Detection of Epstein-Barr Virus in Invasive Breast Cancers

Mathilde Bonnet, Jean-Marc Guinebretiere, Elisabeth Kremmer, Virginie Grunewald, Ellen Benhamou, Genevieve Contesso, Irene Joab

Affiliations of authors: M. Bonnet, I. Joab, Institut National de la Santé et de la Recherche Médicale, EPI 99-32, Pharmacologie Expérimentale et Clinique, Hôpital Saint Louis, Institut de Génétique Moléculaire, Paris, France, and Centre National de la Recherche Scientifique (CNRS) URA 1301, Institut Gustave Roussy, Villejuif, France; J.-M. Guinebretiere, G. Contesso, Department of Pathology, Institut Gustave Roussy; E. Kremmer, Forschungszentrum für Umwelt und Gesundheit GmbH, Institut für Immunologie, Munchen, Germany; V. Grunewald, CNRS URA 1301, Institut Gustave Roussy; E. Benhamou, Department of Biostatistics and Epidemiology, Institut Gustave Roussy.

Correspondence to: Irene Joab, Ph.D., Institut de Génétique Moleculaire, Pharmacologie Expérimentale et Clinique, IFR Saint Louis, 27 Rue Juliette Dodu, 75010, Paris, France (e-mail: i.joab{at}chu-stlouis.fr).


    ABSTRACT
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
BACKGROUND: Epstein-Barr virus (EBV) may be a cofactor in the development of different malignancies, including several types of carcinomas. In this study, we investigated the presence of EBV in human breast cancers. METHODS: We used tissues from 100 consecutive primary invasive breast carcinomas, as well as 30 healthy tissues adjacent to a subset of the tumors. DNA was amplified by use of the polymerase chain reaction (PCR), with the primers covering three different regions of the EBV genome. Southern blot analysis was performed by use of a labeled EBV BamHI W restriction fragment as the probe. Infected cells were identified by means of immunohistochemical staining, using monoclonal antibodies directed against the EBV nuclear protein EBNA-1. RESULTS: We were able to detect the EBV genome by PCR in 51% of the tumors, whereas, in 90% of the cases studied, the virus was not detected in healthy tissue adjacent to the tumor (P<.001). The presence of the EBV genome in breast tumors was confirmed by Southern blot analysis. The observed EBNA-1 expression was restricted to a fraction (5%-30%) of tumor epithelial cells. Moreover, no immunohistochemical staining was observed in tumors that were negative for EBV by PCR. EBV was detected more frequently in breast tumors that were hormone-receptor negative (P = .01) and those of high histologic grade (P = .03). EBV detection in primary tumors varied by nodal status (P = .01), largely because of the difference between subjects with more than three lymph nodes versus less than or equal to three lymph nodes involved (72% versus 44%). CONCLUSIONS: Our results demonstrated the presence of the EBV genome in a large subset of breast cancers. The virus was restricted to tumor cells and was more frequently associated with the most aggressive tumors. EBV may be a cofactor in the development of some breast cancers.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
The Epstein-Barr virus (EBV), a ubiquitous human herpesvirus, causes infectious mononucleosis. It has been associated with the development of different malignancies, such as Burkitt's lymphoma, 40%-50% of Hodgkin's disease, B-cell lymphoma in immunocompromised individuals, and nasopharyngeal carcinoma (NPC) (1). It has also been linked with some cases of other types of malignancies, including gastric carcinoma (2), leiomyosarcoma in immunocompromised individuals (3), T-cell lymphoma (1), and lymphoepithelioma-like carcinoma in the salivary glands, lung, and thymus (4-6). Detection of EBV in some breast tumors was reported in two studies (7,8).

Breast cancer is one of the most prevalent malignancies in Western countries. Although there are several well-established risk factors for breast cancer (early onset of menarche, a late age both for a first complete pregnancy and for menopause, the presence of atypical hyperplasia, a positive family history of breast cancer, and exposure to ionizing radiation), other factors contributing to the development of breast cancer likely exist (7). Breast cancer is a multistep disease in which a virus could play a role (7,9). In some countries, an overlap between regions of high incidence of EBV-associated lymphomas and of a high frequency of male breast cancer has been reported (7,10). Furthermore, EBV-associated lymphomas have been reported to be localized in the breast (11,12). EBV is shed in milk without being an important source for early infection in infants (13,14).

In two polymerase chain reaction (PCR) studies, EBV was observed in 20%-40% of breast tumors assessed (7,8). Labrecque et al. (7) identified EBV-encoded small RNA1 (EBER-1) in a fraction of malignant cells in six different breast tumors. However, Glaser et al. (15) did not detect EBER-1 transcripts in breast cancer, but the investigators did not exclude the presence of EBV in these samples. Immunohistochemical studies on 60 invasive breast cancers by Chu et al. (16) did not detect either Epstein-Barr nuclear antigen 2 (EBNA-2), which is only expressed in lymphoproliferative disorders and lymphomas of immunodeficient patients (1,17), or latent membrane protein 1 (LMP-1), which is only expressed in a few malignancies (4,18-21).

In the studies reported here, we show that EBV is present in breast cancer from results obtained with PCR, Southern blot analysis, and immunohistochemical detection of the viral protein Epstein-Barr nuclear antigen 1 (EBNA-1). This protein is essential for the maintenance of the viral episome in infected cells and is constantly expressed in all EBV infections (1).


    SUBJECTS AND METHODS
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
Patients and tissue samples. One hundred consecutive biopsy specimens of primary invasive breast carcinoma, 30 normal tissues adjacent to breast tumors, and five lymph nodes with metastasis were retrieved, without any preselection, from the Department of Pathology of the Institut Gustave Roussy (Villejuif, France). The clinical data were collected from the medical files and are summarized in Table 1.Go This study was performed in accordance with protocols (for the use of surgical tissues and medical records) previously approved by the local human studies committee. All samples were stored in liquid nitrogen. Ninety-eight of the patients were women, and two were men. Their age at diagnosis ranged from 34 to 85 years. Most of the female patients (73%) were postmenopausal (menopausal status was unknown for two subjects). The diagnosis of invasive breast carcinoma and the histoprognostic Scarff-Bloom and Richardson classification (SBR) were made by use of the criteria described by Contesso et al. (22). The majority of the tumors (i.e., 79) were of the ductal type; 13 were lobular, tubulolobular, or atypical lobular carcinomas; and the remaining eight were distributed among rare histologic types. The histopathologic grade of invasive carcinoma according to the SBR classification was as follows: grade I, 11 tumors; grade II, 50 tumors; and grade III, 38 tumors. In this study, all of the axillary lymph node statuses were identically represented, and the majority of the tumors measured more than 20 mm. Fragments of the tumors were snap-frozen for evaluation of the estrogen and progesterone receptors by an enzyme immunoassay method (Abbott Laboratories, Chicago, IL) (23). In 78% of tumors, estrogen and/or progesterone receptors were detected.


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Table 1. Detection of Epstein-Barr virus (EBV) genome by polymerase chain reaction (PCR) and characteristics of patients and tumors

 
Extraction of DNA. Pulverized frozen tissues were treated for 3 hours at 55 °C with 10 volumes of a solution containing 200 µg/mL of proteinase K (Appligene, Illkirch, France) dissolved in 50 mM Tris-HCl (pH 8.5), 1 mM EDTA, and 0.5% Tween 20 (Sigma Chemical Co., St. Louis, MO). The enzyme was then heat inactivated at 95 °C for 10 minutes, and the samples were stored at -20 °C until used.

Amplification of DNA. DNA samples (10 µL) were subjected to PCR using Taq DNA polymerase (Promega Corp., Madison, WI), specific oligonucleotides listed in Table 2,Go and a DNA Thermal cycle 480 (The Perkin-Elmer Corp., Foster City, CA). The standard cycle procedure was a 5-minute denaturation at 95 °C (followed by the addition of 0.5 U of the enzyme), then 40 cycles of 30 seconds of denaturation at 95 °C, 1 minute of annealing at 58 °C for BZLF1, 55 °C for BNLF1, 60 °C for EBER-2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and a 2-minute extension at 72 °C. Cycling was followed by a 7-minute extension at 72 °C. Two microliters of the BZLF1-PCR product was taken for a second round of PCR by use of internal primers (Table 2Go). The PCR products were analyzed by electrophoresis through agarose gels.


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Table 2. Primers used in polymerase chain reaction amplifications*

 
Restriction fragment length polymorphism (RFLP) analysis. BZLF1-PCR products were digested with BsmI (Life Technologies, Inc. [GIBCO-BRL], Gaithersburg, MD) at 65 °C, and the BNLF1 products were digested with NcoI (Life Technologies, Inc.) at 37 °C for 1 hour. Fragments were sized on a 2% agarose gel (Bioprobe Systems, Montreuil sous Bois, France).

Southern blot hybridization. High-molecular-weight DNA (40 µg for breast tumors and 10 µg for control cells) extracted from biopsy specimens was digested with BamHI (Life Technologies, Inc.), separated in 0.6% agarose gel, and transferred to nylon membrane (Hybond; Amersham Life Science Inc., Arlington Heights, IL). The BamHI W fragment of EBV genome was labeled with [32P]deoxycytidine triphosphate (3000 Ci/mmol) by random priming (Prime, a gene labeling system; Promega Corp.) according to the manufacturer's instructions (Promega Corp.). Hybridization was performed overnight at 42 °C in 50% formamide, 1% sodium dodecyl sulfate (SDS), 10% dextran sulfate, 1 M NaCl, 100 µg/mL salmon sperm DNA, and 100 µg/mL yeast transfer RNA. The filter was washed twice with 2x standard saline citrate (SSC) and 0.1% SDS at room temperature, twice with 0.2 x SSC, 0.5% SDS for 15 minutes at 65 °C, and twice with 0.1 x SSC and 1% SDS for 20 minutes at 65 °C. Autoradiography was performed at -80 °C with intensifying screens.

In situ hybridization. In situ hybridization for the detection of EBV-specific RNAs (EBER) was performed according to the manufacturer's instructions (Dako, Glostrup, Denmark).

Immunohistochemical identification of EBNA-1. Cytospun cells and frozen sections were fixed with 4% paraformaldehyde for 20 minutes. For paraffin-embedded sections, the tumor tissue was fixed in 5% acetic acid, 2% formol, and 75% absolute ethyl alcohol (Sigma Chemical Co.) before paraffin embedding. Paraffin sections were deparaffinized and pretreated in 10 mM Tris and 1 mM EDTA (pH 9.5) for 30 minutes in a microwave oven. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide. After a brief wash with phosphate-buffered saline (PBS), slides were incubated in 10% fetal bovine serum-PBS and then with monoclonal antibodies (MAbs) 1H4 (frozen sections) or 2B4 (paraffin sections) at dilutions of 1 : 10 or 1 : 100, respectively (24). An unrelated rat immunoglobulin G (IgG) MAb of the same isotype served as a negative control. Bound antibody was detected by use of biotinylated rabbit anti-rat IgG (Vector Laboratories, Inc., Burlingame, CA) at a dilution of 1 : 150. A standard immunoperoxidase-staining procedure (Vectastain ABC elite kit; Vector Laboratories, Inc.) was used for detection. Hematoxylin was used for counterstaining. Raji cells (EBV-positive Burkitt lymphoma cell lines) and an NPC biopsy specimen were used as positive controls and DG75 cells (EBV-negative Burkitt lymphoma cell lines) were used as a negative control.

Statistical methods. Proportions were compared with Fisher exact test for unordered (25) or ordered (26) categoric variables. P values were considered to be statistically significant for P<.05 (two-sided tests).


    RESULTS
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
Detection of EBV in Breast Cancer Tissue

Histopathologic types of the breast tumors analyzed as well as the characteristics of the patients are shown in Table 1Go. DNA was amplified by PCR with primers (Table 2Go) covering three different regions of the EBV genome: EBER-2, BZLF1 (BamHI Z Leftward Frame 1), and BNLF1 (BamHI N Leftward Frame 1). The PCR sensitivities for these cases were, respectively, 20, two, and two copies of the Namalwa EBV genome [Namalwa cells contain two integrated copies of EBV genome (27)]. The expected products were 108 base pairs (bp) for EBER-2, 994 bp for BZLF1, and 337 or 307 bp according to BNLF1 polymorphism (Fig. 1Go). The samples were considered to be EBV positive if amplification of all three genes occurred. We were, therefore, able to detect the EBV genome in 51 of 100 biopsy specimens. Fig. 1Go shows the PCR results for six breast tumors: four EBV-positive (8T, 32T, 34T, and 92T) and two EBV-negative (38T and 82T) tumors. GAPDH amplification was used as a positive control for DNA extraction.



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Fig. 1. Detection of Epstein-Barr virus (EBV) genome by polymerase chain reaction analysis of representative breast tumors, healthy breast tissue, and tumor-positive lymph nodes. The primers used in amplification are described in Table 2Go. The 994 base-pair (bp) BZLF1 fragment was obtained with ZES2-ZAS2 in a first-round amplification and with ZES-ZEAS in a second-round amplification. The BNLF1 fragment was obtained with LMP2CS-LMP2CAS. The expected size is 337 or 307 bp (deletion of 30 bp), depending on the polymorphism. The 108-bp EBER-2 fragment was amplified with EBER-2S-EBER-2AS1, and the 157-bp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fragment was amplified with GAPDH3S-GAPDH3AS. T = breast tumors, H = healthy tissue, and L = tumor-positive lymph nodes with metastasis. NPC = nasopharyngeal carcinoma; lane 0 = without DNA; 32T, 8T, 34T, and 92T = positive tumors; 38T and 82T = negative tumors; 92H and 82H = negative healthy breast tissue taken next to the tumor; and 92L = tumor-positive lymph node.

 
BZLF1 and BNLF1 genes exhibited polymorphism allowing RFLP analysis as we recently described (28) and as shown in Fig. 2Go. Seven different patterns were identified in the analyzed breast tumors (Table 3Go). No specific strain appeared to be preferentially associated with breast cancer. The patterns of the EBV genes observed were identical for the same tumor in two independent series of amplification that should exclude dealing with a possible PCR contamination products. Taken together, these results show the presence of the EBV genome in a high proportion of infiltrating breast carcinomas.



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Fig. 2. BZLF1 and BNLF1 polymorphisms by restriction fragment length polymorphism (RFLP) analysis (28). A) RFLP analysis of BZLF1 gene. Genomic DNA was amplified with ZES2-ZAS2 primers in a first round and with ZES-ZEAS in a second round. The 994-base-pair (bp)-amplified fragment was digested with BsmI, separated on 2% agarose gel, and stained with ethidium bromide. Lane 1 = B95-8 strain; lane 2 = absence of BsmI restriction site at position 102407; and lane 3 = P3HR1 strain. B) RFLP analysis in the 3' end of the BNLF1 ORF (open-reading frame). Genomic DNA was amplified with LMP2CS-LMP2CAS. The 337-bp-amplified fragment was digested with NcoI, separated on 2% agarose gels, and stained with ethidium bromide. Lane 1 = B95-8 strain; lane 2 = absence of NcoI restriction site; and lane 3 = deletion of 30 bp.

 

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Table 3. Different patterns of Epstein-Barr virus genome in the 38 analyzed breast tumors*

 
Five lymph nodes with metastasis were analyzed by the same methods, and the EBV genome was detected in four specimens for which the corresponding tumors were also EBV positive (e.g., patient 92 for whom the lymph node [92L] and the tumor [92T] were both positive in Fig. 1Go). Gene patterns for BZLF1 and BNLF1 were identical in the tumor and in the lymph nodes of the same patient. The lymph node from the fifth patient was EBV negative, as was the corresponding primary breast tumor. We can, therefore, conclude that the presence and the strain of EBV are characteristics of a given tumor and its metastasis.

Detection of EBV in Healthy Tissue Adjacent to Tumor

To determine whether the virus is specifically located in the tumor, we analyzed the presence of the EBV genome by PCR in 30 samples of healthy breast tissue taken from next to the tumor as confirmed by pathologic examination. The virus was not detected in 90% of the cases (Fig. 1Go: 92H and 82H). The EBV genome was observed in only three samples. In two of the samples, the EBV genome was also observed in the tumor. In these cases, the detected genome could be located in the lymphocytes. However, we cannot exclude the presence of a few invading tumor cells in the sample. The statistical analysis of the presence of the EBV genome in tumors (51%) and in healthy tissue (10%) suggested that EBV is mainly restricted to the tumor (P<.001).

EBV Detection and Prognostic Factors

Different clinical characteristics of the studied samples and association with the presence of EBV genome, as determined by PCR, are reported in Table 1Go. No association was observed between EBV detection and tumor histology. However, because the number of nonductal carcinomas was low, no definitive conclusion could be drawn. In addition, no association was observed between the presence of EBV and other prognostic factors, such as age at diagnosis, tumor size, and menopausal status. Nevertheless, the proportion of EBV-positive tumors was statistically significantly higher in carcinomas of high histologic grade, with 66%, 44%, and 27% for grade III, II, and I (SBR classification), respectively (P = .03). In steroid hormone receptor-negative tumors, which are known to represent poor prognosis, the EBV-positive samples were more frequent (79%) than in tumors with hormone receptors (45%) (P = .01). EBV detection in primary tumors was also associated with nodal status (P = .01). This association stems largely from the difference between subjects with more than three tumor-positive lymph nodes versus those with less than or equal to three tumor-positive lymph nodes (72% versus 44%).

Detection of EBV by Southern Blot Analysis in Breast Cancer Tissue

In another series of experiments, we looked in seven different breast tumor biopsy specimens for the presence of the EBV genome by use of Southern blot analysis, which is less sensitive than PCR. Following digestion with BamHI, DNA was hybridized with the EBV BamHI W repeated fragment. The EBV genome was detected in DNA from each of the seven breast tumors; three representative samples are shown in Fig. 3Go. The signal observed was independent of the lymphoplasmocytic reaction of the tumor. As seen in Fig. 3Go, the intensity of the bands was similar for mild (lane 2), moderate (lane 3), and high (lane 6) lymphoplasmocytic reactions. Similar results were obtained with DNA extracted from other breast tumors, two mildly and two moderately infiltrated by lymphocytes (data not shown). These results suggest that EBV-infected cells are not rare in the tumors and that the positive signal is not dependent on the proportion of lymphocytes.



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Fig. 3. Southern blot analysis of breast tumor DNA. DNA, digested with BamHI, was probed with a 32P-labeled BamHI W fragment of Epstein-Barr virus (EBV) genome. Lanes 1 and 5 = DNA from the EBV-negative cell line—DG75 as negative control; lanes 4 and 7 = DNA from the EBV-positive cell line—B95-8 as positive control; and lanes 2, 3, and 6 = DNA from breast tumors with mild (lane 2), moderate (lane 3), or marked (lane 6) lymphoplasmocytic reaction.

 
In Situ Hybridization for EBERs

In situ hybridization was performed on three EBV PCR-positive tumors and on one lymph node with metastasis. One NPC biopsy specimen served as a positive control. No signal could be observed in breast tissue (data not shown).

Detection of EBNA-1 by Immunohistochemistry

Identification of EBV-infected cells and viral expression were investigated by targeting the viral protein EBNA-1, which is essential for maintenance of the viral episome and for its replication (1). Immunohistochemical studies were performed on six EBV-negative and on nine EBV-positive tumors with two different EBNA-1 MAbs, 1H4 and 2B4 (24). The cell lines Raji and DG75 served as positive and negative controls, respectively (data not shown), and NPC tumors served as an additional positive control. Paraffin-embedded sections of NPC- and PCR-EBV-positive breast tumors incubated with 2B4 MAb exhibited granular nuclear staining in the tumor cells (Fig. 4Go). The majority of the NPC cells were labeled (Fig. 4Go, A) and a substantial proportion (5%-30%) of stained tumor cells was detected in breast cancer (Fig. 4Go, B and C; data not shown). The proportion of EBNA-1-positive cells varies greatly from one tumor to another. This variation could reflect differences in the duration of fixation of the clinical samples. No labeling was observed if the MAb was omitted (Fig. 4Go, D) or if an unrelated antibody of the same isotype was used in the reaction (data not shown). Normal breast epithelial cells were not labeled by the 2B4 EBNA-1 MAb (Fig. 4Go, E), although some tumor cells of the embolus exhibited granular staining (Fig. 4Go, E, and inset). Frozen sections of PCR-EBV-positive breast tumors were also EBNA-1 positive by use of the 1H4 MAb (Fig. 4Go, F). No labeling was observed when an MAb was omitted (Fig. 4Go, G) or when an unrelated antibody of the same isotype was used (data not shown). PCR-EBV-negative tumors were not stained by 1H4 MAb (Fig. 4Go, H) and by 2B4 MAb (data not shown). No labeling was seen in lymphocytes, even in lymph nodes with metastases (data not shown). These results confirm that the EBV expression is restricted to tumor cells and that an appreciable number of those cells are infected by the virus.



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Fig. 4. Immunohistochemical detection of EBNA-1 in clinical specimens. With monoclonal antibodies 2B4 (A, B, C, and E) or 1H4 (F and H), EBNA-1 expression is observed in nasopharyngeal carcinoma (A) and in breast tumors that were Epstein-Barr virus (EBV) positive by polymerase chain reaction (B, C, and F), whereas no reactivity is seen in a breast tumor that was EBV negative by PCR (H). No signal is seen with breast tumors without primary antibodies (D and G) or with a nonrelevant antibody with the same isotype. No signal is observed in normal epithelial cells (E), while the cells of the embolus were stained (E and inset). Original microscopic magnifications were x200 (E), x400 (A and B), and x640 (C, D, F, and G).

 

    DISCUSSION
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 
EBV has long been established as an important factor in the cause of several human cancers. More recently, it has been associated with Hodgkin's disease and some cases of gastric carcinomas. This article describes evidence of the presence of EBV in invasive breast carcinomas.

With the PCR experiment, we detected the EBV genome in about half of the breast cancers analyzed and, in particular, in those of the most common form, ductal carcinoma. The statistically significant difference in the detection of EBV DNA between malignant and healthy tissues (P<.001) strongly suggests that EBV is restricted to the tumor. It is highly unlikely that PCR-positive results were due to contamination because the results of the positive PCR findings were confirmed in two independent amplifications of three different genes, with investigation of polymorphism of two of them. Moreover, identical patterns were found in the investigated RFLP analysis.

Previous studies (7,8) reported 21% and 41% of breast cancers to be EBV positive. The somewhat higher percentage of EBV-positive tumors, 51%, observed in our study may be due to a higher sensitivity of the technique used. Alternatively, it may reflect differences between the subjects studied.

The crucial question concerns the cell types infected by EBV. Two other experimental approaches, Southern blot analysis and immunohistochemistry for EBNA-1, were performed to confirm the presence as well as the location of the virus. Of the 51 PCR-positive samples, 10 were investigated both by Southern blot analysis and by immunohistochemistry. All 10 samples gave EBV-positive signals with at least one of these methods.

Within breast carcinomas, infiltrating or blood lymphocytes represent a minority of the cell mass, and EBV is known to infect two to 60 lymphocytes per million (29). Under these conditions, viral DNA would not be detected on Southern blot analysis if only lymphocytes were infected by EBV. In addition, the signal obtained with Southern blot analysis is not related to the degree of lymphoplasmocytic infiltration. Therefore, detection of the EBV genome in breast carcinomas by Southern blot analysis strongly suggests that the predominant source of EBV DNA is the tumor epithelial cells.

To identify the EBV-infected cells, direct detection of EBV product had to be addressed. In situ hybridization with EBER-1 probes is widely used for the detection of EBV-infected cells. Labrecque et al. (7) detected EBER-1 transcripts in a proportion of tumor cells, whereas Glaser et al. (15) did not detect these transcripts in breast cancer. In this study, we also did not observe EBER-1 RNA with in situ hybridization in three tumors or in one lymph node with metastasis (data not shown). The regulation of transcription of EBERs remains poorly understood, and their high expression in infected cells might not be universal. NPCs that have varying degrees of differentiation lack expression of EBERs in some areas (30). Takeuchi et al. (31) did not observe any EBER-1 expression in some EBV-positive NPC cases. It is, therefore, possible that EBERs are not expressed in breast cancer or, if they are, they are expressed only at a lower level than in other tissue.

The definitive identification of infected tumor cells was obtained by immunohistochemical studies with two EBNA-1 MAbs. The antibodies distinctly showed nuclear staining of many epithelial tumor cells, while normal cells (including lymphocytes) were not stained. The fact that only a fraction of tumor cells were found to be EBNA-1 positive in breast cancer could reflect low expression or low accessibility of the protein to staining in some cells. One can see that, even in NPC, not all of the tumor cells were stained (Fig. 4Go, A). Alternatively, at this stage of the disease, the virus could be lost in a fraction of those cells. EBNA-1 is able to induce malignancies in transgenic mice by a mechanism that is not yet understood (32). The expression of EBNA-1 in breast tumors might be important in the transformation phenomenon.

For the first time, a statistically significant relationship was observed between the presence of EBV and several poor prognostic factors for breast carcinomas. EBV is detected more frequently in tumors that are negative for steroid hormone receptors, which are associated with a poor outcome. In addition, the EBV genome is preferentially detected in high histologic SBR grade tumors, corresponding to high mitotic index and a lower degree of differentiation. Similarly, the most undifferentiated forms of NPC and gastric carcinomas are more frequently associated with EBV (33). The association with axillary lymph node invasion suggests that the infection by EBV may be related to the high metastatic potential of the tumor. The point in tumor development at which EBV infection occurs remains to be determined. However, the presence of the EBV genome in metastatic lymph nodes, along with EBV detection in the primary tumor, suggests that the tumor cells were infected by EBV before the migration of metastatic cells.

In conclusion, our results show the presence of EBV genome in a large subset of breast cancers. Because it is more frequently associated with the most aggressive tumors, EBV may play a role in their development.


    NOTES
 
Supported by a grant from "Association de Recherche Contre le Cancer" (2017).

We thank Dr. Bosq for providing nasopharyngeal carcinoma sections. We also thank Professor Calvo and Dr. Alberga for their careful reading of the manuscript.


    REFERENCES
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 Notes
 References
 

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Manuscript received December 22, 1998; revised June 11, 1999; accepted June 21, 1999.


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