Infection with certain types of human herpesviruses has been established as a cause of several cancers. These include Epstein-Barr Virus (EBV) for Burkitt's lymphoma and other immunosuppression related lymphomas, Hodgkin lymphoma, and nasopharyngeal cancer [1]; and human herpesvirus 8 (HHV-8) for Kaposi's sarcoma [2]. These cancers are rare responses to the presence of these widespread viruses.

Several human herpesviruses have been linked to other cancers although the totality of evidence is inconclusive. For example herpes simplex virus type 1 (HSV-1) has been associated with oral cancer[3] and herpes simplex virus type 2 (HSV-2) with cervical cancer in women who are co-infected with specific human papillomavirus types[4]. Human herpesvirus type 6 (HHV-6) has been linked to Hodgkin lymphoma [5], acute myeloid leukaemia [6] and oral cancer [7]. In addition it has been suggested that prostate cancer [8] and multiple myeloma [9] may have infectious causes.

Our group previously found that high antibody levels to HHV8 are highly correlated to the diagnosis of Kaposi's sarcoma [2]. We therefore designed a study to examine, in a systematic way, antibody levels to six of the herpesviruses (HSV-1, HSV-2, Varicella Zoster (VZV), EBV, cytomegalovirus (CMV) and HHV-6) in relation to seven cancer groups for which there is some evidence of an infectious cause (oral, cervical, prostate, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma and leukaemia). The study was part of a larger case-control study of the causes of cancer in black South Africans, which was conducted in public hospitals that treat cancer in greater Johannesburg, South Africa [2, 10]. Since most human herpesviruses are highly prevalent, and PCR on biopsy samples is unrealistic in this setting, we used quantitative measures of anti-human herpesvirus antibodies from enzyme linked immunosorbent assays (ELISAs). In addition we examined the relationships between demographic and lifestyle factors and antibody levels against these viruses, as little is known about these viruses in this population.

The results from the analysis of the control samples suggested that for all assays (except for HSV-1 and HSV-2) the most important source of systematic variation was the day on which the plates were assayed, whereas for the HSV-1 and HSV-2 assays it was the plates themselves (data not shown). As the plate and day on which each patient's sample was tested had been recorded, variation between days and between plates was controlled for in the main analyses by adjusting for these factors.

Crude adult prevalence rates in the study, determined using the manufacturer's criteria for positivity, were: 99% for HSV-1; 59% for HSV-2 (52% in males and 68% in females); 97% for VZV; 96% for EBV (EBNA antigen); 99% for CMV; and 90% for HHV-6 (88% in males and 93% in females).

Associations with cancer types

There was statistically significant variation between the nine disease groups in mean log antibody measure for HSV-2 (p < 0.0001), VZV (p < 0.0001), CMV (p = 0.009) and HHV-6 (p < 0.0001) (Figure 1). Patients with multiple myeloma had lower mean log antibody measures for almost all these herpesviruses compared to other patients. When patients with multiple myeloma were excluded, significant heterogeneity in mean log antibody measures between the eight remaining disease groups was still present for HSV-2 (p = 0.0007) and HHV-6 (p = 0.004).

In paired comparisons (with Bonferroni adjustments), patients with multiple myeloma had significantly lower mean log IgG antibody measures for the response to VZV, CMV and HHV-6 compared to patients in the 'other cancer' group (p < 0.0001, 0.02 and p < 0.0001 respectively) and for HSV-2, VZV and HHV-6 compared to patients with cardiovascular diseases (p = 0.01, 0.0003 and <0.0001 respectively). Because of their systematically lower antibody measures, multiple myeloma patients were excluded from further analyses. Patients with non-Hodgkin lymphoma had significantly lower mean log antibody measures in response to HSV-2 compared to the non-cancer patients (p = 0.004). None of the specific cancer groups had significantly higher mean log antibody measures for any of the viruses compared to either control group.

To further investigate the positive associations reported in previous studies (EBV and Hodgkin lymphoma; EBV and non-Hodgkin lymphoma; HSV-1 and oral cancer; HSV-2 and cervical cancer; HHV-6 with oral cancer, Hodgkin lymphoma and acute myeloid leukaemia) the log antibody measures were divided into tertiles according to the levels in allpatientsexcluding those with multiple myeloma. The control group was defined in this way for these analyses because in general mean antibody levels did not vary significantly across disease groups after exclusion of subjects with multiple myeloma (Figure 1), and including more patients increased the power of these analyses. Only oral cancer showed a statistically significant trend of risk with increasing HHV-6 tertiles (p = 0.02), odds ratio for the top tertile compared to all other subjects = 2.21 (95% CI 1.1 – 4.3) (Table 2). All the oral cancer patients were positive for HHV-6 antibodies by the manufacturer's cut-off. Only two subjects had undifferentiated nasopharyngeal cancer so it was not possible to investigate the relationship between this sub-type of oral cancers and EBV. Neither was it possible to separate acute myeloid leukaemias from chronic myeloid leukaemias with the information that was available, but there was some evidence of a decreasing trend in risk of myeloid leukaemia with increasing tertiles of HHV-6 (p = 0.03); odds ratio for the top tertile compared to all other subjects = 0.58 (95% CI 0.3 – 1.0).

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

¹For associations that had been reported previously.

² 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.

Herpesvirus infections are highly prevalent in human populations. They usually produce transient illness or inapparent infection and remain latent in the host. In general, re-activation of the latent infection, re-infection, or viral persistence will cause the established IgG antibody levels to rise. In Burkitt's lymphoma it has been shown that raised levels of IgG were present months before clinical onset [12]. Antibodies to HHV-8 are also found many months prior to the diagnosis or Kaposi's sarcoma [13] and the diagnosis is highly correlated to high HHV-8 antibody levels [2]. It is therefore reasonable to speculate that raised antibody levels to other viruses may be associated with the development of other cancers.

We conducted a systematic study of the relationship between antibodies against human herpesviruses 1–6 and seven cancer groups in adult black South Africans. Our results suggest that, in this population, neither mean ELISA IgG antibody measure to the antigens used nor high antibody measures against these six human herpesviruses were strongly associated with any of the seven cancer groups. Although we do not think that the variability in the quantitative assay results concealed any strong associations, we may not have had sufficient power to detect weak associations (for example, odds ratios <2.0) or associations with a sub-type of cancer if they were present.

Though there was no evidence of a significant association between high EBV-EBNA antibody measure and non-Hodgkin lymphoma or Hodgkin lymphoma, EBV may be a causative factor of certain types of lymphomas only, including Burkitt's lymphoma and some immunosupression associated lymphomas [1]. As mentioned above, the current study was not powered to detect associations in small sub-groups. However, our results suggest that high IgG antibody measures against EBV-EBNA do not appear to be relevant in most lymphomas in this population. Several other associations between herpesviruses and cancers, which have been reported less consistently in the literature, were not clearly evident in the current study including: HSV-1 and oral cancer; HSV-2 and cervical cancer; HHV-6 and Hodgkin lymphoma; and HHV-6 and myeloid leukaemia.

There was evidence of an increased risk of oral cancer for subjects in the highest compared to lowest tertile of antibodies against HHV-6 compared to other subjects (OR = 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 patients with Hodgkin lymphoma, non-Hodgkin lymphoma, oral cancer and healthy controls found that patients with oral cancers had elevated levels of HHV-6 antibodies compared to the healthy controls [7]. As far as we are aware no additional studies have published results on the association between HHV-6 and oral cancer, but since two studies have found a positive relationship further research is warranted. There was also an unexpected small decrease in the risk of myeloid leukaemia in patients with the highest tertile of antibodies against HHV-6 (OR = 0.58, 95% CI 0.3 – 1.0). An increased risk of acute myeloid leukaemia and HHV-6 has been reported previously in some studies [6, 14, 15], although other studies have not found evidence of such an association [16, 17].

The retrospective design on this study means that it is possible that the cancer affected the patient's antibody levels and therefore that the antibody levels reflect a consequence rather than a cause of the cancer. There was evidence of this with respect to the multiple myeloma patients, who had lower than average mean log antibody measures for response to all six herpesviruses. This may be because myeloma is an over-production of a single immunoglobulin class, which could in turn down-regulate production of other immunoglobulins/antibodies. None of the other disease groups had a systematically higher or lower log antibody measures for response to all six of the herpesviruses. However, it remains a possibility that the positive association between raised HHV-6 antibody levels and the risk of oral cancer could have been caused by opportunistic reactivations due to cancer-associated immunosuppression. Future studies should preferably be conducted with prospectively collected blood samples.

Little is known about the determinants of infection with and antibody response to herpesviruses 1–6 in the population studied. In an analysis of associations with age, sex and lifestyle factors there was evidence that, as expected, HSV-2 antibody measures increased with increasing number of sexual partners. In addition, antibody measures to both HSV-2 and CMV were significantly higher in older age groups, presumably reflecting an increasing number of re-infections, and in women. For HHV-6, which is probably transmitted via saliva [18], antibody measures were also found to be higher in women than in men. Several other studies have reported similar findings for HHV-6 [19, 20] although in the study in West Africa and the Caribbean, this was only true for children [19]. Primary HHV-6 acquisition has been found to be associated with female sex [21]. HIV positive patients in our study did not have significantly higher average antibody measures in response to any of these herpesviruses.

As quantitative methods have not previously been widely used in epidemiological studies of herpesviruses we included a quality control analysis to assess the repeatability of the assay results. We found systematic variability that could be attributed to differing laboratory conditions and for HSV-1 and HSV-2 variability attributable to either the binding of the assay components to the plates or calibration control samples that were not effective at reducing the variation between plates. For quantitative assessments improvements to the kit techniques which we used are needed, possibly using different antigens. In the current study we randomised the patients' samples to the plates so that the systematic differences will not have biased our results and took account of these systematic sources of variability in the analyses by adjusting for the plate and day on which the assay was run.

In the black adult population of greater Johannesburg neither mean IgG antibody measure nor high antibody measures against human herpesvirsues 1 – 6 were strongly associated with any of the seven selected cancer groups. We do not think that the variability in the quantitative assay results concealed any strong associations, but we may not have had sufficient power to detect weak associations or associations with a sub-type of cancer. The finding of a small increased risk for oral cancer with increased antibodies to HHV-6 merits further exploration.