Am J Otoiaryngol 11:375-381.1990
Viral Oncogenesis: Epstein-Barr
Virus
MICHAEL J. GAFFEY, MD, AND LAWRENCE M. WEISS, MD
The Epstein-Barr virus (EBV) is a common herpesvirus that has been linked to several human neoplasms. It has been found in over 90% of cases of Burkitt’s lymphoma in endemic regions, although only 15% of cases in nonendemic regions show evidence of infection. The virus has been found in malignant lymphomas in immunocompromised states, including the acquired immunodeficiency syndrome, iatrogenic immunosuppression, such as in posttransplantation patients, and congenital immunodeficiencies. Recently, EBV genomes have been found in approximately 25% of cases of Hodgkin’s disease, where it has been found in the Reed-Sternberg cells. Epstein-Barr virus genomes are found in over 90% of cases of undifferentiated nasopharyngeal carcinoma. In addition, EBV has been identified in undifferentiated carcinomas in other sites in Asians, such as carcinomas of the parotid in Chinese individuals and Eskimos. AM J OTOLARYNGOL 11:375-381. 0 1990 by W.B. Saunders Company. Key words: nasopharyngeal carcinoma, Burkitt’s lymphoma, non-Hodgkin’s lymphoma, Hodgkin’s disease.
diffuse (EA/D) or restricted (EA/R) type, nuclear antigen (EBNA), and the virus capsid antigen (VCA). Virus gains entrance to susceptible cells via the C3d receptor for complement (CR2, CD19), resulting in either active or latent infection.3 During active infection, the virus expresses a large part of its genome, replicates, and ultimately causes cell death. In latent infection, EBV DNA persists indefinitely within the cell in an inactive state, from which it may be reactivated. Latently infected, proliferating cells usually contain multiple copies of EBV DNA, most of which are maintained as covalently closed, circular episomes.4
DEFINITION Epstein-Barr virus (EBV) was originally discovered in 1964 by Dr M. A. Epstein and associates with the ultrastructural demonstration of a herpes-like virus in cultured Burkitt’s lymphoma tumor cells.’ A member of the family Herpesviridae, EBV is an enveloped, icosahedral virus that contains a 172 Kb, double-stranded, linear segment of DNA.’ The primary etiologic agent of infectious mononucleosis (IM), EBV is also linked to a spectrum of neoplastic conditions, including Burkitt’s lymphoma (BL), other non-Hodgkin’s lymphomas (NHL), Hodgkin’s disease (HD), and nasopharyngeal carcinoma (NPC). A discussion of the evidence for the involvement of EBV in tumorigenesis forms the basis of this review.
EPIDEMIOLOGY Serologic evidence of infection is commonplace worldwide and characteristically occurs in the first decade of life among individuals in lower socioeconomic groups and developing countries. In socioeconomically deprived areas, virtually 100% of the population demonstrates serologic evidence of infection by 10 years of age. The vast majority of childhood infections are either asymptomatic or associated with mild, nonspecific upper respiratory tract symptomatology. Primary infection in older children and adults commonly results in classic infectious mononucleosis. The incidence of infection is highest within crowded living conditions, such as university students or military recruits, and seasonal trends in EBV infection have not been noted in the general population. Viral transmission requires intimate salivary contact; large droplet or aerosol-airborne spread is
BIOLOGY Epstein-Barr virus is a B-lymphotropic, polyclonal cellular activator that stimulates cell division and transforms lymphocytes into indefinitely propagating lymphoblastoid cell lines in vitro. Clinically important antigens expressed by infected cells include the early antigens (EA), either
Received May 16, 1990, from the Division of Pathology, City of Houe National Medical Center, Duarte. CA. Accented for publication June 9, 1990. Address correspondence to Lawrence M. Weiss, MD, Division of Pathology, City of Hope National Medical Center, Duark, CA 91010. Reprints are not available. 0 1990 by W.B. Saunders Company. 0196-0709/90/1106-0003$5.00/O 375
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apparently not a significant mechanism of infection. Inadvertent transmission by blood transfusion and transplacental spread has been reported. Virus is secreted orally by patients with both primary and reactivated infection and may be recovered from the oropharnygeal secretions of up to 20% of healthy adults sampled.’ There are no recognized animal reservoirs or vectors for human EBV. BURKIlT’S LYMPHOMA Burkitt’s lymphoma was first described in 1958 by Dr Denis Burkitt, who reported an unusual jaw tumor arising in children in East Africa.’ Burkitt’s lymphoma is a common facial tumor of children in endemic Africa. In non-Africans, these tumors are seen in older children and adults and may present intra-abdominally. Histologically, African and non-African (nonendemic) tumors are identical, showing sheets of monomorphic, undifferentiated lymphoid cells with numerous mitotic figures, foci of necrosis, and interspersed, nonneoplastic macrophages (Fig 1). The tumor is classified in the International Working Formulation as a high-grade, small noncleaved malignant lymphoma.6 Burkitt’s lymphoma demonstrates distinctive chromosomal translocations, and virtually all cases show evidence of c-myc protooncogene (a gene thought to be significant in the regulation of normal cellular growth) translocation from chromosome 8 to within the immunoglobulin heavy chain genes of chromosome 14. Other translocations have been identified, all of which involve the translocation of c-myc to light chain immunoglobulin gene sites on other
Figure 1. An example of BL wth sheets of small. undifferentiated lymphoid cells with numerous mitotic figures and interspersed, tingible body macrophages.
VIRAL ONCOGENESIS:
EPSTEIN-BARR
VIRUS
chromosomes7 The translocation is thought to initiate deregulation of c-myc expression, by either structural alteration or translational enhancement by proximity to an immunoglobulin gene.7 The initial isolation in 1964 of EBV from a BLderived lymphoblastic cell line suggested the association of EBV with BL.ls8 The strong epidemiologic relationship that has since been established between these entities shows considerable geographic variation.8*g In endemic Northern and Central Africa, approximately 90% to 95% of BL cases are associated with seropositivity to EBV.l’ In Europe and the United States, however, where the incidence of BL is low, only 15% of cases show evidence of EBV infection. Laboratory evidence of a causal connection includes the demonstration of EBV DNA by nucleic acid hybridization within the great majority of BL cases examined.‘l-13 The recent demonstration by Southern blot hybridization of a single type of EBV DNA within BL cells supports the existence of viral nucleic acid in the initial lymphomatous cell and a role for EBV in the pathogenesis of this neoplasm.14 The precise mechanism of involvement, however, remains unknown. One possibility is that the observed chromosomal translocations may be directly induced by EBV. Infected tissue culture cells, however, do not demonstrate a high incidence of chromosomal translocations, and those that do occur are different from those observed in BL.15 Most investigators currently favor the hypothesis that EBV infection results in the indefinite propagation of EBV-transformed B-cell clones with an increased probability of genetic rearrangement and resultant c-myc deregulation.
GAFFEYANDWEISS
Because primary infection with EBV appears to be worldwide, the restricted geographic distribution of BL suggests that environmental factors may be important in the reactivation of the EBV genome within latently infected B lymphocytes. The geographic coincidence of endemic BL and malaria is therefore of interest. It has been shown that acute Plasmodium falciparum malaria causes certain derangements in cellular immunity, including cytotoxic surveillance for EBV-infected lymphoid cel1s.l” As the majority of Central Africans show serologic evidence of EBV infection at a young age, suppression of cellular immunity by concomitant malarial infection would theoretically lead to the circumstance of numerous individuals with indefinitely propagating clones of EBVinfected B cells. The high prevalence of affected persons would then increase the probability of genetic translocations. The possible role of other EBV-activating agents in the induction of BL must also be considered. Using a synergistic assay method to investigate the effect of various chemicals on the in vitro reactivation of EBV DNA in latently infected lymphoid cells, several tumor-promoting substances have been identified in the environment.17,18 Extracts of many members of the plant family Euphorbiaceae, commonly located in the “BL lymphoma belt” of Eastern Africa, demonstrate EBVactivating potency. Locally used for medicinal purposes, the leaves, bark, and roots are commonly chewed, applied to wounds, or boiled and drunk as decoctions.‘7 The primarily oral use of these substances may be related to the common occurrence of mandibular BL in this population. MALIGNANT LYMPHOMAS IN IMMUNOCOMPROMISED STATES
Non-Hodgkin’s malignant lymphomas, predominantly of B-cell origin, are common in immunodeficient patients and may be associated with EBV. Affected patients may have either acquired, iatrogenic, or congenital immunodeficiencies, each of which will be discussed separately. The premier example of acquired immunodeficiency is the acquired immunodeficiency syndrome (AIDS), in which NHL is a common complication.lg Tumors are commonly diffuse, extranodal, and aggressive, with variable median survival dependent on histopathologic type. Individuals with AIDS have a profound defect of Tcell immunity to EBV infection, often demonstrating abnormally high numbers of circulating EBVinfected B cells.” Interestingly, the majority of AIDS-associated NHL patients show evidence of c-myc gene rearrangement.lg The introduction of an activated c-myc gene into EBV-infected lym-
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phoblasts from AIDS patients has also been shown to result in their malignant conversion.21 Therefore, similar to the situation with BL, AIDSassociated immunosuppression and concomitant EBV infection may result in the clonal expansion of EBV-infected B cells, increasing the probability of the subsequent genetic alterations responsible for lymphomagenesis. However, only 40% of NHLs in AIDS patients contain detectable EBV genomes.” Therefore, while EBV may be important in lymphomagenesis in 40% of cases, other mechanisms are obviously operative in the majority of cases. Currently, iatrogenic, or induced, immunosuppression is most commonly encountered in allograft recipients, in whom NHL is an occasional complication, with an incidence ranging from 1% to 15sez3 Posttransplant lymphoproliferative disorders (PTLDs) are usually diffuse, large-cell lymphomas of B-cell origin and may be monoclonal, oligoclonal, or polyclonal.24825 Disease may either regress, with a reduction of immunosuppression, or pursue an aggressive, fatal course. Evidence for an association with EBV includes one study in which lymphomatous tissue from 13 of 14 kidney, liver, heart, or heart-lung transplant recipients were positive for EBV genomic material by Southern blot.26 Another report found serologic evidence of primary EBV infection in five of five, and EBV DNA in three of five PTLDs occurring in 53 heart-lung transplantation recipients.27 A recent study of NHL in cardiac allograft recipients demonstrated EBV DNA within the monoclonal lymphomatous population.28 We frequently uncover evidence of EBV genomic material within PTLDs from heart-lung and liver recipients by in situ hybridization techniques as well (unpublished data). The precise role of EBV in the development of these lesions is unknown. Again, available evidence points to clonal expansion of an EBVinfected, progenitor B cell with a predisposition to genetic alterations. Patients with congenital immunodeficiencies also have an extremely high incidence of NHL in comparison to the general population. Four percent develop malignancies of some type; of these 60% are either NHL or lymphocytic leukemia cases.” Appropriately studied tumors are B-cell lineage, large-cell lymphomas with evidence of integrated EBV genomic material. The WiscottAldrich, ataxia-telangiectasia, Duncan’s, and severe combined immunodeficiency syndromes have all been associated with EBV-positive lymphomas. Duncan’s syndrome, an X-linked lymphoproliferative syndrome, has been particularly well studied.30 Affected males have defective T cells unable to recognize EBV-transformed lymphocytes, and commonly develop fatal IM or
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chronic IM progressive to agammaglobulinemia and/or EBV-associated malignant lymphoma.
MALIGNANT LYMPHOMA, T-CELL TYPE The association of EBV with T-cell neoplasia seems tenuous, as mature T cells are not thought to express the C3d receptor, the protein responsible for EBV entry into B cells. Although T lymphocytes transfected with EBV DNA can support sustained viral replication,31 normal T cells are not known to be a site of latent or active EBV infection. Nonetheless, rare T-cell lymphomas have been found to contain EBV DNA.32 A single report documented three such cases in patients with historic and serologic evidence of chronic EBV infection. Their tumor cells had the characteristics of activated helper T cells with T-cell receptor genetic rearrangements (indicating monoclonality) and evidence of EBV genomic material. Interestingly, tumor cells in two of these cases appeared to express the C3d receptor. Occasional cases of angioimmunoblastic lymphadenopathy, a T-cell lymphoproliferative disorder closely related to T-cell lymphoma, have also been found to contain EBV DNA.33
HODGKIN’S DISEASE A large body of epidemiologic and molecular evidence supports a relationship between EBV and HD. Patients with a history of EBV-positive IM are approximately two to four times more likely to develop HD relative to the general population. 34 Patients with HD often demonstrate an altered antibody pattern to EBV at least 3 years prior to diagnosis, evidence that the activation of latent EBV genomic material may directly precede the initiation of disease.35 The recent demonstration of EBV DNA in diseased tissue provides additional support for the idea that EBV can play a causal role in tumorigenesis. In one report, EBV genomic material was identified in four of 21 cases of mixed cellularity and nodular sclerosing HD.36 Southern blot analysis revealed a monoclonal or oligoclonal proliferation of EBV-infected cells in the positive cases. In another study, 29% of HD cases were positive for EBV genomic material.33 Subsequent in situ hybridization studies localized the EBV DNA to the neoplastic Reed-Sternberg cells.37,38 While EBV genomic material is absent from a significant proportion of HD cases, there is strong evidence supporting the causative role of EBV in a significant subset of cases. Whether EBV is present below the current threshold of detection in other cases of HD remains to be determined.
VIRAL ONCOGENESIS: EPSTEIN-BARR VIRUS
NASOPHARYNGEAL CARCINOMA Undifferentiated NPC (lymphoepithelioma; UNPC), a rare tumor in most parts of the world, has an unusually high incidence in the Chinese. In contrast to the geographically related incidence of EBV-associated BL, the association of EBV with NPC has been reported worldwide. Nasopharyngeal carcinoma as a whole, including both keratinizing and undifferentiated variants, accounts for 89% to 90% of the malignant nasopharyngeal neoplasms in the United States and up to 98% in some areas of the Orient.3gr40 Histologically, UNPC demonstrates cytologically malignant cells with indistinct cytoplasmic borders, resulting in a syncytial pattern of growth, and a reactive inflammatory infiltrate composed predominantly of lymphocytes. The neoplastic epithelial cells may present either as well-defined, cohesive cell nests (Regaud pattern) or as isolated cells within a dense lymphocytic infiltrate (Schmincke pattern) (Fig 2). The association of EBV with UNPC was first proposed in 1966 following the observation that sera from UNPC patients contained antibodies directed against lymphoblasts from productive, EBV-infected cell lines.43 High titers of anti-VCA, EA/D, and neutralizing antibodies were subsequently demonstrated in UNPC patients from different parts of the world by immunofluorescence techniques.44-47 Anti-VCA and anti-EA/D titers are also related to total tumor burden, as antibody titers progressively increase with advancing disease stage and are commonly lower in treated, long-term survivors than in untreated patients.45.46 The close link between EBV and UNPC was established by the demonstration of EBV DNA within African UNPC by nucleic acid hybridization in 1970 by zur Hausen et al*’ and, subsequently, by others.‘2,48,4g Viral-specific nuclear antigens (EBNA) were identified in epithelial cells but not lymphoid cells by both immunofluorescence48~50 and nucleic acid hybridization techniques.47 The demonstration in 1986 of a homogeneous EBV episomal population within the epithelial cells of several UNPCs provided direct evidence that this tumor, like BL, may represent an EBV-initiated clonal expansion of a single infected progenitor ce11.14 Formalin-fixed, paraffinembedded UNPC material has also revealed EBV genomic material by in situ hybridization (Fig 2).5l
The reactivation of latent EBV infection in the nasopharynx is accompanied by the production of high titers of anti-VCA and anti-immunoglobulin EA A (IgA) antibodies.44*52 The simultaneous absence of EBV-specific antibody in either
GAFFEY AND WEISS
Figure 2. (Top] An example of an undifferentiated carcinoma (lymphoepithelioma) of the nasopharynx, with sheets of neoplastic epithelioid cells surrounded by reactive lymphocytes (Regaud pattern). (Bottom] An in situ hybridization study of a lymphoepithelioma for EBV using a “S- labeled probe. Numerous grains are present over the tumor, which indicates a large amount of EBV genomes within the neoplastic cells.
., ‘: ‘. I
healthy controls or patients with other carcinomas suggests their use as markers for the early detection of UNPC.44v53A study of 124 North American UNPC patients revealed the IgA antiVCA antibody as a potentially diagnostic marker for the presence of occult disease.54 In some areas of China, IgA anti-VCA is used to detect UNPC in mass screening programs.55-57 One study of 148,029adults revealed 3,533 with IgA anti-VCA titers antibodies, of whom 460 had antibody 31:80.Of these individuals, 55 cases of UNPC were detected and histologically confirmed.57 Since oropharyngeal epithelial cells lack the C3d receptor necessary for viral entry into human lymphocytes, the precise mechanism of viral penetration into these cells is unknown. The demon-
stration of a human epithelial cell surface protein antigenically related to the C3d receptor, however, suggests that EBV may enter cells through a similar, alternative mechanism.5* While the association of EBV with the pathogenesis of UNPC appears firmly established, the precise role of EBV in the initiation and promotion of tumorigenesis in unknown. As in the case of BL, environmental factors geographically related to areas of endemic UNPC may be significant in the reactivation of persistent EBV genomic material. Many members of the Euphorbiaceae and Thymelaeaceae plant families implicated in the causation of BL, including Croton tiglium (the croton oil tree) and Aleurites fordii (tung oil tree), also have a geographic distribution similar to that
380
of endemic UNPC in Southern China. The leaves, bark, and roots of these plants are commonly used by the local population as medicinal herbs for both external and internal use.17 Individuals may also be exposed via the inhalation of pollen or contact with plant extract-contaminated soil. Rare cases of lymphoepithelioma have been reported outside the nasopharynx. Lymphoepithelioma of the parotid in Chinese individuals and Eskimos has been associated with EBV, while at other sites and in other ethnic groups, only a rare association has been noted.50,5g-61 Close histologic mimics of UNPC that occur in the nasopharynx have not been found to be associated with EBV, at least in North American patients.50 The strong ethnic, geographic, anatomic, and histologic influences that condition the association between EBV and lymphoepithelioma remain incompletely understood. CONCLUSION Epstein-Barr virus is a ubiquitous viral pathogen that commonly infects B lymphocytes and oropharyngeal epithelial cells. Primary infection commonly results in IM, a benign, self-limited disease with a characteristic clinical course. Under conditions of immunosuppression, however, EBV infection results in the polyclonal expansion of EBV-infected B lymphocytes with an increased probability of genetic rearrangement and the development of clinical malignancy. The precise role of EBV in the initiation and promotion of such neoplasms as BL, NHL (of both B- and T-cell lineage), HD, and UNPC, however, has yet to be precisely determined. References 1. Epstein MA, Achong BG, Barr YM: Virus particles in cultured lymphoblasts from Burkitt’s lymphoma. Lancet 1964; 1:702-703 2. Lennette ET: Epstein-Barr virus, in Lennette EH, Balows A. Hausler WT. et al (edsl: Manual of Clinical Microbiology, 4th ed. Washington, DC; American Society for MicrobiologyTi985, pp 728-732 3. Fingeroth JD, Weiss JJ, Tedder TF, et al: Epstein-Barr virus receotor of human B lymphocytes is the C3d receptor CR2. Proc Nat1 Acad Sci USA i984; 81:4510-4514 4. Lindahl T. Adams A, Biursell G, et al: Covalentlv closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line. J Mol Biol 1976; 102:511-530 5. BurkItt D: A sarcoma involving the jaws in African children. Br J Surg 1958; 46:218-223 6. Rosenberg SA (Chairman): National cancer institute sponsored study of classifications of non-Hodgkin’s lymphomas. Summary and description of a working formulation for clinical usage. Cancer 1982; 49:2112-2135 7. Croce CM, Nowell PC: Molecular genetics of human B cell neoplasia. Adv Immunol 1985; 38:245-274 8. de-The G, Geser A, Day NE, et al: Epidemiological evidence for a causal relationship between Epstein-Barr virus and Burkitt’s lymphoma: Results of the Ugandan prospective study. Nature 1978; 274:756-761
VIRAL ONCOGENESIS:
EPSTEIN-BARR
VIRUS
9. Lenoir GM: Role of the virus, chromosomal translocations and cellular oncogenes in the aetiology of Burkitt’s lymphoma, in Epstein MA, Achong BG (eds): The Epstein-Barr Virus: Recent Advances. New York, NY, Wiley, 1986, pp 183-205 10. Geser A, Lenoir G, Anvret M, et al:Epstein-Barr virus markers in a series of Burkitt’s lymphomas from the West Nile District, Uganda. Eur J Cancer ClinOncol 1983; 19:1393-1401 11. zur Hausen H. Schulte-Holthausen H. Klein G. et al: EBV DNA in biopsies of Burkitt tumors and anaplastic carcinomas of the nasopharynx. Nature 1970; 228:1056-1058 12. Nonovama M. Huane. CH. Paaano IS. et al: DNA of EDstein-Barr virus detected in tissue aufBurkitt’s lymphoma and nasopharyngeal carcinoma. Proc Nat1 Acad Sci USA 1973; 70:3265-3268 13. Bornkamm GW, Stein H, Lennert K, et al: Attempts to demonstrate virus-specific sequences in human tumors. IV. EB viral DNA in European Burkitt lymphoma and immunoblastic lymphadenopathy with excessive plasmacytosis. Int J Cancer 1978; 17:177-181 14. Raab-Traub N, Flynn K: The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell 1986; 47:883-889 15. Shade M, Woodward MA, Steel CM: Chromosome aberrations acquired in vitro by human B-cell lines. II. Distribution of break uoints. TNCI 1980: 65:101-109 16. Whittle HC, Brown J, Marsh K, et al: T cell control of Epstein-Barr virus-infected B cells is lost during P. falciparum malaria. Nature 1984; 312:449-450 17. Ito Y: Vegetable activators of the viral genome and the causation of Burkitt’s lymphoma and nasopharyngeal carcinoma, in Epstein MA, Achong BG (eds]: The Epstein-Barr Virus: Recent Advances. New York, NY, 1986, pp 207-236 18. Ito Y, Yanase S, Fujita J, et al: A short term in vitro assay for promoter substances using human lymphoblastoid cells latently infected with Epstein-Barr virus. Cancer Lett 1981; 13:29-37 19. Knowles DM, Chamulak G, Subar M, et al: Clinicopathologic, immunophenotypic and molecular genetic analysis of AIDS-associated lymphoid neoplasia. Clinical and biologic implications. Path01 Annu 1988; 23:33-67 20. Birx DL, Redfield RR, Tosato G: Defective regulation of Epstein-Barr virus infection in patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related disorders. N EnglJ Med 1986; 314:874-879 21. Lombardi L, Newcomb EW, Dalla-Favera R: Pathogenesis of Burkitt lymphoma: Expression of an activated c-myc oncogene causes the tumorigenic conversion of EBV-infected human B lymphoblasts. Cell 1987; 49:161-170 22. Subar M, Neri A, Inghirami G, et al: Frequent c-myc oncogene activation and infrequent presence of Epstein-Barr virus genome in AIDS-associated lymphoma. Blood 1988; 72:667-671 23. Cleary ML, Dorfman RF, Sklar J: Failure in immunological control of the virus infection post-transplant lymphomas, in Epstein MA, Achong BG (eds): The Epstein-Barr Virus: Recent Advances. New York, NY, Wiley, 1986, pp 163-181 24. Cleary ML, Warnke R, Sklar J: Monoclonality of lymphoproliferative lesions in cardiac-transplant recipients: Clonal analysis based on immunoglobulin gene rearrangements. N Engl J Med 1984; 310:477-482 25. Shearer WT. Ritz J, Finegold MJ, et al: Epstein-Barr virus-associated B-cell proliferations of diverse clonal origins after bone marrow transplantation in a 12-year-old patient with severe combined immunodeficiency. N Engl J Med 1985; 312:1151-1159 26. Ho M, Miller G, Atchison RW, et al: Epstein-Barr virus infections and DNA hybridization studies in posttransplantation lymphoma and lymphoproliferative lesions: The role of primary infection. J Infect Dis 1985; 152:876-886 27. Randhawa PS, Yousem SA, Paradis IL, et al: The clinical spectrum, pathology, and clonal analysis of Epstein-Barr virusassociated lymphoproliferative disorders in heart-lung transplant recipients. Am J Clin Path01 1989; 92:177-185 28. Cleary ML, Nalesnik MA, Shearer WT, et al: Clonal analysis of transplant-associated lymphoproliferations based on the structure of the genomic termini of the Epstein-Barr virus. Blood 1988: 72:349-352
GAFFEY AND WEISS 29. Spector B, Perry GS, Kersey JH: Genetically determined immunodeficiencies (GDID) and malignancy: Report from the immunodeficiency-cancer registry. Clin Immunol Immunopathol 1978; 11:12-29 30. Purtilo DT: Malignant lymphoproliferative diseases induced by Epstein-Barr virus in immunodeficient patients, including X-linked, cytogenetic, and familial syndromes. Cancer Genet Cytogenet 1981; 4:251-268 31. Stevenson M, Volsky B, Hedenskog M, et al: Immortalization of human T lymphocytes after transfection of EpsteinBarr virus DNA. Science 1986; 233:980-984 32. Jones JF, Shurin S. Abramowsky C, et al: T-cell lymphomas containing Epstein-Barr viral DNA in patients with chronic Epstein-Barr virus infections. N Engl J Med 1988; 318:733-741 33. Staal SP, Ambinder R, Beschorner WE, et al: A survey of Epstein-Barr virus DNA in lymphoid tissue: frequent detection in Hodgkin’s disease. Am J Clin Path01 1989; 91:1-5 34. Gutensohn N, Cole P: Childhood social environment and Hodgkin’s disease. N Engl J Med 1981; 304:135-140 35. Mueller N, Evans A, Harris NL, et al: Hodgkin’s disease and Epstein-Barr virus: Altered antibody pattern before diagnosis. N Engl J Med 1989; 320:689-695 36. Weiss LM, Strickler JG, Warnke RA, et al: Epstein-Barr viral DNA in tissues of Hodgkin’s disease. Am J Pathol 1987; 129:86-91 37. Weiss LM, Movahed LA, Warnke RA, et al: Detection of Epstein-Barr viral genomes in Reed-Sternberg cells of Hodgkin’s disease. N Engl J Med 1989; 320:502-506 38. Anagnostopoulos I, Herbst H, Niedobitek G, et al: Demonstration of monoclonal EBV genomes in Hodgkin’s disease and KI-1 positive anaplastic large cell lymphoma by combined Southern blot and in situ hybridization. Blood 1989; 74:810818 39. Yeh S: A histological classification of carcinomas of the nasopharynx with critical review as to the existence of lymphoepitheliomas. Cancer 1962: 15:895-920 40. Easton JM, Levine PH, Hyams VJ: Nasopharyngeal carcinoma in the United States. Arch Otolaryngoll980; 106:88-91 41. Regaud C, Reverchon L: Sur un cas d’epithelioma epidermoide developpe dams le massif maxillaire superieur. Rev Laryngol Otol Rhino1 (Bord) 1921; 42:369-378 42. Schmincke A: Uber lympho-epitheliale Geschwulste. Beitr Path01 Anat Allg Path01 1921; 68:161-170 43. Old LJ, Boyse EA, Oettgen HF, et al: Precipitating antibody in human serum to an antigen present in cultured Burkitt’s lymphoma cells. Proc Nat1 Acad Sci USA 1966; 56:16991704 44. Henle G, Henle W: Epstein-Barr virus-specific IgA serum antibodies as an outstanding feature of nasopharyngeal _ _carcinoma. Int J Cancer 1976; 17:i-7 45. Henle W. Henle G. Ho HC. et al: Antibodies to EosteinBarr virus in nasopharyngeal carcinoma, other head and neck neoplasms, and control groups. JNCI 1970; 44:225-231 46. Henle W, Ho HC, Henle G, et al: Antibodies to Epstein-
381 Barr virus-related antigen in nasopharyngeal carcinomas: Comparisons of active cases with long term survivors. JNCI 1973; 51:361-369 47. Desgranges C, Wolf H, De-The G, et al: Nasopharyngeal carcinoma. X. Presence of Epstein-Barr genomes in separated epithelial cells of tumors in patients from Singapore, Tunisia, and Kenya. Int J Cancer 1975; 16:7-15 48. Wolf H, zur Hausen H, Becker V: EB viral genomes in epithelial nasopharyngeal carcinoma cells. Nature New Biol 1973; 244~245247 49. Wolf H, zur Hausen H, Klein G, et al: Attempts to detect virus-specific DNA sequences in human tumors. III. EpsteinBarr viral DNA in non-lymphoid nasopharyngeal carcinoma cells. Med Microbial Immunol (Berl) 1975; 161:15-21 50. Huang DP, Ho JHC, Henle W, et al: Demonstration of Epstein-Barr virus associated nuclear antigen in nasopharyngeal carcinoma cells from fresh biopsies. Int J Cancer 1974; 14:580-588 51. Weiss LM, Movahed LA, Butler AE, et al: Analysis of lymphoepithelioma and lymphoepithelioma-like carcinomas for Epstein-Barr viral genomes by in situ hybridization. Am J Surg Path01 1989; 13:625-631 52. Raab-Traub N, Hood R, Yang C-S, et al: Epstein-Barr virus transcription in nasopharyngeal carcinoma. J Virol 1983; 48:580-590 53. Ringborg U, Henle W, Henle G, et al: Epstein-Barr virusspecific serodiagnostic tests in carcinomas of the head and neck. Cancer 1983; 52:1237-1243 54. Pearson RG, Weiland LH, Neel B, et al: Application of Epstein-Barr virus (EBV) serology to the diagnosis of North American nasopharyngeal carcinoma. Cancer 1983; 51:260268 55. Zeng Y, Yuxi L, Chunren L, et al: Application of an immunoenzymatic method and an immunoautoradiographic method for a mass survey of nasopharyngeal carcinoma. Intervirology 1980; 13:162-168 56. Zeng Y, Liu XX, Wei JN, et al: Serological mass survey of nasopharyngeal carcinoma. Acta Acad Med Sin 1979; 1:123126 57. Zeng Y, Zhong J, Li LY, et al: Follow-up studies on Epstein-Barr virus IgAfVCA antibody-positive persons in Zangwu County, China. Intervirology 1983; 20:190-194 58. Young LS, Dawson CW, Brown KW, et al: Identification of a human epithelial cell surface protein sharing an epitope with the C3diEpstein-Barr virus receptor molecule of B lymphocytes. Int J Cancer 1989; 43:786-794 59. Krishnamurthy S, Lanier AP, Dohan P, et al: Salivary gland cancer in Alaskan natives, 1966-1980. Hum Path01 1987; 18:986-996 60. Saw D, Lau WH, Ho JHC, et al: Malignant lymphoepithelioma lesion of the salivary gland. Hum Path01 1986; 17:914-923 61. Butler AE, Colby TV, Weiss LM, et al: Lymphoepithelioma-like carcinoma of the lung. Am J Surg Path01 1989; 13:632-639
Viral Oncogenesis: Epstein-Barr
Virus
MICHAEL J. GAFFEY, MD, AND LAWRENCE M. WEISS, MD
The Epstein-Barr virus (EBV) is a common herpesvirus that has been linked to several human neoplasms. It has been found in over 90% of cases of Burkitt’s lymphoma in endemic regions, although only 15% of cases in nonendemic regions show evidence of infection. The virus has been found in malignant lymphomas in immunocompromised states, including the acquired immunodeficiency syndrome, iatrogenic immunosuppression, such as in posttransplantation patients, and congenital immunodeficiencies. Recently, EBV genomes have been found in approximately 25% of cases of Hodgkin’s disease, where it has been found in the Reed-Sternberg cells. Epstein-Barr virus genomes are found in over 90% of cases of undifferentiated nasopharyngeal carcinoma. In addition, EBV has been identified in undifferentiated carcinomas in other sites in Asians, such as carcinomas of the parotid in Chinese individuals and Eskimos. AM J OTOLARYNGOL 11:375-381. 0 1990 by W.B. Saunders Company. Key words: nasopharyngeal carcinoma, Burkitt’s lymphoma, non-Hodgkin’s lymphoma, Hodgkin’s disease.
diffuse (EA/D) or restricted (EA/R) type, nuclear antigen (EBNA), and the virus capsid antigen (VCA). Virus gains entrance to susceptible cells via the C3d receptor for complement (CR2, CD19), resulting in either active or latent infection.3 During active infection, the virus expresses a large part of its genome, replicates, and ultimately causes cell death. In latent infection, EBV DNA persists indefinitely within the cell in an inactive state, from which it may be reactivated. Latently infected, proliferating cells usually contain multiple copies of EBV DNA, most of which are maintained as covalently closed, circular episomes.4
DEFINITION Epstein-Barr virus (EBV) was originally discovered in 1964 by Dr M. A. Epstein and associates with the ultrastructural demonstration of a herpes-like virus in cultured Burkitt’s lymphoma tumor cells.’ A member of the family Herpesviridae, EBV is an enveloped, icosahedral virus that contains a 172 Kb, double-stranded, linear segment of DNA.’ The primary etiologic agent of infectious mononucleosis (IM), EBV is also linked to a spectrum of neoplastic conditions, including Burkitt’s lymphoma (BL), other non-Hodgkin’s lymphomas (NHL), Hodgkin’s disease (HD), and nasopharyngeal carcinoma (NPC). A discussion of the evidence for the involvement of EBV in tumorigenesis forms the basis of this review.
EPIDEMIOLOGY Serologic evidence of infection is commonplace worldwide and characteristically occurs in the first decade of life among individuals in lower socioeconomic groups and developing countries. In socioeconomically deprived areas, virtually 100% of the population demonstrates serologic evidence of infection by 10 years of age. The vast majority of childhood infections are either asymptomatic or associated with mild, nonspecific upper respiratory tract symptomatology. Primary infection in older children and adults commonly results in classic infectious mononucleosis. The incidence of infection is highest within crowded living conditions, such as university students or military recruits, and seasonal trends in EBV infection have not been noted in the general population. Viral transmission requires intimate salivary contact; large droplet or aerosol-airborne spread is
BIOLOGY Epstein-Barr virus is a B-lymphotropic, polyclonal cellular activator that stimulates cell division and transforms lymphocytes into indefinitely propagating lymphoblastoid cell lines in vitro. Clinically important antigens expressed by infected cells include the early antigens (EA), either
Received May 16, 1990, from the Division of Pathology, City of Houe National Medical Center, Duarte. CA. Accented for publication June 9, 1990. Address correspondence to Lawrence M. Weiss, MD, Division of Pathology, City of Hope National Medical Center, Duark, CA 91010. Reprints are not available. 0 1990 by W.B. Saunders Company. 0196-0709/90/1106-0003$5.00/O 375
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apparently not a significant mechanism of infection. Inadvertent transmission by blood transfusion and transplacental spread has been reported. Virus is secreted orally by patients with both primary and reactivated infection and may be recovered from the oropharnygeal secretions of up to 20% of healthy adults sampled.’ There are no recognized animal reservoirs or vectors for human EBV. BURKIlT’S LYMPHOMA Burkitt’s lymphoma was first described in 1958 by Dr Denis Burkitt, who reported an unusual jaw tumor arising in children in East Africa.’ Burkitt’s lymphoma is a common facial tumor of children in endemic Africa. In non-Africans, these tumors are seen in older children and adults and may present intra-abdominally. Histologically, African and non-African (nonendemic) tumors are identical, showing sheets of monomorphic, undifferentiated lymphoid cells with numerous mitotic figures, foci of necrosis, and interspersed, nonneoplastic macrophages (Fig 1). The tumor is classified in the International Working Formulation as a high-grade, small noncleaved malignant lymphoma.6 Burkitt’s lymphoma demonstrates distinctive chromosomal translocations, and virtually all cases show evidence of c-myc protooncogene (a gene thought to be significant in the regulation of normal cellular growth) translocation from chromosome 8 to within the immunoglobulin heavy chain genes of chromosome 14. Other translocations have been identified, all of which involve the translocation of c-myc to light chain immunoglobulin gene sites on other
Figure 1. An example of BL wth sheets of small. undifferentiated lymphoid cells with numerous mitotic figures and interspersed, tingible body macrophages.
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chromosomes7 The translocation is thought to initiate deregulation of c-myc expression, by either structural alteration or translational enhancement by proximity to an immunoglobulin gene.7 The initial isolation in 1964 of EBV from a BLderived lymphoblastic cell line suggested the association of EBV with BL.ls8 The strong epidemiologic relationship that has since been established between these entities shows considerable geographic variation.8*g In endemic Northern and Central Africa, approximately 90% to 95% of BL cases are associated with seropositivity to EBV.l’ In Europe and the United States, however, where the incidence of BL is low, only 15% of cases show evidence of EBV infection. Laboratory evidence of a causal connection includes the demonstration of EBV DNA by nucleic acid hybridization within the great majority of BL cases examined.‘l-13 The recent demonstration by Southern blot hybridization of a single type of EBV DNA within BL cells supports the existence of viral nucleic acid in the initial lymphomatous cell and a role for EBV in the pathogenesis of this neoplasm.14 The precise mechanism of involvement, however, remains unknown. One possibility is that the observed chromosomal translocations may be directly induced by EBV. Infected tissue culture cells, however, do not demonstrate a high incidence of chromosomal translocations, and those that do occur are different from those observed in BL.15 Most investigators currently favor the hypothesis that EBV infection results in the indefinite propagation of EBV-transformed B-cell clones with an increased probability of genetic rearrangement and resultant c-myc deregulation.
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Because primary infection with EBV appears to be worldwide, the restricted geographic distribution of BL suggests that environmental factors may be important in the reactivation of the EBV genome within latently infected B lymphocytes. The geographic coincidence of endemic BL and malaria is therefore of interest. It has been shown that acute Plasmodium falciparum malaria causes certain derangements in cellular immunity, including cytotoxic surveillance for EBV-infected lymphoid cel1s.l” As the majority of Central Africans show serologic evidence of EBV infection at a young age, suppression of cellular immunity by concomitant malarial infection would theoretically lead to the circumstance of numerous individuals with indefinitely propagating clones of EBVinfected B cells. The high prevalence of affected persons would then increase the probability of genetic translocations. The possible role of other EBV-activating agents in the induction of BL must also be considered. Using a synergistic assay method to investigate the effect of various chemicals on the in vitro reactivation of EBV DNA in latently infected lymphoid cells, several tumor-promoting substances have been identified in the environment.17,18 Extracts of many members of the plant family Euphorbiaceae, commonly located in the “BL lymphoma belt” of Eastern Africa, demonstrate EBVactivating potency. Locally used for medicinal purposes, the leaves, bark, and roots are commonly chewed, applied to wounds, or boiled and drunk as decoctions.‘7 The primarily oral use of these substances may be related to the common occurrence of mandibular BL in this population. MALIGNANT LYMPHOMAS IN IMMUNOCOMPROMISED STATES
Non-Hodgkin’s malignant lymphomas, predominantly of B-cell origin, are common in immunodeficient patients and may be associated with EBV. Affected patients may have either acquired, iatrogenic, or congenital immunodeficiencies, each of which will be discussed separately. The premier example of acquired immunodeficiency is the acquired immunodeficiency syndrome (AIDS), in which NHL is a common complication.lg Tumors are commonly diffuse, extranodal, and aggressive, with variable median survival dependent on histopathologic type. Individuals with AIDS have a profound defect of Tcell immunity to EBV infection, often demonstrating abnormally high numbers of circulating EBVinfected B cells.” Interestingly, the majority of AIDS-associated NHL patients show evidence of c-myc gene rearrangement.lg The introduction of an activated c-myc gene into EBV-infected lym-
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phoblasts from AIDS patients has also been shown to result in their malignant conversion.21 Therefore, similar to the situation with BL, AIDSassociated immunosuppression and concomitant EBV infection may result in the clonal expansion of EBV-infected B cells, increasing the probability of the subsequent genetic alterations responsible for lymphomagenesis. However, only 40% of NHLs in AIDS patients contain detectable EBV genomes.” Therefore, while EBV may be important in lymphomagenesis in 40% of cases, other mechanisms are obviously operative in the majority of cases. Currently, iatrogenic, or induced, immunosuppression is most commonly encountered in allograft recipients, in whom NHL is an occasional complication, with an incidence ranging from 1% to 15sez3 Posttransplant lymphoproliferative disorders (PTLDs) are usually diffuse, large-cell lymphomas of B-cell origin and may be monoclonal, oligoclonal, or polyclonal.24825 Disease may either regress, with a reduction of immunosuppression, or pursue an aggressive, fatal course. Evidence for an association with EBV includes one study in which lymphomatous tissue from 13 of 14 kidney, liver, heart, or heart-lung transplant recipients were positive for EBV genomic material by Southern blot.26 Another report found serologic evidence of primary EBV infection in five of five, and EBV DNA in three of five PTLDs occurring in 53 heart-lung transplantation recipients.27 A recent study of NHL in cardiac allograft recipients demonstrated EBV DNA within the monoclonal lymphomatous population.28 We frequently uncover evidence of EBV genomic material within PTLDs from heart-lung and liver recipients by in situ hybridization techniques as well (unpublished data). The precise role of EBV in the development of these lesions is unknown. Again, available evidence points to clonal expansion of an EBVinfected, progenitor B cell with a predisposition to genetic alterations. Patients with congenital immunodeficiencies also have an extremely high incidence of NHL in comparison to the general population. Four percent develop malignancies of some type; of these 60% are either NHL or lymphocytic leukemia cases.” Appropriately studied tumors are B-cell lineage, large-cell lymphomas with evidence of integrated EBV genomic material. The WiscottAldrich, ataxia-telangiectasia, Duncan’s, and severe combined immunodeficiency syndromes have all been associated with EBV-positive lymphomas. Duncan’s syndrome, an X-linked lymphoproliferative syndrome, has been particularly well studied.30 Affected males have defective T cells unable to recognize EBV-transformed lymphocytes, and commonly develop fatal IM or
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chronic IM progressive to agammaglobulinemia and/or EBV-associated malignant lymphoma.
MALIGNANT LYMPHOMA, T-CELL TYPE The association of EBV with T-cell neoplasia seems tenuous, as mature T cells are not thought to express the C3d receptor, the protein responsible for EBV entry into B cells. Although T lymphocytes transfected with EBV DNA can support sustained viral replication,31 normal T cells are not known to be a site of latent or active EBV infection. Nonetheless, rare T-cell lymphomas have been found to contain EBV DNA.32 A single report documented three such cases in patients with historic and serologic evidence of chronic EBV infection. Their tumor cells had the characteristics of activated helper T cells with T-cell receptor genetic rearrangements (indicating monoclonality) and evidence of EBV genomic material. Interestingly, tumor cells in two of these cases appeared to express the C3d receptor. Occasional cases of angioimmunoblastic lymphadenopathy, a T-cell lymphoproliferative disorder closely related to T-cell lymphoma, have also been found to contain EBV DNA.33
HODGKIN’S DISEASE A large body of epidemiologic and molecular evidence supports a relationship between EBV and HD. Patients with a history of EBV-positive IM are approximately two to four times more likely to develop HD relative to the general population. 34 Patients with HD often demonstrate an altered antibody pattern to EBV at least 3 years prior to diagnosis, evidence that the activation of latent EBV genomic material may directly precede the initiation of disease.35 The recent demonstration of EBV DNA in diseased tissue provides additional support for the idea that EBV can play a causal role in tumorigenesis. In one report, EBV genomic material was identified in four of 21 cases of mixed cellularity and nodular sclerosing HD.36 Southern blot analysis revealed a monoclonal or oligoclonal proliferation of EBV-infected cells in the positive cases. In another study, 29% of HD cases were positive for EBV genomic material.33 Subsequent in situ hybridization studies localized the EBV DNA to the neoplastic Reed-Sternberg cells.37,38 While EBV genomic material is absent from a significant proportion of HD cases, there is strong evidence supporting the causative role of EBV in a significant subset of cases. Whether EBV is present below the current threshold of detection in other cases of HD remains to be determined.
VIRAL ONCOGENESIS: EPSTEIN-BARR VIRUS
NASOPHARYNGEAL CARCINOMA Undifferentiated NPC (lymphoepithelioma; UNPC), a rare tumor in most parts of the world, has an unusually high incidence in the Chinese. In contrast to the geographically related incidence of EBV-associated BL, the association of EBV with NPC has been reported worldwide. Nasopharyngeal carcinoma as a whole, including both keratinizing and undifferentiated variants, accounts for 89% to 90% of the malignant nasopharyngeal neoplasms in the United States and up to 98% in some areas of the Orient.3gr40 Histologically, UNPC demonstrates cytologically malignant cells with indistinct cytoplasmic borders, resulting in a syncytial pattern of growth, and a reactive inflammatory infiltrate composed predominantly of lymphocytes. The neoplastic epithelial cells may present either as well-defined, cohesive cell nests (Regaud pattern) or as isolated cells within a dense lymphocytic infiltrate (Schmincke pattern) (Fig 2). The association of EBV with UNPC was first proposed in 1966 following the observation that sera from UNPC patients contained antibodies directed against lymphoblasts from productive, EBV-infected cell lines.43 High titers of anti-VCA, EA/D, and neutralizing antibodies were subsequently demonstrated in UNPC patients from different parts of the world by immunofluorescence techniques.44-47 Anti-VCA and anti-EA/D titers are also related to total tumor burden, as antibody titers progressively increase with advancing disease stage and are commonly lower in treated, long-term survivors than in untreated patients.45.46 The close link between EBV and UNPC was established by the demonstration of EBV DNA within African UNPC by nucleic acid hybridization in 1970 by zur Hausen et al*’ and, subsequently, by others.‘2,48,4g Viral-specific nuclear antigens (EBNA) were identified in epithelial cells but not lymphoid cells by both immunofluorescence48~50 and nucleic acid hybridization techniques.47 The demonstration in 1986 of a homogeneous EBV episomal population within the epithelial cells of several UNPCs provided direct evidence that this tumor, like BL, may represent an EBV-initiated clonal expansion of a single infected progenitor ce11.14 Formalin-fixed, paraffinembedded UNPC material has also revealed EBV genomic material by in situ hybridization (Fig 2).5l
The reactivation of latent EBV infection in the nasopharynx is accompanied by the production of high titers of anti-VCA and anti-immunoglobulin EA A (IgA) antibodies.44*52 The simultaneous absence of EBV-specific antibody in either
GAFFEY AND WEISS
Figure 2. (Top] An example of an undifferentiated carcinoma (lymphoepithelioma) of the nasopharynx, with sheets of neoplastic epithelioid cells surrounded by reactive lymphocytes (Regaud pattern). (Bottom] An in situ hybridization study of a lymphoepithelioma for EBV using a “S- labeled probe. Numerous grains are present over the tumor, which indicates a large amount of EBV genomes within the neoplastic cells.
., ‘: ‘. I
healthy controls or patients with other carcinomas suggests their use as markers for the early detection of UNPC.44v53A study of 124 North American UNPC patients revealed the IgA antiVCA antibody as a potentially diagnostic marker for the presence of occult disease.54 In some areas of China, IgA anti-VCA is used to detect UNPC in mass screening programs.55-57 One study of 148,029adults revealed 3,533 with IgA anti-VCA titers antibodies, of whom 460 had antibody 31:80.Of these individuals, 55 cases of UNPC were detected and histologically confirmed.57 Since oropharyngeal epithelial cells lack the C3d receptor necessary for viral entry into human lymphocytes, the precise mechanism of viral penetration into these cells is unknown. The demon-
stration of a human epithelial cell surface protein antigenically related to the C3d receptor, however, suggests that EBV may enter cells through a similar, alternative mechanism.5* While the association of EBV with the pathogenesis of UNPC appears firmly established, the precise role of EBV in the initiation and promotion of tumorigenesis in unknown. As in the case of BL, environmental factors geographically related to areas of endemic UNPC may be significant in the reactivation of persistent EBV genomic material. Many members of the Euphorbiaceae and Thymelaeaceae plant families implicated in the causation of BL, including Croton tiglium (the croton oil tree) and Aleurites fordii (tung oil tree), also have a geographic distribution similar to that
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of endemic UNPC in Southern China. The leaves, bark, and roots of these plants are commonly used by the local population as medicinal herbs for both external and internal use.17 Individuals may also be exposed via the inhalation of pollen or contact with plant extract-contaminated soil. Rare cases of lymphoepithelioma have been reported outside the nasopharynx. Lymphoepithelioma of the parotid in Chinese individuals and Eskimos has been associated with EBV, while at other sites and in other ethnic groups, only a rare association has been noted.50,5g-61 Close histologic mimics of UNPC that occur in the nasopharynx have not been found to be associated with EBV, at least in North American patients.50 The strong ethnic, geographic, anatomic, and histologic influences that condition the association between EBV and lymphoepithelioma remain incompletely understood. CONCLUSION Epstein-Barr virus is a ubiquitous viral pathogen that commonly infects B lymphocytes and oropharyngeal epithelial cells. Primary infection commonly results in IM, a benign, self-limited disease with a characteristic clinical course. Under conditions of immunosuppression, however, EBV infection results in the polyclonal expansion of EBV-infected B lymphocytes with an increased probability of genetic rearrangement and the development of clinical malignancy. The precise role of EBV in the initiation and promotion of such neoplasms as BL, NHL (of both B- and T-cell lineage), HD, and UNPC, however, has yet to be precisely determined. References 1. Epstein MA, Achong BG, Barr YM: Virus particles in cultured lymphoblasts from Burkitt’s lymphoma. Lancet 1964; 1:702-703 2. Lennette ET: Epstein-Barr virus, in Lennette EH, Balows A. Hausler WT. et al (edsl: Manual of Clinical Microbiology, 4th ed. Washington, DC; American Society for MicrobiologyTi985, pp 728-732 3. Fingeroth JD, Weiss JJ, Tedder TF, et al: Epstein-Barr virus receotor of human B lymphocytes is the C3d receptor CR2. Proc Nat1 Acad Sci USA i984; 81:4510-4514 4. Lindahl T. Adams A, Biursell G, et al: Covalentlv closed circular duplex DNA of Epstein-Barr virus in a human lymphoid cell line. J Mol Biol 1976; 102:511-530 5. BurkItt D: A sarcoma involving the jaws in African children. Br J Surg 1958; 46:218-223 6. Rosenberg SA (Chairman): National cancer institute sponsored study of classifications of non-Hodgkin’s lymphomas. Summary and description of a working formulation for clinical usage. Cancer 1982; 49:2112-2135 7. Croce CM, Nowell PC: Molecular genetics of human B cell neoplasia. Adv Immunol 1985; 38:245-274 8. de-The G, Geser A, Day NE, et al: Epidemiological evidence for a causal relationship between Epstein-Barr virus and Burkitt’s lymphoma: Results of the Ugandan prospective study. Nature 1978; 274:756-761
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9. Lenoir GM: Role of the virus, chromosomal translocations and cellular oncogenes in the aetiology of Burkitt’s lymphoma, in Epstein MA, Achong BG (eds): The Epstein-Barr Virus: Recent Advances. New York, NY, Wiley, 1986, pp 183-205 10. Geser A, Lenoir G, Anvret M, et al:Epstein-Barr virus markers in a series of Burkitt’s lymphomas from the West Nile District, Uganda. Eur J Cancer ClinOncol 1983; 19:1393-1401 11. zur Hausen H. Schulte-Holthausen H. Klein G. et al: EBV DNA in biopsies of Burkitt tumors and anaplastic carcinomas of the nasopharynx. Nature 1970; 228:1056-1058 12. Nonovama M. Huane. CH. Paaano IS. et al: DNA of EDstein-Barr virus detected in tissue aufBurkitt’s lymphoma and nasopharyngeal carcinoma. Proc Nat1 Acad Sci USA 1973; 70:3265-3268 13. Bornkamm GW, Stein H, Lennert K, et al: Attempts to demonstrate virus-specific sequences in human tumors. IV. EB viral DNA in European Burkitt lymphoma and immunoblastic lymphadenopathy with excessive plasmacytosis. Int J Cancer 1978; 17:177-181 14. Raab-Traub N, Flynn K: The structure of the termini of the Epstein-Barr virus as a marker of clonal cellular proliferation. Cell 1986; 47:883-889 15. Shade M, Woodward MA, Steel CM: Chromosome aberrations acquired in vitro by human B-cell lines. II. Distribution of break uoints. TNCI 1980: 65:101-109 16. Whittle HC, Brown J, Marsh K, et al: T cell control of Epstein-Barr virus-infected B cells is lost during P. falciparum malaria. Nature 1984; 312:449-450 17. Ito Y: Vegetable activators of the viral genome and the causation of Burkitt’s lymphoma and nasopharyngeal carcinoma, in Epstein MA, Achong BG (eds]: The Epstein-Barr Virus: Recent Advances. New York, NY, 1986, pp 207-236 18. Ito Y, Yanase S, Fujita J, et al: A short term in vitro assay for promoter substances using human lymphoblastoid cells latently infected with Epstein-Barr virus. Cancer Lett 1981; 13:29-37 19. Knowles DM, Chamulak G, Subar M, et al: Clinicopathologic, immunophenotypic and molecular genetic analysis of AIDS-associated lymphoid neoplasia. Clinical and biologic implications. Path01 Annu 1988; 23:33-67 20. Birx DL, Redfield RR, Tosato G: Defective regulation of Epstein-Barr virus infection in patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related disorders. N EnglJ Med 1986; 314:874-879 21. Lombardi L, Newcomb EW, Dalla-Favera R: Pathogenesis of Burkitt lymphoma: Expression of an activated c-myc oncogene causes the tumorigenic conversion of EBV-infected human B lymphoblasts. Cell 1987; 49:161-170 22. Subar M, Neri A, Inghirami G, et al: Frequent c-myc oncogene activation and infrequent presence of Epstein-Barr virus genome in AIDS-associated lymphoma. Blood 1988; 72:667-671 23. Cleary ML, Dorfman RF, Sklar J: Failure in immunological control of the virus infection post-transplant lymphomas, in Epstein MA, Achong BG (eds): The Epstein-Barr Virus: Recent Advances. New York, NY, Wiley, 1986, pp 163-181 24. Cleary ML, Warnke R, Sklar J: Monoclonality of lymphoproliferative lesions in cardiac-transplant recipients: Clonal analysis based on immunoglobulin gene rearrangements. N Engl J Med 1984; 310:477-482 25. Shearer WT. Ritz J, Finegold MJ, et al: Epstein-Barr virus-associated B-cell proliferations of diverse clonal origins after bone marrow transplantation in a 12-year-old patient with severe combined immunodeficiency. N Engl J Med 1985; 312:1151-1159 26. Ho M, Miller G, Atchison RW, et al: Epstein-Barr virus infections and DNA hybridization studies in posttransplantation lymphoma and lymphoproliferative lesions: The role of primary infection. J Infect Dis 1985; 152:876-886 27. Randhawa PS, Yousem SA, Paradis IL, et al: The clinical spectrum, pathology, and clonal analysis of Epstein-Barr virusassociated lymphoproliferative disorders in heart-lung transplant recipients. Am J Clin Path01 1989; 92:177-185 28. Cleary ML, Nalesnik MA, Shearer WT, et al: Clonal analysis of transplant-associated lymphoproliferations based on the structure of the genomic termini of the Epstein-Barr virus. Blood 1988: 72:349-352
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381 Barr virus-related antigen in nasopharyngeal carcinomas: Comparisons of active cases with long term survivors. JNCI 1973; 51:361-369 47. Desgranges C, Wolf H, De-The G, et al: Nasopharyngeal carcinoma. X. Presence of Epstein-Barr genomes in separated epithelial cells of tumors in patients from Singapore, Tunisia, and Kenya. Int J Cancer 1975; 16:7-15 48. Wolf H, zur Hausen H, Becker V: EB viral genomes in epithelial nasopharyngeal carcinoma cells. Nature New Biol 1973; 244~245247 49. Wolf H, zur Hausen H, Klein G, et al: Attempts to detect virus-specific DNA sequences in human tumors. III. EpsteinBarr viral DNA in non-lymphoid nasopharyngeal carcinoma cells. Med Microbial Immunol (Berl) 1975; 161:15-21 50. Huang DP, Ho JHC, Henle W, et al: Demonstration of Epstein-Barr virus associated nuclear antigen in nasopharyngeal carcinoma cells from fresh biopsies. Int J Cancer 1974; 14:580-588 51. Weiss LM, Movahed LA, Butler AE, et al: Analysis of lymphoepithelioma and lymphoepithelioma-like carcinomas for Epstein-Barr viral genomes by in situ hybridization. Am J Surg Path01 1989; 13:625-631 52. Raab-Traub N, Hood R, Yang C-S, et al: Epstein-Barr virus transcription in nasopharyngeal carcinoma. J Virol 1983; 48:580-590 53. Ringborg U, Henle W, Henle G, et al: Epstein-Barr virusspecific serodiagnostic tests in carcinomas of the head and neck. Cancer 1983; 52:1237-1243 54. Pearson RG, Weiland LH, Neel B, et al: Application of Epstein-Barr virus (EBV) serology to the diagnosis of North American nasopharyngeal carcinoma. Cancer 1983; 51:260268 55. Zeng Y, Yuxi L, Chunren L, et al: Application of an immunoenzymatic method and an immunoautoradiographic method for a mass survey of nasopharyngeal carcinoma. Intervirology 1980; 13:162-168 56. Zeng Y, Liu XX, Wei JN, et al: Serological mass survey of nasopharyngeal carcinoma. Acta Acad Med Sin 1979; 1:123126 57. Zeng Y, Zhong J, Li LY, et al: Follow-up studies on Epstein-Barr virus IgAfVCA antibody-positive persons in Zangwu County, China. Intervirology 1983; 20:190-194 58. Young LS, Dawson CW, Brown KW, et al: Identification of a human epithelial cell surface protein sharing an epitope with the C3diEpstein-Barr virus receptor molecule of B lymphocytes. Int J Cancer 1989; 43:786-794 59. Krishnamurthy S, Lanier AP, Dohan P, et al: Salivary gland cancer in Alaskan natives, 1966-1980. Hum Path01 1987; 18:986-996 60. Saw D, Lau WH, Ho JHC, et al: Malignant lymphoepithelioma lesion of the salivary gland. Hum Path01 1986; 17:914-923 61. Butler AE, Colby TV, Weiss LM, et al: Lymphoepithelioma-like carcinoma of the lung. Am J Surg Path01 1989; 13:632-639
