Cancer Epidemiology Biomarkers & Prevention Vol. 9, 671-674, July 2000
© 2000 American Association for Cancer Research
Lewis Antigen Alterations in a Population at High Risk of Stomach Cancer1
Julio Torrado,
Martyn Plummer2,
Jorge Vivas,
Jone Garay,
Gladys Lopez,
Simon Peraza,
Elsa Carillo,
Walter Oliver and
Nubia Muñoz
Nuestra Señora de Aránzazu Hospital, University of the Basque Country, San Sebastián, Spain [J. T., J. G.]; International Agency for Research on Cancer, Lyon, France [M. P., N. M.]; and Cancer Control Center of the Tachira State, San Cristobal, Venezuela [J. V., G. L., S. P., E. C., W. O.]
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Abstract
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Anomalous Lewisa antigen and sulfomucin expression are
considered as markers of progression in precursor lesions of gastric
cancer. Additionally, Lewis antigen and secretor phenotype have been
related to Helicobacter pylori infection and gastric
epithelial damage. The two objectives of this study were to correlate
Lewis antigen alterations with histochemical changes and to explore the
relationship between Lewis and secretor phenotypes and gastric
epithelial damage related to H. pylori infection. The
study subjects were selected from a chemoprevention trial in Tachira
State, Venezuela, an area with a high risk of gastric cancer.
Anomalous Lewisa antigen expression in Lewis (a-b+)
phenotype individuals was closely related to the severity of the
histological lesions, especially to dysplasia and type III intestinal
metaplasia lesions. A weak relationship was observed between
nonsecretor individuals and more advanced lesions of IM, but this
association was not statistically significant. There was no
relationship between secretor phenotype and H. pylori
status, atrophy, regenerative activity, erosion, or ulcer.
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Introduction
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Blood group antigens (Lewis and ABH antigens) are carbohydrate
structures originally identified on RBC by Landsteiner. However, these
antigens are widely expressed in many tissues throughout the body,
being especially abundant in the epithelial cells of gastric mucosa.
The secretor status is defined by the presence of ABH antigens in body
fluids and secretions like saliva, gastric juice, and milk.
Lewis (Lewisa, Lewisb) and
ABH antigens are closely interrelated, and in mucosecretory epithelia
they are produced from a common precursor (type 1 precursor) by
the action of different genes. In the gastric foveolar cells, the
expression of Lewis and ABH antigens is fundamentally controlled by the
action of the secretor and Lewis genes (Fig. 1)
. In people who have the secretor and Lewis genes, all of the precursor
substance is transformed in H type 1 antigen, and they express
Lewisb and ABH antigens in the foveolar
epithelium and in the gastric juice as well. People who do not have the
secretor gene cannot produce Lewisb or ABH
antigens and, if they have the Lewis gene, all of the precursor is
transformed into Lewisa antigen. Finally, people
who do not have the Lewis gene do not express
Lewisa or Lewisb in gastric
mucosa or gastric juice, and they express ABH antigens only if they
have the secretor gene (1, 2, 3)
.
Blood group antigens appear to play an important role in cell
recognition, differentiation, and growth regulation and during the
process of malignant transformation these antigens undergo important
alterations (4)
.
Lewis system abnormalities have been described in gastric carcinoma and
precursor lesions. The anomalous expression of
Lewisa antigen in areas of
IM3
of the stomach in Lewis (a-b+) individuals has been considered as a
risk marker in the gastric precancerous process.
To explore the role of the alterations in the expression of Lewis
antigens in the gastric precancerous process, we have studied
histochemical and antigenic anomalies on gastric biopsies from a
population from the Andean region in Venezuela with high risk for
gastric cancer. We have correlated the Lewis and secretor phenotypes
with histological lesions and with Helicobacter pylori
status. The expression of anomalous Lewisa
antigen in relation to the type of IM was also investigated.
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Materials and Methods
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Subjects in this study were selected from a randomized double
blind, placebo-controlled chemoprevention trial, currently being
conducted in Tachira State, Venezuela, the aim of which is to assess
the effect of antioxidant vitamins on progression of precancerous
lesions of the stomach. The results presented here are a
cross-sectional analysis of the subjects who had a histological
diagnosis of IM at baseline. Although follow-up data are available,
analysis of these data has been deferred until the randomization code
is broken and the cohort of subjects receiving placebo can be
identified.
The design of the chemoprevention trial has been described in detail
elsewhere (5)
. Briefly, participants in the trial were
recruited from the Gastric Cancer Control Program of Tachira State.
Eligible subjects were permanent residents of Tachira between 35 and 69
years of age who, after double contrast X-ray, were selected to undergo
gastroscopic examination. At entry into the trial, physical
examination, blood collection, and gastroscopy were performed. Five
biopsies for histological assessment were taken: one from the lesser
curvature of the antrum, 
1 cm from the pylorus; one from the
greater curvature of the antrum, 1 cm above the pylorus; one from a
midportion of the lesser curvature of the antrum; one from the lesser
curvature of the antrum immediately below the incisura; and one from
the middle corpus, 2 cm from the lesser curvature.
Gastric biopsies were fixed in buffered formalin and stained with
H&E and Giemsa to detect H. pylori. The most severe lesion
among all biopsies was considered for the global diagnosis and for the
immunohistochemical studies. Those biopsies positive for IM were also
stained with periodic acid-Schiff-Alcian blue (6)
and high
iron diamine-Alcian blue (7)
to determine subtypes
of IM that were classified according to the method of Filipe and Jass
(8)
as follows. Type I is characterized by the presence of
mature goblet and absorptive cells with a well-defined brush border.
Goblet cells secrete sialomucins. In type II, there is mild
architectural distortion, crypts are lined by goblet cells, absorptive
cells are few or absent, and columnar mucous cells are present
containing a mixture of neutral and acid sialomucins. The goblet cells
secrete sialomucins or occasionally sulfomucins, or both. In type III,
the overall architecture is more disorganized than in type II. Columnar
cells secrete sulfomucins, and goblet cells contain sialo- or
sulfomucins.
Lewis and secretor phenotypes were determined by
immunohistochemistry in nonmetaplastic areas of gastric mucosa, as
previously reported (9)
. Anti-A (A581, Dako; working
dilution, 1:40), anti-B (A582, Dako; working dilution, 1:40), and
anti-H type 2 (A583, Dako; working dilution, 1:40) were used to
determine the secretor phenotype. The presence of ABH antigens in the
foveolar epithelium defined the secretor phenotype; a negative
detection identified the nonsecretor phenotype.
Anti-Lewisa (7LE, Biogenex; working dilution,
1:250) and anti-Lewisb (2.25LE, Biogenex; working
dilution, 1:250) were used to determine the Lewis phenotype. A diffuse
positive reaction for anti-Lewisa and
anti-Lewisb monoclonal antibodies defined the
Lewis (a+b-) and the Lewis (a-b+) phenotypes, respectively; absence
of both positive reactions defined the Lewis (a-b-) phenotype.
Sections for immunochemistry were deparaffinized, rehydrated, and
successively incubated with: 8% (v/v) hydrogen peroxide in distilled
water for 10 min at room temperature; primary antibodies (working
dilution prepared with Primary Antibody Diluent from Biomeda, Foster
City, CA) for 30 min at 37°C; biotinylated rabbit antimouse
immunoglobulin (Biomeda) for 5 min at 37°C; alkaline
phosphatase-labeled streptavidin (Ultra Probe; Biomeda) for 10 min at
37°C; and a freshly prepared solution of 100 mM Tris
base, 200 mM NaCl, 5 mM
MgCl2, 0.35% HCl, and 0.05% Tween 20, pH 9.0,
with naphthol AS-MX phosphatase/Fast Red TR chromogen (Biomeda), for 15
min at room temperature. Sections were faintly counterstained with
Lillie’s hematoxylin and mounted with Crystal-Mount (Biomeda).
Positive and negative controls were stained with every batch of
samples. In the deparaffinized tissues used, the specificity of primary
antibodies is restricted to the mucin glycoprotein fractions of these
antigenic determinants and was previously determined. Working dilutions
were established by serial titration.
We investigated the aberrant expression of Lewisa
antigen in all Lewis (a-b+) patients. This alteration was categorized
in three patterns (see Ref. (9)
for full description and
illustrations): pattern I, anomalous antigen expressed only in some
goblet cells; pattern II, anomalous antigen expressed in goblet cells
and weakly in columnar cells; pattern III, anomalous antigen strongly
positive in both goblet and columnar cells.
For all variables measured at the level of the individual biopsy,
except for H. pylori status, an overall score was calculated
for each subject by taking the most severe of the available biopsy
scores, and this overall score was used in the tabulations and tests.
For H. pylori status, the overall prevalence in all five
biopsies was used for the tabulation, and the difference between
secretors and nonsecretors was tested by Wilcoxon rank sum test, with
the use of the average of the five biopsy scores (rated 1–4 for
negative, difficult to find, easy to find, and abundant, respectively).
Two statistical tests were used to examine the relationships
between cross-tabulated variables. The 
2 test
was used as a global test for any relationship, and a trend test
(10)
was used to look for a smoothly increasing trend if
one of the variables was ordinal.
The data on abnormal Lewisa secretion and
histological diagnosis were examined by applying a log linear Poisson
model to the tabulated data. When controlling for age and other
confounders, a separate multiple logistic regression model was used for
outcomes of IM-II, IM-III, and dysplasia, with IM-I as a common
baseline.
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Results
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Gastric biopsies taken at baseline were studied from 564 subjects
with a global diagnosis of IM or dysplasia. The most severe lesion
among all biopsies was considered as the global diagnosis. The subjects
ranged in age from 34 to 71 (mean, 51), and 269 were men and 295 women.
The secretor status was determined in only 552 patients.
The prevalence of the different histological lesions was 58% for type
I IM, 12% for type II, 13% for type III IM, and 17% for dysplasia.
In dysplastic cases, 81% showed mild, 17% showed moderate, and 2%
showed severe dysplasia.
Table 1
shows the Lewis and secretor phenotype distribution by sex. There is no
significant difference in the distribution of either phenotype by sex.
A high proportion (93%) of Lewis (a-b-) phenotype subjects were
secretors. The prevalence of Lewis (a+b-) and nonsecretor individuals
was lower than that of the European population, a fact observed in
other Andean populations (9)
.
Secretor Status and H. pylori Infection.
Table 2
shows the distribution of histological diagnosis by secretor status.
There is an excess of secretors in the group of subjects with IM-I, but
overall there is no significant difference in the distribution of the
histological diagnoses between secretors and nonsecretors
(P = 0.16; test for trend, P = 0.10).
Table 3
shows the relationship between secretor status and H. pylori
infection, rated on a four point scale (negative, difficult to find,
easy to find, abundant). H. pylori infection is very
common in this population: 94% of the participants in the
chemoprevention trial are infected. There is no significant difference
between secretors and nonsecretors in degree of H. pylori
infection (P = 0.18). Secretor status was also examined
in relation to three markers of cellular or mucosal damage, atrophy,
regenerative activity, and erosion or ulcer, which were rated on a four
point scale for each biopsy (negative, light, moderate, and severe for
atrophy and regenerative activity and negative, superficial, mucosal
penetration, and submucosal penetration for erosion or ulcer). No
relationship was found with any of these markers (tables not shown;
P = 0.77 for atrophy, P = 0.42 for
regenerative activity, and P = 0.57 for erosion or
ulcer. Trend test results, P = 0.32, P = 0.94, P = 1.00, respectively).
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Table 3 Extent of H. pylori infection (overall prevalence in
subjects with complete biopsy information) by secretor status
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In summary, there is weak evidence of a correlation between secretor
status and severity of histological diagnosis, but this could not be
related to H. pylori infection.
Abnormal Expression of Lewisa in Lewis (a-b+)
Individuals, by Type of IM.
Table 4
shows the relationship between abnormal expression of
Lewisa antigen and histological diagnosis in
Lewis (a-b+) individuals. This relationship is shown in Fig. 2
. With IM type I as a baseline, there is a clear increasing risk of
dysplasia, IM type III, and IM type II with an increasing degree of
abnormal expression of Lewisa. The trends are all
strongly significant (P < 0.001 for dysplasia,
P < 0.001 for IM-III, and P = 0.002
for IM-II). The trend for IM-III is stronger than for IM-II
(P = 0.02), but the trend for dysplasia is weaker than
for IM-III.
These results were unchanged after controlling for the confounding
effect of age, sex, years of education (as a marker of socioeconomic
status), and smoking status, which have been previously
identified as risk factors for advanced precancerous lesions in this
population.
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Discussion
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The intestinal or epidemic type of gastric cancer is considered
the end result of a multistep process in which multiple factors are
involved. H. pylori infection, dietary factors, nitroso
compounds, oxidative damage, and lack of antioxidant vitamins produce a
series of changes in the gastric epithelium which include progressive
grades of atrophy, IM, and dysplasia and, finally, a malignant
transformation (11)
. In this dynamic process, progressive
alterations in the mucin components and the expression of anomalous
antigens may be also observed.
Alterations in the expression of blood group antigens have been
extensively described in gastric cancer (12, 13, 14, 15)
. It has
been proposed that these anomalies are produced by blockages in the
normal synthesis of these antigens, resulting in loss of some of these
structures and the appearance of aberrant antigens. The anomalous
expression of Lewisa antigen in lesions of
gastric intestinal IM and dysplasia from Lewis (a-b+) individuals has
been previously reported. In our experience, this abnormality has not
been observed in the earlier lesions of chronic gastritis and gastric
atrophy or in areas of normal gastric mucosa. Previous studies have
demonstrated that the simultaneous expression of anomalous
Lewisa antigen and sulfomucins indicates a
greater risk of preneoplastic progression (9
, 16)
.
Our results show an increasing frequency in the prevalence and grade of
severity of the anomalous Lewisa antigen
expression correlated to the severity of the histological changes and
to the severity of the histochemical alterations (sialo- and sulfomucin
expression). Thus, the most severe pattern of anomalous
Lewisa antigen expression (pattern III) was more
prevalent in dysplastic cases than in type II or type III intestinal
metaplasia.
The role of H. pylori as the most important etiopathogenic
factor in chronic gastritis is well documented. However, its direct
involvement in more advanced lesions (gastric atrophy, IM, dysplasia,
and cancer) is poorly understood. Recently, the
Lewisb and H antigens have been described as the
receptors responsible for the attachment of H. pylori to the
gastric mucosa (17)
. The cytoplasm of the foveolar cells
and the gastric mucus are very rich in these antigens, and this fact
can explain the especial tropism of this bacterium for the gastric
mucosa.
We think that in secretor individuals, the abundant presence of
Lewisb and H antigens in the gastric mucus acts
as a protective mechanism that traps bacteria and prevents their
attachment to the gastric epithelium. This protective mechanism has
been proposed as an adaptive response to infective microorganisms by
Slomiany (18)
. In the present study, there is no
relationship between H. pylori infection and secretor
phenotype. This is not necessarily inconsistent with the theory that
H. pylori is trapped in the gastric mucosa in secretor
individuals. When rating the degree of H. pylori infection,
it was not possible to distinguish between bacteria attached to cells
and bacteria in mucus.
Previously, we have observed a lower epithelial damage, grade of
atrophy, and a lower prevalence of sulfomucin expression in secretor
individuals than in nonsecretors. In this study, there was no
relationship between secretor status and atrophy, regenerative activity
or erosion or ulcer.
A strong relationship between the expression of sulfomucins and
nonsecretor status has been described in precursor lesions of gastric
cancer (19)
and also in Barrett’s esophagus and
Barrett’s adenocarcinoma (20)
. In the present study,
there is a tendency for nonsecretors to have more type III IM and
dysplasia than secretors but, possibly due to the small number of
nonsecretor individuals, the difference was not significant.
In summary, our results indicate that at the same time as the
morphological changes that occur during the process of gastric
carcinogenesis, another series of events occurs. Thus, the anomalous
appearance of Lewisa and sulfomucins in areas of
9intestinal metaplasia appear to behave as immunohistological indicators
of a greater severity of morphological lesions. Further follow-up
studies are required to clarify the role of predictive markers of risk
in precursor lesions of gastric cancer.
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Acknowledgments
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We thank Olga Andrade, Denis Castro, Victor Sanchez, and Elsa
Cano for their work as histotechnicians; and Gloria Moreno, Angel
Chacon, and Jasmin Rangel for their work as endoscopists on this study.
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Footnotes
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Fondo de
Investigacion Sanitaria y la Consejeria de Salud del Gobierno Vasco. 
2 To whom requests for reprints should be
addressed, at IARC, 150 Cours Albert Thomas, F-69372 Lyon, France.
Phone: (+33) 04 72 73 84 46; Fax: (+33) 04 72 73 85 75.
3 The abbreviation used is: IM, intestinal
metaplasia.
Received 11/ 2/99;
revised 3/ 8/00;
accepted 4/13/00.
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Abstract
Introduction
