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[Cancer Research 63, 2338-2343, May 1, 2003]
© 2003 American Association for Cancer Research

Virology

Reactivity with A Monoclonal Antibody to Epstein-Barr Virus (EBV) Nuclear Antigen 1 Defines a Subset of Aggressive Breast Cancers in the Absence of the EBV Genome1

Paul G. Murray2,,3, David Lissauer3, Jia Junying, Gillian Davies, Sukhjinder Moore, Andrew Bell, Judith Timms, David Rowlands, Christopher McConkey, Gary M. Reynolds, Suk Ghataura, David England, Rebecca Caroll and Lawrence S. Young

Department of Pathology [P. G. M., D. L., J. J., G. D., S. M., D. R., S. G., R. C.], Cancer Research United Kingdom Institute for Cancer Studies [A. B., J. T., C. M., L. S. Y.], Liver Research Laboratories [G. M. R.], and Department of Surgery [D. E.], University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have suggested that common breast cancers are associated with EBV. We used a highly sensitive quantitative real-time PCR method to screen whole tumor sections of breast cancers for the presence of the EBV genome. EBV DNA was detected in 19 of 92 (21%) tumors, but viral load was very low in positive samples (mean = 1.1 copy EBV/1000 cells, maximum = 7.1 copies EBV/1000 cells). Importantly, quantitative real-time PCR failed to detect the EBV genome in microdissected tumor cells from any case. Using a monoclonal antibody (2B4-1) reactive against the EBV nuclear antigen-1, we noted strong staining of tumor nuclei in a proportion of those breast cancers that had tested negative for the presence of the EBV genome. Because nuclear staining with the 2B4-1 antibody was previously observed more frequently in poor prognosis breast cancers, we examined a larger series of breast cancers with complete clinical follow-up. Strong punctate staining of tumor cell nuclei was observed in 47 of 153 (31%) breast cancers; 2B4-1-positive tumors were significantly more likely to be ER-negative (P < 0.0001), to be of higher grade (P = 0.001) and larger (P = 0.03), to involve more regional lymph nodes (P = 0.01), and to have higher Nottingham Prognostic Index scores (P = 0.0003). Conclusions are: (a) EBV can be regularly detected in whole sections of breast cancers but viral copy number is very low; (b) in these cases, tumor cells do not harbor virus; and (c) reactivity with the monoclonal antibody 2B4–1 is detectable in the absence of the EBV genome and is strongly associated with ER-negative breast tumors and with prognostically unfavorable disease. Additional studies should be directed to the identification of this protein and to elucidation of its role in breast cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The ubiquitous EBV has already been associated with several epithelial cancers, including NPC4 and NPC-like tumors (lymphoepitheliomas) of the stomach, salivary gland, lung and thymus, and a subset of gastric adenocarcinomas (1) . Initial studies of lymphoepitheliomas of the breast and medullary carcinomas found no evidence of EBV infection in these tumors (2, 3, 4, 5, 6) . Although two previous studies had suggested a possible role for EBV in breast cancer (7 , 8) , widespread interest in the possibility that the common forms of breast cancer might harbor the EBV genome was initiated by the study of Bonnet et al. (9) , who used PCR and Southern blotting of whole sections to identify the EBV genome and immunohistochemistry using the monoclonal antibody 2B4-1 to detect EBNA1 expression. Intriguingly, by this latter approach, they reported the detection of EBNA1 protein more frequently in hormone receptor-negative disease in tumors of high histological grade and also in those tumors with more than three involved lymph nodes. A further study also suggested a potential link between delayed EBV infection and an increased risk of breast cancer (10) . Furthermore, the more recent detection of 2B4-1 staining of tumor cell nuclei and PCR detection of EBV DNA in whole tumor sections of not only breast cancers but also significant proportions of colon, lung, and prostate cancers was interpreted as indicating a more widespread association of EBV with the more common epithelial malignancies (11) .

In contrast, several studies have not been able to demonstrate a significant association between EBV and breast cancer (12, 13, 14) . These conflicting results might have been because of the widely differing methodologies used in these studies (15) . In all of the malignancies associated with EBV to date, the virus or its products can be detected within the tumor cell population. To unequivocally determine whether the EBV genome is present in the tumor cells of breast cancers, we combined Q-PCR that has the sensitivity and specificity to ensure accurate EBV genome detection, with LCM, enabling the isolation of a pure population of tumor cells for analysis. In parallel, we used immunohistochemistry to examine our series of breast cancers for reactivity with the 2B4-1 reagent (16 , 17) .


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DNA Extraction from Whole Tumor Sections.
Paraffin-embedded samples taken at mastectomy or wide local excision were available from 98 patients (84 cases of no special type, 8 cases of ductal carcinoma in situ, 4 cases of medullary carcinoma, 1 atypical medullary carcinoma, and 1 of mucinous type). Q-PCR was initially used to test for the presence of the EBV genome in DNA extracted (using DNeasy Kit; Qiagen Ltd.) from whole sections of each case. Positive and negative controls were sectioned between cases to detect cross-contamination. Controls were paraffin-embedded D674 cells (EBV-negative) and Namalwa cells (Refs. 18 , 19 ; diploid line containing two integrated EBV genomes), as well as EBV-positive NPC and HD specimens.

Real-Time Q-PCR Assays.
Amplification of DNA was by real-time monitoring of changes in fluorescence intensity using dual-labeled fluorogenic Taqman probes (20) . Amplification of the EBV DNA polymerase (Pol) gene in combination with a FAM-labeled probe (Table 1Citation ; Ref. 21 ) and the human ß2m gene with a VIC-labeled probe (22) were detected simultaneously in a multiplex PCR in which ß2m amplification was primer limited to avoid depletion of PCR reagents. Amplification was performed in 50 µl containing 25 µl of Taqman Universal master mix (PE Biosystems), 0.5 µl of forward and reverse Pol primers (20 µM), 1 µl of 5 µM FAM-labeled Pol probe, 1 µl of 3 µM ß2m forward primer, 1 µl of 4 µM ß2m reverse primer, 0.5 µl of 5 µM VIC-labeled ß2m probe, 10.5 µl of water, and 10 µl of test DNA. After activation of the uracil-N-glycosylase (2 min at 50°C) and Amplitaq Gold (10 min at 95°C), amplification was for 40 cycles (15s at 95°C, 60 s at 60°C), and fluorescent signals detected by an ABI Prism 7700 Sequence Detection System (PE Biosystems).


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  		Table 1 Primer and probe sequences

 
Analysis of Q-PCR Data.
FAM and VIC fluorescence intensities were plotted against cycle number and amplification curves used to determine the Ct values defined as the fractional cycle number at which the amplification curve crosses a threshold level (set as 10 times the SD of the baseline). Because this Ct value is proportional to the initial amount of starting material, the number of Pol and ß2m copies in the test samples could be deduced from standard curves. The Pol calibration curve was obtained from the amplification of serial dilutions of Namalwa DNA containing between 2 and 105 Pol copies (assuming diploid Namalwa cells carry two EBV genomes and that each cell contains 6.6pg DNA); a linear calibration curve was generated by plotting Ct values against log10 Pol copy number from which the number of EBV genomes in unknown samples could be determined. Similarly, a ß2m calibration curve was generated from the Namalwa DNA dilutions and used to determine the cell input in each sample (assuming two ß2m genes/diploid cell). Standards, tests, and positive and negative controls (no template) were analyzed in duplicate. Samples were considered negative if the Ct value exceeded 40 cycles.

LCM.
Cases that contained the EBV genome by Q-PCR analysis of whole sections were subject to LCM using the PixCell II system (Arcturus Engineering Inc., Mountain View, CA). followed by Q-PCR. Multiple clusters of tumor cells from different regions of each specimen were microdissected and pooled onto the same cap and DNA extracted as described above. Clusters of tumor cells containing lymphocytes were not harvested.

EBER ISH.
ISH for EBER-1 and EBER-2 was carried out on cases identified as EBV DNA positive by whole section Q-PCR. Digoxigenin-labeled probes were prepared by in vitro transcription of recombinant plasmids (pBSJJJ1 for EBER-1 and pBSJJJ2 for EBER-2) in the presence of digoxigenin-labeled nucleotides and ISH performed as described previously (23) . Sense probes and EBV-positive controls were included in all runs.

Immunohistochemistry Using 2B4-1.
Immunohistochemical assays using the 2B4-1 reagent were initially performed on 25 cases, which included all those in which Q-PCR of whole sections detected EBV. After endogenous peroxidase blocking and microwave antigen retrieval in citric acid buffer (pH 5.8) for 50 min at 750 W, sections were incubated in 2B4-1 monoclonal antibody for 1 h at room temperature. Bound primary antibody was detected by the sequential application of mouse antirat antibody (Dako Ltd.), the StreptABComplex/horseradish peroxidase duet mouse/rabbit kit (Dako Ltd.) and final visualization using the diaminobenzidine reaction.

Because we identified immunoreactivity with the 2B4-1 reagent in tumors that tested negative for the EBV genome and because nuclear staining with the 2B4-1 antibody was previously observed more frequently in estrogen receptor-negative and in poor prognosis breast cancers, we examined a separate series of 153 breast cancers (for which complete clinical data were available) for their reactivity with this reagent. To facilitate the screening of multiple tissue blocks from each patient, tissue arrays (containing between two and four representative samples of tumor/patient) were prepared. Associations between 2B4-1 reactivity and prognostic factors were tested using Pearson’s {chi}2, Fisher’s exact test, and the Mantel-Haenszel {chi}2 test for linear association. The two-sample Wilcoxon test was used to compare median NPI scores (24) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Q-PCR Assay to Quantify EBV Genome Load and Cellular Input.
A typical Pol amplification plot showing changes in fluorescence intensity against cycle number from a series of Namalwa DNA dilutions is shown in Fig. 1ACitation . A calibration curve (Fig. 1B)Citation generated from the same data shows a strong linear relationship between the Ct values and the log10 value of the initial EBV copy number (r2 = 0.99), and the dynamic range extends over at least five orders of magnitude. We could reproducibly detect as little as two EBV genomes using this assay, although, as expected by Poisson distribution, this standard was amplified inconsistently (data not shown). Amplification plots for ß2m obtained using the same Namalwa DNA dilutions showed a similar sensitivity and dynamic range (data not shown). No significant difference was seen in the sensitivity or efficiency of the Pol and ß2m amplifications generated from Namalwa standards diluted in water or in a background of 600 ng of EBV-negative DNA (data not shown).



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  		Fig. 1. Detection of EBV genomes by Q-PCR. Serial dilutions of Namalwa DNA containing 105, 104, 103, 200, 40, 10, 5, and 2 EBV genomes were amplified using primer/probe combination specific for EBV Pol and cellular ß2m sequences in a multiplex PCR and the fluorescent signals detected using an ABI Prism 7700. A shows the change in FAM fluorescent intensity ({Delta}Rn) plotted against cycle number; replicates are omitted for clarity. B shows a calibration curve generated by plotting the Ct value for each sample, defined as the fractional cycle number at which the amplification plot crosses the threshold (solid horizontal line), against the initial number of EBV copies. The Y intercept corresponds to the number of cycles required to detect a single episome and was consistently <40 cycles.

 
Q-PCR Analysis of Whole Sections of Tumor.
All 98 cases were screened by Q-PCR. Six cases were excluded because of unsatisfactory ß2m yield. Of the remaining 92 cases, a mean of 8980 copies of ß2m (or 4490 diploid cells) were detected/reaction. EBV DNA was detected in 19 of 92 cases (21%; Table 2Citation ), but in all cases, the genome copy number was very low (mean of 1.1 copies of EBV/1000 cells; range, 0.1–7.1). In contrast, Q-PCR analysis of HD- and NPC-positive controls revealed higher viral loads (Table 2)Citation . EBV was not detectable in negative controls.


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  		Table 2 Details of samples in which the EBV genome was detected by Q-PCR of whole sections

 
Q-PCR Analysis of Tumor Cells Isolated by LCM.
In the next series of experiments, LCM was used to isolate tumor cells (Fig. 2)Citation from the 19 cases that had detectable EBV DNA by Q-PCR of whole sections. However, subsequent Q-PCR of these LCM procured cells failed to detect EBV DNA in a single case, although amplification of cellular DNA revealed successful extraction (Table 3)Citation . Tumor cells captured from NPC cases showed multiple EBV copies/tumor cell, even when only small numbers of tumor cells were analyzed (Table 3)Citation .



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  		Fig. 2. LCM of a single cluster of tumor cells. a and b show sections before and after capture, respectively. c shows the microdissected cells on the Eppendorf cap after capture. In the analysis of EBV copy number multiple tumor clusters were pooled and analyzed by Q-PCR.

 

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  		Table 3 Nineteen cases of breast cancer analyzed by LCM-Q-PCR

 
ISH for EBERs.
EBER RNAs were not detected by ISH in tumor cells in any of the 19 cases identified to be EBV positive on the basis of Q-PCR on whole sections. By contrast, strong reactivity was seen in positive controls (Fig. 3)Citation .



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  		Fig. 3. a–c, immunoreactivity for the 2B4-1 monoclonal antibody in tumor cells of breast cancers, which lacked the EBV genome. d, EBER ISH on a known EBV-positive case of post-transplant lymphoproliferative disease. Strong nuclear staining can be seen in a proportion of tumor cells. e, example of EBER ISH on breast cancer specimen with no staining of tumor cells.

 
Immunohistochemistry for EBNA-1.
In the initial series, immunoreactivity with the monoclonal antibody 2B4-1 was detected in tumor cells in 18 of 25 cases, including 13 of 19 cases previously positive by Q-PCR of whole tumor sections and 5 of 6 Q-PCR-negative samples. Immunoreactive tumor cells showed punctate nuclear staining (Fig. 3)Citation .

Next, a series of 153 breast cancers for which complete clinical data were available were analyzed for their reactivity with the 2B4-1 reagent. Strong and punctate staining of tumor cell nuclei was observed in a total of 47 of 153 (31%) breast cancers. 2B4-1-positive tumors were significantly more likely to be estrogen receptor negative (P < 0.0001), of higher grade (P = 0.001), larger (P = 0.03), and to involve more regional lymph nodes (P = 0.01; Table 4Citation ). 2B4-1- positive tumors also had significantly higher NPI scores than their negative counterparts (medians: positive 4.7, negative 3.7; P = 0.0003).


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  		Table 4 2B4-1 reactivity in 153 cases of breast cancer

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
There has been considerable interest in the possibility that a subset of common breast cancers might be EBV-associated. Although an indirect role for EBV infection in tumorigenesis, where EBV infection need not occur within tumor cells or their progenitors, cannot be ruled out, in all of the EBV-associated malignancies established to date, the virus genome or its products are detectable in at least a proportion of the tumor cells. Studies investigating the presence of EBV in breast tumors have produced conflicting results, probably because of the widely differing methodologies used (15) . An association between EBV and breast cancer is an attractive possibility; if a viral etiology were established, it would provide potential opportunities for screening, early detection, or alternative therapies. However, to establish such an association, it is necessary to unequivocally demonstrate the EBV genome or its products within the tumor cell population.

Labrecque et al. (7) and Luqmani et al. (8) both used PCR of whole tumor material and detected EBV DNA in 19 of 91 (21%) and 15 of 28 (42%) cases, respectively. Using a similar approach, Bonnet et al. (9) used PCR to detect EBER-2 and LMP2 DNA in 51 of 100 breast cancers but in fewer (10%) controls and also detected viral DNA by Southern blotting in 7 PCR-positive cases, implying high EBV copy number in the samples. However, Chu et al. (13) did not detect EBV DNA by Southern blotting of 6 DNA PCR-positive cases.

Because conventional end point PCR gives no indication of viral copy number, our initial strategy was to screen whole tumor sections from cases by Q-PCR to quantify EBV DNA load. This identified EBV in 21% of cases. However, viral copy number was very low in all samples in stark contrast to the high EBV copy number detected in whole sections of NPC or even HD tumors.

PCR cannot localize EBV to specific cell types. Therefore, we next obtained pure tumor cells from each PCR-positive case by LCM and analyzed these by Q-PCR for EBV DNA. However, we could not detect the EBV genome in any microdissected tumors, although ß2m copy numbers were high. The interpretation of this data is that EBV is not associated with the malignant epithelial cells of breast cancers and that the EBV copies detected by whole section PCR do not derive from the tumor. Similarly, Horiuchi et al. (25) studied 3 PCR-positive breast cancers but could only detect EBV in infiltrating lymphocytes by DNA ISH.

Bonnet et al. (9) observed positive staining of tumor cells with the anti-EBNA-1 monoclonal antibody 2B4-1 in all 9 PCR-positive breast cancers but in none of the PCR-negative samples. In their study, immunoreactivity with the 2B4-1 reagent was also more likely in estrogen receptor-negative tumors, in tumors of high histological grade, and in those tumors with more than three involved lymph nodes. We observed strong staining of tumor cells with the 2B4-1 reagent in 19 of 25 cases; yet in these cases, the EBV genome was absent from the tumor cells. Although not EBNA-1, the protein detected by the 2B4-1 reagent in our samples has a very similar localization to that reported for EBNA-1 (16) and also for related proteins such as the KSHV-encoded protein LANA (26) . This raises the possibility that this protein, whether viral or cellular in origin, might be related to these DNA-binding herpesvirus proteins.

We next studied a separate series of breast cancers for 2B4-1 reactivity and showed that staining was strongly associated with estrogen receptor-negative tumors, with larger and higher grade tumors, a positive lymph node status, and with higher mean NPI scores. These results suggest that this cross-reactive protein is likely to be important in the pathogenesis of the more aggressive forms of breast cancer. Furthermore, the association of 2B4-1 reactivity with poor prognosis tumors suggests that it should be investigated as a candidate prognostic marker in prospective clinical trials of breast cancer patients. Whether 2B4-1 reactivity could be used as a biomarker to help in the earlier identification of ER-negative disease and thereby enable more timely interventions should also be investigated. This could be particularly important in view of the recent development of more rational approaches to the treatment of ER-negative disease, potentially involving agents such as EGFR tyrosine kinase inhibitors.

Recently, the detection of EBV DNA by whole section PCR and 2B4-1 reactivity in not only breast cancer but also in lung, colon, and prostate tumors was interpreted as providing evidence for a role for EBV in the pathogenesis of these common epithelial malignancies (11) . Our results argue against this interpretation and are additionally supported in a recent study by Kijima et al. (27) , who failed to detect evidence of EBV gene expression in a range of epithelial neoplasms excluding those of lymphoepithelioma type.

Our finding of the absence of EBV from microdissected breast cancer cells is in agreement with the report of McCall et al. (28) , who found only 1 of 115 microdissected tumors to be EBV positive by PCR and by EBER and LMP1 expression. In this case, both tumor cells as well as normal ductal epithelium showed evidence of LMP1 and EBER expression, although the localization of LMP1 to the nuclei might suggest methodological difficulties. A further study in which two breast cancers were subject to PCR after LCM (29) suggested EBV might be present in a minority of tumor cells in some cases. Although we cannot rule out this possibility, the fact that we were unable to detect viral DNA from multiple tumor clusters obtained from different regions of each our cases argues against a significant relationship between EBV and breast cancer.

In conclusion, we have shown that the small quantity of EBV detectable during the gross analysis of breast tumor tissue does not originate from the tumor cells. Our observations extend previous studies to show that the absence of the EBV genome is associated with nuclear reactivity for the 2B4-1 reagent and that this as yet unidentified protein is more frequently associated with the more aggressive forms of breast cancer.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 P. G. M. was supported by the Royal Society Back

2 To whom requests for reprints should be addressed, at Department of Pathology, Division of Cancer Studies, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. Phone: 44-0-121-414-4021; Fax: 44-0-121-414-4019; E-mail: p.g.murray{at}bham.ac.uk Back

3 These two authors contributed equally to this study. Back

4 The abbreviations used are: NPC, nasopharyngeal carcinoma; EBNA1, Epstein-Barr virus nuclear antigen 1; LCM, laser capture microdissection; Q-PCR, quantitative real-time PCR; ß2m, ß2 microglobulin; ISH, in situ hybridization; NPI, Nottingham Prognostic Index; HD, Hodgkin’s disease; EBERs, Epstein-Barr encoded RNAs. Back

Received 10/ 9/02. Accepted 2/27/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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J. G. Perrigoue, J. A. den Boon, A. Friedl, M. A. Newton, P. Ahlquist, and B. Sugden
Lack of Association between EBV and Breast Carcinoma
Cancer Epidemiol. Biomarkers Prev., April 1, 2005; 14(4): 809 - 814.
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 Cancer Epidemiol. Biomarkers Prev.Home page
S. L. Glaser, J. L. Hsu, and M. L. Gulley
Epstein-Barr Virus and Breast Cancer: State of the Evidence for Viral Carcinogenesis
Cancer Epidemiol. Biomarkers Prev., May 1, 2004; 13(5): 688 - 697.
[Abstract] [Full Text] [PDF]

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 J. Virol.Home page
J. Huang, H. Chen, L. Hutt-Fletcher, R. F. Ambinder, and S. D. Hayward
Lytic Viral Replication as a Contributor to the Detection of Epstein-Barr Virus in Breast Cancer
J. Virol., December 15, 2003; 77(24): 13267 - 13274.
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 JNCI J Natl Cancer InstHome page
M. Bonnet-Duquennoy, A. Haratech, B. Vandest, F. Lefeu, and I. Joab
RESPONSE: Absence of the Epstein-Barr Virus Genome in Breast Cancer-Derived Cell Lines
J Natl Cancer Inst, August 20, 2003; 95(16): 1254 - 1255.
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