JMD 2001, Vol. 3, No. 1
Copyright © 2001 American Society for Investigative Pathology & Association for Molecular Pathology
Molecular Diagnosis of Epstein-Barr Virus-Related Diseases
Margaret L. Gulley
From the Department of Pathology, University of Texas Health Science Center at San Antonio and the Audie L. Murphy Memorial Veteran’s Hospital, San Antonio, Texas
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Abstract
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Epstein-Barr virus (EBV) is the causative agent of infectious
mononucleosis, and it may also be found in a wide variety of
benign and malignant lesions including oral hairy leukoplakia,
inflammatory pseudotumor, Hodgkin’s disease,
non-Hodgkin’s lymphoma, nasopharyngeal carcinoma, and
gastric carcinoma. Molecular testing is increasingly important in the
diagnosis and monitoring of patients affected by these diseases. In
biopsy tissues, molecular detection of EBV-encoded RNA
transcripts by in situ hybridization remains the gold
standard for proving that a histopathological lesion is EBV-related.
EBV-encoded RNA hybridization and EBV LMP1 immunostains are used
routinely to detect latent EBV in tissues affected by
posttransplant lymphoproliferative disorder (PTLD) or in enlarged nodes
from patients with infectious mononucleosis. Traditional serology
is the best test for evaluating acute versus remote
infection in healthy individuals. High serological titers serve as a
tumor marker for some EBV-related malignancies, but titers are
not a dependable tumor marker in immunocompromised hosts. EBV viral
load testing by quantitative DNA amplification of blood samples is a
promising new laboratory test that has proven useful for early
diagnosis and monitoring patients with PTLD. Recent studies suggest a
role for EBV viral load testing in nasopharyngeal carcinoma,
Hodgkin’s disease, and AIDS patients with brain lymphoma.
Further research is needed to define more fully the clinical utility of
viral load tests in the full spectrum of EBV-associated diseases. Gene
expression profiling is on the horizon as a means to improve
subclassification of EBV-related diseases and to predict response to
therapy.
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Introduction
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Epstein-Barr virus (EBV) was first identified using
electron microscopy of Burkitt’s lymphoma cell cultures in
1964.1
In subsequent decades, EBV has been linked to a
wide variety of benign and neoplastic diseases. Nasopharyngeal
carcinomas and posttransplant lymphoproliferative disorders are nearly
always EBV-associated, whereas several other tumors, such as Hodgkin’s
disease, non-Hodgkin’s lymphoma, lymphoepithelioma-like carcinoma,
gastric adenocarcinoma, and several types of sarcoma, are less
uniformly EBV-associated.2, 3, 4, 5, 6, 7
EBV causes benign transient
lymphoproliferative lesions at the time of primary infection, and it is
found in a benign lesion of the tongue called oral hairy
leukoplakia.8, 9
Patients affected by these benign or
malignant diseases may benefit from laboratory detection of EBV to
confirm their diagnosis or to monitor disease burden after the
initiation of therapy.
Laboratory detection of EBV is accomplished in several ways (Table 1)
, and recent progress has focused on the molecular analysis of viral
DNA and RNA. In situ hybridization has long been considered
the gold standard for detecting tumor-associated viral infection, and
EBV viral load assays are now being adopted for clinical evaluation of
tumor burden in affected patients. This review article summarizes the
pathobiology of EBV infection and describes the clinical laboratory
tests that are used to assist in diagnosis and monitoring of patients
with EBV-related diseases.
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The Pathobiology of EBV Infection
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EBV has a 173-kb DNA genome for which the nucleotide
sequence and predominant transcripts are well characterized. EBV is
capable of infecting B and T lymphocytes, squamous epithelial cells of
the oropharynx and nasopharynx, glandular epithelium of the thyroid,
stomach, and salivary gland, smooth muscle cells, and follicular
dendritic cells. Healthy virus carriers harbor 1 to 50 EBV genomes per
million blood mononuclear cells, with B lymphocytes representing the
major cellular reservoir.10
Beyond B cells, it is nearly
impossible to find infected cells of other lineages in healthy
carriers, but we presume that the other cell types listed above are
capable of being infected based on the identification of EBV DNA in
lesions arising from them. Investigation of patients with EBV-infected
tumors provides reasonable evidence that EBV was present before
neoplastic transformation, raising the still unresolved question of the
extent to which EBV contributes to tumorigenesis.
EBV infects nearly all humans by the time they reach adulthood. Primary
infection results in transient viremia followed by rapid immune
response. The virus persists for life in its human host by cleverly
balancing its ability to hide from the immune system via latent
infection of B lymphocytes with its ability to replicate and shed from
oral mucosa. At any given time, about 20% of carriers are shedding
salivary virions, leading to nearly universal propagation of the virus
in human populations.
EBV infection of B lymphocytes leads to two alternate outcomes
mimicking the physiological effects of antigen stimulation. One outcome
culminates in the production of memory B cells that persist long-term;
the other outcome results in differentiation toward plasma cells that
are destined to die. These two outcomes support latent viral
persistence and lytic viral replication, respectively. Lifelong
infection of the human host relies on these dual phases of infection
whereby the virus hides from the immune system in memory B cells, and a
subset of these cells are diverted to produce thousands of virions that
not only infect more of the host’s own lymphocytes but also are shed
in saliva to infect other individuals. Viral replication is naturally
enriched in the oral mucosa where memory B cells are routinely
stimulated to differentiate after exposure to foreign antigens.
Lytic viral replication is accompanied by expression of about 90 viral
proteins, including BZLF1 (also known as ZEBRA), and complexes
of viral proteins collectively referred to as early antigen and viral
capsid antigen. These lytic antigens elicit a humoral immune response,
resulting in elevated antibody titers that quell rampant lytic virus
production in the healthy carrier.
Latent infection is characterized by abundant production of EBV-encoded
RNA (EBER), but it is important to mention that EBER transcripts remain
untranslated. EBER transcripts are thought to function in controlling
translation. Also expressed in latently infected cells are EBV nuclear
antigen (EBNA) 1 and latent membrane protein (LMP) 2A , neither of
which elicits an effective immune response. EBNA1 functions to ensure
that the viral genome is propagated to daughter cells upon cell
division, whereas LMP2A keeps other viral proteins from being
expressed. Limited protein expression helps avert immune destruction
in vivo.
In vitro where immune surveillance is absent, infected cell
cultures tend to express a broader spectrum of EBV proteins, such as
LMP1, -2A, and -2B, and EBNA2, -3A, -3B, -3C, and -LP. LMP1 and EBNA2
are critical for the unique ability of EBV to immortalize B cells
in vitro. In this immortalization process, EBV can be
cultured by cocultivating virions with B cells from uninfected persons
(usually neonatal umbilical cord lymphocytes). The resulting
lymphoblastoid cell lines are capable of being propagated indefinitely
in culture media. Naturally infected B lymphocytes can likewise be
cultured from the blood of viral carriers. Viral culture represents an
accurate and semiquantitative measure of EBV in clinical samples, but
it is rarely used in clinical laboratories due to high costs and slow
turnaround time.
More practical laboratory tests for EBV rely on detection of viral DNA
and its gene products. In EBV-infected tissues, three different
patterns of latent viral gene expression are seen. Type I latency
refers to a very limited spectrum of latent viral gene expression,
namely EBER transcripts along with EBNA1 and LMP2A proteins. This
pattern is found in circulating lymphocytes of healthy viral carriers,
and it is also characteristic of Burkitt’s lymphoma and gastric
carcinoma. Type II latency, characterized additionally by LMP1 and
LMP2B coexpression, is seen in Hodgkin’s disease, T cell lymphoma, and
nasopharyngeal carcinoma, all of which tend to occur in immunocompetent
hosts. Type III latency refers to the full spectrum of latent viral
gene expression, as found transiently in acute infectious
mononucleosis, and as seen in EBV-driven lymphoproliferations arising
in immunocompromised hosts. Viral genes expressed in Type III latency
include all of the EBNAs (1, 2, 3A, 3B, 3C, LP), the LMPs (1, 2A, 2B),
and EBER.
Although the patterns of gene expression described above are useful for
characterizing various histopathological entities, in practice there is
heterogeneity of expression among different tumors of the same
histological type, and even among cells within a given tumor. For
example, lymphoid tumors arising in AIDS or transplant patients tend to
express more viral products than do their histological look-alikes
arising in immunocompetent hosts. Therefore, the typical expression
patterns described here provide only rough guidelines to assist in the
clinicopathological diagnosis of each entity.
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EBER in Situ Hybridization
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EBER in situ hybridization is considered the gold
standard for detecting and localizing latent EBV in tissue
samples.11
After all, EBER transcripts are consistently
expressed in virtually every EBV-infected tumor, and they are likewise
expressed in lymphoid tissues taken from patients with infectious
mononucleosis, and in the rare infected cell representing normal flora
in healthy virus carriers. The only EBV-related lesion that lacks EBER
is oral hairy leukoplakia, a purely lytic infection of oral epithelial
cells.12
EBER actually represents two RNA species, EBER1 and EBER2, encoded from
two separate but homologous viral genes. EBER transcripts are expressed
in latently infected cells at levels approaching a million copies per
cell.13
Because EBER transcripts are naturally amplified,
they represent a reliable target for detecting and localizing EBV in
tissue sections by in situ hybridization. The literature is
replete with EBER hybridization protocols that rely on either
oligonucleotide DNA probes, RNA probes (riboprobes), or peptide nucleic
acid (PNA) probes.14, 15, 16, 17, 18, 19, 20
Commercially available EBER
probes are labeled with biotin, digoxigenin, or fluorescein (Dako,
Glostrup, Denmark; Enzo Diagnostics, Farmingdale, NY; Kreatech
Diagnostics, Amsterdam, The Netherlands; Novocastra Laboratories Ltd.,
Newcastle, UK; Shandon Lipshaw, Pittsburgh, PA; Innogenex, San Ramon,
CA; Ventana Medical Systems, Tucson, AZ).
EBER in situ hybridization can be accomplished on paraffin
sections or on cytology preparations. A typical 1-day procedure begins
with removal of any paraffin followed by treatment with proteinase K
and detergent to enhance probe entry into the nucleus where EBER
transcripts are located. Any unbound probe is washed away, and then
colorization and counterstaining are performed. Interpretation of EBER
stains relies on microscopic visualization of the nuclear EBER signal
in latently infected cells. Evaluation of cell type and distribution is
helpful in evaluating the clinical significance of the result (Figure 1)
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Figure 1. A: H&E stain of invasive gastric adenocarcinoma surrounded
by normal surface epithelium. B: EBER in situ
hybridization reveals EBER transcripts in the nucleus of the carcinoma
cells, but not in the overlying normal surface epithelium, nor in the
surrounding benign stromal cells. C: EBER is localized to
dysplastic gastric epithelium but not to adjacent normal-appearing
glands, implying that EBV infection is an early event in gastric
carcinogenesis. D: EBER is localized to the nucleus of a
single small lymphoid cell, representing the rare infected lymphocyte
that might be found in any previously infected individual. Original
magnifications, x50 (A and
B), x80
(C), and x150
(D).
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Even though EBER transcripts are usually abundantly produced in
latently infected cells, users are cautioned about the possibility of
false negative EBER hybridization results as a consequence of RNA
degradation. A control hybridization must be run in parallel to ensure
that RNA is preserved and available for probe binding. This control
might target the polyA mRNA tail using a polyT probe, or it might
target a ubiquitous cell-derived transcript such as U6 RNA. U6 RNA is a
particularly appropriate control because it is similar to EBER in size,
abundance, and intranuclear localization, but it is encoded by a
cellular gene that is constitutively transcribed. With such a control,
the likelihood of false negative EBER interpretation is markedly
diminished. Accurate interpretation of results relies on the ability of
the morphologist to distinguish tumor cells from background lymphocytes
or artifact. When proper attention is paid to these quality control
issues, EBER in situ hybridization is the most reliable
method for determining if a lesion is EBV-associated.
The primary advantage of EBER in situ hybridization is its
ability to localize EBV in the context of cytological and
histopathological features of the tissue. Enlarged lymph nodes from
infectious mononucleosis patients typically contain EBER in a high
fraction of lymphoid cells, including small and large lymphocytes and
immunoblasts.21, 22
In contrast, lymphoid tissues from
remotely infected virus carriers harbor EBV in only rare (<0.1%)
scattered small to medium lymphoid cells.21
In EBV-related
Hodgkin’s disease, EBER is localized to the malignant
Reed-Sternberg/Hodgkin’s cells, whereas the background small
lymphocytes are almost completely negative (<0.1%). Likewise, the
remaining EBV-associated malignancies, including carcinomas, sarcomas,
and lymphomas, exhibit EBER signal in virtually all of the tumor cells,
whereas EBER is absent from the adjacent normal tissue, except perhaps
for rare scattered lymphoid cells. Premalignant lesions of the gastric
epithelium and nasopharyngeal epithelium have also been shown to harbor
EBV, suggesting the EBV infection occurs early during carcinogenesis.
EBER hybridizations are used diagnostically in several specific
clinical situations. They are used routinely for confirming a diagnosis
of EBV-driven posttransplant lymphoproliferative disorder
(PTLD).3, 23
PTLD is a potentially fatal complication of
allogeneic transplantation that requires prompt diagnosis and therapy.
About 95% of all PTLDs are EBV-associated, as shown by EBER expression
by tissue-infiltrating lymphocytes and/or immunoblasts. Treatment
involves cutting back or withdrawing immunosuppressives so that natural
immunity is allowed to destroy virally infected tumor cells. In recent
years, therapeutic success has been reported following infusion of
EBV-specific T cells. The occasional EBV-negative PTLD occurs later
(usually >2 years) after transplant and does not respond as well to
withdrawal of immunosuppression.24, 25
PTLD-like tumors occasionally occur in patients who have not undergone
transplant but who are immunosuppressed for other reasons, such as
rheumatoid arthritis patients on methotrexate therapy.26
As with PTLD, these tumors are often EBER-positive and respond
favorably to immune reconstitution.
In biopsies where the differential diagnosis includes infectious
mononucleosis, Hodgkin’s disease, and/or non-Hodgkin’s lymphoma, EBER
hybridization is often helpful in making the correct diagnosis. In
EBV-related Hodgkin’s disease, EBER is largely restricted to
Reed-Sternberg cells and mononuclear variants, whereas infectious
mononucleosis is characterized by a mixture of small and large
EBER-positive cells including immunoblasts rimming necrotic
zones.22
EBER is not expressed in Kikuchi’s
lymphadenitis, a lesion that shares some clinical and histological
features with infectious mononucleosis.27
Nearly half of all classical Hodgkin’s disease and T cell lymphomas
have EBER-positive tumor cells, whereas only 5% of diffuse large B
cell or anaplastic large cell lymphomas express
EBER.4, 5, 28
Certain subsets of these lymphomas are more
likely than others to harbor EBV, such as nasal T/NK lymphoma (Table 2)
. In classes of tumors that are only fractionally associated with EBV,
further investigation of the prognostic value of EBV testing is
warranted.
Selected subtypes of carcinoma express EBER, notably nasopharyngeal
carcinomas and lymphoepithelioma-like carcinomas of the thymus,
thyroid, salivary gland, lung, or stomach.2, 6, 29
The
majority of nasopharyngeal carcinoma patients initially present with
enlarged lymph nodes containing metastatic undifferentiated carcinoma
of unknown primary, and EBER expression is touted as an
indicator of nasopharyngeal origin.
EBER is usually expressed uniformly in all of the tumor cells
comprising an EBV-associated malignancy, although occasional tumors
have only focal EBER expression. Lack of uniform expression could be a
technical artifact related to focal preservation of RNA, or it could
represent true biological variability in EBER levels. John Sixbey and
colleagues have proposed a "hit-and-run" hypothesis whereby the
virus is lost from some or all cells within a tumor.30
Further research on this topic is warranted. In the meantime, it is
prudent to interpret focal EBER hybridization results in conjunction
with control assays for RNA preservation and in conjunction with other
tests for EBV.
There is intriguing geographic variability in the incidence of
EBV-related tumors. For example, Burkitt’s lymphoma is the most common
pediatric cancer in tropical Africa, where it is almost always
EBV-related, whereas it is 50-fold less common in the United States and
only 20% EBV-related. As another example, EBV-related nasopharyngeal
carcinoma is the most common cancer in parts of Southern Asia, where it
is nearly always EBV-related, whereas the tumor is 50-fold less common
in the United States and only 75% EBV-related. There appears to be an
inverse correlation between the incidence of gastric cancer and its EBV
relatedness, unlike what is observed with nasopharyngeal carcinoma.
These striking geographic variations have yet to be fully explained,
but preliminary studies implicate environmental cofactors over genetic
predisposition or oncogenic viral strains.
Once identified in a patient’s tumor, EBER can be used as a marker of
recurrent disease. For example, when looking for recurrence in patients
treated for nasopharyngeal carcinoma, EBER hybridizations can be used
to complement microscopic examination of nasopharyngeal biopsies. As
described below, blood tests for EBV viral load are also useful markers
of tumor burden after therapy.
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In Situ Hybridization to EBV DNA
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Probes targeting the BamHIW internal repeat
sequence, which is reiterated up to 11 times in each EBV genome, can be
used to detect and localize EBV DNA in tissue sections.16
Single-copy viral sequences could also be targeted, but assay
sensitivity is relatively diminished. In a practical sense, there is
little reason to target EBV DNA rather than EBER RNA, except perhaps in
samples where the RNA has been selectively destroyed. In clinical
situations, EBER transcripts remain the more common target for in
situ detection of EBV.
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LMP1 Immunohistochemistry
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The relative merits of immunohistochemistry versus EBER
in situ hybridization deserve attention. In fact, LMP1
immunostains are nearly as effective as EBER in situ
hybridization for identifying EBV in PTLD cases, in Hodgkin’s disease,
and in infectious mononucleosis.31
Such is not the case
for non-Hodgkin’s lymphomas or carcinomas, however, in which
LMP1 is often undetectable even when EBER is clearly positive.
Some important differences are seen in the distribution of EBER
versus LMP1 expression in tumor samples. In PTLD samples,
LMP1 is typically expressed in about 5% of lesional immunoblasts
(range, 0–100%). When the same PTLD samples are stained for EBER, it
becomes apparent that many more lymphoid cells are EBV-infected, but
only a fraction of those cells coexpress LMP1. Immunoblasts are often
the subtype of lymphocyte that coexpress LMP1, whereas small
lymphocytes are more likely to express EBER alone. Occasional PTLDs
lack LMP1 entirely, even though EBER is clearly positive, implying that
EBER is a more reliable target than is LMP1. Nevertheless, LMP1
immunostains are economical and rapid; therefore, they retain a role in
clinical evaluation of suspected PTLD cases.
LMP1 stains reliably identify EBV in Reed-Sternberg/ Hodgkin’s cells,
although sometimes only a fraction of the EBER-positive tumor cells
coexpress LMP1. LMP1 is reliably expressed in lymph nodes from
infectious mononucleosis patients, with some EBER-positive small
lymphocytes failing to coexpress LMP1, but immunoblasts coexpressing
both markers. Therefore, EBER and LMP1 stains appear to be equally
informative in confirming a diagnosis of infectious
mononucleosis.22, 32
LMP1 immunostains can be performed on paraffin sections using
commercially available antibodies (CS1–4 monoclonal cocktail, Dako, or
S12 monoclonal, Organon-Teknika, Boxtel, The
Netherlands).33
True LMP1 signal is granular in character
and is localized to the cytoplasm and surface membrane. Results should
be interpreted by a morphologist who is confident in discerning tumor
cells from other cells in which a false positive signal has been
described, namely eosinophils, plasma cells, cells of the nervous
system, and poorly fixed cells.
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Measuring EBV Gene Expression by Immunohistochemistry, Reverse
Transcriptase-Polymerase Chain Reaction (RT-PCR), and Nucleic Acid
Sequence-Based Amplification (NASBA)
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Detection of viral proteins can be achieved by immunohistochemical
stains of paraffin sections. Common targets include EBNA1, EBNA2,
LMP2A, and BZLF1.34, 35, 36
Of these, BZLF1, also called
ZEBRA, is the only factor that is characteristic of lytic viral
replication. In fact, BZLF1 immunostains are quite useful in confirming
a diagnosis of oral hairy leukoplakia in tongue biopsies from AIDS
patients using commercially available antibody (clone BZ.1, Dako,
Carpinteria, CA). Interpreting pathologists are cautioned that BZLF1
staining is localized to the nucleus of ballooned epithelial cells of
oral hairy leukoplakia, whereas cytoplasmic cross-reactivity of the
antibody should be disregarded.
Alternative approaches to detecting these viral gene products are
RT-PCR and NASBA.37
Though not yet used routinely in
clinical laboratories, there is much to recommend them as
disease-specific markers, especially if they can be applied in
multiplex or array format. In theory, this should facilitate diagnosis
of each class of EBV-associated disease based on the unique expression
profile of viral and cellular genes that characterizes each disease.
Progress continues to be made in profiling the expression pattern of
each EBV-associated disease. For example, a recent study used NASBA to
identify EBV BARF1 transcripts in gastric
carcinomas.38
BARF1 is likewise expressed in
nasopharyngeal carcinomas but apparently not in lymphocytes, implying
that BARF1 might serve as a marker of these epithelial malignancies
without concern for interference from the occasional bystander
lymphocyte that might be infected.39
If validation studies
pan out, it is feasible that quantitative measurement of this and other
viral transcripts will prove useful for diagnosis and monitoring of
affected patients.
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Southern Blot Analysis of EBV DNA
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Southern blot analysis can be used to determine the clonality of
EBV-infected tissues with respect to the structure of EBV DNA. This
assay, first described by Raab-Traub and Flynn in 1986,40
is based on the presence of variable numbers of terminal repeat
sequences at the ends of each EBV DNA molecule. A given cell is
apparently infected only once, and each infecting genome contains up to
20 terminal repeat sequences. The relatively unique terminal repeat
structure that is present in a given cell is passed along to cellular
progeny upon cell division. Analysis of clinical samples has provided
interesting results. Oral hairy leukoplakia, representing an infectious
process, produces polyclonal viral genomes indicative of lytic viral
replication. On the other hand, EBV-associated tumors harbor monoclonal
EBV DNA.
To perform the EBV clonality assay, lesional DNA is first subjected to
digestion by BamHI restriction enzyme, which cuts at
sequences flanking the region where the terminal repeats are located.
After electrophoresis and transfer, a labeled internal probe is applied
to detect the fragment(s) containing the terminal
repeats.14
Analysis of the band pattern distinguishes
monoclonal from oligoclonal, polyclonal, and uninfected tumors, and
also reveals whether the sample contains substantial amounts of linear
EBV genomes as a consequence of active viral replication (Figure 2)
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Figure 2. The EBV clonality assay evaluates clonality with respect to the
structure of the EBV genome. The assay is based on the presence of
variable numbers of tandem repeat sequences
(shown as open
boxes) at the ends of the linear viral
genome. On infection of a cell, these ends join to form an episome by
fusing up to 20 terminal repeat sequences. When an infected cell
undergoes malignant transformation, the same fused terminal repeat
structure is inherited by all progeny of the malignant clone. The
clonality assay is accomplished by Southern blot analysis of DNA
extracted from patient tissue and digested with BamHI
restriction endonuclease (shown by
arrows) to cut the EBV genome at
sites flanking the terminal repeats. This results in restriction
fragments that are recognized by a DNA probe
(black bar).
Examination of the band pattern on Southern blots reveals that
infectious virions produce a ladder array of small bands. In contrast,
a monoclonal tumor exhibits a single band of high molecular weight, and
an oligoclonal tumor has several such bands.
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Application of this clonality assay reveals monoclonal EBV DNA in
nearly all infected carcinomas, sarcomas, and Hodgkin’s and
non-Hodgkin lymphomas.40, 41, 42, 43
A subset of immunocompromised
patients have either oligoclonal or polyclonal lymphoid proliferations,
and these patients apparently have a better prognosis.3, 44
Even so, monoclonal tumors may respond to immune reconstitution,
leading many clinicians to treat their patients the same regardless of
clonality status.
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Amplification of EBV DNA
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Amplification methods have been used by many clinical laboratories
for detecting EBV in blood, body fluid, or tissue samples. For example,
detection of EBV in biopsies of metastatic undifferentiated carcinoma
of unknown primary narrows the differential diagnosis and
focuses attention on the nasopharynx. As another example, a study of
HIV-infected patients with persistent generalized lymphadenopathy
showed that amplifiable EBV DNA was associated with a heightened risk
of developing lymphoma.45
Most remarkably, amplification
of EBV DNA from the cerebrospinal fluid of AIDS patients is nearly
always indicative of a brain lymphoma, leading oncologists to proceed
with lymphoma treatment without the need for brain biopsy (assuming an
appropriate clinical setting and radiographic support for the
diagnosis).46
After treatment, disappearance of EBV DNA
from the cerebrospinal fluid is associated with better
outcomes.47
From a technical standpoint, PCR amplification of EBV DNA is
accomplished using primers spanning conserved EBV sequences, whereas
strain typing relies on amplification of polymorphic regions of the
viral genome. Strain typing will not be discussed in any detail, since
there are no solid clinical indications for such testing. Even
qualitative amplification assays are difficult to justify because of
their inability to distinguish lesion-specific EBV from that
representing normal flora. After all, EBV DNA is present in a small
fraction of lymphoid cells from every healthy virus carriers, which
means that nearly every adult and a substantial fraction of all
children harbor amplifiable EBV DNA. The inability to distinguish EBV
disease from background infection led many laboratory scientists to
abandon PCR in favor of EBER in situ hybridization for the
reliable detection of lesion-associated EBV in biopsy specimens.
Indeed, EBER studies remain a mainstay of diagnostic surgical
pathology. But improvements in quantitative amplification technology
are stimulating a resurgence of interest in amplification strategies
for detecting EBV in patient samples.
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EBV Viral Load Measurement by Quantitative DNA Amplification
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EBV viral load testing involves quantitative measurement of EBV
DNA in patient samples. A typical viral load assay employs PCR to
coamplify EBV DNA and a spiked control sequence in nucleic acid
extracted from blood samples.14
The amount of
amplification product, measured either at the end point of the assay or
in real time, can be used to calculate the EBV viral load in copies per
milliliter of blood.
The EBV viral load assay has several technical and clinical advantages
over other methods of viral detection. First, the test is rapid, with
with a turnaround time of only 1 to 2 days. Second, it appears that
patients with several subsets of EBV-related diseases are massively and
systemically infected by EBV, allowing us to screen for these diseases
by viral load assays of blood or body fluid, potentially alleviating
the need for invasive tissue biopsy. And finally, recent clinical
studies reveal that several EBV-related diseases can be monitored by
sequential measurement of EBV viral load.
EBV viral load testing appears to be more reliable than serology for
evaluating the EBV status of immunocompromised hosts. In fact, recent
studies of transplant patients showed that those affected by EBV-driven
PTLD have extremely high EBV viral loads, sometimes exceeding 1 million
copies per milliliter of blood.48, 49
Furthermore, viral
load rises as early as several months before the clinical onset of
PTLD, suggesting that the assay might be used to screen high risk
populations for purposes of early intervention.50, 51, 52, 53
And
finally, EBV viral load decreases on successful therapy, suggesting
that the assay should be used to monitor therapeutic
efficacy.52, 54
From a technical standpoint, EBV viral load assays have been shown to
be sensitive, specific, and quantitative across a wide dynamic range. A
commercial kit (BioSource International, Camarillo, CA) is available to
facilitate PCR amplification of EBV EBER genomic sequences. After
coamplification of EBV and a spiked competitor using biotinylated
primers, products are detected in an automated enzyme-linked
immunosorbent assay plate system. Comparison between the amount of EBV
product and the amount of control product permits calculation of
EBV viral load in the patient specimen (Figure 3)
.14
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Figure 3. The EBV viral load assay is accomplished by coamplification of EBV DNA
and a control sequence that is spiked into the sample before DNA
extraction. In the experiment shown here, assay linearity was tested on
serial twofold dilutions of EBV DNA. PCR products at the endpoint of
amplification were evaluated by agarose gel electrophoresis. In
lanes 1–8, the EBV product is seen as a 210-bp band at
template levels as low as 5 copies. The control product, visible at 260
bp, ensures that no inhibitors are present, and it also serves as a
gauge by which to extrapolate the amount of EBV template in each
sample. A molecular weight
(MW) marker is shown on
the left, and lanes 13 and 14 represent control
reactions to which no template was added.
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An alternative procedure for EBV viral load measurement involves
real-time measurement of PCR products, a procedure that has the
potential to reduce labor costs, diminish the risk of amplicon
contamination, and reduce turnaround time.49, 51, 55
Additional laboratory strategies will undoubtedly be developed as
molecular technology continues to advance.
In nasopharyngeal carcinoma patients, EBV viral load shows promise as a
marker of tumor burden that will facilitate monitoring of patients
after therapy.56
Because about half of all affected
patients are destined to relapse, further investigation of the impact
of EBV viral load assays is important to distinguish those patients in
long-term remission from those destined to relapse.
In patients with EBV-related Hodgkin’s disease, a recent study
suggests that EBV viral load might likewise serve as a marker of tumor
burden.57
More research is needed on this and other
EBV-related diseases to define more fully the clinical utility of EBV
viral load assays.
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EBV Serology
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No article about laboratory testing for EBV would be complete
without a discussion of serological testing, which is the gold standard
for confirming acute versus remote EBV infection in
immunocompetent hosts. The heterophile test (also known by a commercial
trade name, the Monospot test) was introduced in 1932 as a marker for
infectious mononucleosis, even though it would be several decades
before EBV was discovered as the causative agent. Heterophile tests are
still used today, often in the form of a 2-minute horse red cell
agglutination test (Seradyn Color Slide II, Seradyn, Indianapolis, IN).
EBV-specific serological assays by enzyme-linked immunosorbent assay or
by immunofluorescent assay are used for more accurate confirmation of
acute or convalescent EBV infection.9
Figure 4
displays a typical serological response to EBV infection.
View larger version (22K):
[in this window]
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Figure 4. Primary EBV infection in healthy hosts is accompanied by an
orchestrated serological response. IgM antibody against viral capsid
antigen (VCA) rises
first. Antibodies against EBNA appear at least 1 month after primary
infection and are measured, along with IgG anti-VCA, as markers of
prior infection and as indicators of EBV reactivation. Titers against
early antigen (EA) rise
on primary infection and again in pathological states of EBV
reactivation.
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EBV-associated tumors are often characterized by abnormally high titers
against early antigen and IgG viral capsid antigen with diminished EBNA
titers. However, this pattern is not specific for malignancy and can be
seen in patients with autoimmune diseases or other immune dysfunction,
implying that serology alone is inadequate for diagnosis of EBV-related
malignancy.
Nasopharyngeal carcinoma patients usually have elevated titers against
multiple viral antigens, particularly IgA antibodies against lytic
antigens, reflecting the tumor’s origin in the mucosa of the
nasopharynx.58
In fact, a panel of serological tests is
used fairly successfully to screen for nasopharyngeal carcinoma in high
risk populations, to assign prognosis in those patients who are
affected, and to detect early relapse after therapy.59
Analogous studies are underway in gastric carcinoma patients who
likewise harbor high serological titers against EBV.7
Immunosuppressed patients have inconsistent humoral responses against
EBV; therefore, serology is not as reliable a marker of clinical
status. In these patients, direct detection of viral nucleic acid or
protein is more reliable for identifying clinically relevant EBV
infection.
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Summary
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|---|
Molecular diagnostics is increasingly important for diagnosis and
monitoring of patients affected by EBV-related diseases. These diseases
represent a wide spectrum of clinical manifestations, from transient
benign infection to aggressive malignancies. As virus-specific
treatments continue to be investigated, it becomes even more important
to recognize these EBV-associated diseases so that proper clinical
management decisions can be made.
New molecular tests combined with traditional serological or
histochemical assays are helpful for diagnosis and monitoring of
EBV-related diseases, depending on the clinical setting and the types
of samples available for testing. EBER in situ hybridization
on biopsy samples and, more recently, EBV viral load testing of blood
samples provide an accurate measure of clinical status in PTLD
patients. Investigations are underway to better define the utility of
these assays across the full spectrum of EBV-associated diseases. On
the horizon are gene expression profiling and array technology, which
likely will improve our ability to subclassify these diseases and
predict responses to therapy.
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Footnotes
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Address reprint requests to Margaret L. Gulley, M.D., Department of Pathology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284-7750. E-mail: gulleym{at}uthscsa.edu
Accepted for publication November 22, 2000.
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