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Epstein?Barr Virus Detection in Nasopharyngeal
Tissues of Patients with Suspected
Nasopharyngeal Carcinoma
Sen-Tien Tsai, M.D.1,3
Ying-Tai Jin, M.D.4
Risa B. Mann, M.D.2
Richard F. Ambinder,
BACKGROUND. Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). Detection of EBV in biopsy specimens may serve as a tumor
M.D., Ph.D.
Department of Oncology, the Johns Hopkins
School of Medicine, Baltimore, Maryland.
Department of Pathology, the Johns Hopkins
School of Medicine, Baltimore, Maryland.
Department of Otolaryngology, National Cheng
Kung University Medical College, Tainan, Taiwan.
Department of Pathology, National Cheng
Kung University Medical College, Tainan, Taiwan.
METHODS. To assess the sensitivity and specificity of the polymerase chain reaction
(PCR) and in situ hybridization in the diagnosis of NPC, formalin fixed, paraffin
embedded nasopharyngeal biopsies from patients in Taiwan suspected of having
NPC were studied.
RESULTS. In specimens from 107 patients with NPC, EBV was detected by PCR in
97 cases (90.7%) and by EBER in situ hybridization in 105 cases (98.1%). In specimens from 61 patients without neoplasia, EBV was detected by PCR in 7 cases
(11.5%) and by EBER in situ hybridization in 0 cases.
CONCLUSIONS. These results suggest that although EBV DNA may occasionally be
detected in nonneoplastic nasopharyngeal tissues, cells expressing EBER are not.
EBER in situ hybridization may therefore prove to be a useful adjunct in the
diagnosis of NPC. Cancer 1998;82:1449?53. q 1998 American Cancer Society.
KEYWORDS: nasopharyngeal carcinoma, Epstein?Barr virus, EBER, in situ hybridization, polymerase chain reaction, diagnosis, epithelium.
Supported by the National Science Council
Grant, Taiwan NC-84-2331-B-006-096, NSC852331-B-006-082, R01 CA63532.
Dr. Ambinder is a Leukemia Society of America
Address for reprints: Richard F. Ambinder,
Johns Hopkins Oncology Center, Baltimore, MD
Received September 9, 1997; revision received
November 28, 1997; accepted November 28,
he nearly uniform association of Epstein ? Barr virus (EBV) with
nasopharyngeal carcinoma (NPC) makes detection of EBV in tumor specimens attractive as a candidate surrogate marker for the
detection of malignancy. In high incidence areas, screening for an
EBV serologic profile suggestive of NPC plays an important role in
early diagnosis.1 ? 5 The use of polymerase chain reaction (PCR) to
detect EBV sequences has been found to be clinically useful in the
diagnosis of occult NPC in patients presenting with squamous cell
malignancies in cervical lymph nodes.6 PCR has also been used to
detect occult cervical metastases.7 The detection of EBV in cerebrospinal fluid is also useful in the diagnosis of primary central nervous
system lymphoma in AIDS patients, and PCR to detect rising EBV
levels in peripheral blood mononuclear cells appears to be useful in
the early diagnosis of posttransplant lymphoproliferative disease.11
The abundance of EBV-encoded RNAs, referred to as EBERs, in EBVassociated tumors has facilitated the detection of virus infection by
in situ hybridization.12,13 EBV detection by in situ hybridization has
also found clinical application in the diagnosis of metastatic NPC14 ? 16
and in the diagnosis of occult recurrent NPC after radiation therapy.17
Can EBV detection play a useful role in the early diagnosis of
NPC in a high risk population, or does the ubiquitous nature of the
viral infection and its shedding in saliva preclude any diagnostic util-
q 1998 American Cancer Society
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CANCER April 15, 1998 / Volume 82 / Number 8
ity? To determine whether either PCR or EBER in situ
hybridization have appropriate sensitivity and specificity for this purpose, we studied nasopharyngeal biopsy specimens from a high-risk Taiwanese population.
Nasopharyngeal biopsies were performed on patients
presenting with symptoms possibly indicative of NPC
(tinnitus, epistaxis, or cervical lymphadenopathy) during the period 1989 ? 1992 at the National Cheng Kung
University Hospital, Tainan, Taiwan. Samples were
fixed in 10% formalin and processed for routine histologic examination by embedding in paraffin wax. NPC
specimens were subclassified, according to the World
Health Organization (WHO) classification, into keratinizing squamous cell carcinoma (WHO-I), nonkeratinizing carcinoma (WHO-II), and undifferentiated
carcinoma (WHO-III).18 Nasopharyngeal biopsies
showing no tumor were classified as nonneoplastic
lesions. One nasopharyngeal biopsy taken from a patient with a history of nasopharyngeal carcinoma and
treatment with radiation therapy was excluded from
consideration retrospectively.
Isolation of DNA from Paraffin Sections
Five 10-mm-thick sections were cut from each paraffin
block and placed in 1.5 mL microcentrifuge tubes.
Each sample was deparaffinized with 1 mL of xylene
and pelleted by centrifugation. Xylene was decanted
and 500 mL of absolute ethanol added. The specimen
was centrifuged and decanted. After desiccation, 50
mL distilled water was added to each sample. The tube
was heated at 100 7C for 10 minutes. The supernatant
was used in PCR amplification.
PCR Amplification
PCR was performed using 2.5 units Taq polymerase
(Perkin Elmer Cetus, Foster City, CA) in a 50 mL reaction containing 10 mL genomic DNA; 50 pmol of each
oligonucleotide primer; 1 mM each of dATP, dCTP,
dGTP, and dTTP; 10 mM Tris-HCl, pH 8.3; 50 mM KCl;
1.5 mM MgCl2; and 0.01% gelatin. An initial denaturation was performed at 95 7C for 90 seconds. Each
cycle included denaturation at 95 7C for 30 seconds,
primer annealing at 58 7C for 60 seconds, and extension at 70 7C for 60 seconds. Samples were overlaid
with 1 drop of mineral oil and subjected to 35 cycles
of amplification, including positive and negative controls in all runs. Positive controls consisted of B95-8
DNA, and distilled water was used as the negative control. PCR products were analyzed by electrophoresis
through a 2% agarose gel in 11 transcatheter arterial
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embolization (40 mM Tris-acetate, 2 mM ethylenediamine ? tetraacetic acid), and made visible by ultraviolet illumination after staining with 1% ethidium bromide.
After NaOH denaturation, the amplified DNA was
transferred to a nitrocellulose membrane (BiotransICN), which was U.V. irradiated. It underwent prehybridization for 2 hours and was then hybridized with
P, oligonucleotide probed, and autoradiographed
overnight at 070 7C with a screen. The oligonucleotide
probe (100 pmol) was prepared by end-labeling using
T4 kinase reaction with g32P-ATP to a specific activity
of 1 ? 3 1 109 cpm/mg. Hybridization was carried out
for 2 hours at 46 7C in hybridization solution (0.5 M
Na2HPO4, pH 7.2; 7% sodium dodecyl sulfate; and
1% bovine serum albumin). Hybridization filters were
washed 3 times for 5 minutes each at 46 7C in washing
solution (40 mM Na2HPO4, pH 7.2; 1% sodium dodecyl sulfate).
To ensure the presence of amplifiable DNA, each
sample was first amplified with b-globin primers,
which would amplify a 268 base pair (bp) fragment of
the human b-globin gene. Only samples showing the
expected b-globin bands were selected for further
studies, including EBV PCR amplification and EBER in
situ hybridization.
EBV PCR amplification was performed using an
oligonucleotide pair, BCR-1: 5 *-AGACCGCCAAGATGCGAAAC-3* (B95-8 strain coordinates 10555-10574)
and BCR-2: 5 *-GTGGAATATGTGAGTGGACA-3* (B958 strain coordinates 10806-10825), yielding a product
of 270 bp. A third internal oligonucleotide, BCR-3: 5 *ATGCCGGCGAAACTGGACCAC-3* (B95-8 strain coordinates 10662-10682), was used as a hybridization
EBER In Situ Hybridization
Five-mm paraffin sections of specimens that were PCR
competent were studied. Hybridization was carried
out as previously described using a digoxigenin-labeled antisense EBER1 RNA and alkaline phosphatase
antidigoxigenin monoclonal antibody conjugate.19 The
color reaction was developed with nitroblue tetrazolium and slides were counterstained with eosin. Specimens were scanned to identify fields with EBER hybridization signal. Association of nuclear staining with
less than a single cell per high-power field was disregarded for purposes of analysis.
After extraction from paraffin, PCR amplification was
carried out with b-globin primers. Approximately twothirds of specimens (168 of 254) yielded the expected
268 bp b-globin band and were considered PCR com-
W: Cancer
EBV Detection in Nasopharyngeal Tissues/Tsai et al.
Results of EBER 1 In Situ Hybridization in Nasopharyngeal
EBER: Epstein?Barr virus?encoded RNA; NPC: nasopharyngeal carcinoma.
A representative autoradiogram is shown of Southern blot
hybridization of Epstein?Barr virus sequences amplified by polymerase
chain reaction. Lanes 2, 5, 6, 8, and 10 show nasopharyngeal carcinoma
DNA amplification products; Lanes 3, 4, 7, and 9 show nonneoplastic
nasopharyngeal tissue DNA amplification products; and Lanes 1 and 11
show amplification products of positive and negative control DNA, respectively.
Detection of EBV DNA in Nasopharyngeal Specimens by PCR
NPC: nasopharyngeal carcinoma; PCR: polymerase chain reaction; EBV: Epstein?Barr virus.
petent. These specimens were evaluated by EBV PCR
(Table 1). A representative Southern blot hybridization
of PCR amplification products hybridized with an internal probe is shown in Figure 1. In this population,
PCR yielded a sensitivity of 0.91, a specificity of 0.89,
and a positive predictive value of 0.93. PCR did not
detect EBV in three of four well-differentiated (WHOI) tumors. This PCR negative group included 2 welldifferentiated specimens in which �% of malignantappearing cells were associated with EBER hybridization signal. There were 6 other specimens that were
not well differentiated but in which EBER hybridization was positive in �% of malignant-appearing
In situ hybridization detected EBER expression in
all but two NPC specimens but in none of the specimens from patients without NPC (Table 2). The sensitivity of EBER in situ hybridization was 0.98 and the
specificity 1. The fraction of tumor cells showing EBER
signal varied between 5% and 100%, with a higher
percentage of EBER positive cells in specimens with
undifferentiated or poorly differentiated tumors (Figs.
2A and B). Apparent hybridization was also detected
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FIGURE 2. In situ hybridization of nasopharyngeal carcinoma specimens
is shown. (A) The hybridization signal is associated with occasional malignant epithelial cells in this WHO-I specimen. (B) The hybridization signal
is associated with virtually all of the tumor cells in this WHO-III specimen.
in acini and glandular tissues in some specimens but
proved to be a nonspecific artifact of the alkaline phosphatase conjugate, as this signal disappeared with an
alternate detection system.14 Thus, although a variety
of chronic inflammatory processes may mimic the
symptoms of malignancy and thus precipitate a diagnostic biopsy, these benign lesions are not commonly
associated with EBER expression.
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CANCER April 15, 1998 / Volume 82 / Number 8
This study confirms the general association of EBV with
NPC and demonstrates that both PCR and EBER in situ
hybridization detect the presence of latent EBV in tumor
tissue in most instances. PCR relies on serial enzymatic
amplification of nucleic acid sequences in vitro, and in
optimal circumstances it can yield amplification well in
excess of 1 million-fold. EBER in situ hybridization, on
the other hand, relies on the natural biologic amplification of the target sequence relative to viral genomic
DNA, i.e., abundant expression of the EBER transcripts.
Estimates place the EBER copy number well in excess
of 106 per latently infected cell.20 Immunoenzymatic detection provides further amplification. Our results show
that EBER in situ hybridization was somewhat more
sensitive, detecting EBV in eight specimens in which
viral DNA was not detected by PCR. In addition, we
confirmed previous findings that the EBER signal is usually detected in WHO-I lesions but in a lower percentage
of cells than in WHO-II or -III lesions.14 There were two
cases of NPC that were not detected by EBER in situ
hybridization. The histology in one of these was WHOI and in the other was WHO-II.
It seems possible that with other primer pairs,
nested primers, or a modified amplification protocol,
the sensitivity of PCR might be increased, although we
note that PCR is still somewhat limited in its application to paraffin specimens by the ability to extract amplification-competent DNA. EBER hybridization detected EBV in all but two of the tumor specimens. EBV
was not detected in these specimens by PCR, and it is
possible that they represent tumors not associated
with EBV.
One might presume that any procedure that relied
on sensitive detection of EBV would be severely limited by nonspecificity. However, EBV appears not to
be commonly detected in healthy nasopharyngeal or
lingual epithelium by either PCR or EBER in situ hybridization. This result echoes recent results from several other laboratories.21,23 PCR occasionally detected
EBV in nonneoplastic lesions, whereas EBER in situ
hybridization did not. The failure of in situ hybridization to detect EBV in PCR positive cases may indicate
that 1) EBER positive cells are so sparse that their presence cannot be readily distinguished from hybridization artifact, 2) EBV infected cells are present (either
lymphoid or epithelial) that do not express EBERs such
as have been described,13,24 ? 26 3) virions but not infected cells are present, or 4) there has been degradation of RNA.
The high sensitivity and specificity of EBER in situ
hybridization of nasopharyngeal tissue suggests that
this procedure might be a useful addition to routine
histology in the evaluation of specimens from a popu-
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lation at high risk for NPC. Specimens in which the
results of routine histologic diagnosis and EBER in situ
hybridization are discrepant might warrant further
consideration. Furthermore, screening programs conceptually similar to those employed for the early diagnosis of cervical carcinoma might be undertaken using
EBER in situ hybridization as a first screen.
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