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Clinical significance of plasma Epstein–Barr Virus DNA loads in a large cohort of Malaysian patients with nasopharyngeal carcinoma
Journal of Clinical Virology 55 (2012) 34–39
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Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Clinical significance of plasma Epstein–Barr Virus DNA loads in a large cohort of Malaysian patients with nasopharyngeal carcinoma San Jiun Chai a , Kin Choo Pua b , Amyza Saleh a , Yoke Yeow Yap c , Paul V.H. Lim d , Selva Kumar Subramaniam e , Chee Lun Lum f , Gopala Krishnan g , Wan Rozita Wan Mahiyuddin h , the Malaysian NPC Study Groupk , Soo-Hwang Teo a , Alan S.B. Khoo i , Lee Fah Yap a,j,∗ a
Cancer Research Initiatives Foundation, Subang Jaya, Malaysia Department of Otorhinolaryngology, Hospital Pulau Pinang, Penang, Malaysia c Department of Surgery, Clinical Campus Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Hospital Kuala Lumpur, Kuala Lumpur, Malaysia d ENT Department, Tung Shin Hospital, Kuala Lumpur, Malaysia e Department of Otorhinolaryngology, Head and Neck Surgery, Sarawak General Hospital, Sarawak, Malaysia f ENT Department, Queen Elizabeth Hospital, Sabah, Malaysia g Department of Otorhinolaryngology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia h Epidemiology and Biostatistics Unit, Institute for Medical Research, Kuala Lumpur, Malaysia i Molecular Pathology Unit, Institute for Medical Research, Kuala Lumpur, Malaysia j Dental Research & Training Unit and Oral Cancer Research & Coordinating Centre, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia b
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Article history: Received 5 March 2012 Received in revised form 18 May 2012 Accepted 21 May 2012 Keywords: Nasopharyngeal carcinoma Epstein–Barr Virus Diagnosis Metastasis
a b s t r a c t Background: Nasopharyngeal carcinoma (NPC) is an Epstein–Barr Virus (EBV)-associated cancer that is the fifth most common cancer in Malaysia. Early and accurate diagnoses are critical for patient prognosis. Unfortunately, early detection of NPC is still a challenge and the cost of more accurate imaging protocols is prohibitive in developing countries like Malaysia. Objectives: To evaluate the clinical values of pre-treatment plasma EBV DNA levels in Malaysian NPC patients. Study design: Plasma EBV DNA levels were measured by quantitative PCR (Q-PCR) in a large and multiethnic cohort of Malaysian patients with NPC (n = 459) and 72 control subjects. Results: We show for the first time that, compared to controls, NPC patients with stage I disease had significantly higher levels of EBV DNA (p < 0.001). Further, the median level of plasma EBV DNA in stage IV patients with distant metastasis was >9-fold higher than those without systemic spread (p = 0.001), suggesting plasma EBV DNA measurement could aid in the diagnosis of metastatic disease in advanced cases. Further, using a cut-off value of 8000 copies/mL, we demonstrate that EBV DNA level is a strong predictor for overall survival of NPC patients. Conclusions: Our data show that pre-treatment plasma EBV DNA is a potential biomarker for early stage and metastatic NPC. We conclude that the quantification of plasma EBV DNA is a useful tool in developing countries to stratify patients for MRI or PET/CT scans where such imaging protocol is not routinely applied. © 2012 Elsevier B.V. All rights reserved.
1. Background Nasopharyngeal cancer (NPC) is an Epstein–Barr Virus (EBV)associated cancer that is particularly prevalent in South East Asia
Abbreviations: NPC, nasopharyngeal carcinoma; EBV, Epstein–Barr Virus; Q-PCR, quantitative polymerase chain reaction. ∗ Corresponding author at: Dental Research and Training Unit, Level 9, Postgraduate and Research Tower, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia. Tel.: +60 3 79676471; fax: +60 3 79676456. E-mail address: [email protected] (L.F. Yap). k See Appendix A. 1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.05.017
and Southern China, where it has an incidence rate of 20 per 100,000 persons per year.1 The 5-year survival rate for patients with early stage diseases is very promising (up to 95%),2–4 indicating that protocols to aid early detection would be of great clinical value. Unfortunately, over 75% of NPC patients present with late stage disease,5 and the 5-year overall survival rate declines to 41–63%.6 Further, the invasive diagnostic method to examine the nasopharynx clinically and the occurrence of occult primary tumours add to the challenge in diagnosing the disease early and accurately.7 Despite substantial improvements in treatment for NPC patients, distant metastasis remains the leading cause of death.8
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Occult or microscopic distant metastasis that are already present at the time of diagnosis are often unnoticed and undetected.9 Imaging for distant metastasis is not always performed unless the patient has symptoms that suggest for systemic spread or abnormal baseline investigation findings. Particularly in developing countries, the cost of more accurate imaging modalities such as MRI or PET/CT scan is prohibitive and this often results in delay of diagnosis and treatment. Therefore, a non-invasive and cost-effective method to diagnose NPC more accurately and predict clinical outcome would have a major impact on clinical practice. The strong etiological link between EBV and NPC has been known for decades. Various serological tests for antibodies against EBV antigens have been developed to detect NPC, however, issues of specificity and sensitivity of these methods have limited their clinical application.10,11 The potential diagnostic and prognostic values of plasma cell-free EBV DNA by quantitative real-time PCR (Q-PCR) was first proposed more than 10 years ago.12 However, in Malaysia, its clinical utility is still not routinely applied. In Peninsular Malaysia, NPC is the fifth most common cancers overall and third most common among men.13 Malaysia is a unique multi-ethnic country of 27 million people with several major ethnic groups, including Malays (53.3%), Chinese (26%), Indians (7.7%) and various groups of indigenous people (11.8%).14 While NPC is more common among Chinese, the indigenous Bidayuh people in Sarawak had the highest incidence rate of NPC in the world during 1996–1998.15 In recognition of the problems and challenges related to the management of NPC in Malaysia, the Malaysian NPC Study Group was formed to establish a clinical outcome database and biobank for NPC.5 To the best of our knowledge, this is the largest study evaluating the clinical significance of plasma EBV DNA in NPC to date, and also the first to use such a large multi-ethnic cohort in an endemic region of NPC.
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Table 1 Clinical characteristics of pre-therapy patients with NPC. Variable Hospital Tung Shin Hospital, Kuala Lumpur Sarawak General Hospital, Kuching Hospital Pulau Pinang, Penang Hospital Kuala Lumpur, Kuala Lumpur Queen Elizabeth Hospital, Kota Kinabalu University Malaya Medical Centre, Kuala Lumpur Age (median, 50.5; range, 13–81) ≤50 >50 Ethnicity Chinese Malay Bidayuh Kadazan Iban Indian Others WHO typing WHO type I WHO type II WHO type III Overall staging Stage I Stage II Stage III Stage IV Systemic spread in stage IV Stage IV; M0 Stage IV; M1 Overall survival after treatmenta Alive Dead a
Sample size (%) 103 (26.4) 85 (21.8) 84 (21.5) 62 (15.9) 43 (11.0) 13 (3.3) 196 (50.1) 194 (49.9) 216 (55.4) 86 (22.1) 35 (9.0) 23 (5.9) 14 (3.6) 2 (0.5) 14 (3.6) 6 (1.5) 79 (20.3) 305 (78.2) 19 (4.9) 85 (21.8) 125 (32.1) 161 (41.3) 140 (87.0) 21 (13.0) 129 (82.2) 28 (17.8)
Follow-up data only available in 157 cases.
3.2. EBV DNA extraction from plasma 2. Objectives The aim of this study was to evaluate the clinical significance of pre-treatment plasma EBV DNA levels in a large and multi-ethnic cohort of Malaysian patients with NPC.
5 mL peripheral blood was collected from each study subject and placed in an EDTA-treated blood tube (BD, USA) for the isolation of plasma. Blood samples were centrifuged at 3300 × g at room temperature for 10 min, and the plasma samples were stored in −80 ◦ C until processing. DNA was extracted from the plasma using a QIAamp DNA Blood Kit (Qiagen, Germany).
3. Study design
3.3. Q-PCR
3.1. Cohort
The levels of EBV DNA in plasma were determined by Q-PCR on the BamHI-W region of EBV genome following a protocol described previously.12 DNA samples that showed undetectable level of the target region were subjected to an amplification of the -globin gene to ensure the integrity of the extracted DNA. The Q-PCR was performed in a 96-well reaction plate format using the ABI Prism® 7000 Sequence Detection System (Applied Biosystems, USA). Experiments were performed in triplicate for each sample in the same reaction. Multiple no-template-controls were included in each analysis as negative controls. A standard curve using DNA extracted from an EBV-positive cell line, Namalwa, was generated in each analysis. Copies of EBV per millilitre of plasma (copies/mL) were calculated based on the standard curve. Samples with undetectable EBV BamHI-W were considered 0 copies/mL plasma.
This retrospective study involved a total of 390 previously untreated, biopsy-proven NPC patients recruited from six major NPC referring hospitals across Malaysia. The routine diagnostic procedure included a detailed clinical examination of the head and neck, fibre optic endoscopy, computed tomography of the entire neck from the base of the skull, chest radiography, whole-body bone scanning, abdominal sonography, a complete blood count, and a biochemical profile. Cancer stage was defined according to the 1997 American Joint Committee on Cancer (AJCC) tumour-nodemetastasis staging system.16 Overall survival was calculated from the first day of treatment until death or the last follow-up visit. The clinical characteristics of these patients are listed in Table 1. The control subjects comprised a multi-ethnic cohort of 41 healthy donors and 31 individuals who did not suffer from NPC, but had other non-malignant diseases at the nasopharynx, ranging 18–75 years old. 33 patients with recurrent NPC and 36 patients in remission were also recruited. Ethical approval for this study was obtained from the Medical Research Ethics Committee from the Ministry of Health, Malaysia (KKM/NIHSEC/08/0804/MRG-IMR). Written consents were obtained from all study subjects.
3.4. Statistical analysis Statistical analyses were performed using the SPSS statistical package (Version 16.0, SPSS Inc., Chicago, USA). Data were assessed for normality using Kolmogarov–Smirnov analysis. Plasma EBV copy number in each category was compared using non-parametric tests of Mann–Whitney or Kruskal–Wallis rank sum test. Values
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Fig. 1. Plasma EBV DNA levels reflect disease status. (a) Comparison of plasma EBV DNA levels in healthy donors, non-NPC controls, newly diagnosed NPC, patients in remission and patients with recurrent disease. The lines inside the boxes denote the median, the boxes denote the interquartile ranges, and the I bars indicate the range of EBV levels (minimum to maximum). p-value was calculated by means of Mann–Whitney test. (b) ROC analysis determines a cut-off value of 186 copies/mL to significantly discriminate NPC patients from the control subjects.
of cut-off point, sensitivity and specificity were determined using a receiver operating characteristic (ROC) curve analysis. Survival analyses were performed using the Kaplan–Meier plot and Cox regression model. A p-value of <0.05 was considered being statistically significant. 4. Results 4.1. EBV DNA loads reflect disease status EBV DNA was detected in plasma samples of 89.7% (350 of 390) untreated NPC patients, 12.2% (5 of 41) healthy donors and 6.5% (2 of 31) non-malignant cases. The elevation of the EBV DNA levels in the NPC patients was statistically significant with a median copy number of 6582 copies/mL (interquartile range = 777–47,035 copies/mL) compared to the healthy donors and non-malignant controls (median = 0 copies/mL; interquartile range = 0–0 copies/mL; p < 0.001; Fig. 1a). The levels of EBV DNA were not significantly different between the healthy donors and non-malignant controls (p = 0.459), demonstrating that plasma EBV DNA could be a specific tumour marker for NPC. Among patients with active recurrent disease, the median EBV DNA level was 286 copies/mL (interquartile range = 0–30,246 copies/mL); while EBV DNA levels in patients in remission was undetectable in most cases (33/36), with a median value of 0 copies/mL (interquartile
Fig. 2. Plasma EBV DNA loads correlate with disease staging. (a) Patients with early stage disease (stage I and II) had higher DNA levels compared to those with advanced stage disease (stage III and IV). (b) EBV DNA levels patients with stage I disease were significantly higher than the control subjects. The lines inside the boxes denote the median, the boxes denote the interquartile ranges, and the I bars indicate the range of EBV levels (minimum to maximum). p-value was calculated by means of Mann–Whitney test.
range = 0–0 copies/mL) which was not significantly different compared to the control subjects (healthy donors plus non-malignant cases, n = 72; p = 0.912; Fig. 1a). To determine the diagnostic value of the EBV DNA prior to therapy, the data from 390 new NPC cases and 72 control subjects were analyzed by a receiver operating characteristic (ROC) curve analysis. The results showed that the area under the curve (AUC) was significantly larger than the area expected by chance alone (p < 0.001, 95% CI = 0.917–0.960; Fig. 1b). The optimal cut-off value was 186 copies/mL; with a sensitivity of 86% and a specificity of 96%. 4.2. EBV DNA loads correlate with disease staging and distant metastasis The median EBV DNA level in patients with early stage disease (stage I and II) and advanced stage (stage III and IV) disease was 2780 copies/mL (interquartile range = 227–17,830 copies/mL) and 10,150 copies/mL (interquartile range = 1502–83,460 copies/mL), respectively. These data show that patients with advanced stage disease had approximately 3.7 times higher EBV DNA loads than patients with early stage disease (Fig. 2a). Notably, EBV DNA levels
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Fig. 3. Plasma EBV DNA levels correlate with distant metastasis in patients with stage IV disease. Patients with distant metastasis (M1) had higher EBV DNA loads than those who did not have systemic spread of disease (M0). The line inside the boxes denote the median, the boxes denote the interquartile ranges, and the I bars indicate the range of EBV levels (minimum to maximum). p-value was calculated by means of Mann–Whitney test.
in patients with stage I disease (n = 19) were significantly higher compared to the control subjects (n = 72) (p < 0.001; Fig. 2b). Of the 161 stage IV cases, patients who had systemic spread of disease (M1; n = 21) showed approximately 9.3 times higher EBV DNA levels compared to those who did not have distant metastasis (M0; n = 140; p = 0.001; Fig. 3), being 199,072 copies/mL (interquartile range = 73,061–1,900,000 copies/mL) and 21,411 copies/mL (interquartile range = 2991–180,553 copies/mL) in M1 and M0 cases, respectively. 4.3. EBV DNA loads predict patient overall survival Among the 390 new cases, the overall survival information of 157 patients who received treatment was available. Using a ROC curve analysis, EBV DNA of 8000 copies/mL was determined as a cut-off value to predict the overall survival of the patients (p < 0.001, 95% CI = 0.66–0.90). Survival probabilities of patients subgroups defined by low and high EBV DNA levels: ‘high level’ denotes EBV DNA level ≥8000 copies/mL while ‘low level’ denotes DNA levels <8000 copies/mL. The results showed that within 2 years of diagnosis, 24 out of 82 (29.3%) patients who had high levels of EBV DNA were dead, while mortality was only seen in 4 out of 75 (5.3%) patients who had low EBV DNA level (Fig. 4a). In support of this data, a Kaplan–Meier analysis also showed that patients with low DNA level had a better survival probability compared to those with high DNA level (Fig. 4b). A multivariate analysis by a Cox regression model was used to estimate overall survival of patients with adjustment for age, gender, stage and WHO pathological type. The results confirmed that plasma EBV level was a more powerful prognostic indicator for overall survival (p = 0.002; hazard ratio = 6.0; 95% CI = 1.97–18.58) than AJCC staging (p = 0.045; hazard ratio = 4.4; 95% CI = 1.03–18.87; Table 2).
Table 2 Multivariate Cox regression model analyses. Variable
Hazard ratio (95% CI)
p-value
Age ≥ 50 (vs. <50) Male (vs. female) WHO type I (vs. II or III) Stage I and II (vs. III and IV) Plasma EBV DNA ≥8000 copies/mL (vs. <8000 copies/mL)
0.8 (0.38–1.87) 1.0 (0.36–2.65) 0.2 (0.03–1.90) 4.4 (1.03–18.87) 6.0 (1.97–18.58)
0.681 0.973 0.171 0.045 0.002
CI denotes confidence interval.
Fig. 4. EBV DNA loads predict patient overall survival. (a) Number of deaths in patients with low EBV DNA levels is lesser than those with high DNA levels. (b) Kaplan–Meier estimates of a better overall survival probability in patients with low EBV DNA levels than those with high DNA levels. “Low” denotes EBV DNA levels of less than 8000 copies/mL, and “High” denotes EBV DNA levels of ≥8000 copies/mL.
5. Discussion It was first demonstrated in 1999 that cell-free EBV DNA could be detected in the plasma of NPC patients using Q-PCR and the DNA levels correlated with various clinical parameters.12 This remarkable finding stimulated a number of studies investigating the value of cell-free EBV DNA as a tumour marker for NPC. However, while some reports17–20 supported the results presented by Lo and colleagues,12 others reported variable and inconclusive data.21,22 In Malaysia, the measurement of cell-free EBV DNA in NPC has not been included in routine clinical practice due to the lack of consensus on its usefulness. Using patients from six major NPC referring hospitals across Malaysia, we are able to use the largest cohort of NPC patients studied to date and, as such, represent the first nation-wide, systematic collection of biospecimens for NPC in Malaysia. In addition to the most common study subjects, the Chinese, this unique cohort contains other ethnic groups that have never been studied previously, and these include Malay and indigenous people who also show high incidence rates of NPC. Our results confirmed previous reports that plasma EBV DNA is a specific marker for NPC at the time of diagnosis. Importantly, our cohort provided us sufficient samples to show that NPC patients had significantly higher levels of EBV DNA, even in patients with stage I disease. This result highlights the potential value of EBV DNA as an early detection marker for NPC. The detection rates of EBV DNA in NPC patients have been reported to vary in a range of 69–96%,12,17,20,23–26 our detection rate of 89.7% is in line with these results. The detection rates in six
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participating hospitals were similar (83–93%; Supplementary Table S1), indicating that the sampling and storage procedures in each of the participating hospitals were robust. The variation in detection rates among studies might be due to the difference in the number of samples analyzed, but given that our cohort contains a large number of patients (n = 390) to provide a strong statistical power, we can conclude that the sensitivity of the quantification of cellfree EBV DNA in NPC should be approximately 90%. In the present study, EBV DNA was detected in 3 of 36 patients in remission. All these three patients were subsequently diagnosed with liver metastasis or local recurrence (one confirmed and one suspected), supporting previous data that showed significant elevations in EBV DNA levels before detectable clinical deterioration in patients who subsequently developed tumour recurrence.17,27–29 Late presentation remains the major challenge in managing NPC.30,31 Currently the mainstay of treatment for advanced cases of NPC is concurrent chemoradiotherapy that invariably results in high morbidity and poor quality of life.32 Though rare, distant metastasis remains the major cause of death.8 Previous studies have shown that the median EBV DNA level was higher in patients with distant metastasis compared to those did not have systemic diseases.17,20,25 However, these studies grouped patients without distant metastasis at all stages in one category, even though it is unlikely that these patients would have higher viral loads because patients with early stage disease were included. Here, we performed a more accurate comparison by determining the correlation between EBV DNA levels and distant metastasis only in stage IV patients. We show that stage IV patients with distant metastases had significantly higher EBV DNA levels than those who did not have systemic spread of the disease, indicating plasma EBV DNA load could be a marker to aid the diagnosis of distant metastasis. Further, a ROC analysis determined 8000 copies/mL of plasma EBV DNA as the optimal cut-off point to predict the overall survival of the NPC patients. Both univariate and multivariate analyses further confirmed that patients with EBV DNA level <8000 copies/mL had better survival probability compared to those who had ≥8000 copies/mL. While Le et al.29 have shown that EBV DNA had a limited value in predicting patient survival, our data support other reports demonstrating that pre-therapy circulating EBV DNA load is an independent prognostic factor for overall survival in NPC.17,18 Therefore, further studies to confirm the predictive value of EBV DNA are warranted. In summary, we show that plasma EBV DNA is specific for NPC and reflects tumour burden. Importantly, our data demonstrate for the first time that plasma EBV DNA measurement could aid a more accurate diagnosis for early stage disease, as well as for distant metastases in stage IV patients. We propose that in developing countries where MRI and PET/CT scans are prohibitively expensive for routine use, plasma EBV DNA measurement could be used to stratify patients for a more accurate imaging protocol.
Funding This project was funded by the Ministry of Health, Malaysia (project code: JPP-IMR 06-060) and Cancer Research Initiatives Foundation.
Competing interests None declared.
Ethical approval Ethical approval was obtained from the Medical Research Ethics Committee from the Ministry of Health, Malaysia (KKM/NIHSEC/08/0804/MRG-IMR). Acknowledgements We wish to thank Drs. Dennis Lo and Lisa Chan from the Chinese University of Hong Kong for their technical advice. We would also like to thank the Director of General Health, Ministry of Health, the Director of the Institute for Medical Research, the Director of the Network of Clinical Research Centre and the Directors of all participating hospitals for their support. We wish to acknowledge the contributions of Ms. Yoon Sook Yee, Ms. Jayanthi, otorhinolaryngologists, oncologists, radiotherapists, and pathologists of the participating hospitals, the staff of the Clinical Research Centre of Hospital Kuala Lumpur, Hospital Pulau Pinang, and Sime Darby Medical Centre. Appendix A. The Malaysian Nasopharyngeal Carcinoma Study Group: • Hospital Pulau Pinang: KC Pua, N Punithavati, BS Tan, YS Ee, LM Ong, RA Hamid, M Goh, JCT Quah, J Lim. • Hospital Kuala Lumpur/UPM: YY Yap, FN Lau, PV Kam. • University of Malaya: G Gopala Krishnan, AZ Bustam, LM Looi, P Shahfinaz, O Hashim, CC Ng, O Rahmat, J Amin. • Sarawak General Hospital/UNIMAS: SK Subramaniam, TS Tiong, UH Sim, TW Tharumalingam, D Norlida, M Zulkarnaen. • Queen Elizabeth Hospital: CL Lum, A Nordin, Halimuddin S, Somasundram. • Hospital USM Kubang Kerian: S Hassan, B Biswal, H Nik Fariza, HA Mubassir, AH Suzina Sheikh. • Institute for Medical Research: ASB Khoo. • Cancer Research Initiatives Foundation: SH Teo, LF Yap. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2012.05.017. References 1. Razak AR, Siu LL, Liu FF, Ito E, O’Sullivan B, Chan K. Nasopharyngeal carcinoma: the next challenges. Eur J Cancer 2010;46:1967–78. 2. Cao XP, Lu TX, Ye WJ, Cui NJ. Prospective study on long-term efficacy of external plus intracavitary radiotherapy on stage I–II nasopharyngeal carcinoma. Ai Zheng 2007;26:204–7. 3. Leung TW, Tung SY, Sze WK, Wong FC, Yuen KK, Lui CM, et al. Treatment results of 1070 patients with nasopharyngeal carcinoma: an analysis of survival and failure patterns. Head Neck 2005;27:555–65. 4. Xiao WW, Han F, Lu TX, Chen CY, Huang Y, Zhao C. Treatment outcomes after radiotherapy alone for patients with early-stage nasopharyngeal carcinoma. Int J Radiat Oncol Biol Phys 2009;74:1070–6. 5. Pua KC, Khoo ASB, Subramaniam SK, Ong KA, Gopala Krishnan G, Shahid H, et al. Nasopharyngeal carcinoma database. Med J Malaysia 2008;63:3. 6. El-Sherbieny E, Rashwan H, Lubis SH, Choi VJ. Prognostic factors in patients with nasopharyngeal carcinoma treated in hospital Kuala Lumpur. Asian Pac J Cancer Prev 2011;12:1739–43. 7. Prasad U, Ablashi D, Pratha Kea. Problem of occult primary in nasopharyngeal carcinoma. In: Prasad U, editor. Nasopharyngeal carcinoma: current concepts. Kuala Lumpur: University of Malaya Press; 1983. p. 11–5. 8. O’Sullivan B. Nasopharynx cancer: therapeutic value of chemoradiotherapy. Int J Radiat Oncol Biol Phys 2007;69:S118–21. 9. Teo PM, Kwan WH, Lee WY, Leung SF, Johnson PJ. Prognosticators determining survival subsequent to distant metastasis from nasopharyngeal carcinoma. Cancer 1996;77:2423–31. 10. Tsang RK, Vlantis AC, Ho RW, Tam JS, To KF, van Hasselt CA. Sensitivity and specificity of Epstein–Barr Virus IGA titer in the diagnosis of nasopharyngeal carcinoma: a three-year institutional review. Head Neck 2004;26:598–602.
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Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Clinical significance of plasma Epstein–Barr Virus DNA loads in a large cohort of Malaysian patients with nasopharyngeal carcinoma San Jiun Chai a , Kin Choo Pua b , Amyza Saleh a , Yoke Yeow Yap c , Paul V.H. Lim d , Selva Kumar Subramaniam e , Chee Lun Lum f , Gopala Krishnan g , Wan Rozita Wan Mahiyuddin h , the Malaysian NPC Study Groupk , Soo-Hwang Teo a , Alan S.B. Khoo i , Lee Fah Yap a,j,∗ a
Cancer Research Initiatives Foundation, Subang Jaya, Malaysia Department of Otorhinolaryngology, Hospital Pulau Pinang, Penang, Malaysia c Department of Surgery, Clinical Campus Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Hospital Kuala Lumpur, Kuala Lumpur, Malaysia d ENT Department, Tung Shin Hospital, Kuala Lumpur, Malaysia e Department of Otorhinolaryngology, Head and Neck Surgery, Sarawak General Hospital, Sarawak, Malaysia f ENT Department, Queen Elizabeth Hospital, Sabah, Malaysia g Department of Otorhinolaryngology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia h Epidemiology and Biostatistics Unit, Institute for Medical Research, Kuala Lumpur, Malaysia i Molecular Pathology Unit, Institute for Medical Research, Kuala Lumpur, Malaysia j Dental Research & Training Unit and Oral Cancer Research & Coordinating Centre, Faculty of Dentistry, University of Malaya, Kuala Lumpur, Malaysia b
a r t i c l e
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Article history: Received 5 March 2012 Received in revised form 18 May 2012 Accepted 21 May 2012 Keywords: Nasopharyngeal carcinoma Epstein–Barr Virus Diagnosis Metastasis
a b s t r a c t Background: Nasopharyngeal carcinoma (NPC) is an Epstein–Barr Virus (EBV)-associated cancer that is the fifth most common cancer in Malaysia. Early and accurate diagnoses are critical for patient prognosis. Unfortunately, early detection of NPC is still a challenge and the cost of more accurate imaging protocols is prohibitive in developing countries like Malaysia. Objectives: To evaluate the clinical values of pre-treatment plasma EBV DNA levels in Malaysian NPC patients. Study design: Plasma EBV DNA levels were measured by quantitative PCR (Q-PCR) in a large and multiethnic cohort of Malaysian patients with NPC (n = 459) and 72 control subjects. Results: We show for the first time that, compared to controls, NPC patients with stage I disease had significantly higher levels of EBV DNA (p < 0.001). Further, the median level of plasma EBV DNA in stage IV patients with distant metastasis was >9-fold higher than those without systemic spread (p = 0.001), suggesting plasma EBV DNA measurement could aid in the diagnosis of metastatic disease in advanced cases. Further, using a cut-off value of 8000 copies/mL, we demonstrate that EBV DNA level is a strong predictor for overall survival of NPC patients. Conclusions: Our data show that pre-treatment plasma EBV DNA is a potential biomarker for early stage and metastatic NPC. We conclude that the quantification of plasma EBV DNA is a useful tool in developing countries to stratify patients for MRI or PET/CT scans where such imaging protocol is not routinely applied. © 2012 Elsevier B.V. All rights reserved.
1. Background Nasopharyngeal cancer (NPC) is an Epstein–Barr Virus (EBV)associated cancer that is particularly prevalent in South East Asia
Abbreviations: NPC, nasopharyngeal carcinoma; EBV, Epstein–Barr Virus; Q-PCR, quantitative polymerase chain reaction. ∗ Corresponding author at: Dental Research and Training Unit, Level 9, Postgraduate and Research Tower, Faculty of Dentistry, University of Malaya, 50603 Kuala Lumpur, Malaysia. Tel.: +60 3 79676471; fax: +60 3 79676456. E-mail address: [email protected] (L.F. Yap). k See Appendix A. 1386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2012.05.017
and Southern China, where it has an incidence rate of 20 per 100,000 persons per year.1 The 5-year survival rate for patients with early stage diseases is very promising (up to 95%),2–4 indicating that protocols to aid early detection would be of great clinical value. Unfortunately, over 75% of NPC patients present with late stage disease,5 and the 5-year overall survival rate declines to 41–63%.6 Further, the invasive diagnostic method to examine the nasopharynx clinically and the occurrence of occult primary tumours add to the challenge in diagnosing the disease early and accurately.7 Despite substantial improvements in treatment for NPC patients, distant metastasis remains the leading cause of death.8
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Occult or microscopic distant metastasis that are already present at the time of diagnosis are often unnoticed and undetected.9 Imaging for distant metastasis is not always performed unless the patient has symptoms that suggest for systemic spread or abnormal baseline investigation findings. Particularly in developing countries, the cost of more accurate imaging modalities such as MRI or PET/CT scan is prohibitive and this often results in delay of diagnosis and treatment. Therefore, a non-invasive and cost-effective method to diagnose NPC more accurately and predict clinical outcome would have a major impact on clinical practice. The strong etiological link between EBV and NPC has been known for decades. Various serological tests for antibodies against EBV antigens have been developed to detect NPC, however, issues of specificity and sensitivity of these methods have limited their clinical application.10,11 The potential diagnostic and prognostic values of plasma cell-free EBV DNA by quantitative real-time PCR (Q-PCR) was first proposed more than 10 years ago.12 However, in Malaysia, its clinical utility is still not routinely applied. In Peninsular Malaysia, NPC is the fifth most common cancers overall and third most common among men.13 Malaysia is a unique multi-ethnic country of 27 million people with several major ethnic groups, including Malays (53.3%), Chinese (26%), Indians (7.7%) and various groups of indigenous people (11.8%).14 While NPC is more common among Chinese, the indigenous Bidayuh people in Sarawak had the highest incidence rate of NPC in the world during 1996–1998.15 In recognition of the problems and challenges related to the management of NPC in Malaysia, the Malaysian NPC Study Group was formed to establish a clinical outcome database and biobank for NPC.5 To the best of our knowledge, this is the largest study evaluating the clinical significance of plasma EBV DNA in NPC to date, and also the first to use such a large multi-ethnic cohort in an endemic region of NPC.
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Table 1 Clinical characteristics of pre-therapy patients with NPC. Variable Hospital Tung Shin Hospital, Kuala Lumpur Sarawak General Hospital, Kuching Hospital Pulau Pinang, Penang Hospital Kuala Lumpur, Kuala Lumpur Queen Elizabeth Hospital, Kota Kinabalu University Malaya Medical Centre, Kuala Lumpur Age (median, 50.5; range, 13–81) ≤50 >50 Ethnicity Chinese Malay Bidayuh Kadazan Iban Indian Others WHO typing WHO type I WHO type II WHO type III Overall staging Stage I Stage II Stage III Stage IV Systemic spread in stage IV Stage IV; M0 Stage IV; M1 Overall survival after treatmenta Alive Dead a
Sample size (%) 103 (26.4) 85 (21.8) 84 (21.5) 62 (15.9) 43 (11.0) 13 (3.3) 196 (50.1) 194 (49.9) 216 (55.4) 86 (22.1) 35 (9.0) 23 (5.9) 14 (3.6) 2 (0.5) 14 (3.6) 6 (1.5) 79 (20.3) 305 (78.2) 19 (4.9) 85 (21.8) 125 (32.1) 161 (41.3) 140 (87.0) 21 (13.0) 129 (82.2) 28 (17.8)
Follow-up data only available in 157 cases.
3.2. EBV DNA extraction from plasma 2. Objectives The aim of this study was to evaluate the clinical significance of pre-treatment plasma EBV DNA levels in a large and multi-ethnic cohort of Malaysian patients with NPC.
5 mL peripheral blood was collected from each study subject and placed in an EDTA-treated blood tube (BD, USA) for the isolation of plasma. Blood samples were centrifuged at 3300 × g at room temperature for 10 min, and the plasma samples were stored in −80 ◦ C until processing. DNA was extracted from the plasma using a QIAamp DNA Blood Kit (Qiagen, Germany).
3. Study design
3.3. Q-PCR
3.1. Cohort
The levels of EBV DNA in plasma were determined by Q-PCR on the BamHI-W region of EBV genome following a protocol described previously.12 DNA samples that showed undetectable level of the target region were subjected to an amplification of the -globin gene to ensure the integrity of the extracted DNA. The Q-PCR was performed in a 96-well reaction plate format using the ABI Prism® 7000 Sequence Detection System (Applied Biosystems, USA). Experiments were performed in triplicate for each sample in the same reaction. Multiple no-template-controls were included in each analysis as negative controls. A standard curve using DNA extracted from an EBV-positive cell line, Namalwa, was generated in each analysis. Copies of EBV per millilitre of plasma (copies/mL) were calculated based on the standard curve. Samples with undetectable EBV BamHI-W were considered 0 copies/mL plasma.
This retrospective study involved a total of 390 previously untreated, biopsy-proven NPC patients recruited from six major NPC referring hospitals across Malaysia. The routine diagnostic procedure included a detailed clinical examination of the head and neck, fibre optic endoscopy, computed tomography of the entire neck from the base of the skull, chest radiography, whole-body bone scanning, abdominal sonography, a complete blood count, and a biochemical profile. Cancer stage was defined according to the 1997 American Joint Committee on Cancer (AJCC) tumour-nodemetastasis staging system.16 Overall survival was calculated from the first day of treatment until death or the last follow-up visit. The clinical characteristics of these patients are listed in Table 1. The control subjects comprised a multi-ethnic cohort of 41 healthy donors and 31 individuals who did not suffer from NPC, but had other non-malignant diseases at the nasopharynx, ranging 18–75 years old. 33 patients with recurrent NPC and 36 patients in remission were also recruited. Ethical approval for this study was obtained from the Medical Research Ethics Committee from the Ministry of Health, Malaysia (KKM/NIHSEC/08/0804/MRG-IMR). Written consents were obtained from all study subjects.
3.4. Statistical analysis Statistical analyses were performed using the SPSS statistical package (Version 16.0, SPSS Inc., Chicago, USA). Data were assessed for normality using Kolmogarov–Smirnov analysis. Plasma EBV copy number in each category was compared using non-parametric tests of Mann–Whitney or Kruskal–Wallis rank sum test. Values
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Fig. 1. Plasma EBV DNA levels reflect disease status. (a) Comparison of plasma EBV DNA levels in healthy donors, non-NPC controls, newly diagnosed NPC, patients in remission and patients with recurrent disease. The lines inside the boxes denote the median, the boxes denote the interquartile ranges, and the I bars indicate the range of EBV levels (minimum to maximum). p-value was calculated by means of Mann–Whitney test. (b) ROC analysis determines a cut-off value of 186 copies/mL to significantly discriminate NPC patients from the control subjects.
of cut-off point, sensitivity and specificity were determined using a receiver operating characteristic (ROC) curve analysis. Survival analyses were performed using the Kaplan–Meier plot and Cox regression model. A p-value of <0.05 was considered being statistically significant. 4. Results 4.1. EBV DNA loads reflect disease status EBV DNA was detected in plasma samples of 89.7% (350 of 390) untreated NPC patients, 12.2% (5 of 41) healthy donors and 6.5% (2 of 31) non-malignant cases. The elevation of the EBV DNA levels in the NPC patients was statistically significant with a median copy number of 6582 copies/mL (interquartile range = 777–47,035 copies/mL) compared to the healthy donors and non-malignant controls (median = 0 copies/mL; interquartile range = 0–0 copies/mL; p < 0.001; Fig. 1a). The levels of EBV DNA were not significantly different between the healthy donors and non-malignant controls (p = 0.459), demonstrating that plasma EBV DNA could be a specific tumour marker for NPC. Among patients with active recurrent disease, the median EBV DNA level was 286 copies/mL (interquartile range = 0–30,246 copies/mL); while EBV DNA levels in patients in remission was undetectable in most cases (33/36), with a median value of 0 copies/mL (interquartile
Fig. 2. Plasma EBV DNA loads correlate with disease staging. (a) Patients with early stage disease (stage I and II) had higher DNA levels compared to those with advanced stage disease (stage III and IV). (b) EBV DNA levels patients with stage I disease were significantly higher than the control subjects. The lines inside the boxes denote the median, the boxes denote the interquartile ranges, and the I bars indicate the range of EBV levels (minimum to maximum). p-value was calculated by means of Mann–Whitney test.
range = 0–0 copies/mL) which was not significantly different compared to the control subjects (healthy donors plus non-malignant cases, n = 72; p = 0.912; Fig. 1a). To determine the diagnostic value of the EBV DNA prior to therapy, the data from 390 new NPC cases and 72 control subjects were analyzed by a receiver operating characteristic (ROC) curve analysis. The results showed that the area under the curve (AUC) was significantly larger than the area expected by chance alone (p < 0.001, 95% CI = 0.917–0.960; Fig. 1b). The optimal cut-off value was 186 copies/mL; with a sensitivity of 86% and a specificity of 96%. 4.2. EBV DNA loads correlate with disease staging and distant metastasis The median EBV DNA level in patients with early stage disease (stage I and II) and advanced stage (stage III and IV) disease was 2780 copies/mL (interquartile range = 227–17,830 copies/mL) and 10,150 copies/mL (interquartile range = 1502–83,460 copies/mL), respectively. These data show that patients with advanced stage disease had approximately 3.7 times higher EBV DNA loads than patients with early stage disease (Fig. 2a). Notably, EBV DNA levels
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Fig. 3. Plasma EBV DNA levels correlate with distant metastasis in patients with stage IV disease. Patients with distant metastasis (M1) had higher EBV DNA loads than those who did not have systemic spread of disease (M0). The line inside the boxes denote the median, the boxes denote the interquartile ranges, and the I bars indicate the range of EBV levels (minimum to maximum). p-value was calculated by means of Mann–Whitney test.
in patients with stage I disease (n = 19) were significantly higher compared to the control subjects (n = 72) (p < 0.001; Fig. 2b). Of the 161 stage IV cases, patients who had systemic spread of disease (M1; n = 21) showed approximately 9.3 times higher EBV DNA levels compared to those who did not have distant metastasis (M0; n = 140; p = 0.001; Fig. 3), being 199,072 copies/mL (interquartile range = 73,061–1,900,000 copies/mL) and 21,411 copies/mL (interquartile range = 2991–180,553 copies/mL) in M1 and M0 cases, respectively. 4.3. EBV DNA loads predict patient overall survival Among the 390 new cases, the overall survival information of 157 patients who received treatment was available. Using a ROC curve analysis, EBV DNA of 8000 copies/mL was determined as a cut-off value to predict the overall survival of the patients (p < 0.001, 95% CI = 0.66–0.90). Survival probabilities of patients subgroups defined by low and high EBV DNA levels: ‘high level’ denotes EBV DNA level ≥8000 copies/mL while ‘low level’ denotes DNA levels <8000 copies/mL. The results showed that within 2 years of diagnosis, 24 out of 82 (29.3%) patients who had high levels of EBV DNA were dead, while mortality was only seen in 4 out of 75 (5.3%) patients who had low EBV DNA level (Fig. 4a). In support of this data, a Kaplan–Meier analysis also showed that patients with low DNA level had a better survival probability compared to those with high DNA level (Fig. 4b). A multivariate analysis by a Cox regression model was used to estimate overall survival of patients with adjustment for age, gender, stage and WHO pathological type. The results confirmed that plasma EBV level was a more powerful prognostic indicator for overall survival (p = 0.002; hazard ratio = 6.0; 95% CI = 1.97–18.58) than AJCC staging (p = 0.045; hazard ratio = 4.4; 95% CI = 1.03–18.87; Table 2).
Table 2 Multivariate Cox regression model analyses. Variable
Hazard ratio (95% CI)
p-value
Age ≥ 50 (vs. <50) Male (vs. female) WHO type I (vs. II or III) Stage I and II (vs. III and IV) Plasma EBV DNA ≥8000 copies/mL (vs. <8000 copies/mL)
0.8 (0.38–1.87) 1.0 (0.36–2.65) 0.2 (0.03–1.90) 4.4 (1.03–18.87) 6.0 (1.97–18.58)
0.681 0.973 0.171 0.045 0.002
CI denotes confidence interval.
Fig. 4. EBV DNA loads predict patient overall survival. (a) Number of deaths in patients with low EBV DNA levels is lesser than those with high DNA levels. (b) Kaplan–Meier estimates of a better overall survival probability in patients with low EBV DNA levels than those with high DNA levels. “Low” denotes EBV DNA levels of less than 8000 copies/mL, and “High” denotes EBV DNA levels of ≥8000 copies/mL.
5. Discussion It was first demonstrated in 1999 that cell-free EBV DNA could be detected in the plasma of NPC patients using Q-PCR and the DNA levels correlated with various clinical parameters.12 This remarkable finding stimulated a number of studies investigating the value of cell-free EBV DNA as a tumour marker for NPC. However, while some reports17–20 supported the results presented by Lo and colleagues,12 others reported variable and inconclusive data.21,22 In Malaysia, the measurement of cell-free EBV DNA in NPC has not been included in routine clinical practice due to the lack of consensus on its usefulness. Using patients from six major NPC referring hospitals across Malaysia, we are able to use the largest cohort of NPC patients studied to date and, as such, represent the first nation-wide, systematic collection of biospecimens for NPC in Malaysia. In addition to the most common study subjects, the Chinese, this unique cohort contains other ethnic groups that have never been studied previously, and these include Malay and indigenous people who also show high incidence rates of NPC. Our results confirmed previous reports that plasma EBV DNA is a specific marker for NPC at the time of diagnosis. Importantly, our cohort provided us sufficient samples to show that NPC patients had significantly higher levels of EBV DNA, even in patients with stage I disease. This result highlights the potential value of EBV DNA as an early detection marker for NPC. The detection rates of EBV DNA in NPC patients have been reported to vary in a range of 69–96%,12,17,20,23–26 our detection rate of 89.7% is in line with these results. The detection rates in six
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participating hospitals were similar (83–93%; Supplementary Table S1), indicating that the sampling and storage procedures in each of the participating hospitals were robust. The variation in detection rates among studies might be due to the difference in the number of samples analyzed, but given that our cohort contains a large number of patients (n = 390) to provide a strong statistical power, we can conclude that the sensitivity of the quantification of cellfree EBV DNA in NPC should be approximately 90%. In the present study, EBV DNA was detected in 3 of 36 patients in remission. All these three patients were subsequently diagnosed with liver metastasis or local recurrence (one confirmed and one suspected), supporting previous data that showed significant elevations in EBV DNA levels before detectable clinical deterioration in patients who subsequently developed tumour recurrence.17,27–29 Late presentation remains the major challenge in managing NPC.30,31 Currently the mainstay of treatment for advanced cases of NPC is concurrent chemoradiotherapy that invariably results in high morbidity and poor quality of life.32 Though rare, distant metastasis remains the major cause of death.8 Previous studies have shown that the median EBV DNA level was higher in patients with distant metastasis compared to those did not have systemic diseases.17,20,25 However, these studies grouped patients without distant metastasis at all stages in one category, even though it is unlikely that these patients would have higher viral loads because patients with early stage disease were included. Here, we performed a more accurate comparison by determining the correlation between EBV DNA levels and distant metastasis only in stage IV patients. We show that stage IV patients with distant metastases had significantly higher EBV DNA levels than those who did not have systemic spread of the disease, indicating plasma EBV DNA load could be a marker to aid the diagnosis of distant metastasis. Further, a ROC analysis determined 8000 copies/mL of plasma EBV DNA as the optimal cut-off point to predict the overall survival of the NPC patients. Both univariate and multivariate analyses further confirmed that patients with EBV DNA level <8000 copies/mL had better survival probability compared to those who had ≥8000 copies/mL. While Le et al.29 have shown that EBV DNA had a limited value in predicting patient survival, our data support other reports demonstrating that pre-therapy circulating EBV DNA load is an independent prognostic factor for overall survival in NPC.17,18 Therefore, further studies to confirm the predictive value of EBV DNA are warranted. In summary, we show that plasma EBV DNA is specific for NPC and reflects tumour burden. Importantly, our data demonstrate for the first time that plasma EBV DNA measurement could aid a more accurate diagnosis for early stage disease, as well as for distant metastases in stage IV patients. We propose that in developing countries where MRI and PET/CT scans are prohibitively expensive for routine use, plasma EBV DNA measurement could be used to stratify patients for a more accurate imaging protocol.
Funding This project was funded by the Ministry of Health, Malaysia (project code: JPP-IMR 06-060) and Cancer Research Initiatives Foundation.
Competing interests None declared.
Ethical approval Ethical approval was obtained from the Medical Research Ethics Committee from the Ministry of Health, Malaysia (KKM/NIHSEC/08/0804/MRG-IMR). Acknowledgements We wish to thank Drs. Dennis Lo and Lisa Chan from the Chinese University of Hong Kong for their technical advice. We would also like to thank the Director of General Health, Ministry of Health, the Director of the Institute for Medical Research, the Director of the Network of Clinical Research Centre and the Directors of all participating hospitals for their support. We wish to acknowledge the contributions of Ms. Yoon Sook Yee, Ms. Jayanthi, otorhinolaryngologists, oncologists, radiotherapists, and pathologists of the participating hospitals, the staff of the Clinical Research Centre of Hospital Kuala Lumpur, Hospital Pulau Pinang, and Sime Darby Medical Centre. Appendix A. The Malaysian Nasopharyngeal Carcinoma Study Group: • Hospital Pulau Pinang: KC Pua, N Punithavati, BS Tan, YS Ee, LM Ong, RA Hamid, M Goh, JCT Quah, J Lim. • Hospital Kuala Lumpur/UPM: YY Yap, FN Lau, PV Kam. • University of Malaya: G Gopala Krishnan, AZ Bustam, LM Looi, P Shahfinaz, O Hashim, CC Ng, O Rahmat, J Amin. • Sarawak General Hospital/UNIMAS: SK Subramaniam, TS Tiong, UH Sim, TW Tharumalingam, D Norlida, M Zulkarnaen. • Queen Elizabeth Hospital: CL Lum, A Nordin, Halimuddin S, Somasundram. • Hospital USM Kubang Kerian: S Hassan, B Biswal, H Nik Fariza, HA Mubassir, AH Suzina Sheikh. • Institute for Medical Research: ASB Khoo. • Cancer Research Initiatives Foundation: SH Teo, LF Yap. Appendix B. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jcv.2012.05.017. References 1. Razak AR, Siu LL, Liu FF, Ito E, O’Sullivan B, Chan K. Nasopharyngeal carcinoma: the next challenges. Eur J Cancer 2010;46:1967–78. 2. Cao XP, Lu TX, Ye WJ, Cui NJ. Prospective study on long-term efficacy of external plus intracavitary radiotherapy on stage I–II nasopharyngeal carcinoma. Ai Zheng 2007;26:204–7. 3. Leung TW, Tung SY, Sze WK, Wong FC, Yuen KK, Lui CM, et al. Treatment results of 1070 patients with nasopharyngeal carcinoma: an analysis of survival and failure patterns. Head Neck 2005;27:555–65. 4. Xiao WW, Han F, Lu TX, Chen CY, Huang Y, Zhao C. Treatment outcomes after radiotherapy alone for patients with early-stage nasopharyngeal carcinoma. Int J Radiat Oncol Biol Phys 2009;74:1070–6. 5. Pua KC, Khoo ASB, Subramaniam SK, Ong KA, Gopala Krishnan G, Shahid H, et al. Nasopharyngeal carcinoma database. Med J Malaysia 2008;63:3. 6. El-Sherbieny E, Rashwan H, Lubis SH, Choi VJ. Prognostic factors in patients with nasopharyngeal carcinoma treated in hospital Kuala Lumpur. Asian Pac J Cancer Prev 2011;12:1739–43. 7. Prasad U, Ablashi D, Pratha Kea. Problem of occult primary in nasopharyngeal carcinoma. In: Prasad U, editor. Nasopharyngeal carcinoma: current concepts. Kuala Lumpur: University of Malaya Press; 1983. p. 11–5. 8. O’Sullivan B. Nasopharynx cancer: therapeutic value of chemoradiotherapy. Int J Radiat Oncol Biol Phys 2007;69:S118–21. 9. Teo PM, Kwan WH, Lee WY, Leung SF, Johnson PJ. Prognosticators determining survival subsequent to distant metastasis from nasopharyngeal carcinoma. Cancer 1996;77:2423–31. 10. Tsang RK, Vlantis AC, Ho RW, Tam JS, To KF, van Hasselt CA. Sensitivity and specificity of Epstein–Barr Virus IGA titer in the diagnosis of nasopharyngeal carcinoma: a three-year institutional review. Head Neck 2004;26:598–602.
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