Affiliations of authors: R. Z. Stolzenberg-Solomon (Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics and Cancer Prevention Studies Branch, Division of Clinical Sciences), P. J. Limburg, P. R. Taylor (Cancer Prevention Studies Branch, Division of Clinical Sciences), D. Albanes (Nutritional Epidemiology Branch, Division of Cancer Epidemiology and Genetics), National Cancer Institute, Bethesda, MD; M. J. Blaser, Departments of Medicine and Microbiology, New York University School of Medicine and Department of Veterans Affairs Medical Center, New York; G. Perez-Perez, Departments of Medicine and Microbiology, New York University School of Medicine; J. Virtamo, National Public Health Institute, Helsinki, Finland.
Correspondence to: Rachael Z. Stolzenberg-Solomon, Ph.D., M.P.H., National Institutes of Health, 6120 Executive Blvd., MSC 7026, Rm. 7039, Bethesda, MD 20892 (e-mail: rs221z{at}nih.gov).
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ABSTRACT |
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INTRODUCTION |
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From 1985 through 1988, the Alpha-Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study (7), a randomized controlled trial of -tocopherol and
-carotene supplementation for the prevention of cancer, collected and stored serum for 29 133 participants. We conducted a nested, prospective casecontrol investigation of the relationship between antibodies to H. pylori whole-cell (WC) and CagA antigens and exocrine pancreatic cancer to determine whether carriage of H. pylori or particular strains was associated with risk of acquiring the tumor.
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SUBJECTS AND METHODS |
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The ATBC Study was a double-blind, placebo-controlled, 2 x 2 factorial design primary prevention trial that tested whether -tocopherol or
-carotene could reduce the incidence of cancer among male smokers. Study rationale, design, and methods have been described previously (7,8). From 1985 through 1988, a total of 29 133 eligible men in southwestern Finland, aged 5069 years, who smoked at least five cigarettes per day, were randomly assigned to receive either active supplements or placebo. Men were excluded from the study if they had a history of malignancy other than nonmelanoma cancer of the skin or carcinoma in situ, severe angina on exertion, chronic renal insufficiency, liver cirrhosis, chronic alcoholism, or other medical conditions that might limit long-term participation, if they were receiving anticoagulant therapy, or if they used supplements containing vitamin E (>20 mg/day), vitamin A (>20 000 IU/day), or
-carotene (>6 mg/day). All of the study participants provided written informed consent before randomization, and the study was approved by the institutional review boards of both the National Public Health Institute (Helsinki, Finland) and the U.S. National Cancer Institute (Bethesda, MD).
Participants completed questionnaires on general background characteristics, including medical, smoking, and dietary histories during their prerandomization baseline visit. Diet was assessed with a validated self-administered dietary history questionnaire that determined the frequency of consumption and the usual portion size of 276 food items during the past year, with the use of a color-picture booklet as a guide for portion size (9). The dropout rate during the trial was similar (30.1%31.3%) for all four treatment groups (-tocopherol,
-carotene, ATBC, and placebo) (7).
Ascertainment of Case Subjects and Control Subjects
All cases of pancreatic cancer diagnosed from January 1985 through December 1995 were identified through the Finnish Cancer Registry and death certificates. The Finnish Cancer Registry provides almost 100% case ascertainment in Finland (10,11). Medical records were reviewed centrally by two study oncologists (7) and the histopathologic and cytologic specimens by one or two pathologist(s) for diagnostic confirmation (7). Only cases confirmed by the study physicians as incident primary malignant neoplasms of the exocrine pancreas (International Classification of Diseases, 9th Revision [ICD-9]-157 (12), excluding islet cell carcinomas, ICD-9157.4) were included in this analysis (n = 130). The interval between baseline serum collection and diagnosis was up to 10 years (median follow-up time, 4.6 years [range, 0.0610 years]), and the median age at diagnosis was 64 years (range, 5076 years).
Control subjects were selected from among ATBC Study participants who were alive at the time the matching case subject was diagnosed and free from cancer (except nonmelanoma skin cancer) as of December 1995. Two control subjects were matched to each case subject by age (±5 years), month of baseline blood draw, completion of dietary history, study center, and intervention group assignment.
Of the 130 pancreatic cancer case subjects and the 260 matched control subjects, 123 case subjects and 239 control subjects had sufficient baseline serum for measurement of H. pylori WC and CagA strains; of these, 121 case subjects had matched control subjects (n = 226). Thirty-one percent, 16%, 27%, and 26% of the case and control subjects were in the placebo, -carotene alone,
-tocopherol alone, and
-tocopherol/
-carotene intervention groups, respectively.
Biomarkers
Fasting serum was collected at the prerandomization baseline visit and stored at 70 °C. Frozen baseline serum samples for both case and control subjects were assayed for antibodies to H. pylori WC and CagA antigen with previously described and validated methods (1315). Serology for immunoglobulin G (IgG) antibodies to H. pylori WC was determined by antigen-specific enzyme-linked immunosorbent assay (ELISA), which was prepared from a pool of sonicates of WC antigens from five different clinical H. pylori isolates (13,15). Serum IgG-specific antibodies to the CagA antigen were determined by ELISA by use of a purified truncated recombinant protein (orv220) from Escherichia coli (Peptide Therapeutics, Cambridge, MA). Serum samples were diluted 1 : 800 and 1 : 100, respectively, for the H. pylori WC and CagA antigen determinations, and optical density for each was calculated from the mean reading of duplicate assays of the same sera, run on separate days. Individuals were considered to be seropositive if the optical density of the IgG antibodies for H. pylori WC was greater than or equal to 1.0 or for CagA was greater than or equal to 0.35. Overall disagreement for H. pylori classification between the first and second run was 8%. For those samples with discordant H. pylori classification between the duplicate assays, a third run was performed. After the third assay, the assay with the greatest variation from the other two assays was discarded, and the results of the remaining two assays were averaged. The sensitivity and specificity for the H. pylori WC assay are both more than 92% (13) and for the CagA assay are 94% and 93%, respectively (14).
Case and control specimens were handled in the same standard manner, and the laboratory was blinded to casecontrol status. Matched serum case and control samples were analyzed consecutively as triplets within batches, and blinded replicate quality-control phantom samples (one H. pylori-seropositive and one -seronegative subject) were placed toward the beginning and end of each batch, constituting approximately 10% of each batch. The intrabatch and interbatch coefficients of variation were 8.6% and 19.6%, respectively, for the H. pylori WC assay and 8.2% and 26.6%, respectively, for the CagA assay. The high interbatch coefficient of variation for H. pylori WC and CagA could potentially lead to misclassification of the exposure and influence risk estimates. However, the percent agreement for classifying H. pylori seropositivity based on cut points for the blinded repeated samples was 100%.
Previously measured serum nutrients were evaluated as confounders of the main effects. Baseline serum -tocopherol and
-carotene were measured for all of the participants in the ATBC Study by high-performance liquid chromatography (7), and serum total homocysteine, folate, pyridoxal-5'-phosphate, and vitamin B12 were measured for subjects in this substudy, with the methods described previously (16).
Statistical Analysis
H. pylori serology was defined as negative (having antibodies to neither WC nor CagA antigens), positive (having antibodies to WC and/or CagA+ antigens), positive with CagA-negative (CagA) strain (having antibodies to WC but not to CagA+ antigen), or positive with CagA+ strains (having antibodies to CagA+ antigen). A small proportion of subjects were H. pylori WC negative and CagA+ (three case subjects, 2.5%; eight control subjects,
3.5%), and these were combined with those who were H. pylori WC positive and CagA+ to form the CagA+ category. Other relevant variables examined as potential confounders were age; smoking history; educational level; history of diabetes, ulcer (peptic or duodenal), pancreatitis, or gallstones; dietary nutrients (energy, protein, carbohydrate, fat, saturated fat, fiber, carotenoids, vitamins C, E, B6, and B12, folate, methionine, sodium, nitrate, and nitrite); foods (fruit, citrus fruit, vegetables [fresh, cooked and cruciferous], roots, legumes, sausages, and cold cuts); alcohol and coffee intake; and serum nutrients (
-tocopherol,
-carotene, folate, vitamin B12, pyridoxal-5'-phosphate, and total homocysteine).
Dietary nutrients highly associated with energy were energy adjusted by use of the residual method described by Willett and Stampfer (17), and nutrients not normally distributed were log-transformed for energy adjustment. Selected characteristics of the case and control subjects (and by H. pylori serology among the control subjects) were compared with the use of nonparametric Wilcoxon rank-sum and chi-squared tests. Conditional logistic regression was used to estimate odds ratios and 95% confidence intervals for pancreatic cancer with subjects testing negative for H. pylori as the reference category; indicator variables for H. pylori positive with CagA+ and positive with CagA categories were included in the model. Multivariable models were developed by individually adding covariates to the model; continuous variables were included in the models if they were associated with both the disease and the risk factor, had a chi-squared P value of less than or equal to .20 in the full model, and changed the risk estimate by greater than or equal to 10%.
All statistical analyses were performed by use of Statistical Analysis Software (SAS) software (SAS Institute, Inc., Cary, NC). All statistical tests were two-tailed and were considered to be statistically significant at the .05 level. Because case and control subjects were matched, the median values, proportions, and risk estimates (including those labeled as crude) should be interpreted as being adjusted for the matching factors.
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RESULTS |
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DISCUSSION |
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Our results are consistent with the only published casecontrol study (6) to our knowledge that has examined H. pylori and pancreatic cancer. That study found greater H. pylori seropositivity among 92 pancreatic cancer case subjects (65%) than among 62 control subjects (45%; control subjects had either colorectal cancer or no disease) and showed a similar significant doubling of risk. In that study, there was no evidence of inflammation or H. pylori colonization in the pancreatic tumor or surrounding tissue in a small subset of 20 specimens (6), a finding consistent with processes occurring in the stomach whereby H. pylori disappears as chronic atrophic gastritis and extensive intestinal metaplasia replace normal mucosae (2).
H. pylori has been implicated as a carcinogenic factor for noncardia gastric cancer and causes a persistent inflammatory-proliferative state that evolves from chronic superficial gastritis to precancerous atrophic gastritis, metaplasia, and dysplasia (2). Given recent evidence that chronic pancreatitis has been associated with pancreatic cancer (19), pathologic consequences of H. pylori similar to those observed in gastric tissue could also be postulated for the pancreas. Two studies (20,21), limited by small sample sizes and in populations with low H. pylori seroprevalence, have not shown a difference in the seroprevalence of antibodies to H. pylori between subjects with and without chronic pancreatitis. However, to investigate the observation that subjects with chronic pancreatitis have a high prevalence of duodenal ulcers (2224), Niemann et al. (25) showed a significantly higher H. pylori seroprevalence in subjects with chronic pancreatitis complicated by duodenal ulcer compared with control subjects with nonorganic abdominal pain (P = .002) or subjects with chronic pancreatitis without duodenal ulcer (P = .04) but not compared with control subjects with simple duodenal ulcer without pancreatitis. With regard to adjacent organs, H. pylori has an established role in the etiology of duodenal ulcer (26) and has been identified in liver biopsy specimens of patients with biliary inflammation (27). Whether or not pancreatic colonization by H. pylori exists is unknown and perhaps should be examined in asymptomatic subjects. Other possible mechanisms for the association between H. pylori and pancreatic cancer include changes in gastrin (increased secretion) and somatostatin (low number of antral somatostatin cells) resulting from H. pylori gastritis (2831). For example, elevated gastrin could stimulate pancreatic growth (32), with associated proliferation potentially increasing the susceptibility of the pancreas to carcinogens, and diminished somatostatin could also allow for increased pancreatic cancer growth (33). Pernicious anemia, a condition marked by hypergastrinemia, has been associated with pancreatic cancer (34,35). Alternatively, the increased formation of N-nitroso compounds produced by hypochlorhydria-related gastric bacterial overgrowth could be another carcinogenic mechanism. Finally, H. pylori, especially CagA+ status, may represent a marker of another gastrointestinal colonizing or infecting organism that may be a risk factor for pancreatic cancer.
In our study, persons with prior cholelithiasis or peptic ulcer disease were more likely to be seropositive for H. pylori and CagA+ strains, although the proportion who had a history of either disease was small and the number of case and control subjects with the conditions did not statistically significantly differ. Cholecystectomy (1,36) and duodenal ulcer disease (37) diagnosed up to 20 years earlier have been associated with subsequent pancreatic cancer, although for neither diagnosis are the data consistent (36,37). Subjects who were seropositive for H. pylori are often asymptomatici.e., without dyspeptic symptoms (38). Combined with the current findings, such studies suggest that persons with H. pylori could have subclinical inflammation or disease.
In conclusion, our study suggests that H. pylori carriage may be a risk factor for pancreatic cancer. Strengths of this study include the prospective nature of the blood collection (i.e., 110 years before diagnosis of the cancer), a relatively large sample size, and a control group derived from the same study cohort. The associations between H. pylori and CagA+ strains and pancreatic cancer are temporally related, biologically plausible, and similar to associations observed for cancer of the stomach. Because the subjects in this study were male smokers, our findings may not be generalizable to other nonsmoking populations, and caution is justified in the interpretation of our results. Our observed associations became stronger after controlling for years of smoking, however, and are in accord with those seen in the other published casecontrol study (6). Evaluation of this relationship in additional studies appears warranted to confirm or refute our findings and to understand potential mechanisms.
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NOTES |
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Supported by Public Health Service contracts N01CN45165 and N01CN45035 (National Cancer Institute) and R01DK53707 (National Institute of Diabetes and Digestive and Kidney Diseases) (partial support for M. J. Blaser and G. Perez-Perez and for laboratory supplies), National Institutes of Health, Department of Health and Human Services.
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Manuscript received October 31, 2000; revised April 6, 2001; accepted April 19, 2001.
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