Background The Epstein–Barr virus (EBV) is consistentlydetected in patients with nasopharyngeal carcinoma. To determinewhether EBV infection is an early, initiating event in the developmentof this malignant tumor, we screened nasopharyngeal-biopsy samples,most of which were archival, for preinvasive lesions, includingdysplasia and carcinoma in situ. Preinvasive lesions were foundin 11 samples, which were tested for the presence of EBV.
Methods EBV infection was detected with in situ hybridizationfor EBV-encoded RNAs (EBERs) and by immunohistochemical stainingfor latent membrane protein 1 (LMP-1). The larger samples werealso tested for the EBV genome with the use of Southern blotting.The expression of specific EBV RNAs was determined by the amplificationof complementary DNA with the polymerase chain reaction.
Results Evidence of EBV infection was detected in all 11 tissuesamples with dysplasia or carcinoma in situ. EBERs were identifiedin all eight samples tested, and LMP-1 was detected in all sixof the tested samples. Six of the seven samples tested for theEBV termini contained clonal EBV DNA. Transcription of the latentEBV gene products, EBV nuclear antigen 1, LMP-1, LMP-2A, andthe Bam HI-A fragment, was detected in most of the samples.Viral proteins characteristic of lytic lesions were not detected.
Conclusions Preinvasive lesions of the nasopharynx are infectedwith EBV. The EBV DNA is clonal, indicating that the lesionsrepresent a focal cellular growth that arose from a single EBV-infectedcell and that EBV infection is an early, possibly initiatingevent in the development of nasopharyngeal carcinoma. Preinvasivelesions contain EBV RNAs that are characteristic of latent infectionbut not the viral proteins that are characteristic of lyticinfection. The detection of the EBV-transforming gene, LMP-1,in all the neoplastic cells suggests that its expression isessential for preinvasive epithelial proliferations associatedwith nasopharyngeal carcinoma.
The Epstein–Barr virus (EBV) is an important factor inthe development of nasopharyngeal carcinoma, an epithelial cancer.1,2Nasopharyngeal carcinoma is rare in North American and Europeanwhites, with an age-adjusted incidence of 1 case per 100,000population. In contrast, it is among the most common cancersin southern China and parts of southeast Asia. An age-adjustedincidence of 26 cases per 100,000 has been reported among malesin Hong Kong. Genetic and environmental factors appear to contributeto the elevated risk of nasopharyngeal carcinoma among the Chinese.Patients with nasopharyngeal carcinoma have elevated titersof IgA antibodies to EBV replicative antigens, including theviral capsid antigen. These antibodies, which frequently precedethe appearance of the tumor, serve as a prognostic indicatorof remission and relapse.1
Regardless of whether a patient with nasopharyngeal carcinomalives in an area of endemic or sporadic incidence, all the tumorcells contain EBV DNA.3,4 The
EBV infection is latent, and the viral DNA is a clonal episome.The clonality of EBV DNA in nasopharyngeal carcinoma arisesfrom the clonal expansion of a single EBV-infected cell.5 IfEBV infected a fully formed neoplastic lesion, one would expectto detect an occasional cell that lacked EBV or a polyclonalEBV infection.
Premalignant and preinvasive neoplastic lesions of the nasopharynxin patients in whom nasopharyngeal carcinoma eventually developsare poorly documented and rare.6,7,8,9 To determine whetherEBV infection is an early, possibly etiologic event in the evolutionof nasopharyngeal carcinoma, we screened a large number of nasopharyngeal-biopsysamples for early malignant changes, including dysplasia andcarcinoma in situ. Eleven samples were identified and analyzedfor the presence of EBV.
Methods
Tissue Samples
Most of the biopsy samples we analyzed were from an archiveof nasopharyngeal-biopsy samples at the University of Malayain Kuala Lumpur, Malaysia. Samples were examined microscopicallyfor the presence of dysplastic or preinvasive nasopharyngealmucosal lesions. Carcinoma in situ, as distinguished from dysplasia,was documented if the normal epithelial cells in the topmostlayers of the tissue were absent.
A total of 5326 biopsy samples were screened. Invasive carcinoma,nasopharyngeal carcinoma, and various grades of dysplasia orcarcinoma in situ were detected in 1811 samples, 56 (3 percent)of which contained preinvasive mucosal lesions with adjacentinvasive cancer. In 11 samples (0.6 percent) there was dysplasiaor carcinoma in situ without adjacent invasive carcinoma. Tissuewas available from 8 of these 11 samples for studies of EBV.Cancer was diagnosed at follow-up examinations within 12 monthsafter the tissue samples had been obtained in five of the eightpatients whose samples were evaluated for the presence of EBV.
Three additional samples of dysplastic nasopharyngeal mucosawithout concomitant carcinoma (samples 3, 6, and 9) were obtainedat the Tumor Institute of Sun Yat Sen University in Guangzhou,China; DNA and RNA were extracted from these samples and analyzedwith molecular probes. Thus, 11 samples of dysplasia or carcinomain situ — 8 from Malaysia and 3 from China — wereanalyzed for the presence of EBV.
Because of heightened awareness of nasopharyngeal carcinomaamong Malaysian Chinese people, nasopharyngeal biopsies areperformed in many possibly symptomatic patients. From a collectionof tissue samples obtained from such patients, 60 samples ofnormal nasopharyngeal tissue, identified in the screening, servedas controls. These samples of normal epithelium were obtainedfrom patients in the ear, nose, and throat clinic who had beenfollowed for two years without evidence of cancer.
In Situ Hybridization of EBV-Encoded RNA
Formalin-fixed, paraffin-embedded tissue was placed on slidespretreated with 1x Denhardt's solution (0.02 percent Ficoll,0.02 percent polyvinylpyrrolidone, and 0.02 percent bovine serumalbumin) and 1 percent poly-l-lysine or 2 percent organosilane.The tissue sections were deparaffinized, rehydrated, and hybridizedto a fluorescein-conjugated oligonucleotide probe, obtainedcommercially, which consists of a mixture of the two EBV-encodedRNAs, EBER-1 and EBER-2 (Dako, Carpinteria, Calif.). Hybridizationwas detected by incubation with rabbit antifluorescein antibodytagged with alkaline phosphatase, followed by reaction withthe substrate 5-bromo-4-chloro-3-indoyl phosphate and the colorimetricindicator nitroblue tetrazolium.
Immunohistochemical Identification of Latent Membrane Protein 1
Latent membrane protein 1 (LMP-1) was identified with the useof a pool of commercially available mouse monoclonal antibodies(CS1 through CS4) and a standard immunoperoxidase staining procedure(Dako). The C15 tumor, an EBV-positive nasopharyngeal carcinomapassaged in nude mice, and previously identified, unambiguousexamples of nasopharyngeal carcinoma were used as positive controls.10The specificity of the staining was confirmed by the use ofEBV-negative squamous-cell carcinomas of the skin and the EBV-negativecell line HeLa as negative controls and by the use of stainingwithout the anti–LMP-1 antibody.
Assessment of EBV Clonality
Sufficient tissue was available for molecular analysis of EBVin four samples from the University of Malaya and three fromSun Yat Sen University. DNA from the tumors and dilutions ofDNA from the Raji lymphoid cell line, representing 1, 5, and50 copies of episomal DNA, were digested with BamHI and analyzedby Southern blotting. The blots were hybridized with probesrepresenting the left end (EcoRI-I) or the right end (XhoI-a)of the EBV genome, as described previously.5
Analysis of EBV Transcription by the Polymerase Chain Reaction
To test for the expression of EBV genes, RNA obtained from theseven larger tissue samples was used to prepare complementaryDNA (cDNA). Equal aliquots of RNA were processed with reversetranscriptase or, as a negative control, without reverse transcriptase.The individual EBV messenger RNAs (mRNAs) were identified bythe amplification of cDNA with 20-base oligonucleotide primersthat span a splice specific for each mRNA, as previously described.11Controls containing all the polymerase-chain-reaction (PCR)reagents and water instead of template were included for allreactions. The PCR products were resolved by agarose-gel electrophoresisand transferred to nitrocellulose, followed by hybridizationto 32P–end-labeled oligonucleotide probes.
Results
In Situ Detection of EBV
The preinvasive lesions were analyzed for transcripts of EBVgenes and for EBV proteins by means of in situ hybridizationand immunohistochemical staining. The small nuclear EBERs arethe most abundant RNAs in latently infected cells; approximately1 million copies per cell are consistently detected in tissuesamples from patients with nasopharyngeal carcinoma.12,13 TheEBERs are not expressed in oral hairy leukoplakia, a permissiveEBV infection, and are thus excellent diagnostic markers ofa latent EBV infection.14,15 We detected EBERs in all eighttested samples with dysplasia or carcinoma in situ (Table 1).Of the 56 samples that contained concomitant preinvasive andinvasive lesions, 22 were analyzed for the expression of EBERs.The EBERs were readily detected throughout the invasive andpreinvasive areas of the samples (data not shown). The resultsof EBER hybridization were negative in the 60 samples of normalnasopharyngeal tissue, confirming the results of a recent study.16
Table 1. Data on EBV Infection in 11 Samples of Preinvasive Lesions from Patients with Nasopharyngeal Carcinoma.
A tissue sample with carcinoma in situ (sample 7) is shown inFigure 1A. The immature atypical cells were characterized byan increased nuclear size, prominent nucleoli with occasionalmitotic figures, and an absence of terminal differentiationin the upper layers toward the luminal surface. The basementmembrane was intact, and lymphoid stroma was absent from theunderlying fibrovascular stroma. EBERs were present in all theimmature cells in the lesion but not in the underlying stroma(Figure 1B). EBERs were also found in a tissue sample (sample10) with severe dysplasia of the nasopharyngeal mucosa. Thedysplasia showed evidence of differentiation in the uppermostlayers of the epithelium (Figure 1C); EBERs were detected inmost cells, including some of the differentiated cells (Figure 1D).
Figure 1. Histologic Features of Preinvasive Neoplasia of the Nasopharynx and Detection of EBV-Encoded RNA (EBER).
Panel A shows a polypoid mucosal lesion of the nasopharynx with carcinoma in situ (sample 7). The normal respiratory tract mucosa has been completely replaced by abnormal cells marked by a lack of differentiation, large nuclei with the loss of nuclear polarity, an increased ratio of cellular nuclei to cytoplasm, and prominent nucleoli. The basement membrane is intact, and the underlying stroma is composed of loose fibrovascular tissue and occasional inflammatory cells. There is no evidence of tumor invasion. (hematoxylin and eosin, x630.) Panel B shows the same lesion with dark-brown nuclear staining, indicating the abundant expression of EBER within all nuclei of all cells throughout the full thickness of the epithelial layer (x630). Panel C shows a mucosal lesion with severe dysplasia (sample 10), characterized by hypercellularity and an increased thickness of the mucosal epithelium, with the preexisting epithelial cells replaced by abnormal cells throughout most of the epithelium. There is some squamous maturation of proliferating cells in the topmost layers. The fragmentation of the epithelial layers is an effect of tissue processing. (Hematoxylin and eosin, x1260.) Panel D shows in situ hybridization of EBER throughout this severely dysplastic lesion. EBERs were also detected in most of the superficial, maturing epithelial cells but not in the underlying lymphoid cells (x1260).
LMP-1 is the only EBV protein with transforming properties inrodent cell lines and is essential for immortalization of Blymphocytes in vitro.17,18 This protein has been detected inapproximately 60 percent of tissue samples from patients withnasopharyngeal carcinoma.19,20 With immunohistochemical staining,we found LMP-1 in all cells in all six of the tested samples(Table 1).
In one tissue sample with carcinoma in situ and in one withdysplasia, coexpression of EBERs and LMP-1 was found in mostof the cells in the abnormal epithelium. In a low-power microscopicalview of the sample with carcinoma in situ (sample 5), the positiveEBER hybridization was confined to the abnormal mucosa; occasionalmacrophages in the subepithelial lymphoid stroma stained weaklybecause of endogenous peroxide activity (Figure 2A). In thesame section, strong staining for LMP-1 was found only in theepithelial cells (Figure 2B).
Figure 2. EBV-Encoded RNA (EBER) and Latent Membrane Protein 1 (LMP-1) in Tissue Samples from Patients with Preinvasive Nasopharyngeal Neoplasias.
In panel A, which shows a tissue sample from a patient with carcinoma in situ (sample 5), the overlying atypical epithelial cells are positive for EBER. Much of the superficial mucosa is eroded. Ebers are not present in the underlying lymphoid stroma (x1260). Panel B shows the same sample with brown immunostaining for LMP-1 in the overlying epithelium, which is intense in the superficial layers. LMP-1 is not present in the lymphoid cells of the submucosa, although there is occasional weak staining of macrophages because of endogenous peroxidase activity (x630). Panel C shows a sample with severe dysplasia (sample 4). EBERs are present, in varying intensity, within the nuclei of most of the epithelial cells (x2500). Panel D shows variably intense brown staining for LMP-1 in all the cells from sample 4. The intensity of the stain increases near the luminal surface. The occasional infiltrating small lymphocytes within the epithelial layer, with dark nuclei and clear cytoplasm, are negative for LMP-1 (x1260).
In a sample with severe dysplasia (sample 4), EBERs were detectedin the majority of cells, with some of the abnormal cells apparentlynegative for the expression of EBER (Figure 2C). A similar resulthas been reported in tissue from patients with nasopharyngealcarcinoma.21 However, all the cells within this focus stainedpositively for LMP-1 (Figure 2D). The degree of staining wasparticularly intense in the superficial layers of the epithelium.In contrast, the infiltrating lymphocytes, marked by clear cytoplasmand dark, condensed nuclei, did not express EBERs (Figure 2D).Our studies revealed LMP-1 in all the samples with preinvasiveneoplasia and in all the dysplastic cells, even when EBERs werenot detected.
Clonality of EBV in Preinvasive Neoplasia
In cells latently infected by EBV, the viral genome is an episomeformed by circularization of the linear EBV genome that is presentin viral particles. Restriction-enzyme fragments of the terminiof the circular episomal DNA can be detected by probes fromboth ends of the linear genome.5 In a monoclonal infection,the fused termini yield a single restriction-enzyme fragment,whereas multiple fused fragments suggest a polyclonal infection.Smaller fragments that do not hybridize to both probes representlinear genomes produced by viral replication.5
EBV clonality was evaluated in four samples of tissue with nasopharyngealcarcinoma from the University of Malaya and in three samplesof tissue with dysplastic nasopharyngeal mucosa from Sun YatSen University. A single EBV terminal restriction-enzyme fragmentwas detected in three of the four samples from Malaysia, whichwere also positive for EBER and LMP-1 expression (Table 1 andFigure 3), and in all three samples from China. In sample 6,smaller fragments that might have represented linear DNA weredetected, in addition to a single fused terminal fragment (Figure 3).The presence of linear EBV DNA in this sample was probablydue to viral replication in a small percentage of the cells.The absence of detectable EBV DNA in one (sample 4) that waspositive for EBER and LMP-1 may have been due to an absenceof abnormal cells in the portion of the sample that was processedfor DNA analysis.
Figure 3. Detection of EBV DNA in Five Samples of Tissue with Preinvasive Neoplasia (Samples 3, 5, 6, 9, and 11).
Southern blots prepared with DNA from tissue with atypical hyperplasia, dysplasia, and carcinoma in situ, digested with BamHI, and dilutions of Raji-cell DNA representing approximately 50 copies, 5 copies, and 1 copy of EBV per cell (R50, R5, and R1, respectively) were hybridized to a single-stranded RNA probe representing the XhoI-a fragment. A single restriction-enzyme fragment was detected in most samples, indicating the presence of clonal, episomal EBV DNA. Sample 6 had additional, smaller fragments that probably represented linear DNA.
Analysis of EBV Expression
Latently infected lymphocytes and nasopharyngeal-carcinoma cellsexpress a specific subgroup of EBV genes. Lymphoid cell linesexpress six EBV nuclear antigens, EBNA-1 through EBNA-6, andtwo integral membrane proteins, LMP-1 and LMP-2. In nasopharyngealcarcinoma, EBNA-1, LMP-1, LMP-2, and transcription from theBamHI-A fragment of the EBV genome are detected.22,23,24,25,26,27We used PCR with complementary DNAs to detect the distinctiveRNAs corresponding to the EBNA-1, EBNA-2, LMP-1, and LMP-2 transcripts;BamHI-A transcription; and a replicative gene product, the EBVreplication activator BamHI-Z (ZEBRA).28
Table 1 summarizes the results of these studies. Most of thepreinvasive neoplastic lesions contained the characteristicRNAs of nasopharyngeal carcinoma — that is, EBNA-1, LMP-1,LMP-2, the BamHI-A fragment, and the EBER RNAs (Table 1). Transcriptsencoding EBNA-2, which is typically present with latent infectionin lymphocytes, or ZEBRA, which distinguishes the earliest stageof lytic infection, were not detected in any of the samplesassayed (Table 1). Therefore, the pattern of expression of viralgenes in the preinvasive samples was identical to that in thesamples with nasopharyngeal carcinoma.
Discussion
The results of this study strengthen the evidence that EBV iscritical to the pathogenesis of nasopharyngeal carcinoma. Thedata demonstrate latent EBV infection of all the cells in all11 samples of preinvasive lesions. The presence of a singleclonal form of EBV in these lesions suggests that nasopharyngealdysplasia or carcinoma in situ is a focus of EBV-induced cellularproliferation and that EBV infection precedes the acquisitionof invasiveness by these tumors.
Rare cases of nasopharyngeal mucosal dysplasia have been detectedby nasopharyngoscopy during intensive screening programs inGuangdong province, China.9 The lesions were marked by a thickeningof the epithelial layer, with a loss of normal stratification,abnormal morphologic features, and pleomorphic nuclei.9 However,these samples were not analyzed for EBV infection. In anotherstudy, EBV DNA was detected by in situ hybridization in onlysome cells in a few samples of tissue with carcinoma in situ.29This finding suggests that EBV infection occurred after theinitial neoplastic event. However, we found evidence of EBVinfection in all samples of preinvasive lesions, with the useof several methods. In situ hybridization is considerably moresensitive for the detection of EBER than for the detection ofEBV DNA, because, when expressed, EBERs are present at veryhigh levels. In addition, LMP-1 was detected in all abnormalcells, even in some that were negative for EBER, indicatingthat the lesions were homogeneously infected.
Analysis of the EBV genomic termini and the expression of viralgenes revealed that the preinvasive lesions contained predominantlylatent infections and expressed the same EBV genes as thoseexpressed in nasopharyngeal carcinoma. EBNA-2, which is essentialfor the in vitro transformation of lymphocytes, has been detectedin patients with transplantation lymphoma and in those withleiomyomas.30,31 The absence of the expression of EBNA-2 inthe preinvasive neoplasias is consistent with its absence innasopharyngeal carcinoma and indicates that EBNA-2 is not requiredfor altered epithelial growth.19,20 The absence of the expressionof ZEBRA confirms the latent infection of these lesions by EBV.
Evidence of viral replication has been detected in oropharyngealepithelial cells during primary or reactivated infection withEBV.32 Sporadic reactivation is probably the result of reintroductionof the virus into epithelium from lymphocytes.33 Normally, thistype of reactivation would lead to viral replication and cytolysis.In nasopharyngeal carcinoma, however, the cascade of viral replicationdoes not proceed. Instead, latent EBV genes are expressed, whichprobably induce the infected cells to proliferate, thus forminga preinvasive lesion. The rare detection of preinvasive neoplasia(in 11 of the 1811 samples, or 0.6 percent) and preinvasiveneoplasia with nasopharyngeal carcinoma (in 56 of the 1811 samples,or 3 percent) and the progression to nasopharyngeal carcinomawithin one year in five cases suggest that EBV-induced clonalproliferation can progress rapidly to cancer.
The consistent expression of specific viral genes and the detectionof LMP-1 in all cells in the preinvasive lesions suggest thatcertain EBV gene products contribute to the abnormal proliferationof the cells. The development of latent infection, rather thanlytic infection, in oropharyngeal cells and critical virus-transformingfunctions may be the rare events that lead to the developmentof nasopharyngeal carcinoma. The development of latent infectionin a nasopharyngeal epithelial cell may be influenced by theway in which the virus enters the cell or by the presence ofa preexisting abnormality, perhaps caused by genetic or environmentalfactors.33,34 Genetic changes such as mutations of the p53,retinoblastoma, or ras genes have not been detected in nasopharyngealcarcinoma,35,36 but other genetic changes may develop whilethe tumor is growing and contribute to its progression and metastasis.
Dr. Raab-Traub is the recipient of a grant (CA32979) from theNational Institutes of Health.
We are indebted to Drs. Jian-Jing Chen and Bao Xiang Ou forproviding tissue samples and to Drs. D. Walling, H.S. Earp,and E. Liu for reviewing the manuscript.
Source Information
From the Lineberger Comprehensive Cancer Center (R.P., R.S., N.R.-T.) and the Department of Microbiology (R.S., N.R.-T.), University of North Carolina, Chapel Hill; and the Departments of Pathology (R.P.) and Otorhinolaryngology (U.P.), University of Malaya, Kuala Lumpur, Malaysia.
Address reprint requests to Dr. Raab-Traub at the Lineberger Comprehensive cancer Center, CB#7295, University of North Carolina, Chapel Hill, NC 27599-7295.
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