Malignant Lymphomas Associated with Immunodeficiency States*

Transcription

1 ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 20, No. 3 Copyright 1990, Institute for Clinical Science, Inc. Malignant Lymphomas Associated with Immunodeficiency States* JAMES J. BIEM ER, M.D. St. Joseph s Hospital and University of South Florida, College of Medicine, Tampa, FL ABSTRACT An increased incidence of malignant lymphomas is common to all types of im m unodeficient patients w hether they be of the natural or constitutionally occurring type, acquired as in acquired imm unodeficiency syndrome (AIDS) or of iatrogenic origin as in organ transplantation. Although there is some degree of heterogenity, the most characteristic feature of these immunodeficient states is alteration oft-cell cytotoxic function. The m alignant lymphomas show a variety of relatively common features, notably: rapid onset following the appearance of the immunodeficient state, a high degree of clinical aggressiveness, and a tendency to present in extranodal sites, particularly the central nervous system (CNS) and gastrointestinal tract. The tumors are almost invariably of B-lymphocytic cell origin and while the histologic classifications reflect some diversity, the vast m ajority of tumors are described as Burkitt-like or diffuse large cell type. There appears to be a high degree of correlation with a preceding fulm inent Epstein-Barr virus (EBV) infection resulting in marked B-cell lymphoproliferation in the absence of effective T-cell control. Initially, the B-cell proliferation is clearly polyclonal and reactive in nature, although as tim e evolves, there appears to be selection of oligoclonal and even m onoclonal cell populations. Such cells are latently infected with EBV and may express EBV nuclear protein two and latent m em brane protein, which are characteristically seen in proliferating B-lym phocytes in response to growth transform ation by EBV. W hile desoxyribonucleic acid (DNA) probes may continue to dem onstrate m ultiple lymphoid clonal populations, it is hypothesized that the hyperproliferative state favors genetic alterations which select out a single malignant clone. This transformed clone is evidenced by expression of a translocated, activated c-myc oncogene and decreased evidence of EBV nuclear protein two and latent m em brane protein, that is, characteristics of Burkitt s lymphoma. O ther * Send reprint requests to: James J. Biemer, M.D., St. Joseph s Hospital, 3001 West Buffalo Avenue, Tampa, FL /90/ $02.00 Institute for Clinical Science, Inc.

2 1 7 6 BIEMER large cell malignant lymphoma phenotypes may show similar findings. W hile most studies have continued to suggest that EBV plays a key role in the developm ent of non-hodgkin s lymphoma (NHL) of AIDS patients, some recent studies have suggested a less dom inant role. Therefore, further exploration of the world of molecular biology will be needed to dem onstrate w hether other factors, namely additional viruses and/or oncogenes play a similar or significant role in the lymphomas of immunodeficient patients. Introduction Increasingly, a new diagnosis of malignant lymphoma (ML) now evokes a secondary response, that is, a question as to w hether or not there is another concurre n t pathologic process w hich m ight have induced or allowed that malignant proliferation to occur. W ith even greater frequency, one finds an affirm ative answer to that question, particularly if the ML is of the non-hodgkin s variety, and of the high grade B urkitt-like or large cell immunoblastic type. The comm on th re a d, it appears, is th a t th e affected patient has some obvious or perhaps subtle undetected im pairm ent of im m une surveillance. W hile an association betw een ML and a state of altered immunity has been long suspected and was emphasized by Lukes and Collins53 in their early work relating lymphomas to im m unological functions, the m ore recen t stim uli evoked by w idespread organ transplantation programs and the acquired im m une deficiency syndrome (AIDS) have brought all of this into sharper focus. It is to the broad area of lym phom agenesis in im m une im paired patients that the following review is directed. Lymphomas Associated with Transplantation Perhaps no other area of neoplasia associated with im m unodeficiency has b een studied as intensely as that of organ tra n sp la n tatio n. T he rep o rts of Penn62,63,64 relating to the cum ulative experiences of the Cincinnati Transplant Tumor Registry (CTTR) have shown that after cancer of th e skin and lips, lym phom as co n stitu te th e single largest group of neoplasms. W hile the average tim e from transplantation to diagnosis of all neoplasms was 58 months, it was only 23 m onths w ith Kaposi s sarcoma, 36 months with lymphomas, and 90 months w ith carcinomas of the vulva and p erin e u m. N o n -H o d g k in s ly m p h o m as (NHL) were reported following all types of transplantation and all types of immunosuppression. The neoplasm s w ere mostly large cell lymphomas overwhelmingly presenting in extranodal locations. Most frequently that site was the central nervous system (CNS), raising speculation that these tum ors may be free of norm al im m une su rv eillan ce in the immunologically priviledged sanctuary of the CNS, thus allowing the tum or cells to p ro life rate freely. T he lym phomas also showed a striking tendency to involve the allograph, and there have b e e n several in te restin g exam ples of tum or growth at the site of injection of an tith y m o cy te g lo b u lin.85 L eukem ic m an ifestatio n s w e re in fre q u e n t. In almost all instances, clinical follow-up dem onstrated an aggressive course with a poor prognosis. W hile most of the accum ulated data reflects the experience w ith renal transplants, th ere have been early reports that N H L was even m ore frequent fol

3 MALIGNANT LYMPHOMAS ASSOCIATED WITH IMMUNODEFICIENCY STATES lowing cardiac transplantation.60,63 In 1978, Anderson e t al2 reported an incidence of 16 p ercen t N H L in patients w ith p reced in g cardiom yopathy who rec e iv e d h e a rt tra n sp la n ts and high doses of azo th io p rin e, stero id s and antithymocyte globulin. In 1981, Bieber et al7 reported that the Stanford experience now stood at an incidence of 15.3 percent tum ors, while Lanza et al49 in 1983, reported the South African precyclosporine experience of 10 p e rc e n t malignant neoplasm s with heart transplants, i.e., twice their historical experience w ith renal transplants. It was sugg e ste d at th e tim e th a t th is h ig h er incidence of neoplasms following cardiac transplants might be due to the greater degree to immunosuppression required for h e a rt tra n sp la n ts, or because of greater susceptibility to lymphoma with patients who received transplants for idiopathic cardiomyopathy.2 The advent of cyclosporine-a in 1980 and its use as a potent immunosuppressant allowed a trem endous ressurgence of organ tran sp lan tatio n, particularly heart transplants.38 Almost immediately, reports of neoplasm s began to appear giving rise to fears th at cyclosporine m ight pose a risk tow ard neoplasia greater than that which was already associated w ith other im m unosuppressive agents.7,20 This fear was heightened by the revelation th a t cyclosporine m arkedly facilitates th e outgrow th of B- ly m p h o b lasto id cell lines from both Epstein-B arr virus (EBV) infected and n o n -in fe c te d ly m p h o c y te s of EBV im m une donors in vitro. The results indicated that cyclosporine interfered w ith the lym phocytes normally responsible for m aintaining the lifelong carrier state initiated by primary infection with EBV, allowing outgrow th of the persistently infected cells in the peripheral blood.10 Craw ford e t al20 also showed that cyclosporine prevented a cytotoxic effect to EBV infected B-cells in vitro and suggested that this suppression of m em ory T-cells may contribute to the high incidence of lymphomas w ith cyclosporine. By 1983, th e e x p e rie n c e o f th e CTTR63 included nonmelanomatous skin carcinomas, NHL s and a variety of other carcinom as. The N H L s ftiade up the bulk of the tumors, and, in fact, if the skin cancers w ere excluded, th e lym phom as constituted 81 percent of the reported neoplasms. O f additional concern was the observation that these neoplasms now appeared at an average of only six m onths following initiation of transplant with cyclosporine-a therapy as opposed to an average of thirty-six m onths in th e precyclosporine experience. W hile th e re c o n tin u e d to be reports of N H L appearing in patients whose blood levels of cyclosporine had been carefully m onitored,41 there was a growing consensus that cyclosporine-a was no m ore tum orgenic th an o th er im m unosuppressive agents if ap p ro p r i a t e b lo o d le v e ls w e r e m a i n tain e d.17,22,34,84 W ith suggestions that th e n u m ero u s early rep o rts of lym phomas were related to excessive doses of cyclosporine-a, careful m onitoring of blood trough levels was recom m ended to m aintain a range of 250 to 1000 ng per m L.5,6 These conclusions were supported by Brumbaugh et al12 whose analysis of six lym phom as among 75 surviving h eart and heart-lung transplants suggested a q u a n tita tiv e re la tio n s h ip b e tw e e n degree of immunosuppression and risk of lymphoma. Mean cyclosporine-a levels over the first three m onths after transplant and during periods of T-eell suppression appeared to be the most im porta n t p red icto rs of th e occurrence of lymphomas in this group. No significant association betw een EBV antibody titer seroconversion and lymphoma could be dem onstrated; however, th e com bination of a four-fold rise in tite r to th e

4 1 7 8 BIEMER restricted early antigen of EBV and a high m ean cyclosporine-a level m ight predict an increased risk for lymphomas. Brum baugh and co-workers contended that th e occurrence of lym phom a related to the quantitative degree of im m u nosuppression and the im portance of identifying and m aintaining m inim al effective levels of im m unosuppression was underscored. Cyclosporine A num ber of recent reviews of cyclosporine have su m m arized its use in tra n sp la n tatio n and th e c u rre n t u n d e rs ta n d in g o f its m e c h a n ism s of action.4,17,34,43 Cyclosporine-A, a cyclic endecapeptide with a molecular weight of 1203, is a metabolite of soil fungi. Its immunosuppressive action is related to its effect on T-lymphocytes with B-lymphocytes, m acrophages, granulocytes, and n atu ral killer cell functions un altered. The drug is not myelosuppressive and in vitro growth of bone marrowderived myeloid, erythroid, and B-lymphoid cells is not impaired. Cyclosporine inhibits T -helper cell function required for B-cell activation. That it does not act on B-cells directly has been shown by the fact that agents such as EBV, which directly stim ulate B-cell proliferation, are not inhibited by the presence of cyclosporine. Further, while the administration of cyclosporine at the tim e of antigenic challenge suppresses subsequent antibody production, previously stim ulated ongoing antibody responses are not influenced. Cyclosporine im pairs production of Interleukin-2 (IL-2) by resting as well as activated T-helper cells and also impairs the release of Interleukin-1 (IL-1) by m acrophages presum ably secondary to its effect on T -helper cells. Im paired IL-2 production leads to abrogation of proliferation and developm ent of cytotoxic T-cells. At the same time, there is a relative sparing of suppressor T-cell subpopulations, cyclosporine seem ingly creating an environm ent which uniquely favors production of suppressor T-cell inducing lymphokines. Tim ing of cyclosporine administration is important with maximal benefit generally obtained with initiation of treatm ent at the time of primary antigen exposure, that is, at the tim e of transplantation. In sum m ary, cyclosporine directly in h ib its ly m p h o k in e p ro d u c tio n by b lockage of T -h e lp e r cell fu n ctio n s required for B-cell activation, cytolytic T-cell generation, and further expansion of T-helper/inducer subpopulations. At the same tim e, th ere appears to be a selective expansion of the suppressor T- cell population which may play a role in allograph tolerance. E pstein-b arr Virus (EBV) The role of EBV in lymphomagenesis has received extensive discussion particularly in th e m ilieux of im m unodeficiency. It is appropriate at this point to summ arize the spectrum of clinical m anifestations which are known to occur w ith EBV, which infects virtually all hum an beings by adulthood and m aintains lifetime latency.55,65,66,69 Children, p articularly those of low er socioeconom ic status, are frequently infected with EBV and run a silent or mild nonspecific ferile clinical course. In such cases, before the age of five years, the h eterophil antibody test is frequently n egative and th e diagnosis can be esta b lish e d only by specific EBV a n tibody detection. A dolescent EBV infections, on the other hand, give rise to the typical acute in fectio u s m o n o n u cleosis (IM ) sy n drom e, w ith its signs and sym ptom s largely due to explosive polyclonal T and B lymphocyte proliferation. Thus, one sees fever, pharyngitis, fatigue, cervical lymphadenopathy, hepatosplenomegaly,

5 MALIGNANT LYMPHOMAS ASSOCIATED WITH IMMUNODEFICIENCY STATES atypical lymphocytosis, and polyclonal elevation of serum im m unoglobulins manifesting the immunological struggle betw een the host and the EBV infective agent. The virus infects B-cells through the C3d receptor and may also infect o ro p h ary n g e a l cells, a lth o u g h EBV receptors have not been conclusively dem onstrated on these cells. Defenses against EBV infection include mucus, epithelial barriers, elaboration of interferon by infected cells, natural killer cell a c tiv ity, H L A -re s tric te d and n o n restricted cytotoxic T-cells, and an array of neutralizing antibodies and others that arm antibody-dependent cellular cytotoxicity. Identification of the atypical lym phocytes in the p e rip h eral blood reveal num erous cells bearing the suppressor T-cell phenotype. Concurrently, an inverted T-helper/Tsuppressor lymphocyte ratio is seen, and with it, transient anergy occurs. During acute IM, one B-cell per 104 circulating lym phocytes is in fected w ith EBV, whereas during the latent period, one B- cell per 106 circulating cells is infected.73 T his le v e l a p p a re n tly p e rs is ts in d efin itely. T h u s, m u ltip le im m une responses, especially cytotoxic T-cells, progressively inhibit the EBV induced B-cell proliferation. The morphology of the lymphoproliferative response seen in ly m p h n o d e s an d to n sils has b e e n described by Childs et al.13 The findings include extensive im m unoblastic prolife ra tio n in sh e e ts and n o d u les w ith marked cytologic atypia that sometimes simulates NHL in addition to the better known tendency to form R eed-sternberg-like cells, which make differentiation from H odgkin s disease (H D ) a potential problem. The hyperglobulinem ia seen in a typical case of IM is polyclonal. Heterophilic antib o d ies are of th e IgM type and appear in the serum of approximately 80 percent of patients with the EBV caused mononucleosis syndrom e. Cytomegalovirus (CMV) infections account for seven percent, Toxoplasma Gondii less than one percent, and recently human immunodeficiency virus (HIV) infections have also been implicated as a less frequent cause of the mononucleosis syndrom e.79 The remainder, however, are largely due to EBV and can be proven only by EBV specific antibody studies. After an acute EBV infection, an array of antibodies appears in the patient s serum, including IgM antibodies to viral capsid antigen (VCA) and early antigen (EA), followed by IgG antibodies to VCA and EA and la te -a p p e a rin g (a fte r one to th re e months) antibodies to EBV nuclear-associated antigen (EBNA). Lifelong p resence of IgG an tib o d ies to VCA and EBNA reflect latency of the virus. Acute IM is sometimes followed by an apparent chronic form of the disease characterized by pharyngitis, lymphadenopathy, fever, headache, malaise, neuropsychologic difficulties, and less comm only by arth ritis, skin rashes, and h e p a to sp le n o m e g a ly. T h e p a tie n ts exhibit an increased IgG antibody titer to the EA and VCA, since EBNA may be low. Henle et al39 have investigated the use of five distinct EBNA s which may be useful in the diagnosis of chronic IM, but conclude that a causal role of the virus in these cases remains doubtful. M e rlin 55 s im ila rly c o n c lu d e s th a t because of their nonspecificity, antibody tests in chronic IM may m islead physicians to overlook o th e r significant d ise a s e s in p a tie n ts w ith c h ro n ic vague symptoms. In the im m unocom prom ised individual, however, the spectrum of clinical manifestations to new EBV infections is much more diverse including severe or fatal IM with marked polyclonal B-cell lym phopro life ration, m alignant B-cell lymphomas, virus-associated hemophagocytic syndrom e, aplastic anem ia, o r a c q u ire d h y p o g a m m a g lo b u lin e m ia E pstein-b arr virus is a truly

6 1 8 0 BIEMER T-cell independent B-cell activator. In vitro, it infects and immortalizes B-lymphocytes allowing continuous replication and subculturing of the infected cells when they are provided with nutrients. In the presence of T-lymphocytes from immune donors, however, activation is usually curtailed after 14 to 28 days with rem oval of virally infected B-cells by activated T-lymphocytes generated by secondary in vitro immunization.56 If T- cells are elim inated or if seronegative individuals are used, perm anent lymphoblastoid B-cell lines can be obtained. Additionally, if cyclosporine is added to cultures which include EBV im m une individuals, perm anent B-cell lines are now formed indicating that cyclosporine inactivates T-cells normally responsible for the lim itation of B-lymphoblastoid cell line formation.9 W hether or not an in vivo equivalent of this immortalization process is responsible for the persistence of EBV-infected B-lymphocytes in vivo, or w hether or not the virus passes from infected to uninfected B-lymphocytes in vivo is difficult to assess.55 In any event, the studies of EBV-induced lymphoproliferation in the X-linked lymphoproliferative syndrom e (XLPS) by Purtillo and associates,65,66,68 81 in tam arin s,16 that associated with immunosuppression of organ transplantation, ,37,89 and m ore recently in AIDS35,45,70 are now most compelling. N ature of the L ym phoproliferative Process The nature of the lymphoproliferative process associated w ith im m unosuppression has been a subject of great controversy. C learly, th e re are polyclonal in fla m m a to ry ly m p h o p ro life ra tiv e responses to EBV infection or reactivation w here the clinical course and m orphological p ictu re are truly reactive ra th e r than neoplastic. Many of these examples have been referred to as polym orphic diffuse B -cell h y p erp lasia, p se u d o ly m p h o m a s, polyclonal ly m phom as, or virally induced lym phoproliferative disorders. In support of this, many of the cells of these proliferations have been shown to contain EBV. In addition, analysis of surface markers in such lym phoid proliferations, has fre q u e n tly d e m o n s tra te d a m ix tu re of im m unoglobulins supporting th e polyclonal n a tu re of the proliferation. In m any o th er instances, the separation b etw een reactive and neoplastic has been less clear. It has been shown that typical surface or cytoplasmic immunoglobulin markers could not be dem onstrated in many of these lymphoproliferative tumors, and so questions relative to their mono or p o ly clo n a lity have re m a in e d u n a n sw ered. The clinical course of these questionable cases has, however, been almost invariably clinically malignant so long as the patient rem ained in an im m unosuppressed state. F u rth e r support for the potential reversibility and less than full malignant potential of these lym phom as was p re s e n te d by th e report of 17 post-transplant lymphomas (kidney, liver, heart, and heart-lung), in 1984 by Starzl et al.78 Fifteen of the 17 show ed serological evidence of active EBV infection, seven of eight tested by DNA hybridization contained EBV genom es, 15 w ere classified as N H L (11 large noncleaved cell, three im m unoblastic, one unclassified), and two were called plasmacytoid B-cell hyperplasia. Clonality was assessed by kappa-lambda light chain analysis and show ed th at three w ere monoclonal, six polyclonal, and the rem ainder were mixed or indeterm inant. They went on to demonstrate that drastic reduction or discontinuance of im m unosuppression resulted in resolution of these tumors whereas no other form of therapy altered the lethal course

7 MALIGNANT LYMPHOMAS ASSOCIATED WITH IMMUNODEFICIENCY STATES 181 of these neoplasms if that simple course w ere not taken. They w en t on to conclude that the m uch publicized cyclosp o rin e lym phom as w ere relativ ely innocuous if appropriately treated. Prior to this report, Hanto et al37 had reported the use of Acyclovir in a single case of an EBV induced B-cell lym phom a following renal transplantation. That lym phoproliferative lesion which was originally referred to as polymorphic diffuse B-cell hyperplasia m orphologically and polyclonal by immunoglobulin m arker studies, underw ent regression on two occasions during treatm ent with Acyclovir. Only on the final recurrence when the tum or was now shown to be monoclonal, did it fail to respond. The data were regarded as supportive of the hypotheses that these polyclonal B-cell populations may evolve into monoclonal tum ors w ith enhanced m alignant potential that is no longer responsive to antiviral therapy. U tilizing clonal analysis based on immunoglobulin gene rearrangem ents, Cleary e t al14 reached a different conclusion, that is, that these lymphoproliferative lesions w ere neoplastic at the earliest stages of detectable disease. They rep o rted 10 patients w ith lym phoproliferative disease w hose original diagnoses w ere equally divided betw een cardiomyopathy and atherosclerotic coronary artery disease, and whose im m u n o su p p re ssio n was e q u a lly d iv id e d betw een those who did receive cyclosporine and th o se w ho did not. All tum ors w ere diffuse large cell lym phom as, w ith eight of 10 specifically classified as immunoblastic lymphomas. All but one case contained genomes of EBV, ranging from 5 to 20 genom e equivalents per cell as determ ined by D N A -hybridization. All tum ors w ere positive for one or more B-cell markers (la, B l, T015), b u t only one case had detectable surface or cytoplasmic im m u n o g lo b u lin. H o w e v e r, e v e ry case showed clonal patterns of immunoglobulin-gene rearrangem ent for at least one of the th re e im m unoglobulin chains, indicating that a m ajor portion of the biopsy specim en in each case was composed of a single clone of B-lymphocytes. The authors14 questioned w hether or not the lym phom as they encountered w ith cardiac transplantation w ere differe n t in th e ir earlier m anifestations of monoclonality than the other reported p o ly clo n al lesio n s w hich had b e e n largely associated with renal transplants. Clearly, however, in view of the frequent lack of cytoplasmic or surface im m unoglobulin markers in all of these tumors, conclusions of th eir mono or polyclonality based upon those studies m ust be held in question when compared to the m ore sen sitiv e gen e rearra n g e m e n t assessm ents of clonality using DNA probes. In view of the dem onstrated monoclonality of these lesions, Cleary et al14 advocated an tin eo p lastic therapy, i.e., radiation and chemotherapy at the earliest possible tim e, perhaps in com bination w ith reduced im m unosuppression. The possible role of Acyclovir was regarded as unclear although its benefit a fte r d e m o n s tra te d m o n o clo n a lity seem ed unlikely. L ater in 1984, C leary and Sklar15 reported on five patients whose lym phoproliferative lesions associated with cardiac transplants had been sam pled at several sites or at various times. Surprisingly, each site when analysed for clonal im m unoglobulin gene rearrangem ents for each of the three immunoglobulin gene loci, showed monoclonal but different patterns indicating that the tumors were, in fact, multiclonal or oligoclonal in nature. W hile instances of biclonal sp o n ta n e o u s lym phom as have b e e n described,77 the phenom enon is apparently unusual although perhaps m ore frequent than originally thought.

8 1 8 2 BIEMER The reason for the high incidence of m u lticlo n ality in th e lym phom as of im m unosuppressed transplant patients was thought to be connected with the presence of the EBV in each of these tum ors. E pstein-b arr virus, which is associated with Burkitt s lymphoma and nasopharyngeal carcinoma, is also known to im m ortalize cultured lym phoblasts and to induce experim ental lym phoid tum ors in tam arin s.16 Im m unosuppressive therapy reactivates the latent virus, and, once reactivated, this virus may act as a continuously p rese n t oncogenic agent within these patients. In addition, absence of norm al im m unological surveillance in immunosuppressed patients probably perm its unrestricted dissem i nation of EBV throughout the body and prevents norm al inhibition of newly arising clones of cells. S upport for the importance of defective immunity in the d e v e lo p m e n t of lym phom as am ong im m unosuppressed patients comes from accounts of well docum ented regression of lymphoid tumors in transplant recipients when immunosuppressive therapy is reduced.78 M ultifocal EBV induced m alignant lymphomas produced in tamarins show similar evidence of arising from different B-cell clones.16 These tumors are large cell lym phom as containing m ultiple copies of the EBV genome. Hybridization of tum or DNA with immunoglobulin gene probes reveals th at each lymphoma is oligo or monoclonal in origin and th at individual tum ors from the same animal arise from different B-cell clones. If EBV causes th e tum ors, as seem s beyond question, then it m ust induce m ultiple transformation events in every animal, each event giving rise to separate and distinct clonally derived proliferations in the various organs. The authors felt th a t id en tical processes appear to induce the EBV-associated tum or m asses of im m unosuppressed lymphoma patients, and these, like those of the tam arins, are distinct from polyclonal processes w hen stu d ie d by re fined genetic m ethods for d e te rm in ing clonality. M ore recently, Young et al87 have studied EBV nuclear and laten t m em b ran e proteins which are characteristically expressed in B-lymphocytes proliferating in vitro in response to growth transform ation by EBV. These two proteins are thought to be effectors of lymphocyte growth since they increase the expression of B-lymphocyte activation (C D 23) and c e ll-a d h e sio n (LFA 3 & ICAN1) molecules in vitro. The authors dem onstrated that these two EBV proteins and their associated B-lymphocyte activation or adhesion m olecules are expressed in the infiltrating B-lymphocytes in immunocom prom ised patients with EBV lymphoproliferative disease (five bone m arrow transplant patients and one with AIDS with dem onstrated monoclonal lymphoid proliferation). This p attern is different from B urkitt s lymphom a (BL) w here these proteins and cellular m arkers are down regulated possibly because th e lym phom a cell has evolved through a process that selects for a more malignant phenotype. Presence of these m arkers and absence of the c-myc translocation (characteristically positive in BL), m ight serve, th e authors thought, to separate th e early EBV drive lym phoproliferation from BL w hile it m ight still be am enable to imm unotherapy. T he overw helm ing d ata have now shown that the NHL s associated with im m unosuppression of transplantation re p re s e n t B -lym phocyte expansions which, when tested, almost invariably contain EBV genomes and are presum ably driven to a neoplastic state by that virus. Several exceptions to these generalities have recently been reported. G arvin et al33 describe a 32-year old recipient of a renal graft, who after 10 years of im m unosuppression w ith azathioprine

9 MALIGNANT LYMPHOMAS ASSOCIATED WITH IMMUNODEFICIENCY STATES 183 (AZT) and m ethylprednisolone, develo p e d a m alig n a n t lym phom a of th e m ature T-cell type. The tumor cells w ere positive for the Pan-T-cell markers CD3 and CD2, but were negative for the subset markers CD4 and CD8. The clonal nature of the T-cells was confirmed by dem onstrating rearrangem ent of the T- cell rec e p to r gene by DNA hybridization. W hile serological studies for human T-cell leukemia/lymphoma virus (HTLV- I), and human immunodeficiency virus (HIV) were negative, studies related to EBV w ere apparently not perform ed. Clearly, however, this reportedly first exam ple of a T -cell lym phom a w ith chronic im m unosuppression illustrates th a t o c c a sio n a l n e o p la sm s can b e expected to occur from the suppressed spontaneous T-cell population. T hree additional cases of T-cell lym phomas also reported in 1988 by Jones et al42 b e a r attention. W hile the background of these patients was different in that they w ere not transplant patients and had no evidence of congenital or acquired im m unodeficiency, the lym phom as occurred in association w ith chronic EBV infections m anifested by extraordinarily high titers of E pstein Barr VC A and EA, with low titers to EBNA. M arker studies on the tumors dem onstrated an activated T-cell phenotype (T4, T il, IA positive), and the EBV genom e was found by in situ hybridization in each of the tumors. The DNA digests from two p a tie n ts show ed a monoclonal proliferation of T-cells d e te r m ined on the basis of uniform T-cell receptor gene rearrangem ents and a single band for the joined term ini of the EBV genome. Since infection of the B- cell lym phoid and epithelial cells by EBV is putatively mediated by the C3d (CR2) receptor and since that receptor is found on developing T-cells, the authors suggested that EBV could have infected th e T -cells w hen th a t re c e p to r was p resent (two of their tumors expressed th e C3d recep to r). T hey co n clu d ed th a t EBV may occasionally infect T- cells and contribute to T-cell lym phomas in selected p atien ts w ith severe EBV infections. Acquired Immunodeficiency Syndrome (AIDS) First recognized in 1981, AIDS cases have been increasingly recognized to be at risk for d evelopm ent of m alignant n e o p la sm s W hile Kaposi s sarcoma clearly makes up the majority of such neoplasms, N H L represents the second largest group,28 and its representation may actually be greater than originally suspected following the revised definition of AIDS by the C enter for Disease Control (CDC) to include high grade B-lymphomas in and eventually broadened in These lymphomas have similarly shown a high incidence of extranodal growth with the dom inant sites of presentation rep re senting the CNS, bone marrow, and gastrointestinal tract. Histologically, these n e o p la sm s a re la rg e ly h ig h g ra d e, described as Burkitt-like or immunoblastic lymphomas. Lesser num bers of cases have been described as follicular center cell types of interm ediate grade, while still smaller num bers of low grade lymphomas have been reported. The tumors are almost invariably of B-lymphocyte origin,27,44 although the three cases of chronic lymphocytic leukemia included by Knowles et al44 followed an indolent clinical course and proved to be of suppressor/cytotoxic T-cell subset origin. The relationship of EBV to lymphomagenesis in AIDS has been extensively s tu d ie d ,47 67,68 80 In an early study of 25 homosexual men in 1984 by Crawford et al,21 all were found to be positive for Epstein-B arr VCA, 11 had antibodies to the EA, and 13 of 17 were excreting virus in saliva. The EBNA was not detected in activated im m unoglobu

10 184 BIEMER lin producing B-cells of the peripheral blood nor in lymph node biospies of five patients. It was concluded that while homosexual m en have a high incidence of infection with EBV, the virus was not the cause of the polyclonal activation of B -cells in th e p e rip h e ra l blood nor implicated in the etiology of the lymphadem opathy found in those m en. More recently, several w ell-studied cases of NHL in AIDS patients suggest that EBV does indeed have a causative role in the lymphomagenesis.35,70 Groopman et al35 describe a case of a diffuse large cell lym phom a associated w ith AIDS in a homosexual male. That lymphom a was shown to be of monoclonal B-cell origin by immunoglobulin and B-cell receptor gene studies. Similar to cases of Burkitt s lymphoma unrelated to AIDS, the tum or also had DNA sequences that hybridized to EBV-specific probes, and d em onstrated evidence of c-myc oncogene rearrangem ent. The HIV sequences w ere not detected in the malignant B-cells. T h e ir fin d in g s su g g e ste d th a t EBV potentiates the developm ent of B-cell neoplasia, while HIV sets the im m unosuppressive milieux, but does not appear to infect B-cells directly. The report in 1987 by Rechavi et al70 of two hemophiliac brothers with AIDS and Burkitt s leukem ia is very similar. Their tum or DNA was negative for HIVrelated sequences w hen analyzed by Southern blot using a cloned HIV probe, and, on the other hand, was positive for EBV-specific sequences by hybridization with an EBV probe. Rearranged c-myc genes w ere also dem onstrated in both tumors. In addition, Birx et al11 studied patients with AIDS and AIDS-related disorders showing that these patients have a profound defect of T-cell im m u nity to EBV and have abnormally high n u m b e r s o f E B V -in fe c te d B -c e ll in circulation w hich may predispose th em to th e d e v e lo p m e n t of EBVcontaining lymphomas. In conclusion to th eir study of 105 ly m p h o m as a sso c ia te d w ith A ID S, Knowles et al44,46 hypothesized that lymphom agenesis in AIDS is a m ultistep process in which the oligoclonal B-cell expansions frequently occurring in AIDS re la te d com plex (ARC) h y p erp lastic lymphadenopathy represent a pre-malignant condition for B-NHL. The state of HIV induced im m unosuppression with continuous and massive EBV infection in ARC favors the expansion of m ultiple EBV-transformed B-cell clones increasing the probability of occurrence of additional genetic alterations, i.e., translocation of the c-m yc gene, resu ltin g in malignant transformation of the B-cells. In a separate study,61 it appeared that c-m yc oncogene translocation and/or rearrangem ents w ere virtually a necessary pathogenic feature of AID S-associated B-NHL. Most notably, a single rearran g ed c-m yc band was found in AIDS-associated NHL, while none was d e te c ta b le in th e A ID S -a sso c ia te d ly m p h a d e n o p ath y sy n d ro m e (LAS). These findings suggested that while both B-N H L and LAS nodes contain m ultiple clonal B-cell expansions, only one clone, and only in B-NHL were c-myc genes rearranged. In support of this hypothesis, Lom bardi, et al52 have recently shown that the introduction of an activated c-myc gene into EBV-infected lym phoblasts from patients with AIDS leads to their malignant conversion. In a later study rep o rted in 1988, Subar et al80 questioned their earlier data which supported the almost invariable implication of EBV in NHL s associated with AIDS. C-myc gene rearrangem ents analogus to those observed in sporadic Burkitt lymphomas w ere detected in 12 of 16 cases while only six of the 16 cases had detectable EBV sequences and proteins. None contain ed bcl-2 rearran g em en ts or contained HTLV-I sequences. It was concluded that despite the frequent and

11 MALIGNANT LYMPHOMAS ASSOCIATED WITH IMMUNODEFICIENCY STATES often massive EBV infections associated w ith AIDS, only one-third of AIDS-associated B-cell NHL s had detectable EBV sequences and proteins. Subar and cow o rk ers80 s u b s e q u e n tly q u e stio n e d w h e th e r o r n o t EB V w as d ire c tly involved in the lymphonagenesis of most A ID S -associated N H L s. A group of in v e s tig a to r s 47 h a s a lso r e c e n tly described three cases of AIDS-associated B-NHL s which lacked c-myc gene rearrangements and contained EBV proteins and/or sequences, thus differing from most of their other AIDS-associated lymphomas. Clearly, EBV and c-myc oncogene rearrangem ents are implicated in AIDS-associated B-NHL, b ut w hether or not these factors are operative in most cases as originally suggested or only in a minority of them, must await further definition by additional studies. Burkitt s Lymphoma (BL) Burkitt s lymphoma has been long recognized as a B-NHL of unique morphological appearance, and, prior to its assoc ia tio n w ith o b v io u s cases o f host im m u n o d eficien cy, it was typically divided into the endem ic and sporadic varieties. Endem ic BL affects children and young adults within the malaria belts of equatorial Africa and New Guinea, and has been consistently associated with EBV infection. Sporadic BL, while m o rphologically sim ilar, has no geographic distribution, is not associated w ith a known viral disorder, and represents a typical exam ple of a sporadic tum or to totally unknown etiology. All forms of BL are typically associated with specific chromosomal translocations that result in the juxtaposition of the c-myc locus of chrom osom e 8 with various im m unoglobulin loci. T he m ost common translocation moves the term i nal portion of chromosome 8 containing the c-myc gene to chromosome 14 near the enhan cer of the im m unoglobulin heavy-chain locus. Less frequent are translocations of chromosome 8 to the kappa light-chain locus of chromosome 2 or to the lam bda light chain locus of chrom osom e 22. I t appears th a t relo cation of the c-myc oncogene adjacent to an active im m unoglobulin gene re sults in activation or deregulation of c-m yc causin g one o f th e m u ltip le EBV proliferated B-cell clones to b e come malignant. The studies of Lombardi et al52 where activated c-myc genes were introduced into hum an EBV infected lymphoblastoid cells from in vitro infection of normal cord blood or directly from infected peripheral blood of AIDS patients supp o rt th a t c o n c e p t. Such cells show changes in growth properties typical of tran sfo rm ed cells and acquisition of tu m o rig e n ic ity in im m u n o d e fic ie n t mice. W hether or not those patients who eventually develop endemic BL are, in fact, im m une deficient has long been questioned.40 The dem onstrated epidemiological evidence for a causal relationship betw een EBV and endemic BL in an area w here malaria is holoendem ic has been well dem onstrated.24 M ultiple studies now suggest strongly that chronic heavy m alarial infections do in d eed, cause im m une im pairm ent. W hittle et al86 have shown th a t T-cell subpopulations are radically altered during an attack of Plasmodium falciparum malaria so that, in vitro, B-lymphocytes infected w ith EBV p ro life ra te abnorm ally to secrete large am ounts of im m unoglobulin. In New Guinea, where endemic BL is common, Moss et al57 have shown that re s id e n ts in areas h o lo en d e m ic for malaria have impaired EBV-specific T- cell immunity compared with residents in non-malarious areas. Similarly in Sw eden, patients w ith prim ary Plasmodium falciparum infections have been shown to have a lower proportion of T-helper cells and a lower

12 186 BIEMER T-helper/T-suppressor ratio than normal Sw edes.83 Thus, one m ight speculate that heavy malarial burdens can induce an im m u n o co m p ro m ised sta te, not unlike that associated with AIDS, that allows unrestrained EBV B-cell lymphoproliferation. Subsequently, one can envision genetic translocation of the c-myc oncogene in the state of frenzied lymphoproliferation thus selecting and transforming a malignant clone destined to form endem ic BL. A similar relationship of EBV infection and im m unoincom petence has not been evident in sporadic BL. Naturally O ccurring Immunodeficiency D isease States (NOIDS) Filipovich et al29 reported in 1980, on the incidence of malignancy in naturally o c c u rrin g im m u n o d eficie n c y states (NOIDS) from the retrospective analysis of two can cer reg istries. W hile th e reported conditions were heterogenous, all immunodeficiency states were found to h av e an in c re a s e d in c id e n c e of lym phom as, w ith W isk o tt A lderich Syndrom e (WAS) having th e highest incidence at 15.4 percent, and Ataxia- Telangiectasia (AT) second, approximating 10 to 12 p e rc e n t. In th e ir 1984 report,30 the imm unodeficiency cancer registry at the University of Minnesota (March 1982) included 168 cases of non- H odgkin s lym phom a representing 44 percent of all malignancies. There were 58 lym phom as associated w ith AT, 41 w ith WAS, 37 w ith com m on variable im m unodeficiency disease (CVID), 12 w ith sev ere com bined im m unodeficiency disease (SCID), and 20 with multiple other types of immunodeficiency. Most of the lymphomas were of the large cell variety and the CNS was the m a jo r p r im a r y s ite. L y m p h o m a s occurred in younger patients than those with non-lymphoid tumors and showed a shorter latency period after the onset of im m u n o d e fic ie n c y. T ra n sitio n a l or h y p e rp la stic p re m a lig n a n t lesions w ere observed as w ell as polyclonal tumors that were clinically aggressive. A frequent association with EBV and other possible viruses was noted. Purtillo et al66 have hypothesized that these malignant lymphoproliferative diseases may also be provoked by EBV. Many patients with NOID disorders have dem onstrated defects in T-suppressor function, T-cytolytic function, natural killer function or a combination of defects. W hile a few of the patients reported by Filipovich et al30 appeared to have primarily a B-cell immunodysfunction, clearly th e m ajority of lym phom as occurred in association w ith immunodeficiency syndromes known to have sig n ifican t im p a irm e n t of cell m ediated immunity.40 Also, interestingly on the B-cell end of the spectrum of im m u n o d eficie n c y d isease was th e report of Schwaber et al73 indicating that their patient with a nonsecretory form of common varied agammaglobulinemia, whose immunoglobulin nonsecreting B- lymphocytes lacked receptors for EBV, showed no untoward effects of EBV, and could not become infected in vitro. Among the various prim ary or constitutional forms of immune deficiency, the X-linked Lymphoproliferative Syndrome (XLPS), has served as a prototype for study of lym phoproliferation in association with EBV.65,66 It is stated that in p a tie n ts w ith X LPS, 100 p e rc e n t of affected males develop serious diseases following infection with EBV. Approxim ately tw o -th ird s of th ese p a tie n ts develop a severe frequently fatal form of IM, characterized by a m arked polyclonal B-cell proliferation with elevated serum immunoglobulins. On the other hand, approximately 35 percent of XLPS patients develop m alignant B-cell lym phomas. The majority of these patients

13 MALIGNANT LYMPHOMAS ASSOCIATED WITH IMMUNODEFICIENCY STATES develop their malignant lymphomas de novo, w hile approxim ately o n e-th ird have a history of antecedent IM. Characteristically, the lymphomas tend to be extranodal and most frequently found in the term inal ileum, viscera and CNS. Furtherm ore, they are of B-cell origin and of a high histologic grade ranging from B urkitt-like lymphom as to large cell im m unoblastic sarcomas. Lymphomas in C ancer Patients Receiving Im m unosuppressive T herapy C ancer patients who receive anti-neoplastic radiation and chemotherapy are also known to have an increased incidence of second malignancies. Clearly some of the observed second m alignancies reflect a tum or diathesis w hereby patients with a first neoplasm exhibit a greater tendency to develop a second tum or than patients who have never had cancer. However, particularly in Hodgkins disease (HD), there have been multiple observations of second neoplasms which are specifically relatable to prior therapy.63,64 The most common second m alignancies in H D are A cute Non- Lymphocytic Leukemia (ANLL), NHL, and carcinomas of the urinary bladder.64 In the therapy of H D, increasingly it appears th a t N H L is com parable to ANLL, being diagnosed at a median of four years following the th erap y for H D with an incidence approxim ately four percent. N on-h odgkin s lym phom a has been o b serv ed m ore fre q u e n tly follow ing combined therapy, although it may also occur following radiation or chem otherapy a lo n e.3,18,31,48 T h e ir etio lo g y is unknown although likely considerations include the potential carcinogenic effect of the therapeutic agents or as a result of im m une suppression by them. A lternately, th e recognized im m une a lte r ations known to exist and possibly contribute to the initial developm ent of HD are known to persist in long tim e survivors of HD. Perhaps that same immune suppression allows evolution of N H L as well. The observed histological varieties of the N H L following H D have been predom inately diffuse large cell types, and cell m ark e r stu d ie s have b e e n evenly divided betw een B and T-cell types. This latter feature has not been characteristic of the almost invariable B- cell phenotype of that NHL associated w ith congenital and acquired im m unodeficiency or iatrogenic im m une su p p ressio n. F u rth e rm o re, ev id en ce of EBV involvem ent has not b een p re sented although these citations reflect w ork done p rio r to th e ready availability of DNA p ro b es and analysis for gene rearrangem ents. Role of O ther Agents W hile EBV is currently believed to be the m ost im portant putative agent in lym phom agenesis in im m unodeficient patients, it m ust certainly not be the only agent responsible for these events. The well studied case of B-cell lym phoma in severe combined immunodeficiency reported by Garcia et al32 well illu strates th at point. W hereas m ost recent reports have confirm ed the p resence of EBV in almost all of the B-cell lymphomas associated with some form of immunodeficiency, this one did not. N eith e r EBNA nor EBV-DNA could be detected in tum or tissue by anticomplem en t im m unofluorescence or in situ cytohybridization w ith an EBV-DNA probe. Furtherm ore, EBV-DNA could not be detected by Southern blot hybridization using two EBV-DNA hybridization probes on the same DNA blots that clearly contained the clonal Ig gene rearra n g e m e n t. Sim ilarly, m o re re c e n t reports46,80 cast doubt as to w hether or n ot EBV plays such a key role in the

14 188 BIEMER lym phom agenesis of AIDS p a tie n ts. W hereas those authors dem onstrated c-m yc o n cogene re a rra n g e m e n ts in almost all of the tumors of such patients, EBV sequences and proteins were found in only a third. Clearly, it would appear that other agents, i.e., viruses, oncogenes or as yet u n id e n tifie d fa c to rs, re m a in to b e defined. The HTLV-I is a RNA retrovirus with T-cell trophism which appears geographically lim ited to Southwestern Japan, th e C arib b ean, S o u th eastern U.S., and Africa, and is associated with adult T-cell Leukem ia/lym phom a and s m o ld e rin g T -c ell ly m p h o m a. T he HTLV-I is monoclonally integrated in viral transforming proteins and immune suppressive substances are produced. Im m une deficiency in silent carriers of HTLV-I and in those with smoldering Adult T-Cell Leukem ia/lym phom a suggest that immune surveillance deters the em ergence of Adult T-Cell Leukemia/ Lym phom a.67 A nother lym photrophic virus which has been associated with lymphoproliferative disorders has been described by Salahuddin et al.74 Designated hum an B-lymphotrophic virus (HBLV), it is a herpes-like B-lymphotrophic virus that selectively infects, but does not immortalize freshly isolated human B-cells, and converts them into large refractile mono or binucleated cells with nuclear and cytoplasmic inclusion bodies. Clearly, its possible etiologic role in lymphoproliferative disease of man needs to be further in v e s tig a te d. M ost s tu d ie s do n o t directly implicate HIV in lymphomagenesis.35 Rather its T-helper cell trophism causes lysis of those cells leading to impaired T-cell function which initiates the full blown spectrum of AIDS. The role of oncogenes o th e r than c-myc seem s less clear. The RAS m utations have been seen in the most undifferentiated acute lymphocytic leukemia (ALL) phenotypes58 and have also been shown to induce m alignant transformation and plasma cell differentiation in hum an B-lymphoblasts immortalized by EBV.76 Also, th e bcl-2 oncogene has been shown to have a strong association w ith B -cell lym phom as of follicular center cell origin. Obviously, the role, if any, of these various agents in the lymphoproliferative processes of im m une deficient patients m ust await further definition as the exciting field of molecular biology unfolds. References 1. Ah m e d, T., W o r m se r, G. P., St a h l, R. E., et al. : Malignant lymphomas in a population at risk for acquired im m une deficiency syndrome. Cancer 60: , A n d e r s o n, J. L., B ie b e r, C. P., F o w l e s, R. E., et al.: Idiopathic cardiomyopathy, age, and suppressor-cell dysfunction as risk determinants of lymphoma after cardiac transplantation. Lancet 2: , Ar m it a g e, J. O., D ic k, F. R., G o e k e n, J. A., et al. : Second lym phoid m alignant neoplasms occurring in patients treated for Hodgkin s disease. Arch. Intern. Med. 143: , Be n n e t t, W. M., and N o r m a n, D. J.: Action and toxicity of cyclosporine. Ann. Rev. Med. 37: , B e v e r id g e, T., et al.: Lymphomas and lymphoproliferative lesions developing under cyclosporin therapy. Lancet 1: , Be v e r id g e, T., Cyclosporin-A: An evaluation of clinical results. Transplant. Proc. XV: , B ie b e r, C. P., Reit z, B. A., Ja m ie so n, S. W., et al.: M alignant lym phom a in cyclosporin A treated allograft recipients. Lancet 1:43, B ie b e r, C. P., H u n t, S. A., Sc h w in n, S. A., et al. : Complications in long-term survivors of card iac tr a n s p la n ta tio n. T ra n sp la n t. P roc. XIII: , B ir d, A. G. and M c L a c h l a n, S. M.: Cyclosporin A and Epstein-Barr virus. Lancet 2:418, Bir d, A. G., M c L a c h l a n, S. M., a n d B r itto n, S.: C y c lo sp o rin A p ro m o te s sp o n ta n e o u s o u t g row th in vitro o f E p ste in -B a rr v iru s-in d u ced B- cell lines. N ature 289: , Birx, D. L., Re d f ie l d, R. R., and T o sa to, G.: Defective regulation of Epstein-Barr virus infection in patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related disorders. New Engl. J. Med. 374: , B r u m b a u g h, J., B a l d w in, J. C., St in s o n, E. B., et al.: Quantitative analysis of immunosuppression in cyclosporine-treated heart transplant patients with lymphoma. Heart Transplant. 7V: , 1985.

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