Helicobacter pylori infection assessed by ELISA and by immunoblot and noncardia gastric cancer risk in a prospective study: the Eurgast-EPIC project.
ABSTRACT In epidemiological studies, Helicobacter pylori infection is usually detected by enzyme-linked immunosorbent assay (ELISA). However, infection can spontaneously clear from the mucosa during the progression of atrophy and could lead to substantial under-detection of infection and underestimation of its effect on gastric cancer (GC) risk. Antibodies detected by western blot are known to persist longer after the loss of the infection.
In a nested case-control study from the Eurogast-EPIC cohort, including 88 noncardia GC cases and 338 controls, we assessed the association between noncardia GC and H. pylori infection comparing antibodies detected by western blot (HELICOBLOT2.1) to those detected by ELISA (Pyloriset EIA-GIII(®)).
By immunoblot, 82 cases (93.2%) were H. pylori positive, 10 of these cases (11.4%) were negative by ELISA and only 6 cases (6.8%) were negative by both ELISA and immunoblot. Multivariable odds ratio (OR) for noncardia GC comparing immunoglobulin G positive versus negative by ELISA was 6.8 [95% confidence interval (CI) 3.0-15.1], and by immunoblot, the OR was 21.4 (95% CI 7.1-64.4).
Using a western blot assay, nearly all noncardia GC were classified as H. pylori positive and the OR was more than threefold higher than the OR assessed by ELISA, supporting the hypothesis that H. pylori infection is a necessary condition for noncardia GC.
- [show abstract] [hide abstract]
ABSTRACT: Helicobacter infection is the leading cause of gastric cancer worldwide. Infection with this ubiquitous bacterium incites a chronic active immune response that persists for the life of the host, in the absence of antibiotic-induced eradication. It is the combination of bacterial factors, environmental insults, and the host immune response that drives the initiation and progression of mucosal atrophy, metaplasia, and dysplasia toward gastric cancer. Although it may seem intuitively obvious that removing the offending organism would negate the cancer risk, this approach is neither feasible (half of the world harbors this infection) nor is it straightforward. Most patients are infected in childhood, and present with various degrees of mucosal damage before any therapy. This review outlines the histologic progression of human Helicobacter infection from the early stages of inflammation through the development of metaplasia, dysplasia, and, finally, cancer. The effects of dietary and bacterial eradication therapy on disease progression and lesion reversibility are reviewed within the context of population studies and compared between study designs and populations tested. Eradication studies in the mouse model of infection prevents the formation of gastric cancer, and allows regression of established lesions, providing a useful model to study interaction between bacterium, environment, and host, without the difficulties inherent in human population studies. Recent advances in identifying the bone marrow-derived stem cell as the cell of origin of Helicobacter-induced gastric cancer in the murine model are discussed and interpreted in the context of human disease, and implications for future treatment are discussed.Gastroenterology 09/2007; 133(2):659-72. · 12.82 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: BACKGROUND—The magnitude of the association between Helicobacter pylori and incidence of gastric cancer is unclear. H pylori infection and the circulating antibody response can be lost with development of cancer; thus retrospective studies are subject to bias resulting from classification of cases as H pylori negative when they were infected in the past. AIMS—To combine data from all case control studies nested within prospective cohorts to assess more reliably the relative risk of gastric cancer associated with H pylori infection. To investigate variation in relative risk by age, sex, cancer type and subsite, and interval between blood sampling and cancer diagnosis. METHODS—Studies were eligible if blood samples for H pylori serology were collected before diagnosis of gastric cancer in cases. Identified published studies and two unpublished studies were included. Individual subject data were obtained for each. Matched odds ratios (ORs) and 95% confidence intervals (95% CI) were calculated for the association between H pylori and gastric cancer. RESULTS—Twelve studies with 1228 gastric cancer cases were considered. The association with H pylori was restricted to non-cardia cancers (OR 3.0; 95% CI 2.3-3.8) and was stronger when blood samples for H pylori serology were collected 10+ years before cancer diagnosis (5.9; 3.4-10.3). H pylori infection was not associated with an altered overall risk of cardia cancer (1.0; 0.7-1.4). CONCLUSIONS—These results suggest that 5.9 is the best estimate of the relative risk of non-cardia cancer associated with H pylori infection and that H pylori does not increase the risk of cardia cancer. They also support the idea that when H pylori status is assessed close to cancer diagnosis, the magnitude of the non-cardia association may be underestimated. Keywords: gastric cancer; Helicobacter pylori; cardia cancer; pooled analysis
- [show abstract] [hide abstract]
ABSTRACT: 55 patients with severe ulcer disease and H. pylori infection, successfully treated with antimicrobials, were followed-up with repeated blood samples for up to 32 months. Sera were analysed by enzyme immunoassay (EIA) for IgG and IgA antibodies and by IgG immunoblot. The EIA for IgG antibodies showed a high sensitivity (100%), while IgA antibodies above the cut-off level were found in 55% of the patients. At a median of 77 days after onset of treatment, approximately 50% of the patients showed a significant decrease (> or = 50%) of IgG or had titres below the cut-off level. All patients but 1 had a significant decrease of IgG after 6-12 months. The decrease was slower for IgA. The H. pylori-specific 116 kDa and 19.5 kDa bands were found in all pre-treatment samples, but the decrease in median intensity of the bands was slower than for the IgG EIA. In the 32-months post-treatment samples, both bonds had an intensity still above 50% of the pre-treatment value. The study showed that the IgG EIA is a useful method for monitoring eradication of H. pylori. Immunoblot can detect previous H. pylori infection in EIA negative Individuals.Scandinavian Journal of Infectious Diseases 01/1997; 29(2):147-51. · 1.71 Impact Factor
Annals of Oncology 23: 1320–1324, 2012
Published online 14 September 2011
Helicobacter pylori infection assessed by ELISA and by
immunoblot and noncardia gastric cancer risk in
a prospective study: the Eurgast-EPIC project
C. A. Gonza ´lez1*, F. Megraud2, A. Buissonniere2, L. Lujan Barroso1, A. Agudo1, E. J. Duell1,
M. C. Boutron-Ruault3,4, F. Clavel-Chapelon3,4, D. Palli5, V. Krogh6, A. Mattiello7, R. Tumino8,
C. Sacerdote9, J. R. Quiro ´s10, E. Sanchez-Cantalejo11, C. Navarro12, A. Barricarte13,
M. Dorronsoro14, K.-T. Khaw15, N. Wareham15, N. E. Allen16, K. K. Tsilidis16,
H. Bas Bueno-de-Mesquita17,18, S. M. Jeurnink17,18, M. E. Numans19, P. H. M. Peeters19,
P. Lagiou20, E. Valanou21, A. Trichopoulou21, R. Kaaks22, A. Lukanova-McGregor22,
M. M. Bergman23, H. Boeing23, J. Manjer24, B. Lindkvist25, R. Stenling26, G. Hallmans27,
L. M. Mortensen28, K. Overvad28, A. Olsen29, A. Tjonneland29, K. Bakken30, V. Dumeaux30,
E. Lund30, M. Jenab31, I. Romieu31, D. Michaud32, T. Mouw32, F. Carneiro33, C. Fenge34&
1Unit of Nutrition, Environment and Cancer, Cancer Epidemiology Research Program, Bellvitge Biomedical Research Institute (IDIBELL), Catalan Institute of Oncology,
Barcelona, Spain;2INSERM U853, Bordeaux;3Centre for Research in Epidemiology and Population Health, Institut Gustave Roussy, Villejuif;4Paris South University,
Villejuif, France;5Molecular and Nutritional Epidemiology Unit, Cancer Research and Prevention Institute (ISPO), Florence;6Department of Preventive & Predictive
Medicine, Nutritional Epidemiology Unit, Fondazione IRCCS Istituto Nazionale dei TumoriMilan;7Department Of Clinical And Experimental Medicine, Federico Ii
University, Naples;8Cancer Registry and Histopathology Unit, ‘‘Civile M.P. Arezzo’’ Hospital, Ragusa;9Centre for Cancer Epidemiology and Prevention (CPO
Piemonte), Turin, Italy;10Public Health and Participation Directorate, Health and Health Care Services Council, Asturias;11Andalusian School of Public Health,
CIBER Epidemiologı´a y Salud Pu ´blica (CIBERESP), Granada;12Department of Epidemiology, Murcia Health Council, CIBER Epidemiologı´a y Salud Pu ´blica
(CIBERESP) Murcia, Murcia;13Navarre Public Health Institute, CIBER Epidemiologı´a y Salud Pu ´blica (CIBERESP), Pamplona;14Public Health Division of Gipuzkoa
and Ciberesp, Basque Regional Health Department, San Sebastia ´n, Spain;15Department of Public Health and Primary Care, University of Cambridge, Cambridge;
16Cancer Epidemiology Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK;17National Institute for Public Health and the Environment
(RIVM), Bilthoven;18Department of Gastroenterology and Hepatology, University Medical Centre Utrecht (UMCU), Utrecht;19Julius Center for Health Sciences and
Primary Care, University Medical Center Utrecht, Utrecht, The Netherlands;20WHO Collaborating Center for Food and Nutrition Policies, Department of Hygiene,
Epidemiology and Medical Statistics, University of Athens Medical School, Athens;21Hellenic Health Foundation, Athens, Greece;22Department of Cancer
Epidemiology, German Cancer Research Center, Heidelberg;23Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbru ¨cke, Potsdam,
Germany;24Department of Surgery, Ska ˚ne University Hospital Malmo ¨, Lund University, Malmo ¨;25Department of Internal Medicine, Division of Gastroenterology and
Hepatology, Sahlgrenska University Hospital, Gothenburg;26Department of Medical Biosciences, Pathology, Umea University, Umea, Sweden;27Department of
Public Health and Clinical Medicine, Nutritional Research, Umea ˚ University, Umea ˚, Sweden;28Department of Epidemiology, School of Public Health, Aarhus
University, Aarhus;29Danish Cancer Society, Institute of Cancer Epidemiology, Diet Cancer and Health, Copenhagen, Denmark;30Department of Community
Medicine, University of Tromsø, Tromso, Norway;31International Agency for Research on Cancer (IARC-WHO), Lyon, France;32Department of Epidemiology and
Biostatistics, School of Public Health, Imperial College London, London, UK;33Institute of Molecular Pathology and Immunology of the University of Porto
(IPATIMUP) and Medical Faculty/HS Joa ˜o, Porto, Portugal;34Department of Clinical Pathology, Odense University Hospital, Odense, Denmark;35School of Public
Health, St Mary’s Campus, Imperial College London, London, UK
Received 29 April 2011; revised 6 July 2011; accepted 6 July 2011
Background: In epidemiological studies, Helicobacter pylori infection is usually detected by enzyme-linked
immunosorbent assay (ELISA). However, infection can spontaneously clear from the mucosa during the progression of
atrophy and could lead to substantial under-detection of infection and underestimation of its effect on gastric cancer
(GC) risk. Antibodies detected by western blot are known to persist longer after the loss of the infection.
Methods: In a nested case–control study from the Eurogast-EPIC cohort, including 88 noncardia GC cases and
338 controls, we assessed the association between noncardia GC and H. pylori infection comparing antibodies
detected by western blot (HELICOBLOT2.1) to those detected by ELISA (Pyloriset EIA-GIII?).
*Correspondence to: Dr C. A. Gonza ´lez, Unit of Nutrition Environment and Cancer,
Department of Epidemiology, Catalan Institute of Oncology (ICO), Gran Via s/n
Hospitalet, Barcelona 34-08908, Spain. Tel: +34-932607401; Fax: +34-932607787;
Annals of Oncology
ª The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology.
All rights reserved. For permissions, please email: firstname.lastname@example.org
by guest on May 29, 2013
Results: By immunoblot, 82 cases (93.2%) were H. pylori positive, 10 of these cases (11.4%) were negative by ELISA
and only 6 cases (6.8%) were negative by both ELISA and immunoblot. Multivariable odds ratio (OR) for noncardia GC
comparing immunoglobulin G positive versus negative by ELISA was 6.8 [95% confidence interval (CI) 3.0–15.1], and
by immunoblot, the OR was 21.4 (95% CI 7.1–64.4).
Conclusions: Using a western blot assay, nearly all noncardia GC were classified as H. pylori positive and the OR
was more than threefold higher than the OR assessed by ELISA, supporting the hypothesis that H. pylori infection is
a necessary condition for noncardia GC.
Key words: Helicobacter pylori, noncardia gastric cancer, prospective study, western blot
Helicobacter pylori infection is a well-established cause of
sporadic noncardia gastric cancer (GC) . In epidemiological
studies, H. pylori infection is usually detected by enzyme-linked
immunosorbent assay (ELISA). A combined analysis of 12
case–control studies nested within cohorts  found an overall
odds ratio (OR) of 3.0 [95% confidence interval (CI) 2.3–3.8]
for the risk of noncardia cancer, measuring anti-H. pylori
immunoglobulin G (IgG) antibodies by ELISA. In a meta-
analysis  of 10 case–control studies (using ELISA in 8 studies
and western blot in 2) in which the infection by H. pylori was
assessed by CagA seropositivity, the OR for noncardia GC was
2.71 (95% CI 1.7–4.2).
Severe chronic atrophic gastritis (SCAG) and extended
intestinal metaplasia are believed to create conditions in which
H. pylori is unable to survive, and, as a consequence, there is
a gradual reduction of H. pylori colonization of the gastric
mucosa during the progression of preneoplastic lesions.
Therefore, there is a potential to underestimate the association,
mainly in case–control studies, because of under-detection of
H. pylori infection in blood collected after the diagnosis of GC.
It has been shown that anti-CagA antibodies measured by
western blot analysis (immunoblot) persist longer than specific
IgG antibodies detected by conventional ELISA [4–8], and
therefore, it may be possible to detect previous H. pylori infection
in some ELISA-negative subjects using immunoblot. These studies
have shown that the association between H. pylori infection and
GC is much stronger than considered previously and led to the
hypothesis that H. pylori may be a necessary cause of GC .
In the European Prospective Investigation into Cancer and
Nutrition (Eurgast-EPIC) study, we have previously reported
 that using only antibodies against H. pylori lysate, there
was non-statistically significant association with noncardia GC.
The aim of this study is to assess the magnitude of the risk
associated with H. pylori infection in a new set of noncardia GC
from the Eurogast-EPIC study, comparing antibodies detected
by western blot analysis to those detected by ELISA.
materials and methods
The study subjects belong to the EPIC cohort . Briefly, the EPIC cohort
includes about half million individuals, mostly aged 40–65 years, recruited
between 1992 and 1998 in 23 centres from 10 European countries:
Denmark, France, Greece, Germany, Italy, The Netherlands, Norway, Spain,
Sweden and UK. At enrolment, blood samples were collected from most
participants. Follow-up is based upon population cancer registries in most
countries, except in France, Germany and Greece, where it is mainly
achieved by active contact with study subjects and review of health
insurance and pathology reports.
A nested case–control study within the EPIC cohort (EurGast) was
conducted to analyse the relationship between GC risk and baseline H.
pylori seropositivity status as well as other biomarkers. Each incident
noncardia GC case with an available blood sample was matched by sex, age
group (62.5 years), centre and date of blood collection (645 days) to four
control participants who were randomly selected from the cohort at risk at
the time of diagnosis of the index case. Cases were subjects newly diagnosed
of GC defined by code C16 of the International Classification of Diseases,
10th Revision (ICD-10). Noncardia GC were defined by the code C16.1 to
C16.9. The previous analysis  included 233 GC cases (diagnosed mostly
before 2000) and 910 controls, using only ELISA for detection of H. pylori
antibodies. Cases for this new analysis, using ELISA and immunoblot, were
identified during an update of cancer incidence within the cohort that
included new cases diagnosed in the same EPIC’s participating countries
mostly between 2000 and 2004. An independent panel of pathologists
reviewed the original slides and pathology reports provided by each EPIC
centre for most of the cases, in order to confirm and validate the diagnosis,
tumour site and morphology .
determination of the H. pylori status by ELISA
Helicobacter pylori infection was determined by ELISA using the Pyloriset?
EIA-GIII kit (Orion Diagnostica, Espoo, Finland). Briefly, a 1/200 dilution of
serum in buffer was introduced in H. pylori-coated microtiter wells. After 30
min incubation, the wells were washed and a peroxidase conjugated anti-
human IgG from rabbit was incubated for a further 30 min. After washing, the
tetramethylbenzidine substrate was added for 10 min and the optical density
(OD) measured at 450 nM. The results were expressed as unit per milliliter
according to a calibrator curve. A value ‡20 U/ml was considered as positive.
determination of the H. pylori status and the CagA status
Immunoblot using the HELICOBLOT 2.1 kit (Genelab Diagnostics,
Singapore) was carried out. An H. pylori lysate enriched with recombinant
antigens is electrophoretically prepared and transferred on to the nitrocellular
strips, which are commercially available. Individual strips were incubated
with a 1/100 dilution of serum in blotting buffer on a tray. After 60 min of
incubation, the strip was washed and a goat anti-human IgG conjugated with
alkaline phosphatase was added for a further 60 min. After washing, the 5-
Bromo-4 chloro-3 indolylphosphate and nitrobluetetrazolium substrate was
added for 15 min. The criteria for H. pylori positivity were the presence of
one of the following bands 89 KD,37 KD, 35 KDor both the 30 KDand 19.5
KDbands, and for CagA positivity, the presence of a 116 KDband.
determination of pepsinogen I levels
Pepsinogen I level was determined by ELISA using the kits from Biohit
(Helsinki, Finland). Human pepsinogen-captured antibodies (monoclonal) are
absorbed in microtiter wells. A dilution of 1/5 of the serum was added. After
60 min incubation, a peroxidase conjugated anti-human pepsinogen was added
Annals of Oncology
Volume 23|No. 5|May 2012doi:10.1093/annonc/mdr384 | 1321
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for a further 60 min. After washing, the tetramethylbenzidine substrate was
added for 30 min and the OD was measured at 450 nM. A calibration curve was
used to calculate the pepsinogen concentration. All determinations were carried
out at room temperature. A pepsinogen 1 level <22 lg/l indicated SCAG.
Data are presented as the number and proportion of controls and cases for
each H. pylori assay used. Conditional logistic regression was used to
estimate the OR for noncardia GC risk, adjusting for potential confounders:
education (none, primary, technical/professional, secondary or university),
cigarette smoking (never, former and current) and average daily dietary
intakes (fruit, vegetables and red and processed meat).
During a mean follow-up of 10.65 years (range 0.3–17.6 years),
in the update of cancer incidence from the EPIC cohort, 195
new histologically confirmed adenocarcinoma cases (51 from
the cardia, 88 from the noncardia, 4 mixed and 52 for which
the site was not identified) were diagnosed. The analysis
presented here is based on these 88 noncardia GC cases (of
which 31 were intestinal type, 32 diffuse and 25 mixed or
undefined) and 338 matched controls. In 16 (18.2%) of
these cases (Table 1), SCAG was present according to
serological levels of pepsinogen I. The median serum
concentration of pepsinogen I for all noncardia cases was
64.3. The mean years of follow-up of noncardia cases with
SCAG was relatively similar to those without SCAG (9.0
and 10.7 years, respectively), but cases with SCAG were
significantly older compared with those without SCAG
(mean age at blood extraction 61.3 and 55.3 years, respectively,
P £ 0.001).
The seroprevalence of H. pylori antibodies in noncardia
cases and controls by assay is shown also in Table 1. In 16
cases (18.2%), IgG antibodies detected by ELISA were
negative (eight without SCAG and eight with SCAG). Only six
cases (6.8%) (three with SCAG and three without SCAG)
were negative for both assays, while 82 (93.2%) of noncardia
GC cases were positive for previous H. pylori infection by
immunoblot. Among controls, 188 (55.7%) were positive by
ELISA, while 199 (58.9%) were positive by immunoblot. We
observed 9 controls that were positive by ELISA but negative
by immunoblot and 20 controls negative by ELISA but
positive by immunoblot. Concordance by immunoblot assay
between H. pylori antibodies and CagA antibodies for all cases
and controls was almost complete, and we observed only one
case and four controls reported as negative for CagA but
positive for H. pylori by immunoblot. Out of the 52 cases for
which the anatomical site was undefined, 6 cases (11.5%) (all
without SCAG) were negative for the two assays. The
proportion of cases with SCAG but negative by ELISA (50%)
was higher than those positive by ELISA (11.1%). Similar
results were observed by immunoblot. We did not observe any
differences regarding the proportion of negative cases between
intestinal and diffuse histological subtypes (data not shown).
Table 2 shows the ORs of noncardia GC associated with H.
pylori seropositivity, according to the assay, adjusting for
potential confounders. The OR was 6.8 (95% CI 3.0–15.1)
comparing IgG positive versus negative by ELISA and 21.4
(95% CI 7.1–64.3) comparing IgG positive versus negative by
Our prospective study found that the OR for noncardia GC and
H. pylori seroprevalence is almost fourfold higher by
immunoblot than by ELISA. It supports the evidence that
measuring only IgG by ELISA to classify H. pylori infection
status may lead to a substantial underestimation of the effect of
H. pylori infection in noncardia GC risk [4, 8].
Similar results have been observed in three previous studies
that compared results from ELISA with immunoblot. In
a population-based case–control study from Sweden 
(including 206 noncardia GC and 238 controls), the adjusted OR
for seropositivity of H. pylori infection was 2.2 as measured by
ELISA and 21.0 as measured by immunoblot. In a hospital-based
case–control study from Germany  (including 68 noncardia
GC and 360 controls), the OR increased from 3.7 by ELISA to
18.3 by immunoblot. Finally, in a nested case–control study from
Australia  (including 34 noncardia GC and 134 controls), the
OR increased from 2.3 by ELISA to 10.6 by immunoblot.
We found that the prevalence of seronegativity in noncardia
GC decreased from 18.2% by ELISA to 6.8% by immunoblot,
confirming the underestimation of the prevalence of H. pylori
infection using ELISA. In a meta-analysis  of case–control
studies, which included 10 studies (western blot was used only
in two of these studies), and >1700 noncardia GC cases, which
assessed the relationship between GC and H. pylori by CagA
seropositivity, 37.7% of the noncardia GC cases were negative
for CagA antibodies. On the contrary, using immunoblot in
Table 1. Seroprevalence of Helicobacter pylori IgG antibodies (ELISA) and IgG antibodies (Immunoblot) in noncardia gastric adenocarcinoma cases and
matched controls, in the EurGast-EPIC cohort study
N (%) Immunoblot
PI < 22
lg/ml n (%)
PI < 22
lg/ml n (%)
ELISA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G; PI, pepsinogen I.
Annals of Oncology
1322 | Gonza ´lez et al.Volume 23|No. 5|May 2012
by guest on May 29, 2013
the population-based case–control study from Sweden ,
only 4% of noncardia GC cases were negative, while in the
nested case–control study from Australia , it was 6%,
which are both relatively similar to the proportion observed
in our study. It was recently estimated that the H. pylori
attributable fraction of noncardia GC cases is 74% in
developed countries and 78% in developing countries .
Our results, as well as those from other studies indicate that
this fraction may be higher, and that H. pylori infection could
be a necessary condition of noncardia GC . A necessary
cause is not necessarily a sufficient cause (meaning other
factors are required). It is well known that despite the high
prevalence of H. pylori infection, only a small proportion of
infected subjects develop GC. This underlines the relevance of
the role of other cofactors, such as diet, smoking and genetic
susceptibility, acting as component of a sufficient cause, in
the same or different causal mechanisms, depending of the
relative prevalence of these factors in a population .
Our results suggest that the underestimation of the
prevalence of H. pylori infection by ELISA is greater in cases
with SCAG, which is expected since during progression of
gastric atrophy, the bacterium cleans from the gastric mucosa.
As was expected in our study, noncardia cases with SCAG were
older than cases without SCAG. In our study, SCAG was
defined as pepsinogen I levels <22 lg/l; this cut-off has
previously demonstrated to have relatively high sensitivity
(89.5%) and specificity (91.5%) for the screening of SCAG in
the general population .
We observed 5 cases without SCAG out of 10 noncardia GC
that were H. pylori negative by ELISA but positive by
immunoblot. In a study designed to assess the current mucosal
condition of 64 GC cases through histology and culture , 4
out of 12 cases that were H. pylori negative by ELISA and CagA
positive by immunoblot had no evidence of gastric atrophy. In
addition, no evidence of atrophy was found in 8 out of 10 cases
that were negative by both serological tests. In our study, we
found that three noncardia GC cases were negative for H. pylori
infection by both assays and had no evidence of SCAG. We also
checked the available pathological information for the six
noncardia GC cases that were H. pylori negative, to verify that
there were no errors in the anatomical subsite.
The best method to diagnose H. pylori infection in different
research situations is still not clarified . We used
a commercial immunoblot test (HELICOBLOT 2.1) that has
been shown to have a sensitivity of 100% for current and of
92% for previous H. pylori infection [17, 18], meaning that
some false-negative results can be expected in subjects with
previous infection. We observed that 93.2% of noncardia GC
were positive by immunoblot, which is consistent with the
sensitivity of the method.
Our study has several clinical implications. The immunoblot
might be useful for modelling the relationship between the
decline in H. pylori infection prevalence and declines in gastric
cancer incidence . Our study should not be a basis for
justification of massive eradication therapy, because there is no
evidence yet of the benefit of this approach in the general
population . Furthermore, this could lead to the activation
of antibiotic-resistant strains of other pathogens . However,
it is a good reason to continue efforts for developing vaccines
against H. pylori.
Our study design has several strengths. Most of the cases
were validated by a panel of expert pathologists. The
prospective nature of EPIC allowed H. pylori serology to be
accurately determined in healthy subjects before the onset of
the disease, which is an important strength in comparison to
case–control studies. In addition, as far as we know, this is
the first prospective study in European populations
showing results of both assays in noncardia GC cases.
A limitation is the relatively small sample size of noncardia
GC cases. The length of follow-up is also relatively short and
does not allow to estimate changes in the magnitude of risk
according to the time between blood collection and cancer
In conclusion, we found that the proportion of H. pylori-
negative cases by ELISA is reduced by threefold when results of
western blot are taken into account and accordingly, the OR is
more than threefold higher than that assessed by ELISA. Our
results support the hypothesis that H. pylori infection may be
a necessary cause of sporadic noncardia gastric cancer. Given
that the sensitivity of immunoblot is <100%, an improvement
in the accuracy of tests for the detection of past H. pylori
infection is still needed to clarify if this small fraction of
negative cases is indeed false negative.
The authors want to acknowledge the pathology panel
members: Fatima Carneiro, Hendrik Blaker, Claus Laszlo Igali,
Gabriella Nesi and Roger Stenling for their contribution to the
collection and review of paraffin tumour blocks, slides and
This work was supported by the Health Research Fund (FIS)
of the Spanish Ministry of Health (Exp P10710130), La Caixa
(BM 06-130) and Red Tema ´tica de Investigacio ´n Cooperativa
Table 2. ORaand 95% CI for the association of exposure to Helicobacter
pylori infection detected through IgG ELISA or immunoblot and
noncardia gastric cancer risk in the EurGast-EPIC study
H. pylori infection
H. pylori IgG negative
H. pylori IgG positive
H. pylori IgG negative
H. pyrlori IgG positive
16/150 1.0 (reference)
CI, confidence interval; ELISA, enzyme-linked immunosorbent assay; IgG,
immunoglobulin G; OR, odds ratio.
aConditional logistic regression (matched by age, sex, centre and date of
blood extraction) adjusted by smoking status, school level, red and
processed meat intake and fruit and vegetables consumption.
Annals of Oncology
Volume 23|No. 5|May 2012doi:10.1093/annonc/mdr384 | 1323
by guest on May 29, 2013
en Ca ´ncer (R06/0020) (Spain). The coordination of EPIC is
financially supported by the European Commission
(DGSANCO) and the International Agency for Research on
Cancer. The national cohorts are supported by the Health
Research Fund (FIS) of the Spanish Ministry of Health,
Regional Governments of Andalucı ´a, Asturias, Catalunya,
Basque Country, Murcia and Navarra (Spain); Danish Cancer
Society (Denmark); Liguecontre le Cancer, 3M, Mutuelle
Ge ´ne ´rale de l’Education Nationale, Institut National de la
Sante ´ et de la Recherche Medicale (France); Deutsche
Krebshilfe, Deutsches Krebsforschungszentrum and Federal
Ministry of Education and Research (Germany); Ministry of
Health and Social Solidarity, Stavros Niarchos Foundation
and Hellenic Health Foundation (Greece); Italian Association
for Research on Cancer (AIRC) and National Research
Council (Italy); Dutch Ministry of Public Health, Welfare and
Sports (VWS), Netherlands Cancer Registry (NKR), LK
Research Funds, Dutch Prevention Funds, Dutch ZON (Zorg
Onderzoek Nederland), World Cancer Research Fund
(WCRF), and Statistics Netherlands (The Netherlands);
Norwegian Cancer Society (Norway); Swedish Cancer Society,
Swedish Scientific Council and Regional Government of Ska ˚ne
and Va ¨sterbotten (Sweden); Cancer Research UK, Medical
Research Council, Stroke Association, British Heart
Foundation, Department of Health, Food Standards Agency,
and Wellcome Trust (UK). Some authors are members of
Environmental Cancer Risk, Nutrition and Individual
Susceptibility, a network of excellence funded by the European
Commission (6th EU Framework Programme for Research
The authors declare no conflicts of interest.
1. Correa P, Houghton JM. Carcinogenesis of Helicobacter pylori. Gastroenterology
2007; 133: 659–672.
2. Helicobacter and Cancer Collaborative Group. Gastric cancer and Helicobacter
pylori: a combined analysis of 12 case control studies nested within prospective
cohorts. Gut 2001; 49: 347–353.
3. Huang JQ, Zheng GF, Sumanac K et al. Meta-analysis of the relationship
between cagA seropositivity and gastric cancer. Gastroenterology 2003; 125(6):
4. Ekstro ¨m AM, Held M, Hansson LE et al. Helicobacter pylori in gastric cancer
established by CagA immunoblot as a marker of past infection. Gastroenterology
2001; 121(4): 784–791.
5. So ¨rberg M, Engstrand L, Stro ¨m M et al. The diagnostic value of enzyme
immunoassay and immunoblot in monitoring eradication of Helicobacter pylori.
Scand J Infect Dis 1997; 29(2): 147–151.
6. Mini R, Annibale B, Lahner E et al. Western blotting of total lysate of
Helicobacter pylori in cases of atrophic body gastritis. Clin Chem 2006; 52(2):
7. Enroth H, Kraaz W, Rohan T et al. Does the method of Helicobacter pylori
detection influence the association with gastric cancer risk? Scand J
Gastroenterol 2002; 37(8): 884–890.
8. Mitchell H, English DR, Elliott F et al. Immunoblotting using multiple antigens is
essential to demonstrate the true risk of Helicobacter pylori infection for gastric
cancer. Aliment Pharmacol Ther 2008; 28(7): 903–910.
9. Brenner H, Arndt V, Stegmaier C et al. Is Helicobacter pylori infection
a necessary condition for noncardia gastric cancer? Am J Epidemiol 2004;
10. Palli D, Masala G, Del Giudice G et al. CagA+ Helicobacter pylori infection and
gastric cancer risk in the EPIC-EURGAST study. Int J Cancer 2007; 120(4):
11. Riboli E, Hunt KJ, Slimani N et al. European Prospective Investigation into Cancer
and Nutrition (EPIC): study populations and data collection. Public Health Nutr
2002; 5(6B): 1113–1124.
12. Carneiro F, Moutinho C, Pera G et al. Pathology findings and validation of gastric
and esophageal cancer cases in a European cohort (EPIC/EUR-GAST). Scand J
Gastroenterol 2007; 42(5): 618–627.
13. Parkin DM. The global health burden of infection-associated cancers in the year
2002. Int J Cancer 2006; 118(12): 3030–3044.
14. Buehler JW. Surveillance. In Rothman KJ, Greenland S (eds), Modern
Epidemiology, 2nd edition. Philadelphia, PA: Lippincott Williams & Wilkins 1998.
15. Kekki M, Samloff IM, Varis K, Ihama ¨ki T. Serum pepsinogen I and serum gastrin
in the screening of severe atrophic corpus gastritis. Scand J Gastroenterol Suppl.
1991; 186: 109–116.
16. Ye W, Held M, Enroth H et al. Histology and culture results among subjects with
antibodies to CagA but no evidence of Helicobacter pylori infection with IgG
ELISA. Scand J Gastroenterol 2005; 40(3): 312–318.
17. Monteriro L, Bergey B, Gras N, Me ´graud F. Evaluation of the performance of the
Helico Blot 2.1 as a tool to investigate the virulence properties of Helicobacter
pylori. Clin Microbiol Infect 2002; 8(10): 679–69l.
18. Sima ´n JH, Engstrand L, Berglund G et al. Evaluation of western blot CagA
seropositivity in Helicobacter pylori-seropositive and -seronegative subjects. Clin
Diagn Lab Immunol 2005; 12(2): 304–309.
19. Yeh JM, Goldie SJ, Kuntz KM, Ezzati M. Effects of Helicobacter pylori
infection and smoking on gastric cancer incidence in China:
a population-level analysis of trends and projections. Cancer Causes Control
2009; 20(10): 2021–2029.
20. Herrera V, Parsonnet J. Helicobacter pylori and gastric adenocarcinoma. Clin
Microbiol Infect 2009; 15(11): 971–976.
Annals of Oncology
1324 | Gonza ´lez et al. Volume 23|No. 5|May 2012
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