Infectious Mononucleosis and Malignant Neoplasia - Springer

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Infectious Mononucleosis and Malignant Neoplasia IAN MAGRATH, MB, FRCP.FRCPATH

Introduction Epstein-Barr virus (EBV) infection is ubiquitous, but is usually asymptomatic except in teenagers and young adults, some 50 percent of whom experience the syndrome of acute infectious mononucleosis at the time of primary infection. Although the virus establishes a permissive infection in a number of cell types in the mouth and pharynx, including the ductal cells of the salivary glands, it is the proliferation of latently infected (i.e., nonpermissive for viral replication) B lymphocytes in lymphoid tissue and the resultant cellular immune reaction against these EBV-infected cells which give rise to the syndrome of infectious mononucleosis described elsewhere in this book. EBV gains access to B lymphocytes via the surface receptor for the complement component C3d (CR2) (Nemerow et al. 1987), also known as CD21, and transforms them into lymphoblasts capable of indefinite proliferation in vitro. This capacity is conferred on the cell via a small set of EBV genes which are expressed in the absence of viral replication. In addition to "immortalizing" the cell, however, some of these latent genes induce an immune response against the EBV-infected cells which leads to their destruction. Destruction is accomplished initially by NK cells and nonspecifically reactive T cells which probably respond to EBV-induced activation antigens, and later by specifically reactive cytotoxic T lymphocytes which recognize virally coded proteins on the cell surface in the context of HLA class 1 antigens. The clinical syndrome of infectious mononucleosis, if it occurs at all, is thus limited in D. Schlossberg (ed.), Infectious Mononucleosis © Springer-Verlag New York Inc. 1989

duration and rarely fatal. These two pointsthe potentially unlimited proliferation of EBVinfected B lymphocytes, and their control by immune mechanisms-are crucial and worthy of emphasis; for in the absence of an effective immune response, the stage is set for the progressive and inexorable accumulation of EBVinfected cells which, unchecked, will result in the death of the host. Intuitively, it would seem that the complete lack of an ability to control the proliferation of EBV-infected cells would result in a fatallymphoproliferative process soon after primary EBV infection, whereas lesser degrees of impairment could result in the establishment of a more chronic or more localized process, or possibly of uncontrolled proliferation in immunoprivileged sites such as the brain. Penetration into these special sites may result from the presence of a persistently higher body burden of EBV-infected cells. Rapidly fatal infectious mononucleosis and a variety of EBV-associated lymphoproliferative syndromes do, in fact, occur in immunosuppressed individuals. In the case of acquired immunosuppression developing some time after primary EBV infection, whether due to a virus infection or to the administration of immunosuppressive drugs, any subsequent EBV-associated lymphoproliferative syndrome can only result from a "reactivation" ofEBV infection. In practice, this means that a higher level of EBV replication occurs at sites of productive infection; potentially giving rise to a higher rate of B-cell infection and transformation and a poorer ability to destroy EBV-infected B cells. It is still uncertain whether a pool of latently infected B lymphocytes, regulated in size by T cells, persists

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after primary EBV infection (the currently favored hypothesis), or whether such cells are repeatedly infected during passage through tissues harboring and releasing EBV (e.g., salivary glands), only to be destroyed by cytotoxic T lymphocytes. By whatever means, a balance is struck such that a small number of virus-infected B cells is always detectable in the bloodstream of EBV-seropositive adults, the level of which is regulated by HLA-restricted, EBV-specHic cytotoxic and/or suppressor T cells (Rickinson et al. 1980; Rickinson et al. 1981; Tosato and Blaese 1985). The immune response against EBV-infected B cells is extraordinarily efficient, for while its failure or impairment results in the recrudescence of EBV-induced lymphoproliferation, which at best is incapacitating and at worst fatal, such an occurrence is rare indeed. EBV-infected cells are obviously readily destroyed or suppressed by the normal host. Yet several neoplastic diseases arising in individuals without previously recognized clinical immunosuppression have been shown to be associated with EBV -i.e., EBV genomes are present in the tumor cells themselves (Table 12.1). These include Burkitt's lymphoma, a Bcell neoplasm of small noncleaved cells, and nasopharyngeal carcinoma, a tumor which arises in undifferentiated epithelial cells of the nasopharynx. Even though the viral association of these tumors was recognized many years ago, it is still not known whether EBV has a role in their pathogenesis. An additional enigma is the high frequency of small noncleaved cell lymphomas, frequently associated with EBV, which occurs in patients with HIV infection, and yet

Table 12.1 Neoplastic diseases associated with EBV. Carcinomas Nasopharyngeal carcinoma (undifferentiated and well differentiated) Salivary gland Adenoid-cystic carcinoma from Sjogrens syndrome Undifferentiated lymphoepitheliallesion Carcinoma of the palatine tonsil Supraglottic carcinoma (epithelial cells) Thymic carcinoma Cervical carcinoma (epithelial cells)

Lymphoid Burkitt's lymphoma Polymorphic B-ceillymphoma

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the apparent absence, in such patients, of fatal lymphoproliferations of the kind seen in inherited iatrogenic immunodeficiency. In this chapter we shall examine a number of models and clinical syndromes that shed some light on these issues-issues which are important not only within the context of EBV infection, but which also embrace the more general issue of the nature of neoplastic proliferation. Operationally, the uncontrolled proliferation of EBV-infected cells in an immunosuppressed individual has exactly the same consequences as neoplastic proliferation. Indeed, such cases have often been designated as neoplastic. We might well ask, therefore, whether they can be considered as truly neoplastic, and if not, how they differ from neoplastic processes. Further, it is appropriate to question the value, at the pragmatic level of the clinician, of examining this issue. While hindered by the inconvenience that a widely accepted definition of neoplasia does not exist, recent advances in the understanding of the events which occur in an EBV-infected cell, as well as progress in elucidating the molecular abnormalities present in Burkitt's lymphoma (a disease often associated with EBV and accepted by all as a true neoplasm), have equipped us to examine the differences in the pathogenesis of the various lymphoproliferative syndromes associated with EBV. Although we currently lack a complete understanding of the complex molecular interactions that ultimately result in the clinical syndromes manifested, we can certainly begin to appreciate the presence of multiple pathways leading to unbridled lymphoproliferation. With this aim in mind, it is first necessary to discuss the clinical syndromes themselves, and the circumstances under which they arise. These syndromes include chronic and fatal infectious mononucleosis and EBV-associated lymphomas occurring in allograft recipients and patients with inherited and acquired immunodeficiency diseases, including that caused by human immunodeficiency virus (HIV) (Saemundsen, Bertel, et al. 1981; Saemundsen, Purtilo, et al. 1981; Ragona et al. 1986). Of considerable value in drawing parallels with clinical situations in humans are the lymphoproliferative syndromes induced in animals by a number of herpesviruses, including EBV (ZurHausen 1975; Deinhardt and Deinhardt 1979). These animal

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models, which often strongly resemble human diseases, demonstrate that the borderland between nonneoplastic lymphoproliferation and malignant neoplasia is not always clear, particularly when the investigator's tools include only histopathology and simple immunologic characterization (for this reason, the term "lymphoproliferation" is used throughout this chapter to indicate any form of lymphoid hyperplasia, and does not imply that the process is nonmalignant). The animal models are of additional interest, because although viruses have been demonstrated to cause neoplasia in animals, at the present time no human tumor can be unequivocally stated to have a viral etiology. The spectrum of disease caused by EBV, therefore, provides an important paradigm relevant to gaining an understanding of the factors which govern the development of benign or malignant proliferation as a consequence of the invasion of human lymphocytes by foreign genes- for such is the quintessence of a virus infection.

Herpesvirus-Induced "Neoplasia" in Animals Adenocarcinoma in Frogs In 1938, Lucke was able to induce renal adenocarcinomas of leopard frogs (Rana pipiens) with cell-free extracts obtained from established tumors. Subsequently it has been demonstrated that purified Lucke herpesvirus (LHV) will induce adenocarcinomas in frogs inoculated in the embryonic or larval stage; at which time the developing kidney consists exclusively of pronephros (Naegele and Granoff 1977; Naegele, Granoff, and Darlington 1974). Several features of herpesvirus oncogenesis are evident from the Lucke adenocarcinoma. Firstly, an appropriate target cell must be present at the time of infection. Inoculation of LHV after the formation of mesonephros does not lead to tumors. Secondly, as with all DNA viruses, viral replication is not compatible with survival of the infected cell. LHV replication is temperature-dependent and occurs only in winter. Tumor growth, therefore, occurs only in summer, when the virus is in a "latent stage."

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Marek's Disease of Chickens Marek described a paralytic disease of chickens in 1907, which he believed to be an infectious inflammatory neuropathy. Subsequently it was shown that an important aspect of the disease is lymphoproliferation which occurs in the nerve trunks themselves, as well as in other tissues, and may progress to the point of large solid masses that are thought by many observers to be truly neoplastic, i.e., malignant lymphomas. In the early stages of the disease (reviewed in Biggs 1973; Payne, Frazier, and Powell 1976; and Nazerian 1979), there may be regression of the lymphoid infiltrates, so that Marek's disease appears to be on the border between reversible lymphoproliferation and neoplasia. In 1967, Marek's disease was shown to be caused by a herpesvirus, Marek's disease herpesvirus (MOHV) (Churchill and Biggs 1967). As with other herpesviruses, replication of MOHV (lytic infection) leads ultimately to cell death and hence is incompatible with either lymphoproliferation or neoplasia. Marek's disease normally takes one of two major forms (Biggs 1973; Payne, Frazier, and Powell 1976). In the so-called classic form, neuropathy predominates and chickens develop variable palsies of legs and wings. In the acute form, which is often fatal, lymphoid proliferation is sufficiently marked to produce solid tumors of multiple viscera, including liver, heart, lungs, gonads, and kidneys. Occasionally there is overt leukemia. Pathologically the two forms of the disease merge (Payne, Frazier, and Powell 1976). In both the classic and the acute disease there is lymphoproliferation in nerve trunks associated with varying degrees of demyelination. It is not clear which is the primary insult. Lymphoproliferation is also highly variable in extent. Some virus strains produce the classic, or neuropathic, form of the disease, with relatively few birds developing overt neoplasia, while others characteristically produce the acute, lymphomatous type of disease (Nazerian 1979). Still other strains of MDHV, or the related turkey herpesvirus, are essentially apathogenic, and prior infection with these viruses protects the chicken from the diseases produced by the highly pathogenic viruses. Vaccination against

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Marek's disease is based upon this phenomenon, and whereas the turkey herpesvirus is most often used commercially, tumor formation can also be prevented by MDHV-infected cell membrane extracts (Else 1974; Payne, Frazier, and Powell 1976). A number of parallels can be drawn between infectious mononucleosis (and other EBV-related syndromes) and Marek's disease. Several neurologic diseases, including Landry's paralysis and Guillain-Barre syndrome, have been shown to be associated in some cases with acute infection with EBV (Grose et al. 1975). Remarkably, the microscopic lesions in nerves in such patients are very similar to those seen in Marek's disease. The lymphoproliferative process in Marek's disease is clearly reversible in most chickens, but in others it is fulminant and leads to early death. The outcome of infection depends upon both the virus strain and the degree of susceptibility of the chicken. A considerable body of evidence suggests that the host immune system in chickens with Marek's disease is largely responsible for the control of lymphoproliferation, just as it appears to be in humans infected with EBV (Briles, Stone, and Cole 1977; Sharma and Witter 1975; Sharma, Witter, and Purchase 1975; Purchase and Sharma 1974; Nazerian 1979). In chickens, however, the development of fatal lymphoproliferation - which is considered by most to be true neoplasia on the basis of the gross and microscopic pathologic appearance-occurs much more frequently than in humans, although self-limited lymphoproliferation is a part of primary infection in both species. To date there is no evidence that different strains of EBV produce different kinds of disease. In both Marek's disease and EBV infection in humans, little or no virus replication occurs in lymphoid cells, although these contain viral genomes. Replication, which provides a source of virus for horizontal spread, takes place in the salivary glands in humans (and some other epithelial surfaces such as the uterine cervix) and the feather follicles in chickens. Marek's disease serves to emphasize that the outcome of exposure to virus depends upon both the virus itself and the host's ability either to prevent virus infection or to control the proliferation of virus-infected cells.

Oncogenic Herpesviruses of Primates Primate herpesviruses can be broadly divided into those of Old World primates, including humans, and those of New World primates. Viruses of the Old World primates share a considerable degree of DNA homology with EBV and infect B lymphocytes, whereas those of the New World differ considerably at the DNA level from EBV and are T-cell tropic. Many of these viruses can induce lymphoproliferation and lymphoid neoplasia in species other than the natural host (Table 12.2). The best studied New World viruses are Herpesvirus saimiri (HVS) and Herpesvirus ateles (HVA), while EBV itself is the best studied of the EBV-like Old World virus.

Herpesviruses of New World Primates The epidemiology of HVS and HVA in South American monkeys is remarkably similar to that ofEBVin humans but is much less well studied. Squirrel monkeys born in captivity possess maternal antibodies, then rapidly lose them. Inapparent primary infection leads to the acquisition of antibodies by almost 1()() percent of the animals in their second year (Deinhardt and Deinhardt 1979). This has been shown to be due to horizontal transmission. Animals captured in the wild also invariably have antibodies to HVS. Similar observations have been made with HVA. Both HVS and HVA undergo lytic cycles in cells of the pharynx and appear to be excreted in the saliva, probably throughout the life of the animal (entirely similar to EBV humans) (Falk et al. 1973). Neither HVS nor HVA appears to produce any detectable disease in its natural hosts. Virus can be isolated by cocultivation of circulating lymphocytes obtained from seropositive animals with permissive cells, e.g., owl monkey kidney (OMK) cells (Deinhardt and Deinhardt 1979; Fleckenstein 1979). Both HVS and HVA induce lymphoproliferative syndromes when experimentally inoculated into primate species other than their natural hosts, which provide interesting parallels with EBV-related diseases in humans and a number of insights into herpesvirus oncogenesis. The spectrum of diseases resulting from infection of various primate species with HVS or HVA is

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Table 12.2 Oncogenicity of primate herpesviruses. Induction of lymphomas or fatallymphoproliferation

Virus

Natural host

Herpesvirus saimiri

Squirrel monkey (Saimiri sciureus)

Aotus trivirgatus (owl monkey)

Ateles spp. (spider monkeys) Callithrix jacchus (common marmoset)

Herpesvirus ateles

Spider monkeys (Ateles spp.)

Saguinus spp. (tamarin marmosets) Alouatta caraya (howler monkey) Aotus trivirgatus (owl monkey) Callithrix jacchus (common marmoset)

Saguinus spp. (tamarin marmosets) African green monkey-Macaca spp. (Cercopithecus aethiops)

Cytomegalovirus

(Macacques)

Papio hamadryas (baboon) Cercopithecus aethiops (African green monkey) Herpesvirus papio

Baboons (Papio. spp.)

Epstein-Barr virus

Human (Homo sapiens)

Saguinus spp. tamarin marmosets) Aotus trivirgatus (owl monkey) Saguinus oedipus (cotton-top marmoset)

broad, ranging from subclinical seroconversion to fulminating, rapidly fatal lymphoproliferation, in which uniform sheets of immature lymphoid cells infiltrate a wide variety oftissues, including the bone marrow, with a resultant leukemic blood picture. The consequences of infection depend upon both the type of virus and the host species-a situation strongly reminiscent of Marek's disease. Common marmosets, but not cotton-top marmosets, can be protected against lymphoproliferation by attenuated virus strains (Fleckenstein 1979). Membrane preparations derived from HVS-infected OMK cells have been partially successful in protecting cotton-top marmosets against oncogenic HVS, an interesting parallel with Marek's disease (Pearson and Scott 1977). An important aspect of the lymphoproliferative diseases induced by HVS and HVA in marmosets is their polyclonality. Marmosets are always hemopoietic chimerae, and the proliferating lymphoid cells have been clearly demonstrated to be of both male and female origin (Chu and Rabson 1972; Marczynska 1973). This suggests that the lymphoproliferative syndromes are caused by an inability of the inoculated animal to control the numbers of virus-infected lymphocytes. This contrasts

with the minimal disease caused by HSV and HVA in their natural hosts, paralleling EBV infection in humans. Clearly, millennia of concurrent evolution of virus and host have resulted in a state of equilibrium which does not apply when the virus is artificially inoculated into other animal species. The outcome in the latter animals is more reminiscent ofEBV infection in immunocompromised humans.

Herpesviruses of Old World Primates A number of herpesviruses have been isolated from Old World primates, including Herpesvirus papio from baboon, Herpesvirus pan from chimpanzees, and Herpesvirus gorilla from gorillas (Rabin et al. 1980). Most work, however, has been done on EBV itself, which is also a herpesvirus of an Old World primate. Certain strains of EBV (i.e., B95-8, Kaplan, EB3), two originally derived from patients with infectious mononucleosis and one from a patient with Burkitt's lymphoma, will induce lymphoproliferation, often fatal, in owl monkeys and cotton-top marmosets (Shope, Dechairo, and Miller 1973; Epstein, Hunt, and Rabin 1973). Common marmosets, other Saguinus species


(e.g., white-lipped marmosets), woolly monkeys (Lagothrix lagotricha), squirrel monkeys, and Old World primates are not susceptible to tumor induction by EBV, although lymphocytes of all the New World species and of some of the Old World primates can be transformed in vitro by EBV (Miller 1979). EBV is not as highly oncogenic as HVS, since only some 30 - 40 percent of cotton-top marmosets inoculated with EBV (compared with almost 100 percent inoculated with HVS) will develop tumors. The remainder either undergo silent seroconversion or develop a self-limited lymphoproliferative syndrome (Miller 1979; Miller et al. 1977). The proliferating lymphoid cells in EBV-induced tumors contain multiple copies of the EBV genome but, as expected, do not permit EBV replication (Miller et al. 1977). They bear surface immunoglobulin, but few studies address the question of clonality. Several groups are working on the development of vaccines (usually purified EBV membrane antigens) against EBV-induced lymphoproliferation in primates in the hope of developing a vaccine of value in the prevention of EBV-induced disease in humans. Some success has been achieved, but vaccines are still at an early stage of development (Epstein 1986).

Parallels between Animal Herpesvirus and Human EBV Infections The examples cited of lymphoproliferative syndromes produced by herpesviruses (including EBV) in several animal species raise a number of interesting questions concerning the oncogenicity of EBV in humans and the relationship between infectious mononucleosis and lymphoid neoplasia. All of the viruses discussed are horizontally transmitted and are able to induce lymphoproliferation either in their host species or in other species. In many cases this is reversible, e.g., in many chickens with Marek's disease, in some common marmosets inoculated with HVS in all common marmosets infected with EBV, and in some owl monkeys infected with HVS, J.{VA, or EBV. It would appear that there is a spectrum oflymphoproliferative syndromes, the precise nature of which is dependent upon both virus strain and host factors. The proliferating cells may be polyclonal, as is well demonstrated

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in the case of chimera marmosets. Nonetheless, the more aggressive lymphoproliferations are generally labeled as malignant neoplasms by pathologists - based solely on the morphologic appearance and behavior of the tumors. These lymphoproliferations occur shortly after virus infection and progress rapidly, giving rise to profound lymphadenopathy and widespread tissue infiltration. In these respects they resemble fatal infectious mononucleosis in humans rather than Burkitt's lymphoma. Burkitt's lymphoma, when EBV-associated, may develop two years or more after EBV infection, and genetic changes, namely nonrandom translocations, are an essential component of pathogenesis (see below). It seems clear that, as in primary EBV infection in humans, the crucial determining factor of the immediate outcome of virus infection is the vigor of the host response, i.e., the immunologic reaction against virus-infected cells. Just as infectious mononucleosis is a reversible lymphoproliferation in the vast majority of cases, so too are MDHV-, HVS-, and HVA-induced lymphoproliferations reversible in most, if not all, individuals in the natural host species. In susceptible animals (usually of a different species) the lymphoproliferative process may be fatal, just as infectious mononucleosis may be in immunodeficient humans. Another parallel might usefully be drawn between herpesvirus-induced lymphoproliferative syndromes in primates and acute retrovirus-induced tumors in animals. Here, oncogene-carrying retroviruses induce tumors within a matter of weeks after infection (Scherdin and Holzel 1987). In effect, one or more critical cellular functions are altered because of the introduction into the cell of modified versions of the very genes which mediate these functions-i.e., activated oncogenes which were originally derived from cellular genes. Although operationally similar, retrovirus-induced tumors differ significantly from herpesvirus-induced lymphoproliferation in that herpesviruses do not contain modified cellular genes, picked up by a process of recombination during a prior infectious cycle. Instead, they modify the expression of normal cellular genes involved in proliferation, and in doing so, induce "transformation." This process does not appear to involve structural changes in cellular genes of the kind induced by chromosomal transformation. Oncogenic retroviruses,

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in so far that they contain structurally altered cellular genes, may be better compared with tumor induction as a result of cytogenetic changes rather than infection by a virus which has doubtless been associated with the host species for millions of years. Indeed, it has been shown that retrovirus infection can actually replace the requirement for chromosomal translocation in the induction of mouse plasmacytomas (Potter et al. 1987)

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EBV-specific, HLA-restricted T-cell response develops, which becomes a part of the immunologic memory and can be 'readily detected in seropositive individuals (Tosato et al. 1979; Rickinson et al. 1981; Tosato and Blaese 1985; Rickinson 1986). During acute infectious mononucleosis between 0.5 and 18 percent of circulating B lymphocytes express Epstein-Barr virus nuclear antigens (EBNA) (Klein et al. 1976; Katsuki et al. 1979; Robinson, Smith, and Niederman 1980), and some of these cells are capable of proliferation in vivo as well as in vitro (Katsuki et al. EBV Infection in Humans 1979; Robinson, Smith, and Niederman 1980). Subsequent to the acute process, such cells are The clinical syndrome of infectious mononucle- much more difficult to detect in vivo, although osis is predominantly a disease of middle-class EBV-containing continuous cell lines can be oband upper-class adolescents and young adults tained from peripheral blood B lymphocytes, who have escaped primary EBV infection earlier particularly if T cells are removed or rendered in life (Evans, Niederman, and McCollum functionally inactive by cyclosporin A treat1968). In lower socioeconomic groups, and par- ment. In fact, the prevention of the outgrowth of ticularly in socioeconomically deprived popula- continuous cell lines provides a convenient tions in less developed countries, EBV serocon- assay for the assessment of EBV-specific immuversion occurs at an early age and is usually nologic memory in T -lymphocyte subpopulasubclinical (Diehl et al. 1969; Henle and Henle tions (Rickinson et al. 1981). In normal EBV 1979; Biggar et al. 1978; Fleisher, Henle, et al. seropositive individuals it has been estimated 1979). The absence of clinical consequences of that there are between a hundred and a few early seroconversion contrasts markedly with thousand EBNA-positive lymphocytes circulatthe greater than 50 percent incidence of symp- ing in the peripheral blood-about 1 per 107 or toms when EBV infection occurs in the second 108 cells (Rocchi et al. 1973; Gergely et al. 1979) and third decades of life (Niederman et al. 1970; and 1 to 10 per million B cells capable of develEpstein and Achong 1977; Henle and Henle oping into a continuous cell line when cultivated 1979). The increase in the severity of infectious in vitro (Tosato 1987; Birx, Redfield, and Tomononucleosis with age is accompanied by a sato 1986). The number of EBNA-positive cells corresponding increase in heterophile titers normally residing in peripheral lymph nodes or (Fleisher, Lennette et al. 1979), but remains un- other lymphoid tissue is not well defined beexplained. However, whether or not seroconver- cause of the rarity of opportunities to sample sion is associated with symptoms, the cellular such tissue, but EBNA-positive cells are cerimmune response which develops after infection tainly very infrequent. It is also unknown ensures that the proliferation of EBV-infected whether normal B cell precursors are infected by cells is contained at a low level throughout life EBV in vivo, although such cells can be infected (Svedmyr and Jondal 1975; Rickinson, Craw- in vitro (Ernberg, Falk, and Hansson 1987). ford, and Epstein 1977; Rickinson, Moss, and Pope 1979; Rickinson et al. 1980; Moss, RickinFatal Infectious Mononucleosis son, and Pope 1978, 1979; Tosato et al. 1979; Tosato and Blaese 1985; Rickinson 1986). The initial reaction to primary EBV infection and Death as a result of infectious mononucleosis is proliferation of B cells may involve a large non- a rare event, and may occur from secondary specific component (Klein et al. 1981; Tosato et bacterial infection, splenic rupture, massive heal. 1979; Tosato and Blaese 1985), i.e., a general patic necrosis, encephalitis, or a variety of hereaction against activated B cells rather than matologic complications including aplastic aneagainst EBV-associated antigens expressed on a mia (Finch 1969). We are concerned here, subpopulation of such cells. Subsequently an however, with death consequent upon massive

12. Infectious Mononucleosis and Malignant Neoplasia proliferation of EBV-infected lymphocytes. Fatal infectious mononucleosis of this type occurs much more frequently in certain immunodeficiency states, particularly the X-linked familial syndrome described by Purtilo and others and now generally referred to as X-linked lymphoproliferative syndrome (XLP), which is discussed below (Bar et al. 1974; Purtilo 1974; Purtilo et al. 1975). Sporadic cases offatal infectious mononucleosis associated with overwhelming lymphoproliferation have also been described (Crawford et al. 1979; Virelizier, Lenoir, and Griscelli 1978; Britton et al. 1978; Robinson et al. 1980; Thestrup-Pederson et al. 1980). Some of these have been girls, and none appears to be a member of the sibships described by Purtilo, although several of these patients had congenital abnormalities. Pathologic findings in patients who die from uncontrolled EBV-induced lymphoproliferation have been similar in most cases and consistent with those originally reported by Lukes and Cox (1958). Hepatosplenomegaly is usually prominent, and lymphoid tissue is hypertrophied, with distortion or complete replacement of normal lymphoid cytoarchitecture by proliferating lymphoid cells. In some cases there is invasion of the capsule of lymph nodes-a finding normally associated with malignant lymphoma. There is infiltration of almost all tissues by immature lymphoid cells, showing various degrees of differentiation toward plasma cells. Often, this is accompanied by necrosis, which may be extensive on occasion, and involves the organ parenchyma as well as the lymphoid cells (Frizzera 1987). Bone marrow may be involved, and in cases where peripheral blood has been examined, there is a marked elevation of the fraction of EBNA-positive cells. In some patients, morphologically abnormal circulating cells are seen, and these may lead to elevations in white count and a frankly leukemic blood picture. In one patient a white blood cell count reached 99,900 with 40 percent "atypical lymphocytes," which ranged in appearance from immunoblasts to more mature cells resembling plasma cells (Robinson, Smith, and Niederman 1981; Robinson et al. 1980). In this case, blood mononuclear cells contained an average of 20 EBV genome copies per cell. The expression of surface immunoglobulin with both light chain types on the infiltrating cells in several patients with fatal infectious mononucleosis has clearly established

149 the polYclonal nature of the process (Crawford et al. 1979; Robinson et al. 1980), and the infiltrating cells have also been shown, where tested, to contain EBV DNA and EBNA (Bar et al. 1974; Crawford et al. 1979; Robinson et al. 1980). In some of these patients, tissue extracts have contained virus particles capable of transforming B lymphocytes, demonstrating that in at least some of the infiltrating cells, virus replication was occurring. In none of these patients have the cells been reported to contain chromosomal translocations. It appears that most cases of fatal infectious mononucleosis occurring as a result of uncontrolled lymphoproliferation are the consequence of an abnormal immune response to EBV. Some patients-notably the familial cases-are unable to make antibodies to EBV-associated antigens, particularly EBNA. In such cases documentation of EBV infection has been provided by the demonstration of EBV antigens or DNA in the proliferating cells. Familial cases of fatal infectious mononucleosis have a chronic immunodeficiency syndrome leading to an increased frequency of opportunistic infections (Purtilo et al. 1979). Other cases appear to exhibit a very poor T-cell response or other immunologic abnormalities (Crawford et al. 1979; Robinson et al. 1980; Finlay et al. 1986; Filipovitch in press). One 6-year-old boy with persistent lymphadenopathy, very high anti-VCA antibodies, and 4 percent EBNA-positive cells in his peripheral blood had an abnormality of chromosome 16 in peripheral lymphocytes that was dependent for its demonstration on the presence of interferon in the in vitro culture (ThestrupPederson et al' 1980). His mother, but not his father or sister, had a similar, inducible chromosomal abnormality. The patient also had low levels of T cells and poor natural killer (NK) cell function. He finally died after cytotoxic chemotherapy. Autopsy revealed widespread lymphoid infiltration, and EBV DNA was detected in lymph nodes, liver, and spleen. The morphologic appearance was felt to be consistent with American Burkitt's lymphoma, although previous node biopsies had shown immunoblasts and plasma cells (Pallesen et al. 1980). Subsequently, however, the histology was interpreted as showing fatal infectious mononucleosis (Purtilo, personal communication). It is clear that a number of different kinds of immunologic defects can lead to the same result

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-namely, failure to control the proliferation of EBV-infected cells. It is also apparent that the syndrome of fatal infectious mononucleosis closely resembles the lymphoproliferative syndromes induced in animals by herpesviruses. As in the animal syndromes, there has often been debate about whether the patient has a true neoplastic disease, as determined by histology - an inadequate criterion by todays standards. It is worthy of note that the same histologic hallmarks of "malignancy" that are seen in these cases may also be observed in patients with selflimited infectious mononucleosis (Salvador, Harrison, and Kyle 1971; Gowing 1975). Such criteria are clearly fallible! The spectrum of the appearance of lymphoid cells in fatal infectious mononucleosis is, however, very similar to that of immunoblastic lymphoma. In this histopathologic entity, which is a subcategory oflarge-cell lymphoma in the NCI working formulation (National Cancer Institute 1982) and which was frequently referred to as reticulum cell sarcoma in the past, the cells cytologically resemble lymphocytes transformed by mitogens in vitro. The similarity of cells activated by EBV to immunoblastic lymphoma is not, therefore, surprising. Immunoblastic lymphoma is the predominant type of lymphoma that arises in a variety of conditions associated with immunologic disorders, such as autoimmune diseases, glutensensitive enteropathy, Hashimoto's disease, alpha-chain disease, immunoblastic lymphadenopathy (and the related angioimmunoblastic lymphadenopathy), and immunosuppressed states including iatrogenic and congenital immunodeficiencies (Lukes 1980). Some of these tumors have been demonstrated to be polyclonal rather than monoclonal (Hertel et al. 1977) and to contain EBV DNA. This suggests that such tumors may, at least in some cases, result from a failure to regulate the proliferation of EBV-infected cells, and as such, they differ from neoplasms associated with a genetic change, such as a chromosomal translocation.

The X-Linked Lymphoproliferative Syndrome (XLP) A number of families have been described in which males are particularly susceptible to a spectrum of lymphoproliferative disorders rang-

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ing from fatal infectious mononucleosis to malignant non-Hodgkin's lymphoma. In 1969, Hambleton and Cotton described 2 brothers who developed malignant lymphoma after infectious mononucleosis. In 1973, Falletta et al. described a large kindred in which 17 young boys died of a lymphoproliferative syndrome before the age of 6, and in 1974, Bar described fatal infectious mononucleosis in 3 male maternal cousins. A fourth cousin in this family died oflymphoma of the brain, marrow, and CSF. In another family (the Duncan family), 3 brothers died of infectious mononucleosis, and 2 halfbrothers died of lymphomas of the brain and intestinal tract. The brain lymphoma occurred shortly after infectious mononucleosis (Purtilo et al. 1975). An additional case of malignant lymphoma (B-cell immunoblastic type) following infectious mononucleosis in the Duncan kindred was described later (Pattengale, Taylor, and Pegalow 1981). In yet another family, there were 2 boys who died from infectious mononucleosis and several other deaths in males that may have been due to infectious mononucleosis. Two other brothers died from "American Burkitt's lymphoma" and 5 boys from "immunoblastic sarcoma of B-cells" (Purtilo, DeFlorio, et al. 1977). Many other kindreds suffering from the same disorder have been described, and by 1987, 187 cases in 48 kindreds had been enrolled in an XLP registry (Purtilo 1987). Almost 85 percent of patients are dead by the age of 10 and 100 percent by the fifth decade. Some two-thirds of patients die from infectious mononucleosis, and the remaining third develop either hypogammaglobulinemia or a B-cell lymphoma, designated variously as American Burkitt's lymphoma, immunoblastic sarcoma with B-cell characteristics (i.e., exhibiting differentiation toward plasma cells), diffuse histocytic lymphoma, or plasmacytoma (Purtilo, DeFlorio, et al. 1977; Purtilo, Yang, et al. 1977). There is overlap between these phenotypes, since patients who survive their lymphoma almost invariably have hypogammaglobulinemia, while those who survive infectious mononucleosis develop either hypogammaglobulinemia or B-cell lymphoma. Immunopathologic studies carried out in patients with XLP have revealed dtfpletion of Tcell areas in thymus, lymph nodes, and spleen, and initial immunologic studies have demon-

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strated a number of abnormalities (Purtilo et al. 1980; Purtilo et al. 1979; Purtilo, Yang, et al. 1977). The ability to generate antibodies to EBV is often severely impaired, particularly the antiEBNA response (Sakamoto, Freed, and Purtilo 1980), and defects in NK cell activity, interferon-activated killing, and EBV antigen inhibition of leukocyte migration have been reported (Sullivan et al. 1980; Masucci et al. 1981). There is also a relative increase in T cells expressing the T8 antigen, normally associated with suppressor cells, and a suggestion that there is aberrant cytotoxic activity against a variety of tissues (Seeley et al. 1981; Sullivan et al. 1983), XLP patients appear to have an increased frequency of infections with a variety of microorganisms and nematodes (Purtilo et al. 1980; Purtilo et al. 1979), but considerably more work needs to be done before the immunologic defects are fully understood. It is seemingly paradoxical that in the same families in which lymphoproliferation occurs, some members develop hypogammaglobulinemia or agammaglobulinemia, often following infectious mononucleosis (Provisor et al. 1975; Purtilo, DeFlorio, et al. 1977; Purtilo et al. 1979). B-cell numbers are normal in these patients, and it is likely, therefore, that the explanation for the low level of gammaglobulin is abnormal T-cell regulation of immunoglobulin synthesis. These patients exhibit defective NK activity and impaired cell-mediated immunity to EBV antigens (Masucci et al. 1981). Since hypogammaglobulinemia sometimes precedes infectious mononucleosis (Masucci et al. 1981; Purtilo et al. 1979), residual overactivity of nonspecific suppressor cells following infectious mononucleosis seems an unlikely explanation, and the pathogenesis of the agammaglobulinemia currently remains unknown. Agranulocytosis and aplastic anemia can also occur in the XLP families (Purtilo et al. 1979; Purtilo, Yang et al. 1977). Recently, the demonstration that EBV can infect etythroid precursor cells has provided a possible explanation for the marrow failure state, although a detailed pathogenetic mechanism remains to be determined (Baranski 1988). The broad spectrum of morphologic appearances of the lymphoproliferations occurring in XLP is initially confusing but is reminiscent of herpesvirus-induced lymphoproliferations in animals. Individual lymphomas may manifest a

relatively restricted range of cell types and are most often designated as immunoblastic lymphomas of B cells. However, some have been classified as American Burkitt's lymphoma and others, even plasmacytoma. It is pertinent that the lesions in the same patient have been known to manifest more than one of these appearances. Sometimes at autopsy for fatal infectious mononucleosis, the relatively uniform cellular appearance in some areas has prompted a histologic diagnosis of immunoblastic lymphoma or another type of B-celllymphoma (Bar et al. 1974; Purtilo, DeFlorio, et al. 1977; Purtilo et al. 1979; Purtilo et al. 1981). This is very reminiscent of the findings in the lymphoproliferative syndromes occurring in organ transplant recipients (see below). The observation that the initial control of primary EBV infection involves a major nonspecific component, whereas the subsequent regulation of EBV-infected cells appears to be more EBV-specific, provides one possible explanation for the development of either fatal infectious mononucleosis or lymphoma. Broad immunologic defects, possibly involving NK cells, for example, may result in failure of the nonspecific component and permit development of fatal infectious mononucleosis at the time of primary infection. Patients with defects of the EBV-specific component or less severe immunosuppression may be more likely to develop lymphoma. In addition, continued high levels of benign, EBV-induced lymphoproliferation may predispose to the development of a genetically induced "true" neoplasm, although to date, the chromosomal translocations characteristic of Burkitt's lymphoma have not been described in XLP (Harris and Docherty 1988).

Lymphoproliferative Syndromes in Allograft Recipients Renal Transplant Recipients The increased incidence of malignant neoplasia in allograft recipients has been best documented in patients who receive renal transplants (Hoover and Fraumeni 1973; Penn 1970a, 1970b, 1979). In such patients the commonest tumors are skin and lip cancers, but more than 20 percent are malignant lymphomas. Although

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Hodgkin's disease is easily the commonest lymphoma in the general population, this lymphoma is rare in transplant recipients (Cerilli et al. 1977), among whom the majority of malignant lymphomas have been classified as reticulum cell sarcoma or, more recently, large-cell lymphoma, diffuse histiocytic lymphoma, or immunoblastic sarcoma (these terms encompass similar or identical tumors). Lymphomas occur at least 350 times more frequently in transplant recipients than in the general population (Hoover and Fraumeni 1973) and, in addition, have a predilection for the central nervous system, which is involved in approximately half the cases (Schneck and Penn 1971; Penn 1979). The vast majority of the tumors in transplant recipients are of B-cell origin and express surface immunoglobulin; however, a proportion of tumors in heart transplant recipients apparently lack surface immunoglobulin although other B-cell markers are present (Frizzera 1987). Both kappa and lambda light chains are usually expressed, indicating that the tumors (or the majority of them) are polyclonal (Hanto et al. 1981). In a description of the morphologic findings in six patients with post-renal transplant lymphoproliferative syndromes, Frizzera et al. (1981) emphasized the variation in cell size and the tendency to infiltrate tissues. They suggested that the tumors be called "polymorphic B-cell lymphomas" in the presence of nuclear atypia and necrosis, and "polymorphic diffuse B-cell hyperplasia" otherwise. It should be noted that the presence of features leading Frizzera and colleagues to diagnose lymphoma rather than hyperplasia did not have any prognostic significance. It is, in fact, noteworthy that, as in XLP, a broad spectrum of clinical lymphoproliferative syndromes occur after renal transplantation. Hanto et al (1982) reported 6 patients (the same patients described by Frizzera), of whom 3 were in their second decade and 3 in their sixth decade. The young patients presented within months of the transplant, or in one case shortly after treatment for rejection, with fever, sore throat, and lymphadenopathy. The latter patient had a syndrome consistent clinically and serologically with fatal infectious mononucleosis, and the other 2 patients also died from widespread lymphoproliferation of EBV-DNA-containing cells. The 3 older patients presented sev-

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eral years after transplantation (2%, 5, and 8 years), and 2 had more localized disease. In all 3 patients the lymphoid proliferations were shown to be polyclonal and B cell in type, and there was evidence ofEBV DNA in the lymphoid cells. All were also EBV-seropositive prior to transplantation. In 2 of the 3 patients, in whom chromosomal studies were performed, karyotypic abnormalities were detected (though 14q+ or 8;14 translocations were not seen). Hanto et al. suggested, on the basis of their 6 patients, that there may be two main types of lymphoproliferative syndrome that occur after renal transplantation. The first, a syndrome similar or identical to fatal infectious mononucleosis, arises at times of profound immunosuppression. It is sometimes, but not always, the result of primary EBV infection. The occurrence of this syndrome in renal transplant recipients has also been observed by others (Briggs et aI, 1978; Louie and Schwartz 1978; Marker et al. 1979; Starzl et al. 1984). The cells are cytogenetically normal (Hanto et al. 1985) and, in the rare cases examined, do not have clonal rearrangements of immunoglobulin genes although evolution to a monoclonal lymphoma has been described (Hanto et al. 1988). The second syndrome (designated polymorphic diffuse B-celllymphoma by Frizzera) presents as localized immunoblastic lymphoma, and is usually associated with cytogenetic abnormalities, none of which are consistent (Filipovitch et al. in press; Frizzera 1987). This syndrome probably represents a more restricted expression of EBVinfected cells escaping from immunosurveillance. This possibility is supported not only by the histologic resemblance of such tumors to the infiltrating cells of infectious mononucleosis, but by the documented presence of EBV DNA in the cells. Further, in EBV-seropositive renal transplant recipients there is increased excretion of EBV from the pharynx (Strauch et al. 1974) and increases in the titer of anti-EBV antibodies (Marker et al. 1979; Cheeseman et al. 1980), indicating less effective control of EBV-infected cells. Recently, direct evidence of impairment of EBV-specific T-cell memory in allograft recipients treated with cyclosporin A has been obtained (Crawford et al. 1981). Several lymphomas have arisen in renal allograft recipients treated with cyclosporin A, at least one of which was shown to be EBV-associated (Bieber, Reitz, and Jamieson 1980; Nagington and Gray 1980; Crawford et al. 1980).


Other Organ Allograft Recipients Penn (1979) has reported that in organ transplant recipients in general, 32 percent of the malignant neoplasms are lymphomas, compared with 3 - 4 percent in the general population. Over 60 percent of these were reported as reticulum cell sarcoma, and 42 percent involved the central nervous system. In marrow transplant recipients, levels of anti-EBV antibodies (anti-VCA and anti-EA) are abnormally high after transplantation (Lange et al. 1980), but no clearcut increase in the incidence of lymphomas has been detected yet, although a case of immunoblastic sarcoma arising in donor cells after marrow transplantation has been reported (Gossett et al. 1979), and cases of fatal EBV-associated lymphoproliferation have been observed after treatment of acute graft-versus-host disease with monoclonal antibodies directed against the CD3 T-cell antigen (Martin et al. 1984). In heart transplant recipients, similar syndromes have been seen to those in renal allograft recipients. There has been considerable discussion with regard to the clonality of such tumors, as determined by immunoglobulin gene rearrangements (Purtilo et al. 1985). Most of the tumors are polyclonal or oligoclonal. Frequently, different immunoglobulin gene rearrangements are found at different anatomic sites, although sometimes the same rearrangement is seen at different sites. This is entirely consistent with the behavior of lymphoblastoid cell lines in vitro, which gradually convert from polyclonality through oligoclonality to monoclonality. It might be anticipated that disorders resulting from defects in the control of EBV-infected cells would occur in a variety of immunodeficiency states, since lymphoproliferative syndromes occurring in iatrogenic immunosuppression and XLP appear to be accounted for on this basis. There is evidence that this is indeed the case.

Lymphoproliferative Syndromes in Congenital Immunodeficiency Diseases A number of congenital immunodeficiency syndromes have been associated with a high risk of malignancy. Ataxia telangiectasia, common

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variable immunodeficiency, and Wiskott-AIdrich syndrome, for example, are associated with lymphoma or leukemia in 15 to 25 percent of affected individuals (Bowder and Sedgwick 1963; Perry et al. 1980; Filipovitch et al. in press). Severe combined immunodeficiency is also associated with lymphoproliferation, and cases of lymphoproliferation associated with thymic transplantation have been described. Sex-linked hypogammaglobulinemia (Bruton type) and the di George syndrome appear not to be associated with a higher incidence of malignant neoplasia (Kersey, Spector, and Good 1974, 1975; Spector et al. 1978; Filipovitch et al. in press). Although it has been postulated that abnormalities of nucleic acids, e.g., defective DNA repair mechanisms, and/or chromosomal instability may be as important as or of greater importance than the immunologic defect in the development of malignant lymphoma (Louie and Schwartz 1978), this, in itself, does not explain why 90 percent of all cancers in patients with inherited immunodeficiency syndromes are non-Hodgkin's lymphomas, nor why these lymphomas should be predominantly classified as immunoblastic sarcoma as in allograft recipients. An additional similarity with the lymphomas in allograft recipients is the high frequency of brain involvement. These similarities strongly suggest a common pathogenetic mechanism-namely, a defect in the immunologic regulation of EBV-containing B cells. EBV DNA has been demonstrated in the cells of an abdominal lymphoma in a patient with ataxia telangiectasia. Unfortunately, immunologic studies to establish the clonality of the tumor and cytogenetic analysis were not performed (Saemundsen et al. 1981). Patients with a variety of immunodeficiency syndromes who either died from infectious mononucleosis or showed evidence of abnormal proliferation of EBV-infected cells have been reported (Purtilo et al. 1981; Nonoyama et al. 1974; Purtilo et al. 1985). Patients with immunosuppression from a wide variety of causes (including organ transplantation, sarcoidosis, ataxia telangiectasia, rheumatoid arthritis, and lupus erythematosus) have long been known to have higher mean anti-EBV antibody levels than normal individuals (Berkel et al. 1979, 1981), and many of these groups of patients do indeed have a higher-than-average incidence of lymphoma.

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Lymphoproliferative syndromes involving multiple organs and tissues following transplantation of cultured thymus in 3 children with severe combined immunodeficiency syndrome have been described (Borzy et al. 1979). Surface immunoglobulin of the proliferating cells of 2 of these 3 cases were polyclonal, and the disease was classified histologically in one case as immunoblastic sarcoma. Widespread lymphoproliferation was not observed in 11 other thymic transplant patients seen by the authors, although an additional case of "polyclonal immunoblastic sarcoma" following thymic transplantation and containing multiple EBV genomes was described by Reece (Reece et al. 1981). In any event, children with combined immunodeficiency syndrome are at higher risk of lymphoproliferation (Filipovitch et al. in press). Although the reported patients were characterized as having immunoblastic sarcomas or immunoblastic lymphadenopathy, the descriptions are strongly reminiscent of fatal infectious mononucleosis. All patients had impaired T-cell function prior to grafting, and 3 had polyclonal gammopathy; thus the possibility that the graft itself was not responsible for the subsequent lymphoproliferation (except possibly as the medium for EBV infection ofthe recipient) cannot be totally excluded. Perhaps the most likely explanation is that T-helper factors for B cells outweighed Tsuppressor/cytotoxic activity after the graft, and the EBV-infected B cells were therefore favored. It is of interest that children with combined immunodeficiency treated with allogeneic marrow transplantation have not so far been reported to develop lymphoproliferative syndromes (Reece et al. 1981).

Lymphoproliferative Syndromes in Acquired Immunologic Disorders Collagen Vascular and Chronic Granulomatous Diseases A number of collagen vascular and chronic granulomatous diseases are associated with an increased incidence oflymphoid neoplasia. Such diseases, however, are often treated with immunosuppressive therapy, and it may therefore be difficult to determine whether an increased

Ian Magrath incidence of malignant neoplasia in these circumstances is a complication of the disease, its therapy, or a combination ofthe two. Moreover, good prospective studies of such patients have not been done, and much of the available data are anecdotal or lack a common denominator. Some diseases, e.g., Sjogren's syndrome and autoimmune hemolytic anemia, are known to be associated with lymphomas irrespective of treatment, and multiple pathogenetic mechanisms may apply. However, an increased incidence of reticulum cell sarcoma or immunoblastic lymphoma in these diseases, in the light of the data reviewed so far, raises the possibility that further examples of defective control of EBV-infected cells may be unearthed. There is evidence that the lymphoproliferative process occurring in Sjogren's syndrome passes through a polyclonal stage (frequently called pseudolymphoma since regression of these lesions can occur) but then becomes monoclonal (Zulman, Jaffe, and Talal 1978). This change from a polyclonal to a monoclonal process has been documented in other EBV-associated lymphoproliferative syndromes (Hanto et al. 1982; Krueger, Papadakis, and Schaefer 1987), as well as in immunoblastic lymphadenopathy, which frequently terminates in immunoblastic lymphoma (Lukes and Tindle 1978; Nathwani et al. 1978). In the latter disease the neoplasms which emerge are predominantly of T-cell origin and are not known to be associated with EBV. Although EBV is generally considered to be B-cell-trophic, a T-cell lymphoma containing EBV genomes was recently described (Jones et al. 1988). Of 21 cancers reported in patients with systemic lupus erythematosus treated with immunosuppressive therapy, 6 were reported as reticulum cell sarcoma and 1 as plasmacytoma (Louie and Schwartz 1978). Patients with systemic lupus erythematosus also appear to have an increased incidence of malignant neoplasia in the absence of immunosuppressive therapy other than corticosteroids (Lewis et al. 1976). Another set of premalignant conditions which predispose to malignant lymphoma of the immunoblastic variety consists of the angiocentric lymphomas which arise in such processes as lymphomatoid wanulomatosis and midline granuloma. An association with EBV has not been reported, however, and these tumors ,are predominantly T cell in type (Jaffe 1986).

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Lymphomas Associated with HIV Infection The marked increase in the incidence of nonHodgkin's lymphomas in patients with the acquired immunodeficiency syndrome (AIDS) was recognized in the early 1980s (Ziegler et al. 1982). Interestingly, the commonest tumor occurring in such patients is the small noncleaved lymphoma associated with the same cytogenetic abnormalities present in small noncleaved lymphomas arising in the normal population (Chaganti et al. 1983; Whang-Peng et al. 1984), although large-cell lymphomas also appear to be increased in incidence. The evidence that other lymphoma subtypes, including Hodgkin's disease, occur more frequently in patients with HIV infection is much less convincing. Patients with HIV infection often have chronic lymphadenopathy which precedes the onset of flagrant malignant lymphoma, and there can be little doubt that the immune defects, and B-celllymphoproliferation occurring in these patients, constitute a critical predisposing factor. It has been shown that the T-cell response to EBV-infected cells is impaired in patients with AIDS and AIDS-related complex, such that there is a higher level of circulating EBV-containing cells (Birx, Redfield, and Tosato 1986). The majority of these tumors, however, clearly differ from those in other immunodeficient individuals, since they are monoclonal and have nonrandom cytogenetic abnormalities that result in rearrangements of the c-myc gene similar to those occurring in sporadic Burkitt's lymphoma (Pelicci, Knowles, Arlin, et al. 1986). Moreover, although some 40 percent of such tumors contain EBV in the tumor cells, (Koziner et al. 1984; Subar et al, 1988), it is clear that EBV cannot be implicated in the occurrence of lymphomas in this patient group in all circumstances. It would appear that in HIV infection, the factors which predispose to the development of small noncleaved lymphomas are present to excess, rather than the situation being one simply of failure to control EBV-infected B cells.

Lymphoid Neoplasia Following Cancer Chemotherapy The risk of a second malignancy in patients with cancer varies with the treatment received, and in some cases, with the underlying cancer. The

most common second malignancy in patients undergoing cancer chemotherapy is acute myeloblastic leukemia (Penn 1970b, 1979; Reitz et al. 1987), and here the direct effect of a drug (particularly an alkylating agent) or radiation on a target cell may be more important than immunosuppression as a pathogenetic factor. There is also, however, an increased incidence of nonHodgkin's lymphomas in patients undergoing cancer chemotherapy (Tucker et al. 1988). "Reticulum cell sarcoma" has long been known to occur with increased frequency in certain patients, e.g., those with chronic lymphocytic leukemia or myeloma (Penn 1979), but there is little information regarding EBV association in these tumors. In some cases, e.g., diffuse largecell lymphomas arising in patients with underlying follicular lymphoma and possibly chronic lymphocytic leukemia (Richter's syndrome), the "second" lymphoma is a different phase of the primary disease rather than a new tumor arising as a consequence of immunosuppression.

Uncontrolled Proliferation of EBVInfected B Cells: A Hazard of Immunodeficiency States The frequently occurring lymphoproliferative disorders of immunodeficiency syndromes, whether inherited, acquired, or iatrogenically induced (Table 12.3), bear a remarkable similarity to each other, irrespective of the underlying disease process. They range from fulminating widespread proliferation to localized tumor masses. Morphologically, a range of cell types may be seen, from immunoblast to plasma cell, usually in various mixtures but sometimes in

Table 12.3 Immunodeficiency states associated with defective regulation of EBV-infected B-cell proliferation. X-linked lymphoproliferative syndrome Iatrogenic immunosuppression in allograft recipients Ataxia telangiectasia Wiscott-Aldrich syndrome Severe combined immune deficiency after mismatched marrow transplantation or thymic transplantation Chediak-Higashi syndrome

156 relatively pure populations of a single cell type. The documented presence of EBV in the proliferating cells of fatal. infectious mononucleosis and the lymphoproliferative syndromes occurring after renal allografting (including, in the latter cases, some that fulfill histologic criteria of malignant neoplasia), and in a variety of other immunodeficient patients, indicates that these syndromes are the result of the uncontrolled or only partially controlled proliferation of EBVinfected B cells. The similarity of the cytological polymorphism of these processes and that of the animal herpesvirus-induced lymphoproliferative syndromes is striking. So also is the similarity of the debates about whether these processes can truly be classified as malignant lymphomas. In both cases, however, the temporal relationship to primary virus infection in the fulminating cases (i.e, in humans, fatal infectious mononucleosis) and the documented presence of viral genomes in the proliferating cells, as well as (in animals) the ability to prevent the syndrome by vaccination, leaves no doubt about the importance of the virus in etiology. The determinants of the observed differences in behavior are not known, but probably relate to variations in the defect in regulation of EBV-infected B cells. Interestingly, in the XLP syndrome, in effectively immunosuppressed renal transplant recipients and in HIV-positive individuals there is a decrease in the ratio of CD4-positive (helper) T cells to CD8 (suppressor) T cells (Seeley et al. 1981; Ellis, Lee, and Mohanakumar 1981). While at first sight, it might be presumed that this should result in increased suppression of EBV infected cells, suppressor and cytotoxic T cells also require helper cells in order to function. Thus, a deficit of helper cells is detrimental to all immunologic function. Of considerable interest is the striking similarity between the proliferation of EBV-infected cells in vivo in patients with immunodeficiency and the growth of such cells (derived from normal individuals) in vitro as continuous cell lines. Spontaneous outgrowth of the latter from mixed lymphocyte populations is inhibited by T cells, so that the addition of cyclosporin or anti-T cell antibodies to the cultllfe vessel is a standard technique used to increase the rate of cell line outgrowth. Both manipulations have also been used in vivo and are associated with an increased incidence ofEBV-associated B-celllym-

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phoproliferation (Bieber, Reitz, and Jamieson 1980; Nagington and Gray 1980; Crawford et al. 1980; Martin et al. 1984; Sullivan et al. 1984). The gradual reduction of the number of clones in a continuous cell line and the accumulation of random chromosomal defects also parallel, to a remarkable degree, the findings in patients with lymphoproliferations associated with inherited or acquired immunodeficiency. There seems to be little question that the majority of the lymphoproliferative syndromes arising in such patients originate as uncontrolled or poorly controlled growth of EBV-infected B lymphocytes. One possible exception to this occurs in HIV infections. Such patients are at very high risk to develop Burkitt's lymphoma, and not all cases are EBV-associated. The issues surrounding the EBV association of Burkitt's lymphoma will be addressed in a subsequent section. It seems likely, however, that this and possibly truly neoplastic large-cell lymphomas are predisposed to by underlying increased lymphoproliferation. Even if immunodeficiency diseases usually result in EBV-associated polyclonal lymphoproliferations, true cytogenetically abnormal neoplasms can be superimposed.

Treatment Approaches An understanding of the pathogenesis of the lymphoproliferative syndromes in immunosuppressed patients is of paramount importance to the development of effective prevention or treatment. While many of these tumors behave like malignant lymphomas, since the majority of them appear to arise because of a defect in the immunoregulation of EBV-infected cells, treatment approaches might be more logically directed toward correcting this abnormality than attempting to eliminate the malignant cells with cytotoxic drugs. In patients undergoing immunosuppressive therapy, immunosuppressive drugs should be reduced in dose or completely withdrawn, even if this will result in graft rejection. When immunodeficiency is not iatrogenically induced, therapeutic options are few. While there is justifiable reluctance to use anticancer chemotherapy except when a lymphoproliferative process is known to be neoplastic, the application of such a rule necessitates a very


precise definition of neoplasia. Since in many of the lymphoproliferative processes occurring in immunodeficient patients this is often a question of semantics, the use of such a criterion to determine therapy is arbitrary at best. Nevertheless, wherever possible, tumors should be char. acterized in as much detail as possible, since, ultimately, the tumor characteristics may be of value in defining the best treatment approach. In the modern era, histology cannot be considered adequate, and for the most complete description, cytogenetics, immunophenotype molecular analysis for clonality of immunoglobulin genes, and examination for EBV genomes are necessary. Even if all of these investigations are carried out and the process is shown to be a polyclonal or oligoclonal proliferation of EBVinfected B lymphocytes, optimal therapy has still not been determined. Among the therapeutic possibilities is the use of antiviral agents or lymphokines. Interferon in one child was ultimately unsuccessful (Thestrup-Pederson et al. 1980), but appeared to be valuable in a small number of patients when combined with intravenous immunoglobulin (Filipovitch et al. in press) Acyclovir (9-[2-hydrox yethoxymethyl] guanine), an inhibitor of herpesvirus replication, is also worthy of further testing, either early in the infection or even as prophylaxis in very high risk individuals, since it has been shown to prevent herpes simplex reactivation in bone marrow transplant recipients (Saral et al. 1981). On theoretical grounds, however, such treatment seems unlikely to be able to halt the proliferation of EBV-infected clones. Acyclovir prevents virus replication but does not eliminate latent episomal EBV genomes from the cell. Since the latter appear to be responsible for the proliferative drive, acyclovir therapy is unlikely to be effective. At best it could lessen or prevent the transmission of EBV to uninfected cells. One report of a beneficial effect is obscured by the simultaneous reduction of immunosuppressive therapy (Hanto et al. 1982), and other patients have not appeared to benefit (Sullivan et al. 1984). Moreover, even to exert its effect on linear, replicating EBV molecules, acyclovir must be given continuously. Once it is discontinued, virus can again enter a lytic cycle, and meanwhile, the proliferation of already infected cells would be unabated. Treatment approaches using interleukin-2

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and-or lymphokines are worthy of further investigation. A possibility for the future is the use HLA-compatible (preferably autologous, if enough cells can be obtained from the patient) cloned lymphocytes expanded in vitro by means of interleukin-2, and demonstrated to have specific suppressor or cytotoxic activity against EBV-infected B cells. T-cell clones of this kind have been isolated but have not been used therapeutically (Sugamura, Tanaka, and Hinuma 1981; Rickinson 1986). The practical problems of this approach are likely to be considerable. Cytotoxic therapy may be appropriate in some circumstances, and it is worth emphasizing that its use, even in cancer, is based entirely on empiric observations. Thus, there is no a priori reason to withhold its use in rapidly progressing lymphoproliferative syndromes, even if known to be polyc1onal. Although there is a significant risk of further impairment of immunologic regulatory mechanisms, with resultant exacerbation of lymphoproliferation, if the proliferating cells are responsive to therapy, this may not be an issue. There is likely to be, however, a substantially increased risk of opportunistic infection. In widely disseminated lymphoproliferation the patient will die unless treated, so that in this situation, the risks of cytotoxic therapy are justifiable. Although little information exists regarding choice of therapeutic regimens, it would be rational to treat with a regimen designed for B-cell or large-cell lymphomas (Wilson and Magrath in press).

Burkitt's Lymphoma Ironically, although EBV was discovered in cell lines derived from Burkitt's lymphoma as the result of a specific search for a viral etiology of this tumor (Epstein, Achong, and Barr 1964), the role of EBV in its pathogenesis, if any, remains to be elucidated. Whereas there has been considerable debate about the naturemalignant neoplasia versus hyperplasia-of the polymorphic lymphoproliferative states described above, Burkitt's lymphoma fulfills all of the criteria that can be mustered in attempting to define true malignant neoplasia. It consists of a monomorphic proliferation of undifferentiated lymphoid cells of specific histologic ap-

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pearance (Berard, Q'Conor, and Thomas 1969) that tend to accumulate in large tumor masses, particularly in the jaw and abdomen (Magrath 1987), and may be widespread throughout the body. The tumor cells express surface IgM in nearly all cases, with light chains of either kappa or lambda type, but not both, indicating monoclonality (Gunven et al. 1980). Glucose-6-phosphate isoenzyme phenotypes in female heterozygotes and molecular characterization of genetic rearrangements confirm the monoclonality of the tumors (Fialkow et al. 1973; Pelicci, Knowles, Magrath et al. 1986; Neri et al. 1988; Barriga et al. in press, 1988). Burkitt's lymphoma cells bear a nonrandom chromosomal translocation, one of the breakpoints always being in the terminal region of chromosome 8 - the location of the c-myc protooncogeneand one in one of the immunoglobulin chain loci on chromosome 14 (in 85 percent of cases), 22 (in 10 percent of cases), or 2 (5 percent of cases) (Manolov and Manolova 1972; Zech et al. 1976; Lenoir, Philip, and Sohier 1984). These translocations result in deregulation of the c-myc gene, a gene involved in cell proliferation, and are clearly a critical component of pathogenesis. Burkitt's lymphoma is invariably fatal unless treated with appropriate chemotherapy. Fresh tumor cells from African Burkitt's lymphoma, referred to as endemic Burkitt's lymphoma because of the much higher incidence of the tumor in equatorial Africa than in other parts of the world, contain EBV DNA in about 95 percent of cases, but as in other herpesviruscontaining tumors, virus particles are not seen (Nonoyama et al. 1973; Zur Hausen et al. 1970). In contrast to the endemic tumor, sporadic tumors, occurring in Europe and the USA, are associated with EBV in only some 15 to 25 percent of cases (Andersson et al. 1976; Zur Hausen 1975; Barriga et al. 1988). Tumors occurring in North Africa contain EBV genomes in some 85 percent of cases (Whittle et al. 1984), while in many other parts of the world the frequency of EBV association has not been examined. The occurrence in Africa of a small number (5 -10 percent) of EBV-negative Burkitt's lymphomas in seropositive individuals (Lindahl et al. 1974) is consistent with the possibility that the EBVnegative tumor occurs at low frequency throughout the world, but in Africa it is greatly overshadowed by EBV-positive tumors, which

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occur because of a particular concurrence of environmental and/or racial factors. Similarly, the majority of patients with EBV-negative tumors in the USA are seropositive, but at least some are EBV-negative; and we and others (Purtilo personal communication) have observed patients who have developed infectious mononucleosis after the onset of Burkitt's lymphoma. In such cases EBV cannot be invoked for any role in pathogenesis, but this does not exclude the possibility that EBV is of pathogenetic importance in some or all of the tumors with which it is associated. This particular dilemma - the fact that not all Burkitt's tumors are associated with EBVcreates problems in devising hypotheses to account for its presence in the majority of African Burkitt's lymphomas. It has been suggested that malaria may provide sufficient immunosuppression to permit a higher level of continued proliferation of EBV-containing cells following primary infection but that an EBV containing cell clone only becomes truly neoplastic when it develops the specific 8;14 (or variant) chromosomal translocation (Lindahl et al. 1981). In such a situation, EBV could not be conceived of as a necessary component of tumorigenesis, but would increase - perhaps by a substantial amount - the possibility that a chromosomal translocation giving rise to c-myc deregulation would occur, simply by increasing the target cell population for the cytogenetic event. Support for this hypothesis is provided by the observation that T-cell cytotoxicity directed against EBV-infected B cells is reduced in acute malaria (Whittle et al. 1984) and also by the observation that there is a marked increase in the incidence of Burkitt's lymphoma in HIV-positive individuals, in whom preexisting B-cell hyperplasiaoften oligoclonal- has been clearly documented (Pelicci, Knowles, Arlin et al. 1986). Lenoir and Bornkamm (1987) have proposed an alternative hypothesis in which they suggest that the translocation occurs first, but is itself insufficient for neoplastic transformation. This only occurs if a translocation-bearing cell is subsequently infected by EBV. In this case, EBV is postulated to have a vital role. This hypothesis does not explain the occurrence of translocation-positive, EBV-negative Burkitt's lymphoma. It is of interest that even after many years of in vitro culture, EBV-containing cell


lines derived from patients with infectious mononucleosis or from normal circulating lymphocytes-either transformed by EBV or arising "spontaneously" -do not develop 8;14 translocations (Jarvis et al. 1974), although aneuploidy and monoclonality may occur after lengthy periods of in vitro culture (Nilsson 1979). Thus there is no reason to believe that EBV directly influences the likelihood of a translocation developing. A large prospective epidemiologic study in the West Nile district in Uganda, as well as serial observations in a single child for a period of 18 months prior to the development of his tumor, have clearly established that conversion to EBV seropositivity occurs up to several years before the clinical development of Burkitt's lymphoma (de The 1979; de The et al. 1978; Magrath et al. 1975). The West Nile study also demonstrated that African children acquire EBV at an early age, 100 percent becoming EBV-seropositive by the age of 3 years (de The et al. 1975). This pattern differs considerably from that in socioeconomically privileged countries, where seroconversion occurs much later-frequently not until early adulthood (Henle and Henle 1979). Africans destined to develop Burkitt's lymphoma tend to have higher levels of antibodies against the viral capsid antigen (VeA) than do appropriate controls (de The 1979; de The et al. 1978). This may be due to an even higher-thanaverage (for African children) body pool of EBV-infected B cells. De The calculated that children with anti-VeA titers more than twice as high as the mean of the control population had a 30 times greater risk of developing Burkitt's lymphoma. This would seem to indicate either that EBV has some role in the pathogenesis of the tumor in Africa or that the higher anti-VeA titer reflects an immunologic abnormality which is relevant to the induction of the tumor. Regardless of the possible pathogenetic role for EBV, the nonrandom chromosomal translocations associated with Burkitt's lymphoma clearly set it apart from the polymorphic lymphoproliferative processes associated with congenital immunodeficiency or iatrogenic immunosuppression. The monoclonal nature of African Burkitt's lymphoma reflects the rarity of the occurrence of the necessary chromosomal translocation required to bring about the particular alteration in the expression of the c-myc

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gene which gives rise (possibly in conjunction with other genetic changes) to neoplastic growth. But since all tumors bear a chromosomal translocation, the question of why some are ass0ciated with EBV while the remainder are EBVnegative becomes paramount. It remains possible that EBV simply increases the probability of the development of Burkitt's lymphoma but is not essential for its development, as proposed by Klein. Recent studies at a molecular level, however, have provided new insights into this problem. It has become clear that, whereas at a cytogenetic level 8;14 chromosomal translocations are essentially identical, at a molecular level, several patterns of structural rearrangements can be discerned (Pelicci et al. 1986; Neri et al. 1988; Barriga et al. 1988). We can only conclude either that several different functional alterations in the expression of the c-myc gene can give rise to Burkitt's lymphoma or that a common functional effect (e.g., constitutive expression) can be brought about by several different structural changes. If this is so, it is possible that EBV is a necessary component of the neoplastic state in the presence of a subset of the observed structural changes, but is unnecessary in others. It is even possible that in some EBVcontaining tumors, viral genes have no pathogenetic role-the tumor cells could have been infected after neoplastic transformation. The outgrowth, in vitro, of EBV-positive tumor cell lines from EBV-negative tumors (Magrath et al. 1980) supports this possibility. In addition, one tumor has been described in which only a fraction of the cells (Pizzo, Magrath, and Jay 1981) were EBNA positive. However; it also appears that EBV, or infectious mononucleosis, does not predispose to Burkitt's lymphoma, since, otherwise, EBV-positive tumors rather than EBVnegative tumors would predominate even in nonendemic areas. In fact, in these areas at least 75 percent of patients with Burkitt's lymphoma possess EBV antibodies, even though the tumor cells lack EBV genomes. Thus, it is clear that the relationship of EBV to Burkitt's lymphoma is not simple, and further, that its role is likely to be resolved only by studies at a molecular level. The determination of the precise effects of EBV genes in various cell lines should also help to elucidate the relevance of the ability of EBV to transform B cells to its association with Burkitt's lymphoma. Recent progress in the understand-

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ing of the molecular genetics of EBV and of the c-myc gene has begun to open up new possibilities and pathways for research which promise finally to elucidate this problem.

Molecular Genetics of EBV Infection EBV infection of B lymphocytes results in their activation and immortalization. Since lytic infection (i.e., virus production) is associated with cell death, the viral genes relevant to transformation must be the so-called latent genes which are expressed in all EBV-containing cells. Such genes could affect the cell via transcriptional or posttranscriptional mechanisms. On balance, although it is possible that EBV influences the translation, posttranslational processing, or function of one or more cellular proteins, it seems more likely that the virus acts at a transcriptionallevel. This is because one of the main functions of the latently expressed genes is the control of other viral genes involved in episomal and viral replication. EBNA 1, for example, binds and activates a transcriptional enhancer (oriP) necessary for the replication and maintenance of circular viral molecules - the predominant form oflatent viral genomes (Reisman and Sugden 1986). It is quite probable that some of the EBV latent genes may transactivate cellular genes. Indeed, the ability of EBV to transform B lymphocytes probably stems from its ability to activate cellular genes. One of the latently expressed genes, EBNA 2, which is known to be necessary for the expression of a viral gene - the

latent membrane protein (LMP) - also induces the expression of the cell surface antigen, CD23 (Blast-2). The latter is expressed on activated cells and may be a growth factor receptor (Wang et al. 1987), while LMP is able to immortalize rat fibroblasts (Wang, Liebowitz, and Kieff 1985). It is likely that transformation is dependent upon the expression of growth factor receptors at the cell surface, and their activation by autosecreted growth factors-one of which may be the extracellular portion of CD23 itself (Swendeman and Thorley-Lawson 1987; Gordon et al. 1986). Some ofthe same genes responsible for the activation of B cells, however, among them LMP (Thorley-Lawson and Israelson 1987), provide foreign antigens recognizable by T cells. An apparent consequence of this is that cells transformed by EBV are also obligately recognized by immune effector cells. Abnormal proliferation of such cells can only occur in the presence of a defect in host regulatory mechanisms. This raises the question as to how Burkitt's lymphoma cells containing EBV can escape immunosurveillance. Interesting differences in the expression of the latent viral proteins have been described between Burkitt's lymphoma cells and EBV-transformed lymphoblastoid cell lines (Table 12.4). Prominent among these is the absence or weak expression of EBNA 2 and LMP in some EBVpositive Burkitt's lymphomas, although this is clearly not due to a mutation in the EBNA 2 coding region since the viruses from these tumors can readily transform other lymphocytes which then express EBNA 2 (Rowe et al. 1986;

Table 12.4 Viral gene products expressed in latently infected lymphoblastoid cell lines. Gene EBNA I EBNA2 EBNA3 EBNA5 LMP EBER small RNA's

Reading frame/region BKRFI BYRFI BLRF3/BERFI BAM HI WY BAM HI N

LL

BL

Function

+

+

+ + + +

+

Maintenance and replication of episomal virus via binding to ori-P Necessary for lymphocyte transformation. Induces CD23 and LMP Unknown Unknown Necessary for lymphocyte transformation Unknown. Not translated.

+

LL = lymphoblastoid cell line. BL = Burkitt's lymphoma. EBNA = EBV nuclear antigen. LMP = latent membrane protein. + indicates universal expression. - indicates lack of expression or down regulation in a subset of Burkitt's lymphomas.

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Rowe et al. 1987). Although the lack ofEBNA 2 expression is not a universal phenomenon in cell lines (few biopsies have been examined), these data clearly indicate a difference in the regulation of EBV genes in Burkitt's lymphoma cells compared with lymphoblastoid cell lines, where these antigens are invariably strongly expressed. Not surprisingly, the expression of the activation antigen CD23 parallels that ofEBNA 2 (Rowe et al. 1987)-indicating that this possible growth factor receptor, which could be critical to the transformation of B lymphocytes, may not be involved in the pathogenesis of Burkitt's lymphoma. Perhaps this is because Burkitt's lymphoma cells can proliferate independently of the growth factors required by EBV-transformed lymphoblastoid cell lines-as has been shown by in vitro experiments (Gordon et al. 1986; Gordon et al. 1985). One explanation for this is that the deregulation of c-myc eliminates, or markedly reduces, the requirement for growth factor binding to surface receptors, but alternatively, Burkitt's cells may synthesize adequate quantities of their own growth factor. Whether EBV itself participates in the deregulation of c-myc-possibly via transactivation by EBV latent genes-or whether it provides another, separate, element required for neoplastic transformation is not known. It remains possible that EBV has a direct role in Burkitt's lymphoma, although such a role could only be an absolute requirement in a subset of Burkitt's lymphoma. Preliminary information suggests that this subset may be definable on the basis of breakpoint location on chromosome 8, for the majority of cell lines or tumors associated with EBV lack chromosomal translocation breakpoints within the c-myc gene itself (Barriga et al. 1988). It is also possible, although speculative, that EBV is required in such tumors because of its effect on a mutated c-myc gene. This effect may differ sharply from its effect on a normal c-myc gene. An explanation of why some Burkitt's lymphomas are EBV-positive and others negative could be that some kinds of damage to the regulatory region of c-myc may require the presence of EBV in order to induce constitutive expression of c-myc, whereas other kinds of damage may be sufficient per se to induce constitutive expression. An alternative explanation is that EBV acts on one or more cellular genes other than c-myc, the modified function of

which may be required, in the presence of some kinds of EBV damage, to give rise to neoplastic growth. These considerations demonstrate that whatever the role of EBV in Burkitt's lymphoma, there are likely to be significant differences between its effects in a normal cell and its effects in a neoplastic B cell. Perhaps of critical importance in this regard is the difference in the expression of those EBV genes which account for the ability of T cells to specifically destroy virusinfected cells (operationally called LYDMA, for lymphocyte-defined membrane antigen). Clearly, the expression of such genes (which include EBNA's and LMP) is inconsistent with tumor development in an immunologically normal host. It is not surprising that LYDMA is frequently not expressed in Burkitt's lymphoma cell lines (Rowe et al. 1986). Similarly, down regulation of HLA antigens has also been described in some EBV-containing Burkitt's tumor cells which are also less sensitive to allospecific and nonspecific cytotoxicity (Rooney et al. 1986). Finally, LMP appears to activate leukocyte adhesion molecules, known to be involved in the process of immunologic recognition. Such molecules are expressed at much lower levels in many Burkitt's lymphomas (Rickinson, Gregory, and Young 1987). Perhaps these differences are the consequence of a selection process whereby only cells which are poorly recognized by immunocompetent cells can develop into tumor cells. As the tumor grows larger, the immune reaction may be overcome by sheer weight of numbers of tumor cells and by immunosuppression caused by the tumor burden, so that expression of LYDMA would no longer result in tumor rejection, thus accounting for the presence of LYDMA on some Burkitt's lymphomas. While the phenomenon may be more complex than this, the failure of expression of some latent EBV genes in Burkitt's lymphoma cells may be a requirement for tumorigenesis. This clearly differentiates the neoplastic state from the nonneoplastic.

Infectious Mononucleosis and Hodgkin's Disease A number of epidemiologic studies have linked infectious mononucleosis to Hodgkin's disease (Connelly and Anistine 1974; Rosdahl, Larsen,

162

and Clemmensen 1944; Munoz et al. 1978; Kvale, Hoiby, and Pederson 1979). Over 40,000 patients who were known to have had infectious mononucleosis (with a positive heterophile test) have been traced and the incidence of Hodgkin's disease ascertained. Some, but not all (Newell et al. 1973; Carter et al. 1977), of the studies have found a small excess (twofold to fourfold) in the frequency of Hodgkin's disease in these patients compared with the expected incidence, based on age and sex-specific incidence rates, for the general population. Even if we assume that there is an increased frequency of Hodgkin's disease in individuals who have previously had infectious mononucleosis, the nature of this association may well be indirect. Both Hodgkin's disease and infectious mononucleosis occur preferentially in higher socioeconomic groups, for example (McMahon 1966; Gutensohn and Cole 1981), and the possibility of an independent association on this basis arises. Not all patients with Hodgkin's disease possess antibodies to EBV [infectious mononucleosis following Hodgkin's disease has been reported (Davidson and Lessles 1977)]; and whereas some patients with Hodgkin's disease may have elevated antiVCA and anti-EBNA titres, this characteristic appears to be secondary to immunosuppression rather than indicating a direct association between Hodgkin's disease and EBV infection (Hesse et al. 1977; Mochanko et al. 1979). Neither EBV DNA nor EBNA has been detected in Hodgkin's tissue or cultured Reed-Sternberg cells (Nonoyama et al. 1974; Gallo and Geimann 1981). Most individuals acquire EBV silently, so that a direct association between Hodgkin's disease and EBV infection is very unlikely. It is possible, however, that individuals who develop infectious mononucleosis-presumably because their ability to deal with primary EBV infection is marginally less than that of those who do not develop the disease at the time of seroconversion - have a similar predisposition to Hodgkin's disease, because their immune reactivity is at the lower end of the spectrum for the normal population. The coexistence of infectious mononucleosis and Hodgkin's disease is very rare (Massey, Lane, and Imbriglia 1953; Kenis, Dustin, and Peltzer 1958), and evidence for a simultaneous occurrence rate greater than can be accounted for on the basis of chance alone has not been presented.

Ian Magrath

At the present time, the epidemiologic association between Hodgkin's disease and infectious mononucleosis is weak at best (relative incidence 1.8 when shared risk factors are controlled for, according to Gutensohn and Cole 1981). Available evidence provides no indication of a direct association, and it seems likely that the weak association may be entirely due to shared risk factors rather than shared etiology. At a biological level, EBV has been reported to be associated with some cases of histiocytic hyperplasia and hemophagocytosis (Frizzera 1987; Sullivan et al. 1985), but the relevance of this to the possible association with Hodgkin's disease, possibly a disease of dendritic reticular cells, remains unknown.

Intracellular versus Extracellular Disorders of Lymphoproliferation The disorders involving EBV-infected B cells provide an excellent paradigm for the examination of current concepts of malignant neoplasia. EBV infection may be subclinical, or in immunodeficient individuals, it may cause a fata1lymphoproliferative syndrome. It appears likely that some of the "neoplasms" of allograft recipients also arise as a direct consequence of failure to regulate such cells adequately, but such initially polyclonal processes can also become monoclonal (Hanto et al. 1982). Even monoclonality should not be considered a hallmark of true neoplasia, since in EBV-transformed cell lines cultured in vitro a single clone has usually become predominant within a year or so, and usually develops some degree of aneuploidy. The cells of African Burkitt's lymphoma are readily distinguished from such cells on the grounds of morphology, clonality, and karyotype. Even when EBV-transformed cell lines become aneuploid, they do not develop 8;14 translocations. However, it seems probable that underlying increased lymphoproliferation predisposes to the development of a chromosomal translocationsimply on the basis of an increase in the statistical likelihood that such an aberrant event will occur.. Such lymphoproliferation, at a subclinical level, probably underlies Burkitt's lymphoma arising in previously healthy African individuals, while more overt lymphoproliferation predisposes to Burkitt's lymphoma arising in

163

12. Infectious Mononucleosis and Malignant Neoplasia HIV -infected individuals and possibly in other forms of immunosuppression. These considerations lead to a simple classification of EBV-associated fatallymphoproliferative processes into two major categories: (1) those resulting from defects in immunoregulation, in which the proliferating cell type expresses antigens on its surface such that it would readily be dealt with by the host immune sys-

tern, if intact; and (2) those in which there is a genetic change in the cell such that a gene necessary for cell proliferation becomes constitutively expressed (Figure 12.1). At a more teleological level, certain features of the two main types of lymphoproliferation discussed above make intuitive sense. The narrow host range, ubiquity, and intimacy of the relationship of the herpesvirus with its host favor the

oI

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0-0-0-0 Fig. 12.1. Top left. An outline of the normal B-lymphocyte differentiation pathway. a: The first cell type committed to B-lymphocyte differentiation; i.e., there is immunoglobulin gene rearrangement and, shortly after, the development of cytoplasmic immunoglobulin (predominantly JI. chain) signifying the cell as a pre-B cell. b: the first cell to express surface immunoglobulin (lgM). c: Development of Fc, complement, and EBV receptors such that this and subsequent cells can be infected by EBV. d: A normal lymphocyte can be activated by antigen, mitogen, or EBV through the immunoblast stage (f) to a plasma cell (h). R represents cells (lymphocytes and macrophages) that normally suppress the process of B-cell activation and proliferation. Helper cells are not shown. Bottom left. Development of a lymphoproliferative process because of failure of regulatory cells. EBV-infected cells

appear to be the most likely cells to escape regulation. Differentiation is not necessarily affected, so that the lymphoproliferative process tends to be polymorphic as well as polyclonal, although there may be more restricted morphologic expression, and ultimately the process may become oligoclonal or monoclonal. Top right. EBV-negative Burkitt's lymphoma is probably the neoplastic counterpart of an early B cell that has not yet developed EBV receptors. A differentiation block occurring in a single clone may be the reason for accumulation of a specific cell type. Bottom right. EBV-positive Burkitt's lymphoma is probably the neoplastic counterpart of a B cell that has developed EBV receptors and hence may contain EBV DNA. Whether or not the EBV genome potentiates the development of this kind of tumor is not known.

164

development, on an evolutionary time scale, of minimally incapacitating consequences of virus infection; a uniformly fatal outcome would lead to the extinction of both species. The relationship between avian herpesviruses and poultry is in some regards less evolved than that of primate herpesviruses and their respective hosts. Not only may the natural host range be broader in poultry herpesviruses, but a fraction of the normal birds infected by MDHV may develop severe enough diseases to prohibit reproduction or cause death. Primate herpesviruses appear to cause minimal disease in their natural hosts (usually a single species), but in animals infected by a virus that would never be encountered under natural circumstances, a polyclonal, often fatal, lymphoproliferative process may result from virus infection. The development of monoclonal neoplasia is pathogenetically quite different. A rare genetic abnormality is required, which may even be a random event quite incidental to the normal host-virus relationship. In humans, it appears likely that virus genes may "cooperate" with genetic events in the causation of neoplasia. There is little doubt, for example, that EBV, whether directly or indirectly, influences genes involved in cellular proliferation, since it is able to induce cell transformation. It is possible that its effect on a structurally altered gene, for example, c-myc, differs from its effect on a normal gene. In this case it may provide one of perhaps several components required to cause neoplasia.

Clearly, the EBV-associated diseases provide a unique opportunity to explore the pathophysiologic processes at a molecular level in a broad spectrum of lymphoproliferative syndromes ranging from benign hyperplasia to malignant neoplasia. The acquisition of such information has a potential gain of major practical importance - the development of new approaches to the treatment of these diseases. In the case of genetically induced neoplasia in which EBV has a role, the identification of the molecular mechanisms could lead to approaches designed to reverse the biochemical changes, or to bypass them. Since EBV represents foreign genetic material, it provides the equivalent of a tumor-specific target for therapeutic approaches. While such approaches may seem, in the present state of our knowledge, to be fanciful, they at least provide added incentive to research directed toward the identification of pathogenetic mech-

Ian Magrath

anisms. Only the possession of such information will determine whether such approaches can eventually become a reality.

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12. Infectious Mononucleosis and Malignant Neoplasia Briggs, J., Hamilton, D.N.A., MacSween, R.N.M., et al. (1978). Infectious mononucleosis, herpes simplex infection, and diffuse lymphoma in a renal transplant recipient. Transplant 25:227 - 228. Briles, W.E., Stone, H.A., and Cole, R.K. (1977). Marek's disease: Effects of B histocompatibility alloalleles in resistant and susceptible chicken lines. Science 195:193-195. Britton, S., Andersson-Anvret, M., Gergely, P., et al. (1978). Epstein-Barr virus immunity and tissue distribution in a fatal case of infectious mononucleosis. N. Engl. J. Med. 298:89-92. Carter, C.D., Brown, T.M., Herbert, J.T., et al. ( 1977). Cancer incidence following infectious mononucleosis. Am. J. Epidemiol. 105:30-36. Cerilli, J., Rynasiewicz, J.J., Lemos, L.B., et al. (1977). Hodgkin's disease in human renal transplantation. Am. J. Surg. 133:182-184. Chaganti, R.S., Jhanwar, S.c., Koziner, B., et al. (1983). Specific translocations characterize Burkitt's-like lymphoma of homosexual men with the acquired immunodeficiency syndrome. Blood 61: 1265 -1268. Cheeseman, S.H., Henle, W., Rubin, R.H., et al. (1980). Ann. Int. Med. 93:39-42. Chu, E.W., and Rabson, A.S. (1972). Chimerism in lymphoid cell culture line derived from lymph node. J. Natl. Cancer Inst. 48:771- 775. Churchill, A.E., and Biggs, P.M. (1967). Agent of Marek's disease in tissue culture. Nature 215: 528-530. Connelly, R.R., and Anistine, B.W. (1974). A cohort study of cancer following infectious mononucleosis. Cancer Res. 34: 1172 - 1178. Crawford, D.H., Epstein, M.A., Achong, B.G., et al. (1979). Virological and immunological studies on a fatal case of infectious mononucleosis. J. Infect. 1:37-48. Crawford, D.H., Sweny, P., Edwards, J.M.B., et al. (1981). Lancet 1:10-13. Crawford, D.H., Thomas, J.A., Janossy, G., et al. (1980). Epstein-Barr virus nuclear antigen positive lymphoma after cyclosporin. A treatment in patients with renal allograft. Lancet 1:1355-1356. Davidson, R.J.L., and Lessles, S.E. (1977). Infectious mononucleosis in Hodgkin's disease. Acta Haemat. 57:152-155. Deinhardt, F., and Deinhardt, J. (1979). Comparative aspects: Oncogenic animal herpesviruses. In The Epstein-Barr Virus, M.A. Epstein and B.G. Achong (eds.). Berlin, Springer-Verlag, pp. 373-415. de The, G. (1979). Demographic studies implicating the virus in the causation of Burkitt's lymphoma: Prospects for nasopharyngeal carcinoma. In The Epstein-Barr Virus, M.A. Epstein and B.G. Achong (eds.). Berlin, Springer-Verlag, pp. 417-437. de The, G., Day, N.E., Geser, A., et al. (1975). Sero-

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