Group Title: Journal of Experimental & Clinical Cancer Research 2009, 28:14
Title: Characteristics of Epstein-Barr virus-associated gastric cancer: A study of 235 cases at a comprehensive cancer center in U.S.A
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Title: Characteristics of Epstein-Barr virus-associated gastric cancer: A study of 235 cases at a comprehensive cancer center in U.S.A
Series Title: Journal of Experimental & Clinical Cancer Research 2009, 28:14
Physical Description: Archival
Creator: Truong CD
Feng W
Li W
Khoury T
Li Q
Alrawi S
Yu Y
Xie K
Yao J
Tan D
Publication Date: 39847
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Cancer Research BioMed Central


Case study


Characteristics of Epstein-Barr virus-associated gastric cancer: A
study of 235 cases at a comprehensive cancer center in U.S.A
Camtu D Truongi, Wei Feng2, Wei Li2, T Khoury3, Q Li3, S Alrawi4,
Yingyan Yu5, Keping Xie1, James Yao' and Dongfeng Tan*


Address: 'The University of Texas MD Anderson Cancer Center, Houston, TX, USA, 2University of Texas Health Science Center, Houston, TX, USA,
3Roswell Park Cancer Institute, Buffalo, NY, USA, 4University of Florida at Jacksonville, Jacksonville, FL, USA and 5Shanghai Tongji University
School of Medicine, Shanghai, PR China
Email: Camtu D Truong ctruong@mdanderson.org; Wei Feng wfeng@mdanderson.org; Wei Li wei.li@uth.tmc.edu;
T Khoury thaer.khoury@roswellpark.org; Q Li ql@mdanderson.org; S Alrawi salrawi@jux.uf.edu; Yingyan Yu yingyan3@yahoo.com.cn;
Keping Xie kx@mdanderson.org; James Yao jy@mdanderson.org; Dongfeng Tan* dtan@mdanderson.org
* Corresponding author



Published: 3 February 2009 Received: 2 November 2008
journal of Experimental & Clinical Cancer Research 2009, 28:14 doi: 10.1 186/1756-9966-28-14 Accepted: 3 February 2009
This article is available from: http://www.jeccr.com/content/28/1/14
2009 Truong et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Epstein-Barr virus (EBV) has been shown to be associated with gastric cancer. However,
inconsistent findings have been reported regarding the distribution of EBV infected cells (in normal gastric
epithelium vs. intestinal metaplastic cells vs. in neoplastic cells) and the characteristics of EBV-associated
gastric cancer. Lymph node positive EBV-associated gastric cancer has not been systematically studied. The
aims of this study were to evaluate EBV-associated gastric cancer, to assess the distribution of EBV infected
cells including all positive lymph nodes, and to define the characteristics of EBV-associated gastric cancer.
Design: The study included primary gastric cancer patients who underwent surgical resection with no
preoperative treatment at M.D. Anderson Cancer Center between 1987 and 2006. Formalin-fixed
paraffin-embedded tissue from these resection specimens were assessed for EBV by in situ hybridization,
the gold standard for EBV detection in tissue. EBV status was analyzed along with clinicopathologic
parameters including age, gender, tumor type, lymph node status, and pathologic stage of the tumor.
Results: Among 235 patients, 12 had intranuclear expression of EBV. EBV staining was seen only in tumor
cells and no detectable EBV was observed in normal gastric mucosa, intestinal metaplasia or stromal cells.
Eight of 12 patients with EBV-associated gastric cancer had regional lymph node metastasis. Of note,
metastatic tumor cells in all of the involved lymph nodes of these 8 cases contained EBV. The epidemiologic
data showed I I of the 12 patients with EBV-associated gastric cancer were men, ranging in age from 54
to 78 years (mean age, 60 years; median age, 62.1 years). The age distribution for non-EBV associated
gastric cancer patients ranged from 21 to 93 years (mean age, 67 years; median age, 66.4 years).
Conclusion: Our study demonstrated that EBV is present exclusively in gastric cancer cells. The
detection of EBV in tumor cells in all of the lymph nodes involved with metastatic gastric carcinoma
suggests simultaneous replication of EBV and tumor cells. The predominantly male gender and relatively
younger age observed for the EBV-infected gastric cancer cases suggest an association between this
disease and other factors, such as life style.





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Background
In 1990, Burke et al. [1] used a polymerase chain reac-
tion(PCR) method to detect Epstein-Barr virus (EBV) in a
small group of gastric carcinoma cells that resembled cells
of morphologically undifferentiated nasopharyngeal lym-
phoepithelioma. Subsequently, Shibata et al. [2], using in
situ hybridization, demonstrated that EBV genomes were
uniformly present in gastric carcinoma cells resembling
lymphoepithelioma cells but were not present in reactive
lymphoid infiltrate or normal mucosa. In addition, Shi-
bata and Weiss [3] reported that EBV involvement was
detected not only in lymphoepithelioma-like gastric carci-
noma but also in a subset of ordinary gastric carcinomas.

During the past decade, the role of EBV in gastric carcino-
genesis has been recognized as new evidences have con-
tinued to emerge [4-6]. EBV-associated gastric carcinoma
(EBVaGC) harbors distinct chromosomal aberrations and
is characterized by a unique transcription pattern that
resembles but is not identical to that of nasopharyngeal
carcinomas [7,8]. EBVaGC, compared with EBV-negative
gastric carcinoma, shows distinct clinical features [9].

However, findings from studies in which various tech-
niques were used to detect the presence of EBV in gastric
cancer tissue have been highly controversial and conflict-
ing. Some authors found EBV in only carcinoma cells
[4,10-12], whereas others found EBV in both precursor
lesions (e.g., intestinal metaplasia and dysplasia) and car-
cinoma cells [3,13-15]. Moreover, the status of EBV in the
metastatic EBVaGC lymph nodes has not been investi-
gated.

To further examine the role of EBV in gastric carcinogene-
sis, we systematically and retrospectively studied a large
cohort of patients with gastric cancer in a single compre-
hensive cancer center using EBV-encoded RNA 1 (EBER1)
in situ hybridization technique (the gold standard for
identifying EBV, shown to be superior to EBV DNA in situ
hybridization) [16]. We also utilized immunohistochem-
istry to detect EBV-specific proteins, which are known to
be expressed in some EBV-associated malignancies [16].

Materials and methods
Patient population
For inclusion in this retrospective analysis, patients must
have had a diagnosis of primary gastric carcinoma and
undergone complete surgical resection of the tumor as ini-
tial treatment. The study criteria also included adequate
archival tissue for analysis and the availability of complete
clinicopathologic data. Patients who had received preop-
erative treatment (chemotherapy, radiotherapy, or
chemo-radiotherapy) were excluded from the study. A
total of 249 consecutive patients who had been treated at
the University of Texas M. D. Anderson Cancer Center


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during the period of January 1, 1987 through December
31, 2006 met the study criteria.

The collected clinicopathologic data collected consisted of
age, gender, date of initial diagnosis, tumor type, lymph
node status, pathologic tumor stage, and date of death
from gastric carcinoma or of last clinical follow-up. Histo-
logic diagnosis and grade of differentiation were deter-
mined in accordance with the World Health Organization
criteria for gastric tumors [17]. The M. D. Anderson Can-
cer Center institutional review board approval was
granted to investigate molecular markers relevant to gas-
tric cancer pathogenesis in this study.

Histologic examination and tissue microarray construction
Hematoxylin and eosin-stained slides of gastric carci-
noma tissue were reviewed to confirm the histopathologic
diagnoses and to assess the adequacy of specimens before
being selected for molecular analyses. We retrieved neu-
tral buffered formalin-fixed (10% formalin in water, v/v;
pH 7.4) and paraffin-embedded tissue blocks containing
gastric carcinoma and nonneoplastic gastric tissue from
the Department of Pathology at M. D. Anderson Cancer
Center. One investigator (D.F.T.) identified and marked
the areas containing viable tumor and normal tissue ele-
ments for the construction of tissue microarrays (TMAs).
High-density TMAs were assembled using a tissue
puncher-array system (Beecher Instruments, Silver Spring,
MD), as we described previously [18]. Briefly, specimens
retrieved from selected regions of archived donor tissue
were precisely arrayed onto a new (recipient) paraffin
block. Tissue cores were 1.0 mm in diameter and ranged
in length from 1.0 to 3.0 mm, depending on the depth of
tissue available in the donor block. For all cases, three tis-
sue cores were acquired from each normal and tumor
donor block. The three-core samples were subsequently
inserted (spaced 0.8 mm apart) onto 45- x 20- x 12-mm
recipient blocks. A total of four high-density TMAs were
used in this study.

In situ hybridization
To determine the localization of EBV in all specimens, we
performed in situ hybridization using a digoxigenin-
labeled 30 mer-oligonucleotide probe (EBER kit, Ventana
Medical Systems, Tucson, AZ) (5' AGACACCGTCCT-
CACC ACCCGGGACTTGTA3') complementary to small
nuclear EBER1, as described previously [19,20]. Briefly, 4-
lm-thick sections were cut from paraffin-embedded tis-
sues, mounted on slides coated with 3-(aminopropyl) tri-
ethoxysilane (Sigma Chemical Company, St. Louis, MO),
baked at 60 C for 1 hour, and dewaxed. All sections were
treated with 0.2 N HC1 for 20 minutes, followed with 20
gg/ml proteinase K solution (Boehringer Mannheim,
Mannheim, Germany). Next, the slides were dehydrated
and prehybridized for 2 hours at 370C with mixtures of


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50% deionized formamide, 0.18 mol/1 NaC1, 10 mmol/1
NaH2PO4, 1 mmol/1 ethylenediaminetetraacetic acid,
0.1% sodium dodecyl sulfate, 100 lg/ml of denatured
salmon sperm DNA, 100 lg/ml of transfer RNA, and 10%
dextran sulfate. The slides were then hybridized overnight
at 37C with 0.5 ng of digoxigenin-labeled probe. Foll-
wed the first wash of all sections with 0.5 x saline sodium
citrate, hybridization was detected by antidigoxigenin
antibody-alkaline phosphatase conjugate. Next, all sec-
tions were subjected to a second wash followed by a visual-
izing reaction performed with nitroblue tetrazolium salt
and 5-bromo-4-chloro-3-indolyl phosphate solution in
the dark for 6 to 12 hours. The slides were counterstained
with methyl green and mounted with aqueous medium.
Specimens from a patient with known EBV-positive gas-
tric carcinoma were used as positive control, and a sense
probe to EBER1 was used as negative control for each pro-
cedure.

Immunohistochemical analysis
To detect EBV-specific proteins, which are known to be
expressed in EBV-associated epithelial malignancies [16],
we used monoclonal antibodies against latent membrane
protein 1 (LMP-1). Serial 5-mm-thick tissue sections were
cut from microarrays for immunohistochemical analysis.
These sections were processed within 1 week of cutting to
avoid oxidation of antigens. We stained the initial sec-
tions with hematoxylin and eosin to verify histologic type.
We also used antigen retrieval and avidin-biotin staining
and visualized the antibody with an avidin-biotin-horse-
radish peroxidase complex and diaminobenzidine-hydro-
gen peroxide staining method, as described previously by
investigators from our laboratory [21,22]. Briefly, the sec-
tioned array tissue was processed using steam-heat
retrieval for 30 minutes. A mouse monoclonal antibody
(CS1-4; Dako, Carpinteria, CA) against LMP-1 was
reacted with the array sections for 25 minutes at room
temperature in an automatic immunostainer (Dako). The
array sections were then incubated in a detection kit in
accordance with the manufacturer's instructions. Slides
from the immunohistochemical analysis were independ-
ently reviewed by two investigators, who recorded the
staining as negative or positive. All cells in all the cores
were evaluated. Unequivocal nuclear staining in >5% of
tumor cells was considered as positive response; nuclear
staining in <5% of tumor cells was considered as negative
response.

Statistical analysis
The following variables were examined: age, gender,
tumor type, lymph node status, pathologic stage, and EBV
expression. For all statistical tests, two categories were
analyzed in pairs as positive versus negative and present
versus absent. We analyzed categorical variables using the
Fisher's exact test, McNemar test and the Mann-Whitney


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rank-sum test. The follow-up time was calculated using
the potential follow-up method. Overall patient survival
was defined as the time between the date of surgical diag-
nosis to the date of last follow-up (censored) or the date
of patient death (event). The end of follow-up date of this
study was December 31, 2006. Censored cases included
those cases (n = 6) in which the last follow-up date
occurred before December 31, 2006. Patients who
deceased of causes other than gastric cancer were not
included in the study. We analyzed the differences in sur-
vival times between patient subgroups using the log-rank
test. Survival probabilities were calculated (using the Kap-
lan-Meier method) and compared (using the log-rank
test) [23].

We performed Cox proportional hazards regression anal-
ysis [24] using SAS software (SAS Institute, Cary, NC) to
determine the association between the clinicopathologic
variables and overall patient survival. First, we analyzed
the association between possible prognostic factors
(including age, gender, stage, and node classification) and
death, considering one factor at a time. Second, multivar-
iate Cox analysis was performed on backward (stepwise)
procedures that always forced EBV into the model, along
with all variables that satisfied an entry level ofP < 0.05.
As the model continued to add factors, independent fac-
tors did not exceed an exit level ofP > 0.05.

Results
Clinicopathologic data
Clinicopathologic features of the study subjects are sum-
marized in Table 1. Our study consisted of 88 female
(37%), and 147(63%) male. One hundred eighteen
(50%) patients were older than 65 years, while the other
117 (50%) were 65 years or younger. Eighty-three tumors
(35%) were intestinal type, and 152 (65%) were diffuse
type. One hundred thirty-one patients (56%) had stage I-
II disease, and the remaining 104 patients (44%) had
stage III or IV disease. Sixty patients (27%) had nodal
involvement and 165 (73%) had no nodal metastases.

EBV RNA expression in gastric tissue
We tested 249 gastric carcinoma tissues. Of the 249 tumor
specimens, 235 were fully assessable. The yield after tissue
processing was 94% (235 of 249). Among the 235 tumor
cases, 72 also contained non-neoplastic gastric tissue (9
cases from EBV positive tumor cases and 63 from EBV neg-
ative cases). EBER1 was detected by in situ hybridization.
Positive control samples revealed a distinctive diffuse
nuclear stain. Sections incubated with preabsorbed or pre-
immune rabbit antisera showed no immunostaining.

Overall, 12 of the 235 tumors (5.1%) exhibited positive
EBV expression (Figure 1). The intensity varied slightly
from tumor to tumor but was consistent within the same


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Table I: Clinicopathologic features and EBV expression in gastric cancer


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EBV Expression
Negative


Gender


Lymph node


Type


Stage


Total


Female
Male

<65
>= 65

Negative
Positive


Well/Moderately
Poorly


I or II
III or IV


tumor. No relationship was found between the intensity
of EBER-1 expression and any clinicopathological fea-
tures. EBV expression was noted in both diffuse (includ-
ing lymphepithelial carcinoma) and intestinal type of GC
(Table 1). Expression of EBV was not noted in nonneo-
plastic gastric mucosal, intestinal metaplastic, or stromal
cells (endothelial cells and fibroblasts), or infiltrating
inflammatory cells within the tumor sections. Twelve of
235 gastric tumor cases exhibited EBV expression, while
none of the 72 samples containing non-neoplastic gastric
epithelium displayed EBV expression. The difference
between EBV positivity in carcinoma tissues and corre-
sponding non-neoplastic gastric tissues was statistically
significant (X2 = 9.0407; P = 0.0028). In addition, one rep-
resentative positive lymph node from each metastatic case
was examined. We observed that a fairly uniform expres-
sion of EBER1 in metastatic tumor cells. Among the 12
EBVaGC cases, eight patients displayed lymph node
metastasis. Tumor cells in all eight positive lymph nodes
revealed EBV expression (Figure 2). Ten additional meta-
static cases were randomly chosen and lymph nodes with
tumor cells were examined for EBER1. No tumor cells in
the lymph nodes of the 10 additional cases displayed
EBER1 expression.

LMP-I protein expression in gastric tissue
Positive control, using known LMP-1-positive lymphoid
tissue, revealed a distinctive membranous stain. Negative
control sections were immunostained under the same
conditions, with preabosorbed antisera substituted for the
primary antibody, displaying no immunoreactivity.
Among all 249 tested, 231 were assessable. No expression
of LMP-1 was identified in any gastric cancer or in non-
neoplastic gastric tissue.


To verify the foregoing TMA results, we examined a subset
of 40 whole tissue sections (from 12 patients with
EBVaGC and 28 without EBV) for the expression of EBV
and LMP. The findings were consistent with those from
the TMA cores. EBV was detected only in the EBVaGC sec-
tions; no EBV was observed in nonneoplastic gastric tissue
or in intestinal metaplasia.

Association of EBV expression with clinicopathologic
parameters
Age, gender, tumor type, nodal status, and pathologic
tumor stage were the clinicopathologic parameters ana-
lyzed in our study. After examining the associations
between EBV expression and clinicopathologic variables
(Table 2), we found a statistically significant association
between EBV expression and gender. Eleven of the 12
patients with EBVaGC were male. The difference in EBV
positivity in carcinoma tissues between male and female
patients was significant (P < 0.05). Patients with EBVaGC
were 54-78 years old (mean age, 60 years; median age,
62.1 years), whereas patients with gastric cancer not asso-
ciated with EBV were 21-93 years old (mean age, 67 years;
median age, 66.4 years). Subsequently, we analyzed the
differences in survival times between patient subgroups
using the log-rank test. Survival probabilities were calcu-
lated (using the Kaplan-Meier method) and compared
(using the log-rank test). Compared to those without EBV
expression, patients with EBVaGC displayed a favorable
clinical outcome (Figure 3). However, by multivariate Cox
analysis, only lymph node status and tumor stage were
significantly associated with ultimate patient prognosis
(Table 3).





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87 (37%)
136 (58%)

I (47%)
1 12(48%)

56 (25%)
157 (70%)

79 (34%)
144 (62%)

126 (54%)
97(41%)

223 (95%)


Positive

I (0%)
I I (5%)

7 (3%)
5 (2%)

4 (2%)
8(4%)

4 (2%)
8(4%)

5 (2%)
7 (3%)

12(5%)


Total


88 (37%)
147(63%)

1 18(50%)
117(50%)

60 (27%)
165 (73%)

83 (35%)
152 (65%)

131 (56%)
104 (44%)

235 (100%)







Journal of Experimental & Clinical Cancer Research 2009, 28:14


Figure I
Photomicrographs of Epstein-Barr virus (EBV) expression in gastric cancer. Epstein-Barr virus (EBV)-encoded RNA
I (EBERI) in situ hybridization in a gastric carcinoma reveals specific EBERI transcripts (dark) in the nuclei of the tumor cells.
IA-B: intestinal type of gastric cancer with EBV nuclear expression. Note, all tumor glands were positive for EBV, while stro-
mal cells between the tumor glands were negative. I C-D: diffuse type of gastric cancer with EBV nuclear expression, while
scattered lymphocytes were negative. (Original magnification x 10 in Fig. IA&C, original magnification x 40 in Fig. I B&D)


Discussion
Gastric carcinoma is one of the most common cancers
worldwide and the second most common cause of cancer-
related death, with 876,000 new cases diagnosed annually
[17]. In addition, EBV-positive gastric cancer cases make
up the largest group of EBV-associated malignancies.
Thus, defining the role of EBV in the carcinogenesis of this
widespread malignancy is essential.


Using in situ hybridization technique, we examined 235
cases of primary gastric cancers, which to our knowledge
was the largest study group of this type in the United
States. Specific nuclear EBER1 transcripts were found only
in gastric carcinoma cells. In contrast, EBV was detected in
none of the normal or dysplastic epithelia in the EBVaGC
or EBV-negative cases. Specifically, in 10 of the 12 cases of
EBVaGCs, EBER1 was expressed in almost all carcinoma
cells, suggesting that EBV infection occurs early in onco-


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Figure 2
A. Metastatic gastric adenocarcinoma involving lymph node (magnification x 10). 2B. Metastatic tumor cells are
positive for EBV; germinal center is negative (magnification x 40).


genesis with a subsequent clonal expansion of EBV-con-
taining tumor cells, significant findings which have also
been reported by investigators using molecular genetic
techniques [13,25]. In two cases of EBVaGC, EBER1 was
expressed in a small number of gastric carcinoma cells,
visualized with focal EBER1 staining, indicating that EBV
infection occurs after neoplastic transformation has taken
place.

The EBV nuclear expression was restricted to gastric carci-
noma cells. No expression was found in the presumed
precursor lesions of gastric carcinoma. Our results agree
with those of other studies in which EBER transcripts were
not detected in adjacent precursor lesions, such as intesti-
nal metaplasia [4,26-28]. However, some studies have
described the presence of EBV in dysplasia [3,13], and
others have detected the presence of EBV in intestinal
metaplasia [14,15]. There are several reasons for these dis-
crepancies. First, dysplasia adjacent to carcinomas is diffi-
cult to distinguish from local carcinoma spread [17].
Secondly, variation in the techniques used and methods
of interpretation can lead to inconsistent results. For
example, one study that used both polymerase chain reac-
tion and in situ hybridization indicated that the EBV
genome was detected by polymerase chain reaction in one
case of normal gastric mucosa, but not by in situ hybridi-
zation [19].

Recently, one study examining EBV in gastric carcinomas
and gastric stump carcinomas and found that EBER1/2
transcripts were restricted to the carcinoma cells in both
types of cases [ 12,29 The absence of EBER1 transcripts in


preneoplastic gastric lesions (intestinal metaplasia and
dysplasia) but their presence in two distinct types of gas-
tric carcinoma further supports the theory that EBV can
infect only neoplastic gastric cells.

Our study showed that LMP-1 expression was not found
in EBV-positive carcinomas or their precursor lesions,
which is in line with previous observations [28,30-32].
The absence of LMP-1 expression in EBVaGCs suggests
that LMP-1 may not be necessary for such tumors, at least
not for sustaining their already established malignant
state. Rather, LMP-1 may participate in the earlier stage of
tumor development and may be down-regulated thereaf-
ter. Alternatively, the lack of LMP-1 may reflect the result
of clonal selection of LMP-1-negative tumor cells by
immunologic pressure because EBV-specific cytotoxic T
cells are potentially directed against the viral LMPs rather
than against EBV nuclear antigen 1. Yanai et al. [15]
reported that EBV-LMP-1 was observed in cases of
atrophic gastric mucosa. However, this finding is not
likely to be confirmed due to the inconsistent results from
in situ hybridization and due to the fact that the research-
ers used a biotin method. It has been demonstrated that
cross-reactivity can occur and that the interpretation of
positive immunohistochemical results should always be
done in the context of transcript analysis by reverse tran-
scription polymerase chain reaction [7,28] and EBER1 in
situ hybridization [4].

In this population, a 5.1% prevalence of EBV in gastric
cancer was observed, comparable with the prevalence of
EBV detected in gastric adenocarcinomas worldwide


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1.0-




08-




0 6-




0.4-









00-


I I I I I I
0 50 100 150 200 250


EBV
SAll others

_ Positive


time


Figure 3
Survival graph of EBV associated gastric cancer and non-EBV associated gastric cancer.


Table 2: Association of EBV expression and clinicopathologic variables


Univariate analysis


EBV


Gender


Age


Lymph node


Type


Stage


Negative
Positive

Female
Male

<65
>=65

Negative
Positive

Well/Moderately
Poorly

I or II
III or IV


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95% C.I.


Lower


0.71


0.68


0.61


1.87


1.18


1.5 1


Upper


3.27


1.36


1.22


4.72


2.39


3.03


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Table 3: Multivariate analysis: Association of EBV, lymph node
status and tumor stage of gastric cancer with patient's survival


Multivariate analysis


Lymph node


Stage


RR


Negative 1.00
Positive 1.56

Negative 1.00
Positive 2.47

I or II 1.00
III or IV 1.49


[4,25,33] and indicating that the ol
EBV in gastric carcinomas is indeper
regions [11,29]. Our observations of
and younger patient age are in agree
several previous studies [3,33,34]. Ho
first large study of this type condu
States. Our male-to-female ratio of
highest described so far. A male:fem
reported in one large cohort Dutch st

In short, this study, evaluating the
infected cells in a large cohort of pati
prehensive cancer center in U.S.A, c
restrictly expressed in tumor cells an
younger male patients. Further
infected tumor cells were observed
with metastasis. The detection of EBV
cells in all of the lymph nodes involve
noma suggests simultaneous replic
tumor cells. The predominantly ma
tively younger age observed in our sti
ciation between EBV-infected gastri
factors, such as life style.

Competing interests
The authors declare that they have no

Authors' contributions
CDT and WF carried out the pathol
collection, data review, participated i
coordination. WL, TK, and SA particip
and drafting the manuscript. OL car
YY, KX, and JY participated in study de
and coordination. DT was the princi
the study and participated in all aspe
authors read and approved the final r

Acknowledgements
We thank Mr. Mannie for his assistance in the cc
microarrays, Mrs. Liy for EBV staining and Ms. T;


http://www.jeccr.com/content/28/1/14


editing support. This work is partially supported by an institutional grant of
the University of Texas M.D. Anderson Cancer Center.


References
95% C.. I. Burke AP, Yen TSB, Shekitka KM, et al.: Lymphoepithelial carci-
Lower Upper p noma of the stomach with Epstein-Barr virus demonstrated
by polymerase chain reaction. Mod Pathol 1990, 3:377-380.
2. Shibata D, Tokunaga M, Uemura Y, et at: Association of Epstein-
0.72 3.37 0.26 Barr virus with undifferentiated gastric carcinoma with
intense lymphoid infiltration. Am j Pathol 1991, 139:469-474.
3. Shibata D, Weiss LM: Epstein-Barr virus-associated gastric ade-
nocarcinoma. Am Pathol 1992, 140:769-774.
1.48 4.11 0.01 4. Zur Hausen A, van Rees BP, van Beekj, et al: Epstein-Barr virus in
gastric carcinomas and gastric stump carcinomas: a late
event in gastric carcinogenesis. J Clin Pathol 2004, 57:487-491.
5. Takada K: Epstein-Barr virus and gastric carcinoma. Mol Pathol
1.01 2.20 0.04 2000, 53:255-261.
6. Herrmann K, Niedobitek G: Epstein-Barr virus-associated carci-
nomas: facts and fiction. j Pothol 2003, 199:140-145.
7. zur Hausen A, Brink AA, Craanen ME, et a.: Unique transcription
overall prevalence of pattern of Epstein-Barr virus (EBV) in EBV-carrying gastric
dent of geographic adenocarcinomas: expression of the transforming BARFI
gene. Cancer Res 2000, 60:2745-2748.
male predominance 8. zur Hausen A, van Grieken NC, Meijer GA, et al: Distinct chromo-
ment with those of somal aberrations in Epstein-Barr virus-carrying gastric car-
wever, ours was the cinomas tested by comparative genomic hybridization.
Gastroenterology 2001, 121:612-618.
cted in the United 9. Uozaki H, Fukayama M: Epstein-Barr Virus and Gastric Carci-
9.2 was among the noma Viral Carcinogenesis through Epigenetic Mecha-
ale ratio of 9.8 was nisms. Int Clin Exp Pathol 2008, 1(3):198-216.
ale radio of 9.8 was 10. Ott G, Kirchner T, Muller-Hermelink HK: Monoclonal Epstein-
udy [4]. Barr virus genomes but lack of EBV-related protein expres-
sion in different types of gastric carcinoma. Histopathology
1994, 25:323-329.
distribution of EBV II. Imai S, Koizumi S, Sugiura M, et al: Gastric carcinoma: mono-
ents at a single com- clonal epithelial malignant cells expressing Epstein-Barr
confirms that EBV is virus latent infection protein. Proc Not! Acad Sci USA 1994,
91:9131-9135.
d predominately in 12. Yamamoto N, Tokunaga M, Uemura Y, et al: Epstein-Barr virus
ore, positive EBV- and gastric remnant cancer. Cancer 1994, 74:805-809.
n all lymph nodes 13. Gulley ML, Pulitzer DR, Eagan PA, et a.: Epstein-Barr virus infec-
in all lymp nodes tion is an early event in gastric carcinogenesis and is inde-
in metastatic tumor pendent of bcl-2 expression and p53 accumulation. Hum
ed with gastric carci- Pathol 1996, 27:20-27.
f and 14. Yanai H, Takada K, Shimizu N, et al: Epstein-Barr virus infection
ation EBV and in non-carcinomatous gastric epithelium. J Pathol 1997,
le gender and rela- 183:293-298.
dy suggest an asso- 15. Yanai H, Murakami T, Yoshiyama H, et at: Epstein-Barr virus-asso-
ciated gastric carcinoma and atrophic gastritis. J Clin Gastroen-
c cancer and other terol 1999, 29:39-43.
16. Middeldorp JM, Brink AA, Brule AJ van den, et al.: Pathogenic roles
for Epstein-Barr virus (EBV) gene products in EBV-associ-
ated proliferative disorders. Crit Rev Oncol Hematol 2003,
45:1-36.
competing interests. 17. World Health Organisation: Classification of tumours. Pathology and
genetics, tumours of the digestive system Lyon: IARC; 2000.
18. Deeb G, Wang j, Ramnath N, Slocum HK, Wiseman S, Beck A, Tan
D: Altered E-cadherin and epidermal growth factor receptor
ogy review and data expressions are associated with patient survival in lung can-
cer: a study utilizing high-density tissue microarray and
in study design and immunohistochemistry. Mod Pothol 2004, 17(4):430-9.
'ated in study design 19. Oda K, Tamaru J, Takenouchi T, Mikata A, Nunomura M, Saito H, et
a.: Association of Epstein-Barr virus with gastric carcinoma
ried analyzing data. with lymphoid stroma. Am j Pothol 1993, 143:1063.
sign, data collection 20. Tan D, Deeb G, Wang J, Slocum HK, Winston J, Wiseman S, Beck A,
ple investigation of Sait S, Anderson T, Nwogu C, Ramnath N, Loewen G: HER-2/neu
protein expression and gene alteration in stage I-IlIA non-
cts of this work. All small-cell lung cancer: a study of 140 cases using a combina-
nanuscript. tion of high throughput tissue microarray, immunohisto-
chemistry, and fluorescent in situ hybridization. Diagn Mol
Pathol 2003, 12(4):201-1 I.
21. Tan D, Qiang Li, George Deeb, Ramnath N, Slocum H, Cheney R,
instruction of the tissue Anderson T, Brooks J, Wiseman S, Loewen G: Thyroid Transcrip-
amara K. Locke for her tion Factor- (TTF-l) Expression Prevalence and Its Clinical
Implications in Non-Small-Cell Lung Cancer: A High-


Page 8 of 9
(page number not for citation purposes)








Journal of Experimental & Clinical Cancer Research 2009, 28:14 http://www.jeccr.com/content/28/1/14




Throughput Tissue Microarray and Immunohistochemistry
Study. Human Pathol 2003, 34/6:597-604.
22. Aljada IS, Ramnath N, Donohue K, Harvey S, Brooks JJ, Wiseman SM,
Khoury T, Loewen G, Slocum HK, Anderson TM, Bepler G, Tan D:
Upregulation of the tissue inhibitor of metalloproteinase-l
protein is associated with progression of human non-small-
cell lung cancer. J Clin Oncol2004, 22(16):3218-29.
23. Kaplan EL, Meier P: Nonparametric estimation from incom-
plete observation. j Am Stat Assoc 1958, 53:457-481.
24. Agresti A: Categorical Data Analysis. New York, NY, John Wiley
& Sons; 1990:306-347.
25. Hoshikawa Y, Satoh Y, Murakami M, Maeta M, Kaibara N, Ito H,
Kurata T, Sairenji T: Evidence of lytic infection of Epstein-Barr
virus (EBV) in EBV-positive gastric carcinoma. J Med Virol
2002, 66:351-359.
26. Kijima Y, Ishigami S, Hokita S, Coriyama Akiba S, Eizuru Y, Aikou T:
The comparison of the prognosis between Epstein-Barr virus
(EBV)-positive gastric carcinomas and EBV-negative ones.
Cancer Letters 2003:33-40.
27. Oda Kenji I, Koda Keiji I, Takiguchi Nobuhiro 2, Nunomura Masao
3, Seike Kazuhiro I, Miyazaki Masaru: Detection of Epstein-Barr
virus in gastric carcinoma cells and surrounding lym-
phocytes. Gostric Cancer 2003, 6(3):173-178.
28. van Beek J, zur Hausen A, Klein Kranenbarg E, et a.: EBV-positive
gastric adenocarcinomas: a distinct clinicopathological
entity with a low frequency of lymph node involvement. J Clin
Oncol 2004, 22:664-670.
29. Chang MS, Lee JH, Kim JP, Kim HS, Lee HS, Kim CW, Kim YI, Kim
WH: Microsatellite instability and Epstein-Barr virus infec-
tion in gastric remnant cancers. Pathol Int 2000, 50(6):486-92.
30. Luo Bing, Wang Yun, Wang Xiao-Feng, Liang Hua, Yan Li-Ping, Huang
Bao-Hua, Zhao Peng: Expression of Epstein-Barr virus genes in
EBV-associated gastric carcinomas. World Gastroenterol 2005,
I (5):629-633.
31. Hoshikawa Y, Satoh Y, Murakami M, et a.: Evidence of lytic infec-
tion of Epstein-Barr virus (EBV) in EBV-positive gastric car-
cinoma. J Med Virol 2002, 66:351-359.
32. Herrera-Goepfert R, Akiba S, Koriyama C, Ding S, Reyes E, Itoh T,
Minakami Y, Eizuru Y: Epstein-Barr virus-associated gastric car-
cinoma: Evidence of age-dependence among a Mexican pop-
ulation. WorldJ Gostroenterol2005, I I(39):6096-103.
33. Tokunaga M, Land CE, Uemura Y, et al.: Epstein-Barr virus in gas-
tric carcinoma. Am Pathol 1254, 143:1250-1993.
34. Kijima Y, Hokita S, Takao S, et al.: Epstein-Barr virus involvement
is mainly restricted to lymphoepithelial type of gastric carci-
noma among various epithelial neoplasms. J Med Virol 2001,
64:513-518.













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