Guru Trikudanathan1, Aby Philip1, Constantin A Dasanu2, William L Baker1,3
1Department of Internal Medicine, University of Connecticut Medical Center. Farmington, CT, USA.
2Department of Hematology-Oncology, St. Francis Hospital and Medical Center. Hartford, CT, USA.
3University of Connecticut School of Pharmacy. Storrs, CT, USA
Received November 8th, 2010 - Accepted December 2nd, 2010
Context Infection with Helicobacter pylori (H. pylori) has been implicated in the etiopathogenesis of various malignant conditions. Notwithstanding, its etiological association with pancreatic cancer remains inconclusive. Studies focusing on the relationship between H. pylori infection and pancreatic cancer risk have yielded conflicting results. Objective The aim of this study was to obtain a reliable estimate of the risk of H. pylori infection in causing pancreatic cancer, by performing a meta-analysis of the existing observational studies evaluating the association. Methods/Statistics Observational studies comparing the prevalence of H. pylori infection in patients with pancreatic cancer and healthy controls, conducted in adult populations and published in all languages, were identified through systematic search in the MEDLINE and EMBASE up to April 2010. H. pylori infection was confirmed by serological testing using an antigen-specific enzyme-linked immunosorbent assay. Pooled adjusted odds ratios (AOR) and associated 95% confidence intervals (CI) were obtained by using a DerSimonian and Laird random-effects model. Results Six studies involving a total of 2,335 patients met our eligibility criteria. A significant association between H. pylori seropositivity and development of pancreatic cancer (AOR 1.38, 95% CI: 1.08-1.75; P=0.009) was seen. No significant association was seen on pooled analysis of the three studies assessing the relationship between cytotoxin-associated gene A (CagA) positivity and pancreatic cancer. A cumulative meta-analysis suggested a reducing, albeit statistically significant association as the evidence was accumulated. Conclusions The pooled data suggests an association between infection with H. pylori and the development of pancreatic cancer. Further research is needed to confirm our findings.
Helicobacter pylori; Meta-Analysis as Topic; Pancreatic Neoplasms
AOR: adjusted odds ratio; CagA: cytotoxinassociated gene A
Pancreatic cancer is the fourth leading cause of cancer death in men and women in United States, with an estimated 42,470 new cases and 35,240 deaths in 2009 . There is no effective screening diagnostics; therefore, most patients present with metastatic or advanced disease, not amenable to surgery. Due to the paucity of effective therapeutic options, pancreatic cancer has one of the highest mortality rates of all cancers and a median survival of only 3-6 months . Thus, identification of modifiable risk factors and an effective primary prevention appear critical to reduce the incidence of this aggressive malignancy. Notwithstanding, the risk factors for pancreatic cancer remain largely unknown. Older age and cigarette smoking are consistently reported associations that are commonly accepted . Other potential risk factors include African-American race, chronic pancreatitis, obesity, type 2 diabetes mellitus, and consumption of smoked or processed meat. Genetic mutations, hereditary syndromes and familial aggregation may account for approximately 5-10% of cases . During the last decade the causal role of Helicobacter pylori (H. pylori) in the pathogenesis of peptic ulcer disease has been firmly established . Its etiological association with gastric cancer and gastric lymphoma has also been demonstrated .
Some epidemiologic evidence suggests that H. pylori might be involved in the pathogenesis of pancreatic cancer, although the results have not been consistent across the studies [7, 8, 9, 10, 11, 12]. Risch et al. reported a pooled analysis of these studies in 2010 suggesting a positive association between H. pylori and pancreatic cancer . However, no methods were included in this report and the data they used from prior studies was inconsistent. Thus, we performed an updated meta-analysis of relevant clinical studies as well as a cumulative meta-analysis in order to determine the nature of the association between pancreatic cancer and H. pylori and to see how the evidence accumulated and if the conclusions may have shifted over time.
Data Sources and Searches
We performed a systematic review of the published literature according to the “Meta-Analyses of Observational Studies in Epidemiology” guidelines to identify all studies that provided an effect estimate for a potential association between pancreatic cancer and H. pylori . Two authors (G.T. and A.P.) independently conducted MEDLINE search (1966 to April 2010) and EMBASE (1990 to April 2010) for all relevant articles. We also manually searched references from studies or reviews and major gastroenterology meeting abstracts through March 2010. The following medical subject headings (MeSH) or keywords were used: pancreatic cancer, pancreatic tumor, pancreatic neoplasm, Helicobacter pylori, H. pylori, Campylobacter pylori, and peptic ulcer disease. Citations were limited to those published in the English language.
Two independent reviewers assessed studies for inclusion in a parallel manner using “priori-defined criteria”. To assess the potential association, we selected observational (case control, cohort and crosssectional) studies for inclusion if they: 1) were conducted in adult population, defined as patients greater than 18 years of age; 2) included patients with exocrine pancreatic carcinoma as confirmed by biopsy; 3) diagnosed H. pylori infection by serological testing, including using an antigen-specific enzyme-linked immunosorbent assay (ELISA); 4) included a control group; and 5) reported data on incidence of H. pylori infection in patients with and without pancreatic cancer.
Data Extraction and Quality Assessment
Two reviewers (G.T. and A.P.) used a standardized data extraction tool to independently extract study data. Data obtained from each study included study design, inclusion and exclusion criteria, methodological quality criteria, study population, baseline patient characteristics, diagnostic method of pancreatic cancer, and data related to the primary outcomes. We resolved the few encountered disagreements by mutual discussion and, if required, by consulting a third investigator (W.L.B.).The quality of the individual studies included in this review was assessed independently by two investigators. Quality criteria included the similarity of groups at baseline, methods for selecting participants, methods for measuring exposure variables, extent to which valid primary outcomes were described, as well as analytic methods for control-group matching and control confounding. Each study was given a quality rating of good, fair or poor, based on their risk of bias using the above stated criteria.
Data Synthesis and Analysis
The primary outcome of this analysis was the prevalence of pancreatic cancer in patients with H. pylori infection compared with controls. Pooled adjusted odds ratios (AOR) and associated 95% confidence intervals (CI) were obtained by using a DerSimonian and Laird random-effects model . In addition, we conducted a cumulative meta-analysis whereby studies were pooled chronologically by year of publication, starting with the earliest. Statistical heterogeneity among studies was assessed by using the I2 statistic, with significant heterogeneity defined as an I2<50% . We evaluated the presence of publication and related bias by using funnel plots and Egger test . Statistics were performed using Comprehensive Meta-Analysis, Version 2 (Biostat, Englewood, NJ, USA). A P value of less than 0.05 was considered statistically significant.
Certain host and genetic factors can modify the likelihood of acquisition and persistence of H. pylori infection, which may overturn the balance towards carcinogenesis. Cytotoxin-associated gene A positive (CagA+) strains have been demonstrated to have a greater propensity for inflammation, ulceration and malignant transformation. They have been implicated in etiopathogenesis of gastric cancer . The presence of these strains, but also vacA s1+, vacA m1+ and babA2+ H. pylori strains, may increase the risk of malignant transformation in subjects with a positive family history and/or proinflammatory polymorphisms of the interleukin-1 (IL-1) and tumor necrosis factoralpha (TNF-alpha) genes . Early-life social environment, along with the presence of H. pylori virulent strains, may play a significant role in the development of malignancy five to eight decades later [18, 19]. A subgroup analysis was therefore performed to investigate the relationship between antibodies to CagA antigens and exocrine pancreatic cancer.
Results of Primary Literature Review
Our initial search strategy yielded 32 potential citations for inclusion. Two other additional records were identified through other sources. Of those, 25 were excluded through review of the abstracts due to either not being a report involving human subjects (n=5) or an observational study (n=8), or not evaluating the presence of H. pylori infection (n=12) leaving 9 articles for full publication review. Out of these, two studies were excluded because no patients with pancreatic neoplasms were involved and an additional study by Kosunen et al. , which was included in the earlierpublished meta-analysis by Risch et al. , was excluded from our analysis due to the lack of reliable data on pancreatic cancer; therefore, a total of 6 observational studies (n=2,335 patients) met our eligibility requirements (Figure 1) [7, 8, 9, 10, 11, 12].
The characteristics of the included studies are summarized in Table 1. The number of study subjects ranged from 90 to 1,063, with mean/median ages ranging from 47.5 to 68 years. The proportion of males ranged from 49.5% to 100%. All of the included studies confirmed H. pylori seropositivity using serological analysis. The studies by Raderer et al. , Risch et al. , and Wadstrom et al.  had a casecontrol design, while the others were nested casecontrol studies [8, 9, 10]. Four of the studies were rated as fair quality [7, 8, 10, 11], one as good quality , and one which was published only in abstract form as poor quality . The studies differed somewhat in the covariates that were controlled for within the multivariate analysis. All of the studies, except one  had analysis adjusted for smoking history, four adjusted for age [7, 8, 9, 11], three adjusted for sex [7, 9, 11], and one each adjusted for BMI , alcohol use , educational level , and ELISA plate number .
Synthesis of Results
Our meta-analysis of one good-quality , four fairquality [7, 8, 10, 11], and one poor quality  observational studies showed a significant association between H. pylori seropositivity and development of pancreatic cancer (AOR 1.38, 95% CI: 1.08-1.75; P=0.009) (Figure 2). No significant statistical heterogeneity was seen (I2=13.8%). Visual inspection of the funnel plot (Figure 3) could not rule out the possibility of publication bias, however Egger’s weighted regression analysis suggested a low likelihood (P=0.662). When the three observational studies [8, 10, 11], out of the four with fair-quality, that reported CagA positive status were pooled, no significant association was seen (AOR 1.14, 95% CI: 0.66-1.97; P=0.639).
The cumulative meta-analysis demonstrated that, as studies were published, the association between H. pylori and pancreatic cancer remained statistically significant although the point estimate has been reducing over time (Figure 4). The publication of the most recent study by Risch et al.  did not change the point estimate much and may signal a plateau in the evidence, although additional studies will be required.
In this study, we used meta-analytic techniques to evaluate for a possible etiological association between H. pylori infection, a preventable risk factor, and pancreatic cancer. Our updated pooled analysis of 6 studies, evaluating a total of 2,335 patients, found a significant association between the presence of H. pylori infection and pancreatic cancer (AOR 1.38, 95% CI: 1.08-1.75). Our conclusions are similar to those made by Risch et al.  although we reported more detailed methodology. Additionally, the findings of our cumulative meta-analysis suggested that the strength of this association is reducing over time although is staying statistically significant. This suggests that future studies with more diverse patient populations and larger sample sizes are required to further confirm our findings. It should be pointed out that all of the currently available evidence, including the current report, arises from observational studies and suggest only that an association between H. pylori infection and pancreatic cancer may exist. Studies showing causality are currently not available but are required.
Interestingly, besides its proven association with gastric cancer, previous meta-analyses demonstrated positive associations between H. pylori infection and the development of colorectal , hepatocellular , lung  and laryngeal cancers . Conversely, H. pylori infection was suggested to be protective against esophageal cancer . The precise mechanism by which H. pylori contributes to carcinogenesis has not yet been elucidated. Several theories have been proposed to explain the potential association between H. pylori infection and pancreatic cancer. Thus, Nilsson et al.  detected the 16S ribosomal DNA of gastric H. pylori as well as other enteric Helicobacter species in 75% of tissue samples obtained from patients with pancreatic cancer, as compared with none in the corresponding control group. They theorized that proinflammatory cytokines, reactive oxygen species and other inflammatory mediators associated with chronic H. pylori infection may induce tissue inflammation, increasing genomic DNA damage and cell proliferation. This may lead to an inactivation of tumor-suppressor genes, further facilitating the malignant transformation of pancreatic cells . Takayama et al.  suggested that H. pylori infection of human pancreatic cells may enhance their malignant potential in a similar fashion to the gastric cell cancerogenesis. Significant increases in serum levels of IL-8 and VEGF were seen in H. pylori infected gastric cancer patients [28, 29]. These molecules are known to promote angiogenesis, growth and metastasis of human malignancies. Additionally, activities of nuclear factorkB, activator protein-1, and serum response element of human pancreatic cancer cells were shown to be increased by the H. pylori infection . Moreover, the CagA protein was introduced into the pancreatic cancer cells by the H. pylori infection, which further accentuated serum response element activation. Overall, they demonstrated that H. pylori infection leads to increases in inflammatory cytokine and angiogenic factor secretion, ultimately culminating in malignant transformation of the utilized pancreatic cell line .
The prevalence of H. pylori colonization of the gastrointestinal tract is especially high in developing countries as well as in individuals of lower socioeconomic strata and older persons. Another hypothesis suggested that this gastric antral colonization by H. pylori is associated with increased gastric acid output, leading to uninhibited secretin release from the duodenum . This causes an increased basal pancreatic bicarbonate output and enhanced DNA synthesis, resulting in ductal hyperplasia and later malignancy . Further secretin stimulation has also been proven to accelerate the development and frequency of pancreatic tumors induced by nitrosamines in hamster models .
A third hypothetical pathway involves the H. pylori colonization of the gastric corpus which results in a loss of parietal cells, a decrease in gastric acid output, and basal hypergastrinemia . The resulting hypoacidity facilitates bacterial overgrowth and increased production of N-nitroso compounds, which can be activated in the ductal epithelium after transportation to the pancreas by the circulation. This is further supported by the observation of pernicious anemia commonly associated with pancreatic cancer . Further H. pylori induced gastritis causes a reduced absorption of anti oxidants such as vitamin C . Recent studies show that there is an increased risk of pancreatic cancer in patients with gastric ulcer, but not duodenal ulcer bolstering this hypothesis . Lastly, there appears to be a number of similarities between gastric cancer and pancreatic cancer in terms of epidemiologic and possible etiologic cofactors involved in the two malignancies. These include diets deficient in fresh fruits and vegetables, cigarette smoking, poor socio-economic status, which have also been associated with the H. pylori infection itself .
We also realize that our study has several limitations. Our primary endpoint included pooled data of adjusted OR pulled from the included studies. The variables used to adjust these values were not consistent across studies, which may limit the reliability of the data. Too few studies were identified to allow for subgroup analysis by covariates. Second, as with any metaanalysis, the potential for publication bias is a concern. As such, the results of our study must be interpreted in the context of its possibility. However, visual inspection of the funnel plots were did not identify any significant publication bias. This is further strengthened by the non-significant findings using Egger’s weighted regression statistic. Lastly, although a statistically significant association between H. pylori and pancreatic cancer was seen in our pooled analysis, the AOR is relatively modest. Some epidemiologists have suggested that an odds ratio of greater than 2 is required for clinical relevance. Since the AOR we found was 1.38, the clinical relevance of the association could be debated. This should not discount the association; rather justify the need for causative future studies.
In conclusion, our updated and cumulative metaanalysis of six studies suggests an association between H. pylori infection and the development of pancreatic cancer. Further studies with larger sample sizes evaluating this relationship are warranted to confirm our findings and to unearth the potential underlying mechanisms.