Helicobacter pylori seropositivity and risk of lung cancer.

Abstract

Lung cancer is the leading cause of cancer mortality worldwide. Helicobacter pylori (H. pylori) is a risk factor for distal stomach cancer, and a few small studies have suggested that H. pylori may be a potential risk factor for lung cancer. To test this hypothesis, we conducted a study of 350 lung adenocarcinoma cases, 350 squamous cell carcinoma cases, and 700 controls nested within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC) cohort of male Finnish smokers. Controls were one-to-one matched by age and date of baseline serum draw. Using enzyme-linked immunosorbent assays to detect immunoglobulin G antibodies against H. pylori whole-cell and cytotoxin-associated gene (CagA) antigens, we calculated odds ratios (ORs) and 95% confidence intervals (95% CIs) for associations between H. pylori seropositivity and lung cancer risk using conditional logistic regression. H. pylori seropositivity was detected in 79.7% of cases and 78.5% of controls. After adjusting for pack-years and cigarettes smoked per day, H. pylori seropositivity was not associated with either adenocarcinoma (OR: 1.1, 95% CI: 0.75-1.6) or squamous cell carcinoma (OR: 1.1, 95% CI: 0.77-1.7). Results were similar for CagA-negative and CagA-positive H. pylori seropositivity. Despite earlier small studies suggesting that H. pylori may contribute to lung carcinogenesis, H. pylori seropositivity does not appear to be associated with lung cancer.

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Helicobacter pylori Seropositivity and Risk of Lung
Cancer
Jill Koshiol1*, Roberto Flores1, Tram K. Lam1, Philip R. Taylor1, Stephanie J. Weinstein1, Jarmo Virtamo2,
Demetrius Albanes1, Guillermo Perez-Perez3, Neil E. Caporaso1., Martin J. Blaser3.
1Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, U.S. Department of Health and Human Services, Bethesda,
Maryland, United States of America, 2Department of Chronic Disease Prevention, National Institute for Health and Welfare, Helsinki, Finland, 3Departments of Medicine
and Microbiology, New York University School of Medicine, New York, New York, United States of America
Abstract
Lung cancer is the leading cause of cancer mortality worldwide. Helicobacter pylori (H. pylori) is a risk factor for distal
stomach cancer, and a few small studies have suggested that H. pylori may be a potential risk factor for lung cancer. To test
this hypothesis, we conducted a study of 350 lung adenocarcinoma cases, 350 squamous cell carcinoma cases, and 700
controls nested within the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC) cohort of male Finnish
smokers. Controls were one-to-one matched by age and date of baseline serum draw. Using enzyme-linked immunosorbent
assays to detect immunoglobulin G antibodies against H. pylori whole-cell and cytotoxin-associated gene (CagA) antigens,
we calculated odds ratios (ORs) and 95% confidence intervals (95% CIs) for associations between H. pylori seropositivity and
lung cancer risk using conditional logistic regression. H. pylori seropositivity was detected in 79.7% of cases and 78.5% of
controls. After adjusting for pack-years and cigarettes smoked per day, H. pylori seropositivity was not associated with either
adenocarcinoma (OR: 1.1, 95% CI: 0.75–1.6) or squamous cell carcinoma (OR: 1.1, 95% CI: 0.77–1.7). Results were similar for
CagA-negative and CagA-positive H. pylori seropositivity. Despite earlier small studies suggesting that H. pylori may
contribute to lung carcinogenesis, H. pylori seropositivity does not appear to be associated with lung cancer.
Citation: Koshiol J, Flores R, Lam TK, Taylor PR, Weinstein SJ, et al. (2012) Helicobacter pylori Seropositivity and Risk of Lung Cancer. PLoS ONE 7(2): e32106.
doi:10.1371/journal.pone.0032106
Editor: Masaru Katoh, National Cancer Center, Japan
Received November 5, 2011; Accepted January 23, 2012; Published February 24, 2012
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This research was supported by General Funds from the Intramural Research Program of the National Institutes of Health, National Cancer Institute,
Division of Cancer Epidemiology and Genetics. The Division of Cancer Epidemiology and Genetics reviewed and approved the ATBC study and cleared the
manuscript for publication but had no role in the analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: koshiolj@mail.nih.gov
. These authors contributed equally to this work.
Introduction
Lung cancer kills more people worldwide (over 1 million each
year) than any other cancer [1]. Although smoking is the primary
cause, most smokers ($80%) never develop lung cancer [2],
suggesting that oncogenesis requires additional co-factors. Infec-
tions and immune responses that mediate inflammation may
contribute to lung carcinogenesis [3,4]. Evidence supporting this
hypothesis includes associations of lung cancer with 1) elevated
inflammatory markers, such as C-reactive protein, interleukin (IL)-
6, and IL-8 [5,6]; 2) chronic obstructive pulmonary disease, to
which infections can contribute [7,8]; 3) human leukocyte antigen
polymorphisms in genome-wide association studies [9,10]; and 4)
overt infections like tuberculosis and pneumonia [3,7]. In addition,
Jaagsiekte sheep retrovirus causes ovine pulmonary adenocarci-
noma (OPA), a malignancy occurring in sheep [4]. OPA has
similar histology to human lung adenocarcinoma, bronchiolar-
alveolar adenocarcinoma in particular. In humans, lung adeno-
carcinoma occurs at younger ages more often than squamous cell
carcinoma [4], which is consistent with an infectious origin since
some infection-related cancers occur at younger ages [11,12].
One microbe postulated to play a role in lung cancer is
Helicobacter pylori (H. pylori) [13,14]. H. pylori is a key etiologic agent
in the development of distal stomach cancer [15]. A gram-negative
spiral-shaped bacterium, H. pylori colonizes the gastric mucosa,
inducing local inflammation and a systemic immune response
[16]. H. pylori has been classified as a group 1 carcinogen for
stomach cancer by the International Agency for Research on
Cancer [17]. H. pylori can be broadly categorized into two groups:
type I strains, which express the cytotoxin-associated gene (cagA),
and type II strains, which do not [18]. CagA-positive strains affect
gastric epithelial cell signal transduction, which can affect the
cytoskeleton, inflammatory cascades, and mitogenic pathways
[16,18].
H. pylori could potentially affect the lungs in several ways.
Lipopolysaccharide is the major component of the cell wall of
gram-negative bacteria like H. pylori. Lipopolysaccharide stimu-
lates the production of pro-inflammatory cytokines including IL-1,
IL-6, and tumor necrosis factor (TNF)-alpha. This local inflam-
mation can have systemic effects [16,19,20]. H. pylori persistence
leads to chronic inflammation and immune stimulation, which
could contribute to carcinogenesis or conditions associated with
lung cancer, such as chronic bronchitis [16]. The lungs develop
embryologically from the same endodermal cells that line the
gastrointestinal (GI) tract and contain cells that produce peptide
hormones like gastrin [14]. Therefore, higher plasma levels of
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gastrin due to H. pylori in the stomach might promote cellular
proliferation in the lungs as well [14].
It is also possible that gastric H. pylori colonization could
decrease the risk of lung cancer. H. pylori prevalence has declined
over the last 70 years, accompanied by a marked decrease in
noncardia gastric cancer and an increase in esophageal adeno-
carcinoma [21]. Similar to the esophagus where the proportion of
cancers due to adenocarcinomas are increasing in relation to
squamous cell cancers, the relative proportion of lung adenocar-
cinoma has been increasing [22]. With the observed inverse
association between H. pylori and esophageal adenocarcinoma in
Western countries and the lack of association with esophageal
squamous cell carcinoma, an inverse association between H. pylori
and lung adenocarcinoma but no association with lung squamous
cell carcinoma also could be hypothesized.
Prior assessment of the association between H. pylori and lung
cancer has been limited, with fewer than 75 cases in each of five
case-control studies [13,14,23,24,25]. A recent meta-analysis
including four of these studies calculated a pooled odds ratio
(OR) of 3.2 [95% confidence interval (CI): 1.1–9.5], but the
authors noted marked heterogeneity in the results from these
studies [26]. Using existing H. pylori seropositivity data from
previous studies in the Alpha-Tocopherol, Beta-Carotene Cancer
Prevention Study (ATBC) cohort of male Finnish smokers
[27,28,29,30], we found suggestive evidence of an inverse
association of H. pylori with lung adenocarcinoma, but not
squamous cell carcinoma (see Methods section). Based on these
intriguing preliminary data and the lack of well-powered, high-
quality studies, we designed the first prospective study of H. pylori
seropositivity and lung cancer in ATBC.
Materials and Methods
Study population
The ATBC Study, a randomized, double-blind, placebo-
controlled, primary prevention trial, was designed to test the
hypothesis that daily supplementation with alpha-tocopherol, beta-
carotene, or both would reduce the incidence of lung or other
cancers among male smokers. Details of this study have been
published [31]. In brief, Finnish males between 50 and 69 years of
age were identified through the Central Population Register.
Questionnaires were mailed to those with available addresses. Men
who smoked $5 cigarettes per day and who agreed to participate
were mailed an invitation to a local field station for further
evaluation. After excluding men with proven malignancy (other
than nonmelanoma skin cancer or carcinoma in situ), severe
angina on exertion, chronic renal insufficiency, liver cirrhosis,
chronic alcoholism, current anticoagulant therapy, other medical
problems that could limit participation for 6 years, or current use
of supplements with vitamin E (.20 mg/d) or vitamin A
(.20,000 IU/d=2000 retinol
(.6 mg/d), 29,246 men were randomized. Later, 113 men were
found to be ineligible, leaving 29,133 eligible Finnish male
smokers enrolled in ATBC between 1985 and 1988. Both the US
National Cancer Institute and the Finnish National Public Health
Institute institutional review boards approved the ATBC Study,
and all participants provided written informed consent. The men
continue to be followed as a cohort since the trial ended in 1993.
equivalents) orbeta-carotene
Preliminary evaluation using existing data
We used existing H. pylori seropositivity data from previous
studies in ATBC [27,28,29,30] to conduct a preliminary
evaluation of the association between H. pylori and lung cancer.
Enzyme-linked immunosorbent assay (ELISA) measurements of
H. pylori seropositivity were available from 98 lung cancer cases (11
with documented adenocarcinoma and 33 with squamous cell
carcinoma) and over 1500 subjects without lung cancer. The
preliminary data revealed no apparent association between H.
pylori seropositivity and lung cancer overall (OR: 0.96, 95% CI:
0.61–1.5). However, we observed a trend toward an inverse
association with adenocarcinoma (OR: 0.46, 95% CI: 0.14–1.5),
which was stronger for CagA-positive H. pylori strains (OR: 0.38,
95% CI: 0.07–2.0).
Case and control definition
Lung cancer cases were identified using the International
Classification of Diseases, 9thRevision (ICD-9) [32] code 162,
through the Finnish Cancer Registry, which provides nearly 100%
coverage of all cancer cases in Finland [33]. Histology was
determined using the International Classification of Diseases for
Oncology, 2ndedition (ICD-O-2), and 3rdedition (ICD-O-3).
Squamous cell carcinoma included malignant ICD-O codes of
8070. Adenocarcinoma included malignant ICD-O codes of 8140
(adenocarcinoma), 8250 (bronchioloalveolar carcinoma), and 8260
(papillary carcinoma). Cases were selected from among all first
primary lung squamous cell carcinoma or adenocarcinoma cases
diagnosed at least 1 year after the baseline draw date through 30
July 2007. We randomly selected 350 from 927 eligible squamous
cell carcinoma cases and 350 from 392 eligible adenocarcinoma
cases (Figure 1). Controls were matched one-to-one by age at
baseline serum draw (+/25 years) and date of baseline serum
draw (+/230 days). Controls had to be alive and cancer free at the
date that the corresponding case was diagnosed.
Helicobacter pylori testing
We tested fasting serum specimens collected at baseline and
stored in aliquots at 270uC for immunoglobulin G antibodies
against H. pylori whole-cell and CagA antigens using ELISAs
[34,35]. Eleven samples (four from cases and seven from controls)
lacked sufficient serum for testing. We calculated optical density
ratios relative to co-analyzed laboratory standards and classified
optical density ratios $1.0 for the whole-cell antigen assay and
$0.35 for the CagA antigen assay as positive. We evaluated H.
pylori seropositivity in two ways. First, we examined any H. pylori
seropositivity (whole-cell or CagA seropositivity) compared to
seronegativity for both whole-cell and CagA antigens. Second, we
split H. pylori seropositivity into whole-cell and CagA seropositivity.
Twelve individuals were classified as seropositive for CagA and
seronegative for antibodies against the whole-cell antigen. A
previous study comparing serology with gastric biopsy culture
found that individuals who are negative for H. pylori whole-cell
antibodies but positive for CagA antibodies had CagA-positive H.
pylori organisms present in their stomach by culture [36].
Therefore, we classified individuals who were seronegative for
both whole-cell and CagA antibodies as H. pylori negative,
individuals seropositive for whole-cell but seronegative for CagA
as
H.pylori-seropositive/CagA
strains), and individuals seropositive for CagA, regardless of
whole-cell serostatus, as CagA seropositive (CagA-positive strains).
In addition to the 1389 samples from lung cancer cases and
controls, 3 quality control serum samples aliquoted from a single
large pool were equally distributed among 37 different batches. All
samples were assayed in duplicate in the same batch by
experienced technicians blinded to case-control status. In the case
of indeterminate results (i.e., if the values from the duplicate
sample straddled the seropositivity threshold), a repeat analysis was
conducted on the residual of the original aliquot and averaged
across all results, excluding obvious outliers. 95.9% of the samples
seronegative(CagA-negative
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were resolved with two runs in the whole-cell ELISA, and 94.9%
of the samples were resolved with two runs in the CagA ELISA.
More than 99% of samples were resolved with two or three runs
(99.5% for whole-cell and 99.4% for CagA). The remainders were
resolved with four runs.
Statistical analysis
Based on the observed prevalence of H. pylori seropositivity in a
prior ATBC study with a prevalence of 45% among controls [27],
we designed this study to have 90% power to detect ORs of 0.70
or 1.42 for CagA and all lung cancers combined. Assuming an
overall H. pylori exposure prevalence of 75% among the controls,
the minimal detectable ORs at 90% power were 0.68 or 1.53 for
all lung cases compared to controls. We calculated within-batch
coefficients of variation (CV) for the whole-cell and CagA antigen
assays based on continuous optical density values from the 111
pooled QC samples (three within each batch). The within batch
CVs were 12.9% for the whole-cell assay and 25.5% for the CagA
assay, similar to the CVs of 15% and 20%, respectively, in a
previous study of H. pylori and gastric cancer in ATBC using the
same laboratory [27].
We used conditional logistic regression to calculate separate
ORs and 95% CIs for the association of lung adenocarcinoma and
squamous cell carcinoma with H. pylori seropositivity, using the
categories described above. Results for adenocarcinoma and
squamous cell carcinoma were similar according to the Wald test
for homogeneity [37] (P=0.8), so we also evaluated all lung cancer
patients combined.
Potential confounders included ATBC treatment group (place-
bo, alpha-tocopherol, beta-carotene, or both), age at randomiza-
tion (continuous), baseline pack-years (continuous), baseline
number of cigarettes per day (continuous), tooth loss (continuous),
dentures (yes/no), obesity (ordinal body mass index ,25, 25–30,
30+), asthma (yes/no), emphysema (yes/no), bronchitis (yes/no),
and education (,elementary, elementary, partial junior high,
junior high, senior high, graduate). We evaluated the importance
of these variables by removing each covariate from the model one
by one through backwards modeling and determining whether the
ORs for CagA-negative strains and CagA-positive strains changed
by at least 10% in comparison with the full model. No covariate
met this criterion. However, we included pack-years and smoking
amount (cigarettes per day) in all final models to adjust for
smokingexposure,in accord withseveralrecent studies
[8,38,39,40]. Adjusting for pack-years or amount of smoking
had little effect on the ORs (e.g., 2–3% for any H. pylori
seropositivity).
Sensitivity analyses.
A
seropositivity in Finland found that antibody levels remain
relatively stable over time in this population [41], and in ATBC
the association between H. pylori and gastric cancer was similar
when stratified by time to diagnosis [27]. However, to evaluate the
effect of potential changes in H. pylori antibody levels over time, we
stratified by median time since blood draw. Since microbe-related
cancers often occur at younger ages than those not related to
infection [11,12], we also stratified by median age at diagnosis and
evaluated whether the association between any H. pylori
seropositivity and lung cancer varied by age at diagnosis (i.e.,
whether there were departures from multiplicative joint effects)
using the likelihood ratio test (LRT) for interaction terms [37,42].
previous studyof
H.pylori
Results
The case and control series were similar with regard to age,
median cigarettes per day, and other characteristics (Table 1). As
expected based on the primary results of the trial [43,44],
squamous cell cases were slightly less likely to have received
placebo and slightly more likely to have received beta-carotene
than controls. Cases had a higher number of pack-years than
controls, but adjusting for pack-years did not affect the association
between H. pylori and lung cancer, as described in the Methods
section. Among the 696 cases and 693 controls with sufficient
sample available, 555 cases (79.7%) and 544 controls (78.5%) had
antibodies against either H. pylori whole-cell or CagA antigens
(Table 2). Thirty-seven percent of cases (N=258) and 34.3% of
controls (N=238) were seropositive for CagA-negative strains of
H. pylori, and 42.7% of cases (N=297) and 44.2% of controls
(N=306) were seropositive for CagA-positive strains. Of the 603
CagA-positive individuals, 591 [289 out of 297 cases (97.3%) and
302 out of 306 controls (98.7%)] also were positive for antibodies
against the whole-cell antigens.
H. pylori seropositivity was not associated with lung cancer in
ATBC (Table 2). The OR for any H. pylori seropositivity and all
lung cancers combined was 1.1 (95% CI: 0.86–1.5). The ORs did
not vary by histology (OR: 1.1, 95% CI: 0.75–1.6 for
adenocarcinoma and 1.1, 95% CI: 0.77–1.7 for squamous cell
carcinoma). In addition, there was no notable variability in the
Figure 1. Lung cancer case and control selection for Helicobacter pylori serology testing within the ATBC cohort. *Individuals with
nonmelanoma skin cancer were not excluded. {Eligible cases included all first primary lung squamous cell carcinoma or adenocarcinoma cases
diagnosed at least 1 year after the baseline draw date through 30 July 2007. {Controls were matched one-to-one by age at baseline serum draw (+/
25 years) and date of baseline serum draw (+/230 days). Controls had to be alive and cancer free at the date that the corresponding case was
diagnosed.
doi:10.1371/journal.pone.0032106.g001
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Table 1. Characteristics of randomly selected lung adenocarcinoma and squamous cell carcinoma cases and matched cancer-free
controls from the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study.
CharacteristicSubgroup
Adenocarcinoma Squamous cell carcinoma
Cases Controls*CasesControls*
Median age at baseline serum draw58.058.059.859.6
Median date of baseline serum draw1/23/19871/23/19871/22/19871/23/1987
Treatment group, N (%)
Placebo 84 (24.0)94 (26.9)75 (21.4) 97 (27.7)
Beta-carotene 88 (25.1)85 (24.3) 110 (31.4)87 (24.9)
Alpha-tocopherol89 (25.4) 83 (23.7)80 (22.9)86 (24.6)
Alpha-tocopherol+beta-carotene 89 (25.4)88 (25.1) 80 (22.9) 85 (24.3)
Education level, N (%)
Primary school or lower282 (80.6)275 (78.6) 291 (83.1)288 (82.3)
High school or higher 68 (19.4)75 (21.4) 59 (16.9)62 (17.7)
Obesity, N (%)
BMI{,25 178 (50.9)136 (38.9)157 (44.9) 129 (36.9)
BMI{25–,30144 (41.1)160 (45.7) 141 (40.3)165 (47.1)
BMI{$3028 (8.0) 54 (15.4)52 (14.9) 56 (16.0)
Median cigarettes per day (range)20 (5–55) 20 (5–75)20 (5–60) 20 (5–60)
Median pack-years (range) 42 (1–121)35 (1–101)42 (4–111) 35 (1–123)
Asthma, N (%)14 (4.0) 8 (2.3)19 (5.4) 7 (2.0)
Emphysema, N (%) 32 (9.1)24 (6.9) 36 (10.3)21 (6.0)
Bronchitis, N (%) 41 (11.7)27 (7.7) 47 (13.4)25 (7.1)
Median number of lost teeth (range)4 (1–5)4 (1–5)4 (2–5)4 (1–5)
Dentures, N (%)238 (68.0) 216 (61.9)261 (74.8) 241 (68.9)
*Controls matched with cases on age at baseline serum draw and date of baseline serum draw.
{BMI=body mass index.
doi:10.1371/journal.pone.0032106.t001
Table 2. Association of Helicobacter pylori (H. pylori) seropositivity with risk of lung adenocarcinoma and squamous cell carcinoma
in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study.
Model H. pylori serostatusHistology Cases, N* (%) Controls, N* (%) OR (95% CI){
1H. pylori seronegativeAdenocarcinoma 74 (21.2)79 (22.8)1.0 (referent)
H. pylori seropositive 275 (78.8) 268 (77.2)1.1 (0.75–1.6)
2{
H. pylori seronegative 74 (21.2) 79 (22.8)1.0 (referent)
CagA-seronegative{
125 (35.8)119 (34.3)1.1 (0.71–1.7)
CagA-seropositive{
150 (43.0)149 (42.9)1.1 (0.73–1.7)
1H. pylori seronegativeSquamous cell carcinoma67 (19.3)70 (20.2)1.0 (referent)
H. pylori seropositive280 (80.7)276 (79.8)1.1 (0.77–1.7)
2{
H. pylori seronegative67 (19.3)70 (20.2)1.0 (referent)
CagA-seronegative {
133 (38.3) 119 (34.4) 1.3 (0.82–1.9)
CagA-seropositive {
147 (42.4)157 (45.4)1.0 (0.65–1.6)
1H. pylori seronegative All lung cancer141 (20.3) 149 (21.5)1.0 (referent)
H. pylori seropositive555 (79.7) 544 (78.5) 1.1 (0.86–1.5)
2{
H. pylori seronegative 141 (20.3)149 (21.5)1.0 (referent)
CagA-seronegative {
258 (37.1) 238 (34.3)1.2 (0.87–1.6)
CagA-seropositive {
297 (42.7) 306 (44.2)1.1 (0.79–1.5)
*N’s do not sum to total due to missing values.
{Odds ratios (ORs) and 95% confidence intervals (CIs) were adjusted for baseline pack-years and total number of cigarettes per day.
{Single model for CagA-seropositive and CagA-seronegative versus no H. pylori seropositivity.
doi:10.1371/journal.pone.0032106.t002
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associations for CagA-negative or CagA-positive H. pylori seropos-
itivity (Table 2). Results were similar when limited to heavy
smokers with more than 35 pack-years (e.g., OR: 0.99, 95% CI:
0.63–1.6; LRT p-value: 0.7 for any H. pylori seropositivity and all
lung cancers combined).
Sensitivity analyses
Despite concerns that changes in H. pylori seropositivity over
time may weaken the association between H. pylori seropositivity
and cancer risk, associations were similar by median time between
baseline serum draw and diagnosis in this study (Table 3). ORs
tended to be slightly elevated for cases diagnosed more than 9
years after baseline serum draw, but there was no significant
association. In addition, there was little difference in the
association between H. pylori seropositivity and lung cancer by
median age at diagnosis (age 68). For example, the ORs for any H.
pylori seropositivity and any lung cancer were 1.1 (95% CI: 0.73–
1.6) for cases diagnosed at or before age 68 compared to their
matched controls and 1.2 (95% CI: 0.79–1.8) for cases diagnosed
after age 68 (LRT p-value: 0.7). Similarly, the ORs for CagA-
negative H. pylori seropositivity and CagA-positive H. pylori were
1.0 (95% CI: 0.68–1.6) and 1.1 (95% CI: 0.72–1.7), respectively,
for cases diagnosed at or before age 68 and 1.3 (95% CI: 0.83–2.0)
and 1.1 (95% CI: 0.69–1.7) for cases diagnosed after age 68 (LRT
p-value: 0.7). Results were similar for adenocarcinoma and
squamous cell carcinoma separately.
Discussion
Given our preliminary evidence from existing data in ATBC
supporting an inverse association between H. pylori seropositivity
and lung adenocarcinoma on the one hand, and previous
published studies supporting a positive association on the other,
we carefully designed a large nested case-control study to
thoroughly address the hypothesis that H. pylori is associated with
lung cancer. In this well-powered nested case-control study, we
found no evidence of an association between H. pylori and lung
cancer. Neither overall H. pylori seropositivity nor CagA-specific H.
pylori seropositivity were associated with lung cancer. Results were
equally null for lung adenocarcinoma and squamous cell
carcinoma. Sensitivity analyses by time since diagnosis and age
had little effect.
The results of previous studies of H. pylori seropositivity and lung
cancer have been mixed. Of the five previous small case-control
studies, three found moderate to strong increased risk of lung
cancer associated with H. pylori seropositivity [13,14,25], and two
found only weak, non-statistically significant evidence of a positive
association [23,24]. Two studies provided no information
regarding smoking status and lung histology [14,23], and three
provided limited information on smoking status [13,24,25]. Only
one previous small study of 22 squamous cell carcinoma cases and
21 adenocarcinoma cases provided data on H. pylori seropositivity
by lung histology; the association in that study did not vary by lung
histology [13]. None of these studies evaluated factors like smoking
that might affect the association between H. pylori seropositivity
and lung cancer. Given this lack of adjustment for potential
confounders and the fact that small studies are more likely to be
affected by chance [45] and less likely to publish null findings [46],
it unsurprising that previous, small studies found positive (albeit
imprecise) associations.
This study improves on previous studies in a number of ways.
First, as a case-control study nested within the ATBC cohort, this
study provides the only prospective data on H. pylori seropositivity
and subsequent risk of lung cancer. With 700 lung cancer cases,
this study is nearly 10 times larger than the next largest study and
is well powered to assess differences by histology (adenocarcinoma
and squamous cell carcinoma). In addition, all participants were
smokers at baseline, so there is less variation in smoking habits and
therefore less potential for confounding by smoking. As a well-
characterized epidemiologic cohort, ATBC has extensive infor-
mation about potential confounders, which allowed us to carefully
control for cumulative smoking exposure and thoroughly assess
other potential confounders. Finally, the association between H.
pylori and gastric cancer has already been established in this
Table 3. Association of Helicobacter pylori (H. pylori) seropositivity with risk of lung adenocarcinoma and squamous cell carcinoma
by time to diagnosis in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study.
Comparison Time to diagnosis
OR (95% CI)*
AdenocarcinomaSquamous cell carcinomaAll lung cancer
Any H. pylori seropositivity
versus none
#9 years 0.95 (0.54–1.7)1.1 (0.64–1.9) 1.0 (0.70–1.5)
.9 years 1.3 (0.74–2.2)1.3 (0.72–2.4) 1.3 (0.86–1.9)
CagA-negative seropositivity
versus none
#9 years{
0.96 (0.52–1.8)1.2 (0.64–2.1) 1.1 (0.70–1.6)
.9 years{
1.2 (0.68–2.2) 1.5 (0.79–2.9) 1.4 (0.88–2.1)
CagA-positive seropositivity
versus none
#9 years{
0.93 (0.49–1.8)1.0 (0.57–1.9) 0.99 (0.64–1.5)
.9 years{
1.3 (0.72–2.3) 1.0 (0.53–2.1)1.2 (0.77–1.9)
*Odds ratios (ORs) and 95% confidence intervals (CIs) were adjusted for baseline pack-years and total number of cigarettes per day.
{Derived from a single model for CagA-seropositive and CagA-seronegative versus no H. pylori seropositivity for all cases diagnosed #9 years after baseline blood draw
and their paired controls.
{Derived from a single model for CagA-seropositive and CagA-seronegative versus no H. pylori seropositivity for all cases diagnosed .9 years after baseline blood draw
and their paired controls.
doi:10.1371/journal.pone.0032106.t003
Helicobacter pylori and Lung Cancer
PLoS ONE | www.plosone.org5 February 2012 | Volume 7 | Issue 2 | e32106

Page 6

population using these same assays, demonstrating the adequacy
of these assays.
Although the inclusion of only Finish male smokers is beneficial
in limiting the potential for confounding, it also limits the
generalizability of these findings. This study could not address
the association between H. pylori seropositivity and subsequent risk
of lung cancer in women. In addition, never smokers may have a
different risk of lung cancer associated with H. pylori seropositivity
than ever smokers. Although unlikely, it is possible that the
potential biologic mechanisms described in the Introduction (e.g.,
systemic inflammation from gastric H. pylori infection) may be
gender specific or may only be evident in never smokers.
This study provides the most definitive findings for H. pylori
seropositivity and lung cancer to date. In this population of Finish
male smokers, H. pylori seropositivity was not associated with lung
cancer. Although these findings are not entirely generalizable to
other populations (e.g., never smokers), it seems unlikely that H.
pylori is involved in lung carcinogenesis.
Author Contributions
Conceived and designed the experiments: JK NEC DA PRT SJW JV.
Performed the experiments: GPP MJB. Analyzed the data: JK RF TKL.
Contributed reagents/materials/analysis tools: GPP MJB. Wrote the
paper: JK.
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Helicobacter pylori and Lung Cancer
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