In vitro EBV-infected subline of KMH2, derived from Hodgkin lymphoma, expresses only EBNA-1, while CD40 ligand and IL-4 induce LMP-1 but not EBNA-2

In about 50% of classical Hodgkin lymphomas, the Hodgkin/Reed Sternberg (H/RS) cells carry Epstein-Barr virus (EBV). The viral gene expression in these cells is restricted to EBNA-1, EBERs, LMP-1 and LMP-2 (type II latency). The origin of H/RS cells was defined as crippled germinal center B cells that escaped apoptosis. In spite of numerous attempts, only few typical Hodgkin lymphoma (HL) lines have been established. This suggests that the cells require survival factors that they receive in the in vivo microenvironment. If EBV is expected to drive the cells for growth in culture, the absence of EBNA-2 may explain the incapacity of H/RS cells for in vitro proliferation. In EBV carrying B lymphocytes, functional EBNA-2 and LMP-1 proteins are required for in vitro growth. For analysis of the interaction between EBV and the H/RS cells, we infected the CD21-positive HL line KMH2 with the B958 and Akata viral strains. Only EBNA-1 expression was detected in a few cells in spite of the fact that all cells could be infected. Using a neomycin-resistance-tagged recombinant EBV strain (Akata-Neo) we established an EBV-positive subline that was carried on selective medium. In contrast to the type II EBV expression pattern of H/RS cells in vivo, the KMH2 EBV cells did not express LMP-1. The EBV expression pattern could be modified in this type I subline. LMP-1 could be induced by the histone deacetylase inhibitors TSA and n-butyrate, by 5-AzaC, a demethylating agent, and by phorbol ester. None of these treatments induced EBNA-2. Importantly, exposure to CD40 ligand and IL-4 induced LMP-1 without EBNA-2 expression and lytic replication. The KMH2 EBV cells expressed LMP-2A, but not LMP-2B mRNAs. This result is highly relevant for the type II expression pattern of H/RS cells in vivo, since these stimuli can be provided by the surrounding activated T lymphocytes.     

Epstein‐Barr virus‐positive diffuse large B‐cell lymphoma association is not only restricted to elderly patients

Diffuse large B‐cell lymphoma (DLBCL), the most common group of malignant lymphomas, account for 30% of adult non‐Hodgkin lymphomas. The 2008 World Health Organization (WHO) classification included a new entity, Epstein‐Barr virus (EBV)+ DLBCL of the elderly, affecting patients aged 50 years or older. However, some reports of younger EBV+ DLBCL cases, without evidence of underlying immunosuppression, can be found. The role of EBV in tumor microenvironment composition in DLBCL is still not well understood. Our aim was to assess EBV presence and latency pattern as well as tumor T‐cell population in an adult DLBCL series of Argentina. The study was conducted on biopsies from 75 DLBCL patients. EBERs expression was performed by in situ hybridization, while EBV gene expression was analyzed using real‐time polymerase chain reaction. LMP1, LMP2A, EBNA2, EBNA3A, CD4, CD8 and Foxp3 expression was assessed by immunohistochemistry. Nine percent of cases showed EBV expression, with similar frequency among patients younger than 50 years and 50 years or older (13% and 8%, respectively). T‐cell subsets were not altered by EBV presence. Latency type II was the most frequently observed, together with lytic gene expression in EBV+ DLBCL, with ≥20% of EBERs+ cells. These findings suggest that EBV+ DLBCL in our series was similar to the previously described in Asia and Latin‐America, displaying latency II or III expression profile and no age‐specific characteristics. Finally, EBV+ DLBCL may be an entity that is not only restricted to patients who are older than 50 years of age, in consequence the age cutoff revision may be a current goal.     

Lymphoproliferative disorders in autoimmune diseases in Japan: analysis of clinicopathological features and Epstein-Barr virus infection

Lymphoproliferative disorders (LPD) occasionally develop in individuals with immune deficiencies such as immunosuppressive conditions and autoimmune diseases (AID). In our study, the clinicopathologic features and virus status were analyzed in 53 cases with LPD developing in rheumatoid arthritis (RA) and other AID. AID in only 4 of 53 patients had been treated with some sort of immunosuppressive therapy, including methotrexate. Median age at the diagnosis of LPD in AID was 60 years old with marked female predominance (M/F = 0.4). The median interval between the onset of AID and LPD development was 45 months, and longer in RA patients than in other AID (p < 0.01). The primary site of lymphoma was nodal in 21 cases and extra-nodal in 24, with clinical Stage I in 17, II in 5, III in 13, and IV in 13. Immunohistochemistry showed that 39 cases were B cell type, 10 were T cell type and 4 were Hodgkin lymphoma (HL). Then majority of B cell cases were diffuse large B cell lymphomas, and 2 were diffuse polymorphic type. EBER-1 in situ hybridization for Epstein-Barr virus (EBV) showed positive signals in tumor cells in 16 of 53 (30.2%) cases. The EBV-positive rate in T cell LPD (70%) was much higher than that in B cell LPD (12.8%) (p < 0.01). All 4 cases of HL were EBV-positive. Immunohistochemistry showed a latency II pattern of EBV infection (LMP-1(+) and EBNA-2(-)). Five-year overall survival rate was 33%. Multivariate analysis showed that only type of AID was an independent factor for survival of patients, i.e., LPD in RA showed the most favorable prognosis. In conclusion, LPD in AID generally shared common features with sporadic LPD except for a much higher EBV-positive rate in T cell LPD.     

Antibody titers against EBNA1 and EBNA2 in relation to Hodgkin lymphoma and history of infectious mononucleosis

A role for Epstein Barr virus (EBV) in Hodgkin lymphoma (HL) pathogenesis is supported by the detection of EBV genome in about one-third of HL cases, but is not well defined. We previously reported that an elevated prediagnosis antibody titer against EBV nuclear antigens (EBNA) was the strongest serologic predictor of subsequent HL. For the present analysis, we measured antibody levels against EBNA components EBNA1 and EBNA2 and computed their titer ratio (anti-EBNA1:2) in serum samples from HL cases and healthy siblings. We undertook this analysis to examine whether titer patterns atypical of well-resolved EBV infection, such as an anti-EBNA1:2 ratio ≤ 1.0, simply reflect history of infectious mononucleosis (IM), an HL risk factor, or independently predict HL risk. Participants were selected from a previous population-based case-control study according to their history of IM. We identified 55 EBV-seropositive persons with a history of IM (IM+; 33 HL cases, 22 siblings) and frequency-matched a comparison series of 173 IM history-negative, EBV-seropositive subjects on HL status, gender, age and year of blood draw (IM-; 105 cases, 58 siblings). In multivariate logistic regression models, an anti-EBNA1:2 ratio ≤ 1.0 was significantly more prevalent in HL cases than siblings (odds ratio, 95% confidence interval = 2.43, 1.05-5.65); similar associations were apparent within the IM+ and IM- groups. EBNA antibodies were not significantly associated with IM history in HL cases or siblings. These associations suggest that chronic or more severe EBV infection is a risk factor for HL, independent of IM history.     

Antagonistic effects of c-myc and Epstein-Barr virus latent genes on the phenotype of human B cells

Epstein-Barr virus (EBV) immortalized cells and Burkitt lymphoma cells have a completely different growth pattern and phenotype. EBV immortalized cells express a set of 11 viral genes to accommodate B cell activation and proliferation, whereas EBV-positive Burkitt lymphoma cells highly express the c-myc oncogene that is activated through translocation into 1 of the immunoglobulin loci and EBNA1 as the only viral protein. We have developed a primary human B cell line conditionally immortalized by Epstein-Barr virus in which the viral gene program responsible for the induction of proliferation can be switched on and off by the addition or withdrawal of estrogen (cell line EREB2-5). Starting from this cell line we have generated 2 daughter cell lines that proliferate in a c-myc dependent fashion, 1 with a highly active exogenous c-myc gene (cell line A1) and 1 with a regulatable c-myc gene that can be switched on by withdrawal and switched off by addition of tetracycline (cell line P493-6). The comparison of the 3 cell lines has allowed us to dissect the contribution of c-myc and EBV genes to the regulation of the growth pattern and expression of cell surface molecules. We show that MYC and EBNA2 (and their respective target genes) have opposing effects on the expression of several surface markers involved in B cell activation. We show that MYC contributes to the phenotype of Burkitt lymphoma cells by upregulating CD10 and CD38 and downregulating activation markers. The phenotype of the cells is determined on one hand by the absence of the viral gene products EBNA2 and LMP1 that mediate the phenotype of activated lymphoblasts and to a lesser extent by an active contribution of the c-myc gene.     

Epstein-Barr virus infection and risk of lymphoma: immunoblot analysis of antibody responses against EBV-related proteins in a large series of lymphoma subjects and matched controls

Epstein-Barr Virus (EBV) is consistently associated with distinct lymphoproliferative malignancies and aberrant EBV antibody patterns are found in most EBV cancer patients. We evaluate the detection of an abnormal reactive serological pattern to EBV (ab_EBV) infection and the risk of lymphoma in a multicentric case-control study. Serum samples were collected at study entry from 1,085 incident lymphoma cases from Spain, France, Germany, Czech Republic, Italy and 1,153 age, sex and country matched controls. EBV immunoglobulin G (IgG) serostatus was evaluated through a peptide-based ELISA combining immunodominant epitopes of EBNA1 (BKRF1) and VCA-p18 (BFRF3). Further, immunoblot analysis was performed to evaluate distinct antibody diversity patterns to EBV early antigens (EA), besides EBNA1, VCA-p18, VCA-p40 (BdRF1) and Zebra (BZLF1). Patients with chronic active EBV infection and aberrant EBV activity were characterized as having an abnormal reactive pattern (ab_EBV). Ab_EBV was observed in 20.9% of 2,238 included subjects with an increased proportion of cases presenting ab_EBV as compared to the control population (23.9% vs. 18.0% p = 0.001). Ab_EBV positivity was a risk factor for all lymphomas combined (odds ratio [OR] = 1.42, 95% confidence interval [CI]=1.15-1.74), and specifically for chronic lymphocytic leukaemia (OR = 2.96, 95%CI = 2.22-3.95). Lower levels of ab_EBV were observed for follicular lymphoma (OR = 0.38, 95%CI = 0.15-0.98). EBV may be involved in a larger subset of lymphomas among clinically immunocompetent subjects than previously thought, probably explained by an underlying loss of immune control of EBV latent infection. Ab_EBV is a useful tool to explore EBV imbalances preceding or paralleling possible EBV associated oncogenic events.     

SH2D1A expression in Burkitt lymphoma cells is restricted to EBV positive group I lines and is downregulated in parallel with immunoblastic transformation

The SH2 domain containing SH2D1A protein has been characterized in relation to the X-linked lymphoproliferative disease (XLP), a primary immunodeficiency that leads to serious clinical conditions after Epstein-Barr virus (EBV) infection. The SH2D1A gene is mutated in the majority of XLP patients. We previously detected SH2D1A in activated T and NK cells, but not in B lymphocytes. We have found SH2D1A protein in Burkitt lymphoma (BL) lines, but only in those that carried EBV and had a Group I (germinal center) phenotype. All the EBV-carrying Group III (immunoblastic) and the EBV-negative BL lines tested were SH2D1A-negative. Motivated by these differences, we studied the impact of EBV and the cellular phenotype on SH2D1A expression. We approached the former question with BL sublines after both the loss of the virus and subsequent reinfection. We also tested original EBV-negative BL lines carrying transfected EBV genes, such as EBNA1, EBNA2, EBNA6, EBER1, 2 and LMP1, respectively. In our experiments, no direct relationship could be seen between EBV and SH2D1A expression. We modified the phenotype of the Group I BL cells by LMP1 transfection or CD40 ligation. The phenotypic changes, indicated by expression of immunoblastic markers, e.g., SLAM, were accompanied by downregulation of SH2D1A. It seems, therefore, that the presence of EBV and the phenotype of the cell together regulate SH2D1A expression in the BL cells. It is possible that SH2D1A is expressed in a narrow window of B cell development represented by germinal center cells.     

非免疫缺陷相关性B细胞淋巴瘤与EB病毒的相关性研究

目的探讨非免疫缺陷相关性淋巴结内Ｂ细胞淋巴瘤与ＥＢ病毒感染的关系。方法常规染色进行组织学分类,利用免疫组织化学ＬＳＡＢ法确定肿瘤的免疫表型。采用原位杂交方法检测ＥＢ病毒编码的小核ＲＮＡ（ＥＢＥＲｓ）。结果４０例淋巴结内Ｂ细胞淋巴瘤中,低度恶性２０例（中心母细胞－中心细胞性１２例、中心细胞性４例、淋巴细胞性４例）,高度恶性２０例（中心母细胞性１７例、免疫母细胞性２例、Ｂｕｒｋｉｔｔ样１例）。所有病例中,Ｅ－ＢＥＲｓ阳性３例,占所检病例的７．５％,其中低度恶性１例,占低度恶性病例的５％,高度恶性２例,占高度恶性病例的１０％,阳性细胞约占肿瘤细胞的３０％～６０％。结论非免疫缺陷相关性淋巴结内Ｂ细胞淋巴瘤可能与ＥＢ病毒的感染有关,但不是主要致病因素。感染率与恶性程度无明显关系。     

间变性大细胞淋巴瘤EB病毒基因表达产物的检测及其意义

目的：了解间变性大细胞淋巴瘤与埃泼斯坦－巴尔（ＥＢ）病毒的关系。方法：采用原位杂交及免疫组化ＬＳＡＢ法对１２例间变性大细胞淋巴瘤（ＡＬＣＬ）进行ＥＢＶ编码的ＲＮＡ（ＥＢＥＲｓ）及ＥＢＶ潜伏膜蛋白（ＬＭＰＩ）和潜在膜抗原检测。结果：本组１２例ＡＬＣＬ中ＥＢＥＲｓ阳性率为２５％（３／１２）,１２例ＡＬＣＬ ＬＭＰ１检出率为８．３％,ＥＢＮＡ２检出率为０（０／１２）;１２例ＡＬＣＬ免疫表型,８例为Ｔ源性     

Pyothorax-associated lymphoma (PAL) with biclonal Epstein–Barr virus infection: Characterization of a novel PAL cell line with unique features

Pyothorax-associated lymphoma (PAL) is a representative form of diffuse large B-cell lymphoma associated with chronic inflammation, in which the Epstein–Barr virus (EBV) genome is consistently detectable in the lymphoma cells of all PAL cases. Cell lines and animal models would be useful for understanding better this rare lymphoma, but reports of PAL-derived cell lines are scarce. We report a new PAL cell line, designated Pal-2, with unique phenotypic expression. Pal-2 is the first PAL cell line that carries a biclonal EBV infection with abundant viral genome and that exhibits tumorigenic capacity once injected into nude mice.     

EBV-positive pyothorax-associated lymphoma expresses CXCL9 and CXCL10 chemokines that attract cytotoxic lymphocytes via CXCR3

Epstein–Barr virus (EBV)-positive diffuse large B-cell lymphoma associated with chronic inflammation (DLBCL-CI) develops in the setting of long-standing inflammation. This type of lymphoma may have specific expression profiles of chemokines involved in the pathogenesis of DLBCL-CI. EBV-positive pyothorax-associated lymphoma (PAL) is a prototype of DLBCL-CI and represents a valuable model for the study of this disease category. Using a panel of PAL cell lines, we found that PAL cells expressed and secreted C–X–C motif chemokine ligands 9 and 10 (CXCL9 and CXCL10), the ligands of CXCR3, in contrast to EBV-negative DLBCL cell lines, which did not. Culture supernatants from PAL cell lines attracted CXCR3-expressing CD4⁺ T cells, CD8⁺ T cells, and CD56⁺ natural killer cells from human peripheral blood mononuclear cells. PAL cells injected into mice also attracted CXCR3-positive cytotoxic lymphocytes that expressed interferon-γ. The expression of CXCL9 and CXCL10 was detected in PAL tumor biopsy samples from patients, and CXCR3-positive lymphocytes were abundant in the tissue samples. Collectively, these findings suggest that CXCL9 and CXCL10 are produced by PAL cells and can elicit cytotoxic responses via CXCR3. This chemokine system is also likely to contribute to tissue necrosis, which is a signature histological feature of DLBCL-CI. Further studies are warranted to determine whether the CXCL9–CXCL10/CXCR3 axis exerts antitumor effects in DLBCL-CI.     

结外NK/T细胞淋巴瘤检测EB病毒与疗效关系的研究*

目的：探讨血浆中EB病毒阳性在评价结外NK/T细胞淋巴瘤近期疗效和远期疗效中的临床意义。方法：收集2011年1 月至2014年4 月郑州大学第一附属医院经病理及免疫组化确诊为结外NK/T细胞淋巴瘤的患者109 例,应用qRT-PCR方法检测其血浆中EBV-DNA拷贝数,比较EBV 阴性和EBV 阳性患者对治疗疗效与预后的差异。结果：109 例结外NK/T细胞淋巴瘤患者,53例阳性患者中有34例（64.2%）为晚期（Ⅲ～Ⅳ期）,56例阴性患者中22例（39.3%）为晚期（Ⅲ～Ⅳ期）,EBV 阳性组中伴有B 症状
33例（62.3%）,阴性组患者21例（37.5%）,EBV 阴性患者与EBV 阳性患者在分期及B 症状方面均具有统计学意义（P<0.05）。 34例（60.7%）EBV 阴性结外NK/T细胞淋巴瘤患者达到客观缓解率,显著高于EBV 阳性患者的客观缓解率（22例,41.5%）（P<0.05）。EBV 阴性组与EBV 阳性组相比,其2 年无进展生存期更高（P<0.05）。 结论：检测结外NK/T细胞淋巴瘤患者血浆中EB病毒对评价其近期疗效及2 年无进展生存期具有重要的临床意义。     

Monocytes enhance cell proliferation and LMP1 expression of nasal natural killer/T-cell lymphoma cells by cell contact-dependent interaction through membrane-bound IL-15

Nasal natural killer (NK)/T-cell lymphoma (NNKTL) is an Epstein-Barr virus (EBV)-related malignancy with poor prognosis and has distinct histological features characterized by angiocentric and polymorphous lymphoreticular infiltrates including inflammatory cells such as granulocytes, monocytes, macrophages and lymphocytes. Here, we show that the monocytes enhance proliferation as well as LMP1 expression of NNKTL cells by cell contact-dependent interaction through membrane-bound interleukin (IL)-15. We used two EBV-positive NK-cell lines, SNK6 and KAI3, which originated from two patients-SNK6 from a patient with NNKTL and KAI3 from a patient with a severe mosquito allergy. We cocultured the cell lines with granulocytes or monocytes and examined whether proliferation, survival and LMP1 expression of the cells changed. Although cocultured granulocytes did not affect proliferation, survival or LMP1 expression of the cells, cocultured monocytes enhanced both proliferation and LMP1 expression in a dose-dependent manner. These phenomena were not seen when monocytes were placed in a separate chamber. Moreover, the monocyte-inducible proliferation and LMP1 expression were inhibited by treatment with an antibody against IL-15. Furthermore, production of interferon-gamma-inducible protein (IP)-10 were enhanced by coculture with monocytes and were inhibited by the antibody. Immunohistological studies confirmed that a number of infiltrating CD14-positive monocytes contacted CD56-positive lymphoma cells in all of 20 NNKTL tissues tested. These results suggest that monocytes enhance cell growth as well as LMP1 expression of NNKTL cells by cell contact-dependent interaction through membrane-bound IL-15. In the microenvironment of NNKTL tissue, a positive feedback loop of interaction between lymphoma cells and monocytes may be present and contribute to lymphoma progression.     

Response and survival benefit with chemoimmunotherapy in Epstein‐Barr virus‐positive diffuse large B‐cell lymphoma

Epstein‐Barr virus (EBV)‐positive diffuse large B‐cell lymphoma (DLBCL) is a haematologic malignancy with poor prognosis when treated with chemotherapy. We evaluated response and survival benefits of chemoimmunotherapy in EBV‐positive DLBCL patients. A total of 117 DLBCL patients were included in our retrospective analysis; 33 were EBV‐positive (17 treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R‐CHOP] and 16 with CHOP), and 84 were EBV‐negative (all treated with R‐CHOP). The outcomes of interest were complete response (CR) and overall survival (OS) in EBV‐positive DLBCL patients (R‐CHOP versus CHOP) and in DLBCL patients treated with R‐CHOP (EBV‐positive vs EBV‐negative). There were no differences in the clinical characteristics between EBV‐positive and EBV‐negative DLBCL patients. Among EBV‐positive DLBCL patients, R‐CHOP was associated with higher odds of CR (OR 3.14, 95% CI 0.75‐13.2; P = .10) and better OS (hazard ratio 0.30, 95% confidence interval [CI] 0.09‐0.94; P = .04). There were no differences in CR rate (OR 0.52, 95% CI 0.18‐1.56; P = .25) or OS (hazard ratio 0.93, 95% CI 0.32‐2.67; P = .89) between EBV‐positive and EBV‐negative DLBCL patients treated with R‐CHOP. Based on our study, the addition of rituximab to CHOP is associated with improved response and survival in EBV‐positive DLBCL patients. Epstein‐Barr virus status does not seem to affect response or survival in DLBCL patients treated with R‐CHOP.     

Epstein Barr virus in relation to apoptosis markers and patients' outcome in pediatric B-cell non-Hodgkin lymphoma

In this study, we investigated Epstein Barr virus (EBV) presence, associated to proliferation and apoptosis proteins in pediatric B-cell Non-Hodgkin lymphoma (B-NHL). EBERs, Ki67, active caspase 3, Bax and Bcl2 were analyzed on B-NHL tissue from 40 patients. Forty percent showed EBV expression, significantly higher among patients ?10 years (P = 0.027), and associated with immunosuppression (P = 0.020), but not associated apotosis markers. However, EBV was associated with a worse event-free survival (P = 0.016), particularly under immunosuppression. Even though EBV did not seem to alter apoptotic pathways, it exhibited survival disadvantage and could be an important cofactor in B-cell lymphomagenesis in younger children.     

EBV primary infection in childhood and its relation to B‐cell lymphoma development: A mini‐review from a developing region

In most underdeveloped countries, the initial contact with Epstein Barr virus (EBV) usually happens in the first decade of life and results in an asymptomatic infection, whereas in developed areas, primary infection in adolescence or adulthood is accompanied by infectious mononucleosis in 50% cases. Although it is generally a harmless passenger, in some individuals, it is associated with B‐cell lymphoma. In Argentina, EBV primary infection shows the classical pattern observed in developing populations, given that nearly 70% of patients are seropositive by the age of 2 years. However, EBV association with pediatric Hodgkin and Burkitt lymphoma resembles that observed in developed regions. Concerning diffuse large B‐cell lymphoma, our series demonstrated higher EBV association than other adult ones from either developed or underdeveloped countries. Interestingly, the early EBV primary infection observed, characteristic of an underdeveloped population, together with the statistically significant EBV association with patients ≤10 years old demonstrated in all types of lymphoma studied, suggest a relationship between low age of EBV seroconversion and B‐cell lymphoma development risk.     

Geographic variation in Epstein-Barr virus-associated Burkitt's lymphoma in children from Brazil

In developing countries, BL has a strong association with EBV infection during childhood. In South America, the data have shown an EBV association intermediate between that reported in the United States (30%) and that in equatorial Africa (95%). Early age at EBV infection and lower socioeconomic status have been related to increased EBV-associated BL in developing countries. In Brazil, there are not enough data on childhood BL related to EBV infection. Our aim was to evaluate the clinicopathologic features and EBV association of 44 children with NHL from the state of Rio de Janeiro, situated in the southeast of Brazil. EBV was detected using RNA in situ hybridization in 36 biopsy specimens. DNA from fresh tumor samples and from paraffin-embedded tissues of patients were analyzed by PCR, in which the first reaction included primers for an EBNA-2 common region while the nested reaction amplified the region discriminating between EBV types 1 and 2 in separate reactions. EBV was detected in 21 of 29 BLs (72%), and type 1 virus infected the majority of EBV-positive BLs (18/21). There was a trend for younger age in children with EBV-positive BL compared to EBV-negative BL (median age 4 compared to 6 years, respectively; p = 0.056). Our study confirmed that in the southeast of Brazil BL had an intermediate association with EBV. A higher rate of EBV-associated BL was described in the northeast of Brazil. These differences are probably related to regional socioeconomic status. In conclusion, our study suggests that early infection with EBV in the background of a low socioeconomic condition associated with other environmental factors could contribute to BL in Brazil.     

Epstein-Barr virus seroreactivity among unaffected individuals within high-risk nasopharyngeal carcinoma families in Taiwan

Most adults have been infected with EBV. Many studies have indicated that antibodies against specific EBV antigens, particularly IgA antibodies, can be predictive or prognostic of EBV-associated malignancies, such as NPC. We hypothesized that healthy individuals from families with a history of multiple members affected with NPC (who therefore might be genetically susceptible to NPC themselves) might have an EBV antibody profile that is distinct from that seen in healthy individuals from the community at large. To explore this possibility and examine determinants of anti-EBV antibody levels in healthy, high-risk individuals, we evaluated data from 2 parallel studies of NPC in Taiwan, which included 1,229 healthy members of families in which 2 or more individuals were affected with NPC and 320 controls from the community at large. Blood collected from participants was tested for IgA antibodies against EBV VCA and EBNA-1 and for neutralizing antibodies against EBV DNase using standard assays. We observed evidence of familial aggregation of EBV seroreactivity among individuals from high-risk, multiplex NPC families. Anti-VCA IgA and anti-EBNA-1 IgA antibody seroprevalence in unaffected family members of NPC cases was 5-6 times higher than in members of the community (p < 0.01). This elevated seroprevalence among unaffected individuals from high-risk families was observed regardless of the relationship of the unaffected individual to the closest affected relative (siblings, parents, children or spouses). No sociodemographic or environmental factors examined were found to strongly and consistently correlate with elevated seroprevalence, but patterns emerged of increasing seroprevalence among older individuals and among females. Unaffected individuals from high-risk NPC families have elevated anti-EBV IgA antibody titers. The etiologic and clinical implications of this finding remain to be established.