Expression of EBV-encoded genes in children with asymptomatic infection detected by sensitive methods.

Epstein-Barr virus (EBV) is an usually harmless virus whose oncogenic properties in vitro are related to its ability to transform lymphoid cells, and, in consequence, it can be associated with lymphomas. Since a few studies detected EBV presence in supposedly EBV-negative lymphomas, our aim was to evaluate EBV presence by sensitive gene expression assays in the tonsils from healthy pediatric donors from a region with high incidence of EBV-associated lymphomas. EBERs transcripts were detected by View RNA ISH in all cases, even in cases assessed negative by widely used in situ hybridization. The presence of LMP1 transcripts was proved in 93% of cases, co-expressed with EBNA2 in 30%. In this study, evidence for the expression of different latent and lytic viral genes in a population of young age of primary infection, detected with more sensitive methods, in particular at the germinal center, where most EBV-associated lymphomas originate, was provided.

M1 Macrophage Polarization Prevails in Epstein-Barr Virus-Infected Children in an Immunoregulatory Environment.

Macrophages can be polarized toward a proinflammatory phenotype (M1) (CD68+) or to an anti-inflammatory one (M2) (CD163+). Polarization can be triggered by cytokines such as IFN-γ for M1, or IL-10 and TGF-ß, for M2. In the context of pediatric Epstein Barr virus (EBV) infection, little is known about macrophage polarization in EBV primary or persistent infection. When studying tonsils of patients undergoing primary infection (PI), healthy carrier (HC), reactivation (R), and not infected (NI), M1 profile prevailed in all infection status. However, an increase in M2 cells was observed in those patients with broader expression of latency antigens, in particular EBNA2. Tonsils from primary infected patients showed an increased IL-10 expression, whereas, unexpectedly, TGF-ß expression correlated with M1 marker. Furthermore, an inverse correlation was demonstrated between CD68 and IFN-γ. Therefore, in the context of asymptomatic infection in children, M1 macrophage polarization prevails, even in the presence of IL-10 and TGF-Ꞵ immunomodulatory cytokines, and it might be independent from lymphomagenesis process. Our finding indicates that macrophages may have a significant plasticity in response to different types of extrinsic stimuli, and further studies are required to investigate M1 polarization under anti-inflammatory stimuli. IMPORTANCE Most studies on Epstein Barr virus (EBV) primary infection have been performed in adolescents and young adult populations with Infectious Mononucleosis (IM) in developed countries. Furthermore, studies related to macrophage polarization were assessed in EBV-associated lymphomas, but little is known about macrophage polarization in the context of primary infection at the site of viral entry and replication, the tonsils. Therefore, the aim of this study was to characterize macrophage response in children undergoing EBV primary or persistent infection, in order to enlighten the role of macrophages in viral pathogenesis, in a population with a high incidence of EBV-associated lymphomas in children younger than 10 years old. This study may contribute to explain, at least in part, the asymptomatic viral infection in children from an underdeveloped region, given that M1 polarization pattern prevails, but in a regulatory environment.

Epstein-Barr virus recruits PDL1-positive cells at the microenvironment in pediatric Hodgkin lymphoma.

BACKGROUND: Classic Hodgkin lymphoma (cHL) is a lymphoid malignancy in which the microenvironment, where the neoplastic cells are immersed, contributes to the lymphomagenesis process. Epstein-Barr virus (EBV) presence also influences cHL microenvironment composition and contributes to pathogenesis. An increase in PDL1 expression in tumor cells and at the microenvironment was demonstrated in adult cHL. Therefore, our aim was to assess PD1/PDL1 pathway and EBV influence on this pathway in pediatric cHL, given that in Argentina, our group proved a higher incidence of EBV-associated pediatric lymphoma in children. METHODS: For that purpose, EBV presence was assessed by in situ hybridization, whereas PD1 and PDL1 expressions were studied by immunohistochemistry. PDL1 genetic alterations were analyzed by FISH, and survival was evaluated for PD1 and PDL1 expressions. RESULTS: EBV presence demonstrated no influence neither on PD1 expression at the microenvironment nor on PDL1 expression at HRS tumor cells. Unexpectedly, only 38% pediatric cHL displayed PDL1 genetic alterations by FISH, and no difference was observed regarding EBV presence. However, in EBV-related cHL cases, a higher number of PDL1 + cells were detected at the microenvironment. CONCLUSION: Even though a high cytotoxic environment was previously described in EBV-related pediatric cHL, it might be counterbalanced by an immunoregulatory micro-environmental PDL1 + niche. This regulation may render a cytotoxic milieu that unsuccessfully try to eliminate EBV + Hodgkin Reed Sternberg tumor cells in pediatric patients.

Distinctive EBV infection characteristics in children from a developing country.

BACKGROUND: In developing countries, Epstein-Barr virus (EBV) infection is mostly asymptomatic in early childhood. EBV persistence may lead to different malignancies, such as B cell derived lymphomas. In Argentina, most children are seropositive at three years and an increased association between EBV and lymphoma was proved in children under 10 years old by our group. OBJECTIVE: Our aim was to characterize EBV infection at the site of entry and reactivation of viral infection -the tonsils- in order to better understand the mechanism of viral persistence in pediatric patients. METHODS: A cohort of 54 patients was described. We assessed specific antibodies profiles in sera; viral proteins presence by IHC on FFPE samples and EBV type from fresh tissue. RESULTS: EBV type 1 was prevalent, mostly in the youngest patients. Asymptomatic primary infected patients presented higher viral loads and Latency 0/I or II patterns, whereas the Latency III pattern was observed mostly in healthy carriers. There were no differences between groups in the expression of viral lytic antigens. This study discloses new features in patients undergoing primary infection from a developing population. Low viral inoculum and restricted viral antigen expression may be responsible for the lack of symptoms in children from our country.

M1-like macrophage polarization prevails in young children with classic Hodgkin Lymphoma from Argentina.

The microenvironment in classical Hodgkin lymphoma (cHL) comprises a mixture of different types of cells, which are responsible for lymphoma pathogenesis and progression. Even though microenvironment composition in adult cHL has been largely studied, only few groups studied pediatric cHL, in which both Epstein Barr virus (EBV) infection and age may display a role in their pathogenesis. Furthermore, our group described that EBV is significantly associated with cHL in Argentina in patients under the age of 10 years old. For that reason, our aim was to describe the microenvironment composition in 46 pediatric cHL patients. M1-like polarization status prevailed in the whole series independently of EBV association. On the other hand, in children older than 10 years, a tolerogenic environment illustrated by higher FOXP3 expression was proved, accompanied by a macrophage polarization status towards M2. In contrast, in children younger than 10 years, M1-like was prevalent, along with an increase in cytotoxic GrB+ cells. This study supports the notion that pediatric cHL exhibits a particular tumor microenvironment composition.

Distinctive intrahepatic characteristics of paediatric and adult pathogenesis of chronic hepatitis C infection.

Mechanisms leading to liver damage in chronic hepatitis C (CHC) are being discussed, but both the immune system and the virus are involved. The aim of this study was to evaluate intrahepatic viral infection, apoptosis and portal and periportal/interface infiltrate in paediatric and adult patients to elucidate the pathogenesis of chronic hepatitis C. HCV-infected, activated caspase-3(+) and TUNEL(+) hepatocytes, as well as total, CD4(+), CD8(+), Foxp3(+) and CD20(+) lymphocytes infiltrating portal and periportal/interface tracts were evaluated in 27 paediatric and 32 adult liver samples by immunohistochemistry or immunofluorescence. The number of infected hepatocytes was higher in paediatric than in adult samples (p 0.0078). In children, they correlated with apoptotic hepatocytes (activated caspase-3(+) r = 0.74, p < 0.0001; TUNEL(+) r = 0.606, p 0.0017). Also, infected (p = 0.026) and apoptotic hepatocytes (p = 0.03) were associated with the severity of fibrosis. In adults, activated caspase-3(+) cell count was increased in severe hepatitis (p = 0.009). Total, CD4(+), CD8(+) and Foxp3(+) lymphocyte count was higher in adult samples (p < 0.05). Paediatric CD8(+) cells correlated with infected (r = 0.495, p 0.04) and TUNEL(+) hepatocytes (r = 0.474, p = 0.047), while adult ones correlated with activated caspase-3(+) hepatocytes (r = 0.387, p 0.04). In adults, CD8(+) was associated with hepatitis severity (p < 0.0001) and correlated with inflammatory activity (CD8(+) r = 0.639, p 0.0003). HCV, apoptosis and immune response proved to be involved in CHC pathogenesis of both paediatric and adult patients. However, liver injury in paediatric CHC would be largely associated with a viral cytopathic effect mediated by apoptosis, while in adults it would be mainly associated with an exacerbated immune response.

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.

Epstein-Barr virus BZLF1 gene polymorphisms: malignancy related or geographically distributed variants?

The ubiquitous Epstein-Barr virus (EBV) is related to the development of several lymphoid and epithelial malignancies and is also the aetiological agent for infectious mononucleosis (IM). BZLF1, an immediate early gene, plays a key role in modulating the switch from latency to lytic replication, hence enabling viral propagation. Polymorphic variations in the coded protein have been studied in other geographical regions in a search for viral factors that are inherent to malignancies and differ from those present in benign infections. In the present study, in samples of paediatric patients with benign IM and paediatric patients with malignant lymphomas, we detected previously described sequence variations as well as distinctive sequence polymorphisms from our region. By means of phylogenetic reconstruction, we characterized new phylogenetically distinct variants. Moreover, we described an association between specific variants and the studied pathologies in our region, particularly variant BZLF1-A2 with lymphomas and BZLF1-C with IM. Additionally, length polymorphisms within intron 1 were also assessed and compared between pathologies resulting in an association between 29-bp repeated units and lymphomas. In conclusion, this is the first report to characterize BZLF1 gene polymorphisms in paediatric patients from our geographical region and to suggest the association of these polymorphisms with malignant lymphomas.

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 ⩽10years (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.

Vulvar plasmablastic lymphoma in a HIV-positive child: a novel extraoral localisation.

Plasmablastic lymphoma (PBL) has been characterised by the World Health Organization as a new entity. This report describes an unusual case of PBL in a 3-year-old HIV-infected patient showing a cutaneous vulvar lesion with 9 months of evolution and prolapsed vulvovaginal mucosa. Histopathological examination of a biopsy sample showed diffuse submucosal infiltration by large cells with a cohesive growth pattern, and round and vesicular nuclei with fine chromatin centrally or eccentrically placed with one or more prominent nucleoli. Immunohistochemical staining in neoplastic cells was positive for multiple melanoma oncogene (MUM1), CD138, CD45 and epithelial membrane antigen (EMA). The diagnosis was PBL, stage III. Epstein-Barr virus (EBV) expression was positive by EBV encoded RNAs in situ hybridisation. This is believed to be the third case of paediatric HIV-associated PBL reported in the literature, and the first with vulvar localisation, which is a new anatomical location for this entity.

Evolution of hepatitis C virus hypervariable region 1 in immunocompetent children born to HCV-infected mothers.

Hepatitis C virus (HCV) hypervariable region 1 (HVR1) is the most variable region of the viral genome and its heterogeneity reflects the virus-host interplay during chronicity. Paediatric HCV-infected patients develop liver disease with typical clinical features. Here, the evolution of HVR1 and its adjacent regions were ascertained in plasma samples of two HCV-positive children during a 5-year follow-up period. We report an almost complete conservation of the HVR1 amino acid sequence over time, with underlying nucleotide variability both within and outside HVR1, suggesting some kind of constraint on virus evolution, particularly within HVR1. Although overall d(N)/d(S) rates [rates of nonsynonymous nucleotide substitutions per nonsynonymous site (d(N)) and synonymous nucleotide substitutions per synonymous site (d(S))] were <1 in both patients, a high resolution analysis of selection pressures exerted at the codon level revealed few sites subject to selection and an absolute predominance of invariable positions within HVR1. The HVR1 amino acid sequences showed the antigenic properties expected for this region. Taken together, these data suggest peculiar evolutionary dynamics in our patients, which could be attributed to a mechanism of nucleotide invariability along with purifying selection operating on the HVR1. The lack of HVR1 variability may reflect the adaptation of the virus to a particular environment within each patient or a phenomenon of immune tolerance generated in these immunocompetent patients earlier in life.

High frequency of Epstein Barr virus latent membrane protein-1 30 bp deletion in a series of pediatric malignancies in Argentina.

UNLABELLED: Epstein Barr virus widely infects human populations and remains mostly asymptomatic; however, it has been associated with several malignancies. The EBV-encoded latent membrane protein-1 has been involved in neoplasic transformation; a 30-bp deletion and several mutations in the COOH-terminal domain have been associated with histopathological and clinical disease features. OBJECTIVE: To analyze and correlate the presence of mutations and a 30-bp deletion with the influence of LMP-1 on tumorigenicity in a population of EBV+ pediatric malignancies. METHODS: We studied EBV presence by LMP-1 immunohistochemistry, EBERs in situ hybridization and PCR in fresh and formalin-fixed paraffin-embedded tissue samples from 10 Hodgkin's lymphomas, 6 non-Hodgkin's lymphomas, 4 undifferentiated nasopharyngeal carcinomas. Eighteen out of 20 samples were sequenced. Eight fresh normal lymphoid tissue samples and 3 peripheral blood samples were analyzed. RESULTS: All cases were EBV positive. EBV typing rendered 12 EBV-1 and 8 EBV-2. Del-LMP-1 was detected in 15/20 EBV related malignancies, as well as in 4/11 control tissues. A high percentage of patients showed point mutations previously described. The presence of del-LMP-1 and point mutations failed to correlate with clinical course. CONCLUSIONS: We found a marked incidence of del-LMP-1 (75%) in our series. However, we failed to find any correlation between histological aggressiveness of malignancies and the presence of del-LMP-1 and point mutations.

EBV-associated Hodgkin's disease in an HIV-infected child presenting with a hemophagocytic syndrome.

An 8-years-old boy was admitted with fever of unknown origin, cervical lymphadenopathy and hepatosplenomegaly and weight loss. His mother's HIV infection was diagnosed two weeks before his hospitalization, so he was diagnosed as perinatally acquired AIDS. Serology and serial cultures were negative for viral infections, toxoplasmosis, chagas, tuberculosis and atypical mycobacterium. The patient met clinical and laboratory criteria for hemophagocytic syndrome (HS) that was confirmed on bone marrow aspirate and biopsy. A cervical lymph node biopsy was performed which was diagnosed as Hodgkin's disease (HD) diffuse fibrosis lymphocyte depletion subtype. EBERs in situ hybridization and LMP-1 immunohistochemistry on the lymph node biopsy established the EBV association. On the basis of a sequence of appearance of the clinical, laboratory and histological signs, HIV, EBV or HD may have triggered HS as the last fatal event in this pediatric patient.

[Genotype characterization of hepatitis C virus infection in children]. / Caracterización genotipica de la infección por el virus de hepatitis C en niños.

Hepatitis C virus (HCV) infection in children was assessed by RT-nested PCR of the 5'untranslated region (5'UTR) of the viral genome combined with virus genotyping, performed by restriction fragment length polymorphism (RFLP). We analysed HCV infection in 64 children and in 9 HCV chronically infected mothers corresponding to 10 of them. Thirty two children were positive for serum HCV RNA as determined by RT-nested PCR. The viremia was analysed in consecutive samples of 25 children. Nine children (36%) were always positive for HCV RNA, in 5 (20%) a positive RT-nested PCR turned negative in subsequent samples, other 9 (36%) showed alternating RT-nested PCR results and in 2 (8%) the RT-nested PCR turned positive after an initial negative result. The HCV genotype distribution was studied in 27/32 children and in 9 mothers, and it was similar to that reported in the literature for adult and pediatric patients in our country. Genotype 1 was predominant in our population. HCV genotype did not change in the same patient during the time of this study. HCV genotype was the same in mother-infant pairs. We could not establish a correlation between HCV genotype and vertical transmission of HCV. This study will be helpful to further analyze HCV behavior during pediatric infection and the host's response in the initial stages of it.

Caracterización genotipica de la infección por el virus de hepatitis C en niños. / [Genotype characterization of hepatitis C virus infection in children]

Hepatitis C virus (HCV) infection in children was assessed by RT-nested PCR of the 5untranslated region (5UTR) of the viral genome combined with virus genotyping, performed by restriction fragment length polymorphism (RFLP). We analysed HCV infection in 64 children and in 9 HCV chronically infected mothers corresponding to 10 of them. Thirty two children were positive for serum HCV RNA as determined by RT-nested PCR. The viremia was analysed in consecutive samples of 25 children. Nine children (36

) were always positive for HCV RNA, in 5 (20

) a positive RT-nested PCR turned negative in subsequent samples, other 9 (36

) showed alternating RT-nested PCR results and in 2 (8

) the RT-nested PCR turned positive after an initial negative result. The HCV genotype distribution was studied in 27/32 children and in 9 mothers, and it was similar to that reported in the literature for adult and pediatric patients in our country. Genotype 1 was predominant in our population. HCV genotype did not change in the same patient during the time of this study. HCV genotype was the same in mother-infant pairs. We could not establish a correlation between HCV genotype and vertical transmission of HCV. This study will be helpful to further analyze HCV behavior during pediatric infection and the hosts response in the initial stages of it.

Nasopharyngeal carcinoma in childhood and adolescence: a single-institution experience with combined therapy.

BACKGROUND: A high cure rate may be attained for locally advanced, undifferentiated nasopharyngeal carcinoma (NPC) in children, provided that a combined modality of treatment is employed. Both local and systemic therapies are necessary. Results at a single pediatric institution were analyzed. METHODS: From November 1988 to December 1997, 16 consecutive patients were treated with NPC at the Hospital Garrahan in Buenos Aires, Argentina. The authors were able to evaluate 11 patients (9 boys and 2 girls); their median age was 12 (range, 8-14) years. Chemotherapy consisted of 3 courses, every 3 weeks, of 5-fluorouracil (500 mg/m(2)) plus bleomycin (15 mg/m(2)) daily for 4 days, with cisplatin (100 mg/m(2)) added the last day. External beam radiotherapy was delivered over a median of 52 (range, 45-63) days, to a median cumulative dose to the primary site of 55 (range, 50-61.2) grays (Gy). The median dose for the lower neck area was 45 (range, 45-55.8) Gy. All patients received radiotherapy to the primary site and to the initially involved lymphoid areas, with daily single doses of 1.8 Gy (5 of 7 days per week). RESULTS: The main symptoms at onset were cervical mass (100%), epistaxis (54%), cephalalgia (36%), and trismus (36%). All cases were Stage IV (American Joint Committee on Cancer and International Union Against Cancer TNM system). Complete response was achieved in 45% of patients after initial chemotherapy. With a median follow-up of 63 (range, 23-119) months, disease free survival (with standard error [SE]) and overall survival estimates were 61% (16%) and 91% (9%), respectively, at 75 months. Acute toxicity due to therapy was tolerable. Chronic sinusitis (73%), hypothyroidism (73%), and mild (64%) or moderate (9%) neck fibrosis were detected at follow-up. CONCLUSIONS: Although this series is small, the authors concluded that NPC patients have a good chance of survival in the setting described, in spite of locally advanced disease. Chemotherapy might be useful in preventing the development of systemic metastases.

Paediatric Hodgkin's disease in Spain: association with Epstein-Barr virus strains carrying latent membrane protein-1 oncogene deletions and high frequency of dual infections.

The present report analyses the distribution of 30-base pair (bp) latent membrane protein-1 (LMP-1) oncogene deletions in 24 cases of Epstein-Barr virus (EBV)-positive paediatric Hodgkin's disease (HD) and 39 normal controls. The 30 bp deletion was identified in 19/24 paediatric HD cases (79.2%), of which seven (29.2%) showed the deleted fragment alone, whereas in the remaining 12 (50%) it was accompanied by the nondeleted fragment. Conversely, the deletion was found in 8/22 (36.4%) EBV-positive healthy children, in two (9.1%) of whom the deleted fragment was alone, and was coinfecting with the nondeleted fragment in the other six (27.3%). The LMP-1 deletion was significantly associated with paediatric HD, both including dual infections (P=0.006) or excluding them (P=0.01). Type 2 EBV was carried by 25% of HD children, whereas all controls harboured type 1 EBV. The 30 bp deletion was present in all the paediatric HD specimens that contained type 2 EBV, suggesting that a deleted type 2 EBV strain may be more tumourigenic than a nondeleted type 2 EBV strain. These findings indicate that EBV strains carrying a 30 bp deletion in the third exon of the LMP-1 oncogene may have a more important role in the pathogenesis of paediatric HD than full-length EBV strains. Dual infection by LMP-1 deleted and nondeleted EBV strains is a frequent event both in healthy children and in the paediatric HD population.

Oncogene expression in tumour cells of pediatric Hodgkin's disease in Argentina--correlation with Epstein Barr virus presence.

A new category of oncogenes regulating apoptosis, p53 and bcl-2, and the Epstein Barr virus (EBV) latent membrane protein-1 (LMP-1) have been related to Hodgkin's disease (HD) pathogenesis. We attempt to determine p53, mdm2, p21waf-1, bcl-2 and LMP-1 immunohistochemical expression in tissue sections from formalin-fixed, paraffin-embedded lymph node biopsies of pediatric HD. P53 was detected in the nucleus of Reed Sternberg cells and their variants (H-RS) in 68% of the HD cases. However, there was no statistically significant association with either clinical stages or with histological subtypes. P21waf-1, an indirect marker of p53 functional status, showed nuclear labelling of H-RS in all the studied cases. MDM2 co-expressed with p53 in 62% of the cases, suggesting that both proteins regulate one another, in HD by a self regulatory loop. Bcl-2 cytoplasmatic expression in H-RS was demonstrated in 65% of the cases. There was co-expression of bcl-2 and p53 in 51%, but it failed to correlate with a poor prognosis. LMP-1 labelling was shown in 51% of the cases, disclosing a statistically significant association with the under 6-year group (p = 0.005, Fisher's exact test). Since LMP-1 induces the expression of bcl-2 in vitro, the relation of both proteins was analysed and found to co-express in 15/37 cases, with a statistically significant association only in the under 6-year group (p = 0.001, Fisher's exact test). Abnormal accumulation of these oncoproteins in tumour cells could play a role in the pathogenesis of a subset of pediatric HD.

Epstein-Barr virus-associated Hodgkin's disease: epidemiologic characteristics in international data.

Hodgkin's disease (HD) has long been suspected to have an infectious precursor, and indirect evidence has implicated Epstein-Barr virus (EBV), a ubiquitous herpesvirus, as a causal agent. Recent molecular studies using EBER in situ hybridization or latency membrane protein-I (LMP-I) immunohistochemistry have identified EBV latent infection in up to 50% of HD tumors. However, the epidemiologic features of these cases have not been examined in detail. To explore the epidemiology of EBV-positive HD so as to understand the role of EBV in HD etiology more clearly, this project accumulated patient data from 14 studies that had applied these EBV assays to HD tumors. With information on age at diagnosis, sex, ethnicity, histologic subtype, country of residence, clinical stage and EBV tumor status from 1,546 HD patients, we examined risk for EBV-positive disease using logistic regression. Forty percent of subjects had EBV-positive tumors, and EBV prevalence varied significantly across groups defined by the study variables. Odds ratios (OR) for EBV-associated HD were significantly elevated for Hispanics vs. whites (OR = 4.1), mixed cellularity vs. nodular sclerosis histologic subtypes (OR = 7.3, 13.4, 4.9 for ages 0-14, 15-49, 50+ years), children from economically less-developed vs. more-developed regions and young adult males vs. females (OR = 2.5). These findings suggest that age, sex, ethnicity and the physiologic effects of poverty may represent biologic modifiers of the EBV association and confirm that this association is strongly but variably linked to histologic subtype. The data augment biologic evidence that EBV is actively involved in HD pathogenesis in some cases but describe epidemiologic complexity in this process.