Epstein-Barr Virus Subtypes Research Articles

Assessing molecular subtypes of gastric cancer: microsatellite unstable and Epstein-Barr virus subtypes. Methods for detection and clinical and pathological implications

BackgroundThe molecular classification of gastric cancer recognises two subtypes prone to immune checkpoint blockade: the microsatellite unstable and the Epstein-Barr virus (EBV)-related tumours. We aim to assess the concordance between immunohistochemistry and PCR for microsatellite status evaluation, and explore the value of microsatellite instability (MSI) and EBV as predictive survival factors.Material and methodsWe collected 246 consecutively diagnosed gastric cancer cases in all stages and evaluated the microsatellite status using immunohistochemistry for mismatched repair (MMR) proteins and PCR. EBV expression was studied through in situ hybridisation.ResultsForty-five (18%) cases presented MSI and 13 (6%) were positive for EBV. MSI was associated with female sex, older age, distal location and distal non-diffuse type of the modified Lauren classification. EBV expression was most frequent in proximal location and proximal non-diffuse type. The sensitivity, specificity, positive predictive value and negative predictive value of immunohistochemistry for the microsatellite study were 91%, 98%, 91% and 98%, respectively. In the multivariate analysis, MSI was an independent predictor of favourable tumour-specific survival (TSS) in stages I–III (MSI: HR: 0.37, 95% CI 0.12 to 0.95, p=0.04).ConclusionsThe MSI status and the EBV expression should be incorporated in routine pathological report for two reasons. First, MSI defines a different pathological entity with a better outcome. Second, MSI and EBV may be useful biomarkers to identify patients who will respond to immune checkpoint blockade inhibitors. For this purpose, immunohistochemical study for MMR proteins and in situ hybridisation study for EBV evaluation are feasible and cost-effective methods.

Identification of genomic alterations in nasopharyngeal carcinoma and nasopharyngeal carcinoma-derived Epstein-Barr virus by whole-genome sequencing.

Nasopharyngeal carcinoma (NPC) is a common tumor in southern China with marked ethnic and geographic distributions and concomitant Epstein-Barr virus (EBV) infection. However, the molecular basis of NPC remains largely unknown, and the role of EBV genomic variations in the pathogenesis of NPC is unclear. Whole-genome sequencing of a collection of 12 EBV-positive paired NPC tumor/peripheral blood samples from Hunan Province was performed, and the FBXO11 gene was subjected to further functional analyses. We identified 69 missense mutations in signaling pathways typically altered in cancer, including NF-κB and Wnt/Hedgehog/Notch. Additionally, 122 variations were identified in non-coding regions. Among these, a subset of genes was confirmed as dysregulated in NPC by mining the NPC cDNA microarray database. The randomly selected gene, FBXO11, could promote the malignant progression of NPC in vitro. Full-length EBV genomes from 8 of the 12 patients with NPC were also successfully assembled, and latent EBV infection is a primary cause of NPC. The various subtypes of EBV detected exhibited clear correlations with its geographical distribution. This study has explored novel biological markers and tumorigenic pathways with substantial potential to enhance therapeutic strategies for NPC.

Abstract 4215: Comprehensive investigation of programmed death receptor ligand 1 (PD-L1) expression and associated molecular features in gastric cancer patients

Abstract Purpose We explored PD-L1 expression and analyzed its association with molecular subtypes, clinical features and other molecular markers in Korean gastric cancer (GC) patients. Experimental design As with the Asian Cancer Research Group (ACRG) GC cohort, 300 primary tumor specimens were procured from Samsung Medical Center. Tumor PD-L1 expression was measured by immunohistochemistry (IHC) using the 22C3 PD-L1 antibody pharmDx (Dako) kit. PD-L1 positivity was defined as CPS ≥1%, where CPS is PD-L1+ cells (tumor cells, macrophages, lymphocytes) over the total number of tumor cells, expressed as a percentage. ACRG and The Cancer Genome Atlas (TCGA) molecular subtypes, and other molecular and clinical features including PD-L1 mRNA expression by microarray, tumor mutation burden (TMB) by targeted sequencing, microsatellite instability (MSI) status by IHC, Epstein-Barr virus (EBV) infection by in situ hybridization, Helicobacter pylori (H. pylori) infection by microscope, ERBB2 (HER2) and EGFR overexpression by IHC, signet ring cell and Lauren classification were provided as part of the ACRG collaboration. Spearman correlation test, Wilcoxon rank-sum test, Kruskal-Wallis test were utilized to test the association with PD-L1 expression, and survival was analyzed by Kaplan-Meier method and log-rank test. Results The median age was 64 years (range, 24-86), 101 (34%) were female, and 127 (42%) had stage I/II disease. Prevalence of PD-L1 positivity was 59.3%, with significantly higher expression in stage I samples (P = 0.002). PD-L1 positivity was associated with good prognosis (P = 0.008) especially for early stage (I/II) subjects (N = 127, P = 0.007). PD-L1 mRNA was moderately correlated with protein expression (R = 0.430, P < 0.001). For molecular subtype, PD-L1 expression was associated with ACRG subtype (P < 0.001) with significantly higher expression in MSI subtype (P < 0.001). And for TCGA subtype, the association with PD-L1 expression also existed (N = 238, P < 0.001) with significantly higher expression in MSI and EBV subtype (P < 0.001). H. pylori infection was also associated with higher PD-L1 expression (N = 127, P = 0.001). Signet ring cell carcinoma had significantly lower PD-L1 expression (P < 0.001). PD-L1 expression was also significantly higher in ARID1A or PIK3CA mutant samples (N = 233, FDR adjusted P = 0.038). TMB was found to be weakly correlated with PD-L1 expression (N = 233, R = 0.266, P < 0.001). No significant association was observed between PD-L1 expression and overexpression of ERBB2 or EGFR. Conclusion Our study provides evidence that several molecular features, especially MSI and EBV infection, are significantly associated with higher PD-L1 protein expression, which suggests that PD-L1 IHC may be an appropriate biomarker to screen GC patients for immune checkpoint inhibitors. Citation Format: Xiaoqiao Liu, Jeeyun Lee, Kyoung-Mee Kim, Seung Tae Kim, Se Hoon Park, Won Ki Kang, Razvan Cristescu, Senaka Peter. Comprehensive investigation of programmed death receptor ligand 1 (PD-L1) expression and associated molecular features in gastric cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4215.

Genome-wide analysis of Epstein-Barr virus identifies variants and genes associated with gastric carcinoma and population structure.

Epstein-Barr virus is a ubiquitous virus and is associated with several human malignances, including the significant subset of gastric carcinoma, Epstein-Barr virus-associated gastric carcinoma. Some Epstein-Barr virus-associated diseases are uniquely prevalent in populations with different geographic origins. However, the features of the disease and geographically associated Epstein-Barr virus genetic variation as well as the roles that the variation plays in carcinogenesis and evolution remain unclear. Therefore, in this study, we sequenced 95 geographically distinct Epstein-Barr virus isolates from Epstein-Barr virus-associated gastric carcinoma biopsies and saliva of healthy donors to detect variants and genes associated with gastric carcinoma and population structure from a genome-wide spectrum. We demonstrated that Epstein-Barr virus revealed the population structure between North China and South China. In addition, we observed population stratification between Epstein-Barr virus strains from gastric carcinoma and healthy controls, indicating that certain Epstein-Barr virus subtypes are associated with different gastric carcinoma risks. We identified that the BRLF1, BBRF3, and BBLF2/BBLF3 genes had significant associations with gastric carcinoma. LMP1 and BNLF2a genes were strongly geographically associated genes in Epstein-Barr virus. Our study provides insights into the genetic basis of oncogenic Epstein-Barr virus for gastric carcinoma, and the genetic variants associated with gastric carcinoma can serve as biomarkers for oncogenic Epstein-Barr virus.

EBV and HHV-6 Circulating Subtypes in People Living with HIV in Burkina Faso, Impact on CD4 T cell count and HIV Viral Load.

Epstein Barr Virus (EBV) and Human Herpes Virus 6 (HHV-6) are responsible for severe diseases, particularly in immunocompromised persons. There is limited data of the infection of these opportunistic viruses in Burkina Faso.The purpose of this study was to characterize EBV and HHV-6 subtypes and to assess their impact on CD4 T cell count, HIV-1 viral load and antiretroviral treatment in people living with HIV-1. The study population consisted of 238 HIV-positive patients with information on the CD4 T cell count, HIV-1 viral load and HAART. Venous blood samples collected in EDTA tubes were used for EBV and HHV-6 Real Time PCR subtyping.An infection rate of 6.7% (16/238) and 7.1% (17/238) were found respectively for EBV and HHV-6 in the present study. Among EBV infections, similar prevalence was noted for both subtypes (3.9% (9/238) for EBV-1 vs 4.6% (11/238) for EBV-2) with 2.1% (5/238) of co-infection. HHV-6A infection represented 6.3% (15/238) of the study population against 5.0% (12/238) for HHV-6B. EBV-2 infection was significantly higher in patients with CD4 T cell count ≥ 500 compared to those with CD4 T cell count less than 500 cells (1.65% vs 8.56%, p = 0,011). The prevalence of EBV and HHV-6 infections was almost similar in HAART-naive and HAART-experienced patients.The present study provides information on the prevalence of EBV and HHV-6 subtypes in people living with HIV-1 in Burkina Faso. The study also suggests that HAART treatment has no effect on infection with these opportunistic viruses in people living with HIV-1.

Clinical Significance of Four Molecular Subtypes of Gastric Cancer Identified by The Cancer Genome Atlas Project.

Purpose: The Cancer Genome Atlas (TCGA) project recently uncovered four molecular subtypes of gastric cancer: Epstein-Barr virus (EBV), microsatellite instability (MSI), genomically stable (GS), and chromosomal instability (CIN). However, their clinical significances are currently unknown. We aimed to investigate the relationship between subtypes and prognosis of patients with gastric cancer.Experimental Design: Gene expression data from a TCGA cohort (n = 262) were used to develop a subtype prediction model, and the association of each subtype with survival and benefit from adjuvant chemotherapy was tested in 2 other cohorts (n = 267 and 432). An integrated risk assessment model (TCGA risk score) was also developed.Results: EBV subtype was associated with the best prognosis, and GS subtype was associated with the worst prognosis. Patients with MSI and CIN subtypes had poorer overall survival than those with EBV subtype but better overall survival than those with GS subtype (P = 0.004 and 0.03 in two cohorts, respectively). In multivariate Cox regression analyses, TCGA risk score was an independent prognostic factor [HR, 1.5; 95% confidence interval (CI), 1.2-1.9; P = 0.001]. Patients with the CIN subtype experienced the greatest benefit from adjuvant chemotherapy (HR, 0.39; 95% CI, 0.16-0.94; P = 0.03) and those with the GS subtype had the least benefit from adjuvant chemotherapy (HR, 0.83; 95% CI, 0.36-1.89; P = 0.65).Conclusions: Our prediction model successfully stratified patients by survival and adjuvant chemotherapy outcomes. Further development of the prediction model is warranted. Clin Cancer Res; 23(15); 4441-9. ©2017 AACR.

Associations of Epstein-Barr Virus DNA in PBMCs and the Subtypes with Breast Cancer Risk.

Objective: Epstein-Barr virus (EBV) has been found to be implicated in the development of breast cancer. The purpose of the present study was to identify the associations of EBV DNA and the subtypes in peripheral blood mononuclear cells (PBMCs) with the risk of breast cancer.Material and Methods: A case-control study with 671 breast cancer cases and 859 age-matched controls was conducted in Guangzhou, China. Face-to-face interviews were performed and blood samples were collected immediately after admission to the hospital for patients or after the interview for controls. EBV DNA in PBMCs and the subtypes were detected using Polymerase Chain Reaction (PCR) and restricted fragment length polymorphisms (RFLP). IgA antibodies against EBV VCA-p18 and EBNA-1 were examined using commercial enzyme-linked immunosorbent assay kits. Unconditional logistic regression analysis was applied to evaluate the associations of the DNA positivity and subtypes of EBV with the risk of breast cancer.Results: Among the 1530 subjects, 164 cases (24.4 %) and 206 controls (24.0 %) were positive for EBV DNA in PBMCs and no significant difference occurred between cases and controls. The presence of EBV DNA was related to the positivity of EBV IgA antibodies. Of the DNA positive samples, 71 cases and 109 controls for F/f subtype and 58 cases and 112 controls for C/D subtype were successfully obtained. The D subtype was associated with an increased breast cancer risk compared with the C subtype [OR (95% CI): 2.86 (1.25~6.53)]. We did not find an association of the F/f polymorphism with breast cancer risk.Conclusions: The present study suggested that the presence of EBV DNA in PBMCs may not be an appropriate biomarker for breast cancer risk. The subtype D of EBV was likely to be related to breast tumorigenesis.

A population-based study of 135 lymphomas after solid organ transplantation: The role of Epstein-Barr virus, hepatitis C and diffuse large B-cell lymphoma subtype in clinical presentation and survival

Background. Epstein-Barr virus (EBV) plays a major role in the development of post-transplant lymphoproliferative disorder (PTLD), but there is an increasing awareness of EBV-negative PTLD. The clinical presentation of EBV-negative PTLD has not been as well characterised as EBV-positive cases. Further, there is limited knowledge on the clinical importance of diffuse large B-cell lymphoma (DLBCL) cell of origin subtype post-transplant. Materials and methods. We studied the role of EBV, hepatitis C (HCV) and DLBCL subtype in clinical presentation and survival in 135 post-transplant lymphomas diagnosed 1980–2006 in a population-based cohort of 10 010 Swedish solid organ transplant recipients. The lymphomas were re-evaluated according to WHO 2008, examined for EBV, and clinical data were collected from medical records. Results. Lymphoma incidence rate was 159/100 000 person-years and is also reported by lymphoma subtype. EBV-negative lymphomas constituted 48% and were associated with HCV infection (p = 0.02), bone marrow involvement (p < 0.001), and T-cell phenotype (p = 0.002). Among DLBCL, 78% were of non-germinal centre subtype, which was associated with EBV-positivity (69%, p = 0.001), early occurrence (p = 0.03), heart/liver/lung/pancreas recipients (p = 0.02), anti-T-cell globulin (p = 0.001), and tacrolimus treatment (p = 0.02). DLBCL subtypes had similar overall survival. Five-year overall survival was 42% in all treated patients. Independent poor prognostic factors were older age, B symptoms, ECOG 2-4, kidney/pancreas/heart recipients, T-cell lymphoma, and HCV-infection. Conclusions. With long follow-up, a large part of PTLD is EBV-negative, due to a high proportion of T-cell lymphomas and low of polymorphic PTLD. EBV-negative PTLD have a different clinical presentation. HCV may play an aetiological role in late-onset PTLD and was revealed as a new prognostic factor for inferior survival that needs to be confirmed in larger studies. The heavier immunosuppression in non-kidney transplantations seems to play a role in the development of non-germinal centre DLBCL. DLBCL cell of origin subtype lacks prognostic importance in the transplant setting.

Spontaneous Lytic Replication and Epitheliotropism Define an Epstein-Barr Virus Strain Found in Carcinomas

The Epstein-Barr virus (EBV) is found in a variety of tumors whose incidence greatly varies around the world. A poorly explored hypothesis is that particular EBV strains account for this phenomenon. We report that M81, a virus isolated from a Chinese patient with nasopharyngeal carcinoma (NPC), shows remarkable similarity to other NPC viruses but is divergent from all other known strains. M81 exhibited a reversed tropism relative to common strains with a reduced ability to infect B cells and a high propensity to infect epithelial cells, which is in agreement with its isolation from carcinomas. M81 spontaneously replicated in B cells invitro and invivo at unusually high levels, in line with the enhanced viral replication observed in NPC patients. Spontaneous replication and epitheliotropism could be partly ascribed to polymorphisms within viral proteins. We suggest considering M81 and its closely related isolates as an EBV subtype with enhanced pathogenic potential.

Abstract 2537: Infectious, autoimmune, and allergic diseases and risk of Hodgkin lymphoma in children and adolescents: A Children's Oncology Group (COG) study.

Abstract Relatively little research has been devoted to understanding the causes of Hodgkin lymphoma (HL) in children. HL diagnosed at &amp;lt;15 years has incidence patterns and clinical characteristics distinct from HL in older adolescents and adults. Because of these differences, different underlying causes have also been postulated. Nevertheless, similar to adult HL, an infectious origin for pediatric HL has long been suspected and Epstein-Barr virus (EBV) has been implicated in a subset of cases. Results of genome-wide association studies in adult HL, consistent associations between autoimmune diseases and HL in adults, and increased HL incidence in children with congenital and acquired immunodeficiencies together suggest a role for immune dysregulation in lymphomagenesis. Here we examined associations between personal and family histories of infectious, autoimmune, and allergic diseases and HL in children and adolescents using data from the largest case-control study conducted to date. Healthy control children identified by random digit dialing were matched on sex, race/ethnicity, and date of birth to cases diagnosed in 1989-2003 at 0-14 years at North American COG institutions. Parents of 517 cases and 784 controls completed standardized 1-hour telephone interviews, including items regarding medical histories. Tumor EBV status was determined by detection of EBV-encoded RNAs via digoxigenin-based in situ hybridization. Using multivariate conditional logistic regression, we calculated odds ratios (ORs) and 95% confidence intervals (CIs) for risk of HL overall, and by tumor EBV status (EBV+, EBV-) and HL subtypes (nodular sclerosis, mixed cellularity, lymphocyte predominant) after adjusting for income, education, and birth order. Cases (OR=1.79, 95% CI: 1.04-3.08) were more likely to have had an infection &amp;gt;1 year prior to HL diagnosis, and EBV+ cases were more likely to report prior sore throats (including strep throat, scarlet fever, tonsillitis; OR=2.19, 95% CI: 1.06-4.53) and infectious mononucleosis (OR=9.10, 95% CI: 0.81-102.3). Case siblings were also more likely to have had an infection prior to HL diagnosis (OR=2.04, 95% CI: 1.01-4.14). In index children, autoimmune (OR=0.46, 95% CI: 0.16-1.38) and allergic (OR=1.22, 95% CI: 0.93-1.59) diseases identified &amp;gt;1 year before HL diagnosis were not associated with HL overall or by EBV status or subtype. There were no notable associations for autoimmune or allergic diseases in parents or siblings. These results may indicate a true increased risk for infections and lack of association with history of conditions imparting immune dysregulation, or they may be attributable to inaccurate recall. Future studies should employ biomarkers to understand differences in immune function preceding pediatric HL. Supported by NIH Grants R01 CA047473 and K05 CA157439, and Children's Cancer Research Fund, Minneapolis, MN. Citation Format: Amy M. Linabery, Erik B. Erhardt, Rachel K. Fonstad, Seymour Grufferman, Richard F. Ambinder, Greta R. Bunin, Julie A. Ross, Logan G. Spector. Infectious, autoimmune, and allergic diseases and risk of Hodgkin lymphoma in children and adolescents: A Children's Oncology Group (COG) study. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2537. doi:10.1158/1538-7445.AM2013-2537 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Epstein-Barr virus gene polymorphism in different parts of the same nasopharyngeal carcinoma patient

In most previous studies, comparisons of Epstein-Barr virus (EBV) gene polymorphisms and genotypes were made between strains from tumors and normal throat washings (TWs) from different individuals. However, it remains controversial whether different EBV subtypes are present in different parts of the same NPC patient. In order to address this question, in this study, we compared the genotypes in sets of paired throat washings (TW) and paraffin-embedded tissues of 20 patients with nasopharyngeal carcinoma (NPC), and we found the same genotype in throat washings (TWs) and tumor cells from the same individual in most cases. The subtypes of EBER and EBNA1 genes were furthermore sequenced, and identical EBV strains were identified in tumor tissues and TWs. In conclusion, different sites of the same individual are infected by the same EBV strains, except for a few differences in occasional cases, suggesting that the EBV subtype detected in throat washings is a reasonable guide to the subtype present in the carcinoma tissue.

A Case of Age-Related Epstein–Barr Virus (EBV)-Associated B Cell Lymphoproliferative Disorder, so-called Polymorphous Subtype, of the Mandible, with a Review of the Literature

Epstein-Barr virus (EBV) is known to be associated with the development of lymphomas in immunocompromised patients. Recently, age-related immune impairment has been recognized as a predisposing factor in the development of EBV-driven lymphoproliferative disorders (LPDs) in elderly patients without any known immunodeficiency or prior lymphoma. In approximately 70% of reported cases, the affected sites have been extranodal, such as the skin, lung, tonsil and stomach. However, age-related EBV-associated B cell (EBV + B cell) LPD is extremely rare in the oral cavity. Here we report a 71-year-old Japanese man who developed an EBV + B cell LPD resembling classical Hodgkin lymphoma (CHL)--so-called polymorphous subtype-of the mandible. Histopathologically, infiltration of large atypical lymphoid cells including Hodgkin or Reed-Sternberg-like cells into granulation tissue with marked necrosis was found in the mandibular bone. Immunohistochemical analysis revealed that the large atypical Hodgkin or Reed-Sternberg-like cells were CD3-, CD15-, CD20+, CD30+ and Epstein-Barr virus (EBV)-latent infection membrane protein-1 (LMP-1)+. In situ hybridization (ISH) demonstrated EBV-encoded small RNA (EBER) + in numerous Hodgkin or Reed-Sternberg-like cells. EBNA-2 was detected by polymerase chain reaction (PCR) using an extract from the formalin-fixed, paraffin-embedded specimen. To our knowledge, this is the first reported case of the polymorphous subtype of age-related EBV + B cell LPD affecting the mandible.

Characterization of Epstein–Barr virus (EBV) BZLF1 gene promoter variants and comparison of cellular gene expression profiles in Japanese patients with infectious mononucleosis, chronic active EBV infection, and EBV‐associated hemophagocytic lymphohistiocytosis

Epstein-Barr virus (EBV) genotypes can be distinguished based on gene sequence differences in EBV nuclear antigens 2, 3A, 3B, and 3C, and the BZLF1 promoter zone (Zp). EBV subtypes and BZLF1 Zp variants were examined in Japanese patients with infectious mononucleosis, chronic active EBV infection, and EBV-associated hemophagocytic lymphohistiocytosis. The results of EBV typing showed that samples of infectious mononucleosis, chronic active EBV infection, and EBV-associated hemophagocytic lymphohistiocytosis all belonged to EBV type 1. However, sequencing analysis of BZLF1 Zp found three polymorphic Zp variants in the same samples. The Zp-P prototype and the Zp-V3 variant were both detected in infectious mononucleosis and chronic active EBV infection. Furthermore, a novel variant previously identified in Chinese children with infectious mononucleosis, Zp-V1, was also found in 3 of 18 samples of infectious mononucleosis, where it coexisted with the Zp-P prototype. This is the first evidence that the EBV variant distribution in Japanese patients resembles that found in other Asian patients. The expression levels of 29 chronic active EBV infection-associated cellular genes were also compared in the three EBV-related disorders, using quantitative real-time reverse transcription polymerase chain reaction analysis. Two upregulated genes, RIPK2 and CDH9, were identified as common specific markers for chronic active EBV infection in both in vitro and in vivo studies. RIPK2 activates apoptosis and autophagy, and could be responsible for the pathogenesis of chronic active EBV infection.

Relationship of Cell Bearing EBER and p24 Antigens in Biopsy-proven Lymphocytic Interstitial Pneumonia in HIV-1 Subtype E Infected Children

Lymphocytic interstitial pneumonia (LIP) is an uncommon histopathologic entity characterized by infiltration of the interstitium and alveolar spaces of the lung by lymphocytes and other lymphoid elements. An increased incidence of LIP has been seen in the pediatric population, especially in children with acquired immune deficiency syndrome. Our previous study supports the notion that Langerhans cells (LCs) are reservoirs for Epstein-Barr virus (EBV) in lungs of human immunodeficiency virus (HIV) subtype E-infected pediatric LIP. To further understand the pathogenesis of LIP, we studied the relationship between EBV, the suggested causative agent of LIP and HIV-1 capsid protein p24, which play an important role in the interaction with host proteins during HIV-1 adsorption, membrane fusion, and entry in surgical lung biopsy-proven LIP from 9 vertically HIV subtype E-infected pediatric patients. The dominant microscopic feature of LIP demonstrated widespread widening of alveolar septum by mononuclear inflammatory cell infiltrate, mainly composed of mature lymphocytes and plasma cells surrounding airways and expanding to the lung interstitium. EBV-encoded RNA (EBER) in situ hybridization (ISH) and p24 immunohistochemistry, performed on formalin-fixed, paraffin-embedded tissue from open lung biopsy specimens, revealed positive intranuclear EBER signals and intracytoplasmic immunostains for p24 core protein in all 9 LIP cases. By combining ISH and immunohistochemistry, these results suggest that (i) EBV/p24-carrying cells are likely involved in the development of LIP, either directly or indirectly; (ii) LCs and related dendritic cells are the main reservoir of both EBV and HIV subtype E in pediatric LIP and possibly LCs may play an important role in the recruitment of inflammatory cell infiltrates, especially T cells into these tissues; (iii) coexpression of EBV/p24 in bronchioalveolar epithelium supports the hypothesis that these cells serve as a reactivation source for both viruses to achieve greater quantities in alveolar septum and interstitium around bronchioles. These results indicate a strong association between the presence of HIV core protein p24 and expression of EBV RNA transcripts (EBER). Interactions between LCs and related dendritic cells together with T cells are important for effective HIV and EBV replications. The coexpression of both viruses could be related to the evolution of pediatric LIP in HIV subtype E infection.

Clinicopathologic and Molecular Features of 122 Brazilian Cases of Nodal and Extranodal NK/T-Cell Lymphoma, Nasal Type, With EBV Subtyping Analysis

Extranodal natural killer/T-cell lymphoma, nasal type (NK/TCL) is more prevalent in Asia and in some areas of South and Central America, but it is rarely seen in the United States and Europe. In this study, a series of 122 cases of NK/TCL from Brazil was analyzed with respect to clinicopathologic features. Clinical characteristics and geographic distribution were evaluated in 97 cases of nasal/nasopharyngeal region and 23 cases in extranasal sites including 6 nodal cases. Clinical staging and follow-up information was available in a subset of 21 patients. All cases harbored Epstein-Barr virus (EBV), 95% and 85% expressed cytoplasmic CD3 and CD56, respectively, and all cases were positive for at least 1 marker for cytotoxic granules. The global distribution of EBV subtypes showed predominance of strain subtype A, 89%, and subtype B, 11%. No dual infections were detected. TCR-γ TCR-gene rearrangement was observed in 7 cases; all of them extranodal. Three of TCR-γ(+) cases showed EBV subtype A. Two TCR-γ(+)/CD56(+) cases showed EBV subtype B. Geographic distribution of NK/TCL showed higher frequency in the southeast and northeast regions of Brazil. Striking differences among geographic regions were seen with the vast majority of EBV subtype B (86%) occurring in the south and southeast regions.

Primary Cutaneous Epstein-Barr Virus-Associated T-Cell Lymphoproliferative Disorder-2 Cases With Unusual, Prolonged Clinical Course

Hydroa vacciniforme-like lymphoma and extranodal-type natural killer (NK)/T-cell lymphoma are prototypes of Epstein-Barr virus (EBV)-associated cutaneous T- or NK-cell lymphomas. Hydroa vacciniforme-like lymphoma with systemic spread and extranodal-type NK/T-cell lymphoma are characterized by aggressive clinical course. We describe 2 patients with primary cutaneous EBV-associated T-cell lymphomas who did not satisfy the criteria for well-defined entities and showed unusual, prolonged clinical course. They presented with skin ulcerations and mass lesions confined to the extremities without systemic involvement. Skin biopsies demonstrated dense superficial and deep perivascular and periappendageal lymphoid infiltrates expressing CD3 and CD8, but not CD56. The EBV genomes were found within the tumor cells, and monoclonal T-cell receptor gene rearrangement was present. We suggest that these cases represent a peculiar subtype of EBV+ cutaneous T-cell lymphoma, with a tendency to localize in the skin of the extremities and has an indolent clinical course.

Genotypic Analysis of Epstein-Barr Virus Isolates Associated with Nasopharyngeal Carcinoma in Northern China

Objectives: To characterize the specific Epstein-Barr virus (EBV) polymorphisms from nasopharyngeal carcinoma (NPC) patients and healthy donors in Northern China, and to explore the relationship between the EBV genotypes and NPC. Methods: The genotypes of EBV strains were analyzed by the polymerase chain reaction and restriction fragment length polymorphism. Results: The predominant EBV type was A, C or F in both NPC and healthy individuals. The distributions of the EBV subtypes between NPC and healthy donors were not significantly different (p > 0.05). The frequency of type f strains in NPC was significantly lower in Northern China than in Southern China. Conclusions: No evidence of special EBV genotypes associated with NPC was found, suggesting that EBV strains derived from the NPC patients may reflect geographic distribution rather than being NPC restricted.

Abstract 1199: Global proteomic evaluation of the relationship between Epstein-Barr virus (EBV) and c-myc deregulation in endemic versus sporadic Burkitt lymphoma (BL)

Abstract Endemic BL (eBL) is characteristically positive(100%) for EBV, contrasting with sporadic(sBL) BL(∼30% EBV+). eBL vs sBL have different breakpoint regions within c-myc. The different mechanisms of lymphomagenesis however, remain unknown. Global analysis of proteins expressed in the different subtypes may provide insights into biologic, pathogenetic, and molecular differences. Objectives: To compare the proteomic expression profile and signal transduction pathways of EBV+ eBL with EBV+/- sBL and EBV+/- normal B-cells. Normal B-cells (EBV+/-) were isolated from leukopacks obtained from the NY Blood Center using Magnetic Cell Sorting CD20 MicroBeads (Miltenyi biotec, CA). Whole cell lysates obtained from EBV+ eBL Raji, EBV+ sBL NC37, EBV- sBL Ramos, and EBV± B-cells were digested and labeled with iTRAQ reagents. The peptides were resolved by 2D-LC technique. MS/MS spectra were acquired using an Orbitrap XL Tandem Mass Spectrometer (ThermoFisher). MS/MS data was searched using X! Tandem TPP software against human IPI database (v3.50) appended with decoy sequences. iTRAQ ratios of proteins (ProteinProphet probability of &amp;gt;0.9) were normalized and differentially expressed proteins were selected for further analysis. Over 400 proteins were identified; 827 binary differential protein expressions were established with a False Discovery Rate (FDR) of &amp;lt;0.4. Hierarchical clustering of the expression profiles grouped the 3 lymphoma cell lines and the 2 normal B-cell specimens. Specific cellular functions were implicated by differential protein expressions (with associated proteins in parentheses) including apoptotic signaling (AK2, C1QBP, DIABLO, PCNA, PPIF, SERPIN8&amp;9, SET), viral pathogenesis (DDX3X, SYNCRIP, EIF5A), cell proliferation pathways and oncogenes (ANXA6, DDX5, DEK, GNB2L1, NME2, RAP1A&amp;B, RIPK4, STMN1), c-myc expression regulation (ENO1, FUBP1), heat shock and ubiquitination (HSP90AB1, HSP90B1, HSPA5, UCHL1). There are several proteins with established links to malignancy (RPL15, PSMA4, SLC3A2, TLX2, VIM) including TUBB2C, whose overexpression is found in pediatric BL and potential biomarkers for disease (IGHM, IGSF3, LDHA, LDHB, PPBP, LSP1, LYZ, DEFA1, DEFA3). We identified 41 proteins within the c-myc network, 21 of which are c-myc targets. Our results suggest that there are potentially different mechanisms driving cell proliferation and resistance to apoptosis in the different BL subtypes. Confirmatory studies will be required to establish the correlation between EBV, c-myc, and geographical subtype and how they may be involved in promoting lymphomagenesis. Ultimately, identification of proteins unique to the distinct disease subtypes will serve to establish tumor markers that may enable development of new diagnostic, prognostic, and therapeutic strategies. * Drs. Lim and Cairo are equal senior authors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1199.

Age-related Epstein-Barr virus (EBV)–associated B-cell lymphoproliferative disorders: comparison with EBV-positive classic Hodgkin lymphoma in elderly patients

AbstractAge-related Epstein-Barr virus–associated B-cell lymphoproliferative disorder (aEBVLPD) is a disease group characterized by EBV-associated large B-cell lymphoma in elderly without predisposing immunodeficiency. In nearly one- third of cases, aEBVLPD occurs as a polymorphous subtype with reactive cell-rich components, bearing a morphologic similarity to classic Hodgkin lymphoma (cHL). The aim of this study was to clarify clinicopathologic differences between the polymorphic subtype of aEBVLPD (n = 34) and EBV+ cHL (n = 108) in patients aged 50 years or older. Results showed that aEBVLPD was more closely associated with aggressive clinical parameters than cHL, with a higher age at onset (71 vs 63 years); lower male predominance (male-female ratio, 1.4 vs 3.3); and a higher rate of involvement of the skin (18% vs 2%), gastrointestinal tract (15% vs 4%), and lung (12% vs 2%). aEBVLPD was histopathologically characterized by a higher ratio of geographic necrosis, greater increase (> 30%) in cytotoxic T cells among background lymphocytes, higher positivity for CD20 and EBNA2, and absence of CD15 expression. As predicted by the clinical profile, aEBVLPD had a significantly poorer prognosis than EBV+ cHL (P < .001). The polymorphous subtype of aEBVLPD constitutes an aggressive group with an immune response distinct from EBV+ cHL, and requires the development of innovative therapeutic strategies.

Association between EBNA2 and LMP1 subtypes of Epstein-Barr virus and nasopharyngeal carcinoma in Thais

Background Nasopharyngeal carcinoma (NPC) is an Epstein-Barr virus (EBV)-associated malignancy that is common in Thailand. The association of the EBV nuclear antigen 2 gene ( EBNA2) and the latent membrane protein 1 gene ( LMP1) with NPC is unclear. Objective To determine the association between EBNA2 and LMP1 subtypes of EBV and NPC in Thais. Study design EBV DNA from blood samples of 75 Thai patients with NPC and 44 age-matched controls was examined by PCR for EBNA2 subtypes ( EBV type 1 and type 2) and LMP1 subtypes (wild-type LMP1 ( wt-LMP1) and LMP1 containing a 30-bp deletion ( del-LMP1)). Results The frequencies of EBV type 1 in cases (96.0%) and controls (97.7%) were similar. The frequency of del-LMP1 in cases (58.7%) was higher than in controls (36.4%) ( p = 0.023). Among NPC patients, the frequency of del-LMP1 in stages III and IV (65.6%) was higher than in stages I and II (18.2%) ( p = 0.006). By analyzing nucleotide sequence of the LMP1 region, a new subtype, Thai1, was identified. Conclusion LMP1 subtypes of EBV may be involved in NPC susceptibility. The del-LMP1 variant may be a useful molecular marker for early detection, diagnosis and prognosis of Thai patients with NPC.