BZLF1 controlled by family repeat domain induces lytic cytotoxicity in Epstein-Barr virus-positive tumor cells

BACKGROUND: BZLF1, an EBV (Epstein-Barr virus) immediate early gene, is required for EBV lytic replication that causes the death of host cells. EBNA1, the product of EBV latent gene, binds to the family repeats (FR) of the origin of replication (Orip) regulating EBV replication. MATERIALS AND METHODS: A vector pFR-Z (BZLF1 controlled by FR domain of EBV) was constructed and transfected into EBV-positive 5-8F and-negative HNE3 nasopharyngeal carcinoma cells and BZLF1-induced cytotoxicity was tested. RESULTS: EBNA1 expression was detected in 5-8F but not HNE3 cells and, in agreement, pFR controlled luciferase expression was activated in 5-8F cells but inhibited in HNE3 cells. Gardella gel assay demonstrated that pFR-Z effectively induced EBV lytic replication in 5-8F but not HNE3 cells. The lytic cytotoxicity was confirmed by a diminished cell survival and the induction of lytic proteins EA-D and gp125. The cytotoxicity was also strikingly enhanced by addition of GCV (gancyclovir) that kill cells with lytic form EBV. CONCLUSION: pFR-Z is a specific gene therapy vehicle for EBV-positive carcinomas.     

Induction of lytic Epstein-Barr virus (EBV) infection in EBV-associated malignancies using adenovirus vectors in vitro and in vivo

The consistent presence of EBV genomes in certain tumor types (in particular, AIDS-related central nervous system lymphomas and nasopharyngeal carcinomas) may allow novel, EBV-based targeting strategies. Tumors contain the latent (transforming) form of EBV infection. However, expression of either of the EBV immediate-early proteins, BZLF1 and BRLF1, is sufficient to induce lytic EBV infection, resulting in death of the host cell. We have constructed replication-deficient adenovirus vectors expressing the BZLF1 or BRLF1 immediate-early genes and examined their utility for killing latently infected lymphoma cells in vitro and in vivo. We show that both the BZLF1 and BRLF1 vectors efficiently induce lytic EBV infection in Jijoye cells (an EBV-positive Burkitt lymphoma cell line). Furthermore, lytic EBV infection converts the antiviral drug, ganciclovir (GCV), into a toxic (phosphorylated) form, which inhibits cellular as well as viral DNA polymerase. When Jijoye cells are infected with the BZLF1 or BRLF1 adenovirus vectors in the presence of GCV, viral reactivation is induced, but virus replication is inhibited (thus preventing the release of infectious EBV particles); yet cells are still efficiently killed. Finally, we demonstrate that the BZLF1 and BRLF1 adenovirus vectors induce lytic EBV infection when they are directly inoculated into Jijoye cell tumors grown in severe combined immunodeficiency mice. These results suggest that induction of lytic EBV infection in tumors, in combination with GCV, may be an effective strategy for treating EBV-associated malignancies.     

EB病毒相关胃癌组织中病毒基因的表达

背景与目的：EB病毒(Epstein—Barr virus,EBV)与多种恶性肿瘤的发生有关,在鼻咽癌及淋巴瘤等组织中EBV的存在形式和表达已有报道,研究表明不同肿瘤组织中EBV的存在形式和表达不同,在胃癌组织中EBV某些基因尤其是裂解性基因的表达情况报道较少。为明确胃癌组织中EBV潜伏性基因和裂解性基因的表达情况,本研究从mRNA水平检测胃癌组织中EBV潜伏感染和裂解感染相关基因的表达,从分子水平探讨EBV编码基因与胃癌发生发展的关系。方法：应用PCR—Southern杂交检测185例胃癌及其相应癌旁组织中特异性EBVDNA片段,进一步用原位杂交(ISH)技术检测PCR阳性标本EBV编码小RNA1(EBERl)的表达,癌细胞EBERl阳性者确定为EBV相关胃癌(EBV—associated gastric carcinoma,EBVaGC)。采用RT-PCR和Southern杂交技术检测EBVaGCs组织中EBV核抗原(EBNAs)基因转录启动子(Qp、Cp和Wp)、潜伏性基因(EBNA1、EBNA2基因,潜伏膜蛋白LMP1、LMP2A和LMP2B基因)和裂解性基因(即刻早期基因BZLF1和BRLF1,早期基因BARF1和BHRF1,晚期基因BcLF1和BLLF1)的表达。结果：185例胃癌标本EBV阳性率为7．03％(13／185),癌旁组织均为阴性。13例EBVaGCs组织中均检测到启动子Qp mRNA,而Wp和Cp mRNA则为阴性。潜伏性基因中EBNA1 mRNA均阳性(13／13),而EBNA2、LMP1和LMP2B均未见表达,5例标本(5／13)检测到LMP2A mRNA。裂解性基因中BcLF1有7例(7／13)表达阳性,2例(2／13)BHRF1表达阳性,6例标本(6／13)检测到BZLF1 mRNA,BARF1亦有6例(6／13)表达阳性,而BRLF1和BLLF1mRNA均为阴性。结论：EBVaGC中EBV潜伏类型为Ⅰ型或介于Ⅰ和Ⅱ型之间的独特类型;EBVaGC组织中部分裂解性基因表达阳性,部分胃癌存在BARF1和BHRF1表达,其在胃癌中所起的作用有待进一步研究。     

Epstein-Barr virus B95.8 produced in 293 cells shows marked tropism for differentiated primary epithelial cells and reveals interindividual variation in susceptibility to viral infection

Epstein-Barr virus (EBV), a well-characterised B-lymphotropic agent is aetiologically linked to B cell lymphoproliferations, but the spectrum of diseases the virus causes also includes oral hairy leukoplakia, a benign epithelial lesion, as well as carcinomas of the nasopharynx and of the stomach. However, it is still unclear how EBV accesses and transforms primary epithelial cells. Sixteen samples consisting of primary epithelial cells from the sphenoidal sinus or from tonsils were infected with GFP-tagged recombinant B95.8 EBVs produced in the 293 cell line. The rate of infection was assessed by counting GFP-positive cells and cells expressing viral proteins. Primary epithelial cells from all samples were found to be sensitive to EBV infection but there was a marked interindividual variation among the tested samples (2-48% positive cells). This suggests heterogeneity in terms of sensitivity to EBV infection in vivo and therefore possibly to EBV-associated diseases of the epithelium. The virus showed a preferential tropism for differentiated epithelial cells (p63 negative, involucrin positive). In all cases, infected cells expressed EBV lytic proteins but also the LMP1 protein. The viral tropism for differentiated cells and the permissivity of these cells for virus replication reproduced in vitro cardinal features of oral hairy leukoplakia. We have identified a source of EBV that shows unusually strong epitheliotropism for primary epithelial cells that will allow detailed analysis of virus-cell interactions during virus infection, replication and virus-mediated transformation.     

Molecular analysis of latent membrane protein 1 in patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in Japan

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is considered to be an oncoprotein because it is crucial for B-lymphocyte transformation. Since a 30 base pair (bp) deletion in the carboxy-terminal portion of the LMP1 gene was found in a CAO cell line derived from nasopharyngeal carcinoma containing EBV, an association between EB viral genetic alteration and tumorigenicity has been postulated. In this study we have analyzed LMP1 DNA isolated from 10 Japanese patients with EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH). In all HLH patients, we found the 30 bp deletion and 4-8-tandem repeats of the sequence DNGPQDPDNTD in the LMP1 gene. Furthermore, detailed amino acid (aa) sequence analysis revealed that 7 aa substitutions identical to those found in CAO-LMP1 but not in B95.8 cell line-LMP1 were found in all the HLH cases. NF-kappaB assay revealed that HLH-LMP1 activated NF-kappaB significantly more than that of B95.8-LMP1 (p=0.032). We conclude that EBV from all of our HLH cases shared common genetic characteristics with EBV obtained from the CAO cell line, which is distinct from the wild-type EBV isolated from the B95.8 cell line. These data suggest that the mutational changes of the LMP1 gene may play an important role in the pathogenesis of these fatal EBV-related disorders.     

Association between SV40 and non-Hodgkin's lymphoma

Millions of people worldwide were inadvertently exposed to live simian virus 40 (SV40) between 1955 and 1963 through immunization with SV40-contaminated polio vaccines. Although the prevalence of SV40 infections in humans is not known, numerous studies suggest that SV40 is a pathogen resident in the human population today. SV40 is a potent DNA tumor virus that is known to induce primary brain cancers, bone cancers, mesotheliomas, and lymphomas in laboratory animals. SV40 oncogenesis is mediated by the viral large tumor antigen (T-ag), which inactivates the tumor suppressor proteins p53 and pRb. During the last decade, independent studies using different molecular biology techniques have shown the presence of SV40 DNA, T-ag, or other viral markers in primary human brain and bone cancers and malignant mesotheliomas. Evidence suggests that there may be geographic differences in the frequency of these virus-positive tumors. Recent large independent controlled studies have shown that SV40 T-ag DNA is significantly associated with human non-Hodgkin's lymphoma (NHL). In our study, we analyzed systemic NHL from 76 HIV-1-positive and 78 HIV-1-negative patients, and nonmalignant lymphoid samples from 79 HIV-1-positive and 107 HIV-1-negative patients without tumors; 54 colon and breast carcinoma samples served as cancer controls. We used polymerase chain reaction (PCR) followed by Southern blot hybridization and DNA sequence analysis to detect DNAs of polyomaviruses and herpesviruses. SV40-specific DNA sequences were detected in 64 (42%) of 154 NHL, none of 186 nonmalignant lymphoid samples, and none of 54 control cancers. For NHL from HIV-1-positive patients, 33% contained SV40 DNA and 39% Epstein Barr virus (EBV) DNA, whereas NHLs from HIV-1-negative patients were 50% positive for SV40 and 15% positive for EBV. Few tumors were positive for both SV40 and EBV. Human herpesvirus type 8 was not detected. SV40 sequences were found most frequently in diffuse large B cell and follicular-type lymphomas. We conclude that SV40 is significantly associated with some types of NHL and that lymphomas should be added to the types of human cancers associated with SV40.     

Epstein‐Barr virus and risk of non‐Hodgkin lymphoma in the cancer prevention study‐II and a meta‐analysis of serologic studies

Epstein‐Barr virus (EBV) causes rare, malignant lymphomas. The role of EBV in other non‐Hodgkin lymphomas (NHLs) remains unclear, but mildly reduced immune function could lead to reactivation of EBV and subsequent NHL. We examined the association between prospectively‐collected plasma EBV antibodies and NHL risk in the Cancer Prevention Study‐II (CPS‐II) Nutrition Cohort and conducted a meta‐analysis of our and published results. The CPS‐II study included 225 NHL cases and 2:1 matched controls. No associations were observed between EBV serostatus or antibody levels and risk of NHL overall. However, when including only the three most common types of NHL (diffuse large B‐cell lymphoma, follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma), high compared to low early antigen (EA‐D) diffuse and BZLF1‐encoded replication activator antibodies were associated with approximately 60% higher risk of NHL. Odds ratios (ORs) for EBV nuclear antigen‐1 and viral capsid antigen (VCA)‐p18 were elevated but not statistically significant. In the meta‐analysis, both EA (summary OR = 1.52, 95% confidence interval (CI): 1.16–2.00) and VCA (summary OR = 1.20, 95% CI: 1.00–1.44) were positively associated with NHL risk. These results suggest EBV may be associated with a wider spectrum of NHL subtypes, but further study is needed to confirm and fully understand these associations.     

Association of EBV strains, defined by multiple loci analyses, in non-Hodgkin lymphomas and reactive tissues from HIV positive and HIV negative patients

Epstein-Barr virus (EBV) associated with lymphoid neoplasms demonstrates preferential association with certain viral strains. Previous subtyping studies have however been confined to analysis of sequence variability within a single locus in EBV. Variations have now been reported for several latently expressed EBV genes, including, EBNAs-1, 2 and LMP-1. Variant EBNA-1 strains have been identified in Burkitt's lymphomas and clustering of subtypes for LMP and EBNA-2 have been associated with either malignancy and/or clinical disease. To investigate the linkage between the variability in these three loci in EBV associated with lymphoid malignancies, we subclassified EBV-associated lymphoproliferations (9 reactive and 24 malignant) from HIV-negative and HIV-positive patients by analysis of the EBNA-1, LMP1, and EBNA-2 genes. Our results demonstrate that (1) EBV identical to the prototype B95.8 strain (Type 1 EBNA-2, wild type EBNA-1 and LMP-1) is very rarely associated with tumors. (2) The EBNA-1 variant V-leucine, restricted to malignant lymphomas in immunocompetent patients, was readily identified in non-malignant lesions in HIV infected patients. (3) Variations of EBNA-1 occur independent of variations at other loci.     

Zinc finger E-box binding factor 1 plays a central role in regulating Epstein-Barr virus (EBV) latent-lytic switch and acts as a therapeutic target in EBV-associated gastric cancer

BACKGROUND:

The role of Epstein‐Barr virus (EBV) infection in gastric carcinogenesis remains largely unknown. The authors studied the effects of zinc finger E‐box binding factor 1 (ZEB1) on latent‐lytic switch of EBV infection in gastric cancer and explored the importance of EBV in gastric carcinogenesis.

METHODS:

Loss or gain of ZEB1 function was obtained by ZEB1 small‐interfering RNA (siRNA) knock‐down or forced ZEB1 re‐expression. Cell growth was evaluated by cell viability and colony formation assays, and the cell cycle was assessed by flow cytometry. EBV was detected using quantitative polymerase chain reaction (PCR) and in situ hybridization analyses.

RESULTS:

ZEB1 knock‐down in a latent EBV‐infected gastric cancer cell line (YCC10) increased lytic gene BamHI W leftward reading frame 1 (BZLF1) expression and decreased the expression of latent gene EB nuclear antigen 1 (EBNA1) concomitant with the inhibition of cell viability (P < .05) and S‐phase DNA synthesis (P < .01). ZEB1 depletion combined with ganciclovir revealed a further reduction in cell viability (P < .001). ZEB1 knock‐down induced cell apoptosis and the up‐regulation of caspase 3 and poly(adenosine diphosphate‐ribose) polymerase cleavage. Conversely, ectopic overexpression of ZEB1 in a lytic EBV‐infected gastric cancer cell line (AGS‐EBV) inhibited BZLF1 promoter (Zp) activity, BZLF1 expression, and apoptosis and promoted cell growth. EBV infection was detected in 11.3% (80 of 711) of gastric cancers. The presence of EBV was associated with age, men, and intestinal type cancer.

CONCLUSIONS:

ZEB1 was confirmed as a key mediator of the latent‐lytic switch of EBV‐associated gastric cancer, a distinct subtype with different clinicopathologic features. The current results indicated that inhibition of ZEB1 may be a potential target for EBV‐associated gastric cancer therapy. Cancer 2012;. © 2011 American Cancer Society.     

EB病毒感染鼻咽癌细胞后的增殖周期变化

背景与目的:EB病毒(Epstein-Barrvirus,EBV)在不同组织类型鼻咽癌细胞内的增殖周期特征决定了基因治疗所采取的策略。本研究探讨EBV感染鼻咽癌细胞后的增殖周期特征。方法:利用EBV阳性的B95-8细胞标准株与鼻咽癌细胞CNE2接触培养,通过补体激活的细胞毒性试验去除共培养体系中的B95-8细胞,并采用逆转录聚合酶链式反应及Southern印迹杂交的方法分析鼻咽癌细胞中病毒的初始感染状态。结果:B95-8细胞与CNE2细胞接触共培养的方法使EBV成功感染CNE2细胞,EBV裂解期标志性基因BZLF1转录时间平均为12d,病毒感染标志性基因EBER转录时间平均为22d。结论:EBV侵入CNE2细胞后可能多数病毒开始处于裂解期增殖,之后EBV从裂解期进入了相对稳定的潜伏期,并随着细胞的传代培养病毒逐渐丢失。     

Sequence polymorphisms between latent membrane proteins LMP1 and LMP2A do not correlate in EBV-associated reactive and malignant lympho-proliferations

The latent membrane proteins LMP1 and LMP2A are co-expressed in most malignancies associated with Epstein-Barr virus (EBV). In contrast with the transforming LMP1 oncoprotein, LMP2A is expressed in lymphocytes of healthy EBV carriers and considered to maintain viral latency. Critical for these LMP2A functions are a transmembranous epitope recognized by specific cytotoxic T lymphocytes (CTLs) and the N-terminal immunoreceptor tyrosine-based activation motif (ITAM), blocking B-cell receptor signaling. To characterize ITAM and CTL motifs of LMP2A and to correlate them with C-terminal variants of LMP1 including the 30-bp deletion variant (LMP1delta), comparative sequence analysis was performed on 76 samples from patients with reactive and malignant lympho-proliferation (infectious mononucleosis, n=21; tonsillar hyperplasia, n=16, chronic lympho-proliferation, n = 9; Hodgkin's disease, n = 8; Non-Hodgkin's lymphoma, n = 5; AIDS-related large-cell lymphoma, n=17). The CTL motif was conserved in all but 2 cases (C426-->S). The ITAM motif was characterized by strictly conserved YXXL sequences in all cases, with a sequence polymorphism in between. The B95.8 prototype was found in 17% (13/76) of cases, while in 72% a variant with 3 point mutations (166796 C-->A, 166805 C-->A, 166810 C-->T) was detected; 11% had 1 or 2 of these mutations in addition to G-->A at 166793. In the C terminus of LMP1, a hypervariable region including LMP1delta was described in 61% of cases. There was no significant association of a particular LMP2A variant with either malignant phenotype or LMP1delta, demonstrating that the functional domains of LMP2A are conserved and that the sequence polymorphisms in LMP1 and LMP2A are independent.     

Amino-acid change in the Epstein-Barr-virus zebra protein in undifferentiated nasopharyngeal carcinomas from Europe and North Africa

Different Epstein-Barr-virus(EBV) variants were found to be associated with nasopharyngeal carcinoma (NPC). The type-C variant lacks the BamHI site between the BamHI W1* and I* regions and the type-f variant has an extra BamHI site in the BamHI F fragment. The BNLF1 gene (which encodes the LMP1 protein) from a nude-mouse-passaged CAO strain and from NPC biopsies from Taiwanese patients also exhibits variations resulting in structural and functional differences in the protein. The BZLF1 gene encodes the ZEBRA protein which triggers the EBV lytic cycle. A difference has been observed in 8 amino acids in the ZEBRA sequence in B95-8 (Z95) and P3HR1 (ZP3) cell lines. EBV found in NPC biopsies and peripheral-blood cells from Asians was predominantly of the ZP3 type (72%), while 81% of samples from different EBV-associated diseases and peripheral-blood cells from North Africa or Europe were of the Z95 type. We found that an alanine 206 had been replaced by a serine in the Z95 sequence in 72% of the NPC biopsies from European and North African patients. The Zser206 variant is found in a significantly lower percentage (p < 0.001 of other EBV-positive tissues from individuals in the same region (10\N33%). In contrast, a 30-bp deletion is observed near the 3` end of the LMP1 gene in the majority of EBV (86%) from NPC and peripheral-blood cells from Asians, whereas a significantly lower percentage (p < 0.001) of NPC biopsies from European and North African patients (56%) have this deletion, as do lymphocytes from control individuals from the same region (36 and 55% respectively). Int. J. Cancer 75:497-503, 1998. © 1998 Wiley-Liss, Inc.     

Roles of lytic viral infection and IL-6 in early versus late passage lymphoblastoid cell lines and EBV-associated lymphoproliferative disease

Lytically infected EBV-positive lymphoblastoid cells enhance the growth of early-passage, but not late-passage, EBV-immortalized lymphoblastoid cell lines (LCLs) in SCID mice and have enhanced IL-6 secretion. Here, we have examined the importance of IL-6 for the growth of early-passage LCLs (EPL) in SCID mice, identified lytic EBV proteins that activate IL-6 production and compared viral and cellular differences between early versus late passage LCLs (LPL). IL-6 was required for efficient growth of EPL in SCID mice. The EBV immediate-early (IE) proteins, BRLF1 and BZLF1, each induced IL-6 secretion when transfected into 293 and BJAB cells. Interestingly, the combination of BZLF1 and the latent EBV protein, LMP-1, induced much more IL-6 expression in both 293 and BJAB cells than either protein alone. Both BZLF1 and BRLF1 also enhanced IL-10 production in 293 cells. In comparison to the EPL, LPL had much reduced expression of early lytic viral proteins and cellular IL-6. In contrast, expression of cellular IL-10 was similar in EPL versus LPL, while VEGF secretion was increased in late-passage LCLs. These results suggest that both BRLF1 and BZLF1 contribute to IL-6 secretion in lytically infected cells and that lytically infected cells may promote early lymphoproliferative disease in patients through enhanced IL-6 production.     

Therapies based on targeting Epstein‐Barr virus lytic replication for EBV‐associated malignancies

In recent years, Epstein‐Barr virus (EBV) lytic infection has been shown to significantly contribute to carcinogenesis. Thus, therapies aimed at targeting the EBV lytic cycle have been developed as novel strategies for treatment of EBV‐associated malignancies. In this review, focusing on the viral lytic proteins, we describe recent advances regarding the involvement of the EBV lytic cycle in carcinogenesis. Moreover, we further discuss 2 distinct EBV lytic cycle‐targeted therapeutic strategies against EBV‐induced malignancies. One of the strategies involves inhibition of the EBV lytic cycle by natural compounds known to have anti‐EBV properties; another is to intentionally induce EBV lytic replication in combination with nucleotide analogues. Recent advances in EBV lytic‐based strategies are beginning to show promise in the treatment and/or prevention of EBV‐related tumors.     

Transformation of hairy cell leukemia to EBV genome-containing aggressive B cell lymphoma

Hairy cell leukemia is a preplasmacytic B cell leukemia which is not EBV associated, although elevated titers of Epstein-Barr virus (EBV) antibodies have been seen in this leukemia and chronic lymphocytic leukemia. Hairy cells are not readily susceptible to EBV infection in vitro, even though they are EBV receptor-positive B cells. We have observed a 59-year-old patient who after 9 years of hairy cell leukemia developed a well-differentiated IgG-kappa monoclonal B cell lymphoma without further evidence of hairy cell leukemia. Pathologically, the lymphoma showed plasmacytic differentiation, and in the patient's serum, a 2 g/dl monoclonal IgG-kappa component was present. DNA extracted from the lymphomatous lymph node hybridized with DNA fragments of a reiterated sequence of EBV, IR1. The transformation, with no chemotherapy involved, from a preplasmacytic leukemia into a lymphoplasmacytic lymphoma with monoclonal gammopathy may be related to the entry of EBV into these cells. Studies at the molecular level may help understand mechanisms of malignant transformation or interconversion in lymphoproliferative disorders of the B cell type.     

Cell cycle arrest and lytic induction of EBV-transformed B lymphoblastoid cells by a histone deacetylase inhibitor, Trichostatin A

Latent infection of the Epstein-Barr virus (EBV) is strongly associated with the pathogenesis of several human tumor types. The restricted expression of the latent EBV antigens is critical for EBV-associated tumors to escape from immune surveillance. EBV lytic replication can be triggered by various treatments and the induced lytic genes cause strong cytotoxic T lymphocyte (CTL) responses. Histone acetylation or deacetylation is associated with chromatin remodeling and regulates gene expression. Histone deacetylase (HDAC) inhibitors affect cell cycle progression as well as gene expression in a wide variety of transformed cells. We examined whether an HDAC inhibitor, TSA, can affect cell cycle progression and induce EBV lytic replication in EBV-transformed lymphoblastoid cell lines (LCLs). TSA caused cell cycle arrest at low concentrations and induced apoptosis at higher (>300 nM) concentrations in the LCLs and EBV negative BJAB cells. To clarify the underlying mechanism of TSA-induced cell cycle arrest, expression of cell cycle regulatory factors was examined by RNase protection assay and Western blot analysis. Following TSA treatment, a reduced expression of cyclin D2 and an induction of p21 may have played an essential role for G1 arrest in LCLs, while p21 induction might have arrested BJAB cells in G1 phase. A Cdk inhibitor, p57, was increased by 300 nM TSA in both LCLs and BJAB cells, indicating its role in apoptosis. Moreover, immunofluorescene assay and Western blotting showed that TSA induced EBV lytic replication in LCL cells. These results suggest that TSA may exert an enhanced anti-tumor effect for EBV-associated tumors not only by inducing a cell cycle arrest and apoptosis, but also by triggering an EBV lytic cycle.     

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.