ORIGINAL ARTICLE

Histological risk markers for non-cardia early gastric cancer

Pattern of mucin expression and gastric cancer
Akiko Shiotani & Ken Haruma & Noriya Uedo &
Hiroyasu Iishi & Ryu Ishihara & Masaharu Tatsuta &
Mitutaka Kumamoto & Yukinori Nakae &
Shingo Ishiguro & David Y. Graham
Received: 22 May 2006 / Accepted: 23 August 2006 / Published online: 13 October 2006
# Springer-Verlag 2006
Abstract There are limited data regarding the prognostic
value of the pattern of mucin expression in IM. To examine
the role of the type of IM and pattern of mucin expression
in IM as histological risk markers of gastric cancer, 80
patients with a history of endoscopic mucosal resection
(EMR) for early gastric cancer and 80 sex and age-matched
controls were studied. Serum levels of pepsinogen (PG)
were measured by RIA, and MUC2, MUC5AC and MUC6
were evaluated immunohistochemically. There is a signif-
icant association between types of IM and atrophic scores
or PG levels. The most incomplete IM (type II and III)
preserving gastric mucin is the gastric and intestinal mixed
(GI) type, whereas the complete type is the intestinal (I)
type especially in the corpus lesser curve. Gastric cancer
was most significantly associated with incomplete IM in the
corpus lesser curve (OR=6.4; 95% CI, 2.021, p=0.002).
Asynchronous multiple lesions were associated with in-
complete IM in the corpus greater curve (OR=4.8; 95% CI,
1.416, p=0.01). Classification of IM obtained using fixed-
point biopsy samples may enhance the ability of surveil-
lance programs to detect patients at increased risk of gastric
cancer.
Keywords Early gastric cancer
.
Type III intestinal metaplasia
.
MUC2
.
MUC5AC
.
MUC6
Introduction
The pathogenesis of gastric cancer is a multifactorial
process in which both environmental and host-related
factors play significant roles. The risk of developing gastric
cancer is closely related to Helicobacter pylori-associated
progressive gastric inflammation [2, 5, 26, 32]. The degree
of damage is initially most severe in the antrum, but over
time the damage progresses into the gastric corpus and can
be visualized as an advancing atrophic border, which
involves the lesser curve more rapidly than the greater curve
[2, 8, 16, 22]. Intestinal type gastric cancers are thought to
evolve through a multi-step process starting with superficial
gastritis and progressing through atrophy, followed by the
development of dysplasia, and finally carcinoma [1]. The
role of intestinal metaplasia (IM) in the transition from
superficial gastritis to cancer is unclear. There are data
suggesting that cancer and IM arise from different cell
lineages such that IM may not be a precursor lesion but
rather is likely a marker for increased risk [6, 17, 29].
Virchows Arch (2006) 449:652659
DOI 10.1007/s00428-006-0300-8
A. Shiotani
:
K. Haruma
Department of Internal Medicine, Kawasaki Medical School,
Okayama, Japan
N. Uedo
:
H. Iishi
:
R. Ishihara
:
M. Tatsuta
Department of Gastrointestinal Oncology,
Osaka Medical Center for Cancer and Cardiovascular Diseases,
Osaka, Japan
M. Kumamoto
:
Y. Nakae
Department of Medicine, Aishinkai Nakae Hospital,
Wakayama, Japan
S. Ishiguro
Department of Pathology,
Osaka Medical Center for Cancer and Cardiovascular Diseases,
Osaka, Japan
D. Y. Graham
Department of Medicine,
VA Medical Center and Baylor College of Medicine,
Houston, TX, USA
A. Shiotani (*)
577 Matsushima,
Kurashiki, Okayama Prefecture 701-0192, Japan
e-mail: shiotani@med.kawasaki-m.ac.jp
Gastric IM was classified as complete (small intestine) or
incomplete (colonic) using immunohistochemical staining
techniques and into three types based on the staining pattern
of its mucins: I (complete), and II and III (incomplete). Type I
is the most common type; Paneth cells are present and goblet
cells secrete sialomucins. Types II and III are characterized
by the presence of columnar cells and goblet cells secreting
sialomucins and/or sulfomucins, and columnar cells secret
sialomucins in type II and sulfomucins in type III [12].
Although several studies have suggested that patients with
type III IM may be at a higher risk for development of
gastric cancer [3, 34], the predictive value of type I and
type II IM remains unclear.
Mucins are heavily glycosylated proteins that constitute
the major component of the mucous protective layer above
mucous surfaces. Core proteins for 12 human mucins
(MUC1, 2, 3, 4, 5AC, 5B, 6, 7, 8, 9,11,12) were identified
[15, 23, 30, 39]. The normal gastric mucosa shows cell type
specific expression of MUC1, 5AC, and 6, and does not
express MUC2. Alterations of the expression pattern of
mucins were described in carcinoma and IM. Loss of
expression of MUC5AC and increased mucin heterogeneity
were reported in gastric cancer, whereas under-expression
of MUC1, MUC5AC and MUC6 and de novo expression
of MUC2 were described in IM. IM was also classified
based upon the cell classification status using both gastric
and intestinal phenotypic markers [19, 3638]. In that
system, IM is divided into two major types; a gastric and
intestinal mixed (GI) type, and a solely intestinal (I) type.
MUC5AC and MUC6, galactose oxidase-Schiff, etc. are
markers of gastric phenotype, and MUC2, CD10, Villin,
sucrase, etc. are intestinal phenotypic markers [19, 3538].
In retrospective studies, type III IM was found in 75% to
90% of cases of intestinal type gastric cancer that
underwent surgical resections [13, 24]. The presence of
type III IM was also associated with an increased risk of
developing early gastric cancer [3, 14, 28] but not with
diffuse type gastric carcinoma [20].
IM is a manifestation of atrophic mucosa occurring in
patients with chronic H. pylori gastritis. Type I IM is com-
monly found in random biopsies of patients with H. pylori
infection and the prevalence increases with age. Because
detection of IM in routinely obtained endoscopic biopsy
materials is common, its presence alone is not considered a
reliable marker for identifying patients in whom surveil-
lance is indicated [11]. In addition, biopsies taken repeat-
edly from predetermined points showed that neither the
presence nor the type of IM was constant [7].
There are a number of studies suggesting that the
presence of type III IM may be a risk factor for the
intestinal type gastric cancer. However, the majority of
these studies involved surgical specimens of human
stomach with gastric cancer [8, 18, 24, 25] such that while
it is possible to conclude that type III IM is frequently
present in patients with gastric cancer, little can be said
regarding its predictive value. The same problem is present
regarding the role of altered mucin expression pattern in IM
as most of the studies have come from patients who already
had gastric cancers. There are no data regarding the
potential value of mucin expression pattern as a risk factor
for gastric cancer using fixed-point biopsy samples. We
previously used endoscospic biopsies from predetermined
sites and found that the presence of IM at the corpus lesser
curve was the prevalent finding in patients with early
gastric cancer [32].
The aim of the present study was to examine the
usefulness of typing of IM and the pattern of mucin
expression in IM. We compared the histopathological
findings in patients with a history of EMR for early gastric
cancer with a suitable control group to identify markers
useful to identify patients at increased risk of having or
developing gastric cancer.
Materials and methods
This was a case-control study in patients who previously
had endoscopic mucosal resection (EMR) for early gastric
cancer. The study was performed at the Osaka Medical
Center for Cancer and Cardiovascular Diseases and the
Aishinkai Nakae Hospital in Japan. Patients were enrolled
for the study between November 2003 and November
2005.
Patients
Patients with prior medical EMR for early stage, non-
cardiac intestinal type gastric cancer without lymph node
metastasis were age and sex-matched with control patients
who underwent endoscopy for follow-up of peptic ulcer or
for health screening. Patients were excluded if they had
received eradication therapy for H. pylori infection, used
anti-secretory drugs or non-steroidal anti-inflammatory
drugs (NSAIDs), or had predisposing factors of hemor-
rhagic disease or chronic diseases such as insulin-dependent
diabetes mellitus, cirrhosis or renal failure. Drinking and
smoking were defined as regular intake when respective
consumption was more than 35 g of ethanol and 5
cigarettes per day, respectively.
The study was approved by the Osaka Medical Center
for Cancer and Cardiovascular Diseases Ethical Committee,
and informed consent was obtained from each patient. The
biopsy samples from a subpopulation of these patients (40
controls and 40 gastric cancer patients) were also used in
studies regarding loss of Sonic Hedgehog as an indicator of
H. pylori-induced atrophic gastritis progressing to gastric
Virchows Arch (2006) 449:652659 653
cancer and for histological and serum risk markers for non-
cardia early gastric cancer [31, 32].
Endoscopic finding of gastric cancer
EMR was done in patients with intestinal type cancers in
which the depth of invasion was clinically limited to the
mucosa. Patients were followed up at least once per year after
EMR. Experienced endoscopists performed all endoscopies.
IM and atrophy grading
Two specimens each were obtained from the greater
curvature of the antrum 3 cm proximal to the pylorus and
the mid-point of the greater and lesser curvature of the
gastric body. A 4-point visual analogue scale was used to
grade the mucosal biopsies ranging from zero (absent/
normal) to three (maximal intensity) for IM and atrophy [4].
The grading was determined twice for each slide by a single
pathologist who was blinded to the previous histological
scores and other experimental results. The average histo-
logical scores were for the analyses.
Immunohistochemistry
Three adjacent sections 4 m thick were cut onto each of
three polylysine-coated glass slides. The sections were
deparaffinized, washed in phosphatebuffersaline (PBS)
and autoclaved for 10 min in sodium citrate buffer for
antigenic retrieval. For immunostaining, sections were
incubated with diluted primary mouse monoclonal antibodies
against MUC2, MUC5AC, MUC6 (Novocastra Laboratories,
Newcastle, UK) at a dilution of 1:200, 1:500, 1:500,
respectively, at room temperature for 1 h. After washing in
PBS, the sections were incubated for 1 h with an anti-mouse
immunoglobulin conjugated to horseradish peroxidase dex-
tran labeled polymer using an EnVision TM peroxidase
mouse system (DAKO, Carpinteria, CA, USA). The sections
were washed in PBS, stained in 0.05% diaminobenzidine
hydrochrolide (DAB) solution for 5 min, and counterstained
with haematoxylin. After dehydration with xylene, the
sections were mounted under a glass coverslip.
Subtyping IM
Biopsies were stained with Alcian blue (AB)/high-iron
diamine (HID) to categorise IM as complete type (Type I)
or incomplete type (Type II and III). Slides were immersed
in HID solution for 20 h at room temperature, then rinsed
with deionised water, and stained with 1% AB (pH 2.5) for
2 min. IM subtyping was done according to the system
used by Fillipe et al. [12] as follows: Type I, non-secretory
absorptive cells and sialomucin secreting goblet cells; Type
II, few absorptive cells, columnar cells secreting sialomu-
cin, and goblet cells secreting mainly sialomucin but
occasionally sulphomucin; Type III, columnar cells secret-
ing predominantly sulphomucin and goblet cells secreting
sialomucin or sulphomucin.
Assay for serum levels of pepsinogen (PG) I and PG II
Blood samples were collected just before endoscopic
examination. Serum levels of PG I and PG II were measured
by radioimmunoassay (RIA).
Diagnosis of H. pylori infection
H. pylori infection was diagnosed by specific IgG H. pylori
antibodies analyzed by an enzyme-linked immunosorbent
assay (ELISA) kit (E plate, Eiken Kagaku Inc, Tokyo).
Statistical analyses
Values were expressed as the meanSD or median and 25
75% range whichever was appropriate. MantelHaenszel
chi square analyses, KruskalWallis and the non-parametric
MannWhitney U test were performed to measure differ-
ences. Analysis of proportions among the three different
groups was performed using chi square analyses. Compar-
isons were made between those with cancer and the entire
control group and control subjects with IM. After compar-
ison by KruskalWallis, Dunns methods were used to
isolate the groups that differ from the others.
MantelHaenszel statistics were used to assess the
relationship between gastric cancer and histopathological
findings showing the odds ratio (OR) and 95% confidence
interval (CI). Risk factors for gastric cancer were analyzed
by multivariate logistic regression analyses. A two sided
p value of less than 0.05 was considered statistically signif-
icant. All statistical computations were performed using
SPSS (version 11.0 for Windows, SPSS Inc, Chicago, IL).
Results
The study groups consisted of 80 patients with a prior EMR
for non-cardiac early gastric cancer and 80 age and sex-
matched controls. Demographic and clinical characteristics
of the study groups are shown in Table 1. Multiple
malignant lesions were found in 30 patients (asynchronous
15, synchronous 15); the remaining 50 had single lesions.
Malignant lesions in 42 patients were located in the lower
stomach (antrum or angulus), and 29 patients had lesions in
the upper stomach (corpus). In the remaining nine patients,
malignant lesions were located in the two different
locations. Malignant lesions in 20 patients were depressed
654 Virchows Arch (2006) 449:652659
type and those in 50 patients were protruded type. Finally,
ten patients had both types of malignant lesions.
The IM scores for each region of the stomach were
significantly higher in the cancer group than in the control
group (Fig. 1). There is a significant association between
atrophic scores and types of IM in the corpus lesser curve
(p<0.001). The frequency of type III IM increased along with
the scores of atrophy (Fig. 2). The ratio between the concen-
tration of PG I and PG II (PG I/II) was significantly lower in
the type II and type III groups compared to the group without
IM (p<0.01). The PG I/II ratio was inversely related to the
presence of type III IM and was significantly lower (p<0.05)
in the type III group than in the type I group (Fig. 3).
In the gastric mucosa without IM, MUC5AC expression
was detected in the foveolar epithelium and mucous neck
cells of both the antrum and corpus. MUC6 expression was
detected in the pyloric glands of the antrum and the mucous
cells of the neck zone of the corpus. MUC2 was only
detected in the mucosa with IM. MUC2 expression
displayed a vacuolar staining in most goblet cells. In the
gastric mucosa with type I IM, MUC5AC in the goblet cells
was detected in 46% in the antrum and 13% in the corpus
(Fig. 4). In the gastric mucosa with type II and type III IM,
MUC5AC was detected in 83% and 94% of patients,
respectively, in the antrum and 57% and 90%, respectively,
in the corpus (Figs. 4 and 5). In contrast, expression of
MUC6 in IM in the antrum was present in 46% of patients
with type II IM and 50% of patients with type III (Table 2).
Fig. 2 Correlation between types of IM and atrophic scores in the
corpus lesser curve. The frequency of type III IM increased along with
the scores of atrophy (p<0.001, by KruskalWallis)
Fig. 3 Comparison of the ratio between the concentration of PG I and
PG II (PG I/II). The PG I/II was significantly lower in the type II and
type III groups compared to the control group (p<0.01). The PG I/II
ratio was inversely related to the presence of type III IM and was
significantly lower in the type III group than in the type I group
(p<0.05). *p<0.05, **p<0.01 by Dunns methods
Table 1 Patient demographic and clinical characteristics
Control (n=80) Cancer (n=80) p values
Age mean (SD) 68 (9) 68 (8) 0.85*
Sex male/female 60/20 60/20
Current smokers 31.3% 33.8% 0.80**
Regular alcohol intake 48.9% 41.3% 0.53**
H. pylori positive 82.5% 87.5% 0.51**
p values; *unpaired t test, **by MantelHaenszel chi square analyses
Fig. 1 Comparison of IM scores between patients with gastric cancer
() and control subjects (). The ends of the boxes define the 25th and
75th percentiles with the bold line at the median and error bars define
the 10th and 90th percentiles. *p<0.001; p values by MannWhitney
U test. AG, antrum greater curve; CG, corpus greater curve; CL,
corpus lesser curve
Virchows Arch (2006) 449:652659 655
Gastric cancer was associated with a higher frequency of
incomplete IM (types II and III) and IM expressing
MUC5AC (Table 3). The sensitivity and specificity of
detecting incomplete IM in either point were 86% and 65%
(OR=11, 95% CI 5.125, p<0.001). Those of type III IM
were 55% and 95% (OR=22; 95% CI 7.567, p<0.001),
and IM with MUC5AC expression were 81% and 69%,
respectively (OR=8.9, 95% CI 4.319, p<0.001).
Among the patients with IM, the presence of type III IM
in the antrum and corpus lesser curve, and incomplete and
GI mixed type MUC5AC expressing IM in each part were
Fig. 4 Immunohistochemical staining of serial sections of type I (*) and
II (**) IM in the antrum greater curve (original magnification 40).
a. High-iron diaminealcian blue (HIDAB) staining shows goblet
cells stained in blue (sialomucin) of type I IM (*) and cells stained in
brown (sulphomucin) of type II IM (**). b. MUC5 was detected in the
mucosa without IM and type I IM (*)
Fig. 5 MUC2 and MUC5AC immunohistochemical staining of serial
sections of type III IM in the corpus lesser curve (original
magnification 40). a. High-iron diaminealcian blue (HIDAB)
staining shows goblet cells and columnar cells stained in brown
(sulphomucin). b. MUC5AC was detected in the goblet cells and in
the columnar cells. c. IM does not express MUC6
b
656 Virchows Arch (2006) 449:652659
significant risk factors for gastric cancer (Table 4). How-
ever, in a multivariate analysis among those factors, gastric
cancer was significantly associated with a higher frequency
of incomplete IM (types II or III) in the corpus lesser curve
(OR=6.4; 95% CI, 2.021, p=0.002).
Asynchronous multiple lesions were associated with in-
complete IM in the corpus greater curve (OR=4.8; 95% CI,
1.416, p=0.01). However, there was no significant associa-
tion between types of IM and locations of malignant lesions.
Discussion
In this study, we confirmed that MUC5AC expression was
observed most often in type III IM. Previous studies exam-
ined biopsy samples or surgical specimens taken from the
gastric mucosa adjacent to carcinomas, and MUC1 and
MUC5AC together with MUC2 were expressed in every
case with IM types II and III [27, 33]. A recent study from
Japan examined the pyloric regions of stomachs resected for
gastric cancer and showed that MUC5AC was expressed in
38% of type I glands, 78% of type II, and 91% of type III IM
[35]. These results are similar to those reported here. The
most incomplete IM (type II and III) preserving gastric
mucin is the GI mixed type, whereas the complete type is
the type I IM especially in the corpus lesser curve.
Gastric cancer risk increases with age and is highest in
those with severe atrophic gastritis. We found that incom-
plete IM in the corpus lesser curve is common in patients
with gastric cancer. The presence of incomplete IM at this
location is indicative of the presence of corpus atrophy,
which is a known risk factor for an increased risk of the
intestinal type gastric cancer [31, 32]. The fact that
multivariate analysis showed a better association of the type
of IM with the presence of gastric cancer than the score of
atrophy suggests that, in an examination using a limited
number of biopsy specimens, the type of IMmay better reflect
the severity and extent of atrophy. This notion is consistent
with our results showing an inverse correlation between the
type of IM, the PG I/II and the severity of corpus atrophy.
Using a systematic endoscopic protocol with fixed-point
biopsy samples, we previously showed that the scores for IM
in the corpus greater curve and the scores for atrophy in the
corpus lesser curve were significantly higher in patients with
multiple malignant lesions compared to those with a single
lesion [32]. This study extended those findings showing that
the presence of incomplete IM in the corpus greater curve
was an additional histological risk marker for recurrent new
malignant lesions. Kabashima et al. reported that the inci-
dence of IM expressing gastric phenotype in non-neoplastic
mucosa within 5 mm from the margins of carcinoma cells
was higher in the patients with multiple early gastric cancer
lesions than in the patients with a single lesion [21].
This study suggests that the type and location of IM
provides important additional information regarding the
Table 3 Comparison of histological findings between the cancer
group and the control
Control Cancer p values
Type I/Type II/Type III IM
AG 10/16/3 4/26/28 <0.001
CG 5/4/0 12/18/4 <0.001
CL 15/14/3 8/33/33 <0.001
MUC5AC, MUC2 GI type IM
AG 11/18 7/51 <0.001
CG 8/1 27/9 <0.001
CL 18/15 20/56 <0.001
AG, antrum greater curve; CG, corpus greater curve; CL, corpus lesser
curve. IM, intestinal metaplasia; GI type, gastric and intestinal
mixed type. p values by chi square analyses
Table 2 Correlation of HIDAB staining and expression of MUC5AC
and MUC6
MUC5AC,
MUC2 of
GI type IM
(%)
p
values
MUC6,
MUC2
of GI type
IM (%)
p
values
Antrum* <0.001 <0.001
Type I IM n=13 46 39
Type II IM n=40 83 46
Type III IM n=31 94 50
Corpus * <0.001 <0.001
Type I IM n=40 13 5.0
Type II IM n=65 57 19
Type III IM n=39 90 52
*Antrum, antrum greater curve; Corpus, Corpus lesser and greater
curve. IM, intestinal metaplasia; GI type, gastric and intestinal mixed
type. p values by chi square analyses
Table 4 Comparison of the prevalence of type of intestinal metaplasia
among the subjects with intestinal metaplasia
OR (95% CI) p values
Type III IM
AG 14 (4.049) <0.001
CG
CL 18 (5.262) <0.001
Type II & III IM
AG 7.1 (3.514) <0.001
CG 7.1 (2.322) 0.001
CL 18 (8.241) <0.001
MUC5AC, MUC2 GI type IM
AG 6.6 (3.313) <0.001
CG 10 (1.382) 0.03
CL 10 (4.821) <0.001
AG, antrum greater curve; CG, corpus greater curve; CL, corpus lesser
curve. IM, intestinal metaplasia; GI type, gastric and intestinal
mixed type; OR, odds ratio; CI, 95% confidence interval
Virchows Arch (2006) 449:652659 657
extent and severity of gastric atrophy and thus the risk of
developing gastric cancer. Changes in the type of IM could
also reflect the amount of genetic damage that has taken
place and/or changes in the intragastric environment (e.g.,
development of low acid secretion). For example, hypo-
acidity might allow stem cell differentiation in a particular
direction to IM and independently to cancer [10, 31].
Recent studies investigating relationship between genetic
alterations and cellular phenotypes in cancer and IM have
provided evidence that the phenotypic expression in cancer
and precancerous lesions of the stomach is strongly
dependent on genetic changes [9], and that cancer and IM
may arise from different cell lineages [6, 17, 29, 36].
Tatematsu and colleagues used the classification of IM
using gastric and intestinal cell phenotypic markers, and
hypothesized that IM phenotype shift from GI mixed type
to I type [3537]. Intestinalization progresses from GI
mixed type to I type in non-cancerous and cancerous tissue
independently. However, our results do not support the
concept of IM phenotype shift from mixed type to intestinal
type as incomplete IM appeared to follow complete IM and
was correlated with the PG I/II ratio and with the severity
of corpus atrophy. The pathogenesis of IM and actual
relationship to gastric cancer remains unclear and additional
studies are warranted to explore the relationship.
Our study using targeted biopsies showed that the most
incomplete IM preserving gastric mucin is the GI type,
whereas the complete type is the type I IM especially in the
corpus lesser curve, and that IM type was associated with
the presence of early gastric cancer. These data suggest that
patients with incomplete IM in the corpus lesser curve are
likely to have severe atrophic pangastritis and such patients
are at increased risk of developing gastric cancer. More-
over, the presence of incomplete IM in the corpus greater
curve was an additional histological risk marker for
recurrent new malignant lesions. In conclusion, the classi-
fication of IM by histochemical detection using biopsy
samples obtained from fixed-points may enhance the ability
of surveillance programs to detect patients at increased risk
of either having or developing gastric cancer.
Acknowledgements The authors thank all the laboratory technicians
in the Department of Pathology, Osaka Medical Center for Cancer and
Cardiovascular Diseases for the expert technical assistance.
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