This work was supported by the Hong Kong UGC Area of Excellence (AoE) scheme.
Head and Neck
The role of plasma Epstein-Barr virus DNA in the management of recurrent nasopharyngeal carcinoma
Article first published online: 29 MAY 2013
© 2013 The American Laryngological, Rhinological and Otological Society, Inc.
Volume 124, Issue 1, pages 126–130, January 2014
How to Cite
Chan, J. Y. W. and Wong, S. T.-S. (2014), The role of plasma Epstein-Barr virus DNA in the management of recurrent nasopharyngeal carcinoma. The Laryngoscope, 124: 126–130. doi: 10.1002/lary.24193
The authors have no other funding, financial relationships, or conflicts of interest to disclose.
- Issue published online: 20 DEC 2013
- Article first published online: 29 MAY 2013
- Accepted manuscript online: 20 MAY 2013 02:59AM EST
- Manuscript Revised: 18 APR 2013
- Manuscript Accepted: 18 APR 2013
- Plasma Epstein-Barr virus DNA;
- recurrent nasopharyngeal carcinoma;
- local recurrence;
- systemic metastasis
To study the role of plasma Epstein-Barr virus (EBV) DNA in patients with recurrent nasopharyngeal carcinoma (NPC) after previous chemoradiation.
Sixty patients with recurrent NPC were recruited, and their plasma EBV DNA was checked preoperatively, 1 week postoperatively, and 6 months thereafter. In a pilot group of 30 patients, further testing was performed at 60 minutes after tumor resection. The plasma EBV DNA level was correlated with tumor T classification, resection margin status, and subsequent relapse.
The mean preoperative plasma EBV DNA reflected the tumor load (T1: 48 copies/mL, T2: 316 copies/mL, T3: 890 copies/mL, P = .03). It was significantly higher in patients with positive margins at the time of surgery (722 vs. 126 copies/mL, P = .02) and in those with subsequent systemic metastasis (668 vs. 92 copies/mL, P = .01). However, it failed to predict local recurrence after surgery. Postoperative plasma EBV DNA was undetectable in all patients with positive resection margins. Serial measurements were able to identify 87.5% of local recurrences and 100% of distance metastases.
In patients with recurrent NPC requiring salvage nasopharyngectomy, preoperative plasma EBV DNA identifies patients at high-risk of subsequent distant failure after surgery. Serial measurements of plasma EBV DNA after surgery, especially for those with high preoperative levels, is crucial to allow early detection of local of distant failure. Laryngoscope, 124:126–130, 2014
Nasopharyngeal carcinoma (NPC) differs from other head and neck malignancies in its epidemiology, pathology, and treatment outcome.[1, 2] It is endemic in southern China and Southeast Asia, affecting 10 to 50 per 100,000 people per year. The primary treatment is radiotherapy or concurrent chemoradiation depending on the stage of disease on presentation,[4-7] whereas surgery is reserved for persistent or recurrent tumors after the initial therapy. Over the years, we have been advocating the use of the maxillary swing approach, which provides spacious access to the nasopharynx, allowing resection of tumors with wide margins. In the past 2 decades, we have performed more than 300 salvage nasopharyngectomies using this approach for patients with recurrent NPC. Previously, we have reported our experience in the use of preoperative plasma Epstein-Barr Virus (EBV) DNA level and the fluorine-18 fluorodeoxyglucose (18F-FDG) positron-emission tomography (PET)-computed tomography (CT) scan to predict the chance of curative resection during surgery. The aim of the current study was to investigate the role of plasma EBV DNA level as a prognostic factor for the oncological outcome of surgery.
MATERIALS AND METHODS
The study was approved by the ethics committee of the institutional review board of the University of Hong Kong. Between September 2007 and September 2010, we recruited patients who had nasopharyngectomy performed for recurrent NPC after previous chemoradiation. Some of these patients represented the same cohort in our previous published study on the use of preoperative plasma EBV DNA and PET scan. Before surgery, fiber optic nasopharyngoscopy with nasopharyngeal biopsy was performed to confirm the diagnosis and the EBV encoded RNA (EBER) status. Contrast magnetic resonance imaging (MRI) from the skull base down to the neck was performed to delineate the locoregional extent of the disease. Systemic workup was achieved by contrast CT imaging of the thorax and abdomen and bone scintigraphy. In recent years, whole body 18F-FDG PET-CT scan became the investigation of choice for such purpose.
Preoperative liver/renal function test and plasma EBV DNA level was checked on the day before surgery. Blood (5 mL) was collected and stored in ethylenediaminepentaacetic acid bottles. The blood samples were then centrifuged, after which the plasma portion was removed and stored at −20°C. Plasma EBV DNA copy numbers were measured with real-time quantitative polymerase chain reaction (PCR). This was used to identify the Bam HI-W fragment region of the EBV genome. Patients with tumor positive for EBER but negative for plasma EBV DNA were excluded from the study.
All patients had nasopharyngectomy performed subsequently via the maxillary swing approach. The procedure had been described previously,[12-15] with a few modifications over the years. Through a Weber-Ferguson-Longmire incision, osteotomies were made on the ipsilateral maxilla, which was swung out to gain access to the tumor. Nasopharyngectomy was then performed under direct vision, removing the tumor en bloc with the medial pterygoid muscles, the posterior part of the nasal septum, and the whole posterior wall of the nasopharynx up to the medial edge of the contralateral fossa of Rosenmüller. Further resection was guided by intraoperative frozen section results, until either all the margins were free of tumor or no further resection was possible. The maxillary osteocutaneous unit was then returned and fixed with titanium miniplates and screws. The formal paraffin histology report would be reviewed with the oncologists. For patients with microscopic tumor involvement of the resection margins, they would be considered for further adjuvant treatment.
All patients were followed up regularly at the outpatient clinic, during which clinical assessment and endoscopic examination of the nasopharynx would be performed. For the first year after surgery, the patients were followed up monthly, which would then be spaced out progressively afterward. For the first 30 patients in the series, plasma EBV DNA concentration was measured intraoperatively 1 hour after delivery of the tumor. However, as the plasma EBV DNA level in the majority of this pilot group of patients was not measurable at this time point, intraoperative testing was not repeated in the rest of the study patients. All subjects had plasma EBV DNA concentration checked 1 week after surgery and then every 6 months afterward. MRI of the nasopharynx was performed 6 months after surgery. If tumor recurrence were suspected, endoscopic examination and biopsy for nasopharyngeal lesion or ultrasound-guided fine needle aspiration of cervical lymphadenopathy would be performed. Whole-body 18F-FDG PET-CT scan was used when systemic metastasis was suspected.
Patients' data were retrieved from the head and neck cancer database at the Division of Head and Neck Surgery, Department of Surgery, Queen Mary Hospital, University of Hong Kong. One-way analysis of variance was used to compare the mean plasma EBV DNA among patients with different T classifications of the recurrent tumors before surgery. The pre- and postoperative plasma EBV DNA level was compared between patients with positive and negative resection margins, as well as those with subsequent tumor recurrence. Data were analyzed with Statistical Package for Social Sciences version 18.0 (SPSS, Inc., Chicago IL). A P value of .05 or less was considered as significant.
During the study period, 71 consecutive patients presented to us for management of local recurrence of NPC after previous radiotherapy. All tumors were undifferentiated carcinoma (World Health Organization type III), and they were EBER positive. Preoperative plasma EBV DNA was negative (0 copies/mL) in 11 (15.5%) patients despite a positive tumor EBER status. These patients were excluded from the study. Among the remaining 60 patients, 48 (80%) patients were male. The median age of our patients was 52.5 years (range, 28–71 years). All subjects had biochemically normal liver and renal functions. All patients had previous radiotherapy alone or chemoradiation, 14 for American Joint Committee on Cancer (6th edition) stage I, 25 for stage II, 19 for stage III, and two for stage IV disease on initial presentation, and all had clinically complete response. The median time from the completion of radiotherapy to the detection of tumor recurrence was 22.6 months.
All patients in the study had isolated tumor recurrence locally in the nasopharynx. None of them had regional or systemic metastasis found during preoperative workup. The T classification of the recurrent tumor on presentation was: T1 in 14 patients, T2 in 34 patients, and T3 in 12 patients. The quantity of plasma EBV DNA detected before surgery ranged from 18 to 1,042 copies/mL, with a median of 348 copies/mL. The mean preoperative plasma EBV DNA differed significantly among patients with different T classifications (Table 1). All the patients had subsequent nasopharyngectomy performed via the maxillary swing approach. Macroscopic tumor removal was achieved in all patients, although microscopic tumor clearance was possible only in 49 (81.7%) patients. All patients recovered from their surgery uneventfully, and the mean hospital stay was 8.2 days. There was no in-hospital mortality. In patients with positive resection margins (R1 resection), eight patients received further chemoradiation, which was started after a mean of 6.2 weeks after surgery.
|T1, n = 14||T2, n = 34||T3, n = 12||P Value|
|Mean preoperative plasma EBV DNA, copies/mL||48||316||890||.03|
Table 2 summarizes the results of the pre- and postoperative plasma EBV DNA in patients with positive and negative resection margins, showing that subjects with R1 resection had significantly higher preoperative plasma EBV DNA concentration than those with R0 resection (722 copies/mL vs. 126 copies/mL, P = .02). Among the 30 patients with blood taken at 1 hour after tumor delivery, only one patient had measurable plasma EBV DNA level (4 copies/mL). None of our patients had a measurable circulating EBV DNA level at 1 week after surgery, irrespective of the status of resection (R0 or R1). The mean follow-up duration was 36.8 months. Eight (13.3%) patients subsequently developed local tumor recurrence in the nasopharynx after maxillary swing nasopharyngectomy, among whom seven (87.5%) patients presented with elevated plasma EBV DNA level (mean, 208 copies/mL) at the time of diagnosis. None of our patients suffered from regional failure, but 10 patients were later found to have distant metastasis. Six patients had liver metastasis, two patients had bone metastasis, and two patients had multiple metastases in the bone, liver, and lung, and all of them had raised plasma EBV DNA level (mean, 621 copies/mL). The preoperative plasma EBV DNA levels in patients with local tumor recurrence and distant metastasis were shown in Table 3. There was no significant difference in the preoperative plasma EBV DNA concentration between patients who subsequently developed local recurrence and those who remained in remission (148 copies/mL vs. 92 copies/mL, P = .10). On the other hand, it was significantly higher in patients with subsequent distant metastasis (P = .01).
|Mean Plasma EBV DNA Concentration (Copies/mL)|
|Preoperatively, n = 60||Intraoperatively, n = 30||1 Week Postoperatively, n = 60|
|In Remission, n = 42||Local Recurrence, n = 8||Distant Metastasis, n = 10|
|Mean preoperative plasma EBV DNA, copies/mL||92||148||688*|
Despite the apparent improved outcome of treatment for NPC with intensity-modulated radiotherapy and concurrent chemoradiation,[5-7] the treatment of persistent or recurrent disease remains challenging. Surgical salvage offers better local tumor control and survival[18-23] as well as less post-treatment morbidities than reirradiation.[24-26]
Over the years, we have been using the anterolateral, or the maxillary swing, approach for salvage nasopharyngectomy. By swinging the maxillary antrum laterally, not only the ipsilateral nasopharynx is visualized clearly, the contralateral side can also by approached by removing the posterior part of the nasal septum. With such an adequate exposure, the best oncological outcome can be achieved by en bloc removal of the tumor with wide margins. With improving experience, locally advanced tumor, including those with significant parapharyngeal space invasion and petrosal internal carotid artery encasement, as well as those with skull base erosion, can now be resected with the combined craniofacial approach. There has been no hospital mortality, and the postoperative morbidity was acceptable as shown by our previous quality-of-life study.
In endemic areas such as southern China including Hong Kong, NPC is strongly associated with EBV infection, as evidenced by the raised levels of antibodies against EBV in most patients with NPC, the presence of EBV DNA/RNA in tumor cells, and the finding of EBV in a clonal episomal form and precursor lesion of NPC. Mutirangura et al. demonstrated a correlation between apoptosis in tumor tissue and the presence of EBV DNA in the serum of patients with NPC. Lin et al. further demonstrated that peripheral blood cell free EBV DNA is derived from cancer cells, not from inflammatory cells. Since the description of plasma EBV DNA quantitation by real-time quantitative PCR,[11, 31-33] it has been advocated as a sensitive and reliable biomarker for the diagnosis of NPC at a molecular level.[34-37] Numerous studies have investigated the role of plasma EBV DNA in screening[38, 39] and as a marker for early detection, monitoring and prognostic prediction for previously untreated NPC.[40-42] Hou et al. showed that pretreatment plasma EBV DNA level was significantly associated with tumor volume, and the corresponding post-treatment value predicted distant metastasis and overall survival. Hsu et al. and Wang et al. demonstrated that high pretreatment plasma EBV DNA level was related to poor oncological response after chemoradiation for metastatic NPC. Ferrari et al. showed that prechemoradiation circulating EBV DNA significantly correlated with tumor volume and the probability of subsequence tumor recurrence in a Western population.
The role of plasma EBV DNA in the prediction of surgical outcome for recurrent NPC, however, is not fully investigated. The current study represents the first report in the literature investigating the relationship between the pre- and postoperative plasma EBV DNA concentration and the surgical outcome, including local tumor recurrence and distant metastasis. Previously, we have shown that the preoperative plasma EBV DNA level is significantly higher in patients with positive resection margins during maxillary swing nasopharyngectomy. In the current study, the first important finding is that not all patients with histologically proven recurrent undifferentiated NPC that are EBER positive show elevated plasma EBV DNA. As a matter of fact, only 84.5% of our subjects had a raised DNA level before surgery. This should be checked as part of the routine investigations prior to surgery, as it provides a baseline reference as to whether it can be used as a tumor marker for monitoring of treatment response after surgery. Obviously, patients with negative preoperative plasma EBV DNA cannot rely on the tumor marker for follow-up in the future. In patients with measurable circulating EBV DNA before surgery, the test is a useful tool in the detection of subsequent tumor recurrence. A majority (87.5%) of the patients with local tumor recurrence and 100% of those with systemic metastasis had elevated plasma EBV DNA at the time of relapse, whereas it remained undetectable in all patients who were in remission. This demonstrates that serial measurements after treatment is a reliable way to monitor treatment response.
Lo et al. found that an initial rise in the plasma EBV DNA concentration was observed in all patients during the first week of radiotherapy, which may be explained by the liberation of EBV DNA after therapy-induced cancer cell death. It then decreased rapidly over the second week of treatment and could not be measured, with a median half-life of 3.8 days. Subsequently, To et al. showed that the plasma EBV DNA level dropped even faster after nasopharyngectomy for locally recurrent NPC, with a median half-life of only 139 minutes. Our study showed that only one out of 30 patients had measurable plasma EBV DNA at 60 minutes after tumor resection. Unlike in radiotherapy, where there is progressive cell death induced by radiation energy, leading to the continuous release of the viral DNA into the circulation during the early phase of treatment, surgical resection of the nasopharyngeal tumor results in an abrupt removal of the virus-bearing lesion, stopping any further DNA release. The fact that a majority of the patients had undetectable EBV DNA level only 60 minutes after tumor resection also reflects that fact that the half-life of circulating EBV DNA may be extremely short. Further study with more frequent blood sampling and testing is required to further delineate the kinetics of circulating EBV DNA in the body.
A majority of the patients with a microscopic positive resection margin had undetectable plasma EBV DNA levels at 1 hour and 1 week after tumor resection, reflecting its inadequacy to detect microscopic tumor deposits in the body. The early postoperative plasma EBV DNA also failed to predict subsequent local or systemic relapse. Therefore, circulating EBV DNA after surgery is useful only when measured serially for the purpose of detecting recurrence. At present, histological finding of positive resection margin remains the best predictor for local recurrence after salvage nasopharyngectomy. On the other hand, the preoperative plasma EBV DNA level is significantly higher in patients who developed distant metastasis subsequently after surgery. It is useful to identify the high-risk group of patients who warrant close monitoring after surgery or even consideration of further adjuvant treatment in view of increased risk of subsequent distant failure.
In patients with recurrent NPC requiring salvage nasopharyngectomy, preoperative plasma EBV identifies patients at high risk of subsequent distant failure after surgery. Serial measurements of plasma EBV DNA after surgery, especially for those with a high preoperative level, is crucial to allow early detection of local or distant failure.
- 40Pretherapy quantitative measurement if circulating Epstein-Barr virus DNA is predictive of posttherapy distant failure in patients with early-stage nasopharyngeal carcinoma of undifferentiated type. Cancer 2003;98:288–291., , , et al.