ASSESSMENT OF THE METHODS FOR THE DETECTION OF EPSTEIN-BARR-VIRUS NUCLEIC-ACIDS AND RELATED GENE-PRODUCTS IN HODGKINS-DISEASE

BACKGROUND: Epstein-Barr virus (EBV) is present in the pathogenic cell s of a significant number of cases of Hodgkin's disease, particularly of the mixed cellularity subtype. EBV remains latent and the incidence of detection rate of the genomes and gene products varies greatly wit h the methods employed. EXPERIMENTAL DESIGN: From a pool of 137 cases of Hodgkin's disease previously studied by cold in situ hybridization (ISH) for the presence or absence of EBV DNA and the immunohistochemic al reactivity with anti-latent membrane protein 1 antibody, we selecte d 24 cases (12 EBV DNA-positive, 12 EBV DNA-negative) for Southern blo tting, as well as for study with nonisotopic EBER and BHLF1 oligonucle otide probes and amplification of DNA by polymerase chain reaction. EB V positive cases were further tested with anti-ZEBRA (BZLF1) antibody. RESULTS: The EBV DNA detection rate was found to be lower with Southe rn blotting compared to ISH and IHC methods because 8 of the 12 EBV po sitive cases were positive with BamHI W probe and only 7 (of the 8) wi th XhoI 1.9 kb probe. These 7 cases contained monoclonal episomal circ ular EBV genomes. All EBV DNA-positive cases showed EBER gene transcri ption by ISH. EBER probes also reacted with small lymphocytes in all E BV DNA+ and 9 EBV DNA- cases. These EBER-positive small lymphocytes we re detected neither by BamHI W DNA probe nor by immunohistochemical me thods with anti-latent membrane protein 1 and anti-EBNA2 antibodies. P olymerase chain reaction produced a positive result in all EBV DNA+ ca ses and 2 (of the 9) EBV DNA- cases containing EBER+ small lymphocytes . This discrepancy was attributed to amplification of EBV in reactive lymphocytes. Anti-ZEBRA antibody was positive in 2 cases (one BHLF1+) suggesting infrequent viral replication and probable abortive lytic cy cle. CONCLUSIONS: ISH and immunohistochemical methods are more sensiti ve than Southern blot for detecting EBV in Hodgkin and Reed-Sternberg cells of Hodgkin's disease. Polymerase chain reaction appears to be ve ry sensitive but is less sensitive and specific (amplification of EBV DNA of non-neoplastic lymphocytes) than ISH with non-isotopic EBER oli goprobes, which often detects and localizes EBV in pathogenic cells ev en when they are in small numbers and also in a few small lymphocytes. In addition, this method offers the advantage of being applied to rou tinely processed tissue sections.