Antoinette A. T. P. Brink

Pathogenic roles for Epstein /Barr virus (EBV) gene products in EBV-associated proliferative disorders

Epstein-Barr virus (EBV) is associated with a still growing spectrum of clinical disorders, ranging from acute and chronic inflammatory diseases to lymphoid and epithelial malignancies. Based on a combination of in vitro and in vivo findings, EBV is thought to contribute in the pathogenesis of these diseases. The different EBV gene expression patterns in the various disorders, suggest different EBV-mediated pathogenic mechanisms. In the following pages, an overview of the biology of EBV-infection is given and functional aspects of EBV-proteins are discussed and their putative role in the various EBV-associated disorders is described. EBV gene expression patterns and possible pathogenic mechanisms are discussed. In addition, expression of the cellular genes upregulated by EBV in vitro is discussed, and a comparison with the in vivo situation is made.

High Concordance of Results of Testing for Human Papillomavirus in Cervicovaginal Samples Collected by Two Methods, with Comparison of a Novel Self-Sampling Device to a Conventional Endocervical Brush

ABSTRACT A user-friendly self-sampling method for collecting representative cervical cell material would lower the threshold for women to respond to the invitation for cervical screening. In the present article, we introduce such a device; we have evaluated its sensitivity and specificity to detect high-grade cervical intraepithelial neoplasia (CIN), via high-risk human papillomavirus (hrHPV) detection and liquid-based cytology (LBC), compared to endocervical brush samples obtained by gynecologists. Women who had a cervical smear reading of moderate dyskaryosis or worse or a repeat equivocal Pap smear result in the cervical screening program (n = 64) and healthy volunteers (n = 32) took a self-obtained sample at home prior to their visit to the gynecological outpatient department. At the outpatient department, an endocervical brush smear was taken, followed by colposcopy and biopsy whenever applicable. Both self-obtained samples and endocervical brush samples were immediately collected in Surepath preservation solution and used for LBC and hrHPV testing (by general primer-mediated GP5+/6+ PCR). hrHPV test results showed a good concordance between the two sample types (87%; κ = 0.71), with sensitivities for prevalent high-grade CIN that did not differ significantly (92% and 95%; P = 1.0). The hrHPV test on self-obtained samples proved to be at least as sensitive for high-grade CIN as cytology on endocervical brush samples (34/37 versus 31/37; P = 0.5). LBC showed a poor concordance between self-obtained and endocervical brush samples (60%; κ = 0.27). In conclusion, self-obtained samples taken by this novel device are highly representative of the hrHPV status of the cervix. In combination with hrHPV testing, the use of this device may have implications for increasing the attendance rate for cervical screening programs.

No direct role for Epstein-Barr virus in oral carcinogenesis: a study at the DNA, RNA and protein levels

Reports on the association of EBV with oral squamous‐cell carcinomas (OSCCs) are scarce and inconclusive. To determine the potential role of EBV in oral carcinogenesis, we investigated 36 EBV DNA PCR‐positive OSCCs for the expression of EBV transcripts and proteins. From these EBV DNA‐positive OSCCs, 13 were analysed for the presence of EBV products, either at RNA and/or protein level. EBER transcripts were investigated by RNA in situ hybridisation. EBNA‐1, EBNA‐2, LMP‐1, LMP‐2, BHRF1 and BARF0 transcripts were investigated by RT‐PCR and/or NASBA. EBNA‐1, LMP‐1 and ZEBRA protein expressions were investigated by immunohistochemistry. All 36 OSCCs were positive for EBV DNA, using the highly sensitive BamHI W PCR, and 18 of these (50%) were positive using the less‐sensitive PCR, which targets BNLF‐1. However, virtually all OSCCs tested failed to reveal EBV transcripts, including EBERs and EBNA‐1 transcripts. No ZEBRA and LMP‐1 proteins were found in the neoplastic or any other cells of the OSCCs investigated. Immunohistochemistry using a monoclonal antibody (MAb) raised against EBNA‐1 (2B4) resulted in positive staining in some cases of OSCCs, but these results were non‐specific, since EBV‐negative epithelial tissues showed extensive non‐specific staining and no EBNA‐1‐specific transcripts were detected by RT‐PCR or NASBA. The absence of expression of EBV encoded transcripts and proteins indicate that, with the present knowledge on EBV, an active role in oral carcinogenesis for this virus is unlikely. Int. J. Cancer 86:356–361, 2000.

Epstein–Barr virus is present in neoplastic cytotoxic T cells in extranodal, and predominantly in B cells in nodal T non‐Hodgkin lymphomas

Epstein–Barr virus (EBV)‐positive T non‐Hodgkin lymphomas (T‐NHLs) have been described, but it is at present unknown how EBV infects T lymphocytes. It has been postulated that cytotoxic T cells (CTLs) or natural killer (NK) cells can be infected by EBV during the killing of an EBV‐infected target cell. The objective of this study was therefore to determine whether the neoplastic cells in EBV‐positive T‐NHLs (n=221) of various locations have a cytotoxic phenotype. To identify EBV‐harbouring cells, combinations were used of EBV‐encoded RNA (EBER) in situ hybridization (RISH) and immunohistochemistry for T‐ and B‐cell markers and the cytotoxic proteins TIA‐1 and granzyme B. EBV was detected in the neoplastic cells of all nasal T‐NHLs (n=9), 5/34 gastrointestinal (GI) T‐NHLs, and 2/6 lung T‐NHLs, but not in primary cutaneous T‐NHLs (n=103). Moreover, EBV was found in the neoplastic cells of 2/48 nodal anaplastic large cell lymphomas (ALCLs), but not in neoplastic T cells of other nodal T‐NHLs. However, 5/17 nodal peripheral T‐NHLs not otherwise specified (PTCLs NOS) and 1/4 T‐prolymphocytic leukaemias did contain EBV‐positive non‐T cells. Double staining revealed that in EBV‐positive extranodal T‐NHLs (n=16), the EBER‐positive cells had a cytotoxic phenotype (TIA‐1‐ and/or granzyme B‐positive). In nodal non‐ALCL T‐NHLs, the EBER‐positive cells were not positive for TIA‐1 or granzyme B, nor did they express CD3, CD21 or HECA452. Instead, most of these cells expressed the B‐cell marker CD20. These PTCLs NOS with EBER‐positive cells showed features of AILD‐like T‐NHL. It is concluded that neoplastic cells of EBV‐positive extranodal T‐NHLs always have a cytotoxic phenotype, supporting the view that EBV can infect CTLs. In nodal non‐ALCL T‐NHL, EBV is only found in T‐NHL with AILD‐like features and is present in B cells, where it may contribute to the outgrowth of a malignant B‐cell clone. Copyright

Profiling of Epstein-Barr Virus Latent RNA Expression in Clinical Specimens by Gene-Specific Multiprimed cDNA Synthesis and PCR

We describe a two-step RT-PCR method for simultaneous detection of EBNA-1 (QK and Y3K splice variants), EBNA-2, LMP-1, LMP-2a and -2b, ZEBRA, and BARTs RNA encoded by Epstein-Barr virus. As a control for RNA integrity, the low-copy-number transcript derived from U1A snRNP, a cellular housekeeping gene, is coamplified. Copy DNA (cDNA) for these nine targets is simultaneously synthesized in a gene-specific, multiprimed cDNA reaction, which strongly reduces the amount of required clinical specimen and allows more sensitive detection than random hexamer or oligo-dT priming. For amplification, cDNA synthesis is followed by nine separate PCRs for the mentioned targets. Primers were designed either as intron-flanking, to avoid background DNA amplification, or in different exons, allowing identification of differentially spliced RNA molecules. To increase specificity, PCR products are detected by autoradiography after hybridization with radiolabeled internal oligonucleotide probes. The method described is highly suitable for profiling EBV latent RNA expression in tissue biopsies, cultured or isolated cells, and unfractionated whole blood and for definition of EBV latency type I, II, or III gene expression in these samples.