Prevalence of Antibodies to Four Herpesviruses among Adults with Glioma and Controls

Margaret Wrensch1, Adriana Weinberg2, John Wiencke1, Rei Miike1, Geoffrey Barger3 and Karl Kelsey4

1 Department of Epidemiology and Biostatistics, School of Medicine, University of California, San Francisco, CA.
2 Pediatric Infectious Diseases, Diagnostic Virology Laboratory, University of Colorado Health Sciences Center, Denver, CO.
3 Department of Neurology, Wayne State University, Detroit, MI.
4 Harvard School of Public Health, Boston, MA.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The authors previously reported statistically significant inverse associations between adult onset glioma and histories of chickenpox and shingles among 462 cases and 443 controls in the San Francisco Bay Area Adult Glioma Study (1991–1995) and a suggestive but nonsignificant inverse association with immunoglobulin G antibodies to varicella-zoster virus in a small subset of these cases. This report considers antibodies to four common herpesviruses (varicella zoster, herpes simplex, cytomegalovirus, and Epstein Barr) among 134 cases and 165 controls that represent all subjects for whom usable blood specimens were available. The prevalences of immunoglobulin G antibodies to varicella-zoster virus, herpes simplex virus, cytomegalovirus, and Epstein-Barr virus were 90%, 71%, 57%, and 90%, respectively. After adjustment for age, White versus non-White ethnicity, and gender, glioblastoma cases were less likely than controls to have immunoglobulin G antibodies to varicella-zoster virus (odds ratio = 0.4; 95% confidence interval: 0.1, 0.9). They were also somewhat less likely to have antibodies to Epstein-Barr virus but somewhat more likely to have antibodies to herpes simplex virus and cytomegalovirus. Antibody prevalences to all four herpesviruses were similar between cases with other glioma histologies and controls. These results corroborate our previously suggestive findings of an inverse association of varicella-zoster virus antibodies with adult onset glioma.

antibodies; cytomegalovirus; glioblastoma; herpesvirus 3, human; herpesvirus 4, human; simplexvirus

Abbreviations: CI, confidence interval; OR, odds ratio


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In 1997 (1Go, 2Go), we published findings that adults with glioma were significantly less likely than controls to report a history of chickenpox or shingles. We also presented serologic support for this finding for a subset of cases and controls that showed that cases were less likely than controls to have immunoglobulin G antibodies to varicella-zoster virus, but the findings were not statistically significant. This builds on the previous report and provides results of immunoglobulin G antibodies to four herpesviruses, varicella-zoster virus, herpes simplex virus, Epstein-Barr virus, and cytomegalovirus, among all study cases and controls for whom blood specimens were available. Varicella-zoster virus and herpes simplex virus establish latency in the nervous system, while Epstein-Barr virus and cytomegalovirus may infect the nervous system but without establishing latency. In addition, inclusion of additional samples provided adequate sample size for separate consideration of antibodies to herpesviruses in relation to glioblastoma and other glioma histologies.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Details of subject recruitment and interview have been described elsewhere (1Go). Briefly, 476 eligible adults newly diagnosed with glioma in any of six San Francisco Bay Area counties between August 1, 1991, and April 8, 1994, participated and received pathology review by a single neuropathologist (3Go); 462 age, gender, and ethnicity frequency-matched controls were obtained through random digit dialing. Participation rates were 82 percent for cases and 63 percent for controls (1Go). In-person structured interviews asked information about a variety of potential brain tumor risk factors.

Blood collection was undertaken partway through the study. Up to 30 ml of blood were collected from 187 cases and 171 controls in heparinized green topped tubes. Whole blood was stored at -70°C and transported frozen using dry ice. Because the blood specimens were primarily obtained for polymorphism analyses, some specimens were exhausted prior to these serologic studies. Usable specimens for 134 cases and 165 controls were sent to the laboratory of one of the authors (A. W.) for serologic analyses without identification of case-control status.

Serology was performed using a varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus immunoglobulin G enzyme-linked immunosorbent assay (Gull Laboratories, Inc., Salt Lake City, Utah) and a herpes simplex virus 1/2 immunoglobulin G enzyme-linked immunosorbent assay kit (Incstar Corp., Stillwater, Minnesota) according to the manufacturers' instructions. Briefly, test blood samples and controls were diluted 1:21 for varicella-zoster virus, cytomegalovirus, and Epstein-Barr virus or 1:101 for herpes simplex virus and added to antigen-coated wells in a microtiter plate. Bound antibodies were revealed with alkaline phosphatase- or horseradish peroxidase-conjugated anti-human immunoglobulin G and colorimetric substrate. Absorbances were measured with a spectrophotometer. The sensitivities and specificities of the varicella-zoster virus, Epstein-Barr virus, cytomegalovirus, and herpes simplex virus antibody detection methods were >=93 percent. To validate the immunoglobulin G enzyme immunoassay methods for whole blood, we obtained heparinized and nonanticoagulated blood from 10 healthy volunteers. Paired whole blood and serum samples from these 10 donors were run on all four immunoglobulin G tests in parallel. Antibody determinations in whole blood specimens were identical with the corresponding serum samples, indicating that the tests could be performed on whole blood.

Unadjusted odds ratios comparing cases with controls were computed with the SAS FREQ procedure, and odds ratios adjusting for individual year of age, White or non-White ethnicity, and gender were computed with the SAS LOGISTIC procedure (4Go).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Table 1 shows gender, age, review histologic diagnosis, and ethnicity for the entire study population and the subgroup for whom serologic studies were conducted. The younger average age and lower percentage of glioblastoma among cases for whom serologic studies were conducted are due to the poorer survival among older subjects and among subjects with glioblastoma. Blood was obtained, on average, 7.8 (standard error, 0.4) months (median, 6.7 months) after diagnosis.


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TABLE 1. Comparison of the subjects included in serologic studies with the overall study population of brain tumor cases and controls, San Francisco Bay Area Adult Glioma Study, 1991–1995

 
Table 2 shows the total numbers of subjects positive for immunoglobulin G antibodies to the four herpesviruses overall and stratified by ethnicity, gender, and case-control status and for glioblastoma and other glioma histologies. Although Whites were less likely than non-Whites and although males were less likely than females to have immunoglobulin G antibodies to any of the four herpesviruses, none of these differences reached or approached statistical significance (p > 0.20 for all comparisons). The average ages of subjects positive and negative for immunoglobulin G antibodies to varicella-zoster virus or Epstein-Barr virus were very similar, but subjects who were negative for antibodies to herpes simplex virus or cytomegalovirus were, on average, 11 and 7 years younger, respectively, than those who were positive for these antibodies (p < 0.0001; table 3). Seven subjects (five cases and two controls) who were borderline for positivity for immunoglobulin G to varicella-zoster virus were deleted from the analyses presented here. All analyses were repeated with these subjects included, first as negatives and then as positives, and neither materially altered the conclusions. Moreover, all analyses were conducted for White subjects only, and again the results were not materially altered.


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TABLE 2. Frequencies and percentages of subjects positive for immunoglobulin G antibodies to varicella-zoster virus, herpes simplex virus, cytomegalovirus, and Epstein-Barr virus overall and by gender, ethnicity, case-control status, and histologic type of brain tumor, San Francisco Bay Area Adult Glioma Study, 1991–1995

 

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TABLE 3. Average ages (years) of subjects positive or negative for IgG* antibodies to varicella-zoster virus, herpes simplex virus, cytomegalovirus, and Epstein-Barr virus, San Francisco Bay Area Adult Glioma Study, 1991–1995

 
Overall, without adjusting for matching variables, we found the cases and controls to be about equally likely to have immunoglobulin G antibodies to cytomegalovirus, but cases were somewhat less likely than controls to have antibodies to varicella-zoster virus, herpes simplex virus, or Epstein-Barr virus. Glioblastoma cases were somewhat less likely than controls to have antibodies to varicella-zoster virus (p = 0.04) and Epstein-Barr virus but more likely than controls to have antibodies to herpes simplex virus and cytomegalovirus. Cases with other histologies were as likely as controls to have antibodies to varicella-zoster virus but less likely than controls to have antibodies to herpes simplex virus (p = 0.008), cytomegalovirus, and Epstein-Barr virus. After adjustment for case-control differences in age, ethnicity, and gender, only the comparison of glioblastoma cases with controls for immunoglobulin G to varicella-zoster virus remained statistically significant (odds ratio (OR) = 0.4; 95 percent confidence interval (CI): 0.1, 0.9; p = 0.03). See table 4 for results for other histologies and antibodies to other herpesviruses. Further adjustment of odds ratios for each of the four herpesviruses for the presence or absence of antibodies to the other three herpesviruses did not materially alter the results presented in table 4. In the varicella-zoster virus analyses, inclusion of an indicator variable for the varicella-zoster virus serology assay kit used (a kit from a different manufacturer was used for the previously published varicella-zoster virus serologies) did not alter the results. Similarly, inclusion of an indicator variable for batch (167 subjects analyzed for varicella-zoster virus antibodies in reference 2 or the other 136 subjects) did not alter the results for herpes simplex virus, cytomegalovirus, or Epstein-Barr virus presented in table 4.


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TABLE 4. Odds ratios for presence versus absence of immunoglobulin G antibodies to varicella-zoster virus, herpes simplex virus, cytomegalovirus, and Epstein-Barr virus in cases versus controls, San Francisco Bay Area Adult Glioma Study, 1991–1995

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This report adds results for immunoglobulin G antibodies to varicella-zoster virus among 56 cases and 76 controls to the results previously published for 78 adult glioma cases and 89 controls; this now represents all cases and controls from the San Francisco Bay Area Adult Glioma Study for whom adequate blood specimens were available for serologic analyses. This report further presents data for antibodies to three other common herpesviruses. The results corroborate our original finding that cases were less likely than controls to have immunoglobulin G antibodies to varicella-zoster virus. Moreover, the results show that cases with glioblastoma were significantly less likely than controls to have these antibodies but that cases with other histologies were as likely as controls to have antibodies to this ubiquitous virus. Since varicella-zoster virus infection generally occurs early in life and since we also statistically adjust for any age differences, it seems unlikely that age differences between cases and controls could account for these findings. Furthermore, the observation that, after adjustment for age, ethnicity, and gender, glioblastoma cases were somewhat more likely than controls to have antibodies to herpes simplex virus and cytomegalovirus suggests that a general immunologic depression from the brain tumor or treatments among these cases probably does not explain the lower observed prevalence of antibodies to varicella-zoster virus, a possible concern we expressed in our previous report.

Our previous report showed that cases were less likely than controls to report a history of chickenpox or shingles, diseases caused by varicella-zoster virus (1Go). Since publication of that report, we completed neuropathology review. Using the reviewed cases, we found that the results continue to show that cases (n = 282/396; 71 percent) were less likely than controls (n = 367/433; 85 percent) to give a history of chickenpox or shingles (OR = 0.4; 95 percent CI: 0.3, 0.6); these results are virtually identical to those presented previously (1Go). This interview-based finding was somewhat stronger for proxy-reported than self-reported cases (a finding which seems likely to be due to poorer recall of these diseases by proxies), but it was, nevertheless, statistically significant among self-reported cases versus controls. The finding also was reasonably consistent for cases with glioblastoma and other histologies (adjusting for age, gender, and White vs. non-White ethnicity: OR = 0.4; 95 percent CI: 0.3, 0.6 for glioblastoma cases vs. controls; OR = 0.5; 95 percent CI: 0.4, 0.8 for cases with other histologies vs. controls). Two observations lead us to favor an interpretation that there may be a real association of varicella-zoster virus immunity with brain tumor status. First, the overall case- control odds ratios with a history of chickenpox or shingles and with varicella-zoster virus serology are consistent. Second, the odds ratio for the presence of varicella-zoster virus antibodies among glioblastoma cases versus controls is statistically significant. However, the overall varicella-zoster virus serology odds ratio is compatible with chance. In addition, the interview data indicate an association of history of chickenpox or shingles with nonglioblastoma (as well as glioblastoma) histologies, whereas the serology results indicate an association only with glioblastoma. These issues, combined with the relatively small proportion of subjects from whom we obtained blood and the fact that we used different kits for the varicella-zoster virus serology tests between the previous and current reports, are caveats that limit the certainty of our conclusions.

Since our previous report, a large international study of almost 1,200 glioma cases and 2,500 controls has shown that people with glioma were about 30 percent less likely than controls to have had a history of common infections (colds and flu) and about 70 percent less likely than controls to report a wide variety of allergic conditions (5Go); the authors interpreted these findings to indicate an influence of immunologic factors on the development of glioma. We and others have discussed the previous literature on infections and brain cancer risk (2Go, 5GoGoGo–8Go). Hypotheses have included either that infections decrease the chance of cancer development or kill existing cancer cells; alternatively, infections may be less likely to arise in individuals who are more susceptible to cancer or who are developing cancer. Although it has also been suggested that cases might diminish their prior illnesses after a brain tumor diagnosis, reporting differences between cases and controls could not explain the present serologic findings. Another more speculative explanation is that, if a virus or viruses with some cross-reactivity to varicella-zoster virus influence glioma development, individuals with stronger immunity to varicella-zoster virus might be less susceptible to glioma. Alternatively, nascent tumor cells regardless of etiology might express antigens with cross-reactivity to varicella-zoster virus antigens, and those with immunoglobulin G antibodies to these antigens might be more able to eliminate the nascent tumor cells. Another possibility is that latent varicella-zoster virus infection itself in some individuals might afford some protection against glioblastoma formation; perhaps premalignant or malignant conversion of cells might induce a cytolytic reaction among infected cells.

Clearly, the role of viral and other infections in brain tumor etiology requires further epidemiologic investigation. This is especially important given the extremely poor prognosis of most brain cancers and the paucity of knowledge on which to develop meaningful preventive strategies. Replication of these results in an independent series of cases and controls would provide new avenues of research into the causes of this deadly disease.


    ACKNOWLEDGMENTS
 
This work was supported by grants RO1CA52689 and RO3CA57220 from the National Institutes of Health.


    NOTES
 
Reprint requests to Dr. Margaret Wrensch, Box 1215, University of California, San Francisco, CA 94143-1215 (e-mail: wrensch{at}itsa.ucsf.edu).


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Wrensch M, Lee M, Miike R, et al. Familial and personal medical history of cancer and nervous system conditions among adults with glioma and controls. Am J Epidemiol 1997;145:581–93.[Abstract]
  2. Wrensch M, Weinberg A, Wiencke J, et al. Does prior infection with varicella zoster virus influence risk of adult glioma? Am J Epidemiol 1997;145:594–7.[Abstract]
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  7. Inskip PD, Linet MS, Heineman EF. Etiology of brain tumors in adults. Epidemiol Rev 1995;17:382–414.[ISI][Medline]
  8. Wrensch M, Bondy ML, Wiencke J, et al. Environmental risk factors for primary malignant brain tumors: a review. J Neurooncol 1993;17:47–64.[ISI][Medline]
Received for publication April 5, 2000. Accepted for publication November 29, 2000.