Correlates of anti-EBV EBNA1 IgA positivity among unaffected relatives from nasopharyngeal carcinoma multiplex families

Background: To determine whether non-viral nasopharyngeal carcinoma (NPC) risk factors might be associated with (and mediated through) Epstein–Barr virus (EBV) serological responses linked to NPC risk, we evaluated predictors of risk of anti-EBNA1 IgA seropositivity and other markers among unaffected relatives from a large NPC family study in Taiwan. Methods: Multivariate logistic regression conditioned on family was used to examine the associations between sociodemographic, dietary, lifestyle, and occupational variables and risk of anti-EBV EBNA1 IgA positivity, anti-VCA IgA, and anti-DNase positivity. Results: Among 2393 unaffected relatives from 319 multiplex families, 1180 (49.3%) were anti-EBV EBNA1 IgA seropositive. None of the associations with anti-EBNA1 IgA were statistically significant, except for being 31–50 years of age (vs <30, adjusted ORs 0.51–0.57). For one or more EBV serological markers, there were suggestive associations for older age, GuangDong firm salted fish, betel use, current alcohol use, and male gender. Conclusion: Overall, we found little evidence to suggest that non-viral NPC risk factors significantly alter EBV serological patterns, suggesting that non-viral NPC risk factors act through pathways independent of EBV serological responses.

Nasopharyngeal carcinoma (NPC) is rare, albeit relatively common in southern China, southeast Asia, the Arctic, and North Africa (Chang and Adami, 2006). NPC risk factors include male gender, increased age, southern Chinese ethnicity, less education, consumption of salted fish and other preserved foods containing elevated levels of nitrate/nitrosamines, reduced fruit and vegetable consumption, cigarette smoking, betel nut chewing, alcohol consumption, and occupational exposures (Chang and Adami, 2006). Epstein -Barr virus (EBV), a ubiquitous herpesvirus that infects over 90% of adults worldwide, is believed to be necessary, but not sufficient for developing NPC (Chang and Adami, 2006). Longitudinal studies in China and Taiwan have shown that positivity for anti-EBNA1 IgA, anti-VCA IgA, and anti-DNase antibodies were associated with a significantly elevated NPC risk (Zeng et al, 1983(Zeng et al, , 1985Chien et al, 2001;Ji et al, 2007;Hsu et al, 2009;Ng et al, 2010;Yu et al, 2011).
Whether non-viral risk factors act independently of anti-EBV serological responses or mediate EBV effects on NPC development is not known. To address this question, we evaluated the association between non-viral NPC risk factors and EBV serological responses in a group of healthy individuals from high-risk NPC multiplex families in Taiwan.

METHODS
Participants in the present study were part of the NPC multiplex family study in Taiwan, previously described (Pickard et al, 2004;Yang et al, 2005;Yu et al, 2009). Over 300 NPC multiplex families were identified and recruited, including 659 NPC cases and 2557 unaffected parents, siblings, spouses, and children.
Risk factors were assessed by questionnaire for all individuals by a trained nurse. Sociodemographic characteristics were age, gender, ethnicity, and education. Lifestyle factors included smoking, betel use, and alcohol use. Occupational exposures evaluated included duration of formaldehyde exposure and wood exposure.
Dietary intake during ages 10 -30 (representing consumption during adolescence and adulthood) was assessed by food frequency questionnaire, such as: consumption of salted fish, other preserved foods (salted meat, smoked foods, preserved eggs, fried/fermented bean curd, fermented rice, and fermented flour sauce), and fruits and vegetables. Other preserved foods were combined into a single variable by summing across the variables, and dividing into categories corresponding to the questionnaire diet frequency categories. Serum from participants was tested for the following EBV antibody markers: anti-VCA IgA by the immunofluorescent assay, anti-EBNA1 IgA by enzyme-linked immunosorbent assay, and anti-DNase by an enzyme neutralisation assay as previously described (Pickard et al, 2004). For the present analysis, a total of 2393 unaffected family members from 319 NPC multiplex families with data on EBV serology were included.
All estimates in this analysis were obtained using conditional logistic regression with the SAS PHREG procedure (SAS version 9.2), conditioning on families to account for familial correlations (Pfeiffer et al, 2003). We chose anti-EBNA1 IgA (at a cut-off of OD 405 X0.10) as the primary outcome of interest for this analysis, because this measure was optimised for high sensitivity and most strongly predicted NPC risk in our family study (Yu et al, 2011). In secondary analyses, we evaluated (1) anti-EBNA1 IgA (OD 405 X0.20) with an alternative cut-off used for studies of sporadic NPC, (2) anti-VCA IgA based on the cut-off of 1 : 10 or greater dilution of serum, and (3) DNase neutralising activity, based on a cut-off optimised for high sensitivity in our family study (positivity cut-off ¼ 160 or more neutralising units; Yu et al, 2011). We also evaluated anti-EBNA1 IgA without the more ambiguous group (0.10pOD 405 o0.20), so that a clean, positive group (OD 405 X0.20) was compared with a clean, negative group (OD 405 o0.10). However, the results resembled those of the original outcome (OD 405 X0.20 vs OD 405 o0.20), and therefore, were not presented.
Variables selected for consideration in the adjusted models were based on significant or borderline significant associations (based on the 95% confidence intervals (CI)) with EBNA1 positivity (OD 405 X0.10). Dose response was based on calculating overall Wald P-values for categorical variables.

RESULTS
There were a total of 2393 unaffected family members in this study. The mean age was 46 years (47 in women and 44 in men), and 53% were women.

DISCUSSION
With a couple of notable exceptions discussed below, we saw little evidence to indicate that non-viral NPC risk factors influence anti-EBV seroreactivity. This suggests that non-viral NPC risk factors are unlikely to influence NPC risk by altering anti-EBV serological profiles.
We observed a U-shaped curve for age, with anti-EBNA1 IgA positivity being higher among the youngest and oldest, and lower among the middle age categories. The higher antibody positivity rate observed among older individuals, also seen for anti-VCA IgA, could reflect immunosenescence in older age, leading to more frequent viral lytic reactivation (Agarwal and Busse, 2010). The fact that anti-EBNA1 IgA positivity, but not other markers, was also higher among the youngsters, is not clearly understood.
There was a suggestive elevated risk for all EBV markers (aORs 1.6 -2.7) associated with GuangDong firm salted fish, but not mouldy fragrant fish. In an in vitro study, aqueous extracts of Cantonese salted fish activated EBV lytic replication in Raji cells in a dose-dependent manner by causing cells to express EBV early antigen (Shao et al, 1988). However, it is unclear why one type of GuangDong salted fish would activate EBV, but not another type.
A significant association was observed between betel use and anti-DNase positivity, and was suggestive for anti-EBNA1. Although betel nut use is classified as a group 1 carcinogen in humans, there is no data on whether or not betel use can lead to EBV reactivation (IARC, 2004). Betel nut ingredients have induced inflammation in vitro, supporting the biological plausibility of this association (Jeng et al, 2000(Jeng et al, , 2003Chang et al, 2005).
There may be recall bias of diet between the ages of 10 -30, such that young subjects may be prone to recall their diet in adolescence and older subjects prone to recall diet in their adulthood. EBV serology was measured at only one point in time and may not capture all episodes of EBV lytic replication. We would not have detected associations with other aspects of EBV exposure and/or host response to EBV. Our results may have been affected by the reproducibility for anti-VCA IgA testing, which was modest (agreementB68%, kB0.29 -0.38; Pickard et al, 2004). Our findings for a high-risk population may not represent that of the general population, and associations may be attenuated due to similarity of exposures within high-risk families (Yang et al, 2005). However, by studying unaffected relatives from NPC multiplex families, the population is enriched in terms of EBV IgA antibody positivity, which occurs at much lower frequencies in the general population (Pickard et al, 2004). Additional strengths of the study include the large sample size, recruitment through cases identified from the national cancer registry, availability of detailed risk factor information, as well as multiple EBV serological markers.
In summary, the majority of NPC risk factors were not found to be significantly associated with anti-EBNA1 IgA positivity, the strongest predictor of NPC risk in our study population. In conclusion, our data suggest that non-viral NPC risk factors affect NPC risk via mechanisms other than through effects on EBV reactivation or host antibody responses to such infections.
This work is published under the standard license to publish agreement. After 12 months the work will become freely available and the license terms will switch to a Creative Commons Attribution-NonCommercial-Share Alike 3.0 Unported License.