1 Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, MD.
2 Department of Pediatrics, School of Medicine, Johns Hopkins University, Baltimore, MD.
3 Department of Molecular Microbiology and Immunology, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD.
4 Division of Epidemiology, Statistics, and Prevention Research, National Institute of Child Health and Human Development, Rockville, MD.
Received for publication October 20, 2003; accepted for publication March 2, 2004.
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ABSTRACT |
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BK virus; brain neoplasms; child; leukemia; neoplasms; poliovirus; poliovirus vaccines; simian virus 40
Abbreviations: Abbreviations: CI, confidence interval; ORSA, sampling-adjusted odds ratio; SV40, simian virus 40; VLP, virus-like particle.
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INTRODUCTION |
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During the period when poliovirus vaccines were contaminated with live SV40, inactivated poliovirus vaccine was frequently administered to pregnant women in the United States. Conceivably, mothers acquiring SV40 infection as a result of vaccination during pregnancy could have transmitted the virus to their children, either in utero or shortly after birth. In laboratory animals, SV40 acquired during the newborn period is especially tumorigenic (5). Nonetheless, large-scale follow-up studies of children who received SV40-contaminated poliovirus vaccine as young children (even as neonates) have not revealed them to be at increased cancer risk (68).
Heinonen et al. (9) previously examined the possible association between maternal poliovirus vaccination during pregnancy and childhood cancer, using data on children under age 4 years from the Collaborative Perinatal Project (described in more detail below). These investigators found a strong relation between maternal poliovirus vaccination and risk of neural tumors (relative risk = 12; p < 0.01). They also noted a nonsignificant excess risk of leukemia in children whose mothers had received poliovirus vaccine during pregnancy (relative risk = 1.8; p value not reported). However, these results were based on only eight children with neural tumors and eight children with leukemia. Furthermore, Heinonen et al. considered all exposures to inactivated poliovirus vaccine during pregnancy as the relevant risk factor for childhood cancer, although only pre-1963 inactivated poliovirus vaccine contained SV40 (1). Subsequently, Rosa et al. (10) found SV40 antibodies in only 9 percent of the mothers of the cancer cases studied by Heinonen et al. Thus, while Heinonen et al.s study is the only cohort study to have found a relation between exposure to early poliovirus vaccine and cancer, the small number of cancer cases and the lack of serologic evidence of SV40 infection in the mothers complicate interpretation of its findings.
In the present study, we extended the prior work by Heinonen et al. and Rosa et al. We examined cancer incidence among children in the Collaborative Perinatal Project up to their eighth birthday and included 52 children with cancer, more than twice as many as were evaluated previously (9). Additionally, we separately considered maternal exposures to pre-1963 poliovirus vaccine (both inactivated poliovirus vaccine and oral poliovirus vaccine, both of which were contaminated with SV40) and poliovirus vaccine manufactured in or after 1963 (which was free of SV40). Finally, we measured SV40 serostatus in a case-control study within the Collaborative Perinatal Project, to examine whether SV40 infection could reasonably account for associations between maternal receipt of poliovirus vaccine and subsequent cancer in children.
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MATERIALS AND METHODS |
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At study visits made during pregnancy, information on receipt of poliovirus vaccine was obtained from interview of mothers and a review of clinic records. Poliovirus vaccine type was classified as inactivated, oral, or unspecified. Information on the date of the last menstrual period and the lunar month in which vaccination occurred was used to date vaccine exposures (classified for analysis as pre-1963 vs. 1963+).
Malignancies arising in Project children up to their eighth birthday had previously been identified in several ways (13). First, study identification numbers of children with cancer were obtained through a previous study of childhood cancer in this cohort (14). Second, potential cancer cases were identified in a review of records of all children without life-threatening anomalies who weighed at least 1,500 g at birth and who died after the first week of life. Third, Project diagnostic summary forms, completed at 1 and 7 years of age, were reviewed. Cases were included only if cancer was confirmed by a medical record summary that provided a histologic diagnosis, a clinical course (including treatment) consistent with a cancer diagnosis, or both.
Statistical methods for cohort analysis
We calculated the incidence (number of events per 100,000 person-years) of all cancers together and, separately, the incidence of neural tumors, hematologic malignancies, and miscellaneous tumors (see table 1 for classification). Children were considered to be under follow-up through their last examination (median duration of follow-up, 7.3 years; interquartile range, 6.08.0 years). We calculated incidence separately for children with maternal receipt during pregnancy of pre-1963 poliovirus vaccine (possibly contaminated with SV40), children with maternal receipt during pregnancy of only 1963+ poliovirus vaccine (SV40-free), and children with no maternal receipt of poliovirus vaccine during pregnancy. For each cancer outcome, we plotted Kaplan-Meier curves for these three strata. Proportional hazards regression was used to derive vaccine-associated hazard ratios for these cancer outcomes and to adjust for possible confounding by demographic characteristics. These analyses used a method of Wei et al. (15) that accounted for correlated outcomes in children with the same mother.
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The primary measure of childrens SV40 exposure was maternal SV40 seroconversion during pregnancy, which could be measured for all children with paired maternal serum samples. Nonetheless, we considered that poliovirus vaccination was the most likely route by which mothers could acquire SV40 during pregnancy, so we additionally classified their paired serum samples as "informative" or "uninformative" according to the likelihood of observing SV40 seroconversion related to vaccinationthat is, mothers paired serum samples were considered informative if they bracketed vaccination in an appropriate manner. Specifically, we allowed for a lag of 6 weeks for the development of SV40 antibodies following SV40 infection. Thus, paired serum samples were considered informative for SV40 seroconversion if at least one dose of poliovirus vaccine was administered during the window from 6 weeks prior to the first serum sample to 6 weeks prior to the second serum sample.
By design, the 200 controls were randomly sampled in varying proportions from strata defined by maternal vaccination status and informativeness (table 2). This two-stage sampling strategy allowed us to increase statistical efficiency (i.e., we maximized the expected number of seroconverters by oversampling informative mothers and those who received pre-1963 inactivated poliovirus vaccine) and to have sufficient numbers of control mothers with various vaccine exposures to examine the relation between vaccination and SV40 seroconversion (16). Because of this sampling strategy, crude seroconversion rates and corresponding odds ratios do not reflect the underlying parameters. Therefore, as described below, we incorporated information on the sampling fractions from control strata into the analyses to obtain valid comparisons of seroconversion rates across groups.
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Sera were tested for SV40 antibodies using a previously described virus-like particle (VLP) enzyme immunoassay (17). Briefly, SV40 VLPs are empty capsids, generated by spontaneous self-assembly of the major capsid protein VP1, that retain many immunologic properties of native virions. In rhesus macaques, the SV40 VLP assay previously exhibited 100 percent sensitivity and 100 percent specificity with respect to a plaque neutralization assay (plaque assay) (17). For the present study, we included control serum samples from macaques that were seropositive (n = 29) or seronegative (n = 10) on the plaque assay. In humans, some reactivity measured by the SV40 VLP assay may represent cross-reactivity to the human polyomavirus BK (1719). Therefore, we also tested all specimens for antibodies to BK virus using an analogous BK virus VLP enzyme immunoassay (17). All specimens in both VLP assays were measured in duplicate, and we used the geometric mean of the duplicates in the analyses. SV40 VLP seropositivity was determined using a cutoff absorbance of 0.10 optical density units, obtained from an inspection of the histogram of absorbance results and similar to that used previously (19).
Additionally, we performed a plaque assay for SV40 neutralizing antibodies (20). Sera initially positive (i.e., 80 percent reduction of plaque counts as compared with control wells) at a dilution of 1:10 were titrated further at 1:40 and 1:160 dilutions. Titers were missing for 18 specimens because of fungal contamination of the assay plates. With each batch of approximately 100 serum samples, we included diluted serum from an SV40-infected macaque. This positive control sample inhibited plaque formation consistently at 1:500 and 1:5,000 dilutions and variably at a 1:50,000 dilution.
Statistical methods for case-control analysis
Using paired SV40 VLP results, we classified mothers as seroconverters (negative on the first specimen, positive on the second specimen), seroprevalent (positive, positive), seronegative (negative, negative), or seroreverters (positive, negative). We defined plaque assay seroconversion similarly, considering a titer of 1:40 a positive result. Women whose seroconversion status could not be classified because of missing titers were excluded from analysis. As alternative definitions for seroconversion, we considered other cutoff points (0.08 optical density units for the VLP assay; titers of 1:10 and 1:160 for the plaque assay) and a fourfold increase in titer (plaque assay), but these did not produce qualitatively different results (not shown).
We hypothesized that SV40 seroconversion during pregnancy (i.e., new SV40 infection) would be associated with the highest cancer risk in children, although we considered that seroprevalent maternal status might also convey risk. To address this question, we fitted a logistic regression model to assess whether maternal SV40 seroconversion status (seroconverter vs. seroprevalent vs. other; independent variable) was related to risk of childhood cancer (case/control status; dependent variable). We used software developed by Breslow and Chatterjee (21) to estimate parameters and their variances with a pseudolikelihood method that adjusted for the sampling scheme described above (22).
We also fitted a logistic regression model to assess whether, among control mothers, SV40 seroconversion (dependent variable) was related to vaccination status (receipt of pre-1963 inactivated poliovirus vaccine vs. other poliovirus vaccine/no vaccine; independent variable). We used as weights the sampling fractions from strata defined by vaccination and informativeness. Asymptotic variance estimates were obtained (23).
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RESULTS |
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Overall, 21,649 children (39.5 percent) had mothers who received poliovirus vaccine during pregnancy: 12,334 children (22.5 percent) had mothers who received pre-1963 poliovirus vaccine and 9,315 (17.0 percent) had mothers who received only 1963+ poliovirus vaccine. Maternal receipt of inactivated poliovirus vaccine during pregnancy declined over time (i.e., 3851 percent for children born in 19591962 vs. 2533 percent for children born in 19631966). Maternal receipt of oral poliovirus vaccine during pregnancy was rare (<4 percent) for children born before 1963 and peaked at 19 percent for those born in 1964. Additionally, few mothers (<1 percent) received poliovirus vaccine of unspecified type. Among mothers who received pre-1963 poliovirus vaccine during pregnancy, the mean number of doses was 1.3.
White children were more likely to have mothers who received poliovirus vaccine during pregnancy than were non-White children (48.6 percent vs. 31.8 percent; p < 0.0001). Males were slightly less likely to have vaccinated mothers than were females (39.1 percent vs. 39.9 percent; p = 0.05). In addition, vaccinated mothers were older than unvaccinated mothers (mean age = 25.5 years vs. 24.7 years; p < 0.0001).
Association between maternal receipt of poliovirus vaccine and malignancies in children
Fifty-two malignancies were identified during follow-up (incidence = 15 per 100,000 person-years). These included 18 neural tumors, 22 hematologic malignancies, and 12 miscellaneous tumors (table 1). Overall, we included 16 cancers diagnosed at ages 03 years that were not previously identified by Heinonen et al. (9). In addition, we excluded six cases included in Heinonen et al.s report: two microscopic neuroblastomas diagnosed only at autopsy (readily confused with a normally developing adrenal gland) (24), one ovotestis mistakenly classified as a tumor, and three Wilms tumors that we could not identify (all three were reportedly found in children whose mothers had not received poliovirus vaccine during pregnancy). One case of leukemia was reclassified as a case of non-Hodgkins lymphoma.
Children whose mothers had received pre-1963 poliovirus vaccine during pregnancy had the highest overall risk of cancer, while children whose mothers had received only 1963+ poliovirus vaccine or no vaccine had similar risks (figure 1, panel A). This difference in risk between children whose mothers had received pre-1963 vaccine and those whose mothers had not was significant (hazard ratio = 2.3, p = 0.004; table 3). Similar patterns were observed specifically for neural tumors and hematologic malignancies (figure 1, panels B and C), with significantly elevated risks being present for children whose mothers had received pre-1963 poliovirus vaccine (hazard ratio = 2.6 (p = 0.04) and hazard ratio = 2.8 (p = 0.02), respectively; table 3). Among neural tumors, there were sufficient numbers of cases only of neuroblastoma to examine separately the association with maternal receipt of pre-1963 vaccine (hazard ratio = 8.2, 95 percent confidence interval (CI): 1.6, 43; p = 0.01). The risk of miscellaneous tumors did not differ across groups (figure 1, panel D; table 3). These relations did not change after adjustment for sex, race, and maternal age (table 3).
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Among case and control mothers who received poliovirus vaccine during pregnancy, the proportions with informative paired serum samples (i.e., serum samples that bracketed poliovirus vaccination) were similar (81 percent vs. 87 percent). In addition, the durations of windows defined by paired serum samples were similar in case and control mothers (mean of 24.9 weeks vs. 22.0 weeks; table 2).
SV40 antibody results
SV40 VLP assay results for 500 serum samples (i.e., paired specimens from 250 case and control mothers) are shown in figure 2, panel A. SV40 VLP results were bimodally distributed, with a trough at an absorbance of 0.10. Using this value as a cutoff, 82 serum samples (16 percent) were considered SV40 VLP-seropositive. Using the same cutoff, the SV40 VLP assay identified 28 of 29 SV40 plaque assay-seropositive monkeys (97 percent) as VLP-seropositive, while 10 of 10 plaque assay-seronegative monkeys (100 percent) were VLP-seronegative.
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SV40 seroconversion in mothers of cases and controls
Using the SV40 VLP assay (table 4), SV40 seroconversions were seen in four case mothers (8 percent) and six control mothers (3 percent). Accordingly, case mothers were more likely to seroconvert during pregnancy than control mothers (sampling-adjusted odds ratio (ORSA) = 4.0, 95 percent CI: 1.0, 15.7). Case mothers and control mothers were equally likely to be SV40-seroprevalent at the start of pregnancy (12 percent vs. 13 percent; ORSA = 0.8, 95 percent CI: 0.3, 2.1).
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BK virus antibody results
BK virus VLP reactivity was generally much stronger than SV40 VLP reactivity, and results were less clearly bimodal (figure 2, panel B). BK virus results were moderately correlated with SV40 VLP results (Spearman correlation = 0.40; p < 0.0001) and less strongly correlated with SV40 plaque assay titers (Spearman correlation = 0.20; p < 0.0001).
When we used the same cutoff value as that used for the SV40 VLP assay (absorbance = 0.10), 377 samples (75 percent) were seropositive for BK virus. Most mothers (80 percent of cases, 70 percent of controls) were BK virus-seroprevalent at the start of pregnancy. BK virus seroconversions were observed in two case mothers (4 percent) and seven control mothers (4 percent).
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DISCUSSION |
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In our cohort analysis, the observed association between maternal poliovirus vaccination and neural tumors (hazard ratio = 2.6) was markedly attenuated compared with that reported previously by Heinonen et al. (relative risk = 12) (9). One reason for this difference was that the previous investigators evaluated risk related to all inactivated poliovirus vaccines, regardless of year of receipt, whereas we considered pre-1963 vaccine to be relevant to an analysis of SV40-related cancer risk. Specifically, for children under 4 years of age, they classified one child with astrocytoma as having maternal vaccine exposure, although the mother received inactivated poliovirus vaccine only in 1963. Substantial attenuation also resulted from our inclusion of six additional neural tumors among children aged 03 years (table 1). We do not know why Heinonen et al. were unaware of these cases, but all were children who lacked maternal exposure to pre-1963 poliovirus vaccine. Additionally, among 631 children whose mothers received pre-1963 oral poliovirus vaccine, classified by us but not by Heinonen et al. as having relevant vaccine exposure, none developed a neural tumor. Indeed, we found only a modest association between maternal receipt of pre-1963 poliovirus vaccine and risk of neural tumors in children aged 03 years (hazard ratio = 1.7). Of further note, among children aged 07 years, the association between maternal receipt of pre-1963 vaccine and risk of neural tumors was especially strong for neuroblastoma (hazard ratio = 8.2), even though SV40 has never been detected in neuroblastoma (3, 25). Overall, the validity of our methods for ascertaining cancer outcomes was supported by the close correspondence of the observed number of cases with expected rates (13).
In our case-control study, we found no consistent relation between maternal SV40 seroconversion during pregnancy and cancer in children. With the VLP assay, maternal seroconversion was associated with an increased overall cancer risk in children (ORSA = 4.0), but this association was not observed with the plaque assay (ORSA = 0.9). Importantly, the few SV40 seroconversions that we observed were in mothers of children with diverse malignancies, with no apparent pattern (table 5). Seroconversion in both assays was seen for only one woman whose child developed neuroblastoma. SV40 DNA has been reported by some laboratories in cases of ependymoma, astrocytoma/glioma, Wilms tumor, and non-Hodgkins lymphoma (3, 2628). Additionally, Farwell et al. previously reported an association between maternal poliovirus vaccination during pregnancy and childhood medulloblastoma (29). Even though our study included children with each of these malignancies, maternal seroconversion by either SV40 assay was documented only for one astrocytoma.
Perhaps surprisingly, we did not find an unambiguous association between seroconversion and receipt of pre-1963 inactivated poliovirus vaccine. With the VLP assay but not with the plaque assay, SV40 seroconversions were observed more frequently in mothers who received pre-1963 inactivated poliovirus vaccine than among other mothers. Still, SV40 seroconversions by either assay were distinctly uncommon in women who received pre-1963 inactivated poliovirus vaccine. SV40 neutralizing antibodies can be detected within 25 weeks of human or macaque infection (3032), so our use of a 6-week lag in defining informativeness should have enabled us to detect seroconversion following receipt of pre-1963 inactivated poliovirus vaccine. Furthermore, few women were SV40-seroprevalent at the start of pregnancy (table 4), though many would have received pre-1963 inactivated poliovirus vaccine earlier in life. The proportion of persons who seroconverted to SV40 following one or more doses of pre-1963 inactivated poliovirus vaccine varied widely (892 percent) in previous studies (10, 3335). These prior data and our results indicate that SV40 contamination of the inactivated poliovirus vaccine used in the United States was somewhat uneven, with varying frequencies or levels of contamination across vaccine lots (1).
The generally low amount of SV40 antibody (measured in terms of VLP absorbance value or plaque assay titer) in seropositive mothers further militates against SV40s being a cause of childhood cancer. Following initial SV40 infection in macaques, replication of SV40 leads to a robust antibody response (31, 32). Therefore, our results point to an absence or near absence of productive SV40 infections among mothers in the Collaborative Perinatal Project. If vaccine-related exposures in mothers did not lead to productive infection, transmission of SV40 to children, either in utero or neonatally, would have been unlikely.
The lack of a more frequent or robust immune response against SV40 in our subjects was not likely to have been due to poor assay sensitivity. Although the sensitivity of these assays for human SV40 infection is uncertain, both SV40 assays reliably detect infection in rhesus macaques, the natural host (17, 36), and the plaque assay detected SV40 seroconversion following vaccination in prior studies (3335). Nonetheless, among mothers in our study, the results of the SV40 VLP assay and the results of the plaque assay were not highly correlated. In comparison, BK VLP reactivity was much stronger (figure 2), and results from each SV40 antibody assay were somewhat correlated with BK virus results. Our findings and those of previous investigations (1719, 37) suggest that some low-level antibody responses to SV40 seen in humans are nonspecific and are due to cross-reactive antibodies, perhaps to BK virus. BK virus seroprevalence in Project mothers was similar to that seen in other pregnant women (38), arguing against deterioration of stored serum samples.
An important limitation of our study was the composite nature of the cancer outcomes that we evaluated. For instance, although we found an association between maternal exposure to pre-1963 inactivated poliovirus vaccine and neural tumors, the most common type of neural tumor was neuroblastoma, which has not been linked to SV40 (3). Leukemia was the most frequent hematologic malignancy, yet SV40 DNA sequences have not been detected in leukemia specimens obtained from children (39). Given the somewhat small number of children with cancer, we were precluded from reaching firm conclusions regarding particular tumor types. Additionally, it would have been interesting to examine serum samples or tumor specimens obtained from children at the time that they developed their malignancies, but these specimens were unavailable.
If vertically transmitted SV40 infection does not explain the excess cancer risk in children whose mothers received pre-1963 inactivated poliovirus vaccine during pregnancy, what accounts for this observed association? Transmission of poliovirus itself is unlikely to explain our findings, since poliovirus vaccine used in 1963+ was not linked to an increased risk of cancer (figure 1), even though a substantial proportion of this vaccine was oral poliovirus vaccine (which, unlike inactivated poliovirus vaccine, is a live vaccine). In addition, poliomyelitis survivors are not at increased risk of neural or hematologic malignancies (40). Conceivably, confounding could explain the association between maternal poliovirus vaccination and childhood cancer. For example, changes in cancer incidence over calendar time unrelated to poliovirus vaccination may play a role. Indeed, overall cancer incidence was higher in children born before 1963 than in those born subsequently (20 per 100,000 person-years vs. 11 per 100,000 person-years), and the hazard ratios reported in table 3 were somewhat attenuated after adjustment for calendar year of birth (data not shown). Alternatively, the associations with maternal vaccination might represent confounding by socioeconomic statusthat is, early poliovirus vaccine may have been most widely available to women with relatively high socioeconomic status, who were then more likely to return for follow-up visits when childhood cancers were recorded. Finally, the associations between maternal vaccination and childhood cancer could have been due to chance, although the confidence intervals and related p values argue against this possibility (table 3).
In summary, our results do not support a link between SV40 infection during pregnancy and cancer risk in subsequently born children. Our study expands on prior work by Heinonen et al. (9) and Rosa et al. (10), including additional children with cancer and incorporating state-of-the-art serologic assays. Nonetheless, our conclusion regarding maternal vaccination and childhood cancer in the Collaborative Perinatal Project cohort remains the same as that reached by Rosa et al.: "there appears to be an association between the administration of killed-poliovirus vaccine to mothers and neurologic [and hematologic] tumors in their offspring that is not due to SV40; its basis remains to be clarified" (10, p. 1469). Additional serology-based case-control studies may be useful in elucidating the potential role of SV40 in cancer.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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