1 National Public Health Institute, Helsinki and Oulu, Finland
2 University of Tampere, Tampere, Finland
3 Icelandic Cancer Society, Reykjavik, Iceland
4 University of Iceland, Reykjavik, Iceland
5 Finnish Cancer Registry, Helsinki, Finland
6 Research and Development Centre for Health and Social Welfare, Helsinki, Finland
7 University of Helsinki, Helsinki, Finland
Reprint requests to Dr. Matti Lehtinen, National Public Health Institute, Aapistie 1, Oulu FIN-90520, Finland (e-mail: llmale{at}uta.fi).
Received for publication January 5, 2005. Accepted for publication April 29, 2005.
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ABSTRACT |
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antibodies; case-control studies; child; Chlamydia; Helicobacter pylori; leukemia, lymphocytic, acute; Mycoplasma pneumoniae
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INTRODUCTION |
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Infection with Mycoplasma pneumoniae has been suggested to be associated with childhood ALL (8, 9
). Two other ubiquitous human bacterial pathogens, Helicobacter pylori and the genus Chlamydia, have been associated with lymphoproliferative disorders in adults (10
13
). H. pylori is especially well recognized as an etiologic agent in mucosa-associated lymphoid tissue lymphoma (11
). The evidence for associations of H. pylori and Chlamydia with leukemia is lacking.
To study the role of these three bacterial infections in childhood leukemia, we conducted a case-control study nested within a joint cohort of 550,000 mothers and their offspring. Acute maternal infection was defined by the presence of specific immunoglobulin M (IgM) antibodies to the genus Chlamydia, H. pylori, and M. pneumoniae. The presence of immunoglobulin G (IgG) antibodies to H. pylori was interpreted as evidence of persistent infection (14), with an increased predisposition of the offspring.
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MATERIALS AND METHODS |
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The Rubella Screening Serum Bank at the Department of Virology, University of Iceland (Reykjavik, Iceland), comprises 75,000 serum samples collected during 19751997 from practically all (>95 percent; i.e., 50,000) pregnant women in Iceland at 1214 weeks' gestation. The samples are stored at 20°C. Pertinent data on reproductive history were retrieved from the Icelandic Maternity Registry (6).
The Finnish and Icelandic cancer registries, which are population-based and country-wide, were established in 1952 and 1954, respectively. Various checks have shown that they receive notifications of virtually all histologically confirmed new cases of cancer (16, 17
).
Identification of cases and controls
For the present study, cases of childhood leukemia among young persons registered in the Finnish and Icelandic cancer registries were classified into two categories: ALL and other leukemia (non-ALL). Stratification into four categories by age at diagnosis (<1, 1, 26, and >6 years) was applied to distinguish between infant leukemia cases, cases occurring in the ALL peak period, and other childhood leukemia cases.
Mothers of all children who developed leukemia before 15 years of age were identified through the Finnish and Icelandic national population registries. Final index mothers were those who had serum samples in the Finnish and Icelandic maternity cohorts (402 women altogether). For matching, we applied incidence density sampling; that is, three control mothers in Finland and four control mothers in Iceland with totally cancer-free offspring at the time of childhood leukemia diagnosis were matched with the index mother according to age at serum sampling (±2 years), date of specimen collection (±2 months), and offspring characteristics: date of birth (±2 months) and sex of the child. The matching was performed by country to ensure that differences between the national cohorts did not affect the validity of the study. If three or four control mothers were not found, the matching criteria for age and storage time were expanded stepwise by 1 month until control mothers were found.
The control group comprised 1,212 women altogether. The median and maximum differences in age between the index mothers and the control mothers were 0.3 years and 6.6 years, respectively.
Permissions for linkage between the population, cancer, and maternity cohort data files for identification of index and control pairs and use of the joint cohort data file were obtained from the national data protection authorities, the Finnish Ministry of Health, population registry centers, and the national ethical review boards.
Laboratory methods
To identify maternal infection or offspring susceptibility to perinatal infection, the presence of IgM and IgG antibodies to three human bacterial pathogensChlamydia trachomatis, H. pylori, and M. pneumoniaewas determined according to manufacturers' instructions by means of standard enzyme-linked immunosorbent assays (ELISAs) that used the same batches of purified elementary body (C. trachomatis), bacterial lysate (H. pylori), and P1-adhesin-enriched (M. pneumoniae) antigens. For C. trachomatis, we used IgG and IgM ELISAs (Thermo Labsystems, Helsinki, Finland) with reported sensitivity and specificity of 100 percent. For M. pneumoniae, we used IgG and IgM ELISAs (Thermo Labsystems) with reported sensitivity of 75.7 percent and reported specificity of 98.9 percent. For H. pylori, we used an IgG ELISA (Orion Diagnostica, Espoo, Finland) with reported 100 percent sensitivity and 94.3 percent specificity, as well as an IgM ELISA (Immunobiological Laboratories GmbH, Hamburg, Germany).
The cutoff levels were preassigned following the manufacturers' recommendations relative to internal positive and negative reference sera used on all plates. We further controlled for the specificity of the IgM antibody response by separately considering only IgM-positive mothers who were negative for IgM antibodies to the other two bacteria.
The laboratory analyses were carried out with masked samples, whereafter the data were submitted to the Finnish Cancer Registry for decoding and statistical analysis.
Statistical analyses
Relative risks, expressed as matched odds ratios and their 95 percent confidence intervals, were estimated by conditional logistic regression at the National Public Health Institute. Associations with birth order (firstborn vs. others; dichotomous variable) and sibship size (number of siblings; quantitative variable) by the index pregnancy were considered by both adjustment and interaction analyses as planned a priori. The interactions were studied using observed solitary odds ratios and expected conditional odds ratio (OR) estimates from a multiplicative model, including the interaction between variable A (exposure) and variable B (birth order): ORexpected = OR(A, nonB) x OR(nonA, B) (18). Synergistic interaction was defined as an observed joint odds ratio greater than the expected joint odds ratio, tested by means of likelihood ratio statistics and considered to exist in a situation where most of the risk associated with A existed in the presence of B and vice versa.
The analyses were performed using SPSS for Windows (version 9.1; SPSS, Inc., Chicago, Illinois) and Stata (version 5.0; Stata Corporation, College Station, Texas) statistical software. All p values were two-sided; p < 0.05 was considered statistically significant.
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RESULTS |
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In the matched analyses, acute maternal M. pneumoniae infection, as defined by specific IgM positivity, appeared to be associated with an increased risk of childhood leukemia (OR = 1.6, 95 percent confidence interval (CI): 1.0, 2.5; table 2). However, the statistical significance was lost when specific M. pneumoniae IgM positivity, in the absence of C. trachomatis and H. pylori IgM antibodies, was used for the definition of acute infection (OR = 1.5, 95 percent CI: 0.9, 2.4). Adjusting for sibship size also dropped the lower bound of the 95 percent confidence interval below 1 (OR = 1.6, 95 percent CI: 0.9, 2.6). Moreover, there was no significant interaction between M. pneumoniae and birth order per se, and the observed joint odds ratio (ORobs = 1.1) for M. pneumoniae IgM positivity (solitary OR = 1.6) and birth order (solitary OR = 1.3) did not differ from that expected on the basis of a multiplicative model (ORexp = 2.0; p = 0.8, likelihood ratio statistic).
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DISCUSSION |
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Adult leukemia patients have been reported to have low H. pylori seroprevalence (21), but in general, H. pylori seroprevalence varies greatly by time, location, and ethnic group (22
). The Icelandic mothers of the leukemic children had significantly higher H. pylori seroprevalence than would be expected for their age group. In general, reproductive-age Finnish and Icelandic women appear to have similar background exposures to H. pylori over time (23
, 24
; the present study). However, the described phenomenon could not be found in Finland in any calendar-time strata of the Finnish index child-mother pairs. However, the possibility that a certain subset of ALL cases (for example) could be associated with H. pylori remains open.
H. pylori produces chronic and persistent infection in the gastric mucosa, and IgG antibodies disappear only after a period of several months after eradication of the microorganism (14). Thus, the presence of maternal H. pylori IgG antibodies implies the presence of the bacterium and the opportunity for mother-to-child transmission in very early infancy, which is recognized as an important route of infection for H. pylori (25
). We are not aware of any differences in social class or lactation practices between Icelandic and Finnish women that would affect H. pylori exposure in their offspring, but pertinent questionnaire data are lacking. The sex ratio of the leukemia cases fits the Icelandic and Finnish cancer statistics (6
). If H. pylori exposure really differs between Icelandic and Finnish infants and small children, the resulting early exposure to the microbial antigen is probably important in increasing the risk of childhood leukemia (4
, 5
).
The nested case-control design, which was based on quality-controlled cancer registers (15, 16
), cross-generation linkage, and a long follow-up time from the index pregnancy to the leukemia diagnosis, rules out the possibility that the disease process could have activated H. pylori infection in the pregnant women (26
). It is also noteworthy that no other associations with bacterial IgG antibodies were observed.
Our approach was restricted to serologic analyses of first-trimester maternal serum samples and thus suffered from the common problem of misclassification bias (18). Indirect inferences about the offspring's susceptibility to or protection from infection are especially prone to this bias. On the other hand, the risk associated with H. pylori IgG antibodies was specific for the Icelandic women and their offspring, who were diagnosed with childhood leukemia at less than 6 years of age (i.e., with the bulk of ALL cases). Such a finding is unlikely to have resulted from misclassification, but this possibility cannot be ruled out.
M. pneumoniae has been associated with childhood leukemia in case reports, cross-sectional seroepidemiologic studies, and animal studies (8, 9
, 27
, 28
). However, M. pneumoniae IgM antibody positivity, an indicator of acute maternal infection, lost statistical significance when results were controlled for sibship size and birth order, which are common risk factors for childhood leukemia and lymphoma (3
, 29
). This was also the case when the generally poor specificity of the IgM antibody determination was considered further using a robust method of excluding cases and controls with multiple positive IgM antibody findings.
To our knowledge, we have documented for the first time the possibility of an association between maternal H. pylori infection and risk of childhood leukemia in the offspring. Independent confirmatory studies are needed.
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ACKNOWLEDGMENTS |
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The authors thank Drs. Joakim Dillner and Richard Peto for stimulating discussions. The assistance of Pirjo Kontiokari in performing the laboratory analyses is gratefully acknowledged.
This article is publication no. 32 of the Nordic Biological Specimen Banks Working Group on Cancer Causes and Control (NordForsk, Oslo, Norway).
Conflict of interest: none declared.
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References |
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