Application of flow cytometric in situ hybridization assay to Epstein–Barr virus‐associated T/natural killer cell lymphoproliferative diseases

• Published in: Cancer science Vol. 103; no. 8; pp. 1481 - 1488
• Main Authors: Kawabe, Shinji, Ito, Yoshinori, Gotoh, Kensei, Kojima, Seiji, Matsumoto, Kimikazu, Kinoshita, Tomohiro, Iwata, Seiko, Nishiyama, Yukihiro, Kimura, Hiroshi
• Format: Journal Article
• Language: English
• Published: England John Wiley and Sons Inc 01.08.2012
• Subjects: Adolescent; Adult; Antibodies; Cancer; CD4 antigen; Child; Child, Preschool; DNA probes; Epstein-Barr virus; Epstein-Barr Virus Infections - complications; Epstein-Barr Virus Infections - immunology; Female; Flow cytometry; Flow Cytometry - methods; Fluorescence in situ hybridization; Genes, T-Cell Receptor; Herpesvirus 4, Human - genetics; Herpesvirus 4, Human - immunology; Humans; Immunoproliferative diseases; In Situ Hybridization, Fluorescence - methods; Infant; Killer Cells, Natural - immunology; Lymphocytes B; Lymphocytes T; Lymphoproliferative Disorders - diagnosis; Lymphoproliferative Disorders - pathology; Lymphoproliferative Disorders - virology; Male; Middle Aged; Natural killer cells; Original; Peripheral blood; Polymerase chain reaction; Prospective Studies; RNA; RNA probes; surface antigens; T-cell lymphoma; T-cell receptor; Young Adult
• ISSN: 1347-9032; 1349-7006; 1349-7006
• DOI: 10.1111/j.1349-7006.2012.02305.x

Epstein–Barr virus (EBV) infects various types of lymphocytes and is associated with not only B cell‐origin lymphoma, but also T or natural killer cell lymphoproliferative diseases (T/NK LPD). Recently, we established a novel assay to identify EBV‐infected cells using FISH. Using this assay, dual staining with antibodies to both surface antigens and an EBV‐encoded small RNA (EBER) probe can be performed. In the present study, we applied this recently developed FISH assay to EBV‐associated T/NK LPD to confirm its diagnostic utility. Using FISH, we prospectively analyzed peripheral blood from patients with suspected EBV‐associated T/NK LPD. The results were compared with those obtained using immunobead sorting followed by quantitative PCR. In all, 26 patients were included study. Using FISH, 0.15–67.0% of peripheral blood lymphocytes were found to be positive for EBER. Dual staining was used to determine EBER‐positive cell phenotypes in 23 of 26 subjects (88.5%). In five of seven patients with hydroa vacciniforme‐like lymphoma (an EBV‐positive cutaneous T cell lymphoma), EBER‐positive cells were identified as CD3+CD4−CD8− TCRγδ+ T cells. Furthermore, in a 25‐year‐old male patient with systemic EBV‐positive T cell LPD, two lymphocyte lineages were positive for EBER: CD4+CD8− and CD4−CD8+T cells. Thus, we confirmed that our newly developed assay is useful for quantifying and characterizing EBV‐infected lymphocytes in EBV‐associated T/NK LPD and that it can be used not only to complement the pathological diagnosis, but also to clarify the pathogenesis and to expand the spectrum of EBV‐associated diseases. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02305.x, 2012)