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).
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ABSTRACT |
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INTRODUCTION |
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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).
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SUBJECTS AND METHODS |
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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, 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 2
). The PCR products were analyzed by electrophoresis
through agarose gels.
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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).
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RESULTS |
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Histopathologic types of the breast tumors analyzed as well as the
characteristics of the patients are shown in Table 1. DNA was amplified
by PCR with primers (Table 2
) 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. 1
). 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. 1
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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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. 1: 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 1. 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. 3. The signal observed was independent
of the lymphoplasmocytic reaction of the tumor. As seen in Fig. 3
, 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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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.
4). The majority of the NPC cells were labeled (Fig.
4
, A) and a substantial proportion (5%-30%) of stained
tumor cells
was detected in breast cancer (Fig. 4
, 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. 4
, 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. 4
, E), although
some
tumor cells of the embolus exhibited granular staining (Fig. 4
, E, and
inset). Frozen sections of PCR-EBV-positive breast tumors were also
EBNA-1 positive by use of the 1H4 MAb (Fig. 4
, F). No labeling was
observed when an MAb was omitted (Fig. 4
, 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. 4
, 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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DISCUSSION |
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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. 4, 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.
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NOTES |
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We thank Dr. Bosq for providing nasopharyngeal carcinoma sections. We also thank Professor Calvo and Dr. Alberga for their careful reading of the manuscript.
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Manuscript received December 22, 1998; revised June 11, 1999; accepted June 21, 1999.
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