Comparison of in situ hybridization using different nonisotopic probes for detection of Epstein-Barr virus in nasopharyngeal carcinoma and immunohistochemical correlation with anti-latent membrane protein antibody.
BACKGROUND
The detection of Epstein-Barr virus (EBV) in nasopharyngeal carcinoma (NPC) may be of diagnostic importance, particularly in cases from nonendemic areas. For cellular localization of viral genomes, cold in situ hybridization methods for the demonstration of EBV-associated NPC remain difficult and relatively insensitive for routinely processed tissues.


METHODS
The aim of the present study was to assess the importance of tissue processing and the hybridization targets to improve the sensitivity of the cold in situ hybridization method. In situ hybridization was performed in six cases of NPC using three biotinylated EBV cDNA probes (BamHI W/IR1, BamHI Y/EBNA2, XhoI/latent membrane protein) and two cocktails of EBER and BHLF1 oligonucleotides labeled with fluorescein isothiocyanate on routinely fixed and paraffin embedded sections. In two cases, in situ hybridization was also performed on specially processed (ModAMeX) sections. Immunohistochemistry was used to detect EBV-induced antigens using monoclonal antibodies against latent membrane protein, EBNA2 and ZEBRA (BZLF1).


RESULTS
All cases showed EBV nucleic acids regardless of the tissue preparation with the three cDNA probes and on routinely processed sections with EBER oligonucleotides. By using cDNA probes, the best EBV DNA signal was obtained with BamHI W without heating of slides in tissue sections processed by ModAMeX, which probably gives rise to large amounts of single stranded DNAs. All cases positive with cDNA probes were found to be positive with EBER oligonucleotides and negative with BHLF1. However, on routinely processed paraffin sections, the signals with EBER oligonucleotides were stronger than with BamHI W cDNA probe. Dual labeling with in situ hybridization and immunohistochemistry showed that the hybridization signals were restricted to malignant epithelial cells. Latent membrane protein expression was detectable in four of six EBV nucleic acid-positive cases on both ModAMeX and routinely processed sections. The anti-EBNA2 and anti-ZEBRA antibodies were found to be negative on the two cases processed by ModAMeX.


CONCLUSIONS
Cold in situ hybridization, in particular with EBER oligonucleotides, appears to be more reliable than immunohistochemistry with anti-latent membrane protein antibody to detect EBV in NPC in routine pathology. These findings confirm a distinctive phenotype (latent membrane protein +/-, EBNA2-, ZEBRA-) of EBV-positive NPC. The negative staining for BHLF1 oligonucleotides further supports the viral latency.
Brousset
P
P
Anatomical Pathology Laboratory, Centre Hospitalier Universitaire Purpan, Toulouse, France.
Butet
V
V
Chittal
S
S
Selves
J
J
Delsol
G
G
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral / Q000276:immunology*; D001710:Biotin; D002277:Carcinoma / Q000382:microbiology*; D004247:DNA / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis; D004268:DNA-Binding Proteins / Q000276:immunology; D019309:Epstein-Barr Virus Nuclear Antigens; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D008565:Membrane Proteins / Q000276:immunology*; D009303:Nasopharyngeal Neoplasms / Q000382:microbiology*; D015534:Trans-Activators; D014763:Viral Matrix Proteins; D014764:Viral Proteins / Q000276:immunology
D000911:Antibodies, Monoclonal; D000956:Antigens, Viral; C063676:BZLF1 protein, Herpesvirus 4, Human; D004279:DNA, Viral; D004268:DNA-Binding Proteins; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D008565:Membrane Proteins; D015534:Trans-Activators; D014763:Viral Matrix Proteins; D014764:Viral Proteins; D001710:Biotin; D004247:DNA