Epstein‐Barr virus and gastric carcinoma

Epstein‐Barr virus (EBV) has been accepted as an infective agent causing gastric carcinoma (GC). Epstein‐Barr virus‐associated GC, comprising nearly 10% of all cases of GC, is the monoclonal growth of EBV‐infected epithelial cells, which express several EBV‐latent genes (latency I program). Sequential events in the gastric mucosa could be traced from EBV infection of the pit cells to fully developed carcinomas by EBV encoded small RNA (EBER)‐in situ hybridization. The histological features of the carcinoma consist of a lace pattern of carcinoma cells within the mucosa and the dense infiltration of lymphocytes and macrophages at the invasive site, which might be due to cytokines produced by neoplastic cells. The primary molecular abnormality in EBV‐associated GC is global and non‐random CpG island methylation in the promoter region of many cancer‐related genes. The experimental system of recombinant EBV infection using GC cell lines demonstrated that viral latent membrane protein 2A (LMP2A) is responsible for the promotion of DNA methylation. LMP2A up‐regulates cellular DNMT1 through the phosphorylation of STAT3, causing CpG methylation of a tumor suppressor gene, PTEN. DNA methylation in EBV‐infected stomach cells may be due to overdrive of the cellular defense against foreign DNA, which eventually leads to the development of EBV‐associated GC.     

Cellular normalization of viral DNA loads on whole blood improves the clinical management of cytomegalovirus or Epstein Barr virus infections in the setting of pre‐emptive therapy

Two quantitative duplex real‐time PCR assays were developed for co‐amplification of human albumin and cytomegalovirus (CMV) or Epstein Barr virus (EBV) genes after automated extraction on whole blood, and compared two units for expressing viral DNA loads (copies per ml of blood or per 10⁶ peripheral blood leukocytes (PBLs)) on 1,138 positive samples. Both PCRs were characterized by high sensitivity, reproducibility, and linear range. Automated extraction by a MagNA Pure LC Instrument was shown to be more efficient when peripheral blood cell count was inferior to 5 × 10⁹ PBLs/L. Albumin co‐amplification allows the detection of PCR inhibitors and normalization of viral load according to the number of cells calculated in the sample. The two ways of expressing viral load results were highly correlated, but quantitative differences varied in relation to variations of blood cell count. As these two viruses are highly cell associated, viral loads can be underestimated in patients with leucopenia. In the setting of pre‐emptive strategies during CMV infection, the units in which results are expressed can influence clinical management, as illustrated in this article. J. Med. Virol. 81:90–98, 2009. © 2008 Wiley‐Liss, Inc.     

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.     

Evaluation of nasal and nasopharyngeal swab collection for the detection of Epstein‐Barr virus in nasopharyngeal carcinoma

Epstein‐Barr virus detection using nasopharyngeal swabs has been suggested as a potential screening test that could improve the specificity of current EBV‐based serological assays. However, application requires insertion of the swab deep into the nasopharynx, a procedure not amenable to non‐clinic screening. We reasoned that swabbing the more easily accessible nasal cavity might provide an appealing alternative for NPC detection. Patients > 18 years of age diagnosed with histologically confirmed NPC were recruited from the Otolaryngology Department at the National Taiwan University Hospital. ENT clinicians collected both nasal and nasopharyngeal swabs. EBV DNA and cellular beta‐globulin DNA were quantified using quantitative PCR targeting a highly‐conserved region of the BKRF1 gene. EBV DNA was detectable (non‐zero) in all 34 nasopharyngeal swabs and above the positivity threshold of 1666 EBV copies in 30 (88.2%) patients. EBV DNA was detectable in 50% of 34 nasal swabs and above the positivity threshold in four (11.8%) patients. Average EBV DNA levels were >3‐fold higher (P < 0.001) in nasopharyngeal compared to nasal swabs. Among the 17 NPC patients with detectable EBV DNA in both swab types, we observed correlation (P < 0.01) between EBV DNA measurements. Our data represent the first evaluation of EBV DNA collected from nasal swabs. Given current EBV DNA amplification techniques, nasopharyngeal swabs remain more sensitive than nasal swabs for NPC detection.     

Cytokine expression associated with Helicobacter pylori and Epstein‐Barr virus infection in gastric carcinogenesis

Helicobacter pylori and Epstein‐Barr virus (EBV) infection, and associated cytokines are involved in gastric carcinogenesis. We investigated the expression of these cytokines and their relationship with clinicopathological characteristics. The study included specimens from 207 patients with gastric adenocarcinoma, 56 with chronic gastritis, 32 with metaplasia, and 30 with low‐grade epithelial dysplasia. Tissue microarrays were constructed and immunohistochemical staining for IL‐1β, IL‐6, IL‐10, IL‐17, p16, p21, TNF‐α, and TNFR1 was performed. EBV and H. pylori infection status was determined. IL‐1β, IL‐6, IL‐17, p16, and p21 protein expression was significantly higher in adenocarcinoma cases than in the other cases (p < 0.05). EBV was only noted in adenocarcinoma (13 cases, 6.3%). The H. pylori infection rate in adenocarcinoma was significantly higher than that in the other cases (p < 0.005). IL‐6 expression was associated with improved survival (p < 0.05), whereas IL‐17 expression was associated with decreased survival (p < 0.05). IL‐6 expression was inversely associated with angioinvasion, and disease stage (p < 0.05), whereas IL‐17 expression was associated with disease stage (p < 0.05). IL‐10 expression was correlated with IL‐1β and TNF‐α expression, and p16 expression was correlated with IL‐17 and EBV status. Our results indicate that IL‐6 and IL‐17 are associated with gastric carcinogenesis and may be considered prognostic factors.     

Signaling pathways involved in the T‐cell‐mediated immunity against Epstein‐Barr virus: Lessons from genetic diseases

Primary immunodeficiencies (PIDs) provide researchers with unique models to understand in vivo immune responses in general and immunity to infections in particular. In humans, impaired immune control of Epstein‐Barr virus (EBV) infection is associated with the occurrence of several different immunopathologic conditions; these include non‐malignant and malignant B‐cell lymphoproliferative disorders, hemophagocytic lymphohistiocytosis (HLH), a severe inflammatory condition, and a chronic acute EBV infection of T cells. Studies of PIDs associated with a predisposition to develop severe, chronic EBV infections have led to the identification of key components of immunity to EBV – notably the central role of T‐cell expansion and its regulation in the pathophysiology of EBV‐associated diseases. On one hand, the defective expansion of EBV‐specific CD8 T cells results from mutations in genes involved in T‐cell activation (such as RASGRP1, MAGT1, and ITK), DNA metabolism (CTPS1) or co‐stimulatory pathways (CD70, CD27, and TNFSFR9 (also known as CD137/4‐1BB)) leads to impaired elimination of proliferating EBV‐infected B cells and the occurrence of lymphoma. On the other hand, protracted T‐cell expansion and activation after the defective killing of EBV‐infected B cells is caused by genetic defects in the components of the lytic granule exocytosis pathway or in the small adapter protein SH2D1A (also known as SAP), a key activator of T‐ and NK cell‐cytotoxicity. In this setting, the persistence of EBV‐infected cells results in HLH, a condition characterized by unleashed T‐cell and macrophage activation. Moreover, genetic defects causing selective vulnerability to EBV infection have highlighted the role of co‐receptor molecules (CD27, CD137, and SLAM‐R) selectively involved in immune responses against infected B cells via specific T‐B cell interactions.     

Cytomegalovirus and Epstein–Barr virus coinfection in three toddlers with prolonged illnesses

Cytomegalovirus (CMV) and Epstein–Barr virus (EBV) usually cause primary and latent infections during childhood; thus, coinfection with these viruses occurs occasionally in children. However, its clinical impact has not been established, and may be underestimated. Three cases of coinfection involving these two viruses in toddlers are described: a 14‐month‐old male with infectious mononucleosis, an 18‐month‐old female with hemophagocytic lymphohistiocytosis, and a 13‐month‐old female with acute hepatitis. All three patients had prolonged illnesses. Serial serological testing and quantitation of viral DNA for CMV and EBV using peripheral blood from the patients suggested primary infections with both viruses. In all three cases, the viral load of EBV and CMV in the early stage of disease exceeded 6.4 × 10³ and 8.8 × 10² copies/ml of whole blood, respectively, suggesting that the viruses were associated with the clinical condition. Recognizing that coinfection with these viruses may modulate the clinical course of disease is important. J. Med. Virol. 81:1399–1402, 2009. © 2009 Wiley‐Liss, Inc.     

Gastric carcinoma with osteoclast‐like giant cells. Lymphoepithelioma‐like carcinoma with Epstein–Barr virus infection is the predominant type

Osteoclast‐like giant cells (OGC) are rare in gastric carcinomas. Histopathological study of seven gastric carcinomas with OGC demonstrated three distinct types: lymphoepithelioma‐like carcinoma (LELC), non‐LELC, and giant cell tumor (GCT) types. LELC is a poorly differentiated adenocarcinoma with prominent lymphoid stroma. The LELC type (n= 4) showed similar histology to LELC of the stomach, except that they were accompanied by OGC and granulomatous reaction. Epstein‐Barr virus (EBV) infection was demonstrated by EBV‐encoded RNA (EBER) in situ hybridization (ISH) in all the neoplastic cells. The non‐LELC type (n= 2) consisted of EBV‐negative carcinoma cells with inflammatory infiltrates. OGC and granulomas were frequently observed in the glandular lumens with accumulated mucus. The GCT type (n= 1) was a neuroendocrine carcinoma, containing many OGC with metaplastic bone formation, which showed typical morphological features of OGC in GCT of the bone. In all three types, OGC expressed CD68, but not cytokeratin, indicating that OGC had a reactive histiocytic lineage. Both LELC and non‐LELC types are included in the differential diagnosis of isolated granulomatous gastritis, and EBER‐ISH was useful for the identification of LELC type. Both LELC and no‐LELC types were also suggested to have better prognoses, but the behavior of the GCT type needs to be further characterized.     

A fulminant case of classical Hodgkin lymphoma: A diagnostic dilemma of Epstein‐Barr virus‐positive large B‐cell neoplasms

We report a fulminant case of classical Hodgkin lymphoma (CHL). The patient died only approximately 2 months after the onset of subjective symptoms. Autopsy specimens revealed atypical cells resembling Hodgkin and Reed‐Sternberg (HRS) cells in a rich inflammatory background in various organs. There were marked, characteristic angiodestructive lesions from infiltrating HRS‐like cells and numerous macrophages. The HRS‐like cells were infected with Epstein‐Barr virus (EBV), immunohistochemically positive for PAX5 and CD30, and negative for CD3, CD20, and ALK. Most B‐cell markers other than PAX5 were negative, and the HRS‐like cells also expressed cytotoxic molecules. Monoclonal rearrangement of immunoglobulin heavy chain was detected by PCR analysis. According to the 2016 WHO classification, we diagnosed mixed cellularity CHL. However, EBV‐positive diffuse large B‐cell lymphoma (DLBCL), not otherwise specified and EBV‐positive B‐cell lymphoma, unclassifiable with features intermediate between DLBCL and CHL were considered as differential diagnoses because both tumors are aggressive EBV‐positive large B‐cell neoplasms with reactive inflammatory cells and sometimes contains HRS‐like cells. The clinical condition of the current case was closer to these two entities than to CHL. A diagnosis of EBV‐positive large B‐cell neoplasms was difficult because of overlapping morphological and immunohistochemical characteristics, but should be considered for prognosis.     

Coexistence of primary pulmonary Hodgkin lymphoma and gastric MALT lymphoma associated with Epstein‐Barr virus infection: A case report

We describe a 66‐year‐old woman with Epstein‐Barr virus‐associated lymphoproliferative disorder with lung and gastric tumors. We identified two lung tumors measuring 13 and 20 mm in diameter that consisted of CD30‐, CD15‐, and CD20‐positive Hodgkin‐ and Reed‐Sternberg‐like cells and heterogeneous cellular infiltrates in a pronounced nodular pattern, with necrosis and vasculitis, diagnosed as nodular sclerosis classical Hodgkin lymphoma. A gastric tumor showed low‐grade extranodal marginal zone B‐cell lymphoma of the mucosa‐associated lymphoid tissue type. Neoplastic cells in all tumors expressed Epstein‐Barr virus‐encoded RNA based on in situ hybridization. The present case is a rare composite lymphoma arising from different extranodal organs, associated with EBV infection. Her medical history included gamma‐knife therapy for clinical diagnosis with a suspicion of cerebral lymphoma.     

Prognostic significance of CD20 expression and Epstein‐Barr virus (EBV) association in classical Hodgkin lymphoma in Japan: A clinicopathologic study

To investigate the clinicopathological significance of CD20 expression and Epstein‐Barr virus (EBV) association in Hodgkin and Reed–Sterberg cells of classical Hodgkin lymphoma (CHL), CD20 expression and EBV positivity (by EBER in situ hybridization) were investigated in 389 CHL patients in Japan. They included 74 CD20‐positive cases (19%) and 315 CD20‐negative cases (81%). CD20‐positive cases showed significantly older age at onset (P = 0.018) and higher association with EBV (P = 0.002). Multivariate analysis identified EBV‐positivity (but not CD20‐positivity), presence of B symptoms, thrombocytopenia, elevated serum lactate dehydrogenase and performance status >1 as poor prognostic factors for overall survival (OS). We constructed a new prognostic model with these five factors classifying patients into three groups: low risk, 0–1 adverse factor; intermediate risk, 2–3 factors; high risk, 4–5 factors. This prognostic model could stratify the prognosis of CHL patients (P < 0.0001). For 144 patients (58%) classified into the low‐risk group, the 5‐year OS was 91%. For 92 patients (37%) in the intermediate group, the 5‐year OS was 66%; for 11 patients (5%) in the high‐risk group, the 5‐year OS was 36%. In conclusion, EBV is identified as an independent poor prognostic factor for CHL patients. Therefore, examination of EBV association in CHL is recommended as routine pathologic practice especially in countries where EBV infection prevails.     

Disappearance of the Epstein–Barr virus in a relapse of Hodgkin's disease

Hodgkin's disease (HD) is associated with the Epstein–Barr virus (EBV) in approximately half of cases. This is a report of a case of nodular sclerosing HD of the B-cell type that was associated with EBV in the initial manifestation, but was found to be EBV-negative in the relapse of the tumour. Both tumours displayed similar clinical, pathological, and immunohistochemical features. This finding implies that in a given individual EBV can be lost from malignant tumours and therefore shows that the EBV infection is not required to maintain neoplastic growth of HD tumour cells. © 1997 John Wiley & Sons, Ltd.     

Sequence variations of Epstein‐Barr virus‐encoded BARF1 gene in nasopharyngeal carcinomas and healthy donors from southern and northern China

The BamHI A rightward frame 1 (BARF1) gene of the Epstein‐Barr virus (EBV) is involved in carcinogenesis and immunomodulation of EBV‐associated malignancies. The geographical distributions and the disease associations of BARF1 variants remain unclear. In the current study, the BARF1 variants in nasopharyngeal carcinoma (NPC) cases and healthy donors from southern and northern China, the NPC endemic and non‐endemic areas, as well as in 153 sequenced EBV genomes from diseased and normal people from around the world, were determined and compared among areas and populations. Only 1 consistent coding change, V29A, and several consistent silent mutations were identified. Two BARF1 types (B95‐8 and V29A) and 2 B95‐8 subtypes (B95‐8^t165545c and B95‐8^P) were classified. For Chinese isolates, the B95‐8 type was dominant in both southern and northern China, but the isolates from southern China showed a higher frequency of the B95‐8^t165545c subtype than the isolates from northern China (76.0%, 38/50 NPC cases and 50.7%, 37/73 healthy donors vs 26.4%, 24/91 NPC cases and 7.6%, 6/79 healthy donors, P < .0001). Furthermore, the B95‐8^t165545c subtype was more frequent in NPC cases than healthy donors in both southern China (P = .005) and northern China (P = .001). For EBV genomes, the B95‐8^P subtype was dominant in northern China, Europe, America, and Australia, while V29A was dominant in Africa. The B95‐8^t165545c subtype was only identified in Asia and demonstrated high frequency (81.2%, 26/32) in genomes from NPC cases in southern China. These results further reveal conservation and possibly geographically spread variations of BARF1 and may also indicate the preference of EBV strains with the B95‐8^t165545c subtype in NPC cases, without biological or pathogenic implications.     

Epstein‐Barr virus related post‐transplant lymphoproliferative disorder prevention strategies in allogeneic hematopoietic stem cell transplantation

Epstein‐Barr virus associated post‐transplant lymphoproliferative disorders (EBV PTLD) are recognized as a significant cause of morbidity and mortality in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). The number of patients at risk of developing EBV PTLD is increasing, partly as a result of highly immunosuppressive regimens, including the use of anti‐thymocyte globulin (ATG). Importantly, there is heterogeneity in PTLD management strategies between alloHSCT centers worldwide. This review summarizes the different EBV PTLD prevention strategies being utilized including the alloHSCT and T‐cell depletion regimes and the risk they confer; monitoring programs, including the timing and analytes used for EBV virus detection, as well as pre‐emptive thresholds and therapy with rituximab. In the absence of an institution‐specific policy, it is suggested that the optimal pre‐emptive strategy in HSCT recipients with T‐cell depleting treatments, acute graft vs host disease (GVHD) and a mismatched donor for PTLD prevention is (a) monitoring of EBV DNA post‐transplant weekly using plasma or WB as analyte and (b) pre‐emptively reducing immune suppression (if possible) at an EBV DNA threshold of >1000 copies/mL (plasma or WB), and treating with rituximab at a threshold of >1000 copies/mL (plasma) or >5000 copies/mL (WB). There is emerging evidence for prophylactic rituximab as a feasible and safe strategy for PTLD, particularly if pre‐emptive monitoring is problematic. Future management strategies such as prophylactic EBV specific CTLs have shown promising results and as this procedure becomes less expensive and more accessible, it may become the strategy of choice for EBV PTLD prevention.