CORRESPONDENCE

Re: Detection of Epstein-Barr Virus in Invasive Breast Cancers

Antoinette A. T. P. Brink, Adriaan J. C. van den Brule, Paul van Diest, Chris J. L. M. Meijer

Affiliation of authors: Department of Pathology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.

Correspondence to: Chris J. L. M. Meijer, M.D., Ph.D., Department of Pathology, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands (e-mail: cjlm.meijer{at}azvu.nl).

Recently, the presence of Epstein-Barr virus (EBV) DNA in more than 50% of breast carcinoma samples was reported, as detected by DNA polymerase chain reaction (PCR), Southern blotting, and EBV nuclear antigen 1 (EBNA1) immunohistochemistry by use of the 2B4 and 1H4 rat monoclonal antibodies (MAbs), although EBV-encoded RNAs 1 and 2 (EBER1/2) were not found (1). This finding implicates a novel EBV gene expression pattern (i.e., EBNA1 positive and EBER1 and EBER2 negative) and has important consequences for the use of EBER1/2 RNA in situ hybridization as a diagnostic tool for EBV detection in clinical materials.

We have tested 24 snap-frozen breast carcinoma biopsy samples for the presence of EBV at the DNA, messenger RNA (mRNA), and protein levels, by use of EBV-associated lymphoid and epithelial diseases as positive controls. Results are summarized in Table 1Go. By the use of DNA PCR specific for the large internal BamHI-W repeat of the EBV genome (2), EBV was detected in five of the 24 breast carcinoma samples. Of these five samples, two also tested positive by use of a PCR specific for the single-copy gene BNLF1, which encodes the latent membrane protein 1 (3). Subsequently, we applied reverse transcription–PCR for transcripts encoding EBNA1 and for rightward transcripts from the BamHI-A region of the EBV genome by use of previously described primers (46). The virus needs EBNA1 for maintenance of its latent state (7), and rightward transcripts from the BamHI-A region were shown to be expressed in all types of EBV latency (6). In none of the five DNA PCR-positive breast carcinoma samples could EBNA1 transcripts or rightward transcripts from the BamHI-A region be detected, although the quality of the RNA preparations was good, as shown by the presence of ribosomal RNA bands at the gel level and the amplification of "housekeeping" mRNA encoding small nuclear ribonucleoprotein U1A (5). EBER1/2 RNA in situ hybridization signals in epithelial cells were never shown in any of the breast carcinoma samples; however, in one of the PCR-positive breast carcinoma samples, a small number of lymphoid EBER1/2-positive cells was detected.


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Table 1. Presence of Epstein-Barr virus (EBV) DNA, messenger RNA (mRNA), and protein in breast carcinoma biopsy samples and in established EBV-associated diseases*
 
EBNA1 immunohistochemistry by use of the 2B4 MAb gave granular nuclear staining in the neoplastic epithelium of one PCR-positive, but also of one PCR-negative, breast carcinoma sample. These signals can be considered to be nonspecific because of the absence of the corresponding transcripts. The 2B4 MAb also showed nonspecific staining in various EBV-negative tissue samples, including normal breast tissue, and in various other epithelia, as reported previously by us (3).

In conclusion, our data indicate that there is no evidence for an association between breast cancer and EBV because transcription of EBNA1, EBER1/2, and BamHI-A rightward transcripts in breast carcinoma biopsy samples cannot be detected. The positivity for EBV as found with DNA PCR is most likely caused by the presence of some EBV-infected lymphocytes in the breast carcinoma samples, as confirmed in one case by EBER1/2 RNA in situ hybridization. The use of the EBER1/2 probe for the detection of EBV at the single-cell level remains the gold standard.

REFERENCES

1 Bonnet M, Guinebretiere JM, Kremmer E, Grunewald V, Benhamou E, Contesso G, et al. Detection of Epstein-Barr virus in invasive breast cancers. J Natl Cancer Inst 1999;91:1376–81.[Abstract/Free Full Text]

2 Jiwa NM, Kanavaros P, van der Valk P, Walboomers JM, Horstman A, Vos W, et al. Expression of c-myc and bcl-2 oncogene products in Reed-Sternberg cells independent of the presence of Epstein-Barr virus. J Clin Pathol 1993;46:211–7.[Abstract]

3 Cruz I, van den Brule AJ, Brink AA, Snijders PJ, Walboomers JM, Vanderwaal I, et al. No direct role for Epstein-Barr virus in oral carcinogenesis: a study at the DNA, RNA and protein levels. Int J Cancer. In press 2000.

4 Brooks L, Yao QY, Rickinson AB, Young LS. Epstein-Barr virus latent gene transcription in nasopharyngeal carcinoma cells: coexpression of EBNA1, LMP1, and LMP2 transcripts. J Virol 1992;66:2689–97.[Abstract]

5 Brink AA, Oudejons JJ, Jiwa M, Walboomers JM, Meijer CJ, van den Brule AJ. Multiprimed cDNA synthesis followed by PCR is the most suitable method for Epstein-Barr virus transcript analysis in small lymphoma biopsies. Mol Cell Probes 1997;11:39–47.[Medline]

6 Brooks LA, Lear AL, Young LS, Rickinson AB. Transcripts from the Epstein-Barr virus BamHI a fragment are detectable in all three forms of virus latency. J Virol 1993;67:3182–90.[Abstract]

7 Reisman D, Yates J, Sugden B. A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components. Mol Cell Biol 1985;5:1822–32.[Medline]


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