1 Department of Infectious Disease Epidemiology, National Public Health Institute, Helsinki, Finland.
2 Department of Microbiology, National Public Health Institute, Oulu, Finland.
3 Icelandic Cancer Registry, Icelandic Cancer Society, Reykjavik, Iceland.
4 Medical School, University of Iceland, Reykjavik, Iceland.
5 Department of Clinical Microbiology, Malmö Central Hospital, Malmö, Sweden.
6 Department of Public Health, University of Helsinki, Helsinki, Finland.
7 Microbiology and Tumor Biology Center, Karolinska Institute, Stockholm, Sweden.
8 Finnish Cancer Registry, Helsinki, Finland.
9 Medical School, University of Tampere, Tampere, Finland.
Received for publication August 27, 2002; accepted for publication January 31, 2003.
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ABSTRACT |
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antibodies; child; Epstein-Barr virus infections; herpesvirus 4, human; leukemia, lymphocytic, acute; longitudinal studies; prospective studies
Abbreviations: Abbreviations: ALL, acute lymphoblastic leukemia; CI, confidence interval; EBV, Epstein-Barr virus; ELISA, enzyme-linked immunosorbent assay; Ig, immunoglobulin; OR, odds ratio; PCR, polymerase chain reaction.
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INTRODUCTION |
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We performed a case-control study nested within a joint cohort of 550,000 mothers and their offspring with altogether 7 million years of follow-up. The role of maternal infection in Epstein-Barr virus (EBV) and human herpesvirus 6, known or suggested to be associated with childhood lymphomas (8), was assessed by using technically similar serologic tests. Antibodies to a third ubiquitous human herpesvirus, cytomegalovirus, were determined for control purposes.
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MATERIALS AND METHODS |
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The Rubella Screening Serum Bank at the Department of Virology, University of Iceland, contains 75,000 serum samples collected during 19751997 from practically all (>95 percent, i.e., 50,000) pregnant women in Iceland at 1214 weeks of gestation. The samples are stored at 20°C. Data on reproductive history were retrieved from the Icelandic Maternity Registry.
Population-based and countrywide Finnish and Icelandic cancer registries were established in 1952 and 1954, respectively. Various checks have shown that they are notified of virtually all histologically confirmed new cases of cancer (10).
Study design and identification of cases and controls
For the present study, childhood leukemia in the young registered at the Finnish and Icelandic cancer registries was classified into two categories: ALL and other leukemias (non-ALL). Stratification by age at diagnosis into four categories<1, 1, 26, and >6 yearswas applied to distinguish among infant leukemia cases, cases occurring during the ALL peak, and other childhood leukemia cases.
Mothers of children who developed leukemia before 15 years of age were identified through national population registries. Final index mothers selected were those for whom there were serum samples in the Finnish and Icelandic Maternity Cohorts (n = 403). For matching, we applied incidence density sampling; that is, three Finnish control mothers and four Icelandic control mothers whose offspring were totally cancer free at the time of the childhood leukemia diagnosis were matched with the index mother on age at serum sampling (±2 years); date of specimen collection (±2 months); and, for the offspring, date of birth (±2 months) and gender. The matching was performed by country to ensure that differences between the national cohorts did not affect study validity. If three or four control mothers could not be found, the matching criteria on age and storage time were expanded stepwise by 1 month.
The control group comprised 1,216 women. The median and maximum differences in age between the index mothers and control mothers were 0.3 and 6.6 years, respectively.
Permissions for linkage between Population, Cancer, and Maternity Register data files to identify the index mother-case pairs and the control mother-control pairs, and to use the joint cohort data file, were obtained from the national data protection authorities, Finnish Ministry of Health, Population Registry Centre, and national ethical review boards.
Laboratory methods
To identify maternal infection or offspring susceptibility to perinatal infection, immunoglobulin (Ig)M and IgG antibodies to three human herpesvirusescytomegalovirus, EBV, and human herpesvirus 6were determined according to manufacturers instructions by standard enzyme-linked immunosorbent assay (ELISA) tests that used the same batches of the purified virion antigens (cytomegalovirus and human herpesvirus 6) or virion glycoprotein gp125 (EBV). For cytomegalovirus IgG and IgM and for EBV IgG and IgM, we used Gull ELISA IgG and IgM tests (CME 100, CME 150, EBE 100, and EBE 150; Gull Laboratories, Inc., Salt Lake City, Utah) with reported 96.8 percent and 92.1 percent, and 96.7 percent and 99.4 percent sensitivity and with reported 95.5 percent and 98.3 percent, and 95.7 percent and 100 percent specificity, respectively. For human herpesvirus 6 IgG, we used Advanced Biotechnologies (ABI) human herpesvirus 6 IgG antibody ELISA (15-401-000; ABI, Inc., Columbia, Maryland) with 100 percent reported sensitivity and specificity. For human herpesvirus 6 IgM, we used the PanBIO human herpesvirus 6 IgM ELISA test (H6M-200, PanBio, East Brisbane, Australia) with 95 percent reported specificity. EBV and human herpesvirus 6 IgM positivity of the ELISA-positive sera was further evaluated by the absence of antibodies to the EBV nuclear antigen (Biotest IgG-EBNA; Biotest AG, Frankfurt, Germany) and by the presence of human herpesvirus 6 IgM using the immunofluorescence method (human herpesvirus 6 IgM IF, V17 human herpesvirus 6; Biotrin International Limited, Dublin, Ireland), respectively.
Cutoff levels were preassigned by following the manufacturers recommendations relative to internal positive and negative reference sera used on all plates and slides. Specificity of the IgM antibody response was further controlled for by separately considering IgM positives who were negative for IgM antibodies to the other two herpesviruses.
All case-control strata containing an EBV IgM-positive serum sample were also examined for EBV DNA by using quantitative real-time polymerase chain reaction (PCR) (11). Briefly, DNA was extracted from 50-µl serum samples by using the QiAmp Blood kit (Qiagen Gmbh, Hilden, Germany). A primer pair (forward w44f: 5'-CCCAACACTCCACCACACC-3', reverse w119r: 5'-TCTTAGGAGCTGTCCGAGGG-3') from the BamHI-W fragment region of the EBV genome and defining a 65 base-pair amplimer, and an oligonucleotide probe (5'-CACACACTACACACACCCACCCGTCTC-3') with a fluorescein reporter dye and quencher attached to its 5' and 3' ends, respectively, were purchased from PE-Applied Biosystems (Birchwood Science Park, North Warrington, Cheshire, United Kingdom) and were used as described by the manufacturer (12). Universal PCR Mastermix (PE-Applied Biosystems) and the cycling parameters 50°C for 2 minutes, 95°C for 10 minutes, 50 cycles of 95°C for 15 seconds, and 60°C for 1 minute, as well as the ABI7700 Real-Time PCR Machine (PE-Applied Biosystems), were used. In each analysis, a negative reference sampleDNA extracted from serum samples of EBV-seronegative subjectsand positive reference samples1 (detection limit) to 1,000 copies of EBV genome/µlwere run in triplicate in 10-fold dilutions for a standard curve.
The laboratory analyses were conducted with masked samples. After the analyses were completed, the resulting data were submitted to the Finnish Cancer Registry for decoding and statistical analysis.
Statistical analyses
Relative risks, expressed as odds ratios and their 95 percent confidence intervals, were estimated by using conditional logistic regression. In addition, 99 percent confidence intervals were calculated to control for multiple comparisons and are presented in the text. 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 adjusting and interaction analyses, as planned a priori. The interactions were studied by using observed solitary odds ratios (OR) and expected conditional odds ratio estimates from a multiplicative model, including interaction of variable A (exposure) and variable B (birth order), as follows: expected OR = OR(A, nonB) x OR(nonA, B) (13). Synergistic interaction was defined as observed joint odds ratio > expected joint odds ratio, tested by likelihood ratio statistics and considered to exist when most of the risk associated with A occurred in the presence of B, and vice versa.
Existence of a linear trend in the odds ratio estimates by a cases age was evaluated as an interaction between antibody positivity and the cases (childs) age at diagnosis assuming scores 1, 2, 3, and 4 for those older than age 6 years, those aged 26 years, those aged 1 year, and those younger than age 1 year, respectively.
The statistical analyses were performed by using SPSS for Windows 9.1 (SPSS, Inc., Chicago, Illinois) and Stata 5.0 statistical software (Stata Corporation, Inc., College Station, Texas). All p values were two-sided; p < 0.05 was considered statistically significant.
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RESULTS |
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DISCUSSION |
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Misclassification
EBV viral capsid antigen IgM ELISAs based on viral gp125 are considered highly specific (>95 percent) and sensitive (90 percent) for determining acute infectious mononucleosis; however, in EBV reactivations, the specificity is not as good (15, 16). Determination of IgM antibodies is always subject to misclassification bias. Specificity may be violated by the presence of rheumatoid factor; polyclonal activation, which has also been associated with the risk of ALL (4, 5); other, rare reasons for false-positive IgM findings; or cross-reactivity between the different herpesviruses. To address low specificity, we restricted our analyses to those who were positive for a single EBV IgM antibody test only, a robust routine method to improve specificity of the IgM response (16). Doing so controlled for rheumatoid factor and nonspecific reactivity to other herpesviruses as reasons for false IgM positivity, and it yielded higher and more stable odds ratios. On the other hand, a number of viruses, including EBV, are known to cause polyclonal IgM production. Thus, it is possible that the restricted analysis also excluded true findings, and the resulting lower sensitivity may have biased the point estimates downward. Serum antibodies or DNA is not sensitive to storage temperature (17), and low sensitivity is mostly due to the transient nature of the IgM antibody response (23 months). Unfortunately, the period of serum PCR positivity for EBV, even during a manifest primary infection (infectious mononucleosis), is even shorter (12 weeks) (18), and very little is known about serum PCR positivity in secondary EBV infections or reactivations. Moreover, only the first-trimester serum samples were available. Therefore, even though both the EBV PCR and EBV IgM analyses yielded comparable odds ratios for the risk of ALL, serum PCR analysis did not resolve the problem of possible low sensitivity, which again may have biased both point estimates downward.
Chance observation
Other large, joint cohort studies by the Nordic Biological Specimen Bank Study Group on Cancer Causes and Control have disclosed associations between common infections (Chlamydia trachomatis, human papillomavirus, EBV) and more or less common cancers (cervical cancer, oropharyngeal cancer, Hodgkins disease) (9, 19, 20). Our current study is probably unique in linking the first two generations at the population level and then linking the joint cohort to the population-based cancer registries (21). While these registries lack hematologic data on, for example, the karyotype and immunophenotype of the cases, their strict quality control guarantees that all caseindex mother childhood leukemia pairs appearing in the population during the study period were identified (10, 21, 22). To control for multiple comparisons, we also calculated 99 percent confidence intervals; we noted that the EBV-associated risk would still have been statistically significant if multiplied by 6, the number of original variables. Furthermore, no other statistically significant associations between the maternal cytomegalovirus or human herpesvirus 6 antibodies and ALL or non-ALL were found. On the other hand, no association between EBV and ALL was found in the small Icelandic subsample. Thus, to rule out the possibility of a chance observation, independent studies with large sample sizes are needed.
Causal EBV reactivation
That an infection might be involved in the etiology of childhood leukemia was expected a priori on the basis of the hypotheses of Kinlen and Greaves and the findings of population-mixing studies and different clustering studies (4, 5, 7, 23, 24). In general, however, ecologic studies have not been able to verify the mode of exposure (e.g., congenital) or the specific agents involved (e.g., influenza) (7, 2325). On the other hand, case-control studies and similar associations of birth order, sibship size, and seasonality with both ALL and Hodgkins disease (3, 6, 26) suggest that delayed infections might be their plausible causes. We controlled for seasonality and epidemic outbreaks by matching, and for birth order and sibship size by performing adjusting and interaction analyses, and we found increased point estimates for the association between (nonprimary) maternal EBV infection and risk of ALL in the offspring. The point estimates tended to increase further according to a cases decreasing age and were especially high for the youngest age groups including infant ALL, which would fit with an intrauterine EBV infection.
Subclinical and secondary EBV infections of the female and male genital tracts are common, and, in some risk groups, EBV type 2 infections occur exclusively with sexual transmission (27, 28). It is possible that exposure to EBV (type 2) during pregnancy could have caused secondary infections. EBV is also frequently reactivated during pregnancy (29). The index mothers were negative for human immunodeficiency virus (data not shown), but other causes of immunodeficiency predisposing to both EBV reactivation and ALL were not investigated.
In young-adult Hodgkins disease, the most important causal event is probably delayed exposure to EBV, as suggested by the epidemiologic studies, and demonstration of viral DNA and proteins in the tumor tissue (20, 26, 30, 31). Although the biologic plausibility of transformation of B and T cells infected with EBV and harboring episomal viral DNA is beyond doubt irrespective of cellular differentiation stage (32, 33), the relatively infrequent occurrence (up to 15 percent) of EBV DNA in the malignant cells of a small series of ALL cases (34) makes direct viral transformation unlikely. However, in sporadic Burkitts lymphoma, episomal EBV DNA is sometimes lost from the cells (35), and this loss might also happen in the context of ALL once the critical hit has occurred. The role of EBV in inducing chromosomal translocations in EBV-positive Burkitts lymphoma cell lines is open (36, 37), but recombination activating gene-1 (RAG-1) expression has been described following EBV infection of B cells (38) and at all differentiation stages of precursor lymphoblastic leukemia cells (39). Thus, intrauterine EBV infection could result in genotoxic hits responsible for, for example, the different MLL and the TEL-AML1 gene fusions found in infant leukemia and in common ALL (5, 40).
Noncausal EBV reactivation
Finally, EBV reactivation during pregnancy may be a surrogate for some environmental exposures (e.g., to quinone precursors) that may predispose to childhood leukemia (41). It is possible that once metabolized to quinones, quinone precursors form cleavable complexes with topoisomerase II, which thereafter induces abnormal recombination of the MLL gene in the fetus and coincidentally perturbs normal replication of episomal EBV DNA in the mother (42, 43).
Conclusion
In conclusion, an epidemiologic association between maternal EBV infection and the risk of subsequent development of childhood ALL has been documented. The possibility that this association is limited to certain as-yet unidentified subtypes remains open.
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ACKNOWLEDGMENTS |
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This paper is work number 24 of the Nordic Biological Specimen Banks on Cancer Causes and Control (NBSBCCC).
The authors thank Drs. Göran Gustafsson, Elina Hemminki, and Richard Peto for stimulating discussions. The excellent assistance of Pirjo Kontiokari and Xiaming Huang in performing the laboratory analyses is also gratefully acknowledged.
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NOTES |
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REFERENCES |
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