Helicobacter pylori Infection and Risk of Gastric Cancer in Shanghai,
China: Updated Results Based upon a Locally Developed and
Validated Assay and Further Follow-Up of the Cohort1
Jian-Min Yuan,2Mimi C. Yu, Wei-Wen Xu,
Myles Cockburn, Yu-Tang Gao, and Ronald K. Ross
Department of Preventive Medicine, University of Southern California/Norris
Comprehensive Cancer Center, University of Southern California School of
Medicine, Los Angeles, California 90033-0800 [J.-M. Y., M. C. Y., M. C.,
R. K. R.]; Shanghai Institute of Digestive Diseases, Shanghai 200001, People’s
Republic of China [W.-W. X.]; and Shanghai Cancer Institute, Shanghai
200032, People’s Republic of China [Y.-T. G.]
Infection with Helicobacter pylori has been associated
with an increased risk of gastric cancer in low-risk
populations. However, our previous results (P. M. Webb
et al., Int. J. Cancer, 67: 603–607, 1996) from an ongoing
prospective study in Shanghai, China, a relatively high-
risk population, failed to show an association between
H. pylori infection and the subsequent risk of gastric
cancer. That previous study had a relatively short time
period of follow-up and the enzyme-linked
immunosorbent assay (ELISA) used was based on strains
found in Southern England and without validation among
the Chinese. Either one of these two factors could have
had an impact on the validity of those earlier
observations. An ELISA developed and validated among
Shanghai residents was used in the present study to
reexamine specific antibodies to H. pylori in 188 gastric
cancer patients and 548 control subjects. All of the cases
of gastric cancer were identified during the first 12 years
of follow-up of a cohort of 18,244 men, ages 45–64 years
in Shanghai, from whom blood samples were collected at
enrollment during 1986–1989. For each cancer case, three
cancer-free control subjects were randomly selected from
the cohort and matched to the index cases by age (within
2 years), month and year of sample collection, and
neighborhood of residence. The Shanghai-based ELISA
detected a higher prevalence of serum antibodies to H.
pylori than the English-based assay in both gastric cancer
cases (86 versus 53%) and control subjects (85 versus
56%). Virtually all of the subjects (98%) who were H.
pylori-seropositive by the English-based assay tested
positive by the Shanghai-based assay. On the other hand,
73% of gastric cancer cases and 68% of control subjects
who were seronegative according to the English-based
assay tested positive by the Shanghai-based assay. Using
this alternative assay, combined with increased follow-up,
our latest data contradict our earlier findings and show a
statistically significant association between H. pylori
seropositivity and gastric cancer risk (odds ratio, 1.84;
95% confidence interval, 1.08–3.11). We noted an
increasing rate of seropositivity among cases as the time
interval between cohort enrollment and cancer diagnosis
increased. Among subjects followed for 5 or more years
after enrollment, the odds ratio for gastric cancer related
to H. pylori seropositivity was 3.74 (95% confidence
It has been suggested that infection with the bacterium Heli-
cobacter pylori is a risk factor for gastric cancer (1). The best
evidence in support of this association has come from a number
of case-control studies nested within prospective cohorts, which
have shown a significantly increased risk of gastric cancer with
H. pylori infection as assessed by the presence of specific
antibodies (2–6). Previously, data from an ongoing prospective
cohort study in men in Shanghai, China, a relatively high-risk
population for this malignancy (51.7 per 100,000 person-years;
Ref. 7), did not support a positive association between H. pylori
infection and risk of gastric cancer (8). In that publication, we
raised two possible concerns regarding the validity of those
(a) the specific antibodies to H. pylori were assessed using
an enzyme-linked immunosorbent assay (ELISA) developed in
Southern England, using H. pylori strains obtained from local
patients (9). That English-based ELISA has been validated in
several western populations with varying rates of sensitivity
and specificity. Rates of sensitivity and specificity among
tested subjects in Northern Ireland were 88 and 72%, respec-
tively (10). The corresponding rates among the populations of
Rochester, Minnesota; Copenhagen, Denmark; and Victoria,
Australia were 93 and 96%, 74 and 71%, and 86 and 65%,
respectively (11–13). There is no information on the validity of
this English-based assay in a Chinese population. Given that
strains of H. pylori may vary in different populations, it is
possible that the English-based assay is suboptimal (or even
inappropriate) for detecting infection in a Chinese population;
(b) the cohort had been followed for a relatively short
period of time (an average of 2.4 years between blood collec-
tion and the diagnosis of gastric cancer; Ref. 8). There is
evidence that with the development of advanced gastric disease,
the H. pylori bacterium can be cleared from the stomach,
resulting in seroconversion (from positive to negative for H.
Received 11/25/98; revised 4/12/99; accepted 5/24/99.
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.
1This study was supported by NIH Grants R01 CA43092, R35 CA53890, and
2To whom requests for reprints should be addressed, at Department of Preventive
Medicine, USC/Norris Comprehensive Cancer Center, M/S # 44, University of
Southern California, 1441 Eastlake Avenue, Los Angeles, CA 90033-0800.
Phone: (323) 865-0825; Fax: (323) 865-0136; E-mail: firstname.lastname@example.org.
621Vol. 8, 621–624, July 1999
Cancer Epidemiology, Biomarkers & Prevention
pylori antibodies; Ref. 14). Therefore, gastric cancer patients
(but not control subjects) may have been misclassified as se-
ronegative, even though they were infected in the past. In the
presence of such differential misclassification, estimates of the
association between H. pylori seropositivity and gastric cancer
would have been biased downwards.
In the present study, we used a recently developed and
validated assay that was based on patient materials collected
locally to assess H. pylori seropositivity. Follow-up of the
cohort is now extended to an average of 5.2 years postenroll-
ment. This report presents our latest findings on the H. pylori-
gastric cancer association within the Shanghai Male Cohort.
Subjects and Methods
Subjects. The study design has been described in detail pre-
viously (8, 15). Between January 1986 and September 1989, all
men, ages 45–64 years, living in four small, geographically
defined areas of the city of Shanghai were invited to participate
in a prospective study of diet and cancer. A total of 18,244 men
were recruited, and each subject was interviewed in person to
obtain information on current diet, medical history, and other
life-style variables such as tobacco smoking and alcohol con-
sumption. Each subject also provided blood and spot urine
By March 1998, 195 incident cases of gastric cancer were
identified among the cohort members. For each of the cases,
three cancer-free controls matched to the index cases by age
(within 2 years), month and year of sample collection, and
neighborhood of residence at recruitment were selected from
the cohort (8). The first 85 of these cases and 255 of their
matched controls were tested for serum H. pylori antibodies
using the English-based assay (8). Serum samples on 7 gastric
cancer cases and an additional 16 control subjects not matched
to these 7 cancer cases were depleted after the English-based
serological testing. Therefore, 188 cases and 548 controls were
included in the present study. Seventy-eight of these patients
and 218 of their matched controls were included in the com-
parison of H. pylori positivity rates derived from the English-
based versus the Shanghai-based ELISA.
Laboratory Tests. Blood samples from all of the cohort sub-
jects were processed shortly after collection and stored at
?20°C and ?70°C until analysis. Serum samples in the present
study were taken from the ?20°C aliquots and had been
thawed, once when they were used to test for the presence of H.
pylori antibodies using the English-based assay (8). Serum
samples (which were blinded with respect to case/control status
and other characteristics of the individual subjects) were sent on
dry ice to the Shanghai Institute of Digestive Diseases for H.
pylori antibody testing using an enhanced version of ELISA
developed by Pan et al. (16, 17). All of the serum samples from
a given case-control matched set were tested in the same batch,
which routinely contained two positive and two negative con-
trol sera for quality assurance. Duplicate measurements were
made on 332 (45%) of the 736 tested samples; the remaining
404 samples were measured only once to conserve serum
samples for other biomarker measurements. Only seven (2%)
samples yielded inconsistent duplicate results. A third meas-
urement was made on each of these seven specimens and the
majority finding was recorded (two were antibody-positive, and
five were antibody-negative). We also submitted two independ-
ent serum samples on 10% of study subjects (n ? 74) to further
assure high laboratory quality. Results were consistent for 72 of
these subjects. The two subjects with inconsistent results were
tested a third time, and the majority finding was recorded for
each (one was antibody-negative, and the other was antibody-
The ELISA method of Pan et al. (17) is briefly described
as follows. A bacterial suspension from each of 15 H. pylori
strains obtained locally was sonicated, pooled, and used as a
group antigen. The optimal antigen concentration was 10 ?g/ml
as determined by checkerboard titrations. Testing serum sam-
ples were diluted to 1:200. The absorbance was read at 490 nm
on a spectrophotometer (Denatach Minireader II) after stopping
the reaction with 50 ml of 1 M sulfuric acid. A serum sample
was considered positive when the ratio of its absorbance to that
of the standard negative sera was greater than 1.7.
The Pan et al. ELISA method (17) was validated among
Shanghai residents. Two hundred twenty-five patients with
various gastrointestinal symptoms were recruited. Gastric bi-
opsies and serum samples were collected from all of the study
subjects. Culture and smear of biopsied materials showed that
171 subjects were infected with H. pylori. One hundred sixty-
one of the 171 infected patients were seropositive (sensitivity
rate, 94%), and 47 of the 54 uninfected subjects were seroneg-
ative (specificity rate, 87%; Ref. 16).
The assay used in the present study was an enhanced
version of the ELISA described above (17). An additional 15
strains of H. pylori obtained from local patients were added to
the antigen pool (thus, 30 strains in total). To validate this
enhanced version of the ELISA, a separate group of 152 pa-
tients with gastrointestinal symptoms were recruited. One hun-
dred nineteen (78%) patients were men; their mean age was 44
years (range, 22–62 years). Gastric biopsies and serum samples
were collected from all of the study subjects. Culture and smear
of biopsied materials showed that 117 subjects were infected
with H. pylori. One hundred sixteen of the 117 infected patients
were seropositive (sensitivity rate, 99%), and 33 of the 35
uninfected subjects were seronegative (specificity rate, 94%).
Statistical Analysis. Data were analyzed by standard matched-
pair methods (18). The association between H. pylori infection
and gastric cancer was measured by OR3with 95% CI. We also
examined this association by subsite of gastric cancer and by
time interval between blood sample collection and diagnosis of
gastric cancer. Conditional logistic regression models were
used to examine H. pylori-gastric cancer association with or
without adjustment for other potential confounders. The current
set of possible confounding variables was identical to the one
used in the analysis of our first study (8) and included the level
of education (primary school or less, middle school, college or
3The abbreviations used are: OR, odds ratio; CI, confidence interval.
control subjects by the Shanghai-based and the English-based ELISAs
H. pylori antibody status in gastric cancer patients and matched
Negative (%) Positive (%)
Antibody status among cases
Antibody status among controls
2 (2) 32 (15)
H. pylori and Gastric Cancer in China
higher), cigarette smoking (ever versus never), alcohol con-
sumption (weekly versus less frequently), history of peptic
ulcer (yes versus no), blood group (O, A, B, or AB type), and
consumption of dark leafy vegetables (tertiles), cured meats
(tertiles) and pickled vegetables (ever versus never). Results
from “unadjusted” and “adjusted” analyses were remarkably
similar. The unadjusted results are presented in this report.
The mean age (?SD) of gastric cancer patients at the time of
diagnosis was 63.4 years (?5.6). The average time interval
between blood collection and the diagnosis of gastric cancer
was 5.2 years (range, 0.1–11.8 years).
There were 78 patients with gastric cancer and 218 control
subjects who had H. pylori serological results from both the
English-based and the Shanghai-based assays. Table 1 com-
pares these results. Forty (98%) of the 41 gastric cancer patients
who were antibody-positive according to the English assay
tested positive by the Shanghai assay. On the other hand, 27
(73%) of the 37 gastric cancer patients who were antibody-
negative according to the English assay tested positive by the
Shanghai assay. Similar findings were seen in control subjects.
Almost all (98%) of the positive control subjects and a sub-
stantial proportion (68%) of the negative control subjects by the
English assay were seropositive in the present study.
Table 2 shows the association between H. pylori seropos-
itivity and risk of gastric cancer. One hundred sixty-eight (89%)
of the 188 gastric cancer patients and 451 (82%) of the 548
control subjects were H. pylori-seropositive, yielding a statis-
tically significant OR for gastric cancer (OR, 1.84; 95% CI,
We examined the effect of length of follow-up (i.e., the
time interval between blood collection and diagnosis of gastric
cancer) on the association between H. pylori seropositivity and
the risk of gastric cancer. There was no increase in gastric
cancer risk among those with less than 5 years of follow-up. On
the other hand, a statistically significant, 3.7-fold increase in
risk of gastric cancer associated with H. pylori positivity was
observed among subjects with 5 or more years of time interval
between blood collection and the diagnosis of cancer (Table 2).
The positive association remained unchanged after an adjust-
ment for potential confounding factors (data not shown). When
cases were stratified by subsite of cancer, the risk for cardia
cancer was comparable to that for noncardia cancer, regardless
of length of follow-up (Table 2).
With increased follow-up, we now find H. pylori-infected in-
dividuals in Shanghai to be at substantially increased risk for
gastric cancer. Consistent with the report of Forman et al. (19),
we noted an increasing rate of seropositivity among cases as the
time interval between cohort enrollment and cancer diagnosis
increased. Among cases diagnosed 5 or more years after en-
rollment, 93% were seropositive at recruitment; the comparable
figure for those with less than 5 years of follow-up was 86%.
Using data from three nested case-control studies to ex-
amine the H. pylori-gastric cancer association according to time
interval between blood collection and cancer diagnosis, Forman
et al. reported that the OR for gastric cancer was 2.1 among
subjects with less than 5 years of follow-up, 2.3 in those with
5–9 years of follow-up, 4.4 in those with 10–14 years of
follow-up, and 8.7 in those with 15 or more years of follow-up
(19). As previously reported (8), we find no clear increase in
risk (OR, 1.1) among subjects with less than 5 years of follow-
up. The observed OR of 3.7 among those with 5 or more years
(ranging from 5 to 12 years) of follow-up is compatible with the
Forman et al.’s results (19).
The present study clearly indicates that the English-based
assay is suboptimal in identifying H. pylori-infected individuals
among Shanghai Chinese. Although it had few false-positive
readings relative to the Shanghai-based assay, the English-
based assay exhibited a substantial rate of false negatives.
These differences are likely due to strain-specific differences in
H. pylori bacteria inhabiting the British versus the Chinese
population. A study in Thailand has shown that a locally de-
rived ELISA is more sensitive and specific in identifying H.
pylori-infected individuals than a United States-based assay,
although the discrepancy is not as dramatic as that observed in
the present study between the English- and the Shanghai-based
assay. Whereas the Thai-based assay had sensitivity and spec-
ificity rates of 98% and 76%, respectively, the corresponding
figures for the United States-based assay were 86% and 66%,
There is sparse information on H. pylori seropositivity
rates in Chinese populations based on locally derived and
validated ELISAs. In a rural county in Shandong, China, where
gastric cancer mortality in men is about 70 per 100,000 person-
years (21), Zhang et al. (22) reported an H. pylori seropositivity
prevalence rate of 68% in healthy men, ages 35–64 years, using
an assay derived from local strains; no information was given
regarding the sensitivity and specificity of the assay. It is
unclear whether the difference in rates of H. pylori infection
Table 2H. pylori seropositivity in relation to risk of gastric cancer by time interval between cohort enrollment and cancer diagnosis and subsite of cancer
H. pylori-positive (%)Total
H. pylori-positive (%)
By time interval between cohort enrollment and cancer
?5, by cancer subsiteb
?5, by cancer subsiteb
188 168 (89)548451 (82) 1.84 (1.08–3.11)
aOR for gastric cancer in H. pylori-positive versus H. pylori-negative individuals.
bCases who had an unspecified subsite of gastric cancer were excluded from this analysis.
Cancer Epidemiology, Biomarkers & Prevention
between Shanghai and Shangdong men is real or due to possi- Download full-text
bly lower sensitivity of the Shangdong assay.
Several epidemiological studies have found a statistically
significant association of H. pylori seropositivity with noncar-
dia cancer but not with cardia cancer (3–5). In the present study,
there is no evidence of a differential risk between cardia and
noncardia cancer in relation to H. pylori seropositivity.
In summary, the present data demonstrate a significant,
positive association between H. pylori seropositivity and gastric
cancer in Shanghai, China, a relatively high-risk population,
that was observed only among subjects with at least 5 years of
follow-up. The positive association remains unchanged after
adjustment for potential confounding factors.
We thank Y.-B. Xiang, X.-L. Wang, Y.-L. Zhang, and J.-R. Cheng of the
Shanghai Cancer Institute for their assistance in data collection and management;
the staff of the Shanghai Cancer Registry for their assistance in verifying cancer
diagnosis; and Kazuko Arakawa of the University of Southern California for her
assistance in data management and analysis.
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