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From the Department of Pathology,
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University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Northern California Cancer Center,
Union City, California; University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System,
Little Rock, Arkansas; and the Department of Microbiology and Immunology,
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
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Abstract |
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Introduction |
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The assay that many consider to be the gold standard for identifying tumor-associated virus is EBER in situ hybridization. Results are interpreted morphologically so that latent virus can be localized to malignant versus benign cell types. The accuracy of EBER in situ hybridization has recently been called into question by investigators who showed that EBV was present, based on positive molecular or immunohistochemical assays, in certain EBER-negative tumors.3, 4, 5, 7, 8, 9, 10, 11 To explain some of these discrepancies, the "hit and run" hypothesis has been put forward: it states that segments of EBV DNA or EBV gene products are undetected in certain cells or in entire tumors because portions of the EBV genome have been lost or rearranged or integrated into host chromosomal DNA.4, 5 An alternative possibility is that certain molecular or immunohistochemical assays for EBV are non-specific, resulting in false-positive interpretations of tumor-associated EBV.
In the current study, we developed and validated a battery of new quantitative real-time PCR (Q-PCR) assays targeting five different parts of the EBV genome. To assess their utility in detecting disease-related EBV, these assays were applied in a pilot series of paraffin-embedded tissues on which EBER in situ hybridization had also been performed. Furthermore, the assays were applied to several plasma samples to assess their suitability for use in studies in which patients are screened for an EBV-related disease.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sterile procedure was used during histological sectioning to prevent tissue carryover between cases. Before sectioning each block, the work area and forceps were wiped down with 10% bleach in water and rinsed with deionized water. Disposable blades were either discarded or moved to an unused edge before cutting each block. Two 10-µm thick sections from each block were placed into microfuge tubes for subsequent DNA extraction. Five-µm thick sections were placed on glass slides for histochemical staining procedures.
To extract DNA from paraffin-embedded tissue sections, deparaffination was performed using three washes in xylene for 10 minutes at room temperature followed by two washes in 100% ethanol to remove the xylene. Tissues were dried at 55°C and digested overnight at 55°C in 100 µl of TEN buffer (10 mmol/L Tris-HCl pH 8.0, 1 mmol/L EDTA pH 8.0, 20 mmol/L NaCl) containing 20 mg/ml proteinase K, and then the proteinase was inactivated at 95°C for 10 minutes. Undigested tissue remnants were pelleted by centrifugation at 14,000 rpm for 10 minutes. The supernatant containing extracted DNA was transferred to a new microfuge tube and stored at –20°C until use.
Plasma samples were selected from nine patients, four of whom had EBV-related malignancy as shown by EBER in situ hybridization on biopsy materials, and five with infectious mononucleosis confirmed by heterophile antibody positivity. Plasma was also obtained from nine healthy blood donors. Total DNA was extracted from 200 µl of plasma using the QIAmp Blood Kit (Qiagen Inc., Valencia, CA). Before extraction, the plasma was spiked with 2 µl of IPC (TaqMan Exogenous Internal Positive Control DNA, Applied Biosystems (ABI), Foster City, CA) to control for the effectiveness of extraction and amplification. Purified DNA was eluted into 50 µl of AE buffer (Qiagen).
DNA from the Namalwa cell line (two copies of EBV per cell, equivalent to 3 x 105 copies of EBV/µg DNA) was used as a standard by which EBV genomic DNA was measured (American Type Culture Collection, Rockville, MD). DNA from the Raji Burkitt lymphoma line and the P3HR-1 Burkitt cell line were used as additional EBV-related lymphoma cell line controls (American Type Culture Collection). DNA was extracted from fresh cell pellets using the Purgene DNA Isolation kit (Gentra, Minneapolis, MN). Serial 10-fold dilutions of these DNAs were made in nuclease-free water to examine the linearity of each quantitative PCR assay. Potential cross-reactivity with other viruses or genomic DNA was evaluated by BLAST sequence analysis. Specificity testing was performed on purified cytomegalovirus (CMV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) DNA (Advanced Biotechnologies, Inc., Columbia, MD) and on paraffin-embedded tissue from five patients diagnosed with other herpes family viruses (three with CMV and two with herpes simplex).
Development and Validation of Quantitative PCR Assays
EBV genomic sequences were downloaded from GenBank (www.ncbi.nlm.nih.gov). PCR primers and TaqMan probes targeting conserved portions of the reiterated BamH1W segment and the unique EBNA1, LMP1, LMP2, and BZLF1 genes were designed using Primer Express 2.0 software (ABI). These sequences are shown in Table 1
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PCR was performed and products were detected using an ABI Prism 7900 Real-Time PCR instrument and Sequence Detection System software. Thermocycling conditions were: 50°C for 2 minutes; 95°C for 10 minutes; 95°C for 15 seconds and 60°C for 1 minute for 40 cycles. Each 50-µl reaction contained: 1X TaqMan Universal Master Mix, forward and reverse primer (15 
mol each), and TaqMan probe (10 mol). DNA template volume was 1 µl for paraffin tissues, and 5 µl for plasma samples. The plasma reactions also contained 1X IPC Master Mix (ABI) to co-amplify the spiked IPC sequence. To improve assay sensitivity when targeting LMP1 and BZLF1 genes, 30 mol each of the forward and reverse primers was used. To check for amplicon contamination, every run contained at least two "no template" controls in which nuclease-free H2O was substituted for template. A standard curve was generated using 10-fold dilutions of Namalwa DNA varying from 50,000 to 0.5 copies of EBV DNA, and this curve was considered acceptable if a difference of 3.3 +/–0.3 cycles was demonstrated between each of the 10-fold dilutions, and if the correlation coefficient was at least 0.99. Quantification results for experimental samples were extrapolated from the standard curve. Experimental samples were run in duplicate and a mean viral load was calculated.
EBV viral load in paraffin tissue was calculated based on the ratio of copies of EBV to ApoB in a given volume of extracted DNA, with ApoB representing the number of cells in the sample. The resulting ratio was then multiplied by 100,000 to provide the number of copies of EBV per 100,000 cells. For plasma samples, EBV viral load was expressed in copies per ml of plasma. Plasma results were considered negative for EBV when the spiked IPC control sequence was amplifiable while EBV DNA was not. For purposes of data analysis, samples with no measurable EBV DNA were reported as having a viral load of zero.
Qualitative LMP2 Gene PCR Assay
To further investigate a case in which the LMP2 segment selectively failed to amplify by Q-PCR, a second primer set was designed to amplify a 130-bp region of the LMP2 gene encompassing the 69-bp segment that had been targeted by the standard LMP2 Q-PCR primers. This qualitative PCR used the following primer sequences: LMP2Ext forward 5'-CTGTTTTGCAGCTGAGTCC-3' and LMP2Ext reverse 5'-CAATGTTAAAAGGGCTGCACC-3'. The 50-µl PCR reaction consisted of: 1X PCR Buffer, 2 mmol/L MgCl2 2.5 units Platinum TaqDNA Polymerase (Invitrogen, Carlsbad CA), 0.2 mmol/L dNTPs (ABI), 50 mol of each primer, and nuclease-free water. Reaction conditions were: 95°C for 2 minutes; 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute; and then 72°C for 10 minutes. Products were electrophoresed in a 2% agarose gel containing 0.5 mg/ml ethidium bromide. DNA from Case 7 (EBER-positive AIDS lymphoma) and an EBER-positive gastric carcinoma were used as wild-type controls.
LMP1, LMP2, and BZLF1 Expression by Immunohistochemistry
In selected cases with atypical DNA amplification results, immunostains were used to assess viral gene expression. Immunohistochemical analysis for LMP1 and LMP2 was performed on paraffin sections using the Anti-EBV LMP1-clone CS1–4 cocktail of mouse monoclonal antibodies (Dako, Carpinteria, CA) and the TP/LMP2a-clone E411 rat monoclonal antibody (Asencion, Munich, Germany). Antigen retrieval (Biogenex Citrate Antigen Retrieval Buffer, HK086–9K) was performed for 30 minutes, and endogenous peroxidase was quenched for 10 minutes using Peroxidase Quenching Solution (Zymed, San Francisco, CA). Bound antibody was detected using the Zymed PicTure-Plus Kit with Polymer Detection System (Zymed).12
Common antibody diluent (Biogenex HK156–5K) was used to dilute the LMP1 (1:100) and LMP2 (1 mg/ml) antibodies, and sections were incubated with primary antibody for 40 minutes. Bound antibody was detected with a ready-to-use 
-rat-HRP polymer conjugate for 30 minutes (LMP1) or 20 minutes (LMP2), followed by diamino-benzidine (DAB) chromogen (Dako Liquid Dab and Substrate Chromogen System K3468). Tissues were counter-stained with hematoxylin (Dako). EBER-positive Hodgkin lymphoma slides served as positive controls. Results were interpreted microscopically by looking for cytoplasmic and membrane localization of the chromagen in neoplastic cells.
Immunohistochemical analysis of the EBV BZLF1 protein using the Zebra clone BZ.1 antibody (Dako, 1:25 dilution) was performed as described above except for the following modifications. All washes after antigen retrieval were performed in Automation Buffer (Biomeda Corp, Foster City, CA). Endogenous peroxidase was quenched using Peroxidase Block (Dako) for 10 minutes at 37°C. To reduce non-specfic binding, Avidin and Biotin blocks (Dako) were performed for 15 minutes followed by Protein Block (Biogenex) for 10 minutes at 37°C. Sections were incubated with primary antibody for 30 minutes at 37°C, and bound antibody was detected using the Biogenex StrAviGen Multi Link Kit (Biogenex). Sections were incubated with secondary antibody (1:20) for 8 minutes at 37°C followed by incubation with HRP label (1:20; diluted with Biogenex Streptavidin-Peroxidase Diluent) for 8 minutes at 37°C. An oral hairy leukoplakia section served as a positive control. Results were interpreted microscopically by looking for nuclear localization of the chromagen in neoplastic cells.
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Results |
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Assay Linearity
The BamH1W assay was linear across five orders of magnitude from 0.5 to 50,000 copies of Namalwa EBV DNA. The EBNA1, LMP2, and BZLF1 assays were linear across four orders of magnitude (from 5 to 50,000 copies) and the LMP1 assay was linear across three orders of magnitude (from 50 to 50,000 copies). The assays were efficient as shown by a cycle difference for each 10-fold dilution ranging between 3.3 and 3.4 (representing slope of the standard curve). The assays were linear as shown by a correlation coefficient greater than 0.99 (representing the linearity of the standard curve). Serial dilutions of DNA from the Raji cell line and the P3HR1cell line were also used to examine assay linearity. All five novel real-time PCR assays detected both Raji and P3HR1 EBV in a linear fashion (correlation coefficient >0.99), and cycle difference between each 10-fold dilution averaged 3.5 for Raji and 3.3 for P3HR1.
Application of the Viral Load Assays to Paraffin-Embedded Tissues
EBV viral load was measured on 30 paraffin-embedded tissues representing various EBV-related diseases (17 EBER-positive lymphomas or carcinomas) and controls (13 EBER-negative lymphomas) using each of the five validated real-time PCR assays. ApoB, a cellular gene, was co-amplified and used as a normalizer by which to control for the number of cells tested, and to check for inhibitors of amplification or failed extraction. None of the 30 tumors in this study had to be excluded because of inadequate ApoB control amplification. Results, expressed as copies of EBV per 100,000 cells, are shown in Table 2
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When viral load was high by one assay, it tended to be high by the rest of the assays. Likewise, when viral load was low by one assay it was either low or undetectable by the other assays, suggesting varying lower limits of detection among the assays. The BamH1W assay was most sensitive, presumably because it targets a reiterated sequence that is present at approximately 10 copies per EBV genome. The EBNA1 assay was the next most sensitive to EBV-related malignancy, while the LMP1, LMP2, and BZLF1 assays were more prone to false-negative results.
Partial deletion or polymorphism of the EBV genomes could explain, at least in part, the differential productivity of the viral load assays in a given tumor. Case 8 had dropout of two EBV markers, suggesting that it had defective LMP1 and BZLF1 segments. An even more striking differential was seen in Case 6, where the LMP2 gene was not amplified despite high levels of EBV DNA by the other four assays. When tested using an alternate primer set, Case 6 still had no amplifiable LMP2 DNA, suggesting that this tumor had a global defect in the EBV LMP2 region such as gene deletion or chromosomal integration. Further support for this interpretation comes from immunohistochemical staining which revealed no LMP2 protein expression. Likewise, Case 8 did not express LMP1 or BZLF1 by immunohistochemistry, in keeping with our inability to amplify LMP1 and BZLF1 at the DNA level. While lack of protein expression is consistent with a genetic defect, it is certainly not diagnostic of a genetic defect. Further work is required to explore why selected segments of EBV DNA failed to amplify in these two AIDS lymphomas.
Human Plasma Samples
To test the versatility of these Q-PCR assays for an alternate sample type, we measured EBV viral load in plasma samples from nine patients with various EBV-related diseases and nine healthy donors (Table 3)
. Consistently high EBV loads were seen in the four patients with EBV-related malignancy and in the five infectious mononucleosis patients. In contrast, healthy individuals had no measurable EBV DNA by any of the five assays. It appears that the five Q-PCR assays are equally effective and virtually interchangeable in terms of their correlation with clinical status in this small cohort of plasma samples.
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. No amplification was seen when pure CMV or KSHV DNA served as the template or when either of the herpes simplex lesions was assayed. All three of the CMV-related colon lesions had low-level EBV DNA by at least one assay. Since no amplification was observed with purified CMV DNA, the EBV signal in these colon biopsies could have emanated from rare EBV-infected lymphocytes in the tissue. There is no evidence for cross-reactivity with any of the viruses tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The BamH1W assay appears to be the most sensitive of the five for detecting low-level virus, in keeping with the fact that the target sequence is reiterated within each viral genome. A drawback of using this assay is that the number of reiterated BamH1W sequences varies among EBV strains, typically ranging between 7 and 11 repeats per genome. If the BamH1W copy number of a patient’s EBV strain differs from that of the Namalwa Burkitt lymphoma standard, then we will overestimate or underestimate the viral genome copy number in proportion to that difference. This variability confounds our ability to precisely quantitate the number of viral genomes, but this deficiency might be outweighed by the assay’s exquisite sensitivity to low-level virus, which could help detect early stage disease or help monitor residual disease burden after therapy. The EBNA1 Q-PCR appears to be the next most sensitive assay among the five that were developed; it targets a single copy highly conserved gene that is thought to be essential for maintaining the virus long- term in dividing cells.
The true viral load in a given tumor could not be established because viral load varied, sometimes quite substantially, depending on which assay was used. The explanation for this variability was not always evident. If a partial inhibitor in a given tumor sample is assumed to inhibit all five assays equally, then inhibitors cannot explain the variance in viral loads by assay in a given tumor. The variance is especially striking given that the same Namalwa cell line was used as a standard for each of the five viral load assays. However, Namalwa DNA might contain a sequence variant that selectively affects binding of one primer or probe, thus rendering a given assay differentially efficient compared to the other assays.
Despite our efforts to target highly conserved gene sequences, two tumors (Cases 6 and 8, both AIDS lymphomas) had selective dropout of amplifiable EBV DNA at the BZLF1, LMP1, or LMP2 loci. If a multiplicity of EBV assays had not been used in this study, then these cases might have been falsely characterized as negative for EBV. Furthermore, the selective failure of a given PCR assay suggests that a genetic defect is present, and this defect could potentially contribute to disease pathogenesis. For example, BZLF1 encodes an immediate early lytic protein that is critical in controlling the latent versus replicative phases of viral infection. LMP1 protein has growth-promoting properties, which imply a role in tumor promotion or maintenance. LMP2, also located on the cell surface, acts to promote survival of B cells that are crippled by lack of functional antigen receptors. LMP2 is transcribed across the terminal repeat sequences, an area of the genome known to be involved in integration events. The Q-PCR assays described herein could provide a mechanism to screen for gene abnormalities including deletion, polymorphism, or integration. Such events can be further investigated by designing new up- or down-stream primers sets to map a putative deletion, and by sequencing the defective region to look for polymorphisms that interfere with primer or probe binding.
Another advantage of the Q-PCR assays described herein is that they are rapid and less labor intensive (3.5 hours total; 1 hour technologist time) than manual EBER in situ hybridization (7 hours total; 3 hours technologist time). Q-PCR is automated to facilitate high throughput, and is less subject to amplicon contamination than are traditional PCR assays in which product is detected by gel or by enzyme-linked immunoassay (ELISA). To improve these Q-PCR assays further, we recently showed that all components could be halved to make a 25-µl total reaction volume, and products could be amplified equally well on the ABI 7000 or 7700 instruments (Applied BioSystems) with little if any effect on outcome (data not shown).
Paraffin-embedded tissues representing EBV-related non-Hodgkin lymphomas and carcinomas had considerably higher EBV loads than did the EBER-negative tumors. These results suggest the possibility of using a rapid real-time PCR to screen for EBV, followed by EBER in situ hybridization on equivocal cases to distinguish tumor-associated virus from incidental viral infection. It appears that a threshold value of around 100 EBV copies per 100,000 cells distinguishes EBER-positive from EBER-negative cancers. This screening strategy is less likely to be useful for Hodgkin lymphoma cases, given the paucity of tumor cells and the associated low viral loads that approach the levels found in EBER-negative tumors.
Finally, the applicability of the five Q-PCR assays to plasma samples was demonstrated in a pilot series of cases. While all five assays yielded similar results in the nine cases we examined, we predict that future studies will show preferential amplification in certain strains of EBV, as was found in the biopsy samples we studied. The advantage of testing plasma as opposed to biopsy material is the less invasive sample collection and the ability to screen for lesions that are not yet clinically apparent. Prior studies suggest that EBV viral load testing of plasma samples is useful in at least two clinical situations. First, healthy allogeneic transplant patients usually have plasma levels below 700 copies per ml, whereas higher levels are suggestive of progression to post-transplant lymphoproliferative disorder.13 EBV viral load is often elevated days to months before onset of symptoms, and levels quickly fall once effective therapy is initiated. Secondly, nasopharyngeal carcinoma patients often have elevated plasma levels of EBV, with advanced stage patients having higher levels than patients with localized disease.14 After therapy, EBV is undetectable in patients who remain in remission, while it is measurable in those destined to relapse.14
The cell-free EBV DNA found in plasma or serum of patients with EBV-related neoplasia is thought to be un-encapsidated, suggesting that it arose from apoptosis or necrosis of infected cells.15, 16 On the other hand, many infectious mononucleosis patients have a mixture of virion and un-encapsidated EBV DNA in their plasma, suggesting that viral replication contributes to the process by which EBV DNA accumulates in their plasma.16 Further studies of infectious mononucleosis patients are warranted to determine the relative efficacy of EBV viral load and serological tests in confirming a clinical diagnosis; a recent study showed that EBV viral load was false negative in five of 30 heterophile antibody-positive cases (17%).16
In conclusion, the quantitative nature of the assays described herein helps distinguish EBV-related disease from incidental infection. While the BamH1W and EBNA1 assays seem most suitable for clinical applications, the full battery of assays can be used to screen for viral genomic alterations. Their applicability to DNA extracted from paraffin sections allows them to be used retrospectively and in cases for which fresh tissue is not available. Their use in plasma may obviate the need for biopsy in selected clinical situations. Further work must be done on larger series of cases to confirm the results of this pilot study and to define the criteria for altering management based on viral load results. Furthermore, the incidence of and possible pathogenicity of genetic defects in the targeted gene segments must be explored.
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Acknowledgments |
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Footnotes |
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Supported by grants from the National Cancer Institute (R03 CA101500) and the California Breast Cancer Research Program (9WB-0005).
Accepted for publication June 1, 2004.
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