Correspondence to: Irene Joab, Ph.D., Institut de Genetique Moleculaire, Pharmacologie Experimentale et Clinique, IFR Saint Louis, 27 rue Juliette Dodu, 75010 Paris, France (e-mail: i.joab{at}chu-stlouis.fr).
Brink et al. described detection of Epstein-Barr virus (EBV) DNA by polymerase chain reaction (PCR) in five of 24 snap-frozen breast carcinoma biopsy samples by use of primers that cover a repeat fragment and two of 24 samples with the use of a PCR specific for a single-copy gene. However, in the breast cancer samples that were positive for EBV by PCR, they did not detect either transcripts encoding EBV nuclear antigen 1 (EBNA1) or the rightward transcripts of the BamH1-A region of the EBV genome by reverse transcriptionPCR (RTPCR).
Studies on transcripts of the EBV genome in breast cancer specimens have been carried out in Dr. Griffin's laboratory. They observed BamHI-A region transcripts in a subset of breast cancer biopsy samples (with low lymphocyte infiltration) only after optimizing the PCR conditions (Griffin BE: personal communication). It is possible that the experimental conditions used by Brink et al. did not allow them to detect BamHI-A region transcripts.
With one set of primers, Brink et al. amplified the EBNA1 transcripts directed through both the Cp or Wp promoters, and not only those from the Cp, as specified in their Table 1 above. Furthermore, with a second set they amplified transcripts from the Qp/Fp promoters. Contrary to what is indicated in their Table 1, the primers selected would not permit discrimination between Fp and Qp promoters, since the forward primer is located downstream from both of these promoters.
Moreover, transcripts of the EBNA1 gene are present in low amounts in infected epithelial cells from nasopharyngeal carcinoma xenograft [see Table 1 in (1)].
Using the same primers as those used by Brink et al., Deacon et al. (2) could not detect any RTPCR product from EBNA1 messenger RNA (mRNA) in three of 12 cases of Hodgkin's disease, even though they observed expression of EBV-encoded small nuclear RNAs (EBERs) and latent membrane protein 1 (LMP1) staining showing that EBV is indeed expressed in the EBNA1-negative RTPCR biopsy samples [see Table 1 in (2)]. Similarly, while transcripts of EBERs and Cp-initiated mRNA were detected, Tierney et al. (3) could not detect EBNA1 transcription in mononuclear cells of five of 14 patients with infectious mononucleosis. Gonnella et al. (4) did not observe EBNA1 transcripts in three of six bone marrow samples of healthy blood donors in which LMP1 transcripts were observed. They estimated that RTPCR using these primers that allow Fp/Qp- and Wp/Cp-driven EBNA1 detection is 100- and 1000-fold less sensitive, respectively, than the assay developed for LMP1 detection. It is likely that the lower sensitivity of the RTPCR strategy employed for the detection of EBNA1 expression has led to an underestimation of EBNA1-positive cases.
The EBNA1 transcripts driven by the Cp promoter contain three noncoding exons coming from the BamH1-Y fragment as well as transcripts that do not harbor these exons [see Fig. 3 in (5)]. This last category would not be detected in an RTPCR assay when the forward primer chosen is located in an exon from the BamHI-Y fragment.
We did show (6) that, in breast cancer biopsy specimens, 5%30% of the tumor cells were stained by the EBNA1 monoclonal antibodies. The low percentage of EBV-positive cells makes detection of the transcripts even more difficult to achieve.
Furthermore, the gene encoding the EBNA1 protein presents strain variations (7) that might alter primer annealing, leading to a lower detection capacity of the RTPCR procedure.
In our study (6), we detected staining of tumor cells in paraffin-embedded sections with the anti-EBNA1, 2B4 monoclonal antibody. We also obtained EBNA1 staining with 1H4 monoclonal antibody on sections of snap-frozen biopsy specimens. By use of NPC biopsy specimens as controls, we observed that fewer tumor cells were stained; we also observed a higher background staining in frozen sections than in paraffin-embedded sections. The use of frozen sections, which can lead to lower sensitivity and sometimes to higher background staining, could explain the results obtained by Brink et al.
In addition, in paraffin-embedded sections, we could not detect EBNA1 protein [see Fig. 4 in (6)] in normal cells, whereas an embolus containing tumor cells exhibited EBNA1 staining.
Moreover, results mentioned by Bonnet et al. (6) showed a failure to detect EBERs in a number of specimens that harbored the EBV genome. In biologic systems, there is no absolute gold standard.
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6
Bonnet M, Guinebretiere JM, Kremmer E, Grunewald V, Benhamou E, Contesso G, Joab I. Detection of Epstein-Barr virus in invasive breast cancers. J Natl Cancer Inst 1999;91:137681.
7
Habeshaw G, Yao QY, Bell AI, Morton D, Rickinson AB. Epstein-Barr virus nuclear antigen 1 sequences in endemic and sporadic Burkitt's lymphoma reflect virus strains prevalent in different geographic areas. J Virol 1999;73;96575.
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