- Email: [email protected]
serum concentration of Epstein-Barr virus DNA in nasopharyngeal carcinoma
Dossier: Epstein-Barr virus
Biomed Pharmacother 2001 ; 55 : 362-5 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S075333220100083X/FLA
Prognostic implication of pretreatment plasma/serum concentration of Epstein-Barr virus DNA in nasopharyngeal carcinoma Y.M.D. Lo* Department of Chemical Pathology and Institute of Molecular Oncology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, People’s Republic of China (Received 28 June 2001; accepted 2 July 2001)
Summary – Recently, much interest has been focused on the diagnostic application and biology of tumor-derived DNA in the plasma and serum of cancer patients. Such interest has resulted in the demonstration of Epstein-Barr virus (EBV) DNA in the plasma/serum of patients with nasopharyngeal carcinoma (NPC). Using quantitative real-time polymerase chain reaction (PCR) technology, circulating EBV DNA has been found not only to correlate with disease staging, but also to provide additional prognostic information. Following treatment, circulating EBV DNA analysis has been shown to provide useful information for the monitoring for tumor recurrence. In addition to NPC, it is envisaged that circulating DNA technology will find wide applications in the detection and monitoring of many other types of malignancies. © 2001 Éditions scientifiques et médicales Elsevier SAS circulating DNA / prognostication / tumor marker
Nasopharyngeal carcinoma (NPC) is one of the five most common cancers in Hong Kong. It possesses a specific geographical distribution, being common in Southern China and Southeast Asia, but is rare in the West [27]. NPC has a very strong association with Epstein-Barr virus (EBV) infection [36], with EBV DNA being detectable in almost all of the cases in Hong Kong [7]. As the survival rate following treatment is significantly improved for early- compared with advanced-stage disease [32], there is a keen interest in the development of new tumor markers for NPC. In this review, I shall discuss the development of one such marker for this type of cancer, which is based on circulating EBV DNA analysis, and review its role in the prognostication of this disease.
*Correspondence and reprints. E-mail address: [email protected] (Y.M.D. Lo).
CIRCULATING DNA AS A TUMOR MARKER The first description of circulating DNA in the plasma of human subjects has been attributed to the pioneering work by Mandel and Métais in 1948 [23]. However, this work was largely ignored until the 1960s, when interest in the subject was renewed by the finding of large quantities of circulating DNA in patients with systemic lupus erythematosus [31]. Further interest was developed in the 1970s, when increased concentrations of circulating DNA were found in cancer patients [14]. In 1989, Stroun et al. proposed the interesting hypothesis that part of the circulating DNA present in cancer patients may be released by tumor cells [30]. The conclusive proof of this hypothesis came a few years later when a number of workers demonstrated that tumor-derived genetic alterations could be detected in the plasma and serum of cancer patients [4, 25, 29, 33]. Since then, research
Pretreatment of EBV DNA in nasopharyngeal carcinoma
into the field of circulating DNA as a tumor marker has grown rapidly. Tumor targets, which have been detected in the plasma/serum, include oncogene mutations [1, 29, 33, 35], oncogene amplifications [5] and epigenetic alterations [34]. CIRCULATING EBV DNA IN NPC PATIENTS The successful results obtained in the detection of circulating tumor-derived DNA in the plasma of cancer patients have encouraged workers in the field of NPC to investigate whether this technology may also be applicable to the latter malignancy. The close relationship between EBV and NPC makes EBV DNA an obvious tumor target in the plasma/serum of NPC patients. Thus, Mutirangura et al. demonstrated that EBV DNA was detectable in 31% of the serum of NPC patients [24]. Using real-time polymerase chain reaction (PCR) technology, Lo et al. were able to increase the sensitivity of detection to 96%, while at the same time obtaining the first quantitative data on this phenomenon [20]. During radiation therapy, circulating EBV DNA was found to decrease with a median half-life of 3.8 days [22]. It has been proposed that this half-life is an indication of the radiosensitivity of a particular tumor to such treatment. Following therapy, circulating EBV DNA measurement has also been shown to be of use for detecting tumor recurrence [17, 28].
363
ing reaching only borderline statistical significance. In a second cohort, Lo et al. also demonstrated that both serum EBV DNA and disease stage were independent prognostic factors for long-term survival in patients with this disease [19]. Very similar data have recently been published by Lin et al., who have shown that the detectability of EBV DNA in the cellular fraction of blood from NPC patients is correlated with prognosis [15]. The use of DNA extracted from the cellular elements of blood and the use of a qualitative (instead of a quantitative) detection method are the main differences between the approach of Lin et al. [15] and that of Lo et al. [19]. Lin et al. were of the opinion that the EBV DNA detected in their assay originated from circulating tumor cells. It would be interesting to correlate the results of this cellular assay with the plasma assay, and determine which of these would be more applicable to routine clinical use. Thus, taken together, the data provided by Lo et al. [19] and Lin et al. [15] indicate that the peripheral blood EBV DNA load is a new and powerful prognostic factor for NPC. It could be argued that this parameter should be considered for inclusion in future revisions of the staging system for NPC. Furthermore, it is possible that the use of this new prognostic factor may delineate a subgroup of NPC subjects who are at increased risk of tumor recurrence, and who thus would benefit from a more aggressive course of treatment. This would potentially improve survival in this group of patients.
PROGNOSTIC IMPLICATIONS It has been demonstrated that plasma EBV DNA bears a positive correlation with disease stage in NPC subjects [20]. Thus, advanced stage (American Joint Committee on Cancer, stages III and IV) patients had a median plasma EBV DNA concentration some 7.9 times above those with early stage (I and II) disease. It would thus be important to ascertain whether plasma EBV DNA is an independent prognostic marker for NPC, in addition to disease staging. With this objective in mind, Lo et al. demonstrated that the pretreatment levels of plasma EBV DNA are a powerful predictor of early events, defined as tumor recurrence or metastasis within 1 year of treatment [19]. Indeed, in multivariate analysis in which both plasma EBV DNA and disease staging were included, plasma EBV DNA remained the only significant prognostic factor for early events, with disease stag-
FUTURE DIRECTIONS The demonstration of the prognostic significance of plasma EBV DNA measurement in NPC opens up the possibility that such an analysis might also be useful for other EBV-associated malignancies. In this regard, circulating EBV DNA has been detected in many types of cancer, including Hodgkin’s disease, other lymphomas [8, 10, 13], and gastric carcinoma [21]. It would therefore be worthwhile to investigate whether circulating EBV DNA concentration is also a prognostic factor in these types of cancer. In addition to circulating EBV DNA, cell-free EBV-associated RNA has recently been demonstrated in the plasma of NPC patients [16]. As EBV is a DNA virus, the detected RNA represents a transcriptional product from the EBV genome. It remains to be seen whether the inclusion of an RNA-based
364
Y.M.D. Lo
assay would supply any additional diagnostic or monitoring information to that provided by the DNAbased assay alone. Technically, an RNA-based assay would probably be more difficult to work with, in view of the known lability of RNA. Nonetheless, since this report was published, a number of other tumor-derived RNA targets have also been detected in the plasma/serum of cancer patients [3, 6, 12], thus opening up the possibility of non-invasive gene expression profiling from plasma. Apart from EBV, there are many other viruses which are associated with cancers. It would therefore be of interest to study the role of viral nucleic acid detection in the detection, monitoring and prognosis of these cancers. Indeed, such an approach has recently been described for the detection of human papillomavirus DNA in the plasma/serum of patients with cervical cancer [26] and head and neck cancer [2]. The success of plasma/serum EBV DNA as a cancer detection/monitoring tool demonstrates the efficacy of the quantitative analysis of circulating tumorderived DNA. In this regard, it suggests that quantitative analysis of other tumor-derived DNA species could also yield important clinical and biological information. Quantitative tools suitable for this type of analysis have recently been described, e.g., quantitative methylation analysis [9, 18]. Indeed, the latter tool has recently been used to show that tumor-derived methylation changes in plasma are a prognostic factor [11]. It is likely that this type of analysis will be carried out for a variety of other tumor types in the future, making plasma DNA analysis an important addition to our armamentarium against cancer. ACKNOWLEDGEMENTS The author has been supported by the Innovation and Technology Fund, and is a member of the Hong Kong Cancer Genetics Research Group, funded by the Kadoorie Charitable Foundations. REFERENCES 1 Anker P, Lefort F, Vasioukhin V, Lyautey J, Lederrey C, Chen XQ, et al. K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer. Gastroenterology 1997 ; 112 : 1114-20.
2 Capone RB, Pai SI, Koch WM, Gillison ML, Danish HN, Westra WH, et al. Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV-associated head and neck squamous cell carcinoma. Clin Cancer Res 2000 ; 6 : 4171-5. 3 Chen XQ, Bonnefoi H, Pelte MF, Lyautey J, Lederrey C, Movarekhi S, et al. Telomerase RNA as a detection marker in the serum of breast cancer patients. Clin Cancer Res 2000 ; 6 : 3823-6. 4 Chen XQ, Stroun M, Magnenat JL, Nicod LP, Kurt AM, Lyautey J, et al. Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nat Med 1996 ; 2 : 1033-5. 5 Chiang PW, Beer DG, Wei WL, Orringer MB, Kurnit DM. Detection of erbB-2 amplifications in tumors and sera from esophageal carcinoma patients. Clin Cancer Res 1999 ; 5 : 1381-6. 6 Dasi F, Lledo S, Garcia-Granero E, Ripoll R, Marugan M, Tormo M, et al. Real-time quantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA: a simple blood test to monitor disease in cancer patients. Lab Invest 2001 ; 81 : 767-9. 7 Dickens P, Srivastava G, Loke SL, Chan CW, Liu YT. EpsteinBarr virus DNA in nasopharyngeal carcinomas from Chinese patients in Hong Kong. J Clin Pathol 1992 ; 45 : 396-7. 8 Drouet E, Brousset P, Fares F, Icart J, Verniol C, Meggetto F, et al. High Epstein-Barr virus serum load and elevated titers of antiZEBRA antibodies in patients with EBV-harboring tumor cells of Hodgkin’s disease. J Med Virol 1999 ; 57 : 383-9. 9 Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, et al. MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 2000 ; 28 : E32. 10 Gallagher A, Armstrong AA, MacKenzie J, Shield L, Khan G, Lake A, et al. Detection of Epstein-Barr virus (EBV) genomes in the serum of patients with EBV-associated Hodgkin’s disease. Int J Cancer 1999 ; 84 : 442-8. 11 Kawakami K, Brabender J, Lord RV, Groshen S, Greenwald BD, Krasna MJ, et al. Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma. J Natl Cancer Inst 2000 ; 92 : 1805-11. 12 Kopreski M, Benko FA, Kwak LW, Gocke CD. Detection of tumor messenger RNA in the serum of patients with malignant melanoma. Clin Cancer Res 1999 ; 5 : 1961-5. 13 Lei KI, Chan LY, Chan WY, Johnson PJ, Lo YMD. Quantitative analysis of circulating cell-free Epstein–Barr virus (EBV) DNA levels in patients with EBV-associated lymphoid malignancies. Br J Haematol 2000 ; 111 : 239-46. 14 Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 1977 ; 37 : 646-50. 15 Lin JC, Chen KY, Wang WY, Jan JS, Liang WM, Tsai CS, et al. Detection of Epstein-Barr virus DNA the peripheral-blood cells of patients with nasopharyngeal carcinoma: relationship to distant metastasis and survival. J Clin Oncol 2001 ; 19 : 2607-15. 16 Lo KW, Lo YMD, Leung SF, Tsang YS, Chan LY, Johnson PJ, et al. Analysis of cell-free Epstein-Barr virus associated RNA in the plasma of patients with nasopharyngeal carcinoma. Clin Chem 1999 ; 45 : 1292-4. 17 Lo YMD, Chan LY, Chan AT, Leung SF, Lo KW, Zhang J, et al. Quantitative and temporal correlation between circulating cellfree Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 1999 ; 59 : 5452-5. 18 Lo YMD, Wong IH, Zhang J, Tein MS, Ng MH, Hjelm NM. Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specific polymerase chain reaction. Cancer Res 1999 ; 59 : 3899-903. 19 Lo YMD, Chan ATC, Chan LYS, Leung SF, Lam CW, Huang DP, et al. Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating Epstein-Barr virus DNA. Cancer Res 2000 ; 60 : 6878-81.
Pretreatment of EBV DNA in nasopharyngeal carcinoma
20 Lo YMD, Chan LY, Lo KW, Leung SF, Zhang J, Chan AT, et al. Quantitative analysis of cell-free Epstein–Barr virus DNA in plasma of patients with nasopharyngeal carcinoma. Cancer Res 1999 ; 59 : 1188-91. 21 Lo YMD, Chan WY, Ng EKW, Chan LYS, Lai PBS, Tam JS, et al. Circulating Epstein-Barr virus DNA in the serum of patients with gastric carcinoma. Clin Cancer Res 2001 ; 7 : 1856-9. 22 Lo YMD, Leung SF, Chan LY, Chan AT, Lo KW, Johnson PJ, et al. Kinetics of plasma Epstein-Barr virus DNA during radiation therapy for nasopharyngeal carcinoma. Cancer Res 2000 ; 60 : 2351-5. 23 Mandel P, Métais P. Les acides nucléiques du plasma sanguin chez l’homme. CR Acad Sci Paris 1948 ; 142 : 241-3. 24 Mutirangura A, Pornthanakasem W, Theamboonlers A, Sriuranpong V, Lertsanguansinchi P, Yenrudi S, et al. Epstein-Barr viral DNA in serum of patients with nasopharyngeal carcinoma. Clin Cancer Res 1998 ; 4 : 665-9. 25 Nawroz H, Koch W, Anker P, Stroun M, Sidransky D. Microsatellite alterations in serum DNA of head and neck cancer patients. Nat Med 1996 ; 2 : 1035-7. 26 Pornthanakasem W, Shotelersuk K, Termrungruanglert W, Voravud N, Niruthisard S, Mutirangura A. Human papillomavirus DNA in plasma of patients with cervical cancer. BMC Cancer 2001 ; 1 : 2. 27 Rickinson AB, Kieff E. Epstein-Barr virus. In: Fields BN, Knipe DM, Howley PM, Eds. Fields virology. Third edition. Philadelphia: Lippincott-Raven Publishers; 1996. p. 2397-446. 28 Shotelersuk K, Khorprasert C, Sakdikul S, Pornthanakasem W, Voravud N, Mutirangura A. Epstein-Barr virus DNA in serum/plasma as a tumor marker for nasopharyngeal cancer. Clin Cancer Res 2000 ; 6 : 1046-51.
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29 Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik DJ, Yao SL. Soluble normal and mutated DNA sequences from singlecopy genes in human blood. Cancer Epidemiol Biomarkers Prev 1994 ; 3 : 67-71. 30 Stroun M, Anker P, Maurice P, Lyautey J, Lederrey C, Beljanski M. Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology 1989 ; 46 : 318-22. 31 Tan EM, Schur PH, Carr RI, Kunkel HG. Deoxyribonucleic acid (DNA) and antibodies to DNA in the serum of patients with systemic lupus erythematosus. J Clin Invest 1966 ; 45 : 1732-40. 32 Tatsumi-Tamori A, Yoshizaki T, Miwa T, Furukawa M. Clinical evaluation of staging system for nasopharyngeal carcinoma: comparison of fourth and fifth editions of UICC TNM classification. Ann Otol Rhinol Laryngol 2000 ; 109 : 1125-9. 33 Vasioukhin V, Anker P, Maurice P, Lyautey J, Lederrey C, Stroun M. Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia. Br J Haematol 1994 ; 86 : 774-9. 34 Wong IH, Lo YMD, Zhang J, Liew CT, Ng MH, Wong N, et al. Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients. Cancer Res 1999 ; 59 : 71-3. 35 Yamada T, Nakamori S, Ohzato H, Oshima S, Aoki T, Higaki N, et al. Detection of K-ras gene mutations in plasma DNA of patients with pancreatic adenocarcinoma: correlation with clinicopathological features. Clin Cancer Res 1998 ; 4 : 1527-32. 36 zur Hausen H, Schulte-Holthausen H, Klein G, Henle W, Clifford P. EBV DNA in biopsies of Burkitt’s tumors and anaplastic carcinomas of the nasopharynx. Nature 1970 ; 228 : 1056-8.
Biomed Pharmacother 2001 ; 55 : 362-5 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S075333220100083X/FLA
Prognostic implication of pretreatment plasma/serum concentration of Epstein-Barr virus DNA in nasopharyngeal carcinoma Y.M.D. Lo* Department of Chemical Pathology and Institute of Molecular Oncology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, New Territories, Hong Kong Special Administrative Region, People’s Republic of China (Received 28 June 2001; accepted 2 July 2001)
Summary – Recently, much interest has been focused on the diagnostic application and biology of tumor-derived DNA in the plasma and serum of cancer patients. Such interest has resulted in the demonstration of Epstein-Barr virus (EBV) DNA in the plasma/serum of patients with nasopharyngeal carcinoma (NPC). Using quantitative real-time polymerase chain reaction (PCR) technology, circulating EBV DNA has been found not only to correlate with disease staging, but also to provide additional prognostic information. Following treatment, circulating EBV DNA analysis has been shown to provide useful information for the monitoring for tumor recurrence. In addition to NPC, it is envisaged that circulating DNA technology will find wide applications in the detection and monitoring of many other types of malignancies. © 2001 Éditions scientifiques et médicales Elsevier SAS circulating DNA / prognostication / tumor marker
Nasopharyngeal carcinoma (NPC) is one of the five most common cancers in Hong Kong. It possesses a specific geographical distribution, being common in Southern China and Southeast Asia, but is rare in the West [27]. NPC has a very strong association with Epstein-Barr virus (EBV) infection [36], with EBV DNA being detectable in almost all of the cases in Hong Kong [7]. As the survival rate following treatment is significantly improved for early- compared with advanced-stage disease [32], there is a keen interest in the development of new tumor markers for NPC. In this review, I shall discuss the development of one such marker for this type of cancer, which is based on circulating EBV DNA analysis, and review its role in the prognostication of this disease.
*Correspondence and reprints. E-mail address: [email protected] (Y.M.D. Lo).
CIRCULATING DNA AS A TUMOR MARKER The first description of circulating DNA in the plasma of human subjects has been attributed to the pioneering work by Mandel and Métais in 1948 [23]. However, this work was largely ignored until the 1960s, when interest in the subject was renewed by the finding of large quantities of circulating DNA in patients with systemic lupus erythematosus [31]. Further interest was developed in the 1970s, when increased concentrations of circulating DNA were found in cancer patients [14]. In 1989, Stroun et al. proposed the interesting hypothesis that part of the circulating DNA present in cancer patients may be released by tumor cells [30]. The conclusive proof of this hypothesis came a few years later when a number of workers demonstrated that tumor-derived genetic alterations could be detected in the plasma and serum of cancer patients [4, 25, 29, 33]. Since then, research
Pretreatment of EBV DNA in nasopharyngeal carcinoma
into the field of circulating DNA as a tumor marker has grown rapidly. Tumor targets, which have been detected in the plasma/serum, include oncogene mutations [1, 29, 33, 35], oncogene amplifications [5] and epigenetic alterations [34]. CIRCULATING EBV DNA IN NPC PATIENTS The successful results obtained in the detection of circulating tumor-derived DNA in the plasma of cancer patients have encouraged workers in the field of NPC to investigate whether this technology may also be applicable to the latter malignancy. The close relationship between EBV and NPC makes EBV DNA an obvious tumor target in the plasma/serum of NPC patients. Thus, Mutirangura et al. demonstrated that EBV DNA was detectable in 31% of the serum of NPC patients [24]. Using real-time polymerase chain reaction (PCR) technology, Lo et al. were able to increase the sensitivity of detection to 96%, while at the same time obtaining the first quantitative data on this phenomenon [20]. During radiation therapy, circulating EBV DNA was found to decrease with a median half-life of 3.8 days [22]. It has been proposed that this half-life is an indication of the radiosensitivity of a particular tumor to such treatment. Following therapy, circulating EBV DNA measurement has also been shown to be of use for detecting tumor recurrence [17, 28].
363
ing reaching only borderline statistical significance. In a second cohort, Lo et al. also demonstrated that both serum EBV DNA and disease stage were independent prognostic factors for long-term survival in patients with this disease [19]. Very similar data have recently been published by Lin et al., who have shown that the detectability of EBV DNA in the cellular fraction of blood from NPC patients is correlated with prognosis [15]. The use of DNA extracted from the cellular elements of blood and the use of a qualitative (instead of a quantitative) detection method are the main differences between the approach of Lin et al. [15] and that of Lo et al. [19]. Lin et al. were of the opinion that the EBV DNA detected in their assay originated from circulating tumor cells. It would be interesting to correlate the results of this cellular assay with the plasma assay, and determine which of these would be more applicable to routine clinical use. Thus, taken together, the data provided by Lo et al. [19] and Lin et al. [15] indicate that the peripheral blood EBV DNA load is a new and powerful prognostic factor for NPC. It could be argued that this parameter should be considered for inclusion in future revisions of the staging system for NPC. Furthermore, it is possible that the use of this new prognostic factor may delineate a subgroup of NPC subjects who are at increased risk of tumor recurrence, and who thus would benefit from a more aggressive course of treatment. This would potentially improve survival in this group of patients.
PROGNOSTIC IMPLICATIONS It has been demonstrated that plasma EBV DNA bears a positive correlation with disease stage in NPC subjects [20]. Thus, advanced stage (American Joint Committee on Cancer, stages III and IV) patients had a median plasma EBV DNA concentration some 7.9 times above those with early stage (I and II) disease. It would thus be important to ascertain whether plasma EBV DNA is an independent prognostic marker for NPC, in addition to disease staging. With this objective in mind, Lo et al. demonstrated that the pretreatment levels of plasma EBV DNA are a powerful predictor of early events, defined as tumor recurrence or metastasis within 1 year of treatment [19]. Indeed, in multivariate analysis in which both plasma EBV DNA and disease staging were included, plasma EBV DNA remained the only significant prognostic factor for early events, with disease stag-
FUTURE DIRECTIONS The demonstration of the prognostic significance of plasma EBV DNA measurement in NPC opens up the possibility that such an analysis might also be useful for other EBV-associated malignancies. In this regard, circulating EBV DNA has been detected in many types of cancer, including Hodgkin’s disease, other lymphomas [8, 10, 13], and gastric carcinoma [21]. It would therefore be worthwhile to investigate whether circulating EBV DNA concentration is also a prognostic factor in these types of cancer. In addition to circulating EBV DNA, cell-free EBV-associated RNA has recently been demonstrated in the plasma of NPC patients [16]. As EBV is a DNA virus, the detected RNA represents a transcriptional product from the EBV genome. It remains to be seen whether the inclusion of an RNA-based
364
Y.M.D. Lo
assay would supply any additional diagnostic or monitoring information to that provided by the DNAbased assay alone. Technically, an RNA-based assay would probably be more difficult to work with, in view of the known lability of RNA. Nonetheless, since this report was published, a number of other tumor-derived RNA targets have also been detected in the plasma/serum of cancer patients [3, 6, 12], thus opening up the possibility of non-invasive gene expression profiling from plasma. Apart from EBV, there are many other viruses which are associated with cancers. It would therefore be of interest to study the role of viral nucleic acid detection in the detection, monitoring and prognosis of these cancers. Indeed, such an approach has recently been described for the detection of human papillomavirus DNA in the plasma/serum of patients with cervical cancer [26] and head and neck cancer [2]. The success of plasma/serum EBV DNA as a cancer detection/monitoring tool demonstrates the efficacy of the quantitative analysis of circulating tumorderived DNA. In this regard, it suggests that quantitative analysis of other tumor-derived DNA species could also yield important clinical and biological information. Quantitative tools suitable for this type of analysis have recently been described, e.g., quantitative methylation analysis [9, 18]. Indeed, the latter tool has recently been used to show that tumor-derived methylation changes in plasma are a prognostic factor [11]. It is likely that this type of analysis will be carried out for a variety of other tumor types in the future, making plasma DNA analysis an important addition to our armamentarium against cancer. ACKNOWLEDGEMENTS The author has been supported by the Innovation and Technology Fund, and is a member of the Hong Kong Cancer Genetics Research Group, funded by the Kadoorie Charitable Foundations. REFERENCES 1 Anker P, Lefort F, Vasioukhin V, Lyautey J, Lederrey C, Chen XQ, et al. K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer. Gastroenterology 1997 ; 112 : 1114-20.
2 Capone RB, Pai SI, Koch WM, Gillison ML, Danish HN, Westra WH, et al. Detection and quantitation of human papillomavirus (HPV) DNA in the sera of patients with HPV-associated head and neck squamous cell carcinoma. Clin Cancer Res 2000 ; 6 : 4171-5. 3 Chen XQ, Bonnefoi H, Pelte MF, Lyautey J, Lederrey C, Movarekhi S, et al. Telomerase RNA as a detection marker in the serum of breast cancer patients. Clin Cancer Res 2000 ; 6 : 3823-6. 4 Chen XQ, Stroun M, Magnenat JL, Nicod LP, Kurt AM, Lyautey J, et al. Microsatellite alterations in plasma DNA of small cell lung cancer patients. Nat Med 1996 ; 2 : 1033-5. 5 Chiang PW, Beer DG, Wei WL, Orringer MB, Kurnit DM. Detection of erbB-2 amplifications in tumors and sera from esophageal carcinoma patients. Clin Cancer Res 1999 ; 5 : 1381-6. 6 Dasi F, Lledo S, Garcia-Granero E, Ripoll R, Marugan M, Tormo M, et al. Real-time quantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA: a simple blood test to monitor disease in cancer patients. Lab Invest 2001 ; 81 : 767-9. 7 Dickens P, Srivastava G, Loke SL, Chan CW, Liu YT. EpsteinBarr virus DNA in nasopharyngeal carcinomas from Chinese patients in Hong Kong. J Clin Pathol 1992 ; 45 : 396-7. 8 Drouet E, Brousset P, Fares F, Icart J, Verniol C, Meggetto F, et al. High Epstein-Barr virus serum load and elevated titers of antiZEBRA antibodies in patients with EBV-harboring tumor cells of Hodgkin’s disease. J Med Virol 1999 ; 57 : 383-9. 9 Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, et al. MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 2000 ; 28 : E32. 10 Gallagher A, Armstrong AA, MacKenzie J, Shield L, Khan G, Lake A, et al. Detection of Epstein-Barr virus (EBV) genomes in the serum of patients with EBV-associated Hodgkin’s disease. Int J Cancer 1999 ; 84 : 442-8. 11 Kawakami K, Brabender J, Lord RV, Groshen S, Greenwald BD, Krasna MJ, et al. Hypermethylated APC DNA in plasma and prognosis of patients with esophageal adenocarcinoma. J Natl Cancer Inst 2000 ; 92 : 1805-11. 12 Kopreski M, Benko FA, Kwak LW, Gocke CD. Detection of tumor messenger RNA in the serum of patients with malignant melanoma. Clin Cancer Res 1999 ; 5 : 1961-5. 13 Lei KI, Chan LY, Chan WY, Johnson PJ, Lo YMD. Quantitative analysis of circulating cell-free Epstein–Barr virus (EBV) DNA levels in patients with EBV-associated lymphoid malignancies. Br J Haematol 2000 ; 111 : 239-46. 14 Leon SA, Shapiro B, Sklaroff DM, Yaros MJ. Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res 1977 ; 37 : 646-50. 15 Lin JC, Chen KY, Wang WY, Jan JS, Liang WM, Tsai CS, et al. Detection of Epstein-Barr virus DNA the peripheral-blood cells of patients with nasopharyngeal carcinoma: relationship to distant metastasis and survival. J Clin Oncol 2001 ; 19 : 2607-15. 16 Lo KW, Lo YMD, Leung SF, Tsang YS, Chan LY, Johnson PJ, et al. Analysis of cell-free Epstein-Barr virus associated RNA in the plasma of patients with nasopharyngeal carcinoma. Clin Chem 1999 ; 45 : 1292-4. 17 Lo YMD, Chan LY, Chan AT, Leung SF, Lo KW, Zhang J, et al. Quantitative and temporal correlation between circulating cellfree Epstein-Barr virus DNA and tumor recurrence in nasopharyngeal carcinoma. Cancer Res 1999 ; 59 : 5452-5. 18 Lo YMD, Wong IH, Zhang J, Tein MS, Ng MH, Hjelm NM. Quantitative analysis of aberrant p16 methylation using real-time quantitative methylation-specific polymerase chain reaction. Cancer Res 1999 ; 59 : 3899-903. 19 Lo YMD, Chan ATC, Chan LYS, Leung SF, Lam CW, Huang DP, et al. Molecular prognostication of nasopharyngeal carcinoma by quantitative analysis of circulating Epstein-Barr virus DNA. Cancer Res 2000 ; 60 : 6878-81.
Pretreatment of EBV DNA in nasopharyngeal carcinoma
20 Lo YMD, Chan LY, Lo KW, Leung SF, Zhang J, Chan AT, et al. Quantitative analysis of cell-free Epstein–Barr virus DNA in plasma of patients with nasopharyngeal carcinoma. Cancer Res 1999 ; 59 : 1188-91. 21 Lo YMD, Chan WY, Ng EKW, Chan LYS, Lai PBS, Tam JS, et al. Circulating Epstein-Barr virus DNA in the serum of patients with gastric carcinoma. Clin Cancer Res 2001 ; 7 : 1856-9. 22 Lo YMD, Leung SF, Chan LY, Chan AT, Lo KW, Johnson PJ, et al. Kinetics of plasma Epstein-Barr virus DNA during radiation therapy for nasopharyngeal carcinoma. Cancer Res 2000 ; 60 : 2351-5. 23 Mandel P, Métais P. Les acides nucléiques du plasma sanguin chez l’homme. CR Acad Sci Paris 1948 ; 142 : 241-3. 24 Mutirangura A, Pornthanakasem W, Theamboonlers A, Sriuranpong V, Lertsanguansinchi P, Yenrudi S, et al. Epstein-Barr viral DNA in serum of patients with nasopharyngeal carcinoma. Clin Cancer Res 1998 ; 4 : 665-9. 25 Nawroz H, Koch W, Anker P, Stroun M, Sidransky D. Microsatellite alterations in serum DNA of head and neck cancer patients. Nat Med 1996 ; 2 : 1035-7. 26 Pornthanakasem W, Shotelersuk K, Termrungruanglert W, Voravud N, Niruthisard S, Mutirangura A. Human papillomavirus DNA in plasma of patients with cervical cancer. BMC Cancer 2001 ; 1 : 2. 27 Rickinson AB, Kieff E. Epstein-Barr virus. In: Fields BN, Knipe DM, Howley PM, Eds. Fields virology. Third edition. Philadelphia: Lippincott-Raven Publishers; 1996. p. 2397-446. 28 Shotelersuk K, Khorprasert C, Sakdikul S, Pornthanakasem W, Voravud N, Mutirangura A. Epstein-Barr virus DNA in serum/plasma as a tumor marker for nasopharyngeal cancer. Clin Cancer Res 2000 ; 6 : 1046-51.
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