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1 ) , ) 3 f. I i VoL. 13, No. 7, J'uLY 1977 without recognized symptoms, primary infec- tion~ is accompanied by heterophil-positive infectious mononucleosis in the other half (29-32). Despite the unclear pathogenesis of the in- fectious mononucleosis syndrome, infectious (transforming) EBV can be regularly re- covered from throat washings of patients with the disease (33-36). During the acute phase, the peripheral blood of these patients con- tains specific killer T lymphocytes that can lyse EBV-genome-positive but not EBV- negative target cells (37). In parallel, large blast cells appear in the B cell fraction, containing the EBV-determined nuclear anti- gen EBNA (38). Part of the infectious mono- nucleosis syndrome may reflect an acute re- jection reaction against virally converted lymphocytes. Two human cancers, nasopharyngeal car- cinoma (NPC) and African Burkitt s lym- phoma show a remarkably consistent associa- tion with~ EBV, both by serology (39, 40) and by EBV genome tests (41-43). Africaw Burkitt's lymphoma may be regarded as the neoplastic proliferation of an EBV-genome- carrying clone (44) in 97% of cases (45). The very rare cases of Burkitt's lymphoma occurring outside the highly endemic regions of Africa do not, as a rule, show a similar association with EBV either by serology (46), or EBV genome tests (47). Recently, how- ever, some EBV-genome-carrying European and American Burkitt's lymphomas have been found. As do the EBV-carrying established' lines, biopsy specimens of African Burkitt's lym- phoma carry multiple copies of the viral genome per cell (41-43). Part of these genomes exists in a covalently closed, free circular form (48),,whereas another part ap- pears to be integrated with the cell genome. The biopsy specimens containi the same type of circles as established lines (49). EBV-carrying Burkitt's lymphoma cells EBV AND MALIGNANCY' grow into established in vitro lines more readily than explants from infectious mono- nucleosis or normal seropositive donors. Im the majority of the cases studied, the clonal! characteristics of established lymphoma-de- rived lines correspond to the in vivo clone (50), but contaminating EBV-positive B cells occasionally can overgrow the lympho- ma cells (5l)~. Representative lymphoma lines differ from "lymphoblastoid lines," i.e., in vitro EBV-transformed cells and lines de- rived from nonlymphomatous sources, with regard to a number of morphologic, func- tional and growth characteristics (10). Lym- phoma lines are relatively uniform, but lymphoblastoid lines show great heterogene- ity. EBV-carrying African Burkitt's lympho- mas are already uniclonal in vivo (44), how- ever., whereas lines derived from normal donors are polyclonal (52). This diversity may explain some of the differences. Alter- natively, lymphoma development may in- volve the appearance of a special neoplastic cell type, not present in EBV-transformed normal lymphocyte populations. The latter possibility is supported by the recent ob- servation (53-55) that a highly specific chromosome 14 translocation may be found in biopsy, specimens of Burkitt's lymphoma and derived lines but is not observed in EBV-carrying lymphoblastoid lines of non- lymphoma origin. The implications of these findings are discussed in more detail below. Some EBV-genome-negative B-type 1ym- phomas have been established' as continuous lines (56), but only with considerable dif- ficulty. The easy overgrowth of EBV-carry- ing normal cells is one of the main problems. EBV-negative lymphoid lines have never been established from normal tissues, how- ever. Human lymphocytesprobably can grow as established lines only if they are derived from a lymphoma or if they carry the EBV genome or both. African Burkitt's lymphoma is the only known condition in which 717

1 ) ) ) ) I EPSTEIN-BARR VIRUS, INFECTIOUS MONONUCLEOSIS, BURKITT'S LYMPHOMA AND NASOPHARYNGEAL CARCINOMA GEORGE KLEIN Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden Epstein-Barr virus (EBV) is a lymphotropic herpesvirus in man (1), its main target being the human B lymphocyte (2). Only B lymphocytes and most if not all T lympho- cytes have specific EBV receptors (3). Re- cent evidence suggests that the complement receptor of the B lymphocyte is either identi- cal to or closely associated with the EBV receptor. EBV can convert normal lymphocytes. which have a limited life span in virro, into permanently growing cell lines (4-9). Such "immortalized" lines have a diploid or near- diploid' karyotype (10), carry multiple copies of the viral genome in each (1 1-1'4) and ex- press EBV-specific nuclear antigen (EBNA) (15). This antigen is a virally determined or virally altered chromosomal' protein, the only known viral product expressed in all EBV- DNA-carrying cells, independent of virus production. Similar EBV-DNA- and EBNA- carrying lines can be established from the peripheral blood or lymph nodes of EBV- seropositive donors, but not from those of seronegative donors (16; 1i7). At least some of the EBV-DNA-carrying "immortalized" cell lines and the lines de- rived from normal seropositive donors (18- 20) can grow as malignant tumors after heterotransplantation into immunologically deficient animals. This implies that the lines have a malignant potentiali at least under these relatively artificial conditions. EBV can also transform the B lymphocytes of cer- tain simian hosts (21, 22). Some of the derived lines can grow progressively after reimplantation and' kill the original, autoch- thonous host (23). Im marmosets (23-25)' and owl monkeys (26), the virus also has a direct oncogenic activity. The induced lym- phomas carry the viral genome an& contain EBNA. EBV infects the majority of all adult hu- man populations in alli countries (27. 28). Its seroepidemiology resembles that of other hori- zontally transmitted viruses, with the regular presence of passively transmitted antibody in the newborn, its subsequent decline, and~ the reappearance of' actively indltced anti- body after infection. The timing and extent of seroconversion; are strongly related to socioeconomic status. In low socioeconomic groups. infection occurs during early child- hood, as a rule. It is not accompanied'~ by any recognized disease, and the route of trans- mission is unknown. Only a minority of young children become infected'in high socio, economic groups where a later infection, during the teens, pred'ominates. Although at least half' the teen-age infections appear 716

) 3 ) 3 1 . G. KLEIN' lymphoma derivation and EBV-po.;itive sta- tus coincide. This unique position of Burkitt's lympho- ma as the only known EBV-carrying lympho- proliferative neoplasia in man, together with the known transforming and oncogenic prop- erties of the virus, make EBV an important etiologic candidate for oncogenesis. Before accepting that a causative association may exist, alternatives must be considered, how- ever. Numerous early researchers have favored the "passenger hypothesis." This idea implies that lymphoma cells arising in EBV-carrying persons for EBV-unrelated reasons pick up the virus as a passenger, just as do normal lymphocytes, carry it along as they proliferate, and thereby increase the antigenic load and induce antibody produc- tion: However, the very fact that non-Burkitt lymphomas that arise in EBV-seropositive patients do not pick up the virus is in itself a strong argument against this idea. It may be argued that such lymphoma cells are in- sensitive to EBV infection. However, some of the EBV-genome-negativeJymphoma lines, derived from EBV-negative Burkitt-like lymphomas that have arisen in seropositive patients could be infected with EBV in rirro (57), followed by permanent conversion into EBV-DNA- and EBNA-carrying lines (58). This finding confirms that EBV-sensitive lymphoma cells that arise in seropositive pa- tients do not necessarily become infected' by horizontal virus spread in vivo, presumably owing to the regular presence of neutralizing antibodies. It also suggests that EBV-genome- positive Burkitt's lymphomas originate from a genome-carrying cell. If this speculation is accepted, we are left with essentially two interpretations, which may be referred to as the immunologic and co-factor hypotheses. Arguments can be found for both inter- pretations. As already mentioned: lines de- rived from in vitro EBV-transformed nor- mal lymphocytes can have a neoplastic po- 718 ISRAEL 1.. t4tFD.. $Cl. tential, as demonstrated by the ability of some of them to grow progressively and l'ili xenogeneic (e.g„ nude mice) and' autologpus (simian) hosts. On the other hand, the re- cent findings of Nilsson and' Poten (10) show that EBV-carrying lymphoblastoid lines dc- rived' from normal donors or from benign conditions, such as infectious mononucleosis, differ from EBV-carrying. Burkitt's-lympho- ma-derived lines with regard to a whole series of morphologic, functional and growth characteristics. Perhaps even more important is the discovery of Manolov and Manolova (53), recently confirmed and extended by Jarvis et al. (54) and by Zech et al. (55), that definite chromosome differences exist between normal and lymphoma-derived lines. Normal diploid' lines were only found among EBV-carrying lymphoblastoid lines derived from non-Burkitt's lymphoma donors. In contrast, all lymphoma lines so far examined were characterized by various chromosomal anomalies. The majority contained the char- acteristic chromosome 14 marker, recently identified as an 8-14 translocation (55). Thus, EBV is fully capable of "immortaliz- ing" B lymphocytes with a normal diplbid karyotype in ritro„ but EBV-carrying lympho- mas in rivo involve genetic changes in addi- tion, probably of a: rather specific kind. Such an interactioni of viral transformation and cytogenetic changes is not as unique as it may appear at first sight. There are many experimental examples showing that knowni oncogenic viruses do not transform all or even the majority of appropriate target cellk.. Transformability is dependent on an obscure but probably specific "state of competence." Competence may be detenmined by the dif- ferentiation state or the genetic constitution of the cell, or both. The importance ofl the genetic constitution is emphasized by increas- ing evidence (59-64) that both chemically' and virally induced tumors may display high- ly specific chromosomal changes, different.

) 1 ! VoL. 13, No. 7;,JULY 1977 for tumors induced by different agents. Some forms of viral oncogenesis may require specific genetic changes as a pre- requisite for full development of cancer in rr„o. Cells with a normal diploid karyotype possess regulatory mechanisms that can coun- teract the neoplastic change, even in cells that contain integrated genomes of a po- tentially oncogenic virus. The temperature- sensitive transformants of Renger and Basil= ico (65) exemplify a situation in which a cellular function can influence the phenotype of virally transformed cells. The recent ex- periments of' Melero et al. (66) on DNA binding proteins in normal and transformed cells are another case in point. Somatic cell hybridization experiments (67-69) have shown that the highly malignant behavior of established, polyoma-induced or other tumors can be suppressed by fusion with normal diploid cells. The importance of cellular genetics for virus-induced neoplastic transformation is also illustrated by the wholly unexpected finding (70-77) that simian virus 40' (SV40)) transforms with a higher efficiency if the target cells are derived from hosts with a known tendency for increased chromosomal variation or mitotic anomalies (e.g., Fanr coni's or Klinefelter's syndromes, ataxia- telangiectasia, or xeroderma pigmentosum). The increased SV40 transformability of nor- mal dipioid human fibroblasts with aging and accumulating chromosomal aberrations may be a further example. Zech et al. (55) have recently found that the Burkitt's lymphoma-associated chromo- some 14 translocation was also present in some EBV-negative Burkitt's and non-Bur- kitt's lymphomas, but never in EBV-trans- formed cells derived from normal' donors or from patients with infectious mononucleosis. This cytogenetic change may somehow pro- mote transformation to a malignant lympho- ma. A relation between abnormal lympho- EBV AND MALIGNANCY cyte growth and structural rearrangement of the long arm of chromosome 14 is also sug- gested by recent studies of cells from pa- tients with ataxia-telangiectasia (78). Since a minority of African EBV-carrying Burkitt's fymphomas lack the chromosome 14 trans- location (53, 55) (although they have other chromosomal anomalies), the visible mani- festation of this particular translocation is not an absolute requirement for the develop- ment of full'~fledged lymphomas. A similar situation may exist in chronic myelogenous leukemia where the Ph„ chromosome (an- other translocation) is present in the majori- ty of cases. Studies on the genetics of experimental car- cinogenesis provide ample evidence (79, 80) that genetic factors may influence the proba- bility of neopl'astic transformation at the level of the target cell itself. Thus, a given genetic change-here expressed by the chro- mosome 14 translocation-may influence the probability of lymphoma induction by EBV and also by other„ as yet unknown, agents. This idea does not preclude the possibility that EBV and other agents may occasionally induce lymphomas in the absence of chromo- some 14 translocation. A very, different kind of interaction, involv- ing EBV and C-type viruses;, is often men- tioned in recent discussions concerning the etiology of Burkitt's lymphoma (81). Al- though there are many examples of inter- actions between genetic factors and oncogenic viruses bringing about neoplastic transfor- mation, there are few examples of oncogenic interactions between two viruses. The dem- onstration of C-type-virus-related informa- tion in Burkitt's lymphoma and otherlympho-r or myelopoietic malignancies does not con- clusively support the idea, because C-type viruses are ubiquitous an& most of them doo not, appear to be at all oncogenic (82). The mere demonstration of C-type-related sequences is therefore noC necessarily an im- 719

>. ). ) G. Kt.EtN portant fact without more directly compelling evidence. In view of its demonstrated direct oncogenicity in monkeys and its highly ef- ficient transforming ability in rirro. EBV hardly needs another virus to help it along, It is very likely that it requires a special form of cellular competence, perhaps a ge- netic deficiency in some regulatory mecha- nism. before fully autonomous cancer cells can emerge. A discussion about the possible role of EBV in human cancers would not be com- plete without considering the relation be- tween EBV and NPC. In contrast to Burkitt's lymphoma, there appears to be no major geographic variation: in the EBV-carryingg status of NPC (42, 83, 84), but there is a striking histologic restriction. To date, only poorly differentiated or anaplastic tumors have been found to carry the viral genome. In contrast to earlier interpretations, it is now clear (85-87) that the viral genomes are not carried by the tumor-infiltrating lymphocytes-largely T cells (88)-but by the carcinoma cells themselves. They also express the EBNA antigen (85. 86, 89). Recent preliminary evidence suggests that the NPC-associated genome may be slightly different from the Burkitt's-lymphoma-as- sociated genome. Pagano (90) found that certain sequences were missing from the EBV-DNA of a Tunisian with NPC, as com- pared' with a Burkitt's-lymphoma-derived viral probe. In two nude mouse carriers of NPC purified from infiltrating human lympho- cytes by heterologous passage, Kaschka-Die- rich et al. (91) found covalently closed EBV circles. It remains to be established whether these circles have the same characteristics as the Burkitt's-lymphoma-associated circles. If they are different, this may be due to a variation in the virus strains associated with the different individual donors, in analogy with the molecular variations between dif- ferent herpes simplex isolates (92), or there 720 IsRAEL 1. ~ MED. Sct:.L may be some disease-associated importance. At this stage, the NPC-EBV relation raises many interesting questions. The exclitsive and regular presence of the viral genome in one histologically distinct tumor type, inde- pendent of geographic location and' high or low endemicity, strongly suggests that the association must have some etiologic rel- evance, but it is impossible to state a pref- erence for a causative vs. a promoting, rela- tion. Genetic factors are known to play an important role in some highly endemic ethnic groups, the Chinese in particular (93). Inn addition to the possible existence of an NPC- associated viral subtype, it would be im- portant to obtain some information about the EBV susceptibility of' the normal progen- itor cell in the nasopharyngeal epithelium. It would not be surprising if a cooperative interaction of viral transformation and host- cell genetics were eventually to emerge. If this is the case, the picture may resemble the above-postulated situation for Burkitt's lymphoma-at least in principle-although the details may be quite different. REFERENCES' L EPSTEIN MA. ActtoNC BG' and BARR YM. Virus particles in cultured lymphoblasts from Burkitt's lymphoma. Lancer 1: 702,, 1964. 2:, JONDAL M and KLEIN G. Surface mark'ersof human B and T lymphocytes. 11. Presence of Epstein-Barr virus receptors on B lympho- cytes. J Esp Mcd 138: 1365, 1973. 3. GREAVEs:FM, BROWN G and RICKINSON AB. Epstein-Barr virus binding sites on hmpho- cyte subpopulation and the origin of lympho- blasts in culture lvmphoid cell lines and in the blood of patients with infectious mono- nueleosis. Clin lmmuno! Immunopathol' 3:. 514, 1975: 4. MILLER G. Human k•mpfioblastoid cell liness and Epstein-Barr virus: a review ofl their interrelationships and' their relevance to the etiology of Ieukoproliferative states in man. Yale J Biol Mcd'43: 358. 1971. 5: POPE JH. HORNE MK and Scorr W. Identi- fication of the filtrable Itukocyte-transform- ing factor of QIMR-WIL cells as herpes- like virus. hn J Cancer 4: 225. 1969. 6,. GCRnER'.. P,. WItAN-PENG J and MONROE JH. Transformation and chromosome changes in- duced by Epstein-Barr virus in normal

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