A rapid and reliable enzyme immunoassay PCR-based screening method to identify EBV-carrying gastric carcinomas.
Epstein-Barr virus (EBV) is associated with a substantial number of gastric adenocarcinomas worldwide, as confirmed by EBER1/2-RNA in situ hybridization (RISH). In the present study, we developed a rapid and sensitive PCR-based prescreening method for the detection of EBV in gastric carcinomas to reduce the amount of laborious EBER1/2-RISH assays to be performed. The method was evaluated by testing gastric adenocarcinomas (n = 242) using both BamHI W PCR-enzyme immunoassay (EIA) and EBER1/2-RISH, in combination with appropriate DNA and RNA quality controls. Seventy-four percent of the paraffin-embedded gastric adenocarcinomas had good DNA quality as shown by beta-globin polymerase chain reaction (PCR) after proteinase K and boiling pretreatment, whereas after DNA purification this was increased to 90%. Thirty-two percent of all cases were EBV-DNA positive after PCR-EIA, whereas 10% of these gastric cancers contained EBV transcripts in the neoplastic cells as confirmed by EBER1/2-RISH. Interestingly, only samples with high optical density (OD) 405/630 values in PCR-EIA, equivalent to the maximum reading of the assay as determined by the positive control, contained EBV-positive tumor cells in the EBER1/2-RISH. In contrast, the weak positive samples, as determined by low OD readings in the PCR-EIA were EBER1/2-RISH negative. In conclusion, high OD values in EBV PCR-EIA are very valuable to prescreen EBV-carrying gastric carcinomas as confirmed by EBER1/2-RISH. Only these samples and those with poor DNA quality will require testing in the EBER1/2-RISH, thereby reducing the amount of laborious RISH assays with 85%.
Department of Pathology, Vrije Universiteit Medical Center, Amsterdam, The Netherlands.
Josine
J
van Beek
Axel
A
zur Hausen
Elma Klein
EK
Kranenbarg
Ralph J
RJ
Warring
Elisabeth
E
Bloemena
Mikael E
ME
Craanen
Cornelis J H
CJ
van de Velde
Jaap M
JM
Middeldorp
Chris J L M
CJ
Meijer
Adriaan J C
AJ
van den Brule
D000199:Actins / Q000032:analysis; D000230:Adenocarcinoma / Q000175:diagnosis / Q000821:virology; D004279:DNA, Viral / Q000032:analysis*; D020031:Epstein-Barr Virus Infections / Q000821:virology; D004854:Herpesvirus 4, Human / Q000302:isolation & purification*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D007150:Immunohistochemistry; D017403:In Situ Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis; D016601:RNA-Binding Proteins / Q000032:analysis; D012269:Ribosomal Proteins; D012680:Sensitivity and Specificity; D013274:Stomach Neoplasms / Q000175:diagnosis / Q000821:virology; D014412:Tumor Virus Infections / Q000821:virology*
D000199:Actins; D004279:DNA, Viral; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012269:Ribosomal Proteins; C067169:RPL22 protein, human