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From the Division of Pathology, City of Hope National Medical Center, Duarte, California
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Introduction |
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Breast carcinoma is the most common malignant tumor and the leading cause of cancer death in women in Western countries. Although the etiology of breast carcinoma is not completely understood, genetic background and hormonal effects are believed to play important roles in its development. In recent years, researchers have questioned whether EBV may play a role in the development of breast carcinoma. This hypothesis is based on several observations. First, a subtype of breast carcinoma known as medullary carcinoma has lymphoepithelioma-like features with prominent lymphocytic infiltration, and lymphoepithelioma-like carcinomas in other organs (especially from foregut sites) are often positive for EBV.8,10 Second, latently EBV-infected mothers shed EBV into breast milk.11,12 Third, EBV-associated lymphomas have been described in the breast.13 Studies examining an association between breast carcinoma and EBV have yielded disparate results. Some studies have shown between 20 and 50% of breast carcinoma cases with molecular evidence of the EBV genome.14-16 However, other studies have found no molecular or immunohistochemical evidence for an association between EBV and the development of breast carcinoma.17-20
In an attempt to resolve this dispute, we studied the incidence of EBV infection in 48 cases of invasive breast carcinoma by immunohistochemistry [using anti-EBNA-1, latent membrane protein (LMP)-1, and ZEBRA monoclonal antibodies], in situ hybridization (EBER-1), polymerase chain reaction (PCR) (using primers detecting EBV EBNA-4 and LMP-1), and Southern blot methods (terminal repeat probe).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We searched the Tumor Bank of the Department of Anatomical
Pathology at City of Hope National Medical Center for cases of invasive
breast carcinoma from the years 1985 to 1999. We retrieved all cases
for which both frozen tissues and paraffin-embedded tissues were
available and this numbered 48 cases. Fresh tumor tissues had been
snap-frozen at the time of surgery and were kept in a -70°C freezer.
Tissues had also been routinely fixed in 10% neutral formalin and
embedded in paraffin. One paraffin tissue block with tumor was selected
from each case. The breast carcinomas were graded as I, II, and III,
based on histological pattern, nuclear pleomorphism, and mitotic rate
(Bloom and Richardson Grading System).21
The cases were
also examined for unusual numbers of lymphocytes (including
plasmacytoid lymphocytes), which we defined as clusters or sheets of
small lymphoid cells accounting for 
10% of the tumor volume, either
within the tumor or at the infiltrating edges. Clinical data were
obtained from the medical record.
EBER-1 in Situ Hybridization
The in situ hybridization study methods have been previously described.22 Briefly, we used a probe from a region of the EBV genome that is actively transcribed in latently infected cells, a 30-base oligonucleotide complementary to a portion (bp 69 to 98) of the EBER-1 gene. The sequence was 5'-AGA CAC CGT CCT CAC CAC CCG GGA CTT GTA-3' (Operon Technologies, San Pablo, CA). The probe was labeled with biotin at its 3' end. Paraffin sections were deparaffinized and digested with pronase (nuclease-free). Sections were incubated with prehybridization solution and then hybridized with sheared salmon sperm and yeast tRNA along with the appropriate amount of probe. The probe was used at a concentration of 0.25 ng/µl with overnight hybridization. Sections were then incubated in a solution of avidin-alkaline phosphatase conjugate, washed for 3 minutes, incubated in McGadey’s substrate, briefly washed in distilled water, air-dried, and coverslipped. No counterstain was used. A poly d(T) was used as a control for total RNA preservation, and a known EBV-positive case of NPC was used as a positive control. A case was considered positive if the nucleus, or nucleus and cytoplasm, of a tumor cell stained dark blue or black.
EBNA-1, LMP-1, and ZEBRA Immunohistochemistry
Dr. Grasser (Abteilung Virologie, Institut fur Medizinische Mikrobiologie und Hygiene and Institut fur Pathologie, Universitaatakliniken des Saarlandes, Homburg, Germany) kindly provided the rat monoclonal antibody clone 2B4 to EBV EBNA-1.23 We also used two other antibodies, mouse monoclonal antibody clone CS1–4 to LMP-1 protein (DAKO Corporation, Carpinteria, CA) and clone BZ.1 to ZEBRA (BamHI Z fragment, Epstein-Barr-Replication Activator) protein (DAKO). EBNA-1, LMP-1, and ZEBRA immunohistochemistries were performed in all 48 cases of breast carcinoma. Paraffin sections were deparaffinized and rehydrated in a graded alcohol series. Two of the antibodies required heat-induced epitope retrieval using 100 mmol/L ethylenediaminetetraacetic acid buffer (pH 8.0) or 10 mmol/L citrate buffer (pH 6.0), for EBNA-1 and ZEBRA, respectively, in a steamer (Black and Decker, Shelton, CT) at 100°C for 20 minutes. The sections were then incubated with 2B4 at 1:500 dilution at room temperature overnight, with CS1-4 at 1:320, or with BZ.1 at 1:20 dilution at room temperature for 40 minutes and washed three times (5 minutes each) with phosphate-buffered saline (PBS) buffer. The sections were then incubated with a biotinylated goat, anti-rat antibody (Vector Laboratories, Burlingame, CA) (for EBNA-1) at a dilution of 1:150, or biotinylated goat, anti-mouse/anti-rabbit antibody (Ventana Medical Systems, Tucson, AZ) (for LMP-1 and ZEBRA) at a dilution of 1:8, followed by application of two washes (5 minutes each) of PBS buffer, followed by avidin-biotin complex (Vector). The slides were counterstained with hematoxylin. Sections of known EBV-positive classical Hodgkin’s disease were used as positive controls for EBNA-1 and LMP-1, and tissue sections of infectious mononucleosis were used as a positive control for ZEBRA. Positive staining was interpreted as nuclear or granular nuclear (EBNA-1), membrane and cytoplasmic (LMP-1), or nuclear (ZEBRA) in the tumor cells.
PCR Studies for EBNA-4 and EBV-LMP-1
Viral genomic DNA was extracted from fresh frozen tumor tissues, using 0.2 mg/ml proteinase K digestion buffer overnight, followed by denaturation by boiling. The PCR studies were performed with 2 µl of extracted DNA in a 30-µl mixture containing 50 mmol/L KCl, 10 mmol/L Tris buffer (pH 8.3), 50 µm of each deoxynucleotide triphosphate, 2.5 mmol/L MgCl2, 1 U of Taq polymerase (Perkin Elmer, Foster City, CA), and 20 pmol of each primer. We used primers for EBNA-4 that flank the DNA region coding for epitopes of 399 to 408 and 416 to 424 of the prototype B95.8 EBV virus, using the nucleotide positions 96541 to 96540 and nucleotide positions 96770 to 96751 (EBV GenBank Accession Number V01555), respectively: EBNA-4 + 5'-GAG GAG GAA GAC AAG AGT GG-3' and EBNA-4–5'-GAT TCA GGC GTG GCT CTT GG-3'. The expected EBNA-1 PCR product size was 230 bp. We also used primers for the EBV-LMP-1 gene that flank the site of the characteristic 30-bp deletion of LMP-1 gene, using the nucleotide positions 168350 to 168331 and nucleotide positions 168190 to 168209 (EBV GenBank Accession Number V01555), respectively: LMP-1 + 5'-CGG AAG AGG TTG AAA ACA AA-3' and LMP-2–5'-GTG GGG GTC GTC ATC ATC TC-3'. The expected LMP-1 gene product size was 161 bp. After initial denaturation for 5 minutes at 95°C, 45 amplification cycles were performed as follows: denaturing at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 40 seconds. A final extension at 72°C for 7 minutes completed the PCR amplification. The PCR setup and the work after PCR were performed in separate laboratories to minimize the possibility of contamination. Primers flanking ß-globin gene were used as a positive control for DNA preservation (expected PCR product size was 268 bp), whereas a known EBV-positive case of T/NK cell lymphoma was used as a positive control.
Southern Blot
Frozen tissues were cut into 5-µm thin sections, to which proteinase K was added up to 50 µg/ml in TE buffer, then incubated at 50°C overnight. Genomic DNA was extracted with phenol for three times and precipitated with ethanol. The genomic DNAs were then cut with BamHI and EcoRI restriction enzymes (New England BioRad, Boston, MA). The cut DNA was run in 10% polyacrylamide gel overnight at 34 V, each lane loaded with 16 µg of DNA. The positive control consisted of 0.2 to 3.3 µg of DNA extracted from Raji cells. DNA was transferred to nitrocellulose paper with 0.4 N NaOH, blotted with P-32 labeled EBV probes that flanked the EBV terminal repeat. The film (Kodak B10MAX MR; Eastman-Kodak, Rochester, NY) was exposed for up to 8 days.
Statistical Methods
Clinical and pathological measurements were compared between patients whose tumors showed EBV gene expression and those whose tumors did not, using Pearson’s chi-square test.
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Results |
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All of the patients were women. The ages ranged from 24 to 90 years old, with a median age of 59 years. The tumor size ranged from 1.0 cm to 12 cm, with a median size of 3 cm. Forty-three cases were invasive ductal carcinoma and five cases were invasive lobular carcinoma. Two cases were histological grade I, 36 were grade II, and 10 were grade III. Thirty-seven of 48 cases had axillary lymph node dissections, with an average of 23 nodes examined in each case. Of the 37 cases with axillary lymph node dissection, 11 cases had no nodal disease. Six patients had a total of one to three nodes with carcinoma and 20 cases had more than three lymph nodes with metastatic breast carcinoma.
In Situ Hybridization for EBER-1
All 48 cases of breast carcinoma were hybridized with poly d(T)
and EBER-1. All 48 cases showed strong nuclear and cytoplasmic
positivity for poly d(T) (control for RNA preservation). Five of the 48
(10%) cases showed nuclear and cytoplasmic positivity for EBER-1
(Figure 1A)
. The positive cells were
tumor cells, which were grouped together topographically, but comprised
<0.1% of total tumor cell population in all five cases. None of the
EBER-1-positive cases contained an unusual number of infiltrating
lymphocytes or lymphoplasmacytoid cells. None of the cases had EBER-1
signal in nonneoplastic cells.
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Twelve of the 48 cases of breast carcinoma were positive for
EBNA-1, showing a granular nuclear staining. Although scattered
EBNA-1-positive tumor cells were seen in some cases (Figure 1B)
, the
majority of EBNA-1-positive cells were clustered together (Figure 1C)
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The percentage of positive tumor cells was low—<1% of total tumor
cells in any positive case. EBNA-1 did not stain any lymphocytes,
endothelial cells, or stromal cells in any of the cases. None of the
breast carcinoma cases stained for LMP-1 and ZEBRA proteins.
EBV-EBNA-4 and LMP-1 PCR Studies
Strong ß-globin (control for DNA preservation) amplified bands
were identified from the DNA of all 48 cases, indicating that the
quality and quantity of the purified DNAs were adequate. Amplified
EBNA-4 bands and amplified EBV-LMP-1 bands (Figure 2)
were each identified from 5 of 48
cases (10%), including three cases that co-expressed EBV-LMP-1 and
EBNA-4. An additional two cases each expressed either EBV-LMP-1 or
EBNA-4. Only one of the five (20%) EBV-LMP-1-positive cases showed a
30-bp LMP-1 gene deletion.
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DNA from the seven positive PCR EBV-LMP-1 or EBNA-4 cases was
extracted for Southern blot studies. Case 7 (Table 2)
did not contain
enough DNA for Southern blot study. The other six cases showed no
detectable bands (Figure 3)
even with
loading of a much greater quantity (16 µg) of DNA than was used for
the positive control (3.3 µg) and despite the fact that the film was
exposed to autoradiography for up to 8 days. Sufficient DNA was
available in three cases to perform dilution studies up to 0.2 µg of
control DNA. Despite a clearly visible band at the most dilute
concentration of control (at a sensitivity of 
1 copy/cell), the
three cases showed no detectable bands (data not shown).
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We identified no statistically significant difference in EBV gene expression based on tumor grade, tumor size, lymph node status, or patient age.
Correlation of EBV Gene Expression with Lymphocytic Infiltration
Fifteen of the 48 (31%) cases showed prominent lymphoplasmacytic
infiltration that was present primarily at the periphery or the
infiltrating edges of tumors. Five of seven cases (71%) that were
EBV-positive by PCR studies showed prominent lymphocytic infiltration
(Table 2)
. In contrast, 10 of 41 (27%) EBV-negative cases by PCR
showed prominent lymphocytic infiltration. This difference was
statistically significant (P = 0.013).
Co-Expression of EBV Genes in Breast Carcinoma
A total 19 of 48 (39%) cases were found to be positive for EBV by
at least one methodology test. However, only 6 of the 19 cases (31%)
were positive by more than one EBV marker (Table 2)
. Only one case
(case 1) was positive for both EBNA-1 and EBER-1. In this case, the
localization of EBNA-1 and EBER-1 was compared, and we observed
different cell populations expressing the EBNA-1 protein and EBER-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression of EBV viral genes in tumor cells can be detected by in situ hybridization (viral RNA expression), PCR and Southern blot methods (viral genomic DNA), and immunohistochemistry (viral protein expression). The gold standard for the identification of a significant association of EBV with a given neoplasm has been in situ hybridization demonstration of EBERs within all or virtually all of the neoplastic cells.24 This methodology has high sensitivity and high specificity and has proven useful in the study of EBV in lymphoid malignancies as well as carcinomas. Polymerase chain reaction studies are generally of limited use because they may be overly sensitive and detect EBV DNA in host cells. Thus, PCR studies alone do not provide definitive evidence of EBV within neoplastic cells, as one cannot exclude the possibility that EBV within bystander lymphocytes contributes the positive signal. Indirect evidence for an association of EBV with a given neoplasm is provided by a Southern blot study that shows a high quantity of EBV genomes, particularly when terminal repeat probe analysis shows that the EBV is present within a monoclonal population of cells. Immunohistochemical demonstration of EBNA-1 expression may provide additional evidence that a given tumor is EBV-positive, but there is much less experience with this methodology, which is also less sensitive.17
Several groups have examined breast carcinoma tissues for evidence of EBV and have reported widely divergent findings. Using EBER in situ hybridization, Iezzoni and colleagues8 did not detect EBV in 52 cases of infiltrating breast carcinoma with medullary features (lymphoepithelioma-like carcinoma). Chu and colleagues18 tested breast carcinoma samples in a high-incidence EBV-associated NPC population and found that none of 60 Taiwanese breast carcinoma cases were positive for EBV, using EBER in situ hybridization and EBNA-2 and LMP-1 immunohistochemistry. Using EBER-1 in situ hybridization, Glaser and colleagues19 also failed to demonstrate any positive cases in 107 breast carcinoma tissues from the United States. Recently, Brink and colleagues17 studied 24 cases of European breast carcinoma and used DNA PCR to detect large internal BamHI-A rightward transcripts of the EBV genome and the single-copy gene BNLF1, which is also known as the LMP-1 gene. They found 5 of 24 (21%) positive for the former transcript, of which two also tested positive for the latter. No cases were positive only for BNLF-1 (LMP-1). However, when they applied reverse transcriptase-PCR for transcripts encoding EBNA-1, they found none of the five DNA PCR-positive breast carcinoma samples to have EBNA-1 transcripts. By EBNA-1 immunohistochemistry (using the 2B4 clone), they found one PCR-positive and one PCR-negative breast carcinoma to be positive. EBER-1 and EBER-2 RNA in situ hybridization signals were not detected in any of the breast carcinoma cells, but one of the PCR-positive cases had a small number of EBER-positive lymphocytes. They concluded that there was no evidence for an association between breast carcinoma and EBV because transcription of EBNA-1, EBER-1, EBER-2, and BamHI-A transcripts in breast carcinoma samples could not be detected. The EBV positivity detected by DNA PCR methods was thought to be most likely caused by the presence of EBV-infected lymphocytes in the breast carcinoma samples.
In contrast, Labrecque and colleagues15 found 19 of 91 (21%) British breast cancers to be EBV-positive using DNA PCR to detect EBER-1 and EBER-2. Luqmani and colleagues16 also found 15 of 28 (42%) British breast cancers to be positive using a PCR method. However, none of their PCR-positive cases were positive using EBER in situ hybridization. Using DNA PCR to detect EBER-2 and LMP-2, Bonnet and colleagues14 found 51 of 100 (51%) French breast carcinoma cases to be EBV-positive. They also used Southern blot analysis to look for the presence of the EBV genome in seven of their breast tumor specimens. The EBV genome was detected in DNA from all seven EBV PCR-positive breast tumors and observation of the signal was independent of any lymphoplasmacytic reaction in the tumor. By EBNA-1 immunohistochemical study with the use of clones 2B4 (paraffin sections) and 1H4 (frozen sections), they tested nine EBV-positive and six EBV-negative breast carcinomas. All nine EBV PCR-positive breast tumors exhibited granular nuclear positivity, but none of six EBV PCR-negative tumors were positive. No staining was observed in normal breast epithelial cells and lymphocytes. The authors concluded that the EBV-infected cells comprise a large percentage of breast tumor cells and that the positive signal was not dependent on the presence and proportion of lymphocytes. However, EBV EBER in situ hybridization did not show any signal in three of three EBV PCR-positive breast tumors or one lymph node with EBV-positive metastasis.14
In the current study, we found that 19 of 48 (39%) breast carcinoma cases had evidence of EBV, by at least one method. However, only 6 of 48 (12.5%) were positive with more than one assay. We identified EBER-1-positive breast carcinoma cells in 5 of 48 (10%) cases. However, in all five cases, only a small subset (<1 in 1000 cells) of the neoplastic cells were EBV-positive. This is in contrast to other EBV-associated malignancies, such as NPC, gastric carcinoma, and malignant lymphoma (including Hodgkin’s disease), for which all or virtually all of the neoplastic cells are positive within a given case. Peripheral T-cell lymphoma represents a notable exception in which a subset of cases have EBER positivity in only a subset of the neoplastic cells. Although one may postulate that there could have been a more widespread infection with eventual elimination of the virus, a more likely explanation is that EBV infects rare tumor cells after neoplastic transformation has occurred. In addition, when latent state infection switches to lytic state infection, EBV-infected cells may be negative for EBERs. Such a switch is mediated by ZEBRA protein. Therefore, lytic EBV-infected cells are frequently positive for ZEBRA protein.25,26 However, none of our cases (including 19 cases with discrepant EBV gene expression) showed immunohistochemical positivity for ZEBRA. In addition, the LMP-1 antibody cocktail used in this study contains a mixture of LMP antibodies, at least one of which recognizes a LMP band expressed late in the virus productive cycle and therefore may detect lytic proteins in addition to latent cycle proteins.27 Therefore, we conclude the discrepant expression of EBV gene products in 19 cases of breast carcinoma most likely did not result from lytic state infection. We also considered the remote possibility that EBER-1 in situ hybridization may show false-negative results, which has been shown to be extremely low in many studies. A recent report on hepatocellular carcinoma contained three cases that were positive for EBV by Western blotting studies and reverse transcriptase-PCR analysis but were negative for EBV EBER by in situ hybridization.28 However, this study needs confirmation with a larger series.
Twelve of 48 (25%) cases were positive by EBNA-1 immunohistochemical
studies, including nine cases positive by this modality alone. We used
an antibody (2B4) produced against a 
-cro/lacZ-EBNA-1 fusion protein
that has been shown to react in all forms of EBV
latency.23
This antibody has been shown to have nearly
equal sensitivity in paraffin or frozen section immunohistochemistry,
and equal or superior sensitivity to another anti-EBNA-1 antibody
termed 1H4.14,23
Both the 2B4 and 1H4 antibodies were also
used by Bonnet and colleagues,14
for whom EBNA-1
immunohistochemistry was one of the primary study methodologies.
Although the sensitivity of the EBNA-1 antibodies may be high, the lack
of confirmatory evidence of EBV by other modalities in the majority of
cases studied in the current report suggests that the antibody
specificity may not be absolute. In fact, there may be cross-reactivity
to antibodies directed against other epitopes of the EBNA-1 protein.
Studies have shown that the irregular GGGGCAGGA repeat motifs of the
latent infection cycle EBNA-1 gene are interspersed in cellular DNA.
Therefore, monoclonal antibodies to the irregular glu and ala repeating
peptide motifs of EBNA-1 may cross-react with cell
proteins;29,30
we have observed 2B4 EBNA-1 nuclear
staining in normal hepatocytes (data not shown). We also considered the
possibility that our use of an ethylenediaminetetraacetic acid-based
buffer for immunohistochemistry antigen retrieval led to increased
sensitivity of antigen detection, as has been described by other
investigators.31
However, our staining conditions were
optimized to minimize any background staining, and false-positives or
false-negatives were not seen in any of our control tissues. Therefore,
at the current time, we do not consider EBNA-1 immunohistochemical
positivity as evidence of EBV infection in the absence of any other
confirmatory studies.
EBV is the most ubiquitous viral infection with >90% of the adult population worldwide infected by the virus.32 EBV is known to infect 2 to 60 lymphocytes per million blood lymphocytes, leading to a lifetime latent infection. By PCR, healthy EBV-seropositive donors have been shown to have 104 to 106 EBV-positive B-cells.33,34 Other studies of normal lymphoid tissues have shown between 1 in 1000 to 1 in 10,000 infected cells. Therefore, when DNA is extracted from fresh tissue for PCR studies, the contaminated B-cell latent EBV DNA is also potentially amplified, which may lead to a positive PCR result. The amount of amplified EBV DNA is dependent on the degree of tissue blood contamination, lymphocytic infiltration, and the viral copy numbers in the EBV-positive B-cells. Seven of our 48 (15%) cases were positive for EBV by PCR studies for LMP-1 and/or EBNA-4. All but one of these cases were also studied by Southern blot studies, which revealed no evidence of EBV, despite dilution studies showing sensitivity of at least one copy of EBV DNA per cell. A prominent lymphocytic infiltration was found in five of the seven cases, suggesting that rare EBV-positive lymphocytes were genesis of the positive PCR results. Sampling considerations, including peripheral blood contamination of surgically removed tissues, may explain why our PCR-positive cases failed to reveal EBER-positive lymphoid cells.35
Geographical variation of EBV infection has been observed in many EBV-associated neoplasms, including NPC, Burkitt lymphoma, and Hodgkin’s disease. For example, the incidence of EBV infection in African Burkitt lymphoma is much higher than in other parts of the world and the incidence of EBV-associated Hodgkin’s lymphoma is higher in Latin America than in developed countries.10,36 The studies claiming an association between EBV and breast carcinoma cells were all from European populations.14-16 In contrast, another report from Europe failed to demonstrate an association of EBV with breast carcinoma.17 Therefore, it is unlikely that geographical variation alone can explain the high incidence of EBV-positive breast carcinomas found in some series.
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Footnotes |
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Accepted for publication April 19, 2001.
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