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Journal of Clinical Oncology, Vol 24, No 34 (December 1), 2006: pp. 5414-5418 © 2006 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.07.7982 Plasma Epstein-Barr Viral Deoxyribonucleic Acid Quantitation Complements Tumor-Node-Metastasis Staging Prognostication in Nasopharyngeal Carcinoma
From the Department of Clinical Oncology, Sir YK Pao Centre for Cancer, Centre for Clinical Trials, and Department of Chemical Pathology, The Chinese University of Hong Kong, Hong Kong, People's Republic of China Address reprint requests to Anthony T.C. Chan, MD, Department of Clinical Oncology, Prince of Wales Hospital, Shatin, Hong Kong, China; e-mail: anthonytcchan{at}cuhk.edu.hk
PURPOSE: To evaluate the effect of combining circulating Epstein-Barr viral (EBV) DNA load data with TNM staging data in pretherapy prognostication of nasopharyngeal carcinoma (NPC). PATIENTS AND METHODS: Three hundred seventy-six patients with all stages of NPC were studied. Pretreatment plasma/serum EBV DNA concentrations were quantified by a polymerase chain reaction assay. Determinants of overall survival were assessed by multivariate analysis. Survival probabilities of patient groups, segregated by clinical stage (I, II, III, or IV) alone and also according to EBV DNA load (low or high), were compared. RESULTS: Pretherapy circulating EBV DNA load is an independent prognostic factor for overall survival in NPC. Patients with early-stage disease were segregated by EBV DNA levels into a poor-risk subgroup with survival similar to that of stage III disease and a good-risk subgroup with survival similar to stage I disease. CONCLUSION: Pretherapy circulating EBV DNA load is an independent prognostic factor to International Union Against Cancer (UICC) staging in NPC. Combined interpretation of EBV DNA data with UICC staging data leads to alteration of risk definition of patient subsets, with improved risk discrimination in early-stage disease. Validation studies are awaited.
Although the concept of incorporation of biomarkers as an integral part of cancer staging is appealing, this has only been realized for protein markers in the case of testicular germ cell tumors and gestational trophoblastic tumors.1 The detection of tumor-derived genetic materials, such as tumor-associated DNA, in the peripheral circulation has opened up new possibilities of tumor monitoring.2 To date, the most mature example of such application is the use of circulating Epstein-Barr viral (EBV) DNA in monitoring of nasopharyngeal carcinoma (NPC).3 The marker is detectable in as much as more than 95% of patients with NPC at diagnosis by quantitative polymerase chain reaction (q-PCR) systems.4,5 A cumulating body of data suggests that the quantified marker reflects tumor burden and is useful for monitoring of progress of the disease.4,6,7 These observations suggest that EBV DNA data could be of value in pretherapy risk stratification and cancer staging. The previous studies on the prognostic value of pretherapy EBV DNA were linked to clinical outcome in the early follow-up period6-9; the typical median follow-up time in those studies was approximately 2 years. The present study addresses the prognostic effect of combining EBV DNA and International Union Against Cancer (UICC) staging data with respect to overall survival at longer term follow-up, with special focus on whether the risk definition of patient subsets would be altered in a manner that may lead to change in therapy decisions.
Patients This study combines the data from two cohorts of patients in two tumor marker studies. The consideration for combining two cohorts of patients is to maximize the sample size for a prognostication study. The first cohort of patients comprised 133 patients recruited in the period from 1993 to 1994 (cohort A), and the second cohort comprised 243 patients recruited in the period from 1997 to 2000 (cohort B). Both cohorts were staged and managed with consistent work-up and therapy protocols. In both cohorts, consecutive consenting patients with all stages of NPC were recruited at the respective periods before commencement of oncologic treatment, from the oncology clinic of the authors' institution. The characteristics of the two cohorts were comparable (Table 1), apart from use of chemotherapy in a small proportion of patients in cohort B, mostly within a clinical trial setting (also see Oncologic Treatment). The studies were approved by the institution's clinical research ethics committee, and written informed consent was obtained from all patients at recruitment.
Clinical Staging Protocol TNM stage designation was according to the definitions of the fifth (1997) edition of the UICC–American Joint Committee on Cancer staging criteria.10,11 All patients in both cohorts were subject to the same staging protocol as follows. Tumor stage was determined by computed tomography of the nasopharynx and skull base, nasoendoscopy, and cranial nerve examination. Node stage was determined by clinical palpation (size and location of all nodes were routinely recorded on purpose-specific diagrams, allowing the assignment of 1997 node stage for patients recruited before 1997). To maintain consistency in staging in this study, all patients were restaged by the 1997 staging criteria excluding any available magnetic resonance imaging information because magnetic resonance imaging had been performed for some but not all patients. Chest radiograph and serum alkaline phosphatase level were obtained. Imaging screening for distant metastases was performed for patients with N3b stage disease by bone scintigram and upper abdominal imaging. Patients with distant metastatic disease at presentation (M1 stage) were excluded from the present study. The stage distribution within the two cohorts is detailed in Table 1. The histologic subtypes of the tumors were WHO type II or III12 in all patients, except for two patients with type I tumor.
Oncologic Treatment
Plasma EBV DNA Assay
Clinical Outcome Assessment
Statistical Analysis
The median follow-up time of the combined cohort was 5.8 years.
Overall Survival The 5-year survival probabilities of different clinical stages are listed in Table 2, and the corresponding Kaplan-Meier plots are shown in Figure 1. A trend of increasing prognostic segregation between adjacent stages was noted with advancing clinical stage. The difference in survival probabilities between stages I and II was not significant (P = .27), the difference between stages II and III was marginal (P = .05), and the difference between stages III and IV was significant (P = .0002).
Survival probabilities of patient subgroups defined by low and high EBV DNA levels, with respect to early-stage disease (stages I and II) and advanced-stage disease (stages III and IV) are also listed in Table 2. Within stages I and II disease, high EBV DNA levels strongly predict inferior survival (P = .0003), in contrast to the lack of significant discrimination by clinical stage alone (P = .27). The high EBV DNA group, which included 6% of patients with stage I disease and 38% of patients with stage II disease, had a survival probability similar, if not inferior, to that of stage III patients (Table 2). The low EBV DNA group, which included most patients with stage II disease, had a survival probability similar to stage I patients. Figure 2 shows the Kaplan-Meier plots of these two groups of patients segregated by EBV DNA levels in stages I and II disease. Analysis on stage II disease alone gave the same conclusions. The 5-year survival rates for the low and high EBV DNA groups in stage II disease were 90% (95% CI, 82% to 97%) and 63% (95% CI, 48% to 77%), respectively. Analysis of stage I disease alone would not be meaningful in view of the very small number of patients with high EBV DNA levels with stage I disease. Within stages III and IV disease, high EBV DNA level also predicts inferior survival (P = .03), but the discrimination was not more powerful than that by UICC stage alone (P = .0002). Analysis on stage III disease alone and on stage IV disease alone did not show significant differences in survival between the high and low EBV DNA groups.
DSS All analyses were repeated using DSS as the end point, and the same conclusions as for overall survival were obtained (data not shown).
DMFS
LFFS
The present study examined two attributes of a biomarker that are prerequisites for consideration of integration with TNM staging. First, the marker should be an independent determinant to TNM staging in predicting survival on long-term follow-up. Second, the combined biomarker and staging data should allow alteration in risk definition of patient groups that have bearing on management decisions.14 The present study is the first one to confirm these attributes with reference to long-term clinical follow-up data for EBV DNA as a biomarker for NPC. The current therapy for NPC is dichotomized between poor-risk disease, which is commonly defined as stages III and IV disease and treated with chemoradiotherapy,15 and good-risk disease, which is treated by radiation therapy alone. The present study on NPC is based on data in an era when combined chemoradiotherapy was only applied to a small subset of patients within an investigative protocol,13 rather than as standard therapy for advanced-stage disease. The failure to demonstrate the prognostic impact of chemotherapy in the present study is probably a result of the limited statistical power associated with the small number of patients who received chemotherapy treatment within a study including all stages of NPC. The publication of results of confirmatory randomized trials13,16,17 and meta-analyses18,19 in recent years has led to adoption of combined chemoradiotherapy as standard therapy for patients with stage III and IV disease. The optimal treatment for patients with stage II disease, an intermediate-risk group,20 is less well defined, partly because of different versions of stage classification for advanced-stage disease used in the different randomized trials. The value of a biomarker for better characterization of risks within early-stage disease is obvious. The results of the present study suggest that the majority of stage II patients belong to a good-risk group with high survival probability after treatment by radiation therapy alone, whereas a poor-risk group characterized by high EBV DNA load had a much less favorable prognosis. It is also worth noting that, with the introduction of IMRT in the treatment of NPC, a significant improvement in local tumor control in the early follow-up period has been observed.21 As a result, the pattern of recurrence is further skewed towards one predominated by distant metastatic recurrence.21 It is of interest to note that, although EBV DNA predicts overall survival, it seems to be a better prognosticator for distant metastatic recurrence than local recurrence in NPC.22 This has two implications in the IMRT-based chemoradiotherapy era. First, EBV DNA may assume even greater prognostic importance when the pattern of failure is distant failure dominated. Second, in future randomized trials on therapeutic options to reduce distant failure, a marker that has independent prognostic effect on survival to clinical stage and that is sensitive for distant failure prediction would be valuable to define prognostically uniform patient groups for entry into trials. These considerations are relevant to future review of the UICC staging system. Although the sample size of the present study was one of the largest reported for EBV DNA prognostication to date, the borderline prognostic discrimination between some clinical stages, such as between stages II and III, suggest that a even larger sample size is preferred for validation studies. In fact, previous studies on EBV DNA prognostication did not consistently show prognostic differences between clinical stages, very probably because of the smaller sample sizes involved. Data on longer follow-up would also be of interest to assess whether high EBV DNA loads could predict any late distant failure. We are looking towards the exciting prospect of successful validation that could lead to establishing the first example of a nucleic acid–based biomarker being integrated into cancer staging. Within and beyond the model of NPC, there has been an expanding body of data on the evaluation of other peripheral blood–based molecular markers, including genetic and epigenetic markers.23,24 With the accelerating pace of accumulation of such data, the biologic dimension is likely to assume increasing presence in cancer staging in the near future.
The authors indicated no potential conflicts of interest.
Supported in part by the Hong Kong Research Grants Council, the Kadoorie Charitable Foundation, and the Michael Kadoorie Cancer Genetics Research Programme. Presented in part at the American Association for Cancer Research International Conference on Molecular Diagnostics in Cancer Therapeutic Development, September 12-15, 2006, Chicago, IL. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
1. Sobin LH, Wittekind CH Gestational trophoblastic tumours, in: UICC TNM Classification of Malignant Tumours (ed 5). New York, NY, Wiley-Liss, 1997, pp 174-177 2. Nawroz H, Koch W, Anker P, et al: Microsatellite alterations in serum DNA of head and neck cancer patients. Nat Med 2:1035-1037, 1996[CrossRef][Medline] 3. Mutirangura A, Pornthanakasem W, Theamboonlers A, et al: Epstein-Barr viral DNA in serum of patients with nasopharyngeal carcinoma. Clin Cancer Res 4:665-669, 1998[Abstract] 4. Lo YMD, Chan LYS, Lo KW, et al: Quantitative analysis of cell-free Epstein-Barr virus DNA in plasma of patients with nasopharyngeal carcinoma. Cancer Res 59:1188-1191, 1999 5. Heid CA, Stevens J, Livak KJ, et al: Real time quantitative PCR. Genome Res 6:986-994, 1996 6. Chan AT, Lo YM, Zee B, et al: Plasma Epstein-Barr virus DNA and residual disease after radiotherapy for undifferentiated nasopharyngeal carcinoma. J Natl Cancer Inst 94:1614-1619, 2002 7. Lin JC, Wang WY, Chen KY, et al: Quantification of plasma Epstein-Barr virus DNA in patients with advanced nasopharyngeal carcinoma. N Engl J Med 350:2461-2470, 2004 8. Lo YM, Chan AT, Chan LY, et al: Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating Epstein-Barr virus DNA. Cancer Res 60:6878-6881, 2000 9. Leung SF, Chan AT, Zee B, et al: Pretherapy quantitative measurement of circulating Epstein-Barr virus DNA is predictive of posttherapy distant failure in patients with early-stage nasopharyngeal carcinoma of undifferentiated type. Cancer 98:288-291, 2003[CrossRef][Medline] 10. Sobin LH, Wittekind CH Pharynx, in: UICC TNM Classification of Malignant Tumours (ed 5). New York, NY, Wiley-Liss, 1997, pp 26-30 11. Fleming ID, Cooper JS, Henson DE, et al (eds): AJCC Cancer Staging Manual (ed 5). Philadelphia, PA, Lippincott-Raven, 1997, pp 31-39 12. Shanmugaratnam K, Sobin LH: World Health Organization. International Histological Classification: Histological Typing of Tumors of the Upper Respiratory Tract and Ear (ed 2). Berlin, Germany, Springer-Verlag, 1991 13. Chan AT, Leung SF, Ngan RK, et al: Overall survival after concurrent cisplatin-radiotherapy compared with radiotherapy alone in locoregionally advanced nasopharyngeal carcinoma. J Natl Cancer Inst 97:536-539, 2005 14. Sobin LH: TNM: Principles, history, and relation to other prognostic factors. Cancer 91(suppl):1589-1592, 2001[CrossRef][Medline] 15. Al-Sarraf M, LeBlanc M, Giri PG, et al: Chemoradiotherapy versus radiotherapy in patients with advanced nasopharyngeal cancer: Phase III randomized Intergroup study 0099. J Clin Oncol 16:1310-1317, 1998 16. Lin JC, Jan JS, Hsu CY, et al: Phase III study of concurrent chemoradiotherapy versus radiotherapy alone for advanced nasopharyngeal carcinoma: Positive effect on overall and progression-free survival. J Clin Oncol 21:631-637, 2003 17. Kwong DL, Sham JS, Au GK, et al: Concurrent and adjuvant chemotherapy for nasopharyngeal carcinoma: A factorial study. J Clin Oncol 22:2643-2653, 2004 18. Huncharek M, Kupelnick B: Combined chemoradiation versus radiation therapy alone in locally advanced nasopharyngeal carcinoma: Results of a meta-analysis of 1,528 patients from six randomized trials. Am J Clin Oncol 25:219-223, 2002[CrossRef][Medline] 19. Langendijk JA, Leemans CR, Buter J, et al: The additional value of chemotherapy to radiotherapy in locally advanced nasopharyngeal carcinoma: A meta-analysis of the published literature. J Clin Oncol 22:4604-4612, 2004 20. Chua DT, Sham JS, Kwong DL, et al: Treatment outcome after radiotherapy alone for patients with stage I-II nasopharyngeal carcinoma. Cancer 98:74-80, 2003[CrossRef][Medline] 21. Lee N, Xia P, Quivey JM, et al: Intensity-modulated radiotherapy in the treatment of nasopharyngeal carcinoma: An update of the UCSF experience. Int J Radiat Oncol Biol Phys 53:12-22, 2002[CrossRef][Medline] 22. Leung SF, Lo YM, Chan AT, et al: Disparity of sensitivities in detection of radiation-naive and postirradiation recurrent nasopharyngeal carcinoma of the undifferentiated type by quantitative analysis of circulating Epstein-Barr virus DNA1,2. Clin Cancer Res 9:3431-3434, 2003 23. Wong TS, Kwong DL, Sham JS, et al: Quantitative plasma hypermethylated DNA markers of undifferentiated nasopharyngeal carcinoma. Clin Cancer Res 10:2401-2406, 2004 24. Chang HW, Chan A, Kwong DL, et al: Evaluation of hypermethylated tumor suppressor genes as tumor markers in mouth and throat rinsing fluid, nasopharyngeal swab and peripheral blood of nasopharyngeal carcinoma patient. Int J Cancer 105:851-855, 2003[CrossRef][Medline] Submitted June 8, 2006; accepted September 20, 2006.
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Copyright © 2006 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
4,000 copies/mL), age, sex, and cohort identity (cohort A or B). Kaplan-Meier plots of overall survival were established for patient groups of different UICC stages (stages I to IV) and for patient subgroups with high and low EBV DNA levels within early (stages I and II) and advanced (stages III and IV) UICC stages. The consideration for segregating patient subgroups by EBV DNA within early and advanced stages separately is a pragmatic one, in view of the different therapies being used for early-stage and advanced-stage NPC. Any shift of risk groups across the therapeutic decision boundary would be of clinical relevance. Log-rank tests were performed to assess the difference in survival probabilities between patient subsets. Analyses were repeated for the following end points: DSS, DMFS, and LFFS. All statistical tests were two sided, and a P < .05 was considered to indicate statistical significance. Analyses were performed with the use of SAS software (version 8.2; SAS Institute, Cary, NC). 
