Confidential: For Review OnlyHelicobacter pylori treatment, vitamin and garlic
supplementation for the prevention of gastric cancer: 22.3-year follow-up of a randomized intervention trial
Journal: BMJ
Manuscript ID BMJ-2019-050223
Article Type: Research
BMJ Journal: BMJ
Date Submitted by the Author: 13-Apr-2019
Complete List of Authors: Li, Wenqing; Peking University Cancer HospitalZhang, Jing-Yu; Peking University Cancer HospitalMa, Junling; Peking University Cancer HospitalLi, Zhe-Xuan; Peking University Cancer HospitalZhang, Lian; Peking University Cancer HospitalZhang, Yang; Peking University Cancer HospitalGuo, Yang; Peking University Cancer HospitalZhou, Tong; Peking University Cancer HospitalLi, Jiyou; Peking University Cancer HospitalShen, Lin; Peking University Cancer HospitalLiu, Wei-Dong; Peking University Cancer HospitalHan, Zhong-Xiang; Peking University Cancer HospitalBlot, William; International Epidemiology InstituteGail, Mitchell; Division of Cancer Epidemiology & Genetics, National Cancer Institute, National Institutes of HealthPan, Kai-Feng; Peking University Cancer HospitalYou, Wei-cheng; Peking University Cancer Hospital
Keywords: cancer epidemiology, gastric cancer, cancer prevention, Helicobacter pylori, vitamin, garlic
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Helicobacter pylori treatment, vitamin and garlic supplementation for the prevention of gastric
cancer: 22.3-year follow-up of a randomized intervention trial
Wen-Qing Li1*, Jing-Yu Zhang1*, Jun-Ling Ma1, Zhe-Xuan Li, Lian Zhang1, Yang Zhang1,
Yang Guo1, Tong Zhou1, Ji-You Li1, Lin Shen1, Wei-Dong Liu2, Zhong-Xiang Han2, William J.
Blot3, Mitchell H. Gail4, Kai-Feng Pan1, Wei-Cheng You1
* WQL and JYZ contributed equally to this paper.
Author Affiliations: 1 Key Laboratory of Carcinogenesis and Translational Research (Ministry
of Education/Beijing), Department of Cancer Epidemiology, Peking University Cancer Hospital
& Institute, Beijing, China; 2 Linqu County Public Health Bureau, Shandong, China;
3International Epidemiology Institute, Ltd., Rockville, MD, and Vanderbilt University Medical
Center, Nashville, TN; 4Division of Cancer Epidemiology and Genetics, National Cancer
Institute, Bethesda, MD, United States.
Corresponding author:
Wei-Cheng You, Key Laboratory of Carcinogenesis and Translational Research (Ministry of
Education/Beijing), Department of Cancer Epidemiology, Peking University Cancer Hospital &
Institute, 52 Fu-cheng Road, Haidian District, Beijing 100142, China. Phone: 86-10-88196866,
Fax: 86-10-88122437, E-mail: [email protected].
Kai-Feng Pan, Key Laboratory of Carcinogenesis and Translational Research (Ministry of
Education/Beijing), Department of Cancer Epidemiology, Peking University Cancer Hospital &
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Institute, 52 Fu-cheng Road, Haidian District, Beijing 100142, China. Phone: 86-10-88196937,
Fax: 86-10-88122437, E-mail: [email protected]
Short title: Effects of H. pylori treatment, vitamin and garlic supplementation
Word count: Abstract (340); Text (3518).
Number of tables: 3 Number of figures: 4
Number of Supplementary tables: 2
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Abstract
Objective: During 14.7-years’ follow-up in the Shandong Intervention Trial, 2-week treatment
for Helicobacter pylori (H. pylori) resulted in statistically significant reduction in gastric cancer
(GC) incidence. Results for GC mortality and for the effects of garlic and vitamin
supplementation, though promising, were not statistically significant. Longer follow-up was
needed to determine the effects of H. pylori treatment, vitamin and garlic supplementation on
GC incidence and cause-specific mortality.
Design: A blinded randomized factorial placebo-controlled trial.
Setting: Linqu county, a well-defined high-risk area for GC in China.
Population: 3365 participants were included.
Interventions: H. pylori treatment, using amoxicillin and omeprazole for 2 weeks (among H.
pylori positive participants); vitamin (C, E and selenium) and garlic (extract and oil)
supplementation for 7.3 years (1995-2003).
Main Outcomes and Measures: Cumulative GC incidence was identified via scheduled
endoscopies in 1999 and 2003, and active clinical follow-up through 2017. GC mortality and
causes of death were obtained from death certificates and hospital records.
Results: During 1995-2017, we identified 151 incident GC cases and 94 GC deaths. A
protective effect of H. pylori treatment on GC incidence persisted 22 years post-intervention
(odds ratio [OR]=0.48, 95% confidence interval [CI]=0.32-0.71). Vitamin, but not garlic
supplementation, also decreased GC incidence significantly (OR=0.64, 95% CI=0.46-0.91). All
three interventions significantly reduced GC mortality, with hazard ratios (95% CIs) of 0.62
(0.39-0.99) for H. pylori treatment, 0.48 (0.31-0.75) for vitamin, and 0.66(0.43-1.00) for garlic
supplementation. Effects of H. pylori treatment on both GC incidence and mortality and of
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vitamin supplementation on GC mortality appeared early, but effects of vitamins and garlic on
GC incidence and of garlic on GC mortality only appeared later. We did not find statistically
significant associations of interventions with other types of cancer or cardiovascular disease
(secondary outcomes).
Conclusion: Short-term H. pylori treatment and 7.3-year vitamin and garlic supplementation
each significantly reduced GC mortality 22.3 years after randomization. H. pylori treatment and
vitamin supplementation also reduced GC incidence statistically significantly. The findings offer
many opportunities for GC prevention.
Trial registration: NCI PDQ database (trial No. NCI-OH-95-C-N029; available at
http://www.cancer.gov/clinicaltrials).
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Introduction
Gastric cancer (GC) remains the third leading cause of cancer deaths worldwide, with almost
half of the estimated deaths from GC occurring in China in 20181. Linqu County, a rural area in
Shandong province in northeastern China, has one of the highest GC mortality rates in the
world2. Epidemiologic studies have provided solid evidence linking Helicobacter pylori (H.
pylori) infection to the progression of precancerous gastric lesions and development of GC,
whereas diets rich in vitamin and garlic consumption may protect against GC in this high-risk
area3-5. In 1995, the Shandong Intervention Trial (SIT, clinicaltrials.gov identifier:
NCT00339768) was initiated in Linqu, to evaluate the effects of three interventions to prevent
the progression of precancerous gastric lesions and occurrence of GC6-9. The interventions
included H. pylori treatment for 2 weeks, vitamin supplementation, and garlic supplementation,
each for 7.3 years. After 14.7 years’ follow-up (1995-2010), SIT reported a statistically
significant reduction in GC incidence and non-statistically significant reduction in GC mortality
associated with H. pylori treatment, and was recognized as the first single trial to show a clear
reduction in GC incidence from H. pylori treatment8 10 11. Although both garlic and vitamin
supplementation showed favorable trends for decreased GC incidence and mortality, these
effects were not statistically significant8.
While SIT suggested a potential role of H. pylori treatment in GC prevention in the near
term, there was a need for additional follow-up to determine whether the reductions in GC
incidence from H. pylori treatment would persist and lead to a demonstrable reduction in GC
mortality. It also remained unknown whether vitamin and garlic supplementation would yield a
statistically significant reduction in GC incidence and mortality with additionally extended
follow-up. We therefore extended the follow-up of the SIT participants to 22.3 years after
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randomization to ascertain GC incidence and mortality. In addition, to clarify the entire
spectrum of effects of these interventions, we also examined their associations with other
cause-specific mortality rates as secondary outcomes.
Methods
Ethics statement
The original trial and study extension were approved by institutional review boards of Peking
University Cancer Hospital, and the US National Cancer Institute (NCI), and subjects provided
written informed consent.
Study population
Details of the SIT, including study population, trial design and randomization, and treatments
have been described previously6-8. In brief, a total of 3411 subjects aged 35-64 years in Linqu
were randomly assigned on July 23, 1995 (Figure 1). After excluding 46 subjects due to
violations of eligibility, 3365 eligible subjects remained. 2258 H. pylori-seropositive subjects, as
determined by IgG serology from enzyme-linked immunoassay in 1994, were randomly
assigned to three interventions (2-week of H. pylori treatment, and/or 7.3 years of garlic
supplementation and/or 7.3 years of vitamin supplementation) or their placebos in a 2×2×2
factorial design. A total of 1107 H. pylori-seronegative subjects were randomly assigned in a
2×2 factorial trial of vitamin and garlic supplementation, and also received placebo for
antibiotics to preserve masking. Randomized treatment assignments were generated at Westat
(Rockville, MD). Both the participants and the investigators were masked to treatment
assignment.
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From September 15 to November 29, 1995, H. pylori-seropositive subjects received the
combination of amoxicillin (1 g) and omeprazole (20 mg) (n=1130) or placebo (n=1128) twice
daily for 2 weeks for H. pylori treatment. A repeat 2-week course of active treatment was
implemented on 382 subjects for whom the initial active therapy did not eradicate H. pylori, as
determined by 13C urea breath test from January to March 1996. To preserve masking, 383
subjects in the placebo group matched on village, age and sex were offered re-treatment with
placebo. The vitamin and garlic oral supplements were taken twice daily between November 30,
1995 and March 31, 2003, a duration of 7.3 years. The vitamin supplement contained vitamin C
(250 mg), vitamin E (100 IU), and selenium (37.5μg) (n=1677) or placebo (n=1688). From
December 1995 to May 1996, the vitamin supplement also contained beta-carotene (7.5 mg).
The reason for the removal of beta-carotene has been detailed previously6. The garlic
supplement (n=1678) contained 400 mg of aged garlic extract and 2 mg of steam-distilled garlic
oil or placebo (n=1687). The trial was registered at the NCI PDQ database (trial No.
NCI-OH-95-C-N029; available at http://www.cancer.gov/clinicaltrials).
Follow-up and assessment of study endpoints
Participants were followed from the date of trial randomization (July 23, 1995). During the
period of active treatment through March 31 of 2003, participants were visited monthly to
distribute supplements. The initial trial follow-up ended on May 1, 2003. The extended
follow-up was continued through Dec 1, 2017.
The primary outcomes were GC incidence and mortality. We also assessed other
cause-specific mortality as secondary outcomes. Incidence of GC was ascertained either from
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scheduled gastroscopies in 1999 and 2003 for all subjects, with biopsies at seven standard sites;
from gastroscopies conducted from May 2, 2003 to Dec 1, 2017 for those diagnosed with
moderate or severe dysplasia (DYS) at any biopsy site or with mild DYS at two or more sites in
2003; or from cancer registry or autopsy reports, which were confirmed by reviewing medical
records. Details of gastroscopy and biopsy procedures and histopathologic criteria were
described elsewhere2 12. Causes of death were obtained from the Reporting System managed by
the Chinese Center for Disease Control and Prevention, summarizing the information from
Disease Surveillance Points. This internet-based Reporting System integrates death certificates
from hospitals, as well as police and judicial departments in Linqu. To avoid any missed record
of new GC cases or deaths due to delayed reporting, a village doctor supervised the follow-up in
each village and documented the vital status and occurrence of cancer and major chronic
diseases. In addition, staff from Peking University Cancer Hospital & Institute visited each
village every 3 months after 2003 to gather information on GC incidence and cause-specific
mortality. Related medical records with pathological reports were reviewed for confirmation.
Statistical analysis
In the primary analyses, we examined the risk of GC incidence and mortality associated with the
three interventions. Because many GC cases were diagnosed at scheduled gastroscopies in 1999
and 2003, we estimated odds ratios (ORs) of GC cumulative incidence and corresponding 95%
confidence intervals (CIs). The conditional logistic analysis was stratified on baseline
histopathology, categorized as moderate chronic atrophic gastritis (CAG) or less severe, severe
CAG or superficial intestinal metaplasia (IM), deep IM, or DYS. For analysis of GC mortality,
we used the Cox proportional hazards models on the scale of time since randomization to
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estimate hazard ratios (HRs) and 95% CIs, stratified on baseline histopathology. The
proportional hazards assumption within strata was tested by including an interaction term
between time and treatment (P>0.05 for all tests). In the multivariate-adjusted models,
conditional logistic or Cox regression analyses were further adjusted for age, gender, smoking
and alcohol intake. Sensitivity analyses were conducted by including indicators for the other
treatments in addition to the treatment being analyzed in the regression models. In secondary
analyses, we used the Cox model to assess associations of interventions with all-cause mortality
and with cause-specific mortality, including deaths from any cancer, from individual cancers
with at least 10 deaths among baseline H. pylori positive participants, and from cardiovascular
disease (CVD).
Kaplan-Meier survival curves were plotted to compare time to all-cause mortality and to
GC mortality for each intervention. We further examined the temporal changes in the effects of
interventions on cumulative GC incidence and on mortality by dividing the follow-up period
into three separate time intervals: the initial trial period (participant randomization to May 1,
2003)7, extended follow-up period I (May 2, 2003 to August 1, 2010)8 9, and extended follow-up
period II (August 2, 2010 to December 1, 2017) (Figure 1).
We stratified analyses to determine whether the effects of interventions varied by
participants’ baseline histopathology or age. Heterogeneity of ORs or HRs across strata was
evaluated using Q statistics for meta-analysis. To clarify potential effect modification between
interventions, we also tested the 2-way interactions between H. pylori treatment, vitamin
supplementation and garlic supplementation on GC incidence and mortality.
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All analyses were performed using the intention-to-treat approach. All P values were
2-tailed. All statistical calculations were performed with SAS statistical software, version 9.4
(SAS institute, Cary, NC).
Patient and public involvement
No patients were involved in setting the research question or the outcome measures, nor were
they involved in developing plans for recruitment, design, or implementation of the study. No
patients were asked to advise on interpretation or writing up of results. Study results will be
reported by publication, presentation at scientific meetings, and at public events to popularize
cancer prevention and control held by our institution. The results will also be disseminated using
social media platforms.
Results
A total of 3365 participants were included. As shown in Supplementary Table 1, blocked
random intervention assignments6 ensured balanced distribution in baseline characteristics
between active treatment and placebo groups for all three interventions. During 22.3 years’
follow-up since trial randomization (Figure 1), we identified 151 incident GC cases and 94 GC
deaths, which accounted for 12% of all deaths. Of them, 119 GC cases and 76 GC deaths
occurred among the baseline H. pylori positive participants (Table 1).
We first evaluated GC incidence associated with the three interventions (Table 2). H. pylori
treatment was inversely associated with risk of GC incidence, even after multivariate-adjustment
(OR=0.48, 95% CI=0.32-0.71). We also found statistically significant decrease in GC incidence
associated with vitamin supplementation (OR=0.64, 95% CI=0.46-0.91), and a non-significant
decrease in GC incidence associated with garlic supplementation (OR=0.81, 95% CI=0.57-1.13)
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(Table 2).
The risk of GC mortality was also examined as a primary outcome (Table 3). A statistically
significant protective effect on GC mortality was observed for all three interventions; the fully
adjusted HR (95% CI) was 0.62 (0.39-0.99) for H. pylori treatment, 0.48 (0.31-0.75) for vitamin
supplementation and 0.66 (0.43-1.00) for garlic supplementation (Table 3). Kaplan-Meier
curves showed that the cumulative protective effect on GC mortality became evident after about
8 years for H. pylori treatment and vitamin supplementation and after about 12 years for garlic
supplementation (Figure 2). For both GC incidence and mortality, sensitivity analyses that
adjusted for other interventions yielded similar findings (data not shown).
Separate analyses for the three follow-up periods did not demonstrate statistically
significant treatment effect heterogeneity in ORs for GC incidence or HRs for GC mortality
(Figure 3), perhaps because of limited power. However, the garlic supplementation effects were
negligible in the initial trial period both for GC incidence and mortality, which only became
apparent by year 14.7 and persisted through year 22.3 (Figure 3). The treatment effect for
vitamin supplementation was also negligible for GC incidence in the initial trial period, but not
for GC mortality (Figure 3).
In secondary analyses (Table 3), vitamin supplementation was marginally associated with
decreased all-cause mortality (HR=0.87, 95% CI: 0.76-1.01, P=0.07). We did not find
statistically significant associations of the interventions with all cancer mortality or with
esophageal cancer (EC)-specific mortality. H. pylori treatment was marginally associated with a
decrease in colorectal cancer mortality (P=0.07) and a non-significant increase in liver cancer
death (P=0.15). The associations with other specific causes of death were also not statistically
significant (Table 3).
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There was no statistically significant evidence of heterogeneity in the effects of H. pylori
treatment or garlic supplementation for GC incidence or mortality between participants with
baseline normal gastric mucosa, SG, or CAG versus those with IM or DYS, nor among different
age groups (Figure 4). Although tests for heterogeneity were only marginally significant for
vitamin supplementation, the data suggest greater preventive effect for those with favorable
baseline histology (normal, SG, CAG) and those under age 45 years, both for GC incidence and
mortality (Figure 4).
Tests for two-way interactions among treatment effects were not statistically significant.
Among baseline H. pylori seronegative participants, there was a non-significant indication that
those only taking vitamin supplements had a lower GC incidence than those taking both vitamin
and garlic supplements (P-interaction=0.08) (Supplementary Table 2).
Discussion
Based on a total follow-up of 22.3 years in the SIT, the preventive effect of short-term H. pylori
treatment on risk of developing GC continued 22 years post-treatment and resulted in
significantly lower GC mortality. We also found significantly decreased long-term risk of
developing GC associated with vitamin supplementation and reduced GC mortality for those
taking vitamin or garlic supplements.
Implications of findings
The effect of H. pylori treatment on GC has been examined in prior intervention trials7-9 13-18,
and recent studies19 20. Although H. pylori treatment is now recognized as a potential strategy for
GC prevention11 21, major uncertainties are yet to be clarified before the strategy can be
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implemented at the community level. The duration of effectiveness of H. pylori treatment needs
to be studied by long-term follow-up, because the development of GC involves progression
through multiple histologic stages22. Our findings confirm the statistically significant reduction
in GC incidence and the favorable, but not statistically significant reduction in GC mortality
after 14.7 years8, indicating even greater beneficial effect of H. pylori treatment on GC
incidence than we previously reported at 14.7-year (OR=0.61, 95% CI=0.38-0.96)8. The
reduction in GC incidence in SIT was also larger than in recent meta-analyses of intervention
trials23 24. With more GC deaths during longer follow-up, the reduction in GC mortality became
statistically significant 22.3 years after intervention, although the effect became visible in
Kaplan-Meier curves after roughly 8 years.
Gaps remain regarding the full range of beneficial and adverse effects from H. pylori
treatment. An inverse association of H. pylori infection with esophageal adenocarcinoma (EAC)
has been reported11 25 26. Although uncertainty remains for esophageal squamous cell carcinoma
(ESCC), a meta-analysis found decreased ESCC risk associated with H. pylori infection in
Eastern populations26. SIT provided few events and little power to detect effects of H. pylori
therapy on EC mortality and cannot distinguish ESCC from EAC; there is little indication of
adverse risk, however. Increased risk of colorectal cancer associated with H. pylori infection has
also been reported27. In our study, there was an indication of reduced colorectal cancer deaths
with H. pylori treatment, but the number of deaths was again too small to conclusively
demonstrate this effect. In addition, we found a non-significant excess of liver cancer deaths
with H. pylori treatment. It is worth noting that these comparisons were not adjusted for
multiple comparisons. Despite prolonged follow-up, SIT’s sample size for H. pylori treatment
(n=2242) limits our ability to estimate the benefits and risks for many health outcomes.
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Whether a point of no return exists in H. pylori-associated gastric carcinogenesis after
which treatment would be too late to reduce GC risk is still under debate, as H. pylori infection
causes early precancerous histopathologic changes12. The Maastricht V/Florence Consensus
Report stated that H. pylori eradication results in significant improvement of gastritis and gastric
atrophy but not of IM28. A trial also showed that H. pylori eradication did not reduce the
incidence or IM or decrease its histological severity29. However, the Consensus Report also
recognized that the progression of IM can be halted by H. pylori eradication28, as supported by
our intervention trial7.Advanced gastric lesions occur more frequently at old ages2 12, which
raises concerns about the effectiveness of prophylactic H. pylori eradication among those with
advanced gastric lesions and older individuals. Such concerns have been partly addressed by
evidence that H. pylori therapy reduces metachronous GC incidence20 30, but other reports on
new GC with precancerous lesions or metachronous GC have been contradictory15 31, and some
experts recommend using H. pylori treatment only before gastric atrophy or IM develops15. Our
long-term data confirm the earlier SIT finding9 that H. pylori treatment reduces GC incidence
and mortality also in subjects with IM and DYS at baseline and in subjects aged 55-71 years at
baseline. Thus, H. pylori infection may promote late-stage neoplastic progression. It is also
possible that H. pylori treatment has eliminated non-H.pylori bacteria crucial for progression to
GC9 32, supporting the need to explore other microbiota underlying gastric carcinogenesis. In
any case, H. pylori treatment is potentially useful for old as well as young people and for those
with advanced as well as early precancerous gastric lesions.
Few intervention trials have been conducted in populations with nutritional deficiencies.
The Linxian Nutrition Intervention Trial (NIT) showed a durable beneficial effect of 5.25-year’s
supplementation with “factor D”, a combination of selenium, vitamin E, and beta-carotene, on
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total and GC mortality, which lasted up to 10 years post-trial but waned later on33-35. In Linqu,
where SIT was conducted, the mean serum levels of vitamin C and selenium were well below
reference ranges3 36. Observational studies showed that GC risk was inversely associated vitamin
C intake in this nutritionally deprived population5. A high level of serum vitamin C was also
associated with a decreased risk of histologic progression to DYS and GC in Linqu3. Although
no effects of vitamin supplementation were seen on gastric histopathology or GC incidence in
SIT in the initial trial period7, follow-up for 14.7 years revealed noteworthy, but not statistically
significant, reductions in GC incidence and mortality. Thus, the statistically significant SIT
findings of reductions in GC incidence and mortality after 22.3 years are supported by earlier
clinical trial data and observational studies. There is a marginally significant indication that
vitamin supplementation is more effective in those with mild histopathology at baseline and age
under 45 years. Similarly, only participants younger than 55 years benefited from factor D in the
NIT34.
Observational data show decreased risk of GC with elevated consumption of garlic and
allium vegetables in Linqu12 and elsewhere37. Garlic and its derivatives have antioxidative,
antimicrobial, and immune modulation properties38 39. However, there are few long-term
randomized placebo-controlled intervention trials utilizing dietary or supplemental allium
vegetables for cancer prevention. One trial indicated that aged garlic extract prevented
metachronous colorectal adenomas40, while another trial revealed a statistically significant
protective effect of synthetic diallyl trisulfide (allitridum) combined with selenium on GC
incidence in men, but not in women41. After 22.3 years, garlic supplementation led to a
non-statistically significant reduction in GC incidence and a statistically significant reduction in
GC mortality, confirming favorable, but non-statistically significant trends seen at 14.7 years8.
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The time course and duration of protective effects is of interest. H. pylori treatment retarded
the progression of precancerous gastric lesions 7 and evidence began to accumulate for
protective effects on GC incidence and mortality in the initial trial period. The beneficial effects
on GC incidence and mortality persisted through year 14.78 and again through year 22.3 (Figure
3). In contrast, neither vitamin nor garlic supplementation retarded histopathologic progression
during the initial trial ending at 20037. The favorable effects for garlic supplementation came
later than for H. pylori treatment, and the favorable effects for vitamin supplementation on GC
incidence also appeared later than for H. pylori treatment (Figure 3). The effects of these
interventions on GC mortality became visible in Kaplan-Meier plots after about 8 years for H.
pylori treatment and vitamin supplementation and after about 12 years for garlic
supplementation. The mechanisms for these different time patterns in terms of treatment effects
on the carcinogenic process remain to be elucidated.
Strengths and limitations of study
Strengths of SIT included excellent treatment compliance and long-term follow-up, with
virtually complete ascertainment of GC cases and cause-specific deaths in a well-defined high-
risk population. There are several limitations. First, SIT included 3365 participants, and despite
long-term follow-up, the numbers of events were too small to be convincing for some of causes
of death in secondary analyses and also restricted more detailed subgroup analyses for GC.
Second, we cannot disentangle the effect of particular components of vitamin and garlic
supplements or characterize a dose-response relationship. Third, we did not have information
concerning participants’ H. pylori infection status or non-trial treatments for H. pylori or
nutritional or garlic supplementation after the active trial ended in 2003. Although there have
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been dramatic social and economic changes in China since 2003 and some participants may
have changed their diets or lifestyles, it is unlikely that these changes would have differed by
initial intervention assignment, because the SIT was a randomized, placebo-controlled blinded
trial. Fourth, we routinely conducted endoscopic screening during the trial period only, but any
bias in the diagnosis of endoscopically detectable GC since 2003 would be non-differential
between intervention groups. Fifth, our findings might not generalize to a well-nourished
population or a population with low GC risk.
Conclusions
In conclusion, our study found statistically significantly decreased risk of GC incidence and
mortality with short-term H. pylori treatment during 22.3 years after active treatment,
demonstrating the longest durable beneficial effect among the major randomized trials of H.
pylori treatment. Multiyear vitamin supplementation yielded statistically significant reductions
in GC incidence and mortality. Garlic supplementation also yielded a statistically significant
decrease in GC mortality and a promising, but not statistically significant, decrease in GC
incidence. These finding offer many opportunities for GC prevention. However, before major
public health campaigns for GC prevention are launched utilizing antibiotic-based H. pylori
therapy or nutritional regimens, further large-scale intervention trials are warranted to delineate
the full range of beneficial and adverse effects of H. pylori treatment, to confirm the preventive
effects of vitamin and garlic supplementation, and to identify possible risks from nutritional
regimens.
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Acknowledgment: We thank Dr. Joseph F. Fraumeni Jr (National Cancer Institute) for his
assistance with the study before and during the trial period. We are grateful to the residents,
field staff, and government of Linqu county for supporting this trial. We thank Wakunaga of
America, Ltd, for performing assays of S-allyl cysteine and providing garlic supplements,
Shanghai Squibb for providing vitamin supplements, and Astra Corporation for providing
amoxicillin and omeprazole. These study sponsors attended a planning meeting in April 2005 at
which elements of the protocol and provision of intervention materials were discussed, but these
sponsors did not write or approve the protocol, participate in data collection (apart from pro-
viding assays for S-allyl cysteine), interpret the data, participate in writing this article, or
influence the decision to submit this article for publication. We also thank members of the Data
Safety and Monitoring Committee for guidance and oversight during the trial.
Contributors: WQ-L, KF-P and WC-Y had had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the data analysis. WQ-L,
JY-Z, Y-G, KF-P and WC-Y were responsible for the overall study design, data analysis, and
interpretation of data. WQ-L and JY-Z wrote the initial draft of the manuscript. All the authors
were involved in preparing this manuscript. WQ-L, JY-Z, MH-G, WJ-B, KF-P, and WC-Y
contributed to the critical revision of the manuscript.
Funding: This research was supported by the Intramural Research Program of the National
Institutes of Health, National Cancer Institute, and in part by National Cancer Institute Contracts
NO2-CP-71103 and NO2-CP-21169. Additional support was from the National Basic Research
Program of China (973 program: 2004CB518702 and 2010CB529303). The funding sources had
no role in study design; in the collection, analysis, and interpretation of data; in the writing of
the report; or in the decision to submit the article for publication.
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Competing interests: All authors have completed the Unified Competing Interest form at
www.icmje.org/coi_disclosure.pdf (available on request from the corresponding author) and
declare: no support from any organisation for the submitted work; no financial relationships
with any organisations that might have an interest in the submitted work in the previous three
years; no other relationships or activities that could appear to have influenced the submitted
work.
An exclusive license: The corresponding author has the right to grant on behalf of all authors
and does grant on behalf of all authors, an exclusive licence (or non exclusive for government
employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit
this article (if accepted) to be published in BMJ editions and any other BMJPGL products and
sublicences to exploit all subsidiary rights, as set out in our licence
(http://resources.bmj.com/bmj/authors/checklists-forms/licence-for-publication).
Transparency declaration: On behalf of all coauthors, the corresponding authors affirm that
the manuscript is an honest, accurate, and transparent account of the study being reported; that
no important aspects of the study have been omitted; and that any discrepancies from the study
as planned (and, if relevant, registered) have been explained.
Ethical approval: The original trial and study extension were approved by institutional review
boards of Peking University Cancer Hospital, and the US National Cancer Institute (NCI), and
subjects provided written informed consent.
Data sharing: Further information on the intervention trial available upon request.
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19. Leung WK, Wong IOL, Cheung KS, et al. Effects of Helicobacter pylori Treatment on Incidence of Gastric Cancer in Older Individuals. Gastroenterology 2018;155(1):67-75. doi: 10.1053/j.gastro.2018.03.028 [published Online First: 2018/03/20]
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35. Wang SM, Taylor PR, Fan JH, et al. Effects of Nutrition Intervention on Total and Cancer Mortality: 25-Year Post-trial Follow-up of the 5.25-Year Linxian Nutrition Intervention Trial. Journal of the National Cancer Institute 2018 doi: 10.1093/jnci/djy043 [published Online First: 2018/04/05]
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39. Pan Y, Lin S, Xing R, et al. Epigenetic Upregulation of Metallothionein 2A by Diallyl Trisulfide Enhances Chemosensitivity of Human Gastric Cancer Cells to Docetaxel Through Attenuating NF-kappaB Activation. Antioxid Redox Signal 2016;24(15):839-54. doi: 10.1089/ars.2014.6128 [published Online First: 2016/01/24]
40. TANAKA S, HARUMA K, KUNIHIRO M, et al. Effects of aged garlic extract (AGE) on colorectal adenomas a doubleblinded study. Hiroshima J MedSci 2004;53(3.4):39-45.
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Figure 1. The flow diagram of trial design and participant follow-up
Figure 2. The plotted Kaplan-Meier survival estimates for GC mortality (A-C) and total mortality (D-F) by Helicobacter pylori treatment, vitamin and garlic supplementation. Follow-up time begins at trial randomization.
Figure 3. The association of Helicobacter pylori treatment, vitamin and garlic supplementation with gastric cancer incidence (ORs) and mortality (HRs) in three separate time periods. The analyses were conducted adjusting for baseline histology (
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Table 1. The number of incident gastric cancer cases and deaths by three intervention groups during the trial and extended follow-upa
No. of incident gastric cancers No. of deaths due to gastric cancer
Treatment groupsNo. ofsubjects Trial
period
Extended follow-up period I
Extended follow-up period II
Total Trial
period
Extended follow-up period I
Extended follow-up period II
Total
H. pylori treatment 2258 47 39 33 119 18 24 34 76
Active 1130 19 15 7 41 8 10 11 29
Placebo 1128 28 24 26 78 10 14 23 47
Vitamin 3365 59 47 45 151 21 26 47 94
Active 1677 29 19 12 60 9 8 14 31
Placebo 1688 30 28 33 91 12 18 33 63
Garlic 3365 59 47 45 151 21 26 47 94
Active 1678 30 19 20 69 12 9 18 39
Placebo 1687 29 28 25 82 9 17 29 55
aEvents during the trial period included those from the date of random assignment (July 23, 1995), to the
initial follow-up end (May 1, 2003). The follow-up extension included two periods, the extended follow-up
period I (May 2, 2003 to August 1, 2010) and extended follow-up period II (August 2, 2010 to December 1,
2017).
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Table 2. The odds ratios (ORs) and 95% confidence intervals (CIs) for gastric cancer incidence
associated with Helicobacter pylori treatment, vitamin and garlic supplementation
Adjusted for baseline histology only Fully adjusteda
InterventionsPlacebo(n/n)b
Treatment (n/n)b
OR (95% CI) P Placebo(n/n)b
Treatment (n/n)b
OR (95% CI) P
H. pylori treatment
78/1123 40/1119 0.48 (0.33-0.72)
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Table 3. The hazard ratios (HRs) and 95% confidence intervals (CIs) for cause-specific death according to Helicobacter pylori treatment, vitamin and garlic
supplementation
Adjusted for baseline histology only Fully adjusteda
InterventionsPlacebo (n)
Treatment (n)
HR (95% CI) P Placebo (n)Treatment
(n)HR (95% CI) P
H. pylori treatment 1123 1119 1086 1086
Total deaths 272 264 0.98 (0.83-1.16) 0.79 254 245 1.00 (0.84-1.19) 0.96
All cancerb 141 115 0.81 (0.63-1.04) 0.10 131 105 0.81 (0.62-1.05) 0.10
Gastric cancer 47 29 0.62 (0.39-0.99) 0.045 45 29 0.62 (0.39-0.99) 0.046
Esophageal cancer 12 14 1.17 (0.54-2.53) 0.69 10 11 1.12 (0.47-2.64) 0.80
Liver cancer 11 21 1.93 (0.93-4.01) 0.08 10 18 1.78 (0.82-3.86) 0.15
Colorectal cancer 12 3 0.25 (0.07-0.89) 0.03 10 3 0.30 (0.08-1.10) 0.07
Lung cancer 40 33 0.83 (0.52-1.31) 0.42 38 31 0.82 (0.51-1.32) 0.42
Other cancer 19 15 0.79 (0.40-1.56) 0.50 18 13 0.73 (0.36-1.50) 0.40
Cardiovascular Disease 96 106 1.11 (0.84-1.47) 0.45 90 100 1.17 (0.88-1.55) 0.29
Other deaths 35 43 1.24 (0.79-1.93) 0.35 33 40 1.27 (0.80-2.01) 0.31
Vitamin 1679 1665 1627 1610
Total deaths 423 370 0.86 (0.75-0.99) 0.04 392 345 0.87 (0.76-1.01) 0.07
All cancerb 187 166 0.89 (0.72-1.09) 0.26 175 152 0.88 (0.71-1.09) 0.23
Gastric cancer 62 31 0.49 (0.32-0.76) 0.001 61 29 0.48 (0.31-0.75) 0.001
Esophageal cancer 18 15 0.84 (0.42-1.67) 0.62 16 12 0.77 (0.36-1.63) 0.50
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Liver cancer 21 25 1.20 (0.67-2.15) 0.53 18 24 1.38 (0.75-2.55) 0.30
Colorectal cancer 11 10 0.92 (0.39-2.17) 0.85 10 8 0.81 (0.32-2.06) 0.66
Lung cancer 49 56 1.16 (0.79-1.70) 0.46 44 53 1.23 (0.83-1.84) 0.30
Other cancer 26 29 1.13 (0.66-1.91) 0.66 26 26 1.02 (0.59-1.76) 0.94
Cardiovascular Disease 168 141 0.84 (0.67-1.05) 0.12 156 132 0.84 (0.67-1.06) 0.14
Other deaths 68 63 0.93 (0.66-1.32) 0.70 61 61 1.02 (0.71-1.45) 0.92
Garlic 1678 1666 1631 1606
Total deaths 406 387 0.96 (0.83-1.10) 0.53 383 354 0.90 (0.78-1.04) 0.17
All cancerb 185 168 0.91 (0.74-1.12) 0.36 175 152 0.87 (0.70-1.08) 0.19
Gastric cancer 55 38 0.69 (0.46-1.04) 0.08 54 36 0.66 (0.43-1.00) 0.049
Esophageal cancer 18 15 0.82 (0.42-1.64) 0.58 16 12 0.78 (0.37-1.65) 0.51
Liver cancer 22 24 1.11 (0.62-1.98) 0.73 19 23 1.26 (0.69-2.32) 0.46
Colorectal cancer 11 10 0.93 (0.40-2.19) 0.87 11 7 0.63 (0.24-1.64) 0.34
Lung cancer 54 51 0.95 (0.65-1.39) 0.79 51 46 0.91 (0.61-1.36) 0.66
Other cancer 25 30 1.22 (0.72-2.07) 0.47 24 28 1.21 (0.70-2.08) 0.50
Cardiovascular Disease 152 157 1.04 (0.83-1.30) 0.74 142 146 1.04 (0.83-1.31) 0.72
Other deaths 69 62 0.90 (0.64-1.26) 0.53 66 56 0.86 (0.60-1.23) 0.40aThe fully adjusted analyses were adjusted for baseline histology (
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Figure 1. The flow diagram of trial design and participant follow-up
106x134mm (300 x 300 DPI)
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Figure 2. The plotted Kaplan-Meier survival estimates for GC mortality (A-C) and total mortality (D-F) by Helicobacter pylori treatment, vitamin and garlic supplementation. Follow-up time begins at trial
randomization.
1243x636mm (144 x 144 DPI)
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Figure 3. The association of Helicobacter pylori treatment, vitamin and garlic supplementation with gastric cancer incidence (ORs) and mortality (HRs) in three separate time periods. The analyses were conducted
adjusting for baseline histology (
Confidential: For Review Only
Figure 4. The association of Helicobacter pylori treatment, vitamin and garlic supplementation with gastric cancer incidence and mortality, stratified by baseline gastric lesions and age. The analyses were conducted, adjusting for baseline histology (
Confidential: For Review Only
Supplementary Table 1. Baseline characteristics of study subjects
H. pylori treatment Vitamin supplementation Garlic supplementationGroup Active
(n=1130) Placebo (n=1128)
Active(n=1677)
Placebo (n=1688)
Active (n=1678)
Placebo(n=1687)
Age (Mean ± SD) 46.8±9.2 46.9±9.2 47.0±9.2 47.1±9.2 47.0±9.2 47.1±9.2
Sex (%)
Female 50.4 49.7 48.8 48.6 48.8 48.6
Male 49.6 50.3 51.2 51.4 51.2 51.4
Baseline histology (%)
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Supplementary Table 2. The 2-way interactions between interventions on gastric cancer
incidence and mortality
Gastric cancer incidencea Gastric cancer mortalityaSubgroups
n/nb OR (95% CI) P for interactionc n/nb HR (95% CI) P for interactionc
Baseline H. pylori seropositive participants
Joint effect of vitamin supplementation and H. pylori eradication
Placebo 44/549 Reference 0.72 28/549 Reference 0.56
H. pylori treatment only 25/543 0.50 (0.30-0.85) 20/543 0.69 (0.39-1.22)
Vitamin only 32/537 0.69 (0.42-1.13) 17/537 0.60 (0.33-1.11)
H. pylori treatment and vitamin 15/543 0.30 (0.16-0.55) 9/543 0.31 (0.15-0.66)
Joint effect of garlic supplementation and H. pylori eradication
Placebo 40/546 Reference 0.52 28/546 Reference 0.52
H. pylori treatment only 23/546 0.54 (0.31-0.92) 16/546 0.55 (0.30-1.01)
Garlic only 36/540 0.93 (0.57-1.50) 17/540 0.59 (0.32-1.07)
H. pylori treatment and garlic 17/540 0.38 (0.21-0.69) 13/540 0.44 (0.23-0.84)
Joint effect of garlic and vitamin supplementation
Placebo 41/548 Reference 0.15 31/548 Reference 0.20
Vitamin only 22/544 0.50 (0.29-0.87) 13/544 0.41 (0.22-0.79)
Garlic only 28/544 0.66 (0.39-1.10) 17/544 0.53 (0.29-0.95)
Vitamin and garlic 25/536 0.59 (0.35-1.00) 13/536 0.41 (0.21-0.78)
Baseline H. pylori seronegative participants
Joint effect of garlic and vitamin supplementation
Placebo 14/268 Reference 0.08 8/268 Reference 0.73
Vitamin only 4/271 0.29 (0.09-0.90) 2/271 0.28 (0.06-1.36)
Garlic only 6/267 0.43 (0.16-1.16) 5/267 0.71 (0.22-2.28)
Vitamin and garlic 7/259 0.51 (0.20-1.32) 1/259 0.12 (0.02-1.00)
aThe fully adjusted analyses were adjusted for baseline histology (
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