The Role of CDX2 in Intestinal Metaplasia Evaluated Using Immunohistochemistry.

Abstract

Intestinal metaplasia (IM) has been regarded as a premalignant condition. This study evaluated the role of the transforming factor CDX2 according to the severity and type of IM.
This analysis was performed on 383 subjects with IM in the antrum and/or body, with diagnoses that were categorized as controls, dysplasias, and gastric cancers. The IM grades were classified into four groups as negative, mild, moderate or severe using the updated Sydney scoring system. The IM subtypes were categorized as type I, type II, and type III using high iron diamine and alcian blue (pH 2.5) staining. The CDX2 expression in the IM foci was evaluated using immunohistochemistry in specimens from the antrum and/or body.
CDX2 expression increased according to IM severity (p=0.001) but was not associated with the IM subtype (p=0.881) in the antrum specimens. Similarly, CDX2 expression increased according to the IM grade (p=0.001) but was not associated with the IM subtype (p=0.755) in the body specimens. CDX2 expression was also increased according to baseline disease in the antrum, especially dysplastic and GC group (p=0.003), but not in the body (p=0.582). However, status of Helicobacter pylori infection was not associated with CDX2 expression in the antrum (p=0.692) and body (p=0.271).
These results show that CDX2 expression is associated with the IM grade regardless of the IM subtype and that it was more frequent in the dysplasia group. These results suggest that CDX2 expression might play an important role in the progression of IM in various environments that can affect neoplastic change.

Full text

Page 1

ORiginal Article
Gut and Liver, Vol. 6, No. 1, January 2012, pp. 71-77
The Role of CDX2 in Intestinal Metaplasia Evaluated Using
Immunohistochemistry
Byoung Hwan Lee*, Nayoung Kim*,†, Hye Seung Lee‡, Jung Mook Kang†, Hyun Kyung Park*, Hyun Jun Jo*, Cheol Min
Shin*, Sang Hyub Lee*, Young Soo Park*,†, Jin Hyeok Hwang*,†, Jin-Wook Kim*,†, Sook-Hyang Jeong*,†, Dong Ho Lee*,†,
Hyun Chae Jung†, and In Sung Song†
*Department of Internal Medicine, Seoul National University Bundang Hospital, Seongnam, †Department of Internal Medicine and Liver
Research Institute, Seoul National University College of Medicine, Seoul, and ‡Department of Pathology, Seoul National University Bundang
Hospital, Seongnam, Korea
Background/Aims: Intestinal metaplasia (IM) has been re-
garded as a premalignant condition. This study evaluated the
role of the transforming factor CDX2 according to the severity
and type of IM. Methods: This analysis was performed on
383 subjects with IM in the antrum and/or body, with diag-
noses that were categorized as controls, dysplasias, and gas-
tric cancers. The IM grades were classifi ed into four groups
as negative, mild, moderate or severe using the updated
Sydney scoring system. The IM subtypes were categorized as
type I, type II, and type III using high iron diamine and alcian
blue (pH 2.5) staining. The CDX2 expression in the IM foci
was evaluated using immunohistochemistry in specimens
from the antrum and/or body. Results: CDX2 expression
increased according to IM severity (p=0.001) but was not as-
sociated with the IM subtype (p=0.881) in the antrum speci-
mens. Similarly, CDX2 expression increased according to the
IM grade (p=0.001) but was not associated with the IM sub-
type (p=0.755) in the body specimens. CDX2 expression was
also increased according to baseline disease in the antrum,
especially dysplastic and GC group (p=0.003), but not in the
body (p=0.582). However, status of Helicobacter pylori infec-
tion was not associated with CDX2 expression in the antrum
(p=0.692) and body (p=0.271). Conclusions: These results
show that CDX2 expression is associated with the IM grade
regardless of the IM subtype and that it was more frequent in
the dysplasia group. These results suggest that CDX2 expres-
sion might play an important role in the progression of IM in
various environments that can affect neoplastic change. (Gut
Liver 2012;6:71-77)
Key Words: Helicobacter pylori; Intestinal metaplasia; CDX2;
Correspondence to: Nayoung Kim
Department of Internal Medicine, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam 463-707, Korea
Tel: +82-31-787-7008, Fax: +82-31-787-4051, E-mail: nayoungkim49@empal.com
Received on June 10, 2011. Accepted on September 13, 2011.
pISSN 1976-2283 eISSN 2005-1212 http://dx.doi.org/10.5009/gnl.2012.6.1.71
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0)
which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Grade; Subtype
INTRODUCTION
Gastric cancer (GC) is one of the most common malignancies
in Korea. The risk factors for GC are known to be Helicobacter
pylori infection, atrophic gastritis (AG), and intestinal metapla-
sia (IM). IM is defined as the replacement of gastric columnar
epithelial cells by cells with intestinal morphology, which ap-
pears in the multi-step progression of GC, especially in the
intestinal type GC as proposed by Correa.1 According to Correa
hypothesis, H. pylori was thought to play a crucial role in the
formation and progression of IM. That was generally accepted
in the epidemiologic and population-based study,2,3 though
arguing data were existed.4 In addition, there was not com-
mon consensus about the effect of H. pylori on the subtype or
progression of IM in these epidemiologic studies. Furthermore,
IM was considered to be irreversible change in spite of H. pylori
eradication.5 Therefore, the beneficial effect of H. pylori eradica-
tion was not clear in the area of already formed IM.
In regard to the property of IM itself, it is important to search
the high risk of IM in the gastric carcinogenesis and frequent
surveillance may be needed in this IM. Many investigations
have researched the molecular and genetic mechanisms of IM,
and have attempted to classify IM according to the risk for
developing GC. Jass and Filipe6 suggested a classification of
IM based on morphology and classic mucin staining, and the
incomplete type (type II and III) was considered to have a higher
risk for GC than the complete type (type I).7,8 Another classifica-
tion of IM was suggested according to the gastric phenotype as
well as the intestinal phenotype such as gastric (G) type, gastric-

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72 Gut and Liver, Vol. 6, No. 1, January 2012
and-intestinal (GI) mixed type, and intestinal (I) type.9 Many
arguments still existed in the classification of IM for the cancer
risk in spite of these efforts.10 Furthermore, the extensive IM was
thought to be more important as a gastric carcinogenesis than
subtype.11
CDX2, a member of the caudal-related homeobox gene family
and intestinal-specific transcriptional factor, plays an important
role in the development of small and large intestine.12 Aberrant
expression of CDX2 was observed in the gastric IM and vari-
ous site of adenocarcinoma.13,14 The crucial role of CDX2 in the
formation of IM has been identified in the transgenic mice and
gastric carcinoma was observed in the CDX2 transgenic mice.15
In our previous study, CDX1 and CDX2 were found to play an
important role in the formation of IM and in the progression to
dysplasia and GC in human gastric specimens using real-time
polymerase chain reaction (RT-PCR) method.16 However, this is
only gene level that it is necessary to be proved by protein level.
Nevertheless, there have not been well-known about the roles of
CDX2 transcriptional factor in the subtype or progression of IM
until now. In addition, there were few data regarding the role of
CDX2 in the progression of gastric carcinogenesis related with
H. pylori infection by the immunohistochemistry (IHC) method.
From this background, we tried to investigate the role of the
CDX2 transcriptional factor in the formation and progression of
IM among the diverse gastric disorders such as gastritis, dyspla-
sia, and GC using IHC method.
MATERIALS AND METHODS
1. Study subjects
Three hundred and eighty-three patients were enrolled from
2003 to 2007 in Seoul National University Bundang Hospital.
Approximately 50% of the patients had gastrointestinal symp-
toms within 3 months, but most of them received gastroscopy
for the GC screening. The subjects were categorized into three
groups, mainly depending on the gastroscopy findings and his-
tological diagnosis; a control group and two different disease
groups (dysplasia and GC). As CDX2 expression usually appears
in the presence of IM, the study pool for IHC of CDX2 was se-
lected when there was microscopic IM finding in either antrum
or body after H&E staining. In GC group, poorly differentiated
or signet ring cell type GC was excluded because these types
of GC are usually thought to be not associated with multistep
progression of GC. Subjects with a history of gastric surgery, H.
pylori eradication therapy or systemic diseases requiring chronic
medication were excluded. The Institutional Review Board at
Seoul National University Bundang Hospital approved this
study, and written informed consent was obtained from all of
the participants.
2. H. pylori testing and histology
To determine the presence of current H. pylori infection, 10
biopsy specimens were obtained for the three types of H. pylori
testing (histology, Campylobacter like organism [CLO] test [Delta
West, Bentley, Australia], and culture). Among them, two biopsy
specimens (one each from the greater curvature and lesser cur-
vature) of the antrum and body were fixed in formalin, respec-
tively, and used for evaluation of the presence of gastric atrophy
(loss of appropriate glands including both metaplastic and non-
metaplstic atrophy) and IM (by H&E staining). The presence of
atrophy on any of two specimens in the antrum and body were
averaged regarding gastric atrophy, and the same method was
applied to IM. For determination of H. pylori, modified Giemsa
staining was performed for two specimens from each of the an-
trum and body, and histological features of gastric mucosa were
recorded using the updated Sydney scoring system. All biopsies
were examined independently by two experienced patholo-
gists (H.S.L. and Hee Eun Lee), who were unaware of the patient
details. In the event of disagreement, the biopsies were reex-
amined by these two pathologists until agreement was reached.
One specimen from each of the lesser curvature of the antrum
and body was used for CLO test. Two specimens from each of
the antrum and body were used for culture. If any one of these
three H. pylori tests were positive, then the host was regarded
as having an ongoing H. pylori infection. The anti-H. pylori im-
munoglobulin G (Genedia H. pylori ELISA; Green Cross Medical
Science Co., Eumsung, Korea) was also determined when the
three above-mentioned H. pylori tests were negative.17 Korean
strain was used as antigen in this H. pylori antibody test. When
the H. pylori serology was positive, these subjects were regarded
as having past H. pylori infection and classified as H. pylori-
positive.
3. HID-AB2.5 staining for metaplasia subtyping
Sulfated (brown) and acidic non-sulfated (blue) mucosub-
stances were simultaneously stained with high iron diamine
and alcian blue (pH 2.5) (HID-AB2.5). The IM was classified as
follows: type I, mature absorptive cells and goblet cells, the lat-
ter secreting sialomucins; type II, few or absent absorptive cells,
presence of columnar ‘intermediate’ cells in various stages of
differentiation secreting sialomucins or, occasionally, sulfomu-
cins, or both; type III, columnar intermediate cells secreting pre-
dominantly sulfomucins and goblet cells secreting sialomucins
or sulfomucins, or both.18 If more than one HID subtype of IM
was present, it was classified as the dominant IM phenotype. In
addition, the pathologists (H.S.L. and H.E.L) were blinded to the
clinical information of the patients.
4. IHC for CDX2
Tissues from the antrum and body were fixed in 10% neutral
buffered formalin, paraffin-embedded, and then cut into sec-
tions (4 μm). IHC was performed by the standard avidin-biotin
complex method. A monoclonal antibody for CDX2 (BioGenex;
San Ramon, CA, USA; dilution 1:200) was used as a primary

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Lee BH, et al: Role of CDX2 in Intestinal Metaplasia 73
antibody and anti-mouse immunoglobulin G (Sigma, St Louis,
MO, USA) labeled with biotin was used as a secondary antibody.
Negative controls were treated similarly with the exception of
the primary antibodies. The whole areas of slides were evalu-
ated and positive CDX2 IHC was defined by at least more than
10% staining of the IM foci (Fig. 1).19 Among 383 subjects, the
specimen of 42 in the antrum and that of 155 in the body were
excluded from CDX2 staining due to no observable foci of IM,
respectively. In addition, 49 and 24 subjects with IM foci could
not be used in CDX2 IHC, mainly due to loss of paraffin block.
Finally, the specimen of the antrum (293 subjects) and body (204
subjects) were used in CDX2 IHC.
5. Statistical analysis
All statistical analyses were performed using SPSS software
version 15.0 (SPSS Inc., Chicago, IL, USA). The Pearson chi-
square test was used to compare pairs of groups and Student’s
t-test was used to compare the mean value of each group. p-
value below 0.05 was considered as a statistically significant.
RESULTS
1. Baseline characteristics
The 383 subjects with histological IM was classified to three
groups: control group (n=165), dysplastic group (n=67), and GC
group (n=151). The baseline characteristics are shown in Table
1. The mean age and the proportion of male were higher in the
dysplastic and GC group, respectively (p<0.001). The severity of
AG was higher in dysplasia and GC group than control group in
the body specimens (p<0.001), but has no significant difference
in the antrum specimens (p=0.769) (Table 1).
2. Pathological features of IM
The distribution and pathologic features of IM that were de-
termined by an updated Sydney classification were shown in
Table 2. One antrum specimen was not obtained for histology
in control group and one body specimen in GC group. There-
fore, 164 antrum specimens were used for the analysis of IM
grade and extent in control group and 150 body specimens in
GC group (Table 2). More extended distribution of IM was ob-
served in the dysplasia and GC groups than the control group
Table 1. Baseline Characteristics
Characteristic
Control
(n=165)
Dysplasia
(n=67)
Gastric
cancer
(n=151)
p-value
Age, yr
Sex
Male
Female
H. pylori positivity
H. pylori positive
H. pylori negative
Atrophic gastritis
Antrum
None
Mild
Moderate
Severe
Body
None
Mild
Moderate
Severe
58.6±12.663.3±8.465.0±9.4 <0.001*
<0.001†
93 (56.4)
72 (43.6)
52 (78.8) 114 (75.5)
15 (21.2) 37 (24.5)
0.326†
146 (88.5)
19 (11.5)
63 (94.0) 128 (84.8)
4 (6.0) 23 (15.2)
0.769†
34 (31.8)
46 (43.0)
20 (18.7)
7 (6.5)
11 (32.4)
10 (29.4)
11 (32.4)
2 (5.9)
32 (39.0)
25 (30.5)
15 (18.3)
10 (12.2)
<0.001†
63 (64.3)
16 (16.3)
18 (18.4)
1 (1.0)
11 (28.9)
10 (26.3)
13 (34.2)
4 (10.5)
26 (32.1)
19 (23.5)
29 (35.8)
7 (8.6)
Data are presented as mean±SD or number (%). Bold indicates statis-
tical significance.
*One-way ANOVA test; †Chi-square test.
Fig. 1. Immunohistochemistry of CDX2. (A) Negative, (B) weak positive, and (C) strong positive (×200).

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74 Gut and Liver, Vol. 6, No. 1, January 2012
(p<0.001). That is, dysplasia and cancer group showed IM in
both of antrum and body, 65.7% and 62.7%, respectively, but
control group showed 31.1%. In addition, The IM grade deter-
mined by an updated Sydney classification was more severe in
the dysplastic and GC groups than in the control group, in both
antrum (p=0.001) and body (p<0.001). However, there was no
significant difference when comparing according to the sub-
types of IM in both the antrum and body (Table 2).
3. CDX2 IHC
The specimen of the antrum (293 subjects) and body (204
subjects) were stained by CDX2 IHC. CDX2 expression was
demonstrated in 219 (74.7%) and 169 (82.8%), respectively.
CDX2 expression was increased with IM grade in the antrum
(p=0.001) and body (p=0.001). However, CDX2 expression was
not associated with IM subtype in the antrum and body (Table 3).
In addition, CDX2 expression was higher in the dysplasia and
GC group than control group in the antrum (p=0.003), but not
in the body (p=0.582) (Table 3).
4. H. pylori status
There was no significant difference between H. pylori-positive
and -negative group in terms of IM grade, IM subtype, and
CDX2 expression in both of antrum and body (Table 4).
DISCUSSION
In this study, we demonstrated that CDX2 expression was as-
sociated with IM grade, but not subtype. These results suggest
that CDX2 plays a role in the formation of IM. The biology of
gastric IM has been investigated regarding variant molecular
and genetic factors such as Shh, BMP4, CDX1, CDX2, and C-
myc et al., but the formation and progression of IM is still dif-
ficult and undisclosed area.20 Among the efforts, the transgenic
mice model has shown that CDX transcriptional factors play
Table 2. The Relationship between Underlying Disease Groups and
IM Extension, Grade, and Subtype
VariableControlDysplasia
Gastric
cancer
p-value*
Extension of IM
One side
Both side
Grade of IM
Antrum
None
Mild
Moderate
Severe
Body
None
Mild
Moderate
Severe
Subtype of IM
Antrum
Type I
Type II
Type III
Body
Type I
Type II
Type III
<0.001
113 (58.9)
51 (27.0)
23 (12.0)
44 (23.3)
56 (29.2)
94 (49.7)
0.001
23 (56.1)
75 (51.7)
55 (38.2)
11 (21.2)
8 (19.5)
20 (13.8)
24 (16.7)
15 (28.8)
10 (24.4)
50 (34.5)
65 (46.9)
26 (50.0)
<0.001
90 (59.6)
41 (40.6)
30 (31.3)
4 (11.8)
15 (9.9)
21 (20.8)
21 (21.9)
10 (29.4)
46 (30.5)
39 (38.6)
45 (46.9)
20 (58.8)
0.427
18 (50.0)
45 (42.1)
39 (38.2)
4 (11.1)
20 (18.7)
13 (12.7)
14 (38.9)
42 (39.3)
50 (49.0)
0.150
21 (39.6)
29 (28.7)
11 (37.9)
14 (26.4)
20 (19.8)
3 (10.3)
18 (34.0)
52 (51.5)
15 (51.7)
Data are presented as number (%). Bold indicates statistical signifi-
cance.
IM, intestinal metaplasia.
*Chi-square test.
Table 3. IM Grade, Subtype, and Disease Status according to CDX2
Expression
Variable CDX2 negativeCDX2 positive p-value*
Grade of IM
Antrum
Mild
Moderate
Severe
Body
Mild
Moderate
Severe
Subtype of IM
Antrum
Type I
Type II
Type III
Body
Type I
Type II
Type III
Disease group
Antrum
Control
Dysplasia+GC
Body
Control
Dysplasia+GC
0.001
43 (34.7)
26 (20.3)
5 (12.2)
81 (65.3)
102 (79.7)
36 (87.8)
0.001
26 (28.6)
8 (9.8)
1 (3.2)
65 (71.4)
74 (90.2)
30 (96.8)
0.881
9 (25.7)
22 (22.9)
23 (23.7)
26 (74.3)
74 (77.1)
74 (76.3)
0.755
7 (13.7)
9 (9.3)
5 (18.5)
44 (86.3)
88 (90.7)
22 (81.5)
0.003
39 (34.8)
35 (19.3)
73 (65.2)
146 (80.7)
0.582
12 (19.4)
23 (16.2)
50 (80.6)
119 (83.8)
Data are presented as number (%). Bold indicates statistical signifi-
cance.
IM, intestinal metaplasia; GC, gastric carcinoma.
*Chi-square test.

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Lee BH, et al: Role of CDX2 in Intestinal Metaplasia 75
a crucial role in the formation of IM.21 Furthermore, GC was
developed in the CDX2 transgenic mice model.15 In the present
study, the CDX2 expression was found to be 74.7% and 82.8%
in the antrum and body, which are quite similar to the previ-
ous data, about 80%.22-24 IHC for CDX2 was performed for the
positive IM specimen, our data suggest that IM could be present
without CDX2 in human stomach, and the CDX transcriptional
factors might not be the unique factors in the IM formation.
That is, other factors such as bile reflux, genetic or environmen-
tal factor might intermediate IM formation, especially in case of
CDX-2 negative subjects.
There has been a great diversity of opinions in the role of
CDX2 in gastric carcinogenesis. Some reports even claimed that
CDX2 transcriptional factor may act as a tumor suppressor.25,26
In the present study, CDX2 expression was increased in the neo-
plastic groups (dysplasia and GC) in the antrum, but not in the
body, suggesting that CDX2 is not directly with gastric carci-
nogenesis. Instead, CDX2 looks like to be important in the pro-
gression of IM to the more severe and widespread form. There is
a high chance of development of dysplasia and intestinal type
of GC in this environment. In our previous study, we preformed
quantitative analysis using RT-PCR for the evaluation of the
role of CDX1 and CDX2 in the formation of IM and the progres-
sion to dysplasia and GC.16 In this study which is not identical
pool with the present study, CDX2 transcriptional factor was
associated with both IM grade and subtype. Taken together,
quantitative study such as RT-PCR might be a more powerful
tool regarding the role of CDX2 in the research of IM biology.
However, RT-PCR is based on transcriptional level but not on
the translational level that IHC research could be very definite
in supporting data. In the present study, CDX2 expression by
IHC was correlated with IM grade, which is the identical finding
of RT-PCR. Autoregulatory effect of CDX2 transcriptional factor
has been assumed for this relationship. That is, CDX2 could au-
toregulate itself by binding CDX2 promotor area.27 We also tried
to perform the IHC of CDX1 but we could not find the adequate
antibody for human study in spite of several times of trial.
The direct relationship of CDX2 expression and H. pylori
infection was not confirmed.22,28-30 However, disappearance of
CDX2 expression was observed by H. pylori eradication in the
level of gastritis in the absence of definitive IM.29,30 However,
H. pylori eradication did not cause any effects on the CDX2
expression when IM was already documented.22,28 In addition,
a recent study has shown that low degree of CDX2 expres-
sion was observed more than 50% in subjects with normal and
healthy gastric mucosa without evidence of H. pylori infection,
and bile reflux is one of the assumptive mechanisms in that
case.30,31 In the present study, the prevalence of H. pylori was
not associated with IM grade, IM subtype and CDX2 expres-
sion. However, CDX2 expression was significantly higher in the
H. pylori-positive group than -negative group when RT-PCR
method was used in our previous study.16 There could be several
reasons. First, CDX2 IHC method may have limitation as the
interpretation is not simple, which is different from quantitative
study. Another reason could be study design. That is, the present
study was not age and sex-matched study, which is different
from the previous study. In addition, the prevalence of H. pylori
in the control group of the present study was 88.5%, which was
not different from dysplasia or GC group. This prevalence of H.
pylori in the control group is much higher than 51.4% in com-
parison to the population-based study during similar periods.32
The main reason of high prevalence of H. pylori, 88.5% is selec-
tion criteria of the present study. That is, as CDX2 expression
usually appears in the presence of IM the study pool for IHC of
CDX2 was selected when there was microscopic IM finding in
either antrum or body regardless of control, dysplasia, and GC
in the present study. This result suggests that H. pylori plays an
important role in the formation of IM.
Incomplete type (type II and III) of IM was considered to be
more important in the carcinogenesis than complete type (type I),
but statistical significance was usually marginal.7,8 Instead, a lot
of studies demonstrated that the extension of IM was important
risk for the gastric carcinogenesis.11,33-35 In the present study, IM
grade and extension were more important than IM subtype with
GC progression in the background IM. This finding is consis-
tent with the results of prior studies demonstrating that the IM
subtype was less important than the extension or grade of IM.11
There have been few data regarding the IM grade as a high risk
Table 4. IM Grade, Subtype, and CDX2 Expression according to H.
pylori Status
Variable
H. pylori
negative
H. pylori
positive
p-
value*
Grade of IM
Antrum
None/Mild/Moderate/Severe
Body
None/Mild/Moderate/Severe
Subtype of IM
Antrum
Type I/II/III
Body
Type I/II/III
CDX2 expression
Antrum
Negative
Positive
Body
Negative
Positive
0.512
6/14/17/9 35/131/127/43
0.768
17/11/12/6 134/90/84/28
0.619
6/15/1130/92/91
0.505
7/12/6 46/89/23
0.692
9 (12.2)
23 (10.5)
65 (87.8)
196 (89.5)
0.271
6 (17.1)
20 (11.8)
29 (82.9)
149 (88.2)
Data are presented as number (%).
IM, intestinal metaplasia.
*Chi-square test.

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76 Gut and Liver, Vol. 6, No. 1, January 2012
factor for the gastric carcinogenesis. However, the present study
suggests that IM grade might be an important factor as IM ex-
tension for the gastric carcinogenesis.
In conclusion, CDX2 expression was associated with IM grade
regardless of IM subtype, and it was frequent in the dysplasia
group. These results suggest that CDX2 expression might play
an important role in the progression of IM which environments
provide neoplastic change.
CONFLICTS OF INTEREST
No potential conflict of interest relevant to this article was
reported.
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