© 2004 Foundation for Promotion of Cancer Research
Serum EBV DNA as a Biomarker in Primary Nasopharyngeal Carcinoma of Indian Origin
1 Division of Cancer Research, Regional Cancer Center, 2 Department of ENT, Medical College Hospital, Trivandrum, 3 Division of Cytopathology, Regional Cancer Center and 4 Division of Cancer Research, Regional Cancer Center, Kerala, India
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Background: Nasopharyngeal carcinoma (NPC) is a unique tumor due to its etiology and endemic distribution. Ethnic and regional factors are found to strongly influence the risk of disease; however, there have been no well-conducted studies on Indian patients. The present study assesses the relationship between Epstein–Barr Virus (EBV) and sporadic Indian NPC and the role of serum EBV DNA in NPC detection.
Methods: Primers directed against non-polymorphic Epstein–Barr nuclear antigen-1 (EBNA-1) gene were used to detect the presence of EBV DNA from fresh tissue and serum in NPC, using PCR.
Results: EBV DNA was detected in 69% of the biopsies and 58% of the serum of the NPC patients. With respect to histology, WHO Type III NPC, WHO Type II tumors and WHO I tumors showed 100%, 72.2% and 33% EBV positivity, respectively. EBV positivity was also observed in 23% (6/26) of benign samples. All biopsies of patients with positive serum samples were positive for EBV DNA.
Conclusion: EBV infection was found in sporadic NPC of South Indian origin, which confirms the etiological role of EBV in NPC. Detection of EBNA-1 in the serum and corresponding tissues of NPC patients suggests that the serum EBV DNA originates from NPC and also indicates the benefit of circulating viral DNA as an early marker in the diagnosis of NPC. Serum DNA-PCR methods can be extrapolated to follow-up studies involving tumor regression or to assess the response to various therapies.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Nasopharyngeal carcinoma (NPC) is a unique subset of head and neck squamous cell carcinoma (HNSCC). Marked geographical variation is shown in incidence. Although NPC is rare worldwide, it is one of the most common cancers in Southeast Asian countries as well as in China, where it has an incidence of 20 to 50 per 100 000 individuals (1). The incidence of NPC in the Indian subcontinent is not well documented, but it seems to show geographical variation. NPC is generally rare in India as compared to other Southeast Asian countries, with the exception of some north-eastern states, mainly Nagaland (2). The mongoloid race in north-eastern Indian states has shown an increase in NPC incidence, which is reported to be linked mainly to the consumption of smoked foods. Studies on NPC have shown that NPC tumorigenesis is closely related to the consumption of smoked and preserved foods, exposure to soot and dust and occupational exposure to formaldehyde and various herbal oils containing Epstein–Barr Virus (EBV) activating compounds (3).
Viral etiology of NPC with EBV holding the pivotal position has been reported (4). A serological test for EBV associated antibodies was proposed as a screening test in NPC (5). Quantitative EBV DNA analysis has also been shown to be a sensitive molecular tool for detection and confirmation of NPC (6). An alternative approach to detect tumor markers in samples collected directly from the nasopharyngeal region via a non-invasive procedure of nasopharyngeal brushing or swabs (7) is also being employed in some high-risk populations. It has been recently demonstrated that cell-free EBV DNA can be found in the plasma and serum of NPC patients (8,9,10). So far, no study has been conducted on the Indian population to assess the etiology of sporadic NPC and to investigate the role of EBV in NPC tumorigenesis. The association of EBV with NPC and the relationship between pretreatment circulating EBV DNA and clinicopathological events has been explored in this study.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Clinical Specimens and Sample Preparation
Tissue samples from 29 patients with primary NPC prior to any treatment were collected from the ENT Department, Medical College Hospital, Trivandrum, Kerala. The representative samples were fixed in buffered formalin and embedded in paraffin for routine histopathological analysis. Biopsies and serum from these individuals were also collected and stored at –20°C. Biopsy and serum samples from 26 patients with nasal polyps were also included as a source of benign tissue. Demographic and clinical information was obtained by interviewing the patients using a structured proforma.
Histology
The first and last sections (5 µm) of the paraffin embedded tissues were stained with hematoxylin and eosin and histologically examined for conformation of diagnosis and estimation of the percentage of normal and neoplastic cells and inflammatory infiltrate. The 29 primary NPCs in this study were grouped by a pathologist, according to the WHO classification. It included 6 WHO type I, 18 WHO type II and 5 WHO type III tumors.
DNA Isolation
Fresh biopsy and serum samples were incubated with digestion buffer containing proteinase K overnight at 37°C. DNA was extracted by the standard phenol/chloroform method and ethanol precipitation. Control of the quality of tissue DNA was determined by ß-globin PCR as has been previously described (11).
Detection of EBV
Primer pairs that amplified the non-polymorphic Epstein–Barr nuclear antigen-1 (EBNA-1) gene (Table 1) were constructed, and the presence of EBV was confirmed by a 262 bp product (12). PCR reaction consisted of initial denaturation at 95°C for 3 min and 45 cycles of denaturation at 94°C for 30 s, annealing at 54°C for 30 s, extension at 72°C for 40 s and a final extension at 72°C for 3 min. Negative controls consisted of a mixture of all the reagents used in the PCR mixture preparation with the addition of 1 µl of sterile distilled water instead of DNA. Special care was taken to avoid cross contamination by using disposable tubes and aerosol filter tips and by carrying out the different PCR steps, such as sample preparation, electrophoresis and PCR solution preparation in separate rooms.
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Statistical Analysis
The statistical analysis of the EBV status in relation to clinical and histological data was performed by the Chi-square test. All P values were two-sided and considered significant at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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Patient Details
The clinicopathological details of the patients with respect to age, sex, tumor, characteristics, tobacco and alcohol habits and exposure to kitchen smoke, soot, etc. are given in Table 2. Tobacco and alcohol habits and exposure to smoke and soot are grouped under the variable habits. From a total of 29 histologically proven primary NPC cases, 26 benign lesions and their corresponding sera were analyzed in this study. Majority of NPC patients were above 20 years of age (22 cases) (Table 2), whereas patients with benign lesions were less than 20 years of age. Males predominated (18 cases) as compared to females (11 cases). Among the NPC cases, 27 had habits such as cigarette smoking, alcohol drinking, "betel" chewing, exposure to kitchen smoke, alone or any of these habits in combination. Details regarding oral hygiene were available only in 22 cases, of which 15 cases had poor oral hygiene. Among the analyzed cases, 17 had advanced disease (stage II and III) and 12 had early-stage disease (stage I and II), 17 had lymph node involvement and 21 had either local or regional recurrence of the disease. The maximum follow-up period was 3 years, at the end of which 18 patients were alive (12 in the EBV-positive group and six in the EBV-negative group) (Table 2). EBV positivity showed a significant association with only the age and sex of the patients.
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EBV Detection
Tissues
Of the 29 primary NPC tissues analyzed, 20 (69%) showed EBV PCR product. Of the six WHO Type I NPC, two (33%) were positive for EBV. Thirteen cases (72.2%) and 5 cases (100%) were positive for EBV in WHO Type II and III, respectively (Table 3, Fig. 1).
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Twenty-six benign nasal polyps were analyzed and only six (23%) were positive for EBNA-1 gene amplification product. EBV positivity was found to be statistically significant in NPC cases as compared to benign control by Chi-square test (Table 3).
Serum
Serum samples corresponding to the tissues of the benign lesions were available in only 12 cases, and of these, two cases (16.6%) showed an EBV PCR product, which was very feeble, indicating low copy numbers of the viral genome (Table 4, Fig. 1). This was in contrast to the high EBV positivity in 15 of 20 NPC serum samples (75%). The difference was statistically significant by the Chi-square analysis (P = 0.004). The odds ratio of EBV positivity, an indicator of cancer, was 12.5 (CI 2.089–74.808). Six serum samples were available in WHO Type I NPC, of which one was excluded from the study due to the lack of an adequate amount of DNA and two were positive for EBNA-1. Fifteen serum DNA samples of WHO Type II NPC were available, of which five were excluded due to insufficient DNA and eight showed EBV PCR amplification products. All the serum samples belonging to WHO Type III were positive for EBNA-1.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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NPC is often difficult to diagnose because of the non-specific nature of its clinical symptoms and the difficulty in visualizing the nasopharynx (13). Submucosal primary lesions often escape endoscopic examination (14). Most of the tumors remain undiagnosed until they present as metastasis to the cervical lymph nodes, often without overt pathology at the primary site (10). Koppikar et al. have reported that the 5-year actuarial disease-free survival of Indian patients is 13% (15), which indicates the need for an early diagnosis and aggressive treatment strategies to improve survival. Early detection of NPC should improve cure rate and reduce morbidity and metastasis. This requires a battery of diagnostic tests and an effective screening system.
EBV is a large herpes virus carried by almost all human adults. The EBV genome is large, exceeding 172 kb of ds-DNA and encoding more than 100 genes. Approximately 10 genes are known to play a role in virally mediated cell growth. EBNA-1 is expressed in type-1 latency of EBV, allowing for replication and maintenance of the viral genome during cell division (16). EBV infections are common worldwide, with over 90% of adults being seropositive. EBV is usually latent and infrequently produces infectious progeny with a persistent and active lytic infection that persists for several years. After the primary infection, EBV can diffuse across the basal membrane and cause latent infection in B-lymphocytes, which often disseminates the infection to the distal epithelial surfaces, mainly in the nasopharynx (17). Analysis of the terminal repeat length of EBV DNA in NPC suggests that the tumors are clonal, arising from a single infected cell (4).
Most epidemiological studies reveal that EBV infection is associated with the WHO II and III subtypes but not with the well-differentiated WHO I NPC (18,19,20). Patients with an undifferentiated form of NPC show consistent positivity of the EBV genome in tumors and have elevated IgG and IgA responses to the EBV capsid antigen (VCA) as well as to the diffuse type of early antigen (EA-D) (21). WHO Type I is reported to have serological patterns similar to those of the control populations and show no specific association with EBV infection (22). This finding has been supported by several other studies (23–26). All these results imply a non-association of well-differentiated keratinized NPC with EBV.
Overall, Indian NPC showed 69% EBV positivity, with all the histological subtypes showing the presence of EBV. The highest EBV positivity was observed in WHO III (100%) followed by WHO II (72.2%). The keratinizing type (WHO I) tumor showed the least EBV positivity of 33%. These results are consistent with those of Raab-Traub et al. (27) and Chang et al. (28), who detected EBV DNA only in low copy numbers in all histological subsets of NPC, with the undifferentiated NPC cells containing higher EBV copy numbers than the other two types. Sam et al. (29) reported that patients with all three forms of NPC have elevated IgA titers to VCA.
Normal nasopharynx is not an EBV reservoir (8,9,28) and previous studies have demonstrated the absence of EBV in normal nasopharyngeal epithelium and serum (30,31). Studies conducted by Hording et al. (32) in exfoliated nasopharyngeal cells of normal subjects in a high-risk population showed the presence of EBV in 13% of the subjects, which can be attributed to former exposure to EBV (33). The results of the present study show the presence of EBV in 23% (6/26) of the benign samples. The controls included in this study were mainly below the age of 20 years and showed the presence of benign adenoid lesions. Previous reports showed that in normal adults, 1–50 B cells per million in the circulation are infected with EBV, and the number of latently infected cells within a person remains stable over years (34). The presence of EBV in benign adenoid lesions suggests that further environmental and genetic factors other than viral presence are necessary for tumor initiation and progression.
Recent studies have demonstrated the use of detecting tumor DNA in plasma or serum in several cancers (35) as a potential diagnostic marker. In this study, two of the twelve DNA samples from the controls showed amplified fragment for the EBNA-1 gene in low copy numbers as evidenced by low amplification signal. Further, two of the five WHO Type I and eight of the ten WHO Type II NPC cases and all the five serum DNA samples of the WHO Type III cases showed serum DNA positivity for EBNA-1 DNA (Table 4). Samples which showed positivity for serum DNA were also positive for tissue DNA, which suggests that the serum EBV DNA originated from NPC and confirms its use as a marker for tumor DNA.
This is the first study evaluating the association of EBV with Indian NPC patients. The results show a positive association in 69% of the cases and also suggest the potential use of serum EBV DNA detection as an early marker of NPC. Studies involving a larger sample size, especially for WHO type 1 NPC, would be able to confirm these findings and this marker could find application as both a diagnostic and a prognostic marker in the clinical management of NPC.
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Acknowledgments |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONAcknowledgmentsREFERENCES |
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The authors thank the Indian Council of Medical Research for providing financial support for this project.
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FOOTNOTES |
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+ For reprints and all correspondence: Prabha Balaram, Associate Professor and Head, Research Division, Regional Cancer Center, Trivandrum 695 011, Kerala, India. E-mail: prabhabalaram{at}yahoo.co.in
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Received January 19, 2004; accepted March 22, 2004
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