Clinical Significance of Quantitative Analysis of EBV DNA in Plasma of NPC Patients After Curative Radiotherapy

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1 Clinical Significance of Quantitative Analysis of EBV DNA in Plasma of NPC Patients After Curative Radiotherapy *Jin-gao Li, M.D., *Xiao-chang Gong, M.D., *Xia Yuan, M.D., Xue-sen Zhou, M.Sc., Wen-xue Chen, M.Sc., *Fan Ao, M.D., *Meng Wu, M.D. Department of *Radiation Oncology, Molecular Medicine, Jiangxi Provincial Cancer Hospital, Nanchang, Jiangxi Province, , P.R.China. Purpose: To study the value of plasma EBV DNA analysis in monitoring recurrence and distant metastasis of nasopharyngeal carcinoma after curative radiotherapy. Methods and Materials: 360 NPC patients after curative radiotherapy were included in this study. Plasma EBV DNA was analyzed using Real- Time PCR technique. The results were compared with clinical work ups. Results: Plasma EBV DNA was positive in 87 and negative in 273 of the 360 patients. Among the 87 EBV DNA positive patients, 70 had their disease progressed including 25 recurrences and 45 metastases. Among the 273 plasma EBV DNA negative cases, 21 had their disease progressed including 17 recurrences and 4 metastases. So the Positive Predictive Value and Negative Keywords: Polymerase chain reaction; Nasopharyngeal carcinoma; Epstein-Barr virus (EBV) DNA; Recurrence; Distant metastasis Predictive Value was 80% and 92%, and the sensitive, specific, false positive, false negative, and accurate rate were 77%, 94%, 6%, 23% and 89% respectively for plasma EBV DNA analysis in detecting tumor progression. Altogether, There were 42 recurrent, 49 metastatic, and 269 disease-free cases. Plasma EBV DNA was positive in 25 of 42 cases with recurrence, 45 of 49 cases with metastases and 17 of 269 disease-free cases. The positive ratios and median concentrations of EBV DNA were 60%, 4700copies/ml; 92%, copies/ml, 77%, 38000copies/ml; and 6%, 0copy/ml respectively for recurrent, metastatic, progression and disease-free group. The difference was statistically significant. Conclusion: Plasma EBV DNA analysis is useful in surveillance of NPC following radiotherapy. INTRODUCTION Nasopharyngeal carcinoma is rare in most parts of the world but is common in southern China, Southeast Asia, North Africa and parts of the Middle East. 1 The mainstay treatment of NPC is radiotherapy, with promising result for early stage disease. 2 For more advanced disease, Concurrent chemo-radiotherapy is the most effective treatment and 5 year progression free survival rate is about 60%, with localregional recurrence and/or distant metastasis being the main factor of treatment failure. 3 Correspondence to: Jin-gao Li, M.D., Department of Radiation Oncology, Jiangxi Provincial Cancer Hospital, 519 East Beijing Road, Nanchang, , P.R.China. Tel: (+86) , Fax: (+86) , lijingao@hotmail.com 285

2 Jin-gao Li The extent of involvement by the tumor at the time of salvage treatment is an important determinant factor of survival for recurrent patients. 4 For distant metastatic cases, the outcome of treatment is often poor. But a small part of patients still may achieve long-time survival after chemotherapy or multiple-modality therapy. 5, 6 It is raisonable to postulate that the treatment outcome of recurrence or metastasis may be improved if they could be detected in an early stage when the tumor bourden is small and amenable to radical salvage treatment. Up to date, the diagnosis of tumor progression relies on the timely follow-up, usually by a complete medical history, physical examination especially of the head and neck region, radiological study of the primary and the suspected distant sites, and if practical, pathological study of the disease. Although contrast-enhanced computed tomography (CT) and magnetic resonance imaging (MRI) can provide anatomic information with high spatial resolution and can easily identify morphologic abnormalities, the appearance of edema, scar tissue, and radiation induced tissue changes have limit its value in the surveillance of recurrence after radiotherapy. 7,8 Radiological procedures about monitoring distant metastasis after treatment of nasopharyngeal carcinoma have not been well established. Studies from staging investigations suggest that systemic imaging by chest X-ray, bone scan and liver ultrasonography has limited value in asypmtomatic cases due to its low sensitivities. 9 For these reasons, the treatment of recurrent or metastatic disease diagnosed by the conventional methods remains discouraging. 5 New image techniques, such as FDG-PET, are more accurate in evaluating tumor responses to treatment and could permit early detection of tumor recurrence in patients with head and neck carcinoma, 10, 11 but their prohibitive costs make repeated examinations unfeasible for disease monitoring. IgA antibodies against EBV capsid antigen and early antigen have been used as serologic marks in screening and diagnosing of nasopharyngeal carcinoma. 12 The titers of VCA/IgA and EA/IgA drop with the success removal of the tumor, but they could be higher than those of normal positive populations in the first several years after curative radiotherapy, 13 which limits its value in monitoring recurrence or metastasis after radiotherapy. Plasma EBV DNA could be detected in most of nasopharyngeal carcinoma patients. 14 Concentrations of EBV DNA in the plasma of patients with NPC by quantitative fluorescent PCR have been correlated with the tumor burden. 15 They drop rapidly after removal of the tumor by surgery or radiotherapy, 16, 17 which theoretically makes it an ideal tool to detect residual, recurrent or metastatic disease in an early stage. Studies have shown that patients with recurrent tumors often have elevated EBV DNA levels before their recurrence, whereas those with continous remission disease have low or undetectable level of EBV DNA. 18 Yet the true value of plasma EBV DNA test in posttreatment surveillance of recurrence and metastasis has not been fully evaluated. The sensitivity, specifisity, positive predictive value and negative predictive value of plasma EBV DNA test in the diagnosis of tumor progression after treatment of nasopharyngeal carcinoma have never been reported in the literature. In this study, we analysed the plasma EBV DNA by quantitative real-time PCR, and prospective correlate the results with the clinical and radiological workups based on the hypothesis that real-time quantitative PCR analysis of plasma EBV DNA could serve as an early surrogate, and improve the accuracy for detection of recurrent and metastatic disease after curative radiotherapy in nasopharyngeal carcinoma. MATERIALS AND METHODS Patient characteristics From December10 to November 12, follow-up patients after curative radiotherapy were recruited in this prospective study. The study was approved by the Ethics Committee of the hospital. Written informed consent was received from all patients. There were 360 patients, including 257 male and 103 female. The median age was 48 (from 19 to 75). The median interval from the beginning of radiotherapy to the study was 10 months of (from4 to 147months). All patients were diagnosed nasopharyngeal carcinoma before radiotherapy with WHO type II or III. They all achieved clinical complete remission (CR) after radiotherapy. 30 patients also received concurrent chemotherapy of weekly cis-platin, with a dose of 40mg/m 2 surface body areas. Study Design Clinical work up included complete medical history, physical examination of head and neck region including nasopharyngoscopy, computed tomography (CT) or magnetic resonance imaging (MRI ) of head and neck from 3cm up the base of scull to the clavicle level, chest radiography, abdominal ultrasound. If pulmonary or hepatic metastasis is suspected but cannot be confirmed, computed tomography is performed to complete the radiological procedure. Bone scintigram was performed only if bone pain or bone tenderness was complained or if there is an elevation of the alkaline phosphatase and bone metastasis is suspected clinically. Local recurrence should be confirmed by histopathology and node recurrence should be confirmed by histopathology or at lease by cytology with fine needle aspiration. Detectability and concentrations of EBV by Austral - Asian Journal of Cancer ISSN , Vol. 7, No.5, December 2008pp

3 quantitative fluorescent PCR were compared with the clinical work-up results, which were used as golden standard to diagnose recurrence or metastasis. Blood samples were taken for plasma EBV DNA analysis at each follow-up time, but only the results of first sample analysis were used in this study. DNA Extraction and Real-Time Plasma EBV DNA Analysis Peripheral blood (3ml) was obtained from each patient, placed in an EDTA containing tube, and centrifuged at 2000 g for 5minutes. The plasma was then taken, transferred into plain polyproplene tubes and stored at 20 0 C for further processing. DNA from plasma was extracted as reported in the literature. 17 The amount of Plasma samples Austral - Asian Journal of Cancer ISSN , Vol. 7, No.5, December 2008pp

4 Jin-gao Li Table1. Plasma EBV DNA detectability and tumor control state EBV DNA tumor tumor total detectability progression control positive negative total Among the 87 cases with positive EBV DNA in their plasma, there were 17 cases with tumor control, and among the 273 cases with negative EBV DNA there were 21 cases with tumor progression, so the false positive rate and false negative rate were 6% (17/269) and 23% (21/91) respectively. Altogether, there 322 cases whose detectabilities of EBV DNA were in accordance with clinical or radiological results, thus the accurate rate was 89% (322/360). The differences of the positive rate and concentrations of plasma EBV DNA between loco-regional recurrence and distant metastasis, tumor control and tumor progression patients (Table 2) Among the 91 patients with tumor progression, there were 42 loco-regional recurrent cases and 49 distant metastatic cases. Among the 42 recurrent patients, 25 with positive plasma EBV DNA, so the positive rate was 60%. The concentrations of EBV DNA in the recurrent patients were from 0copy/ml to copies/ml with median Table2. Median concentrations and positive rate of plasma EBV DNA for different tumor control and tumor progression groups, loco-regional recurrence and metastasis groups Number of median positive patients concentrations* rate Tumor control % Tumor progression % recurrence % metastasis % *The plasma EBV DNA concentrations between tumor control and tumor progression groups, loco-regional recurrence and distant metastasis groups were compared twice: The first one use 0 to replace less than detection limit, and the second one use the detection limit ( ) to replace less than detection limit. Both have the same results(p=0.000). concentrations of copies/ml. Among the 49 patients with distant metastases, there were 45 with positive plasma EBV DNA, so the positive rate was 92%. The EBV DNA concentrations were from 0 copy/ml to copies/ml with median concentrations of copies/ml. The difference of the EBV DNA positive rates and its concentrations between the locoregional recurrent patients and distant metastasis cases were statistically significant ( both P=0.000). Among the 91 cases with tumor progression, 70 cases with positive plasma EBV DNA, so the positive rate was 77%. The plasma EBV DNA were from 0copy/ml to copies/ml, with median concentrations of copies/ml. Among 269 tumor control patients, 17 with positive plasma EBV DNA, so the positive rate was 6%. The EBV DNA concentrations were from 0copy/ml to copies/ml.the median concentrations were 0 copy/ml. The difference of the EBV DNA positive rates and its concentrations between the tumor progression patients and tumor progression cases were statistically significant ( both P=0.000). The first site of metastasis of the 49 patients with metastatic diseases was as follows: 21 with bone metastasis, 13 with lung metastasis, 7 with liver metastasis and 7 with metastases at multiple sites: metastases that involved at least any two of the bone, liver or lung. One patient has metastasis to the skin, and this patient was excluded from further analysis. There were 2 patients with negative plasma EBV DNA in the bone metastasis patients, and one patient each with negative plasma EBV DNA in the lung and liver metastasis patients. The median concentrations of plasma EBV DNA for bone, lung, liver and multiple site metastasis were copies/ml (from 0 to copies/ml), copies/ml (from 0 to copies/ml), copies/ml (from 0 to copies/ml), and copies/ml (from 0 to copies/ml). There were no statistically significant differences between any two groups and among the above groups in regards of the plasma EBV DNA concentrations (all have P>0.10). The follow-up results of tumor control patients with different EBV DNA detectability 27 patients with positive and 257 patients with negative EBV DNA in their plasma but without tumor progression at the time of the test after complete clinical, radiological and pathological examination were followed for a median of 4 months (from 2 to 10 months). 10 patients have their disease progressed including 2 loco-regional recurrence and 8 distant metastases in the EBV DNA positive group and 5 Austral - Asian Journal of Cancer ISSN , Vol. 7, No.5, December 2008pp

5 Clinical Significance of Quantitative Analysis of EBV DNA in Plasma... disease progression including 3 recurrences and 2 distant metastases were confirmed in the EBV DNA negative group. The probabilities of having progression disease of clinical tumor control patients with positive and negative EBV DNA were 37% and 2% respectively during a median period of 4 months. The difference was statistically significant (P=0.000 by chi-square test). The median EBV DNA concentrations of the 10 patients with tumor progression and the 17 patients without tumor progression during the follow-up period were copies/ml and copies/ml respectively, showing a tendency of higher EBV DNA in the tumor progression patients but without statistical significance (P=0.15 by Wisconxon rank-sum test). DISCUSSION A simple, reliable, sensitive and specific blood test for tumor progression after treatment would be valuable in the serveillance of tumor recurrence and metastasis after curative treatment. Unlike the so-called conventional clinical surveillance, Blood test permits a whole body screen rather than relying on symptoms of the site of the disease, and it is less resource intensive than the whole body screen methods such as PET scan. Repeated measurements of serum CEA in patients with colorectal cancer and PSA in patients with prostate cancer have provided an early surrogate for evaluating effects of new treatment modalities, become an useful tool for detecting treatment failure, 19, 20 and have associated with a reduction in all cause mortality in colorectal cancer. 19 But for most other types of cancer, there are currently no established blood test methods in monitoring recurrence and metastasis, mostly because of a lack of specific and sensitive tumor markers. Although the exact carcinogenic effect of EBV remains unclear, it is generally agreed that EBV infection is a major risk factor for nasopharyngeal carcinoma. 21 This is supported by the finding that EBV could be detected consistently in all undifferentiated nasopharyngeal carcinoma tissues. 21 Elevated titers of antibodies, such as IgA antibodies against EBV capsid antigen and early antigen(vca/iga and EA/ IgA) and IgG antibodies against EBV capsid antigen, early antigen, and soluble complement-fixing antigen can also be detected in patients with NPC. 22, 23 IgA antibodies against EBV capsid antigen and early antigen have been used as serologic marks in screening and diagnosing of nasopharyngeal carcinoma. 12 Although the titers of VCA/ IgA and EA/IgA drop with the success removal of the tumor, they could be higher than those of normal positive populations in the first several years after curative radiotherapy, 13 which limits its value in monitoring recurrence or metastasis after radiotherapy. It has been reported that plasma EBV DNA could be detected in most radiation-naïve nasopharyngeal carcinoma patients but seldom detected in healthy controls. 14 Concentrations of EBV DNA in the plasma of patients with NPC by quantitative fluorescent PCR have been correlated with the tumor burden. 15 It has also been shown that plasma EBV DNA rapidly declined shortly after surgical remove of the tumor with a median half-life of 139 mins, and shortly after the start of radiotherapy with a median half-life of 3.8 days. 16, 17 Thereafter, plasma EBV DNA is a potential marker for surveillance of nasopharyngeal carcinoma after treatment. Using the clinical and radiological examinations as the golden standard for diagnosing recurrence or metastasis, our study shows that the positive prognostic value of plasma EBV DNA test is 80% and the negative prognostic value is 92%. This means that plasma EBV DNA analysis correlates well with the routine clinical surveillance, especially when the EBV DNA is negative. Most importantly, Even the routine surveillance is negative at the time of PCR analysis, the possibilities of tumor progression are as high as 37% after a median interval of 4 months when the plasma EBV DNA is positive, whereas the possibilities of tumor progression are only 2% when the EBV DNA is negative. This suggests that when the plasma EBV DNA is positive after radiotherapy, more complete workup, such as PET is justified. If the complete examination is negative, closer surveillance is still needed. When the plasma EBV DNA is negative, the possibility of tumor progression in 4 months is so small that routin follow-up interval, usually 6 months is safe and rational. There are limited yet controversal reports about the positive rate between the loco-regional recurrent patients and distant metastatic patients. Li et al 24 compared the positive rate of plasma EBV DNA between 8 patients with local recurrence and 22 patients with distant metastases. The positive rates were 100% and 98% respectively. Recently, Shao et al. 25 also reported a 100% positive rate of plasma EBV DNA in 8 patients with recurrent diseases. Similar with the Hong et al s report, 26 we found that patients with distant mestastatic disease after radiotherapy have significant higher positive rate of plasma EBV DNA than those with loco-regional disease. The positive rate of plasma EBV DNA of metastatic patients is similar to that of the radiation-naïve patients reported in the literature, whereas the positive rate of the recurrent patients is lower than that of the radiation-naïve patients. 12 The most possible reason is that radiation induced stromal fibrosis, which is a common Austral - Asian Journal of Cancer ISSN , Vol. 7, No.5, December 2008 pp

6 Jin-gao Li complication following high dose radiotherapy, could decrease the microcirculation, 27 and partially blocks the release of EBV DNA from the tumor tissue. The second reason is that cancer cells in the blood system can be killed by various mechanisms, so EBV DNA is released directly to the circulation system. Other reasons, although less likely, include changed release machinery after irradiation and relatively smaller tumor burden of recurrent patients than that of the metastatic cases. Regardless of the reasons, our study indicates that plasma EBV DNA analysis was superior to the conventional follow-up methods for patients with distant metastasis. For patients with locoregional recurrent disease, plasma EBV DNA analysis was not sensitive and its value in this group of patients needs further study. To further study if this blood test could indicate the specific metastatic sites, we compared the concentrations of plasma EBV DNA in patients with bone, lung, liver and multiple site metastases. None of the differences attains statistically significant level. This is in difference with the Hong s and Shao s study. In Hong s study, 26 patients who had liver or bone metastases had more concentrations of EBV DNA compared with patients who had lung metastases. Whereas in Shao s study, 25 patients with metastatic disease in the liver had the most concentrations of plasma EBV DNA, followed by lung metastasis. The patients with bone metastasis had the lest concentrations of plasma EBV DNA. The authors argued that differences of the metastatic tumor burden or the ease of release of EBV DNA into the blood system account for this results. Because regular test of plasma EBV DNA was not adopted as a tool in monitoring recurrence or metastasis of nasopharyngeal carcinoma in our hospital until the end of this study, as was in the Hong s study, 26 our group of patients with metastatic diseases may be diagnosed in more advanced stage. The differences of the tumor burdens among patients with different metastatic sites were not so significant as to lead to different concentrations of plasma EBV DNA. Another reason that the plasma EBV DNA concentrations were not different among patients with different metastatic sites would be related to the metastatic patterns of nasopharyngeal carcinoma. As reported by Leung et al, 48% of patients with metastatic nasopharyngeal carcinoma concomitantly developed metastatic disease in multiple sites including bone, live and lung. 28 Thus the first metastatic sites recorded in our group of patients may only reflect a diagnosis bias, that is to say, some of the patients with metastasis confined to a single organ may still have or will eventually have multiple site metastatic diseases: the concentrations of plasma EBV DNA in any metastasis group may not exactly reflect the concentrations of the specific organs but rather reflect the concentrations of multiple site metastases. This could also explain why the concentrations of the plasma EBV DNA of the patients with multiple site metastatic diseases were not different to those of any group of patients with single metastatic site. In conclusion, based on our data, conclusive association between the concentrations of plasma EBV DNA and the specific metastatic site cannot be confirmed. The accurate rate of EBV DNA test in diagnosing tumor progression could be higher than that of our study. The first is the relatively short follow-up time after the EBV DNA test. Lo et al 15 reported that plasma EBV DNA could be detected 6 months before the tumor progression. As the follow-up continues, more recurrence or metastasis will be confirmed and the accurate rate could be higher. The second is the ignorance of the plasma EBV DNA detectability of our patients before radiotherapy. Because a small part of patients have negative plasma EBV DNA before radiotherapy, 11 the plasma EBV DNA of these patients will most probably be negative even they have progressed disease. If the plasma EBV DNA was tested before the initiation of the treatment and only the patients with positive EBV DNA were followed by this means, the accurate rate could be further higher. Plasma EBV DNA test has a high specificity (94%) but relatively low sensitivity (77%). This means that surveillance of nasopharyngeal patients following radiotherapy by EBV DNA test alone has a low misdiagnosis rate but will miss about one-fourth progressed tumors. Clinical and radiological examinations should still be the basis of surveillance. Although plasma EBV DNA test is a useful means in the surveillance of patients with nasopharyngeal carcinoma after radiotherapy, several questions remain to be resolved 17 : 1) Is the tumor visible with FDG-PET if the plasma EBV DNA is positive but the routine clinical and radiological examinations is negative? 2) If FDG-PET is positive, could early treatment of these patients have better treatment outcome? 3) If FDG-PET is negative, Is the systemic treatment justified? Further studies may answer these questions. REFERENCES 1 Yu MC and Yuan JM. Epidemiology of nasopharyngeal carcinoma. Seminars in Cancer Biology 2002;12: Chua DT, Sham JS, Kwong DL, et al. 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