Epstein-Barr Virus Expression Research Articles

Epstein–Barr virus expression in plasma cell neoplasms and its association with plasmablastic morphologic features

Plasma cell neoplasms include plasma cell myeloma, plasmacytoma, primary amyloidosis, and light and heavy chain deposition diseases. Epstein–Barr virus (EBV) expression has rarely been reported to be associated with plasma cell neoplasms except for patients with a previous history of transplantation or severe immunodeficiency. In contrast, EBV expression has been reported in the majority of plasmablastic lymphoma, a subtype of diffuse large B cell lymphoma. We report three cases with a plasma cell neoplasm (two plasma cell myelomas and one case of plasmacytoma), where EBV infection was diffusely detected by in situ hybridization. All three patients were under the age of 40 years, and one of the three patients was an HIV-positive individual while the other two patients did not have any identifiable source for immunosuppression. All three cases showed multifocal plasmablastic features, which is concordant with the most recent reports about the possible association of EBV expression with plasmablastic morphologic features and increased proliferative activity.

Epstein-Barr virus infection and expression of latent membrane protein 2A in Hodgkin's lymphoma of Uygur patients

To investigate Epstein-Barr virus(EBV) infection in Hodgkin's lymphoma (HL) of Uygur patients and related clinicopathological characteristics. EBV-encoded small RNA (EBER) was detected in 40 cases of HL and 20 cases of lymphoid reactive hyperplasia by in-situ hybridization. Expression of LMP2A in HL was investigated by immunohistochemistry. EBV was detected in 26/40 (65.0%) of HL and 5/20 of lymphoid reactive hyperplasia (P < 0.05). The expression level of EBER showed significant difference among various histological subtypes of HL (P < 0.05) and between patients with and without B symptom (P = 0.02). However, no difference was found in relation to gender, clinical stage and tumor burden. The expression of LMP2A in the mixed cellularity and nodular sclerosis classical HL associated with EBV infection was 57.7% (15/26). Expression of LMP2A was not detected in lymphoid reactive hyperplasia cases. Uyghur patients with Hodgkin's lymphoma have a high infection rate of EBV and distinct clinicopathologic characteristics.

Application of a new human cell line, F2N78, in the transient and stable production of recombinant therapeutics

Host cell lines developed by genetic engineering sometimes show instabilities in maintaining their genetically acquired phenotypes. Previously, a hybrid host cell line, designated as hybrid of kidney and B cells (HKB), capable of retaining selected phenotypes originally existing in the parental cells was developed via fusion of 293 cells and HH514-16 cells. Although HKB did indeed successfully preserve several favorable phenotypes, the expression of Epstein-Barr virus (EBV) specific nuclear antigen 1 (EBNA1), which should be constitutively expressed for host cells to utilize oriP expression vector in transient production of therapeutic proteins, was observed to be unstable. Here, in an attempt to obtain stable expression of EBNA1, a cell type that contains an integrated EBV genome, rather than HH514-16 cells, which harbor an episomal EBV genome, was applied for fusion with 293 cells. Fusion of 293 cells with Namalwa cells led to the creation of a new type of hybrid, F2N, which was able to stably express EBNA1 while not producing EBV particles. One of the F2N clones, F2N78, was observed to maintain EBNA1 expression for more than 1 year under serum-free suspension culture conditions along with human specific glycosyl phenotypes observed previously in HKB. In addition, F2N78 was demonstrated to be an appropriate host cell line for both the transient and stable production of recombinant therapeutics with the features of safety expected of production cell lines for human use.

The Influence of Toll-Like Receptor Stimulation on Expression of EBV Lytic Genes

Epstein-Barr virus (EBV) establishes latency in the resting memory B-cell compartment. It has been recently suggested that maintenance of chronic infection is dependent on periodic reactivation. Although the stimuli for EBV reactivation in vivo during natural infections are largely unknown, there is evidence indicating that heterologous infections could trigger herpesviruses reactivation. The purpose of this work was to identify the influence of Toll-like receptors stimulation on EBV replication in EBV latently infected Burkitt lymphoma cells (P3HR-1, Raji and Namalwa). The cells were stimulated with Pam3CSK4 (synthetic triacylated lipoprotein), PolyI:C (synthetic analog of dsRNA), LPS (lipopolysaccharide from E.coli), measles virus (MeV) and PMA (phorbol myristate acetate). Non-stimulated cells (NS) served as control. EBV expression was investigated at mRNA level for three viral lytic genes: BZLF1 (immediate early, ZEBRA), BALF2 (early, EA) and BcLF1 (late, VCA). Additionally, the effect of stimulation on NF-kBp65 and inflammatory cytokines (IL-lb, IL-6, IL-8, IL-10, IL-12p70, and TNF) was investigated. Stimulation of TLRs led to limited changes in EBV expression manifesting as increase of ZEBRA at mRNA level in cells treated with PolyI:C and Pam3CSK4. Stimulation with PolyI:C, Pam3CSK4 and LPS also lead to considerable increase of NF-kBp65, while increased levels of inflammatory cytokines were observed for IL-8, TNF and IL-6 in cells treated with PMA and MeV. In conclusion, the results of our experiments support the suggestion that TLRs stimulation with microbial ligands influences EBV virus replication.

BZLF1 Expression of EBV is correlated with PARP1 Regulation on Nasopharyngeal Carcinoma Tissues

Nasopharyngeal carcinomas (NPC) is a cancer that arises in the epithelial tissue that covers the inside of the nasopharyngeal mucosa and nasopharynx. Infected Epstein Barr Virus (EBV) cell in a latent infection associated with the expression of nine latent proteins. Latent Membrane Protein 1 (LMP1) is one of latent proteins, and mayor EBV oncoprotein, with functions including virus growth, and to activate BamHI-Z Leftward Reading Frame 1 (BZLF1)-EBV, which can inhibit p53 to induce apoptotic resistance, metastasis, and immune modulation. The body will respond to the expansion of EBV infection with activation of Poly(ADP-ribose)Polymerase-1 (PARP1). The objective of study is to observe the expression of BZLF1 and determine PARP1 regulation in nasopharyngeal tissues. NPC-T2, NPC-T3 and polyp tissues slides are from Ulin Hospital, Banjarmasin. To characterize the necrotic cells such as pyknosis, karyorrhexsis, and karyolysis, histological slides were stained by HE that the necrotic cells measured by using a BX-53 microscope (Olympus) with CellSens Standard software. Tissues slides were stained by using immunofluorohistochemistry with EBV-BZLF1 antibody-Mouse anti-EBV monoclonal antibody against Goat anti-mouse IgG-FITC and anti-PARP1 antibody (MC-10) against Goat anti-mouse IgG labeled Rhodamin. The expression intensities were measured by Confocal Laser Scanning Microscope (Olympus). The percentage number of necrotic cells and BZLF1 and PARP1 expression intensity were analyzed using SPSS 16.0 by one-way ANOVA test with α = 0.05, beside that we use correlate and regression analyze. The research showed that the amount of karryorhexis higher than pyknosis and karyolysis in both tissues. BZLF1 expression 1.79 INT/sel (in polyp), 2.76 INT/sel (NPC Type 2) and 4.36 INT/sel (NPC Type 3), PARP1 expression 2.25 INT/sel (in polyp), 3.31 INT/sel (NPC Type 2), dan 5.93 INT/sel (NPC Type 3).The high of intensity of expression BZLF1 induced the increasing of PARP1 expression, but not correlated with percentage of necrotic cell. Interestingly, in all tissues (NPC-T1, NPC-T2 and polyp) is increased number of karyolysis cells properly, but not in pyknosis and karryorrhexis cells. The conclusion of this study indicated that the BZLF1 induce PARP1 to repair DNA damage against EBV infection.Keywords: BZLF1, CLSM, EBV, nasopharyngeal carcinoma, PARP1.

Poikilodermatous mycosis fungoides with a CD8+ CD56+ immunophenotype: a case report and literature review

Mycosis fungoides (MF) represents the most common type of cutaneous lymphoma. MF shows varieties in both its clinical presentation and immunophenotype. We herein report one case of poikilodermatous MF with a CD8+ CD56+ immunophenotype and present a literature review. A 20-year-old Japanese woman presented with a 10-year history of multiple poikilodermatous and reddish or brownish patches with mild pruritus on the chest, abdomen, back, buttock and thighs. Histopathologically, small- to medium-sized atypical lymphocytes infiltrated into the epidermis, indicating epidermotropism, along the basal layer, and distributed in band-like appearance in the papillary dermis. Immunohistochemically, atypical lymphocytes expressed CD3, CD8, CD56, T-cell intracellular antigen (TIA)-1, granzyme B and beta F1 but lacked expression of CD4, CD20, CD30 and Epstein-Barr virus (EBV) latent membrane protein 1. An EBV-encoded small non-polyadenylated RNA-1 (EBER-1) signal was not detected. On the basis of these findings, the diagnosis of CD8+ CD56+ MF was established. Poikilodermatous MF with a CD8+ CD56+ immunophenotype, as presented herein, is extremely rare. Although further investigation is needed to fully clarify the nature of this aberrant phenotype of MF, we stress that it is important to recognize this rare immunophenotype of MF to distinguish it from aggressive cytotoxic cutaneous lymphomas.

Early Response to Methotrexate, Vincristine and Procarbazine Chemotherapy-Adapted Strategy for Primary CNS Lymphoma: Omitted Radiotherapy for Patients Achieving Complete Response in Interim Analysis.

AbstractAbstract 2719The aim of this study was to evaluate treatment outcomes and prognostic factors of high-dose MVP (methotrexate, vincristine and procarbazine) chemotherapy and response adapted intensification therapy in primary central nervous system lymphoma (PCNSL). We evaluated the expression of Epstein-Barr virus (EBV)-encoded RNA in tissue and pretreatment EBV-DNA in whole blood. Forty patients with PCNSL who treated with high-dose MVP chemotherapy were retrospectively reviewed. Additional radiotherapy or autologous stem cell transplantation were performed for the patients who could not achieve complete response (CR) at interim response (Figure 1). The median age was 55 years. CR rate was 50.0% and objective response rate was 80.0%. Two-year overall survival (OS) was 59.8% and 2-year progression free survival (PFS) was 47.1%. Grade 3 or 4 neutropenia was seen in 19 of 40 (47.5%) patients and thrombocytopenia was developed in 13 (32.5%) patients. Treatment-related mortality was occurred in 4 patients. Delayed neurotoxicity was occurred in 4 patients. Poor performance status, elevated cerebrospinal fluid protein level and whole blood EBV-DNA positivity showed inferior OS (P=0.036, P=0.022, P=0.013, respectively, Figure 2). We detected whole blood EBV-DNA in 10 (29.4%) of 34 patients. It was related with poor OS and PFS in high risk patients according to International Extranodal Lymphoma Study Group scores (P=0.015, P=0.027, respectively). This study suggests that high-dose MVP chemotherapy without intrathecal chemotherapy and response adapted strategy may be an effective regimen for the newly diagnosed PCNSL. Whole blood EBV-DNA positivity could predict the inferior outcome in high-risk patients. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Epstein–Barr virus presence in pediatric diffuse large B‐cell lymphoma reveals a particular association and latency patterns: Analysis of viral role in tumor microenvironment

Non-Hodgkin's lymphoma represents 6-10% of pediatric malignancies, and diffuse large B-cell lymphoma (DLBCL) is one of the three major subtypes. The 2008 WHO classification included a new entity, Epstein-Barr virus (EBV)-positive DLBCL of the elderly, affecting patients >50 years. It has been demonstrated that EBV may play a role in tumor microenvironment composition, disturbing antitumor immune response and disease progression. As most studies were performed in adults, our aim was to assess EBV presence and latency pattern, as well as T-cell microenvironment in a pediatric DLBCL series of Argentina. The study was conducted on formalin-fixed paraffin-embedded biopsies from 25 DLBCL patients. EBV-encoded small nuclear early regions (EBERs) expression was performed by in situ hybridization, whereas EBV gene expression was analyzed using real-time PCR. Epstein-Barr virus latent membrane proteins (LMP)1, LMP2A, CD3, CD4, CD8 and Foxp3 expression were assessed by immunohistochemistry (IHC). Forty percent of cases showed EBV expression, with a significantly higher incidence among patients <10 years (p = 0.018), and with immunosuppressed (p = 0.023). T-cell subsets were not altered by EBV presence. Full EBV latency antigen expression (latency type III) was the most frequently pattern observed, together with BZLF1 lytic gene expression. One patient showed II-like pattern (LMP1 without LMP2A expression). Based exclusively on IHC, some patients showed latency II/III (EBERs and LMP1 expression) or I (EBERs only). These findings suggest that EBV association in our series was higher than the previously demonstrated for elderly DLBCL and that EBV latency pattern could be more complex from those previously observed. Therefore, EBV could be an important cofactor in pediatric DLBCL lymphomagenesis.

Epstein-Barr Virus-Negative Aggressive Natural Killer-Cell Leukaemia with High P-Glycoprotein Activity and Phosphorylated Extracellular Signal-Regulated Protein Kinases 1 and 2

Aggressive natural killer-cell leukaemia (ANKL) is a rare type of disease with fulminant course and poor outcome. The disease is more prevalent among Asians than in other ethnic groups and shows strong association with Epstein-Barr virus (EBV) and P-glycoprotein (P-gp) expression associated with multidrug resistance. Here we present a case of a 47 year old Caucasian female with a prior medical history of azathioprine treated ulcerative colitis who developed EBV-negative form of ANKL. The patient presented with hepatosplenomegaly, fever and nausea with peripheral blood and bone marrow infiltration with up to 70% of atypical lymphoid cells positive for cCD3, CD2, CD7, CD56, CD38, CD45, TIA1 and granzyme B, and negative for sCD3, CD4, CD5, CD8, CD34 and CD123 indicative of ANKL. Neoplastic CD56+ NK-cells showed high level of P-glycoprotein expression and activity, but also strong expression of phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) MAP kinase. The patient was treated with an intensive polychemotherapy regimen designed for treatment of acute lymphoblastic leukaemia, but one month after admission developed sepsis, coma and died of cardiorespiratory arrest. We present additional evidence that, except for the immunophenotype, leukaemic NK-cells resemble normal NK-cells in terms of P-gp functional capacity and expression of phosphorylated ERK1/2 signalling molecule. In that sense drugs that block P-glycoprotein activity and activated signalling pathways might represent new means for targeted therapy.

Epstein-Barr virus infection and expression of B-cell oncogenic markers in HIV-related diffuse large B-cell Lymphoma.

Epstein-Barr virus (EBV)-mediated lymphomagenesis in the setting of HIV infection has been widely accepted. However, little is known about how EBV impacts prognosis. We investigated the hypothesis that EBV infection is associated with expression of specific B-cell oncogenic markers in HIV-related diffuse large B-cell lymphoma (DLBCL) and examined the prognostic use of detecting EBV infection. HIV-related DLBCL cases diagnosed between 1996 and 2007 within Kaiser Permanente California were identified. Immunohistochemical staining was used to analyze the expression of selected markers that are cell-cycle regulators, B-cell activators, and antiapoptotic proteins among others. EBV infection was determined by in situ hybridization of EBV RNA. Correlations between EBV and marker expression were examined using Spearman correlation coefficient. The prognostic use of EBV status was examined in multivariable Cox model adjusting for International Prognostic Index (IPI). Receiver-operating characteristics (ROC) analysis was used to evaluate improvement in model discrimination. Seventy HIV-related DLBCL cases were included (31% EBV±). EBV+ tumor was associated with increased expression of BLIMP1 and CD30 and reduced expression of BCL6 and LMO2. EBV+ tumor was independently associated with elevated 2-year overall mortality [HR, 3.3; 95% confidence interval (CI), 1.6-6.6]. Area under the ROC curve showed improved model discrimination when incorporating tumor EBV status with IPI in the prediction model [0.65 vs. 0.74 (IPI only)]. Our results suggest that EBV infection was associated with expression of several tumor markers that are involved in the NF-κB pathway and that detecting tumor EBV status may have prognostic use in HIV-related DLBCLs.

Epstein‐Barr virus frequency in primary central nervous system lymphomas in Turkey

Primary central nervous system (CNS) lymphomas are rare neoplasms which are usually not associated with Epstein-Barr virus (EBV) in immunocompetent patients. The aim of this study was to investigate the incidence of EBV in primary CNS lymphomas in the Turkish population. Thirty-two primary CNS lymphomas diagnosed according to the WHO 2008 criteria were included in this study. The presence of EBV small ribonucleic acids was investigated by in situ hybridization using EBV encoded small RNA oligonucleotides. The expression of CD10, Bcl-6, MUM-1/IRF-4, Bcl-2, and Ki-67 were evaluated using immunohistochemistry. The patient cohort included 20 male patients and 12 female patients with a median age of 53.5 years (range 13-75). Seven (22%) of cases were classified as germinal center (GC) and 25 (78%) cases as non-GC phenotype according to the Hans criteria. Twenty-six (81%) of the cases showed strong Bcl-2 expression and the median Ki-67 index was 78%. EBV expression was observed in four primary CNS lymphoma cases (12.5%). Most primary CNS lymphomas show non-GC phenotype with high Bcl-2 expression and high proliferative rate. The incidence of EBV in primary CNS lymphomas from Turkey appears to be higher than that observed in the Western countries.

ARID1A expression loss in gastric cancer: pathway-dependent roles with and without Epstein–Barr virus infection and microsatellite instability

The AT-rich interactive domain 1A gene (ARID1A), which encodes one of the subunits in the Switch/Sucrose Nonfermentable chromatin remodeling complex, carries mutations and is responsible for loss of protein expression in gastric carcinoma, particularly with Epstein-Barr virus (EBV) infection and a microsatellite instability-high phenotype. We used immunohistochemistry to investigate the significance of ARID1A loss in 857 gastric carcinoma cases, including 67 EBV(+) and 136 MLH1-lost gastric carcinomas (corresponding to a microsatellite instability-high phenotype). Loss of ARID1A expression was significantly more frequent in EBV(+) (23/67; 34%) and MLH1-lost (40/136; 29%) gastric carcinomas than in EBV(-)MLH1-preserved (32/657; 5%) gastric carcinomas (P < 0.01). Loss of ARID1A correlated with larger tumor size, advanced invasion depth, lymph node metastasis, and poor prognosis in EBV(-)MLH1-preserved gastric carcinoma. A correlation was found only with tumor size and diffuse-type histology in MLH1-lost gastric carcinoma, but no correlation was observed in EBV(+) gastric carcinoma. Loss of ARID1A expression in EBV(+) gastric carcinoma was highly frequent in the early stage of gastric carcinoma, although EBV infection did not cause downregulation of ARID1A: EBV-positive nasopharyngeal carcinomas (n = 8) and lymphomas (n = 15) failed to show loss of ARID1A, and EBV infection did not cause loss of ARID1A in gastric carcinoma cell lines. Taken together, loss of ARID1A may be an early change in carcinogenesis and may precede EBV infection in gastric epithelial cells, while loss of ARID1A promotes cancer progression in gastric cancer cells without EBV infection or loss of MLH1 expression. Loss of ARID1A has different and pathway-dependent roles in gastric carcinoma.

실험계획법을 이용한 HEK293 및 Namalwa 세포배양 특성 규명

Various human host cell lines, which are more effective than the other original human cell lines, have been developed and used. Highly efficient human cell line can be obtained from the fusion between human embryonic kidney 293 (HEK293) and human Burkitt's lymphoma cells (Namalwa). Fused cell line has the advantages of both cell lines such as the high transfection efficacy of HEK293 cells and the constitutive expression of Epstein-Barr virus (EBV) genome which is related with high expression of target protein and anti-apoptotic growth of Namalwa cells. In this study, characterization of two original cell lines was performed by using design of experiment (DOE) considering cell maintenance, media development, optimization of culture condition, and scale-up. The formation of aggregates was apparent with high glutamine concentration at more than 6 mM. Supplementation of hydrolysates showed positive effects on the growth performances of HEK293 cells. On the contrary, Namalwa cells showed negative results. It was confirmed that Namalwa cells were more sensitive to lower temperature at <TEX>$35^{\circ}C$</TEX> and hyperosmotic condition over 260 mOsm/kg. In addition, both cell lines showed limited growth in 3-L bioreactor due to shear stress.

Epstein-Barr virus lytic gene expression is tightly linked to ER stress but not cytotoxicity with bortezomib or nelfinavir

Epstein-Barr virus (EBV) is associated with AIDS-related lymphomas and other malignancies. We have previously shown that the proteasome inhibitor bortezomib is an activator of EBV lytic gene expression and that these effects are mediated by ER stress and the unfolded protein response (UPR) [1]. We investigated the relationship between the induction of UPR and EBV lytic gene expression with a variety of UPR inducers, as well as the association of their viral and antitumor effects in a variety of tumor cell lines. Bortezomib, thapsigargin, and tunicamycin activate the UPR and EBV lytic cycle. Recently, nelfinavir has also been reported to lead to ER-stress and the UPR. We found a dose-dependent relationship with bortezomib and with nelfinavir for the induction of the UPR markers (Bip, ATF4, XBP1s, and CHOP10), EBV lytic gene expression (measured as ZTA RNA), and cell toxicity in Burkitt’ sl ymphoma cell lines.. Blocking ER stress and UPR activation, by cycloheximide (CHX) treatment or by Bip knockdown, diminished ZTA induction but had no effect on cellular toxicity. We also studied EBV lymphoblastoid cell lines (LCLs). In contrast to the BL cell lines, bortezomib did not induce ER stress, activate the UPR or lead to EBV lytic gene expression but was nonetheless toxic to LCLs. These results indicate that bortezomib and nelfinavir both induce ER stress and UPR leading to EBV lytic reactivation in BL cells. UPR induction corresponds with EBV lytic gene induction but appears to be distinct from cellular toxicity. Our findings suggest that ER stress, UPR and viral activation are closely linked but may be separable from the cytotoxic effects of some pharmacologic inducers. Bortezomib and nelfinavir may serve as laboratory and clinical tools for manipulating viral gene expression in EBV associated malignancies.

Oral dysplasia and squamous cell carcinoma: Correlation between increased expression of CD21, Epstein-Barr virus and CK19

Epstein-Barr virus is an orally transmitted human gammaherpesvirus that infects B lymphocytes and epithelial cells. Although most primary infections are asymptomatic, long term carriage of the virus can be associated with either lymphoid or epithelial malignancies. The association of EBV with oral squamous cell carcinomas is sporadic and it is uncertain if the virus is involved in initiation of the tumor or, possibly, in its progression. Complement receptor type 2, CR2 or CD21, is a receptor for the major attachment protein of EBV, which significantly enhances epithelial cell infection, but its expression on normal tissues is restricted to tonsil and adenoid epithelium. As cells become dysplastic they are reported to express higher levels of CK19. We sought to evaluate whether CD21 and CK19 expression change as oral epithelial cells outside Waldeyer's ring become dysplastic. Epithelial cells were isolated by laser capture microdissection and levels of CD21, CK19 and EBV RNA were measured by quantitative reverse transcriptase PCR. We report that expression of CD21 increases in frequency and intensity as oral epithelial cells become more dysplastic and that expression correlates with an increase in infection by EBV. Tumors or dysplastic lesions that carry EBV also generally express higher levels of CK19 than those that do not. The findings suggest that dysplasia may make cells more susceptible to infection by EBV and that infection by the virus may alter the phenotype of the infected cell in a manner which could affect prognosis.

Expression of Epstein-Barr Virus–Encoded Proteins in Extranodal NK/T-cell Lymphoma, Nasal Type (ENKL): Differences in Biologic and Clinical Behaviors of LMP1-Positive and -Negative ENKL

Extranodal NK/T-cell lymphoma, nasal type (ENKL) is closely associated with Epstein-Barr virus (EBV). To elucidate its pathogenetic role, we examined the expression profiles of EBV-encoded proteins, especially focusing on latent membrane protein 1 (LMP1). Immunohistochemistry was carried out using clinical samples from ENKL cases, which were diagnosed between 1996 and 2010 at our institution. We statistically assessed the correlation between LMP1 positivity and the clinicopathologic data and further examined phosphorylation status of NF-κB RelA and Akt in ENKL cell lines. Most of the 30 examined cases showed pleomorphic morphology, natural killer cell immunophenotype, and a localized disease. Immunohistochemistry detected EBERs, but not EBNA2, in all cases. LMP1 and LMP2A were positive in 22 (73.3%) and 12 cases (40.0%), respectively. LMP1-positive cases tended to show a localized disease (P = 0.060, the Fisher exact test). Nuclear localization of phosphorylated RelA and detection of phosphorylated Akt were predominantly observed in LMP1-positive cases (P = 0.002 and P < 0.001, respectively, the Fisher exact test). RNA silencing experiments of LMP1 in Hank1 cells suggested a positive correlation between LMP1 expression and phosphorylation of RelA and Akt. With a median follow-up period of 26.7 months (range, 0.2-142.3 months), the 2.5-year overall survival rates for LMP1-positive and -negative cases were estimated at 78.3% and 12.5%, respectively (P = 0.001, log-rank test). LMP1 expression shows correlations with phosphorylation of RelA and Akt and possibly has a favorable impact on clinical outcome in ENKL.

Soluble factors produced by activated CD4+T cells modulate EBV latency

Following infection with Epstein-Barr virus (EBV), the virus is carried for life in the memory B-cell compartment in a silent state (latency I/0). These cells do not resemble the proliferating lymphoblastoid cells (LCLs) (latency III) that are generated after infection. It is of fundamental significance to identify how the different EBV expression patterns are established in the latently infected cell. In view of the prompt activatability of CD4(+) T cells in primary EBV infection, and their role in B-cell differentiation, we studied the involvement of CD4(+) T cells in the regulation of EBV latency. Lymphoblastoid cell lines (LCLs) were cocultured with autologous or allogeneic CD4(+) T cells. Activated T cells influenced the expression of two key viral proteins that determine the fate of the infected B cell. EBNA2 was down-regulated, whereas LMP1 was unregulated and the cells proliferated less. This was paralleled by the down-regulation of the latency III promoter (Cp). Experiments performed in the transwell system showed that this change does not require cell contact, but it is mediated by soluble factors. Neutralizing experiments proved that the up-regulation of LMP1 is, to some extent, mediated by IL21, but this cytokine was not responsible for EBNA2 down-regulation. This effect was partly mediated by soluble CD40L. We detected similar regulatory functions of T cells in in vitro-infected lymphocyte populations. In conclusion, our results revealed an additional mechanism by which CD4(+) T cells can control the EBV-induced B-cell proliferation.

Prognostic Factors for Survival in 60 Patients with Classical Hodgkin Lymphoma From Peru

Abstract 4863 Introduction:Hodgkin lymphoma (HL) is an uncommon lymphoma with a good prognosis. However, the characteristics of HL in Peruvian patients have not been previously reported. HL is thought to be related to chronic Epstein Barr virus (EBV) infection, and new biological markers such as EBV, CD68 and FOXP3 expression could be of potential biological interest in this entity. Our study aims to identify prognostic factors for survival in Peruvian patients with classical HL. Methods:This study was approved by the IRB at our institution. Between January 2001 and December 2009, patients diagnosed with classical HL were selected for this study. HIV-negative patients were excluded. Clinical data were reviewed retrospectively and patient’s biopsies were analyzed for the immunohistochemical expression of CD20, PAX5, CD30, CD15, FOXP3 and CD68. Samples were also analyzed for the presence of EBV-encoded RNA (EBER) using an in situ hybridization (ISH) technique. Clinicopathological characteristics will be presented using descriptive statistics. Overall survival (OS) estimates were calculated using the Kaplan-Meier method and compared using the log-rank test. For the multivariate survival analysis, Cox proportional-hazard regression test was used. P-values <0.05 were considered statistically significant. RESULTS:A total of 60 patients with a pathological diagnosis of classical HL were included in our study. The median age was 55 years (range: 18–98 years) with a 7:3 male predominance. Advanced stages (stage 3 and 4) were seen in 52%, ECOG performance status >1 in 15% and B symptoms in 67%. White blood cell (WBC) counts >15,000 cells/mm3 was seen in 7%, lymphocyte count <600 cells/mm3 in 12%, albumin levels <4 g/dl in 70% and hemoglobin <10.5 g/dl in 40% of the patients. FOXP3 expression >25% was seen in 45%, CD68 >25% in 48% and EBER 2–3+ in 53%. ABVD was the preferred therapy, accounting for 90% of the cases and a complete response to ABVD was obtained in 78%. With a median follow-up 34 months, the median overall survival (OS) has not been reached and the 5-yr OS was 80%. In the univariate analysis, ECOG >1 (p=0.0001), elevated LDH levels (p=0.04) and lymphocyte count <600 cells/mm3 (p=0.05) were adverse prognostic factors. Age, sex, B symptoms, WBC, albumin and hemoglobin levels were not associated with survival. In the multivariate analysis, only ECOG >1 (p=0.01) was an independent adverse prognostic factor in Peruvian patients with classical HL. CONCLUSION:ECOG is a best prognostic factor in Peruvian patients with a diagnosis of classical Hodgkin lymphoma. CD68 expression, EBV status and FOXP3 expression by the malignant cells were not prognostic factors for OS in our cohort of patients. Disclosures:Castillo:GlaxoSmithKline: Research Funding; Millennium Pharmaceuticals: Research Funding.

Epstein-Barr virus BART9 miRNA modulates LMP1 levels and affects growth rate of nasal NK T cell lymphomas.

Nasal NK/T cell lymphomas (NKTCL) are a subset of aggressive Epstein-Barr virus (EBV)-associated non-Hodgkin's lymphomas. The role of EBV in pathogenesis of NKTCL is not clear. Intriguingly, EBV encodes more than 40 microRNAs (miRNA) that are differentially expressed and largely conserved in lymphocryptoviruses. While miRNAs play a critical role in the pathogenesis of cancer, especially lymphomas, the expression and function of EBV transcribed miRNAs in NKTCL are not known. To examine the role of EBV miRNAs in NKTCL, we used microarray profiling and qRT-PCR to identify and validate expression of viral miRNAs in SNK6 and SNT16 cells, which are two independently derived NKTCL cell lines that maintain the type II EBV latency program. All EBV BART miRNAs except BHRF-derived miRNAs were expressed and some of these miRNAs are expressed at higher levels than in nasopharyngeal carcinomas. Modulating the expression of BART9 with antisense RNAs consistently reduced SNK6 and SNT16 proliferation, while antisense RNAs to BARTs-7 and -17-5p affected proliferation only in SNK6 cells. Furthermore, the EBV LMP-1 oncoprotein and transcript levels were repressed when an inhibitor of BART9 miRNA was transfected into SNK6 cells, and overexpression of BART9 miRNA increased LMP-1 protein and mRNA expression. Our data indicate that BART9 is involved in NKTCL proliferation, and one of its mechanisms of action appears to be regulating LMP-1 levels. Our findings may have direct application for improving NKTCL diagnosis and for developing possible novel treatment approaches for this tumor, for which current chemotherapeutic drugs have limited effectiveness.

Fatigue and herpesvirus latency in women newly diagnosed with breast cancer

Fatigue is a notable clinical problem in cancer survivors, and understanding its pathophysiology is important. The current study sought to determine biomarkers of fatigue that exist before cancer treatment. Relationships between the expression of latent Epstein-Barr virus (EBV) and cytomegalovirus (CMV) and fatigue were examined in 158 women newly diagnosed with breast cancer or awaiting a positive diagnostic result. Higher CMV antibody titers, but not EBV antibody titers, were associated with a greater likelihood of being fatigued. Associations between fatigue and higher CMV antibody titers remained after controlling for alcohol use, smoking, comorbidities, depressive symptoms, age, BMI, cancer stage, and sleep problems. More sleep problems and higher levels of depressive symptoms were also associated with a greater likelihood of being fatigued. CMV antibody titers, but not EBV antibody titers, were associated with higher levels of C-reactive protein (CRP), but CRP was not associated with fatigue. When the cellular immune system is compromised, reactivation of latent herpesviruses may fuel chronic inflammatory responses. Prior work has suggested that fatigue may be related to inflammation and its associated sickness behaviors; accordingly, our findings may be tapping into this same physiological substrate.