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Antibodies against six human herpesviruses in relation to seven cancers in black South Africans: A case control study

Antibodies against six human herpesviruses in relation to seven cancers in black South Africans:... Background: Infections with certain human herpesviruses have been established as risk factors for some cancer types. For example, Epstein-Barr Virus is considered a cause of Burkitt's lymphoma and other immunosuppression related lymphomas, Hodgkin lymphoma, and nasopharyngeal cancer. Several other human herpesviruses have been linked to cancers but the totality of evidence is inconclusive. Methods: We conducted a systematic sub-study from within an ongoing case control study of adult black South Africans to investigate the relationship between antibodies to six human herpesviruses and seven cancer groups that may be caused by infectious agents. Subjects had incident cancers of the oral cavity(n = 88), the cervix(n = 53), the prostate(n = 66), Hodgkin lymphoma(n = 83), non-Hodgkin lymphoma(n = 80), multiple myeloma(n = 94) or leukaemia(n = 203). For comparison, patients with other cancers(n = 95) or cardiovascular disease(n = 101) were randomly selected from within the study. Patients were interviewed and their blood was tested for IgG antibodies against HSV-1, HSV-2, VZV, EBV- EBNA, CMV and HHV-6 using enzyme linked immunosorbent assays. Because these viruses are highly prevalent in this population, optical density results from the assays were used as an indirect, quantitative measure of antibody level. Results: There was significant variation in the mean log antibody measures for HSV-2, VZV, CMV and HHV-6 between the disease groups. However, none of the specific cancer groups had significantly higher mean log antibody measures for any of the viruses compared to either control group. In a more detailed examination of seven associations between cancers and herpesviruses for which there had been prior reports, two statistically significant associations were found: a decreasing risk of myeloid leukaemia and an increasing risk of oral cancer with increasing tertiles of antibodies against HHV-6 compared to all other patients (p-trend = 0.03 and 0.02, respectively). Odds ratios for the top tertile compared to the bottom tertile were 0.58 (95%CI 0.3 – 1.0) for myeloid leukaemia and 2.21 (95% CI 1.1 – 4.3) for oral cancer. Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 Conclusion: In this population, using these tests for IgG, neither mean antibody measure nor high antibody measure against human herpesviruses 1–6 was strongly associated with any of the seven cancer groups. However, we may not have had sufficient power to detect weak associations or associations with a sub-type of cancer if they were present. diagnosed cancer at tertiary government hospitals in Background Infection with certain types of human herpesviruses has Johannesburg (Chris Hani-Baragwanath, Hillbrow, and been established as a cause of several cancers. These Johannesburg General Hospitals). A standard question- include Epstein-Barr Virus (EBV) for Burkitt's lymphoma naire, administered in the language of the patient (usually and other immunosuppression related lymphomas, an Nguni or Sotho group language), was used. Questions Hodgkin lymphoma, and nasopharyngeal cancer [1]; and were asked about socio-demographic factors and behav- human herpesvirus 8 (HHV-8) for Kaposi's sarcoma [2]. ioural characteristics including age, sex, birthplace, resi- These cancers are rare responses to the presence of these dence, level of education, tobacco and alcohol use, and widespread viruses. reproductive and lifetime sexual history. Several human herpesviruses have been linked to other Blood was collected from 84% of patients at the time of cancers although the totality of evidence is inconclusive. interview and prior to commencing treatment. The For example herpes simplex virus type 1 (HSV-1) has been remainder were too ill, had collapsed veins, or refused associated with oral cancer[3] and herpes simplex virus consent. All interviewed patients with oral cancer (n = type 2 (HSV-2) with cervical cancer in women who are co- 88), Hodgkin lymphoma (n = 83), non-Hodgkin lym- infected with specific human papillomavirus types[4]. phoma (n = 80), multiple myeloma (n = 94) and leukae- Human herpesvirus type 6 (HHV-6) has been linked to mia (n = 203) and a random sample of subjects with Hodgkin lymphoma [5], acute myeloid leukaemia [6] and cervical (n = 53) and prostate cancer (n = 66) were oral cancer [7]. In addition it has been suggested that pros- included in the study if they had provided a blood sam- tate cancer [8] and multiple myeloma [9] may have infec- ple. Controls were from two groups of patients attending tious causes. the same hospitals: a group of patients with other cancers (n = 95) and a group with cardiovascular diseases (n = Our group previously found that high antibody levels to 101). The controls were selected randomly within sex and HHV8 are highly correlated to the diagnosis of Kaposi's age bands, and frequency matched to the cancer cases as a sarcoma [2]. We therefore designed a study to examine, in whole according to five-year age-groups and sex. Diag- a systematic way, antibody levels to six of the herpesvi- noses of cancer were established, where appropriate, by ruses (HSV-1, HSV-2, Varicella Zoster (VZV), EBV, histology, haematology, or cytology. The study was cytomegalovirus (CMV) and HHV-6) in relation to seven approved by the Committee for Research on Human Sub- cancer groups for which there is some evidence of an jects (Medical) of the University of the Witwatersrand, infectious cause (oral, cervical, prostate, Hodgkin lym- and informed consent was obtained from all patients. phoma, non-Hodgkin lymphoma, multiple myeloma and leukaemia). The study was part of a larger case-control Laboratory methods study of the causes of cancer in black South Africans, After coagulation, specimens were centrifuged to obtain which was conducted in public hospitals that treat cancer serum. The serum specimens were aliquotted and stored in greater Johannesburg, South Africa [2,10]. Since most at -20° to -30°C. HIV testing was carried out by the Serol- human herpesviruses are highly prevalent, and PCR on ogy Laboratory of the South African Institute for Medical biopsy samples is unrealistic in this setting, we used quan- Research (now the National Health Laboratory Service), titative measures of anti-human herpesvirus antibodies using commercial ELISA kits. Early testing was for HIV-1 from enzyme linked immunosorbent assays (ELISAs). In only; later testing for both HIV-1 and HIV-2. Patients with addition we examined the relationships between demo- borderline results were considered to be HIV negative. graphic and lifestyle factors and antibody levels against these viruses, as little is known about these viruses in this Herpes virus antibody testing was done at the South Afri- population. can National Institute for Virology (now the National Institute for Communicable Diseases) from January to April 2000. Throughout the testing period specimens were Methods Study Participants kept at 4°C. The commercially available kits used were: The study population has been described previously Eurogenetics for HSV-1, HSV-2 and CMV; Clark Laborato- [2,10]. Briefly, between March 1995 and February 1999 ries for VZV and EBV nuclear antigen-1(EBNA); and Pan- trained nurses interviewed adult black patients with newly Bio for HHV-6. These were selected following Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 apreliminary study which demonstrated good quantita- Since little is known about risk factors for infection with tive performance with South African sera. The test for human herpesviruses in this population, we examined the EBNA IgG used "recombinant EBNA-1 antigen". All other relationship between antibody measure and a number of kits used unspecified antigens. socio-demographic variables using analysis of variance with adjustment for each of these variables as well as can- Patients' sera were randomly allocated to the 96-well cer group, day of assay, and assay plate. Due to the microplates to minimise potential bias due to systematic number of comparisons that were made these analyses differences between the plates or day on which the plates were examined at the 0.01 significance level. were run. Each specimen was run in a single well. There were a total of 12 plates for each assay and in general two All analyses were carried out using SAS statistical software plates were run each day. Repeatability of each assay was [11]. investigated through replicate measures on a pool of sera samples with high antibody measures against each virus. Results Test results were initially in optical density units which are In total 667 patients with the seven specific cancers of proportional to the concentration of antibody i.e. the interest were included in the study. A further 95 patients antibody titre. The CMV kit had a calibration curve giving with other types of cancer and 101 patients with cardio- final results in antibody units/ml. The remaining kits con- vascular diseases were included as control subjects. tained standards used to determine a cut-off for positivity. Demographic characteristics of the study members are Results are reported as ratios to the manufacturer's cut-off shown in Table 1. value. Throughout this paper we use the general term "antibody measure" to refer to the manufacturer's result, The results from the analysis of the control samples sug- regardless of whether it is in AU/ml or "times cut-off". gested that for all assays (except for HSV-1 and HSV-2) the most important source of systematic variation was the day Statistical Analysis for the control samples on which the plates were assayed, whereas for the HSV-1 There were several potential sources of variability in each and HSV-2 assays it was the plates themselves (data not assay such as antigen binding within or between plates, shown). As the plate and day on which each patient's sam- washing conditions between binding steps and laboratory ple was tested had been recorded, variation between days conditions such as the temperature, humidity, and light. and between plates was controlled for in the main analy- For each of the herpesviruses the logarithm (log) antibody ses by adjusting for these factors. measure was computed for the control pool and analysis of variance was used to estimate the magnitude of the Crude adult prevalence rates in the study, determined components of variance for each of the sources of variabil- using the manufacturer's criteria for positivity, were: 99% ity. for HSV-1; 59% for HSV-2 (52% in males and 68% in females); 97% for VZV; 96% for EBV (EBNA antigen); Statistical Analysis of the Patient Data 99% for CMV; and 90% for HHV-6 (88% in males and To examine the relation between antibodies against the 93% in females). human herpesviruses 1–6 and cancer the mean log anti- body measures for each of the nine disease groups were Associations with cancer types There was statistically significant variation between the compared using analysis of variance. Least square mean log antibody measures were calculated for each disease nine disease groups in mean log antibody measure for group with adjustment for age, sex, HIV status, and the HSV-2 (p < 0.0001), VZV (p < 0.0001), CMV (p = 0.009) day and the plate on which the assay was run. Paired com- and HHV-6 (p < 0.0001) (Figure 1). Patients with multi- parisons were conducted between each cancer group and ple myeloma had lower mean log antibody measures for each of the two control groups (other cancers and cardio- almost all these herpesviruses compared to other patients. vascular disease patients) with Bonferroni adjustments for When patients with multiple myeloma were excluded, sig- multiple comparisons. Where positive associations had nificant heterogeneity in mean log antibody measures previously been found (see Introduction [1-7]), the spe- between the eight remaining disease groups was still cific hypotheses were investigated in more detail by com- present for HSV-2 (p = 0.0007) and HHV-6 (p = 0.004). paring the specified cancer group to all the other patients combined with respect to tertiles of log antibody measure. In paired comparisons (with Bonferroni adjustments), Odds ratios were calculated using logistic regression with patients with multiple myeloma had significantly lower adjustment for age, sex, HIV status and the day and the mean log IgG antibody measures for the response to VZV, plate on which the assay was run. CMV and HHV-6 compared to patients in the 'other can- cer' group (p < 0.0001, 0.02 and p < 0.0001 respectively) and for HSV-2, VZV and HHV-6 compared to patients Page 3 of 9 (page number not for citation purposes) Table 1: Demographic characteristics of patients by disease group Cancer group Lip, oral cavity and pharynx Cervix Prostate Hodgkin lymphoma non-Hodgkin lymphoma Multiple myeloma Leukaemia Other cancers Cardiovascular disease n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%) Males 64 (73) 0 (0) 66 (100) 46 (55) 45 (56) 52 (55) 92 (45) 45 (47) 45 (45) Females 24 (27) 53 (100) 0 (0) 37 (45) 35 (44) 42 (45) 111 (55) 50 (53) 56 (55) Age <35 yrs 6 (7) 15 (28) 0 (0) 33 (40) 22 (28) 5 (5) 57 (28) 23 (24) 17 (17) Age 35–49 yrs 17 (19) 12 (23) 6 (9) 35 (43) 25 (31) 23 (24) 54 (27) 26 (27) 29 (29) Age 50+ yrs 65 (74) 26 (49) 60 (91) 15 (18) 33 (41) 66 (70) 92 (45) 46 (48) 55 (55) HIV +ve 3 (3) 5 (9) 1 (2) 9 (11) 18 (23) 4 (4) 8 (4) 6 (6) 11 (11) Total 88 (100) 53 (100) 66 (100) 83 (100) 80 (100) 94 (100) 203 (100) 95 (100) 101 (100) Includes cancers of the digestive organs, respiratory organs, bone, skin, soft tissue, breast, female and male genital organs, bladder, brain, thyroid and of unspecified sites. Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 Distribution Figure 1 of mean log antibody measures (and 95% CI) by disease group for herpesviruses 1–6 Distribution of mean log antibody measures (and 95% CI) by disease group for herpesviruses 1–6. with cardiovascular diseases (p = 0.01, 0.0003 and analyses because in general mean antibody levels did not <0.0001 respectively). Because of their systematically vary significantly across disease groups after exclusion of lower antibody measures, multiple myeloma patients subjects with multiple myeloma (Figure 1), and including were excluded from further analyses. Patients with non- more patients increased the power of these analyses. Only Hodgkin lymphoma had significantly lower mean log oral cancer showed a statistically significant trend of risk antibody measures in response to HSV-2 compared to the with increasing HHV-6 tertiles (p = 0.02), odds ratio for non-cancer patients (p = 0.004). None of the specific can- the top tertile compared to all other subjects = 2.21 (95% cer groups had significantly higher mean log antibody CI 1.1 – 4.3) (Table 2). All the oral cancer patients were measures for any of the viruses compared to either control positive for HHV-6 antibodies by the manufacturer's cut- group. off. Only two subjects had undifferentiated nasopharyn- geal cancer so it was not possible to investigate the rela- To further investigate the positive associations reported in tionship between this sub-type of oral cancers and EBV. previous studies (EBV and Hodgkin lymphoma; EBV and Neither was it possible to separate acute myeloid leukae- non-Hodgkin lymphoma; HSV-1 and oral cancer; HSV-2 mias from chronic myeloid leukaemias with the informa- and cervical cancer; HHV-6 with oral cancer, Hodgkin tion that was available, but there was some evidence of a lymphoma and acute myeloid leukaemia) the log anti- decreasing trend in risk of myeloid leukaemia with body measures were divided into tertiles according to the increasing tertiles of HHV-6 (p = 0.03); odds ratio for the levels in allpatientsexcluding those with multiple mye- top tertile compared to all other subjects = 0.58 (95% CI loma. The control group was defined in this way for these 0.3 – 1.0). Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 1 2 Table 2: Odds ratios (OR) and 95% confidence intervals (CI) for specific cancers versus all other subjects (controls) by tertile of virus antibody measure in the controls Virus and cancer group Control tertiles Cases/Controls OR 95% CI P-trend HSV-1 and Oral cancer 0–5.04 23/200 1.00 5.05–6.07 26/199 1.06 (0.6 – 2.0) 6.08+ 31/199 1.28 (0.7 – 2.3) 0.40 HSV-2 and Cervical cancer 0–1.08 11/91 1.00 1.09–2.17 11/91 0.90 (0.4 – 2.2) 2.18+ 23/90 1.82 (0.8 – 4.1) 0.10 EBV-EBNA and non-Hodgkin lymphoma 0–3.81 24/208 1.00 3.82–5.29 31/203 1.32 (0.7 – 2.4) 5.30+ 22/211 0.87 (0.4 – 1.8) 0.80 EBV-EBNA and Hodgkin lymphoma 0–3.87 33/211 1.00 3.88–5.31 18/208 0.49 (0.3 – 0.96) 5.32+ 21/208 0.66 (0.3 – 1.4) 0.19 HHV-6 and Oral cancer 0–1.59 16/195 1.00 1.60–2.43 28/194 1.83 (0.9 – 3.6) 2.44+ 31/195 2.21 (1.1 – 4.3) 0.02 HHV-6 and Hodgkin lymphoma 0–1.58 21/172 1.00 (excluding oral cancers) 1.59–2.40 17/174 0.87 (0.4 – 1.7) 2.41+ 28/172 1.32 (0.7 – 2.5) 0.39 HHV-6 and Myeloid Leukaemia 0–1.61 47/131 1.00 (excluding oral cancers) 1.62–2.59 52/134 1.16 (0.7 – 1.9) 2.60+ 26/131 0.58 (0.3 – 1.0) 0.03 For associations that had been reported previously. The control group in these analyses included all other subjects, except patients with multiple myeloma, because in general mean antibody levels did not vary significantly across disease groups after exclusion of subjects with multiple myeloma (Figure 1), and including all patients increased the power of these analyses. All analyses are adjusted for age group, sex, day of assay and assay plate. Association with age, sex and socio-demographic factors ical onset [12]. Antibodies to HHV-8 are also found many Possible determinants of high antibody measures to these months prior to the diagnosis or Kaposi's sarcoma [13] herpesviruses, such as age and sex and socio-demographic and the diagnosis is highly correlated to high HHV-8 anti- factors, were investigated in an analysis of variance using body levels [2]. It is therefore reasonable to speculate that data from all the patients except those with multiple mye- raised antibody levels to other viruses may be associated loma (Table 3). No risk factors were identified as signifi- with the development of other cancers. cantly influencing high log antibody measures in response to HSV-1. For HSV-2 mean log antibody meas- We conducted a systematic study of the relationship ures increased with age (p-trend = 0.003), were higher in between antibodies against human herpesviruses 1–6 and women (p < 0.0001), increased with number of sexual seven cancer groups in adult black South Africans. Our partners (p-trend = 0.01) and were lower in single people results suggest that, in this population, neither mean (p-heterogeneity = 0.005). No statistically significant ELISA IgG antibody measure to the antigens used nor high associations were found for VZV, or EBV-EBNA. Mean log antibody measures against these six human herpesviruses antibody measure for response to CMV increased signifi- were strongly associated with any of the seven cancer cantly with age (p-trend = 0.006) and was also higher in groups. Although we do not think that the variability in females than in males (p < 0.0001). Similarly, the mean the quantitative assay results concealed any strong associ- log antibody measure for HHV-6 antibodies was signifi- ations, we may not have had sufficient power to detect cantly higher in females than males (p = 0.0002). weak associations (for example, odds ratios <2.0) or asso- ciations with a sub-type of cancer if they were present. Discussion Herpesvirus infections are highly prevalent in human Though there was no evidence of a significant association populations. They usually produce transient illness or between high EBV-EBNA antibody measure and non- inapparent infection and remain latent in the host. In gen- Hodgkin lymphoma or Hodgkin lymphoma, EBV may be eral, re-activation of the latent infection, re-infection, or a causative factor of certain types of lymphomas only, viral persistence will cause the established IgG antibody including Burkitt's lymphoma and some immunosupres- levels to rise. In Burkitt's lymphoma it has been shown sion associated lymphomas [1]. As mentioned above, the that raised levels of IgG were present months before clin- current study was not powered to detect associations in Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 Table 3: Mean log antibody measure (and standard error) for human herpesviruses 1–6 according to age at diagnosis, sex and other socio-demographic factors. Risk factor Virus HSV-1 HSV-2 VZV EBV-EBNA CMV HHV-6 Age <20 1.55 (0.11) -0.19 (0.20) 1.06 (0.12) 1.52 (0.15) 1.79 (0.21) 0.64 (0.15) 20–39 1.61 (0.04) 0.25 (0.08) 1.06 (0.05) 1.44 (0.07) 2.19 (0.12) 0.70 (0.06) 40–59 1.61 (0.04) 0.36 (0.07) 1.07 (0.05) 1.40 (0.07) 2.26 (0.07) 0.74 (0.06) 60+ 1.60 (0.05) 0.48 (0.08) 1.11 (0.06) 1.47 (0.07) 2.41 (0.09) 0.66 (0.05) p-value 0.92 0.003 0.41 0.77 0.006 0.52 Sex Males 1.59 (0.05) 0.05 (0.08) 1.03 (0.06) 1.43 (0.07) 1.98 (0.09) 0.59 (0.06) Females 1.60 (0.05) 0.40 (0.08) 1.11 (0.06) 1.48 (0.07) 2.34 (0.10) 0.78 (0.06) p-value 0.91 <0.0001 0.09 0.44 <0.0001 0.0002 Smoking Never 1.60 (0.04) 0.21 (0.08) 1.03 (0.05) 1.45 (0.07) 2.16 (0.09) 0.67 (0.06) Ever 1.59 (0.05) 0.24 (0.09) 1.11 (0.06) 1.46 (0.08) 2.17 (0.10) 0.70 (0.06) p-value 0.91 0.56 0.07 0.96 0.94 0.34 Education < Grade 3 1.59 (0.05) 0.30 (0.09) 1.10 (0.06) 1.41 (0.08) 2.18 (0.10) 0.74 (0.07) Grade 3–7 1.64 (0.05) 0.24 (0.08) 1.09 (0.05) 1.47 (0.07) 2.13 (0.09) 0.69 (0.06) Grade 8+ 1.55 (0.05) 0.14 (0.09) 1.04 (0.06) 1.49 (0.08) 2.18 (0.10) 0.64 (0.07) p-value 0.53 0.09 0.26 0.18 0.99 0.14 Area of birth Urban 1.61 (0.05) 0.28 (0.09) 1.10 (0.06) 1.46 (0.07) 2.14 (0.10) 0.70 (0.06) Rural 1.57 (0.04) 0.18 (0.08) 1.05 (0.05) 1.45 (0.07) 2.19 (0.09) 0.67 (0.07) p-value 0.29 0.13 0.16 0.67 0.48 0.45 Area living Urban 1.59 (0.04) 0.19 (0.07) 1.08 (0.05) 1.45 (0.07) 2.17 (0.09) 0.71 (0.06) Rural 1.59 (0.05) 0.27 (0.09) 1.07 (0.06) 1.46 (0.08) 2.15 (0.11) 0.67 (0.06) p-value 0.99 0.35 0.83 0.76 0.85 0.53 HIV +ve 1.53 (0.06) 0.27 (0.11) 1.12 (0.07) 1.43 (0.09) 2.22 (0.12) 0.71 (0.08) -ve 1.66 (0.04) 0.18 (0.07) 1.02 (0.05) 1.48 (0.06) 2.11 (0.08) 0.67 (0.05) p-value 0.03 0.35 0.10 0.52 0.34 0.66 No. of 0–2 1.60 (0.05) 0.12 (0.08) 1.02 (0.06) 1.47 (0.07) 2.12 (0.09) 0.64 (0.06) sexual 3–5 1.58 (0.05) 0.25 (0.08) 1.05 (0.06) 1.41 (0.07) 2.18 (0.09) 0.75 (0.06) partners 6+ 1.60 (0.05) 0.31 (0.09) 1.15 (0.06) 1.49 (0.08) 2.19 (0.11) 0.67 (0.07) p-value 0.96 0.01 0.03 0.96 0.34 0.26 Marital Single 1.58 (0.04) 0.06 (0.08) 1.04 (0.05) 1.45 (0.07) 2.03 (0.09) 0.65 (0.06) status Married 1.62 (0.05) 0.22 (0.08) 1.11 (0.05) 1.43 (0.07) 2.16 (0.09) 0.67 (0.06) Widowed 1.59 (0.06) 0.20 (0.11) 1.04 (0.07) 1.41 (0.09) 2.17 (0.12) 0.66 (0.08) Separated 1.58 (0.07) 0.42 (0.12) 1.10 (0.08) 1.54 (0.10) 2.29 (0.14) 0.77 (0.09) p-value 0.64 0.005 0.45 0.52 0.26 0.34 Multivariate analysis of variance excluding patients with multiple myeloma and adjusted for all factors above plus disease group, plate and day of assay. P-values are for trend tests where appropriate and otherwise for heterogeneity. small sub-groups. However, our results suggest that high patients with oral cancers had elevated levels of HHV-6 IgG antibody measures against EBV-EBNA do not appear antibodies compared to the healthy controls [7]. As far as to be relevant in most lymphomas in this population. Sev- we are aware no additional studies have published results eral other associations between herpesviruses and cancers, on the association between HHV-6 and oral cancer, but which have been reported less consistently in the litera- since two studies have found a positive relationship fur- ture, were not clearly evident in the current study includ- ther research is warranted. There was also an unexpected ing: HSV-1 and oral cancer; HSV-2 and cervical cancer; small decrease in the risk of myeloid leukaemia in HHV-6 and Hodgkin lymphoma; and HHV-6 and mye- patients with the highest tertile of antibodies against loid leukaemia. HHV-6 (OR = 0.58, 95% CI 0.3 – 1.0). An increased risk of acute myeloid leukaemia and HHV-6 has been reported There was evidence of an increased risk of oral cancer for previously in some studies [6,14,15], although other stud- subjects in the highest compared to lowest tertile of anti- ies have not found evidence of such an association bodies against HHV-6 compared to other subjects (OR = [16,17]. 2.21, 95% CI 1.1 – 4.3), and a trend of increasing risk across the tertiles (p = 0.02). A similar study in India of The retrospective design on this study means that it is pos- patients with Hodgkin lymphoma, non-Hodgkin lym- sible that the cancer affected the patient's antibody levels phoma, oral cancer and healthy controls found that and therefore that the antibody levels reflect a conse- Page 7 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 quence rather than a cause of the cancer. There was evi- ures against human herpesvirsues 1 – 6 were strongly dence of this with respect to the multiple myeloma associated with any of the seven selected cancer groups. patients, who had lower than average mean log antibody We do not think that the variability in the quantitative measures for response to all six herpesviruses. This may be assay results concealed any strong associations, but we because myeloma is an over-production of a single immu- may not have had sufficient power to detect weak associ- noglobulin class, which could in turn down-regulate pro- ations or associations with a sub-type of cancer. The find- duction of other immunoglobulins/antibodies. None of ing of a small increased risk for oral cancer with increased the other disease groups had a systematically higher or antibodies to HHV-6 merits further exploration. lower log antibody measures for response to all six of the herpesviruses. However, it remains a possibility that the Authors' contributions positive association between raised HHV-6 antibody lev- VB and FS conceived of and designed the study. MU was els and the risk of oral cancer could have been caused by involved in the design of the plate template for serological opportunistic reactivations due to cancer-associated testing, in co-ordination of the serological testing, and in immunosuppression. Future studies should preferably be verification of patient data. NB participated in the study conducted with prospectively collected blood samples. design, selection of test kits and design of the plate tem- plate for serological testing as well as supervision of the Little is known about the determinants of infection with serological testing. MH provided pathology reports. MP, and antibody response to herpesviruses 1–6 in the popu- PR, and RS supplied patients for this study. AB conducted lation studied. In an analysis of associations with age, sex the statistical analysis and drafted the manuscript. VB, and lifestyle factors there was evidence that, as expected, MU, RN and FS were involved in the statistical analysis HSV-2 antibody measures increased with increasing and critical revision of the manuscript. number of sexual partners. In addition, antibody meas- ures to both HSV-2 and CMV were significantly higher in Acknowledgements During the study period the Cancer Epidemiology Research Group was older age groups, presumably reflecting an increasing supported by the South African Medical Research Council, the National number of re-infections, and in women. For HHV-6, Health Laboratory Service (formerly South African Institute for Medical which is probably transmitted via saliva [18], antibody Research), the University of the Witwatersrand, the Cancer Association of measures were also found to be higher in women than in South Africa and Cancer Research UK. men. Several other studies have reported similar findings for HHV-6 [19,20] although in the study in West Africa We are indebted to: Sr. Gloria Mokwatle and colleagues for carrying out and the Caribbean, this was only true for children [19]. the interviews; Ms. F. Mngomezulu and Ms. H. Mathabatha for data coding Primary HHV-6 acquisition has been found to be associ- and entry; Mrs. Lettie Bester for specimen preparation; Ms. Diana Bull for case selection and specimen randomisation; Ms. Lorraine Cranston and Ms. ated with female sex [21]. HIV positive patients in our Jane Franz at the South African National Institute for Communicable Dis- study did not have significantly higher average antibody eases (formerly National Institute for Virology) for carrying out the labora- measures in response to any of these herpesviruses. tory tests; the clinicians and nursing staff at Chris Hani Baragwanath, Hillbrow, and Johannesburg Hospitals for granting access to clinics, wards, As quantitative methods have not previously been widely and records; and the patients for their participation in this study. used in epidemiological studies of herpesviruses we included a quality control analysis to assess the repeata- References bility of the assay results. We found systematic variability 1. International Agency for Research on Cancer: Epstein-Barr virus and Kaposi's sarcoma herpesvirus/human herpesvirus 8 Lyon: World Health that could be attributed to differing laboratory conditions Organization; 1997. and for HSV-1 and HSV-2 variability attributable to either 2. Sitas F, Carrara H, Beral V, Newton R, Reeves G, Bull D, Jentsch U, the binding of the assay components to the plates or cali- Pacella-Norman R, Bourboulia D, Whitby D, Boshoff C, Weiss R: Antibodies against human herpesvirus 8 in black South Afri- bration control samples that were not effective at reducing can patients with cancer. NEJM 1999, 340:1863-1871. the variation between plates. For quantitative assessments 3. Schildt EB, Eriksson M, Hardell L, Magnuson A: Oral infections and dental factors in relation to oral cancer: a Swedish case–con- improvements to the kit techniques which we used are trol study. Eur J Cancer Prev 1998, 7:201-206. needed, possibly using different antigens. In the current 4. Smith JS, Herrero R, Bosetti C, Munoz N, Bosch FX, Eluf-Neto J, Cas- study we randomised the patients' samples to the plates so tellsague X, Meijer CJ, Van Den Brule AJ, Franceschi S, Ashley R: Her- pes simplex virus-2 as a human papillomavirus cofactor in that the systematic differences will not have biased our the etiology of invasive cervical cancer. J Natl Cancer Inst 2002, results and took account of these systematic sources of 94:1604-1613. 5. Alexander FE, Daniel CP, Armstrong AA, Clark DA, Onions DE, variability in the analyses by adjusting for the plate and Cartwright RA, Jarrett RF: Case clustering, Epstein-Barr virus day on which the assay was run. Reed-Sternberg cell status and herpes virus serology in Hodgkin's disease: results of a case-control study. Eur J Cancer 1995, 31A:1479-1486. Conclusion 6. Gentile G, Mele A, Ragona G, Faggioni A, Zompetta C, Tosti ME, In the black adult population of greater Johannesburg nei- Visani G, Castelli G, Pulsoni A, Monarca B, Martino P, Mandelli F: ther mean IgG antibody measure nor high antibody meas- Human herpes virus-6 seroprevalence and leukaemias: a Page 8 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 case-control study. GIMEMA (Gruppo Italiano Malattie Ematologiche dell' Adulto). Br J Cancer 1999, 80:1103-1106. 7. Shanavas KR, Kala V, Vasudevan DM, Vijayakumar T, Yadav M: Anti- HHV-6 antibodies in normal population and in cancer patients in India. J Exp Pathol 1992, 6:95-105. 8. Strickler HD, Goedert JJ: Sexual behavior and evidence for an infectious cause of prostate cancer. Epidemiol Rev 2001, 23:144-151. 9. Gao SJ, Alsina M, Deng JH, Harrison CR, Montalvo EA, Leach CT, Roodman GD, Jenson HB: Antibodies to Kaposi's sarcoma-asso- ciated herpesvirus (human herpesvirus 8) in patients with multiple myeloma. J Infect Dis 1998, 178:846-849. 10. Sitas F, Pacella-Norman R, Carrara H, Patel M, Ruff P, Sur R, Jentsch U, Hale M, Rowji P, Saffer D, Connor M, Bull D, Newton R, Beral V: The spectrum of HIV-1 related cancers in South Africa. Int J Cancer 2000, 88:489-492. 11. SAS Institute Inc: SAS. Cary, NC, USA 1999, version 8.2:. 12. De-Thé G, Geser A, Day NE, Tukei PM, Williams EH, Beri DP, Smith PG, Dean AG, Bornkamm GW, Feorino P, Henle W: Epidemiolog- ical evidence for causal relationship between Epstein-Barr virus and Burkitt's lymphoma from Ugandan prospective study. Nature 1978, 274:756-761. 13. Gao S-J, Kingsley L, Hoover DR, Spira TJ, Rinaldo CR, Saah A, Phair J, Detels R, Parry P, Chang Y, Moore PS: Sero conversion to anti- bodies against Kaposi's sarcoma-associated herpesvirus- related latent nuclear antigens before the development of Kaposi's sarcoma. NEJM 1996, 335:233-241. 14. Clark DA, Alexander FE, McKinney PA, Roberts BE, O'Brien C, Jar- rett RF, Cartwright RA, Onions DE: The seroepidemiology of human herpesvirus-6 (HHV-6) from a case-control study of leukaemia and lymphoma. Int J Cancer 1990, 45:829-833. 15. Salonen MJ, Siimes MA, Salonen EM, Vaheri A, Koskiniemi M: Anti- body status to HHV-6 in children with leukaemia. Leukemia 2002, 16:716-719. 16. Levine PH, Ablashi DV, Saxinger WC, Connelly RR: Antibodies to human herpes virus-6 in patients with acute lymphocytic leukemia. Leukemia 1992, 6:1229-1231. 17. Schlehofer B, Blettner M, Geletneky K, Haaf HG, Kaatsch P, Michaelis J, Mueller-Lantzsch N, Niehoff D, Winkelspecht B, Wahrendorf J, Sch- lehofer JR: Sero-epidemiological analysis of the risk of virus infections for childhood leukaemia. Int J Cancer 1996, 65:584-590. 18. Mukai T, Yamamoto T, Kondo T, Kondo K, Okuno T, Kosuge H, Yamanishi K: Molecular epidemiological studies of human her- pesvirus 6 in families. J Med Virol 1994, 42:224-227. 19. Cleghorn FR, Maybank KA, Jack N, Pate E, Mingle J, Levine PH, Manns A: Comparison of HHV-6 antibody titers in West Africa and the Caribbean. Ann Epidemiol 1995, 5:497-500. 20. Linhares MI, Eizuru Y, Tateno S, Minamishima Y: Seroprevalence of human herpesvirus 6 infection in Brazilian and Japanese pop- ulations in the north-east of Brazil. Microbiol Immunol 1991, 35:1023-7. 21. Zerr DM, Meier AS, Selke SS, Frenkel LM, Huang ML, Wald A, Rhoads MP, Nguy L, Borenemann R, Morrow RA, Corey L: A population- based study of primary human herpesvirus 6 infection. N Engl J Med 2005, 352(8):753-5. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Infectious Agents and Cancer Springer Journals

Antibodies against six human herpesviruses in relation to seven cancers in black South Africans: A case control study

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Springer Journals
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Copyright © 2006 by de González A et al; licensee BioMed Central Ltd.
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Biomedicine; Cancer Research; Infectious Diseases; Oncology
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1750-9378
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10.1186/1750-9378-1-2
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17150131
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Abstract

Background: Infections with certain human herpesviruses have been established as risk factors for some cancer types. For example, Epstein-Barr Virus is considered a cause of Burkitt's lymphoma and other immunosuppression related lymphomas, Hodgkin lymphoma, and nasopharyngeal cancer. Several other human herpesviruses have been linked to cancers but the totality of evidence is inconclusive. Methods: We conducted a systematic sub-study from within an ongoing case control study of adult black South Africans to investigate the relationship between antibodies to six human herpesviruses and seven cancer groups that may be caused by infectious agents. Subjects had incident cancers of the oral cavity(n = 88), the cervix(n = 53), the prostate(n = 66), Hodgkin lymphoma(n = 83), non-Hodgkin lymphoma(n = 80), multiple myeloma(n = 94) or leukaemia(n = 203). For comparison, patients with other cancers(n = 95) or cardiovascular disease(n = 101) were randomly selected from within the study. Patients were interviewed and their blood was tested for IgG antibodies against HSV-1, HSV-2, VZV, EBV- EBNA, CMV and HHV-6 using enzyme linked immunosorbent assays. Because these viruses are highly prevalent in this population, optical density results from the assays were used as an indirect, quantitative measure of antibody level. Results: There was significant variation in the mean log antibody measures for HSV-2, VZV, CMV and HHV-6 between the disease groups. However, none of the specific cancer groups had significantly higher mean log antibody measures for any of the viruses compared to either control group. In a more detailed examination of seven associations between cancers and herpesviruses for which there had been prior reports, two statistically significant associations were found: a decreasing risk of myeloid leukaemia and an increasing risk of oral cancer with increasing tertiles of antibodies against HHV-6 compared to all other patients (p-trend = 0.03 and 0.02, respectively). Odds ratios for the top tertile compared to the bottom tertile were 0.58 (95%CI 0.3 – 1.0) for myeloid leukaemia and 2.21 (95% CI 1.1 – 4.3) for oral cancer. Page 1 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 Conclusion: In this population, using these tests for IgG, neither mean antibody measure nor high antibody measure against human herpesviruses 1–6 was strongly associated with any of the seven cancer groups. However, we may not have had sufficient power to detect weak associations or associations with a sub-type of cancer if they were present. diagnosed cancer at tertiary government hospitals in Background Infection with certain types of human herpesviruses has Johannesburg (Chris Hani-Baragwanath, Hillbrow, and been established as a cause of several cancers. These Johannesburg General Hospitals). A standard question- include Epstein-Barr Virus (EBV) for Burkitt's lymphoma naire, administered in the language of the patient (usually and other immunosuppression related lymphomas, an Nguni or Sotho group language), was used. Questions Hodgkin lymphoma, and nasopharyngeal cancer [1]; and were asked about socio-demographic factors and behav- human herpesvirus 8 (HHV-8) for Kaposi's sarcoma [2]. ioural characteristics including age, sex, birthplace, resi- These cancers are rare responses to the presence of these dence, level of education, tobacco and alcohol use, and widespread viruses. reproductive and lifetime sexual history. Several human herpesviruses have been linked to other Blood was collected from 84% of patients at the time of cancers although the totality of evidence is inconclusive. interview and prior to commencing treatment. The For example herpes simplex virus type 1 (HSV-1) has been remainder were too ill, had collapsed veins, or refused associated with oral cancer[3] and herpes simplex virus consent. All interviewed patients with oral cancer (n = type 2 (HSV-2) with cervical cancer in women who are co- 88), Hodgkin lymphoma (n = 83), non-Hodgkin lym- infected with specific human papillomavirus types[4]. phoma (n = 80), multiple myeloma (n = 94) and leukae- Human herpesvirus type 6 (HHV-6) has been linked to mia (n = 203) and a random sample of subjects with Hodgkin lymphoma [5], acute myeloid leukaemia [6] and cervical (n = 53) and prostate cancer (n = 66) were oral cancer [7]. In addition it has been suggested that pros- included in the study if they had provided a blood sam- tate cancer [8] and multiple myeloma [9] may have infec- ple. Controls were from two groups of patients attending tious causes. the same hospitals: a group of patients with other cancers (n = 95) and a group with cardiovascular diseases (n = Our group previously found that high antibody levels to 101). The controls were selected randomly within sex and HHV8 are highly correlated to the diagnosis of Kaposi's age bands, and frequency matched to the cancer cases as a sarcoma [2]. We therefore designed a study to examine, in whole according to five-year age-groups and sex. Diag- a systematic way, antibody levels to six of the herpesvi- noses of cancer were established, where appropriate, by ruses (HSV-1, HSV-2, Varicella Zoster (VZV), EBV, histology, haematology, or cytology. The study was cytomegalovirus (CMV) and HHV-6) in relation to seven approved by the Committee for Research on Human Sub- cancer groups for which there is some evidence of an jects (Medical) of the University of the Witwatersrand, infectious cause (oral, cervical, prostate, Hodgkin lym- and informed consent was obtained from all patients. phoma, non-Hodgkin lymphoma, multiple myeloma and leukaemia). The study was part of a larger case-control Laboratory methods study of the causes of cancer in black South Africans, After coagulation, specimens were centrifuged to obtain which was conducted in public hospitals that treat cancer serum. The serum specimens were aliquotted and stored in greater Johannesburg, South Africa [2,10]. Since most at -20° to -30°C. HIV testing was carried out by the Serol- human herpesviruses are highly prevalent, and PCR on ogy Laboratory of the South African Institute for Medical biopsy samples is unrealistic in this setting, we used quan- Research (now the National Health Laboratory Service), titative measures of anti-human herpesvirus antibodies using commercial ELISA kits. Early testing was for HIV-1 from enzyme linked immunosorbent assays (ELISAs). In only; later testing for both HIV-1 and HIV-2. Patients with addition we examined the relationships between demo- borderline results were considered to be HIV negative. graphic and lifestyle factors and antibody levels against these viruses, as little is known about these viruses in this Herpes virus antibody testing was done at the South Afri- population. can National Institute for Virology (now the National Institute for Communicable Diseases) from January to April 2000. Throughout the testing period specimens were Methods Study Participants kept at 4°C. The commercially available kits used were: The study population has been described previously Eurogenetics for HSV-1, HSV-2 and CMV; Clark Laborato- [2,10]. Briefly, between March 1995 and February 1999 ries for VZV and EBV nuclear antigen-1(EBNA); and Pan- trained nurses interviewed adult black patients with newly Bio for HHV-6. These were selected following Page 2 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 apreliminary study which demonstrated good quantita- Since little is known about risk factors for infection with tive performance with South African sera. The test for human herpesviruses in this population, we examined the EBNA IgG used "recombinant EBNA-1 antigen". All other relationship between antibody measure and a number of kits used unspecified antigens. socio-demographic variables using analysis of variance with adjustment for each of these variables as well as can- Patients' sera were randomly allocated to the 96-well cer group, day of assay, and assay plate. Due to the microplates to minimise potential bias due to systematic number of comparisons that were made these analyses differences between the plates or day on which the plates were examined at the 0.01 significance level. were run. Each specimen was run in a single well. There were a total of 12 plates for each assay and in general two All analyses were carried out using SAS statistical software plates were run each day. Repeatability of each assay was [11]. investigated through replicate measures on a pool of sera samples with high antibody measures against each virus. Results Test results were initially in optical density units which are In total 667 patients with the seven specific cancers of proportional to the concentration of antibody i.e. the interest were included in the study. A further 95 patients antibody titre. The CMV kit had a calibration curve giving with other types of cancer and 101 patients with cardio- final results in antibody units/ml. The remaining kits con- vascular diseases were included as control subjects. tained standards used to determine a cut-off for positivity. Demographic characteristics of the study members are Results are reported as ratios to the manufacturer's cut-off shown in Table 1. value. Throughout this paper we use the general term "antibody measure" to refer to the manufacturer's result, The results from the analysis of the control samples sug- regardless of whether it is in AU/ml or "times cut-off". gested that for all assays (except for HSV-1 and HSV-2) the most important source of systematic variation was the day Statistical Analysis for the control samples on which the plates were assayed, whereas for the HSV-1 There were several potential sources of variability in each and HSV-2 assays it was the plates themselves (data not assay such as antigen binding within or between plates, shown). As the plate and day on which each patient's sam- washing conditions between binding steps and laboratory ple was tested had been recorded, variation between days conditions such as the temperature, humidity, and light. and between plates was controlled for in the main analy- For each of the herpesviruses the logarithm (log) antibody ses by adjusting for these factors. measure was computed for the control pool and analysis of variance was used to estimate the magnitude of the Crude adult prevalence rates in the study, determined components of variance for each of the sources of variabil- using the manufacturer's criteria for positivity, were: 99% ity. for HSV-1; 59% for HSV-2 (52% in males and 68% in females); 97% for VZV; 96% for EBV (EBNA antigen); Statistical Analysis of the Patient Data 99% for CMV; and 90% for HHV-6 (88% in males and To examine the relation between antibodies against the 93% in females). human herpesviruses 1–6 and cancer the mean log anti- body measures for each of the nine disease groups were Associations with cancer types There was statistically significant variation between the compared using analysis of variance. Least square mean log antibody measures were calculated for each disease nine disease groups in mean log antibody measure for group with adjustment for age, sex, HIV status, and the HSV-2 (p < 0.0001), VZV (p < 0.0001), CMV (p = 0.009) day and the plate on which the assay was run. Paired com- and HHV-6 (p < 0.0001) (Figure 1). Patients with multi- parisons were conducted between each cancer group and ple myeloma had lower mean log antibody measures for each of the two control groups (other cancers and cardio- almost all these herpesviruses compared to other patients. vascular disease patients) with Bonferroni adjustments for When patients with multiple myeloma were excluded, sig- multiple comparisons. Where positive associations had nificant heterogeneity in mean log antibody measures previously been found (see Introduction [1-7]), the spe- between the eight remaining disease groups was still cific hypotheses were investigated in more detail by com- present for HSV-2 (p = 0.0007) and HHV-6 (p = 0.004). paring the specified cancer group to all the other patients combined with respect to tertiles of log antibody measure. In paired comparisons (with Bonferroni adjustments), Odds ratios were calculated using logistic regression with patients with multiple myeloma had significantly lower adjustment for age, sex, HIV status and the day and the mean log IgG antibody measures for the response to VZV, plate on which the assay was run. CMV and HHV-6 compared to patients in the 'other can- cer' group (p < 0.0001, 0.02 and p < 0.0001 respectively) and for HSV-2, VZV and HHV-6 compared to patients Page 3 of 9 (page number not for citation purposes) Table 1: Demographic characteristics of patients by disease group Cancer group Lip, oral cavity and pharynx Cervix Prostate Hodgkin lymphoma non-Hodgkin lymphoma Multiple myeloma Leukaemia Other cancers Cardiovascular disease n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%) n (%) Males 64 (73) 0 (0) 66 (100) 46 (55) 45 (56) 52 (55) 92 (45) 45 (47) 45 (45) Females 24 (27) 53 (100) 0 (0) 37 (45) 35 (44) 42 (45) 111 (55) 50 (53) 56 (55) Age <35 yrs 6 (7) 15 (28) 0 (0) 33 (40) 22 (28) 5 (5) 57 (28) 23 (24) 17 (17) Age 35–49 yrs 17 (19) 12 (23) 6 (9) 35 (43) 25 (31) 23 (24) 54 (27) 26 (27) 29 (29) Age 50+ yrs 65 (74) 26 (49) 60 (91) 15 (18) 33 (41) 66 (70) 92 (45) 46 (48) 55 (55) HIV +ve 3 (3) 5 (9) 1 (2) 9 (11) 18 (23) 4 (4) 8 (4) 6 (6) 11 (11) Total 88 (100) 53 (100) 66 (100) 83 (100) 80 (100) 94 (100) 203 (100) 95 (100) 101 (100) Includes cancers of the digestive organs, respiratory organs, bone, skin, soft tissue, breast, female and male genital organs, bladder, brain, thyroid and of unspecified sites. Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 Page 4 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 Distribution Figure 1 of mean log antibody measures (and 95% CI) by disease group for herpesviruses 1–6 Distribution of mean log antibody measures (and 95% CI) by disease group for herpesviruses 1–6. with cardiovascular diseases (p = 0.01, 0.0003 and analyses because in general mean antibody levels did not <0.0001 respectively). Because of their systematically vary significantly across disease groups after exclusion of lower antibody measures, multiple myeloma patients subjects with multiple myeloma (Figure 1), and including were excluded from further analyses. Patients with non- more patients increased the power of these analyses. Only Hodgkin lymphoma had significantly lower mean log oral cancer showed a statistically significant trend of risk antibody measures in response to HSV-2 compared to the with increasing HHV-6 tertiles (p = 0.02), odds ratio for non-cancer patients (p = 0.004). None of the specific can- the top tertile compared to all other subjects = 2.21 (95% cer groups had significantly higher mean log antibody CI 1.1 – 4.3) (Table 2). All the oral cancer patients were measures for any of the viruses compared to either control positive for HHV-6 antibodies by the manufacturer's cut- group. off. Only two subjects had undifferentiated nasopharyn- geal cancer so it was not possible to investigate the rela- To further investigate the positive associations reported in tionship between this sub-type of oral cancers and EBV. previous studies (EBV and Hodgkin lymphoma; EBV and Neither was it possible to separate acute myeloid leukae- non-Hodgkin lymphoma; HSV-1 and oral cancer; HSV-2 mias from chronic myeloid leukaemias with the informa- and cervical cancer; HHV-6 with oral cancer, Hodgkin tion that was available, but there was some evidence of a lymphoma and acute myeloid leukaemia) the log anti- decreasing trend in risk of myeloid leukaemia with body measures were divided into tertiles according to the increasing tertiles of HHV-6 (p = 0.03); odds ratio for the levels in allpatientsexcluding those with multiple mye- top tertile compared to all other subjects = 0.58 (95% CI loma. The control group was defined in this way for these 0.3 – 1.0). Page 5 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 1 2 Table 2: Odds ratios (OR) and 95% confidence intervals (CI) for specific cancers versus all other subjects (controls) by tertile of virus antibody measure in the controls Virus and cancer group Control tertiles Cases/Controls OR 95% CI P-trend HSV-1 and Oral cancer 0–5.04 23/200 1.00 5.05–6.07 26/199 1.06 (0.6 – 2.0) 6.08+ 31/199 1.28 (0.7 – 2.3) 0.40 HSV-2 and Cervical cancer 0–1.08 11/91 1.00 1.09–2.17 11/91 0.90 (0.4 – 2.2) 2.18+ 23/90 1.82 (0.8 – 4.1) 0.10 EBV-EBNA and non-Hodgkin lymphoma 0–3.81 24/208 1.00 3.82–5.29 31/203 1.32 (0.7 – 2.4) 5.30+ 22/211 0.87 (0.4 – 1.8) 0.80 EBV-EBNA and Hodgkin lymphoma 0–3.87 33/211 1.00 3.88–5.31 18/208 0.49 (0.3 – 0.96) 5.32+ 21/208 0.66 (0.3 – 1.4) 0.19 HHV-6 and Oral cancer 0–1.59 16/195 1.00 1.60–2.43 28/194 1.83 (0.9 – 3.6) 2.44+ 31/195 2.21 (1.1 – 4.3) 0.02 HHV-6 and Hodgkin lymphoma 0–1.58 21/172 1.00 (excluding oral cancers) 1.59–2.40 17/174 0.87 (0.4 – 1.7) 2.41+ 28/172 1.32 (0.7 – 2.5) 0.39 HHV-6 and Myeloid Leukaemia 0–1.61 47/131 1.00 (excluding oral cancers) 1.62–2.59 52/134 1.16 (0.7 – 1.9) 2.60+ 26/131 0.58 (0.3 – 1.0) 0.03 For associations that had been reported previously. The control group in these analyses included all other subjects, except patients with multiple myeloma, because in general mean antibody levels did not vary significantly across disease groups after exclusion of subjects with multiple myeloma (Figure 1), and including all patients increased the power of these analyses. All analyses are adjusted for age group, sex, day of assay and assay plate. Association with age, sex and socio-demographic factors ical onset [12]. Antibodies to HHV-8 are also found many Possible determinants of high antibody measures to these months prior to the diagnosis or Kaposi's sarcoma [13] herpesviruses, such as age and sex and socio-demographic and the diagnosis is highly correlated to high HHV-8 anti- factors, were investigated in an analysis of variance using body levels [2]. It is therefore reasonable to speculate that data from all the patients except those with multiple mye- raised antibody levels to other viruses may be associated loma (Table 3). No risk factors were identified as signifi- with the development of other cancers. cantly influencing high log antibody measures in response to HSV-1. For HSV-2 mean log antibody meas- We conducted a systematic study of the relationship ures increased with age (p-trend = 0.003), were higher in between antibodies against human herpesviruses 1–6 and women (p < 0.0001), increased with number of sexual seven cancer groups in adult black South Africans. Our partners (p-trend = 0.01) and were lower in single people results suggest that, in this population, neither mean (p-heterogeneity = 0.005). No statistically significant ELISA IgG antibody measure to the antigens used nor high associations were found for VZV, or EBV-EBNA. Mean log antibody measures against these six human herpesviruses antibody measure for response to CMV increased signifi- were strongly associated with any of the seven cancer cantly with age (p-trend = 0.006) and was also higher in groups. Although we do not think that the variability in females than in males (p < 0.0001). Similarly, the mean the quantitative assay results concealed any strong associ- log antibody measure for HHV-6 antibodies was signifi- ations, we may not have had sufficient power to detect cantly higher in females than males (p = 0.0002). weak associations (for example, odds ratios <2.0) or asso- ciations with a sub-type of cancer if they were present. Discussion Herpesvirus infections are highly prevalent in human Though there was no evidence of a significant association populations. They usually produce transient illness or between high EBV-EBNA antibody measure and non- inapparent infection and remain latent in the host. In gen- Hodgkin lymphoma or Hodgkin lymphoma, EBV may be eral, re-activation of the latent infection, re-infection, or a causative factor of certain types of lymphomas only, viral persistence will cause the established IgG antibody including Burkitt's lymphoma and some immunosupres- levels to rise. In Burkitt's lymphoma it has been shown sion associated lymphomas [1]. As mentioned above, the that raised levels of IgG were present months before clin- current study was not powered to detect associations in Page 6 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 Table 3: Mean log antibody measure (and standard error) for human herpesviruses 1–6 according to age at diagnosis, sex and other socio-demographic factors. Risk factor Virus HSV-1 HSV-2 VZV EBV-EBNA CMV HHV-6 Age <20 1.55 (0.11) -0.19 (0.20) 1.06 (0.12) 1.52 (0.15) 1.79 (0.21) 0.64 (0.15) 20–39 1.61 (0.04) 0.25 (0.08) 1.06 (0.05) 1.44 (0.07) 2.19 (0.12) 0.70 (0.06) 40–59 1.61 (0.04) 0.36 (0.07) 1.07 (0.05) 1.40 (0.07) 2.26 (0.07) 0.74 (0.06) 60+ 1.60 (0.05) 0.48 (0.08) 1.11 (0.06) 1.47 (0.07) 2.41 (0.09) 0.66 (0.05) p-value 0.92 0.003 0.41 0.77 0.006 0.52 Sex Males 1.59 (0.05) 0.05 (0.08) 1.03 (0.06) 1.43 (0.07) 1.98 (0.09) 0.59 (0.06) Females 1.60 (0.05) 0.40 (0.08) 1.11 (0.06) 1.48 (0.07) 2.34 (0.10) 0.78 (0.06) p-value 0.91 <0.0001 0.09 0.44 <0.0001 0.0002 Smoking Never 1.60 (0.04) 0.21 (0.08) 1.03 (0.05) 1.45 (0.07) 2.16 (0.09) 0.67 (0.06) Ever 1.59 (0.05) 0.24 (0.09) 1.11 (0.06) 1.46 (0.08) 2.17 (0.10) 0.70 (0.06) p-value 0.91 0.56 0.07 0.96 0.94 0.34 Education < Grade 3 1.59 (0.05) 0.30 (0.09) 1.10 (0.06) 1.41 (0.08) 2.18 (0.10) 0.74 (0.07) Grade 3–7 1.64 (0.05) 0.24 (0.08) 1.09 (0.05) 1.47 (0.07) 2.13 (0.09) 0.69 (0.06) Grade 8+ 1.55 (0.05) 0.14 (0.09) 1.04 (0.06) 1.49 (0.08) 2.18 (0.10) 0.64 (0.07) p-value 0.53 0.09 0.26 0.18 0.99 0.14 Area of birth Urban 1.61 (0.05) 0.28 (0.09) 1.10 (0.06) 1.46 (0.07) 2.14 (0.10) 0.70 (0.06) Rural 1.57 (0.04) 0.18 (0.08) 1.05 (0.05) 1.45 (0.07) 2.19 (0.09) 0.67 (0.07) p-value 0.29 0.13 0.16 0.67 0.48 0.45 Area living Urban 1.59 (0.04) 0.19 (0.07) 1.08 (0.05) 1.45 (0.07) 2.17 (0.09) 0.71 (0.06) Rural 1.59 (0.05) 0.27 (0.09) 1.07 (0.06) 1.46 (0.08) 2.15 (0.11) 0.67 (0.06) p-value 0.99 0.35 0.83 0.76 0.85 0.53 HIV +ve 1.53 (0.06) 0.27 (0.11) 1.12 (0.07) 1.43 (0.09) 2.22 (0.12) 0.71 (0.08) -ve 1.66 (0.04) 0.18 (0.07) 1.02 (0.05) 1.48 (0.06) 2.11 (0.08) 0.67 (0.05) p-value 0.03 0.35 0.10 0.52 0.34 0.66 No. of 0–2 1.60 (0.05) 0.12 (0.08) 1.02 (0.06) 1.47 (0.07) 2.12 (0.09) 0.64 (0.06) sexual 3–5 1.58 (0.05) 0.25 (0.08) 1.05 (0.06) 1.41 (0.07) 2.18 (0.09) 0.75 (0.06) partners 6+ 1.60 (0.05) 0.31 (0.09) 1.15 (0.06) 1.49 (0.08) 2.19 (0.11) 0.67 (0.07) p-value 0.96 0.01 0.03 0.96 0.34 0.26 Marital Single 1.58 (0.04) 0.06 (0.08) 1.04 (0.05) 1.45 (0.07) 2.03 (0.09) 0.65 (0.06) status Married 1.62 (0.05) 0.22 (0.08) 1.11 (0.05) 1.43 (0.07) 2.16 (0.09) 0.67 (0.06) Widowed 1.59 (0.06) 0.20 (0.11) 1.04 (0.07) 1.41 (0.09) 2.17 (0.12) 0.66 (0.08) Separated 1.58 (0.07) 0.42 (0.12) 1.10 (0.08) 1.54 (0.10) 2.29 (0.14) 0.77 (0.09) p-value 0.64 0.005 0.45 0.52 0.26 0.34 Multivariate analysis of variance excluding patients with multiple myeloma and adjusted for all factors above plus disease group, plate and day of assay. P-values are for trend tests where appropriate and otherwise for heterogeneity. small sub-groups. However, our results suggest that high patients with oral cancers had elevated levels of HHV-6 IgG antibody measures against EBV-EBNA do not appear antibodies compared to the healthy controls [7]. As far as to be relevant in most lymphomas in this population. Sev- we are aware no additional studies have published results eral other associations between herpesviruses and cancers, on the association between HHV-6 and oral cancer, but which have been reported less consistently in the litera- since two studies have found a positive relationship fur- ture, were not clearly evident in the current study includ- ther research is warranted. There was also an unexpected ing: HSV-1 and oral cancer; HSV-2 and cervical cancer; small decrease in the risk of myeloid leukaemia in HHV-6 and Hodgkin lymphoma; and HHV-6 and mye- patients with the highest tertile of antibodies against loid leukaemia. HHV-6 (OR = 0.58, 95% CI 0.3 – 1.0). An increased risk of acute myeloid leukaemia and HHV-6 has been reported There was evidence of an increased risk of oral cancer for previously in some studies [6,14,15], although other stud- subjects in the highest compared to lowest tertile of anti- ies have not found evidence of such an association bodies against HHV-6 compared to other subjects (OR = [16,17]. 2.21, 95% CI 1.1 – 4.3), and a trend of increasing risk across the tertiles (p = 0.02). A similar study in India of The retrospective design on this study means that it is pos- patients with Hodgkin lymphoma, non-Hodgkin lym- sible that the cancer affected the patient's antibody levels phoma, oral cancer and healthy controls found that and therefore that the antibody levels reflect a conse- Page 7 of 9 (page number not for citation purposes) Infectious Agents and Cancer 2006, 1:2 http://www.infectagentscancer.com/content/1/1/2 quence rather than a cause of the cancer. There was evi- ures against human herpesvirsues 1 – 6 were strongly dence of this with respect to the multiple myeloma associated with any of the seven selected cancer groups. patients, who had lower than average mean log antibody We do not think that the variability in the quantitative measures for response to all six herpesviruses. This may be assay results concealed any strong associations, but we because myeloma is an over-production of a single immu- may not have had sufficient power to detect weak associ- noglobulin class, which could in turn down-regulate pro- ations or associations with a sub-type of cancer. The find- duction of other immunoglobulins/antibodies. None of ing of a small increased risk for oral cancer with increased the other disease groups had a systematically higher or antibodies to HHV-6 merits further exploration. lower log antibody measures for response to all six of the herpesviruses. However, it remains a possibility that the Authors' contributions positive association between raised HHV-6 antibody lev- VB and FS conceived of and designed the study. MU was els and the risk of oral cancer could have been caused by involved in the design of the plate template for serological opportunistic reactivations due to cancer-associated testing, in co-ordination of the serological testing, and in immunosuppression. Future studies should preferably be verification of patient data. NB participated in the study conducted with prospectively collected blood samples. design, selection of test kits and design of the plate tem- plate for serological testing as well as supervision of the Little is known about the determinants of infection with serological testing. MH provided pathology reports. MP, and antibody response to herpesviruses 1–6 in the popu- PR, and RS supplied patients for this study. AB conducted lation studied. In an analysis of associations with age, sex the statistical analysis and drafted the manuscript. VB, and lifestyle factors there was evidence that, as expected, MU, RN and FS were involved in the statistical analysis HSV-2 antibody measures increased with increasing and critical revision of the manuscript. number of sexual partners. In addition, antibody meas- ures to both HSV-2 and CMV were significantly higher in Acknowledgements During the study period the Cancer Epidemiology Research Group was older age groups, presumably reflecting an increasing supported by the South African Medical Research Council, the National number of re-infections, and in women. For HHV-6, Health Laboratory Service (formerly South African Institute for Medical which is probably transmitted via saliva [18], antibody Research), the University of the Witwatersrand, the Cancer Association of measures were also found to be higher in women than in South Africa and Cancer Research UK. men. Several other studies have reported similar findings for HHV-6 [19,20] although in the study in West Africa We are indebted to: Sr. Gloria Mokwatle and colleagues for carrying out and the Caribbean, this was only true for children [19]. the interviews; Ms. F. Mngomezulu and Ms. H. Mathabatha for data coding Primary HHV-6 acquisition has been found to be associ- and entry; Mrs. Lettie Bester for specimen preparation; Ms. Diana Bull for case selection and specimen randomisation; Ms. Lorraine Cranston and Ms. ated with female sex [21]. HIV positive patients in our Jane Franz at the South African National Institute for Communicable Dis- study did not have significantly higher average antibody eases (formerly National Institute for Virology) for carrying out the labora- measures in response to any of these herpesviruses. tory tests; the clinicians and nursing staff at Chris Hani Baragwanath, Hillbrow, and Johannesburg Hospitals for granting access to clinics, wards, As quantitative methods have not previously been widely and records; and the patients for their participation in this study. used in epidemiological studies of herpesviruses we included a quality control analysis to assess the repeata- References bility of the assay results. We found systematic variability 1. International Agency for Research on Cancer: Epstein-Barr virus and Kaposi's sarcoma herpesvirus/human herpesvirus 8 Lyon: World Health that could be attributed to differing laboratory conditions Organization; 1997. and for HSV-1 and HSV-2 variability attributable to either 2. Sitas F, Carrara H, Beral V, Newton R, Reeves G, Bull D, Jentsch U, the binding of the assay components to the plates or cali- Pacella-Norman R, Bourboulia D, Whitby D, Boshoff C, Weiss R: Antibodies against human herpesvirus 8 in black South Afri- bration control samples that were not effective at reducing can patients with cancer. NEJM 1999, 340:1863-1871. the variation between plates. For quantitative assessments 3. Schildt EB, Eriksson M, Hardell L, Magnuson A: Oral infections and dental factors in relation to oral cancer: a Swedish case–con- improvements to the kit techniques which we used are trol study. Eur J Cancer Prev 1998, 7:201-206. needed, possibly using different antigens. In the current 4. 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Zerr DM, Meier AS, Selke SS, Frenkel LM, Huang ML, Wald A, Rhoads MP, Nguy L, Borenemann R, Morrow RA, Corey L: A population- based study of primary human herpesvirus 6 infection. N Engl J Med 2005, 352(8):753-5. Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." Sir Paul Nurse, Cancer Research UK Your research papers will be: available free of charge to the entire biomedical community peer reviewed and published immediately upon acceptance cited in PubMed and archived on PubMed Central yours — you keep the copyright BioMedcentral Submit your manuscript here: http://www.biomedcentral.com/info/publishing_adv.asp Page 9 of 9 (page number not for citation purposes)

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