wp, Cp, LMP1p, and LMP2Ap.121 In contrast, Because of limited understanding of this newly defined
the methylation does not control Qp. Several studies re- entity, treatment of EBVaGC is the same as that of their
vealed that the activity of Cp, LMP2Ap, and Qp can be counterpart, EBVnGC. In other words, the therapy for
influenced by histone H3 and H4 acetylation.122,123 These gastric carcinoma is ignored of the EBV status, and just
epigenetic modifications of EBV genome help the infected depended on other clinicopathologic characteristics, such as
cell to escape immune surveillance, and may be involved in the pTNM staging. Since EBV plays an important role in
transformation of gastric epithelial cells. EBVaGC, based on the current understanding of the virus-
host interactions in EBVaGC, several therapeutic ap-
proaches are worthwhile to be introduced. One promising
LOCAL IMMUNITY IN EBVaGC approach is to use demethylating agents, such as 5-aza
The presence of EBV in EBVaGC cells can provoke cytidine, to recover the lytic infection of EBV, leading to
systemic immune response as well as local immune response. the cell lysis of the infected cells.133 This approach may

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Chen et al J Clin Gastroenterol  Volume 46, Number 4, April 2012

have particular merit in EBVaGC, in which methylation of 12. Ushiku T, Chong JM, Uozaki H, et al. p73 gene promoter
the tumor suppressor gene is also a key abnormality. An- methylation in Epstein-Barr virus-associated gastric carcino-
other possible therapeutic approach is to activate viral ma. Int J Cancer. 2007;120:60–66.
thymidine kinase by a proteosome inhibitor, such as bor- 13. Wu MS, Shun CT, Wu CC, et al. Epstein-Barr virus-associated
gastric carcinomas: relation to H. pylori infection and genetic
tezomib.134 The reactivation coupled with the admin- alterations. Gastroenterology. 2000;118:1031–1038.
istration of a radiolabeled substrate would theoretically 14. Sudo M, Chong JM, Sakuma K, et al. Promoter hyper-
result in specific targeting of the radioactive end-product to methylation of E-cadherin and its abnormal expression in
the EBVaGC.134 However, the efficiency and the side effects Epstein-Barr virus-associated gastric carcinoma. Int J Cancer.
of these agents are possible drawbacks and should be 2004;109:194–199.
studied further. 15. van Beek J, zur Hausen A, Klein Kranenbarg E, et al. EBV-
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entity with a low frequency of lymph node involvement. J Clin
CONCLUSIONS Oncol. 2004;22:664–670.
EBVaGC is a distinct entity, comprising about 10% of 16. Levine PH, Stemmermann G, Lennette ET, et al. Elevated
all cases of gastric carcinoma in the world. EBVaGC shows antibody titers to Epstein-Barr virus prior to the diagnosis of
some distinct clinicopathologic characteristics, such as male Epstein-Barr-virus-associated gastric adenocarcinoma. Int J
predominance, predisposition to the proximal stomach, and a Cancer. 1995;60:642–644.
high proportion in diffuse-type gastric carcinomas. EBVaGC 17. Imai S, Koizumi S, Sugiura M, et al. Gastric carcinoma:
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global and nonrandom CpG-island methylation of the pro- virus latent infection protein. Proc Natl Acad Sci USA. 1994;
91:9131–9135.
moter region of many cancer-related genes, which causes 18. Galetsky SA, Tsvetnov VV, Land CE, et al. Epstein-Barr-
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