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Technical Briefs |
Departments of1 Chemical Pathology and2 Clinical Oncology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong SAR, China;
aaddress correspondence to this author at: Department of Chemical Pathology, 1/F, Clinical Sciences Bldg., Prince of Wales Hospital, 30-32 Ngan Shing Street, Shatin, New Territories, Hong Kong SAR; fax 852-2636-5090, e-mail loym{at}cuhk.edu.hk
Monitoring of Epstein–Barr virus (EBV) DNA load in the circulation is clinically valuable for the management of EBV-associated diseases, including nasopharyngeal carcinoma (NPC) and certain lymphomas (1)(2). Many studies have used lymphocytes or peripheral blood mononuclear cells as materials for EBV DNA detection in such patients (3)(4)(5)(6). However, cell-free EBV DNA has also been reported in the plasma and serum of patients with NPC and certain lymphoid malignancies (1)(7). These reports raise fundamental questions on whether EBV DNA exists predominantly in a cellular or cell-free form in the peripheral blood of such patients and whether the relative distribution of these forms of EBV DNA differs in different types of EBV-associated disorders.
To study these issues, it is important to ensure that the EBV DNA present in the plasma is truly acellular and that the isolated cellular EBV DNA is free from contamination from the cell-free form that may be present in the plasma. Previous reports on cell-associated EBV DNA have not addressed the impact of the thoroughness of the washing of the isolated circulating cells during sample processing. The first objective of the current study was to investigate the efficiencies of different peripheral blood cell (PBC) clean-up procedures in producing data that would truly reflect the concentration of the cell-associated form of EBV DNA. We then studied the relative distribution profiles of cell-free and cell-associated EBV DNA in blood samples from patients with NPC and lymphoid malignancies.
For this study, we recruited 49 patients with NPC and 14 patients with Hodgkin disease, natural killer (NK)/T-cell lymphoma, or Burkitt lymphoma managed at the Department of Clinical Oncology at the Prince of Wales Hospital, Hong Kong. Patients gave informed consent, and ethics approval was received from the Institutional Review Board. EDTA blood (3 mL) was centrifuged at 1600g for 10 min, followed by microcentrufugation of plasma at 16 000g for 10 min. We then resuspended 1 mL of the buffy coat layer and washed it three times with 1 and 10 mL of phosphate-buffered saline (PBS), respectively. We collected 400 µL of the plasma, the washing supernatant from each washing round, and washed PBCs for DNA extraction.
DNA was extracted by use of the QIAamp Blood Kit (Qiagen), according to the "blood and body fluid protocol" (8). Real-time PCR was performed in an Applied Biosystems 7700 Sequence Detector. EBV DNA concentrations were measured by quantitative PCR analysis of the BamHI-W fragment region of the EBV genome as described previously (9). All DNA samples were also quantified for the ß-globin gene, which served as a control for the amplifiability of DNA. Multiple negative water blanks were included with each set of samples. The theoretical and practical aspects in the expression of EBV DNA concentration in plasma have been described (1). This approach was adopted for documenting the EBV DNA concentration in the plasma and washing supernatants derived in the clean-up study. For comparison of the relative content of cell-free and cell-associated EBV DNA, the calculation of the concentration of the EBV DNA was modified and expressed in copies/mL of whole blood (see the Data Supplement that accompanies the online version of this Technical Brief at http://www.clinchem.org/content/vol50/issue10/). The relative distribution profiles of the cell-free and cell-associated EBV DNA in the blood is expressed as the cell-free percentage (%CF), which was derived by dividing the blood concentration of the cell-free EBV DNA by the total concentration of the both cell-free and cell-associated forms.
Six newly diagnosed NPC patients, before receiving therapy, were recruited for the study of clean-up efficiency. Their plasma EBV DNA concentrations ranged from 2630 to 17 100 000 copies/mL. Sample clean-up efficiency, expressed as the fractional retention (% retention) was calculated by dividing the EBV DNA concentration of the washing supernatant by that of the corresponding plasma sample. Lower fractional retention values were observed when the cellular components were washed with 10 mL of PBS rather than with 1 mL of PBS (Table 1
). No measurable EBV DNA was detected in the washing aliquots recovered from the last-washing supernatants from all these cases except case 3, for which the plasma EBV DNA concentration was very high (17 100 000 copies/mL; Table 1
). The presence of EBV DNA in the washing supernatant suggested inadequate removal of the residual plasma-derived EBV DNA; the source of EBV DNA detected in the corresponding PBC samples therefore could not be ascertained. Thus, the reported presence of EBV DNA in the PBCs or unfractionated whole blood in previous studies (10)(11)(12)(13) might be attributable, at least in part, to the EBV DNA in the plasma. We modified the washing procedure to include a fourth washing step with 10 mL of PBS for all subsequent analyses; no EBV DNA was measurable in the fourth washing supernatant (data not shown).
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We then investigated the distribution profiles of circulating cell-free and cell-associated EBV DNA in a cohort of patients with NPC and EBV-associated lymphoid malignancies. Forty-nine NPC patients with International Union Against Cancer stage I–IV NPC were recruited: 34 newly diagnosed patients, 11 posttherapy follow-up patients, and 4 patients undergoing radiotherapy. The median (interquartile range) blood cell-free EBV DNA concentrations of the newly diagnosed patients and patients receiving radiotherapy were 1120 (129–5300) copies/mL, and 0 (0–17) copies/mL, respectively. Among the 11 posttherapy patients, the 2 patients with distant metastases had a high circulating EBV DNA concentration in their plasma (range, 20 200–50 200 copies/mL). The remaining 9 patients with clinical remission had no detectable cell-free EBV DNA. No cell-associated EBV DNA was detected in any of the 49 NPC patients.
For the 14 lymphoma patients, 4 of the 10 patients (7 with NK/T-cell lymphoma; 3 with Hodgkin disease) who stayed in clinical remission and 1 with relapsed disease (NK/T-cell lymphoma) had both cell-free and cell-associated EBV DNA in the blood. Strikingly, the concentrations [median (interquartile range)] of both cell-free and cell-associated forms in the clinical remission group [131 (61–272) and 47 200 (38 300–155 000) copies/mL, respectively] were higher than those in the relapsed patient (36 and 186 copies/mL), with an inverse relationship being found in the calculated cell-free percentage. The cell-free percentage for the patient with relapsed disease (3.7%) was almost 18-fold greater than that of the clinical remission group (0.2%), suggesting a relative predominance of cell-free EBV DNA in the circulation of the patient with active disease compared with those in stable remission. On the other hand, only cell-free EBV DNA was detectable in one of the three newly diagnosed (NK/T-cell lymphoma, Hodgkin disease, and Burkitt lymphoma) patients. No EBV DNA was detected in either blood compartment of the remaining nine cases.
Serial measurements of blood EBV DNA were performed in seven lymphoma patients. For the three patients with newly diagnosed lymphoma, EBV DNA could not be detected in any pre- and posttherapy blood samples in two of them. The other patient showed a pretherapy cell-free EBV DNA concentration of 1110 copies/mL. The cell-free EBV DNA concentration decreased during radiotherapy but subsequently increased at the end of the therapeutic course, which corresponded to the clinical finding of a partial response to radiotherapy (Fig. 1A
). No cell-associated EBV DNA was found in any of the serial blood samples collected over 20 weeks of follow-up. The patient died of the disease 5 weeks after treatment. For the three patients in clinical remission, both cell-free and cell-associated EBV DNA were found concurrently in the blood circulation during the period of follow-up study (see Fig. 1 in the online Data Supplement). The median (interquartile range) concentration of cell-free EBV DNA was relatively low [126 (46–272) copies/mL] compared with the high concentration of the cell-associated form [23 000 (660–155 000) copies/mL], with a correspondingly low cell-free percentage [0.2 (0.08–0.69)%].
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The patient with relapsed disease demonstrated a multiphasic profile in the concentrations of both cell-free and cell-associated EBV DNA during disease progression (Fig. 1B
). There was a parallel increase in both the cell-free EBV DNA concentration, from a baseline of 27 to 619 copies/mL, and the cell-associated EBV DNA concentration, from 400 to 73 600 copies/mL, over the first 60 days, followed by a subsequent decrease in both forms after the patient received chemotherapy. Notably, we observed a second phase of increase in cell-free EBV DNA as the disease progressed and a decrease during the period in which the patient received chemotherapy, whereas the cell-associated form was either not detected or was present at a comparatively low concentration. The calculated cell-free percentage also exhibited a wider dynamic range, from 0% to 100%, during disease progression, in contrast to the stable low cell-free percentage observed in patients with clinical remission. These data indicate that the distribution of EBV DNA exhibits a more heterogeneous pattern in the blood of patients with lymphoid malignancies.
EBV resides in epithelial cells of the pharynx and B cells of seropositive people (14). An increased risk of lymphoproliferative disorders has been well documented in patients treated with immunosuppressive agents after organ transplantation (5) and in patients with AIDS (15). Similarly, EBV-associated lymphomas that develop in immunosuppressed patients are suggested to be linked to the degree of competence of the immune system (16)(17). Clearly, such cases emphasize the role of immunosuppression in certain aspects of lymphomagenesis, irrespective of the cause of the immune defect. It has previously been suggested that EBV-associated epithelial and lymphoid malignancies involve the clonal expansion of a single EBV-infected progenitor cell (18)(19).
The mechanisms leading to the release of EBV DNA from tumor cells into the circulation are unclear. Several workers have suggested that the high concentrations of serum EBV DNA might be explained by increased EBV replication at sites other than the tumor (20), but such a proposition cannot account for the temporal changes and the rapid decrease in circulating EBV DNA in patients with NPC and lymphoid malignancies during the course of therapy (21). Recently it has been reported that short circulating DNA molecules are released into the circulation, possibly by apoptosis of cancer cells (22). In the present study, we compared the circulating EBV DNA in the blood compartments of patients with NPC and lymphoid malignancies by deriving an index, the cell-free percentage (%CF), to describe this dynamic phenomenon. A %CF value of 100% was found for all NPC cases, underscoring the exclusive existence of circulating cell-free EBV DNA in patients with this EBV-associated tumor. This is in sharp contrast to the very low %CF (0.2%) observed in lymphoid malignancies. The inclusion of %CF in future large-scale clinical studies on such EBV-associated disorders is thus warranted.
Acknowledgments
This work was supported by an Earmarked Research Grant (CUHK 4086/02M) from the Hong Kong Research Grants Council.
References
The following articles in journals at HighWire Press have cited this article:
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  M. L. Gulley, H. Fan, and S. H. Elmore Validation of Roche LightCycler Epstein-Barr Virus Quantification Reagents in a Clinical Laboratory Setting J. Mol. Diagn., November 1, 2006; 8(5): 589 - 597. [Abstract] [Full Text] [PDF]
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, cell-associated EBV DNA. Patients in continuous clinical remission are shown in Fig. 1 of the online Data Supplement.