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American Journal of Otolaryngology–Head and Neck Medicine and Surgery 30 (2009) 95 – 100 www.elsevier.com/locate/amjoto
Molecular diagnosis of nasopharyngeal carcinoma using detection of Epstein-Barr virus latent membrane protein-1 gene in cervical metastatic lymph nodes☆ Bijan Khademi, MD a , Jalal Mahmoudi, MD a , Ahmad Monabati, MD b , Behzad Maghsoudi, MD c , Mohammad J. Ashraf, MD b , Mohammad Mohammadianpanah, MD d,⁎, Narjes Tabibi, BSc b , Behnaz Valibeigi, BSc b , Elham Abedi, BSc b a
Department of Otolaryngology, Shiraz University of Medical Sciences, Shiraz, Iran b Department of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran c Department of Anesthesiology, Shiraz University of Medical Sciences, Shiraz, Iran d Department of Radiation Oncology, Shiraz University of Medical Sciences, Shiraz, Iran Received 16 November 2007
Abstract
Background: Cervical lymphadenopathy could be a manifestation of occult nasopharyngeal carcinoma (NPC). Epstein-Barr virus (EBV) is frequently detected in NPC, and its malignant transformation is associated through the action of the oncoprotein latent membrane protein-1 (LMP-1). Purpose: The aim of this study was to investigate whether a primary nasopharyngeal origin could be localized by detection of EBV LMP-1 gene in cervical metastatic lymph nodes. Materials and methods: In this prospective study, 32 paraffin-embedded tissues of various head and neck carcinomas and 20 normal tonsil specimens were examined for the presence of LMP-1 gene, using polymerase chain reaction. Results: Ten of 12 nasopharyngeal biopsies and 8 of 10 metastatic lymph nodes of the same NPC were positive for LMP-1 gene. The LMP-1 gene was detected in metastatic lymph nodes of NPC, with a sensitivity of 80%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 91%. On the contrary, the LMP-1 gene was not detected in any of the samples of other head and neck carcinomas and their metastatic nodes as well as in tonsillar specimens. There was a significant association between the presence of LMP-1 gene and tumor location in the nasopharynx (P b .0001). Conclusion: The presence of LMP-1 gene in metastatic cervical lymph nodes is significantly associated with nasopharyngeal origin of the carcinoma. Meanwhile, EBV has no role in the tumorigenesis of carcinomas arising from other head and neck regions. Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved.
1. Introduction ☆
Conflicts of interest: The authors disclose any commercial or other associations that might pose a conflict of interest in connection with submitted manuscript. ⁎ Corresponding author. Department of Radiation Oncology, Nemazee Hospital, Shiraz 71936-13311, Iran. Tel.: +98 711 6260135; fax: +98 711 6260135. E-mail address:
[email protected] (M. Mohammadianpanah).
The Epstein-Barr virus (EBV) is a widespread human herpesvirus associated with the development of both lymphoid and epithelial tumors. It affects approximately 95% of the world's adult population, and after primary infection, the individual remains a lifelong carrier. However,
0196-0709/$ – see front matter Crown Copyright © 2009 Published by Elsevier Inc. All rights reserved. doi:10.1016/j.amjoto.2008.02.013
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the virus can contribute to oncogenesis as evidenced by its frequent detection in certain tumors, classically, Burkitt lymphoma, posttransplant B-cell lymphomas, Hodgkin disease, and nasopharyngeal carcinoma (NPC), and by its unique ability to efficiently transform resting B cells in vitro into permanently growing lymphoblastoid cell lines (LCLs). These transforming effects are associated with the restricted expression of EBV genes such that only a subset of socalled latent virus proteins is expressed in virus-infected tumors and in LCLs. Distinct forms of EBV latency are manifest in the different tumors, and these appear to be a vestige of the pattern of latent gene expression used by the virus during the establishment of persistent infection within the B-cell pool. This review summarizes our current knowledge of EBV latent gene function and how this relates to the role of the virus in the etiology of different tumors [1-3]. This virus induces malignant cell transformation through the activation of oncoprotein latent membrane protein-1 (LMP-1). However, after wide application of polymerase chain reaction (PCR), EBV genomes are discovered to be harbored in almost every NPC with high sensitivity and specificity, although its role in tumorigenesis in metastatic lymph nodes of patients with NPC and other head and neck cancers remains unclear [2-6]. This study was designed to determine whether EBVderived LMP-1 gene present in metastatic lymph nodes of patients with NPC can be used as a diagnostic marker for predicting the primary site of the tumor and to evaluate the presence of LMP-1 in carcinomas arising from any location in the head and neck region, other than the nasopharynx. 2. Materials and methods 2.1. Patients This study was approved by the Ethics Committee of Shiraz Medical School, and written informed consent was obtained from the participants. Subjects were recruited during the study period from March 2005 to September 2006 and included: A. those subjects with proven NPC who had associated cervical lymph node metastasis; B. patients with proved head and neck carcinoma other than NPC who had tissue-proven cervical lymph node metastasis; and C. 20 subjects of tonsillectomy-driven tissue from patients with tonsillar hyperplasia. Tissue samples were obtained from the primary source of NPC and the related lymph node metastasis as the study specimens. Control specimens were obtained from the lymph node metastasis of other head and neck carcinomas and tonsillar tissue of tonsillectomy patients. All samples were obtained during surgical interventions, which were performed under general anesthesia.
2.2. Sample preparation The tissue samples were prepared from paraffinembedded tissue blocks by cutting 5-μm-thick sections. Specific precautions were used to avoid possible viral crosscontamination, including use of fresh microtome blades for each specimen, use of fresh gloves, and diligent cleaning of all instruments with 70% alcohol and disinfectant materials. 2.3. DNA extraction and purification Five to 10 tissue sections were put into a sterile 2-mL Eppendorf tube and then 1 mL xylol was added to each tube and incubated for 30 minutes. Then it was centrifuged at 14 000 rpm for 10 minutes at room temperature, and the supernatant was discarded. These steps were repeated one more time. Later, 1 mL ethanol 100% was added to the tubes and incubated for 30 minutes and centrifuged for l0 minutes. The supernatant was then discarded. After that, 1 mL ethanol 70% was added to each of the tubes and centrifuged at 14 000 rpm for 10 minutes, and the supernatant was then discarded. After this stage, 500 mL phosphate-buffered saline was added to the solutions and centrifuged for 15 minutes, and the supernatant was discarded. This process was repeated, and finally, 500 μL lysis buffer (proteinase K, 25 μL; Iris-HCl, 10 mL [pH 8:5]; EDTA, 1 μL; sodium dodecyl sulfate 10%, 50 μL; distilled deionized water, 419 μL) was added to the tubes and incubated at 52°C overnight. At the next step, 500 μL phenol/chloroform/isoamyl (25:24:1) was added to the tubes and shaken by vortex and centrifuged at 12 000×g for 10 minutes, and the supernatants were then transferred to new tubes, after which 1 vol chloroform was added to the tubes and mixed by vortex and centrifuged at 12 000×g for 5 minutes. Then the upper aqua was removed, and the supernatant was carefully transferred to fresh tubes. Subsequently, 0.1 vol sodium acetate (3 M) was added to the tube and mixed by vortex; 1 vol isopropanol was added to the tube and incubated at −20°C overnight. On the next day, the tubes were centrifuged at 12 000×g at 4°C for 10 minutes, and the supernatant was discarded and washed. With 0.5 mL ethanol 78% centrifuged at 12 000×g for 5 minutes, DNA was collected and dried completely and was then dissolved in 50 μL distilled deionized water. 2.4. DNA amplification For PCR amplification, oligonucleotide primers for detecting LMP-1 gene (sense Bam HIW1 and antisense Bam HIW2; the sequence is shown later) were used to examine the presence of extracted DNA. The PCR was performed on a total volume of 25 μL, consisting of extracted DNA 1 μL, sense and antisense primers (10 pmol) 2 μL, Tag-DNA polymerase 5 μL, dNTP 0.5 μL, MgCl2 0.75 μL, H2O 13.25 μL, PCR buffer 2.5 μL. The prepared samples were amplified as follows: initial denaturation step at 94°C for 5 minutes and 35 next cycles as DNA denaturations at 94°C for 1 minute, annealing 60°C for
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1 minute, and DNA extension at 72°C for 1 minute. The final extension of DNA at 72°C for 5 minutes was done in a programmable thermal controller. The product was a 129-bp fragment that was specific for LMP-1 gene of EBV. The product was examined on 2.5% agarose gel electrophoresis in l× Tris–boric acid–ethylenediaminetetraacetic acid solution and stained with ethidium bromide to determine the presence or absence of EBV-genomic DNA. 2.5. Detection of the presence of LMP-1 gene We chose to amplify the regions of the EBV LMP-1 gene (sense primer Bam HIW1: 5′-CCA GAC AGC AGC CAA TTG-TC -3′; antisense primer Bam HIW2: 5′-GGT AGA AGA CCC CCT CTT- AC-3′) for identification of the LMP1 gene. Negative control samples containing water instead of DNA were always processed in a manner parallel to the patients' samples. DNA from LCL-P17 cell line was used as the EBV-positive control sample. 2.6. Statistical analysis The χ2 test was used to measure the association between the presence of LMP-1 gene and various tissue locations. Data were processed using SPSS version 15.0 software (SPSS, Chicago, IL). 3. Results The patients consisted of 52 subjects (32 males, 20 females), with an age range of 18 to 82 years. During the study period, 12 cases of NPC and 20 cases of other head and neck carcinomas with tissue-proven squamous cell type of carcinoma who had associated lymph node metastasis were referred to Khalili Hospital for surgical management. Also, 20 cases of tonsillar hyperplasia were obtained from tonsillectomy specimens during the same period (Table 1).
Table 1 The results of study according to demographic characteristics and presence of LMP-1 gene in various head and neck carcinomas No. NPC 12 Sex Male Female Age (mean ± SD), y PCR of tumor LMP-1 (+) LMP-1 (−) PCR of lymph node LMP-1 (+) LMP-1 (−)
Head and neck carcinoma 20
Tonsillectomy specimens 20
32 20
9 13 3 7 (43 ± 13.87) (61.5 ± 11.42)
10 10 (16.5 ± 7.5)
10 42
10 2
0 20
0 20
8 44
8 4
0 20
0 20
No. indicates number of patients.
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Table 2 The characteristics of patients according to histopathology, site, and presence of LMP-1 gene in various head and neck carcinomas
Oral cavity Ca Buccal Tongue Hard palate Floor of mouth Lower lip Larynx Scalp Nasopharynx Tonsillectomy specimen
Case no.
LMP-1 gene positive (no.)
Pathology
9 1 5 1 1 1 10 1 12 20
0 0 0 0 0 0 0 0 10 0
All SCC
All SCC SCC All NKC (WHO type II) All lymphoid hyperplasia
Ca indicates carcinoma; NKC, nonkeratinizing carcinoma; WHO, World Health Organization.
Among the NPC group of patients, an amplified product of LMP1 gene was detected in 10 of 12 specimens derived from the primary site and 8 of 10 specimens derived from their associated metastatic lymph nodes. In 2 patients, the associated lymph nodes had no malignant involvement and were diagnosed as reactive hyperplasia. Based on these results, the following data were calculated for the association of LMP-1 gene in primary NPC and associated metastatic lymph nodes: sensitivity, 80%; specificity, 100%; positive predictive value, 100%; and negative predictive value, 91%. Amplified sequence of LMP-1 gene was not detected in any of the samples from other head and neck carcinomas (oral cavity, laryngopharyngeal, and scalp) or tonsillectomy specimens. The PCR results are shown in Table 2. The statistical analysis showed a highly significant association (χ 2 = 21.68, P b .0001) only between the presence of LMP-1 gene and tumor localization in the nasopharynx. No correlation was found between the presence of LMP-1 gene and the histopathologic type of carcinoma. 4. Discussion Unknown primary squamous cell carcinoma (SCC) of the head and neck is relatively uncommon and offers a challenging diagnostic and therapeutic problem. The majority of these tumors arise from the upper aerodigestive tract. After comprehensive diagnostic work-up, a primary site will be detected in approximately 40% of patients, and the base of the tongue and tonsillar fossa consist of approximately 80% of these unknown primary sites. The rest of occult primary sites are mainly detected in the hypopharynx, nasopharynx, and larynx [7-9]. Nasopharyngeal carcinoma is a common malignancy in most part of Asia, in particular China, but it occurs sporadically in the west. Cervical lymphadenopathy is the most common presenting symptom of the NPC [10]. There is
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now increasing evidence supporting the association between EBV and NPC [2,11]. Almost 100% of poorly differentiated or undifferentiated NPCs contain EBV genomes and express EBV protein [2]. Epstein-Barr virus has distinct oncogenic potential, and EBV-associated malignancies are a leading cause of cancer morbidity and mortality worldwide [3]. Humans are infected with EBV by contact with oral secretion, and almost all seropositive individuals actively shed the virus in the saliva; therefore, this theory was suggested that EBV is implicated in the tumorigenesis of upper aerodigestive tract tissue [4]. Patients with NPC have elevated levels of immunoglobulin A (IgA) antibodies to EBV replicative antigens including IgA anti-viral capsid antigen. These antibodies that frequently precede the appearance of the neoplasm serve as a prognostic indicator of remission and relapse. Although these antibodies can predict the occurrence of NPC, the use of serologic titers as a screening method for NPC has been disappointing in some studies [12]. Wei et al, in a prospective study, showed that only 7 (5.4%) of 130 of asymptomatic patients with elevated IgA titer against EBV had subclinical NPC. According to this finding, the authors suggest nasopharyngoscopy for early detection, immediate administration of treatment, and increase chance of eradication of NPC in high-risk regions [13]. Regardless of geographic area and whether a patient with NPC survives, all the tumor cells contain EBV DNA [2,4]. The definition of peripheral virus reservoirs has a critical role for describing the natural history of EBV infection and persistence. Many studies have suggested that B lymphocytes are the most likely site of EBV persistence, and this is consistent with the high density of EBV receptors (CD21) and virus-triggered transformation pathways in these cell [14]. Latently infected lymphocytes and NPC cells express a specific subgroup of EBV genes [4,14]. Lymphoid cells express 6 nuclear antigens, Epstein-Barr nuclear antigen (EBNA)-1 through EBNA-6, and 2 integral membrane proteins, LMP-1 and LMP-2 [4,14,15]. EBNA-2 is essential for the in vitro transformation of lymphocytes. However, its absence in NPC indicates that it is not required for altered epithelial growth [4,15]. The role of EBNA-1 and LMP-2 proteins in EBV-infected cells is the maintenance of latent infection [14]. LMP-1 is the primary EBV oncogene that is detected in approximately in 50% to 70% of patients with NPC [14,16,17]. Although its expression is not uniform in all NPC samples using immunohistochemical methods, the screening of NPC with the advent of PCR is refined to improve the sensitivity and specificity by demonstrating LMP-1 gene directly. Hao et al demonstrated the presence of EBV-specific genomic DNA by PCR in nasopharyngeal swab specimens with a predicted sensitivity of 94.7% and specificity of 100%. In another study, it was shown that LMP-1 gene sensitivity was 87.3% and specificity was 98.4%, and detection of LMP-1 gene was able to differentiate recurrent NPC from osteoradionecrosis [12,17,18]. In another report, Tsang et al [18] showed that
the sensitivity and specificity rates for identifying NPC by the PCR method were 95.6% and 98.7%, respectively. In our findings, we could not find any correlation between the presence of LMP-1 gene and pathological type, but there was significant association (P b .0001) between the presence of LMP-1 gene and tumor location in the nasopharynx. Previous studies evaluating nasopharyngeal biopsies for identifying EBV-genomic DNA provided varying results. Sam et al found that 23 (88%) of 26 samples from EBVseropositive Chinese Malaysians with symptoms similar to NPC but with a negative biopsy were EBV-negative by using PCR, and in 3 EBV-positive patients, further analysis confirmed NPC [18]. Hao et al found EBV LMP-1 gene in 0.2% (7/256) samples of nasopharyngeal swabs of patients without NPC. According to this study, the probability of detecting EBVgenomic DNA in the nasopharynx of the normal general population is low, although seroepidemiological studies have indicated that 90% of all adults have been infected by EBV and are chronic carrier [12,14,17-19]. These data are in agreement with the experience of Liavaag et al [14], but contrary to others. Tugwood et al [20] evaluated nasopharyngeal biopsies of 18 healthy individuals, and EBV-specific DNA was detected in all of them. Hoerding et al found EBV-specific DNA in 35% (6/17) of nondiseased Danes and 81% (44/55) of healthy Greenland Eskimos. Thus, they concluded that EBV infection leads to a chronic carrier state in the nasopharynx [21]. The variety of findings is remarkable, and occasional cross-contamination of samples with viral DNA, or probably during PCR, cannot be ruled out in some reports with a high proportion of positive samples [12,14,18,22]. There are several reports that EBV-derived LMP-1 gene is a classic oncogene and enhances the metastatic property of NPC [23-25]. However, there are very few studies regarding the sensitivity and specificity rate of several laboratory methods available to detect the presence of EBV LMP-1 gene in metastatic lymph nodes of NPC [26-28]. Deamant et al identified EBV RNA in l0 of 19 samples of nonneoplastic lymph nodes of Peruvian patients. In another study, Liavaag et al did not find any EBV-genomic DNA in 8 nonneoplastic lymph nodes of fine needle aspirations using PCR [14]. Further analysis of our data demonstrated that the sensitivity rate of our PCR method for detecting LMP-1 gene in metastatic lymph nodes of patients with NPC is 80%, and it seems that PCR could be regarded as a reliable method for detecting EBV-derived LMP-1 gene in NPC cells and its metastatic lymph nodes. Despite the intrinsic sensitivity of PCR, identifying LMP1 gene in the oral cavity, oropharyngeal, and laryngeal carcinoma remains rare [14,18,22,29]. Khabie et al showed that only 1.9% of all tonsillar carcinoma specimens were positive for EBV DNA by PCR vs 11.3% of the noncancerous tonsillectomy samples [14,18]. By performing PCR on 231 biopsies of the upper
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aerodigestive tract, as well as from salivary gland tissue and neck nodes of healthy individuals, Liavaag et al found EBVgenomic DNA in only 2 specimens [14]. In a prospective study using PCR, Tsang et al investigated the incidence of the presence of LMP-1 gene in various head and neck cancers and indicated that the LMP-1 gene was not detected in any of the samples from oral cavity, laryngopharyngeal carcinoma, nasopharyngeal lymphoid hyperplasia, or tonsillectomy specimens except in only 1 case of tonsillar carcinoma [18]. Goldenberg et al, using quantitative PCR on 300 head and neck carcinoma samples, found 1% of the specimen to be overtly positive for the EBV genome [29]. The lack of EBV-positive samples, except in the nasopharynx, in our study does not support the theory that a high proportion of the population are carriers of EBV in their salivary glands, tonsils, neck nodes, or the mucosal epithelium of the upper aerodigestive tract (Table 2). According to the above-mentioned studies and current series, EBV DNA is rare in the upper aerodigestive tract tissue and it is not the principal EBV reservoir [14,18,29]. There is mounting evidence that demonstrates the relationship between EBV, a probable etiology of NPC, with other head and neck carcinomas, especially oral SCC [18,30,37]. On the other hand, a large number of reports have refuted these data (Table 3). The diversity of results in different studies remains unexplained, but the following reasons are suggested: small sample sizes, different sampling methods, sensitivity and specificity of the method used, primer sequences, deparaffinized management, PCR methods, cross-contamination with viral DNA and during PCR, amplification of cycles, source of tumoral tissue, and so on. Some of these drawbacks can be prevented by strict measures, such as consistent use of fresh blade, fresh latex gloves, plugged pipette tip, multiple PCRs, and concomitant DNA-free control reactions to avoid and check for cross-contamination. Table 3 Summary of studies detecting EBV DNA in oral SCC by PCR Reference
Year of study
No. of EBV positive samples/No. of tested (%)
Tumor tissue source
Mao and Smith [37] van Heerden et al [36] Curz et al [34] Maeda et al [33] D'costa et al [32] Mizugaki et al [35] Gonzales-Moles et al [30] Shimakage et al [31] Tsang et al [18]
1993
7/30 (23%)
Paraffin
1995
11/45 (24%)
Paraffin
1997 1998 1998
36/36 (100%) 29/45 (64.4%) 25/103 (25%)
Frozen Paraffin Paraffin
1998
0/30 (0%)
Frozen
2002
15/78 (19.2%)
Paraffin
2002
26/36 (72%)
Paraffin
2003
0/6 (0%)
Paraffin
99
5. Conclusions In the current study, we investigated whether a primary nasopharyngeal origin could be localized by detection of EBV LMP-1 gene in cervical metastatic lymph nodes. We conclude that the presence of LMP-1 gene is significantly associated with tumor location especially in the nasopharynx. The presence of LMP-1 gene in various head and neck carcinomas except NPC is rare, and the oral cavity, oropharynx, and laryngopharynx are not the main reservoirs of EBV. Our PCR method is a rapid and sensitive method for diagnosis of NPC. Polymerase chain reaction can be used as a potential tool for screening of NPC, especially in conditions in which neck metastasis is clinically present with unknown origin. Acknowledgment This investigation was financially supported by grant number 83-2331 from the vice-chancellery for research at Shiraz University of Medical Sciences. The authors would like to thank Dr M.A. Mosleh-Shirazi for editorial assistance and Dr N. Zarei, the Center for Development of Clinical Research of Nemazee Hospital, for statistical assistance, and special thanks are due to all the colleagues of the authors in private pathology laboratories for the help in preparing the specimens. References [1] Pathmanathan R, Prasad U, Raab-Traub N, et al. Undifferentiated non keratinizing and squamous cell carcinoma of the nasopharynx. Variants of Epstein-Barr-virus–infected neoplasia. Am J Pathol 1995; 146:1355-67. [2] Cohen JI. Epstein-Barr virus infection. N Engl J Med 2000;343:481-92. [3] Dickson RI, Flores AD. Nasopharyngeal carcinoma. An evaluation of 134 patients treated between 1971-1980. Laryngoscope 1985;95: 276-83. [4] Pathmanathan R, Prasad U, Raab-Traub N, et al. Clonal proliferation of cells infected with Epstein Barr virus in pre invasive lesions related to nasopharyngeal carcinoma. N Engl J Med 1995;333:693-8. [5] Goldenberg D, Golz A, Netzer A, et al. Epstein-Barr virus and cancers of the head and neck. Am J Otolaryngol 2001;22:197-205. [6] Vasef MA, Ferlito A, Weiss LM. Nasopharyngeal carcinoma, with emphasis on its relationship to Epstein-Barr virus. Ann Otol Rhinol Laryngol 1997;106:348-56. [7] Mendenhall WM, Mancuso AA, Amdur RJ, et al. Squamous cell carcinoma metastatic to the neck from an unknown head and neck primary site. Am J Otolaryngol 2001;22:261-7. [8] Califano J, Westra WH, Koch W, et al. Unknown primary head and neck squamous cell carcinoma: molecular identification of the site of origin. J Natl Cancer Inst 1999;91:599-604. [9] Aslani M, Sultanem K, Voung T, et al. Metastatic carcinoma to the cervical nodes from an unknown head and neck primary site: is there a need for neck dissection? Head Neck 2007;29:585-90. [10] Chan AT, Teo PM, Johnson PJ. Nasopharyngeal carcinoma. Ann Oncol 2002;13:1007-15. [11] Chang YS, Tyan YS, Liu ST, et al. Detection of Epstein-Barr virus DNA amplification. J Clin Microbiol 1990;28:2398-402. [12] Hao SP, Tsang NM, Chang KP. Screening nasopharyngeal carcinoma by detection of LMP-1 gene from nasopharyngeal swab. Cancer 2003; 97:1909-13.
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