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      Epstein-Barr Virus (EBV) detection and typing by PCR: a contribution to diagnostic screening of EBV-positive Burkitt's lymphoma

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          Abstract

          Background

          Epstein-Barr virus (EBV) is associated to the etio-pathogenesis of an increasing number of tumors. Detection of EBV in pathology samples is relevant since its high prevalence in some cancers makes the virus a promising target of specific therapies. RNA in situ hybridization (RISH) is the standard diagnostic procedure, while polymerase chain reaction (PCR)-based methods are used for strain (EBV type-1 or 2) distinction. We performed a systematic comparison between RISH and PCR for EBV detection, in a group of childhood B-cell Non-Hodgkin lymphomas (NHL), aiming to validate PCR as a first, rapid method for the diagnosis of EBV-associated B-cell NHL.

          Methods

          EBV infection was investigated in formalin fixed paraffin-embedded tumor samples of 41 children with B-cell NHL, including 35 Burkitt's lymphoma (BL), from Rio de Janeiro, Brazil, by in situ hybridization of EBV-encoded small RNA (EBER-RISH) and PCR assays based on EBNA2 amplification.

          Results

          EBV genomes were detected in 68% of all NHL. Type 1 and 2 accounted for 80% and 20% of EBV infection, respectively. PCR and RISH were highly concordant (95%), as well as single- and nested-PCR results, allowing the use of a single PCR round for diagnostic purposes. PCR assays showed a sensitivity and specificity of 96% and 100%, respectively, with a detection level of 1 EBV genome in 5,000–10,000 EBV-negative cells, excluding the possibility of detecting low-number EBV-bearing memory cells.

          Conclusion

          We describe adequate PCR conditions with similar sensitivity and reliability to RISH, to be used for EBV diagnostic screening in high grade B-NHL, in "at risk" geographic regions.

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          Most cited references 35

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          A revised European-American classification of lymphoid neoplasms: a proposal from the International Lymphoma Study Group.

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            B cells under influence: transformation of B cells by Epstein-Barr virus.

             Ralf Küppers (2003)
            Epstein-Barr virus (EBV) is an extremely successful virus, infecting more than 90% of the human population worldwide. After primary infection, the virus persists for the life of the host, usually as a harmless passenger residing in B cells. However, EBV can transform B cells, which can result in the development of malignant lymphomas. Intriguingly, the three main types of EBV-associated B-cell lymphoma - that is, Burkitt lymphoma, Hodgkin lymphoma and post-transplant lymphomas - seem to derive from germinal-centre B cells or atypical survivors of the germinal-centre reaction in most, if not all, cases, indicating that EBV-infected germinal-centre B cells are at particular risk for malignant transformation.
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              Molecular diagnosis of Epstein-Barr virus-related diseases.

              Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis, and it may also be found in a wide variety of benign and malignant lesions including oral hairy leukoplakia, inflammatory pseudotumor, Hodgkin's disease, non-Hodgkin's lymphoma, nasopharyngeal carcinoma, and gastric carcinoma. Molecular testing is increasingly important in the diagnosis and monitoring of patients affected by these diseases. In biopsy tissues, molecular detection of EBV-encoded RNA transcripts by in situ hybridization remains the gold standard for proving that a histopathological lesion is EBV-related. EBV-encoded RNA hybridization and EBV LMP1 immunostains are used routinely to detect latent EBV in tissues affected by posttransplant lymphoproliferative disorder (PTLD) or in enlarged nodes from patients with infectious mononucleosis. Traditional serology is the best test for evaluating acute versus remote infection in healthy individuals. High serological titers serve as a tumor marker for some EBV-related malignancies, but titers are not a dependable tumor marker in immunocompromised hosts. EBV viral load testing by quantitative DNA amplification of blood samples is a promising new laboratory test that has proven useful for early diagnosis and monitoring patients with PTLD. Recent studies suggest a role for EBV viral load testing in nasopharyngeal carcinoma, Hodgkin's disease, and AIDS patients with brain lymphoma. Further research is needed to define more fully the clinical utility of viral load tests in the full spectrum of EBV-associated diseases. Gene expression profiling is on the horizon as a means to improve subclassification of EBV-related diseases and to predict response to therapy.
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                Author and article information

                Journal
                Diagn Pathol
                Diagnostic Pathology
                BioMed Central (London )
                1746-1596
                2006
                7 August 2006
                : 1
                : 17
                Affiliations
                [1 ]Bone Marrow Transplantation Center (CEMO), Instituto Nacional de Câncer (INCA), Praça Cruz Vermelha 23, 20230-130, 6 th floor, Rio de Janeiro, RJ, Brazil
                [2 ]Department of Pathology, Botucatu School of Medicine, Universidade Estadual Paulista, São Paulo, SP, Brazil
                [3 ]Hematology Service, Instituto Nacional de Câncer (INCA), Praça Cruz Vermelha 23, 20230-130, Rio de Janeiro, RJ, Brazil
                [4 ]Genetics Division, Instituto Nacional de Câncer (INCA), Rua André Cavalcanti 37, 4 th floor, 20231-050, Rio de Janeiro, RJ, Brazil
                Article
                1746-1596-1-17
                10.1186/1746-1596-1-17
                1559641
                16893464
                Copyright © 2006 Hassan et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                Categories
                Methodology

                Pathology

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