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© 2001 American Society for Clinical Oncology Detection of Epstein-Barr Virus DNA in the Peripheral-Blood Cells of Patients With Nasopharyngeal Carcinoma: Relationship to Distant Metastasis and SurvivalFrom the Institute of Clinical Medicine, College of Medicine, and Department of Biochemistry and Center for Cellular and Molecular Biology, School of Life Science, National Yang-Ming University; Department of Radiation Oncology, Taichung Veterans General Hospital; Taipei Veterans General Hospital, Taipei; Cancer Center, Department of Basic Medicine, Hung Kuang Institute of Technology; and School of Public Health, China Medical College, Taichung, Taiwan. Address reprint requests to Yau-Huei Wei, PhD, Department of Biochemistry and Center for Cellular and Molecular Biology, School of Life Science, National Yang-Ming University, No 155, Li-Nong St, Sec 2, Taipei, 112 Taiwan; email: joeman{at}ym.edu.tw
PURPOSE: Nasopharyngeal carcinoma (NPC) has been proved to be an Epstein-Barr virus (EBV)-associated cancer. By use of nested polymerase chain reactions (PCRs), we examined whether the presence of EBV DNA in the peripheral-blood cells (PBC) can serve as a prognostic indicator for NPC. PATIENTS AND METHODS: Peripheral blood from 124 patients with NPC who had no evidence of distant metastasis and 114 healthy volunteers with serologically positive findings for EBV infection was collected prospectively. Plasma and erythrocytes were separated. DNA was extracted from PBCs and analyzed by a nested PCR using primers specific to Epstein-Barr virus nuclear antigen 1 (EBNA-1). All patients were treated by radiotherapy with or without chemotherapy. Clinical parameters and status of EBNA-1 in PBCs were used for survival analysis using the Kaplan-Meier method and the Cox proportional hazards model. RESULTS: Positive rates of EBNA-1 DNA in PBCs of NPC patients and healthy volunteers are 71% and 14%, respectively (P = .001). No significant difference was observed with regard to the clinical characteristics of patients who were EBNA-1–positive (n = 88) and those who were EBNA-1–negative (n = 36). After a median follow-up period of 38 months (range, 24 to 56 months), 29 of 88 EBNA-1–positive patients and only one of 36 EBNA-1–negative patients developed distant metastases (P = .00015). Kaplan-Meier estimates of overall survival (P = .0010), metastasis-free survival (P = .0004), and progression-free survival (P = .0004) were significantly lower for the patients in the EBNA-1–positive group than for those in the EBNA-1–negative group. Multivariate Cox analysis confirmed the same results. CONCLUSION: The presence of EBNA-1 DNA in PBCs is a novel, important risk factor for patients with NPC that indicates a significantly higher risk of developing distant metastasis as well as a lower survival rate.
Nasopharyngeal carcinoma (NPC) is distinguished from other cancers of the head and neck by its epidemiology, histopathology, clinical characteristics, and therapy. It is a geographically endemic, Epstein-Barr virus–associated carcinoma of epidermoid origin. It has poorly differentiated or undifferentiated pathology with a higher incidence of neck lymph node metastasis and greater radiosensitivity and chemosensitivity. Radiotherapy is the primary treatment of NPC. Recent trials support the use of concomitant chemoradiotherapy for patients with advanced NPC.1-3 Treatment failures in the past have been due to high rates of local recurrence and distant metastasis. Because of recent advances in radiation oncology, however, the failure pattern has changed to predominantly distant metastasis.1-8 Distant metastasis has been demonstrated to be the most important determinant of patient survival. Although systemic chemotherapy usually is recommended for patients with distant metastasis, the cure rate for patients with metastatic NPC is very low, with a 2-year survival rate of less than 10%.9,10 The effect of chemotherapy is proportional to the tumor burden. Careful selection of high-risk patients who need aggressive early treatment may improve treatment outcome. Because the clinical staging system cannot fully reflect the prognosis of patients with NPC, recent studies have focused on seeking helpful biologic predictors by using basic technology. NPC has been proved as an EBV-associated cancer for a long time. It has been demonstrated that EBV is harbored in almost every NPC tumor, regardless of the degree of differentiation and geographic distribution.11-14 Using Epstein-Barr virus nuclear antigen 1 (EBNA-1) as a target gene, we established a simple, nested PCR assay to detect EBV DNA from peripheral-blood cells (PBCs) in 124 patients with NPC. We investigated the relationship between the presence of EBV DNA in PBCs, distant metastasis, and patient survival.
Patients A total of 124 patients with biopsy-proved NPC (121 previously untreated patients and three with local relapse) and no evidence of distant metastasis were enrolled onto this prospective study between April 1996 and November 1998. The routine staging work-up included a clinical examination of the head and neck region, fiber nasopharyngoscopy, a computed tomographic or magnetic resonance imaging scan from the skull base to the whole neck, chest radiography, a whole-body bone scan, an abdominal sonogram, a complete blood count with differential count, a platelet count, a biochemical profile, and an EBV serology. Chest computed tomographic scans and bone marrow biopsies were not routine but were selectively performed when lung metastasis was suspected on the basis of chest x-ray and an abnormal blood routine was noted. The patient’s cancer stage was defined according to the 1992 American Joint Committee on Cancer TNM staging system.15 In addition, 114 healthy volunteers were recruited to serve as control subjects. Informed consent was obtained from each individual.
Clinical Treatment
Blood Processing and DNA Extraction
Oligonucleotide Primers
Nested Polymerase Chain Reaction (PCR)
Quality Control and Evaluation Criteria
Statistical Analysis
Life-table estimation was done according to the method of Kaplan-Meier.16 Univariate comparison of survival curves was performed by use of the log-rank test.17 The multivariate Cox proportional hazards model was used to estimate the hazard ratios and 95% confidence intervals.18 Variables in the model included sex, age, performance status, pathologic type, T stage, N stage, and EBNA-1 status in PBCs. The statistical significance of association between EBNA-1 status in PBCs and characteristics of the patients was assessed by using
Detection System The first round and nested PCRs for the EBNA-1 gene yielded 609 base pair (bp) and 308 bp products ( Figs 1A and 1B), respectively. The sensitivity test of our nested PCR system demonstrated that the EBNA-1 gene could be detected at the 0.1-pg level by visual examination of the PCR product in the gel stained with ethidium bromide (Fig 1C). Similar results were obtained in two independent experiments. Sequencing of the PCR products from randomly selected positive samples confirmed the presence of EBNA-1 gene in all tested samples (data not shown). The detection rates of EBNA-1 DNA in PBCs from patients with NPC and healthy volunteers were 71% (88 of 124) and 14% (16 of 114), respectively (P = .001; odds ratio, 14.97).
Patient Characteristics Table 1 lists the patient characteristics according to the presence or absence of EBNA-1 DNA in PBCs. We observed no significant differences in these characteristics between the groups with regard to age, sex, performance status, pathologic type, T stage, N stage, overall stage, mode of radiotherapy, and adjuvant chemotherapy.
Follow-Up Outcomes The median follow-up was 38 months (range, 24 to 56 months). We recorded 35 treatment failures: five primary recurrence alone, 28 distant metastasis alone, and two combined primary recurrence and distant metastasis. Twenty-nine (33.0%) of 88 EBNA-1–positive patients were proved to have developed distant metastasis later, whereas only one patient with distant metastasis was found among 36 EBNA-1–negative patients (P = .00015; odds ratio, 17.2) ( Fig 2). Among 85 patients at the N2/N3 stage, 28 patients (32.9%) developed distant metastasis, whereas only two of 39 patients at the N0/N1 stage developed distant metastases (P = .00055; odds ratio, 9.08). Among 62 patients who were both EBNA-1 DNA-positive and at the N2/N3 stage, 27 patients (43.6%) developed distant metastases. Thus, the combined presentation of EBNA-1 DNA and N2/N3 stage may better predict distant metastasis of NPC than either of these presentations alone.
Survival Analysis Kaplan-Meier estimates demonstrated that both EBNA-1 status and N stage significantly affected patients’ metastasis-free survival, overall survival, and progression-free survival rates. The 3-year overall survival rates for EBNA-1–negative and EBNA-1–positive patients were 97.2% and 65.5%, respectively (P = .0010) ( Fig 3A). The metastasis-free survival rates at 3-year follow-up was 97.1% for those who had a negative EBNA-1 finding and 64.6% for those who were EBNA-1–positive (P = .0004) (Fig 3B). The 3-year progression-free survival rates for EBNA-1–negative and EBNA-1–positive patients were 94.3 and 59.7%, respectively (P = .0004) (Fig 3C). The overall survival, metastasis-free survival, and progression-free survival rates of patients at the N0/N1 stage were significantly better than those of patients at the N2/N3 stage (89.2% v 67.7%, P = .0132, Fig 4A; 94.6% v 64.7%, P = .0007, Fig 4B; and 91.8% v 59.7%, P = .0005, Fig 4C, respectively).
Cox Multivariate Analysis Results similar to those reported above were obtained using multivariate analysis by the Cox proportional hazards model ( Table 2). EBNA-1 status, N stage, and performance status were significant prognostic factors in metastasis-free survival, overall survival, and progression-free survival rates after adjustment for other variables.
N Stage Versus EBNA-1 Status in the Prediction of Subsequent Distant Metastasis Because 62 (70.5%) of 88 EBNA-1–positive patients were at the N2/N3 stage and 23 (63.9%) of 36 EBNA-1–negative patients were at the N2/N3 stage, the presence of EBNA-1 DNA could not be used to predict nodal staging (P = .475,  2 test). Among 85 patients at the N2/N3 stage, the crude prediction rates of subsequent distant metastasis for EBNA-1–positive patients and EBNA-1–negative patients were 43.6% (27 of 62) and 4.4% (one of 23), respectively (P < .001, 2 test). Thus, EBNA-1 status does predict which nodal patients will relapse distantly. When we modified multivariate models such as the Cox survival model or logistic regression analysis by stepwise forward selection criteria, the variable of EBNA-1–positive status entered the model first, followed by N2/N3 stage. Hence, N stage is not a better predictor of distant metastasis than EBNA-1–positive status according to the results of statistical analyses. We may say that nodal positivity may predict distant metastasis as well as but not better than EBNA-1–positive status does. By considering both EBNA-1–positive status and N2/N3 stage, one may better predict distant metastasis in patients with NPC.
Survival Analysis of 82 Patients Who Received Concurrent Chemoradiotherapy
With the recent advances in molecular biology, some useful biologic markers have been identified to predict prognosis and aid in clinical oncologic decision making in the following regards: tumors with greater angiogenesis have higher rates of distant metastasis19,20; the presence of occult cytokeratin-positive cells in bone marrow increases the risk of relapse in breast cancers21; and tumor suppressor gene p53 alterations predict tumor response to neoadjuvant chemotherapy in head and neck cancers.22 These molecular diagnostic methods will improve the validity of clinical staging in the future. The etiologic factors identified for NPC include EBV, environmental risk factors, and genetic susceptibility. A close association between EBV and NPC has been established previously by the following findings: the presence of DNA, RNA, and proteins of EBV in almost all cells of almost all tissue samples of patients with NPC,11-14 evidence that tumor cells are clones derived from a single EBV-infected cell,23 and high levels of EBV protein antibodies in healthy individuals in whom NPC later develops and in patients with primary or recurrent carcinoma. EBV also has been detected in premalignant (preinvasive) nasopharyngeal lesions, including carcinoma in situ and dysplasia.24 Latent infection by EBV does not occur in normal nasopharyngeal epithelial cells, however.25 The PCR-based technique makes it possible to detect a very small amount of biomolecules in a wide array of biologic samples. Using the EBNA-1 gene as a target, we established a nested PCR system to detect EBV DNA in pretreatment PBCs of patients with NPC and evaluated its clinical significance. Because the PCR-based technique is very sensitive, a critical issue is the judgment regarding a positive result. If the relative proportion of the target molecule is abundant, repeated tests will always produce positive results. When the relative proportion of the target molecule is below the detection threshold, however, all duplicate tests will be negative. Inconsistent results of several tests from the same samples are possible if the relative proportion of the target molecule is within a certain range. In our study, we considered the final outcome to be positive if at least one of several tests from the same sample was positive for the target gene. This concept has seldom been discussed in the field of molecular detection when inconsistent results of replicate tests have occurred, even though it is a generally accepted rule of pathologic diagnosis. For example, if two or more slices were taken from a resected lymph node in a cancer patient, the result would be reported as lymph node metastasis if any one of the slices were positive. This result would be justified if the experiments were performed carefully to avoid contamination. According to our experience, the amount of extracted DNA from the PBC pellets of 10 mL blood is between 0.1 and 1.0 mg, with an average yield of 0.5 mg. Only 50 ng DNA is needed for each PCR test under our assay condition. Thus, each patient sample is enough for 10,000 PCR tests. If the final result of qualitative PCR testing is obtained from one or two tests, false negatives may occur. During our experimental setup, DNA extracted from each of the blood samples of 114 healthy volunteers who were serologically positive for EBV infection was assayed five times. The cumulative positive rates increased as the number of individuals tested increased: 0% in one test, 3.5% in duplicate tests, 11.4% in triplicate tests, 13.2% in four tests, and 14.0% in five tests, respectively. From the trend of increasing detection rates, we judged that at least three tests should be performed for each sample to avoid sampling error. Too many tests are unnecessary and time-consuming, however. We thus performed five tests on the blood sample of each patient. Under our assay system, the detection rate for EBNA-1 was significantly higher in patients with NPC than that in healthy control subjects. This finding is further evidence that EBV is closely associated with NPC. In Taiwan, almost 100% of adults have been infected by EBV, which may establish a persistent infection in the host. Recent studies have demonstrated that EBV-infected cells in vivo are B cells with an inactivated phenotype.26-28 These resting B cells are the major site of latent infection and are important in the dissemination of infection to distal epithelial surfaces. Using a PCR and modified Gardella gel system, Decker et al.27 demonstrated that intact, full-length episomal EBV DNA was detected in PBCs of all five healthy donors. The frequency of EBV-carrying cells in PBCs of normal donors ranged from 1 in 2 x 105 to 107 whole mononuclear cells or from 1 in 2 x 104 to 106 B cells. Using quantitative, competitive PCRs, the EBV genome copy number in latently infected adults was estimated to be less than 0.1 copy/105 lymphocytes.29 Stevens et al30 found that 46% of PBC samples of healthy donors were EBV DNA-positive by quantitative, competitive PCR testing. In most samples, viral load was less than 2000 EBV copies/mL blood. The variations in EBV DNA detection rates reported by different groups may be ascribed to the use of different assay systems. Molecular detection of EBV genes in PBCs or in the sera or plasma of patients with NPC is rarely reported in the literature. Using capsid protein gp220 (Bam HI L region) as a target and 30-cycle PCR, Kuo et al31 reported that EBV DNA was detected in 91.3% of tumor tissue from 69 patients with NPC and in 16.7% of nasopharyngeal tissue from 18 healthy individuals. Nevertheless, no EBV DNA was detected in the mononuclear cells of PBC in 69 patients with NPC and 18 normal control subjects. Using EBNA-2 as a target gene and 40-cycle PCR, Mutirangura et al32 demonstrated that 13 (31%) of 42 patients with NPC were positive for EBV DNA in their sera and that all 82 normal control subjects were negative. They deduced that EBV DNA in serum can originate from dead NPC cells. In a later study, they demonstrated higher sensitivity (58.7%) in the sera or plasma of 167 patients with NPC by nested PCR testing, but 10 (13%) of 77 normal blood donors in that study had positive results.33 Using primers designed from either the Bam HI-W region or the EBNA-1 gene, Lo et al34 detected cell-free EBV DNA in plasma by real-time quantitative PCR testing in 55 (96%) of 57 patients with NPC and in three (7%) of 43 control subjects. In a series of studies, they demonstrated that circulating EBV DNA levels correlated with NPC staging,35 tumor recurrence,36 and patient survival.37 Their results suggest that EBV DNA in serum or plasma can serve as a good tumor marker for the detection and monitoring of NPC. Instead of using serum/plasma samples, we used PBC DNA for EBV detection. In our study, 71% of NPC patients had EBV DNA in PBCs. These EBV-carrying cells may arise from disseminated cancer cells or from resting B cells latently infected by EBV. Kaplan-Meier survival analysis and Cox multivariate analysis demonstrated that EBNA-1 status in PBCs is an important independent prognostic factor for metastasis-free, progression-free, and overall survival rates. Follow-up data pointed to the presence of EBV DNA in PBCs as an ominous predictor of subsequent distant metastasis. In order to define the origin of the EBV DNA we detected, whole blood was collected from 10 patients with newly diagnosed NPC. Mononuclear cells were isolated by Ficoll-Hypaque density-gradient centrifugation and then subjected to magnetic cell separation for B-cell isolation (Miltenyi Biotec, Bergisch Gladbach, Germany). We obtained untouched B cells by the magnetic depletion of non-B cells. Using primers specific to the Bam HI-W region34 and DNA extraction from B cell and non-B cell fractions, we performed real-time quantitative PCR and found no detectable DNA after 40 cycles of amplification. By qualitative nested PCR developed in this study, 6 of 10 patients were found to be EBNA-1-positive in non-B-cell DNA and EBNA-1-negative in B-cell DNA. One patient was EBNA-1-positive in both non-B-cell and B-cell fractions. The remaining three patients were negative in both non-B-cell DNA and B-cell DNA. We concluded that the major source of viral DNA detected in PBCs was circulating tumor cells. We further compared EBNA-1 sequence variation from fresh frozen primary tumor tissue with PBC DNA. Direct sequencing of nested PCR products from both primary tumors and PBCs revealed the match mutation pattern in codons 487, 499, 502, 524, 528, and 533 (V-val variant subtype) as compared with the sequence of B95-8 cell line (P-ala prototype). The clinical analyses demonstrated that patients with NPC who had EBV DNA in their PBCs were at significantly higher risk of developing distant metastasis as determined from the results of either all 124 patients or the 82 patients who received uniform concurrent chemoradiotherapy. Except for well-established prognostic factors (N stage and performance status), the results of our study clearly identified a novel biologic factor that contributes to the clinical management of patients with NPC. After median follow-up of 38 months, 29 (33.0%) of 88 patients with detectable EBNA-1 DNA in their PBCs developed distant metastasis. According the 1992 American Joint Committee on Cancer TNM staging system15 and our own clinical experience (unpublished data), we may be able to designate a group of patients with NPC who are at high risk of distant failure if one of the following criteria is met: (1) N3 patients (nodal size greater than 6 cm), (2) supraclavicular node metastasis, (3) T4N2 disease, (4) N2 disease with at least one nodal size greater than 4 cm, or (5) residual disease after radiotherapy. Among the 124 patients with NPC who were enrolled in this study, combining both EBNA-1 DNA positivity and these clinical factors, more than 50% of patients (27 of 47) are expected to fail at distant site(s) soon. Thus, we recommend that these patients be started on aggressive systemic chemotherapy. For patients with little possibility of distant failure, systemic chemotherapy should be prohibited to avoid unnecessary morbidity and waste of medical resources.
Supported by grants NSC89-2320-B-075A-007-M08 and NSC89-2316-B-010-012 from the National Science Council, Taiwan, Republic of China.
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Copyright © 2001 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
, patients with distant metastasis.
N2, M0) undifferentiated nasopharyngeal carcinoma: A positive effect on progression-free survival—International Nasopharynx Cancer Study Group: VUMCA I trial. Int J Radiat Oncol Biol Phys 35:463-469, 1996
