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Lymphoma complicating immunodeficiency disorders
Annals of Oncology 5 (Suppl. 2): S29-S35, 1994. O 1994 Kluwer Academic Publishers. Primed in the Netherlands.
Original article Lymphoma complicating immunodeficiency disorders A.M. Levine University of Southern California School of Medicine, Los Angeles, California, USA
involvement, and short survival. However, certain biologic and clinical differences are also apparent. Thus, while the transplantation-associated lymphomas are uniformly associated with the Epstein-Barr Virus (EBV), this association is Background: An increased incidence of lymphoma is seen in not seen in all systemic AIDS-related lymphomas, nor in all various types of immune deficiency or dysregulation, includ- congenital immune deficiency disorders. Likewise, while ing congenital immune deficiency diseases, organ transplan- c-myc activation has been described in certain types of HTVtation with iatrogenic immunosuppression, autoimmune dis- related lymphomas, c-myc dysregulation has not been reorders, endemic Burkitt's lymphoma, and AIDS-associated ported in association with organ transplantation. lymphoma. Conclusions: The study of lymphomatous disease in variResults: The lymphomas seen in various immunodefi- ous states of immune dysregulation may allow an underciency disorders share certain features. Thus, all consist of standing of the diverse pathogenic mechanisms operative in high-grade, B-cell tumors, with predilection for extranodal the development of malignant lymphoma in man. Summary
similarities and differences among them. Comparisons with various animal models will also be discussed, in an attempt to formulate hypotheses regarding the etiologic and pathogenic mechanisms of lymphomagenesis in man.
Animal models
Cotton-top tamarins Inoculation of 10 53 lymphocyte-transforming doses of Epstein-Barr virus into 8 cotton-top tamarins has been shown to result in lymphomatous tumor masses in all. The tumors in four animals regressed spontaneously, while four developed terminal disease. Multifocal immunoblastic or large-cell lymphomas were found in both lymph nodes and visceral organs at the time of autopsy. All lymphomas were associated with EBV, with 2-25 EBV genome equivalents per lymphoma cell. Cell suspensions from each lymphoma generated Epstein-Barr nuclear antigen (EBNA) positive cell lines, which produced infectious EBV. No consistent chromosomal translocations were seen. The tumors were all genotypically oligo- or monoclonal as demonstrated by Southern blot analysis. Of interest, different immunoglobulin gene rearrangements were found at different sites of lymphomatous disease [1].
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Lymphomas are seen with increased frequency in several settings, all of which are associated with underlying deficiency or dysregulation of the immune system. These disorders include certain congenital immune deficiency diseases, iatrogenic immunosuppression associated with allogeneic organ transplantation, autoimmune diseases, endemic Burkitfs lymphoma, and the AIDS-associated lymphomas. All of these disorders have certain features in common, including history of immune dysregulation and ongoing, chronic antigenic stimulation. In addition, all are similar in terms of the pathologic types of lymphoma associated with them, namely, those consisting of high-grade, B-cell tumors. The lymphomas which occur tend to be widespread at initial diagnosis, with involvement of extranodal organs and predilection for the brain in some. Further, all are characterized by an aggressive clinical course, with short subsequent survival. While similar in many respects, notable differences also exist in the lymphomas which develop in these various settings. Thus, while the Epstein-Barr virus (EBV) genome is present within the tumor cell in many of the disorders, this is not uniformly the case. Further, the clonality of the lesions is diverse, as is the expression of certain oncogenes, such as c-myc. This paper will review the specific entities associated with increased risk of lymphoma, with emphasis on the
30 Transgenic mice: c-myc driven by immunoglobulin enhancers
HIV-1 infection of EBV-positive human B lymphocytes
Laurence and Astrin exposed peripheral blood B lymphocytes from EBV-positive, HTV-negative donors to HTV-1 in vitro, with resultant immortalization of lymphoblastoid cell lines [5]. These lines were found to contain approximately 1 copy/cell of HTV, when analyzed by PCR. Further, the lines expressed high levels of various EBV gene products, including latent membrane protein (LMP), CD23, and the EBV receptor (CD21). Logarithmic, continuous growth occurred for over 40 weeks in media containing only 1% FBS, and colonies formed in soft agar. When these cell lines were injected into SCID mice, Effects of combined EBV infection and c-myc activation invasive Burkitt's lymphoma developed by week 8 in 2 of 4 animals. The lymphoma tissue demonstrated a Lombardi and colleagues [3] employed expression vecmarked up-regulation of c-myc transcripts and protein, tors containing constitutively expressed c-myc genes. and in EBV-DNA and RNA. These were transfected into EBV-infected human B-lymphoblastoid cells from normal cord blood or peripheral blood of patients with HTV infection. By Summary: In vitro experiments and animal models Northern blot and RNA-ase protection analyses, constitutive expression of exogenous c-myc was found, The above experiments indicate that EBV itself may which caused down-regulation of expression of endo- lead to lymphoma in certain genetic species, such as the genous c-myc. When injected subcutaneously into cotton-top tamarin. In this regard, LMP has been athymic nude mice, subcutaneous tumors occurred shown to have transforming and oncogenic potential within two to four weeks in all, consisting of undiffer- [6]. Furthermore, the dysregulated expression of c-myc entiated B-cell lymphomas, which lacked the clear fea- may transform human B-lymphoblastoid cells in vitro, tures of Burkitt's lymphoma and contained normal while transgenic mice carrying Ig-myc chimeric conkaryotypes. The transfected B-lymphoblastoid cells structs have an increased incidence of lymphoma [2]. also demonstrated an altered growth pattern in semi- FLTV-l infection of immortalized B-cell lines can lead solid media, typical of that seen in Burkitf s lymphoma to up-regulation of c-myc transcripts, and HTV may lines, and also demonstrated decreased dependency on also directly effect cellular c-myc expression [7]. Infection of Cynomolgus monkeys by SIV can lead to spegrowth factors. cific T-cell depletion and eventual development of This work has suggested that the expression of EBV-positive B-cell lymphomas [4]. c-myc may normally be controlled by negative feedIt is clear from these data that altered expression of back loops, which, if altered, could eventuate in maligcertain EBV proteins, along with dysregulated expresnant transformation. sion of c-myc, may be sufficient for the development of malignant lymphoma in various animal models. The Cynomolgus monkeys (Macaca fascicularis) inoculated presence of underlying states of abnormal immunity withSW may provide the circumstances necessary for altered expression of these potentially oncogenic genes and Feichtinger and colleagues [4] inoculated 26 Cynomol- proteins. gus monkeys with 10 x 105 infectious doses of simian immunodeficiency virus (SIV^). All animals became infected, and all developed immunodeficiency, as evi- Naturally occurring immunodeficiency syndromes denced by CD4 cell depletion. By day 464, 9 (38%) developed lymphoma, consisting of high-grade tumors The overall risk of cancer is increased approximately in 8 (Burkitt-like in 6; immunoblastic in 2). All lympho- 100 times in patients with various congenital immune mas were of B-cell phenotype, and all exhibited im- deficiency diseases (NOLDS), and lymphoma communophenotypic monoclonality. The tumors were prises approximately half of all cancers which develop
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Using transgenic mice (C57 BL x SJL), various constructs of the c-myc oncogene with different enhancers were implanted by Adams and colleagues [2]. Specific immunoglobulin enhancers (E^-myc or EkSV myc) were most likely to result in a malignant phenotype, with the E^-myc construct leading to disseminated lymphoma in 13 of 15 animals. The lymphomas were of B-lymphoid origin in all and were genorypically monoclonal, or occasionally oligoclonal. Tumor masses at different sites were found to have the same immunoglobulin gene rearrangement. Transplantation of these lymphomas into syngeneic mice resulted in frank tumors in all, extending over four generations. The B-cell lymphomas which occurred were not stage-specific, and both pre-B and mature B phenotypes were encountered. Of note, high expression of c-myc RNA was found within lymphoma tissues.
negative for SFV proteins or genome, although EBV sequences were present in some. Of interest, an additional 26 Cynomolgus monkeys were inoculated with HTV-2. While all became infected, all remained healthy, with no evidence of CD4 depletion or immune deficiency. No lymphoma developed in these animals.
31 [8]. Of interest, full immune reconstitution by bone marrow transplantation may prevent the subsequent development of lymphoma in these individuals. The biologic characteristics of the lymphomas which develop in the setting of NOIDS are not well characterized, although certain differences between them are already apparent. X-linked lymphoproliferative syndrome
Wiskott-Aldrich syndrome Wiskott-Aldrich syndrome is an X-linked recessive disorder, characterized by immunodeficiency, eczema, and thrombocytopenia. Various immune abnormalities have been described, including occasional reports of defective anti-EBNA response to EBV, as well as NK cell defects after EBV infection. Lymphoma develops in approximately 14% of children with Wiskott-Aldrich syndrome, consisting primarily of large-cell immunoblastic lymphoma. These are of B-lymphoid origin, and are primarily extranodal at the time of initial presentation. The central nervous system is involved in approximately 25% [8,11]. The lymphomas which occur are uniformly associated with EBV. Both polyclonal and monoclonal lymphomas have been described [10,11]. Ataxia telangiectasia
Ataxia telangiectasia (AT) is an autosomal recessive disorder, characterized by cerebellar ataxia, oculocuta-
Lymphomas complicating allogeneic organ transplantation
The relative risk of lymphoma in the setting of organ transplantation and iatrogenic immunosuppression is approximately 25-49 times greater than expected [12]. The incidence of lymphoma is approximately 2% in renal transplant recipients, and 5% in cardiac recipients, in whom the most common underlying cardiac disorder is idiopathic cardiomyopathy [13]. Increasing degrees of immunosuppression are associated with increased risk of lymphoma. Thus, history of multiple transplants, use of cyclosporin A, and use of monoclonal antibodies for T-cell depletion have all resulted in an increased risk of post-transplant lymphoproliferative disease [14]. Of interest, decrease or withdrawal of immunosuppressive therapy has resulted in spontaneous regression of these lymphomas in approximately 25%-50%ofcases[15]. Post-transplant lymphomas comprise three groups of lymphoproliferative disorders [10,16]. Polymorphic diffuse B-cell hyperplasia is associated with polyclonal phenotype, a clinical syndrome associated with significant systemic B symptoms, and a polymorphic pathologic picture, consisting of plasma cells, small cleaved cells, typical large cells, and single-cell necrosis. Polymorphic diffuse B-cell lymphoma would be classified as diffuse mixed lymphoma pathologically, characterized by atypical immunoblasts, small cleaved cells, and extensive 'geographic' necrosis. The allograft may be involved in 20% of cases, GI tract in 25%, and CNS in approximately 14%. Polyclonal tumors have been described in approximately 36% of cases, with monoclonal lymphomas in 29%. True monomorphic largecell immunoblastic lymphomas may also evolve in the setting of organ transplantation [17]. Phenotypically, these lymphomas may be nonmarking, poly-, or monoclonal. By Southern blot analysis, monoclonality has been confirmed, with different clones in different anatomic sites of disease [18]. Extranodal disease is expected in the majority, with CNS involvement in up to 77%. Post-transplant lymphomas are uniformly associated
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X-linked lymphoproliferative syndrome (XLP) is an X-linked recessive disorder characterized by defective immune response to EBV. When infected by EBV, affected boys may develop chronic or fatal infectious mononucleosis; aplastic anemia; hypogammaglobulinemia; or malignant lymphoma [9]. Numerous immune defects have been described in XLP including incomplete EBV-specific antibody production, with low or absent anti-EBNA response, poor or decreased activity of EBV-specific memory T cells, abnormal levels and ratios of CD4 and CD8 cells, loss of NK activity after EBV infection, and thymic epithelial destruction after EBV infection [9,10]. Approximately 35% of boys with XLP develop lymphoma. These lymphomas are often extranodal, with prominent involvement of the ileocecal area and the central nervous system. Microscopically, the lymphomas are high-grade tumors of B-cell origin, consisting of either small noncleaved or immunoblastic types [10]. The EBV genome is present in virtually all cases, with presence of EBV within tumor cells. Abnormal karyotypes have been described, with the few cases studied demonstrating t(8;14) or t(8;22). Evolution from polyclonal to monoclonal phenotype has been observed, although careful study of immunophenotype or genotype has not yet been accomplished.
neous telangiectasia, and several immune defects. The immunologic abnormalities described in AT include the occurrence of multiple chromosome breaks with a decreased ability for DNA repair, as well as multiple defects in cell mediated immunity. Chronic, active EBV infection has been described following primary EBV infection, and defective anti-EBNA antibody response may be seen, in addition to NK cell defects [11]. Approximately 10% of children with AT develop lymphoma over time. These lymphomas are large cell in type and usually present in nodal sites. EBV genome is usually not present, although several EBV-associated cases have been described. Both polyclonal and monoclonal lymphomas have been seen [8,11].
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with EBV [10, 12, 16]. Abnormal c-myc expression is not seen. Lymphoma complicating autoimmune disease(s)
AIDS-related lymphoma Clinicopathologic characteristics
The AIDS-related lymphomas are high-grade B-cell tumors, consisting of large-cell immunoblastic or small noncleaved-cell (Burkitt's or Burkitt-like) lymphomas in approximately 80%-90% of cases [27]. Intermediate-grade, large noncleaved-cell lymphomas have also been described, and are considered AIDS-defining. While low-grade B-cell lymphomas, various T-cell lymphomas, and Hodgkin's disease have been diagnosed in HTV-infected patients, their incidence has not increased with the AIDS epidemic, and they are not considered AIDS-defining conditions. Clinically, the AIDS lymphomas present in extranodal sites, occurring in approximately 80%-90% of Endemic Burkitt's lymphoma cases, in distinction to 'usual' lymphomas, in which Endemic African Burkitt's lymphoma has been de- extranodal presentation is expected in approximately scribed in areas of the world which are endemic for 40% of cases [27]. Common sites of involvement inmalaria. While not classically considered an example of clude the central nervous system (33%), gastrointesimmunodeficiency-related lymphoma, several aspects tinal tract (25%), bone marrow (25%), and other diverse sites. of lymphomatous disease do suggest a relationship. Lymphoma primary to the central nervous system Immune deficiency has been demonstrated secondary to chronic malarial infection. Thus, Whittle and col- (P-CNS lymphoma) has a particularly poor prognosis, leagues demonstrated a decrease in CD4 cells, with a and far-advanced underlying HTV-related immunodefidecrease in CD4:CD8 ratios during acute malarial in- ciency is expected. Thus, the median CD4 cell count at 3 fection in Africa [23]. Decreased CD4 cells, with de- diagnosis is 30 mm , and approximately 75% of pacreased CD4:CD8 ratios have been described in Swe- tients have been diagnosed with AIDS prior to the dedish patients with primary P. falciparum infection [24]. velopment of P-CNS lymphoma [28]. Median survival Further, impaired EBV-specific T-cell immunity has is approximately two to three months despite therapy, been described in residents of areas which are holo- and death is often due to opportunistic infection. All such lymphomas are of large-cell or immunoblastic endemic for malaria [25]. type, and EBV genome is uniformly present within Prospective studies of large numbers of children livlymphoma cells. Expression of certain latent EBV proing in the malarial belt of Africa have demonstrated teins, such as EBER, has been demonstrated in 21/21 that EBV infection, with high titers of anti-VCA antisuch cases, while LMP has been found in 45% [29]. body, may predict for the development of Burkitt's lymphoma [26]. Thus, EBV infection precedes the occurrence of lymphoma in such children. Moreover, BL is Pathogenesis of disease uniformly associated with EBV, with EBV genome The mechanisms of lymphomagenesis in AIDS lymdemonstrated within tumor cells in all cases [26]. Pathologically, the lymphomas which develop are phoma are not yet fully understood, although ongoing small noncleaved-cell lymphomas, of B-cell origin. B-cell proliferation and stimulation are integral com-
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Malignant lymphoma has been described with increased frequency in various types of autoimmune disorders, including rheumatoid arthritis (RA), and others. Employing a nationwide tumor registry in Finland, and linking this with data from patients with RA, Hakulinen and colleagues found a two-fold increase in lymphoma among patients with RA [19]. In a retrospective casecontrol study of 238 patients with RA, half of whom received cyclophosphamide, an excess of lymphoma was seen among the patients taking this drug [20]. In studying patients with miscellaneous autoimmune disorders, given various forms of immunosuppressive therapy (azathioprine in 68%, cyclophosphamide in 28%, chlorambucil in 4%), the relative risk of lymphoma was found to be 11 times greater than that expected in the general population. The lymphomas which develop in patients with autoimmune disease tend to be large-cell or immunoblastic lymphomas, of B-cell origin [21]. Extranodal disease is seen in many, and lymphoma confined to the site(s) of prior immune disease has been described [21]. Association with EBV has been confirmed in one case of lymphoma developing in a patient with RA, treated with cyclosporine [22].
Extranodal involvement is common, with lymphoma found prominently in the jaw, ileocecal area, ovaries, and other sites. Central nervous system involvement is rather unusual, occurring in 2%-3% of cases. Endemic Burkitt's lymphoma is a monoclonal tumor. Specific chromosomal translocations have been well described, including t(8;14), t(8;22), and t(8;2), ah1 involving the immunoglobulin heavy- or light-chain genes, as well as the myc oncogene, present on chromosome 8. In this regard, c-myc dysregulation has been described in all cases. Prognosis depends upon the extent of disease, and cure has been possible with single-agent cyclophosphamide, given in high dose.
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ponents of chronic HTV infection. While HTV per se is not present within lymphoma-cell DNA, HIV does induce ongoing B-cell expansion and activation, resulting in the development of reactive B-cell hyperplasia in lymphoid tissues, known as persistent generalized lymphadenopathy (PGL) [30,31]. These reactive lymphoid tissues harbor large quantities of HTV during the course of asymptomatic HIV infection [32]. The relative risk of lymphoma in patients with PGL is increased 850 times over expected [33]. Cytokine networks
EBV In addition to cytokine pathways, EBV may be implicated in the pathogenesis of AIDS lymphoma, perhaps related to the impaired immunosurveillance of EBVinfected cells in patients with AIDS [38,39]. The precise role of EBV in the pathogenesis of AIDS-related lymphoma is somewhat controversial. Shibata and colleagues studied the reactive lymphadenopathy tissues from 35 HTV-infected patients by PCR and in situ hybridization, and found EBV DNA in 13 of 35 (35%); the presence of detectable amounts of EBV DNA in these 13 reactive biopsies was associated with an increased incidence of concurrent lymphoma at another site or with development of lymphoma over time [40]. While it is apparent that AIDS-related primary CNS lymphoma is uniformly associated with latent EBV proteins, this has not been the case in AIDS-related systemic lymphoma, where EBV expression has been reported in 3 of 7 cases (42%) in one study [41], and in only 6 of 16 (38%) cases in another study [42]. Of interest, however, in analyzing 10 of these EBV-positive systemic ATDS-lymphoma cases, Neri and colleagues found evidence of clonal EBV infection in all.
Abnormal DNA rearrangements The DNA rearrangements which occur normally during B-cell differentiation may provide vulnerable, abnormal recombination-prone sites, leading to chromosomal translocations involving the Ig heavy- or lightchain genes. These recombinase errors could lead to the specific chromosomal translocations which have been described in AIDS lymphoma, including t(8; 14), t(8;22), or t(8;2) [45]. Consistent with this hypothesis, Pelicci and colleagues [46] described the development of multiple clonal rearrangements of immunoglobulin genes within reactive PGL tissues; the subsequent selection and growth advantage of one such clone could explain the eventual development of monoclonal B-cell malignancy from ongoing polyclonal B-cell response. Most series have clearly documented the genotypic and/or immunophenotypic monoclonality of AIDSrelated lymphoma [27]. Recently, however, Shiramizu and colleagues employed Southern blot analysis on DNA, as well as frozen-section immunohistochemical analysis from 40 cases of AIDS-related lymphoma, noting that approximately 40% were polyclonal in genotype [47]. While multiple oligoclonal expansions have been documented prior to the development of monoclonal lymphoma [46], the report of polyclonal lymphomas, especially comprising such a large proportion, is most unusual, and its significance awaits confirmation.
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The activation of various cytokine networks may contribute to the chronic B-cell proliferation which characterizes HTV disease. B cells from HTV-infected patients with hypergammaglobulinemia constitutively express TNF-a and IL-6 [34]. High levels of IL-6 gene expression have been demonstrated in both HIV-positive and HTV-negative cases of large-cell lymphoma, independent of EBV status [35]. The number of IL-6expressing cells has been shown to be 17 times higher in lymphoma cases versus control nodal tissues; and seven times higher in large-cell lymphomas containing a high proportion of immunoblasts [35]. Of interest, elevated serum levels of IL-6 have been documented prior to the development of large-cell lymphoma in a group of patients with symptomatic HTV infection [36]. While not unique to AIDS lymphoma, then, IL-6 may play a role in the pathogenesis of diverse types of B-cell neoplasia, in which terminally differentiated cells predominate. Aside from IL-6, IL-10 may also be operative in the development of HTV-related lymphoma [37].
Thus, EBV integration occurred before clonal B-cell expansion, which would be consistent with an etiologic role of EBV [42]. The differences between EBV expression in primary CNS versus systemic lymphoma may be a function of the site of disease, or may be related to differing histologic types, with large-cell or immunoblastic lymphoma more likely to be associated with EBV, as opposed to small noncleaved-cell lymphoma. Thus, EBV genome has been detected by in situ hybridization in 11 of 17 cases of systemic immunoblastic lymphoma (65%), versus only 1 in 5 cases of Burkitt's lymphoma [43]. However, employing Southern blot analysis, PCR and in situ hybridization in 59 cases of systemic AIDS lymphoma, Shibata and colleagues have demonstrated presence of EBV in the majority of all cases, including 85% of immunoblastic, 55% of large-cell, and 57% of small noncleaved-cell lymphomas [44]. Similarly, Emillie noted EBV genome in 66% of small noncleaved-cell, 75% of immunoblastic, and 60% of systemic large-cell lymphomas [35].While these discrepancies may be related to differing interpretations of pathologic material, it is clear that EBV cannot be the 'sole' cause of lymphoma in the setting of AIDS. Still, in those cases which are EBV-positive, mechanisms are in place which could eventuate in malignant transformation [5].
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C-myc deregulation and other genetic abnormalities
Pathogenesis The lymphomas arising in the setting of AIDS thus involve multiple factors, including underlying immunosuppression; chronic B-cell proliferation (due to HTV, cytokine release, and/or EBV); expression of certain latent EBV viral proteins associated with cellular transformation; occurrence of recombinase errors, leading to chromosomal translocations; and subsequent dysregulation of c-myc and/or other oncogenes, as well as the inactivation of tumor-suppressor gene(s). While some of these factors may occur in the majority of cases, heterogeneity has already been encountered, indicating that distinct mechanisms of lymphomagenesis may occur in different pathologic types or sites of disease.
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The chromosomal translocations described in AIDSrelated lymphoma could result in dysregulation of the c-myc oncogene present on chromosome 8 [48]. Of note, however, c-myc dysregulation has not been uniformly documented in AIDS lymphoma, occurring in 100% of small noncleaved-cell lymphomas in one series [41], and in only a minority of large-cell or immunoblastic lymphomas in another study [49]. Moreover, the specific mechanisms leading to c-myc dysregulation are diverse in the AIDS lymphomas studied to date, with c-myc translocation, similar at the molecular level to sporadic Burkitt's lymphoma, in some series [42], while point mutations were documented in another well-studied case [50]. Furthermore, HTV-1 infection of immortalized B-cell lines, can itself result in up-regulation of c-myc transcripts [5], while HTV may directly affect cellular c-myc gene expression [7]. Regardless of the mechanism of dysregulation, myc protein is a potent regulator of cell proliferation. Dysregulation of c-myc has been shown to contribute to transformation of human B cells in vitro, and may cause B-cell lymphoma in transgenic animals carrying Ig- myc chimeric constructs [2,3]. It is apparent, then, that c-myc dysregulation may provide another mechanism for the development of lymphoma in the setting of AIDS. Mutations or losses of p53 have also been reported in approximately 60% of AIDS lymphoma cases, again restricted to the small noncleaved-cell subtype [49]. Other genetic abnormalities, including 6q deletions [51], and Ras gene mutations [49] have been described as well.
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Correspondence to: Alexandra M. Levine, M.D. Professor of Medicine Chief, Division of Hematology USC School of Medicine Room 801 1441 Eastlake Avenue Los Angeles, California 90033 USA
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Original article Lymphoma complicating immunodeficiency disorders A.M. Levine University of Southern California School of Medicine, Los Angeles, California, USA
involvement, and short survival. However, certain biologic and clinical differences are also apparent. Thus, while the transplantation-associated lymphomas are uniformly associated with the Epstein-Barr Virus (EBV), this association is Background: An increased incidence of lymphoma is seen in not seen in all systemic AIDS-related lymphomas, nor in all various types of immune deficiency or dysregulation, includ- congenital immune deficiency disorders. Likewise, while ing congenital immune deficiency diseases, organ transplan- c-myc activation has been described in certain types of HTVtation with iatrogenic immunosuppression, autoimmune dis- related lymphomas, c-myc dysregulation has not been reorders, endemic Burkitt's lymphoma, and AIDS-associated ported in association with organ transplantation. lymphoma. Conclusions: The study of lymphomatous disease in variResults: The lymphomas seen in various immunodefi- ous states of immune dysregulation may allow an underciency disorders share certain features. Thus, all consist of standing of the diverse pathogenic mechanisms operative in high-grade, B-cell tumors, with predilection for extranodal the development of malignant lymphoma in man. Summary
similarities and differences among them. Comparisons with various animal models will also be discussed, in an attempt to formulate hypotheses regarding the etiologic and pathogenic mechanisms of lymphomagenesis in man.
Animal models
Cotton-top tamarins Inoculation of 10 53 lymphocyte-transforming doses of Epstein-Barr virus into 8 cotton-top tamarins has been shown to result in lymphomatous tumor masses in all. The tumors in four animals regressed spontaneously, while four developed terminal disease. Multifocal immunoblastic or large-cell lymphomas were found in both lymph nodes and visceral organs at the time of autopsy. All lymphomas were associated with EBV, with 2-25 EBV genome equivalents per lymphoma cell. Cell suspensions from each lymphoma generated Epstein-Barr nuclear antigen (EBNA) positive cell lines, which produced infectious EBV. No consistent chromosomal translocations were seen. The tumors were all genotypically oligo- or monoclonal as demonstrated by Southern blot analysis. Of interest, different immunoglobulin gene rearrangements were found at different sites of lymphomatous disease [1].
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Lymphomas are seen with increased frequency in several settings, all of which are associated with underlying deficiency or dysregulation of the immune system. These disorders include certain congenital immune deficiency diseases, iatrogenic immunosuppression associated with allogeneic organ transplantation, autoimmune diseases, endemic Burkitfs lymphoma, and the AIDS-associated lymphomas. All of these disorders have certain features in common, including history of immune dysregulation and ongoing, chronic antigenic stimulation. In addition, all are similar in terms of the pathologic types of lymphoma associated with them, namely, those consisting of high-grade, B-cell tumors. The lymphomas which occur tend to be widespread at initial diagnosis, with involvement of extranodal organs and predilection for the brain in some. Further, all are characterized by an aggressive clinical course, with short subsequent survival. While similar in many respects, notable differences also exist in the lymphomas which develop in these various settings. Thus, while the Epstein-Barr virus (EBV) genome is present within the tumor cell in many of the disorders, this is not uniformly the case. Further, the clonality of the lesions is diverse, as is the expression of certain oncogenes, such as c-myc. This paper will review the specific entities associated with increased risk of lymphoma, with emphasis on the
30 Transgenic mice: c-myc driven by immunoglobulin enhancers
HIV-1 infection of EBV-positive human B lymphocytes
Laurence and Astrin exposed peripheral blood B lymphocytes from EBV-positive, HTV-negative donors to HTV-1 in vitro, with resultant immortalization of lymphoblastoid cell lines [5]. These lines were found to contain approximately 1 copy/cell of HTV, when analyzed by PCR. Further, the lines expressed high levels of various EBV gene products, including latent membrane protein (LMP), CD23, and the EBV receptor (CD21). Logarithmic, continuous growth occurred for over 40 weeks in media containing only 1% FBS, and colonies formed in soft agar. When these cell lines were injected into SCID mice, Effects of combined EBV infection and c-myc activation invasive Burkitt's lymphoma developed by week 8 in 2 of 4 animals. The lymphoma tissue demonstrated a Lombardi and colleagues [3] employed expression vecmarked up-regulation of c-myc transcripts and protein, tors containing constitutively expressed c-myc genes. and in EBV-DNA and RNA. These were transfected into EBV-infected human B-lymphoblastoid cells from normal cord blood or peripheral blood of patients with HTV infection. By Summary: In vitro experiments and animal models Northern blot and RNA-ase protection analyses, constitutive expression of exogenous c-myc was found, The above experiments indicate that EBV itself may which caused down-regulation of expression of endo- lead to lymphoma in certain genetic species, such as the genous c-myc. When injected subcutaneously into cotton-top tamarin. In this regard, LMP has been athymic nude mice, subcutaneous tumors occurred shown to have transforming and oncogenic potential within two to four weeks in all, consisting of undiffer- [6]. Furthermore, the dysregulated expression of c-myc entiated B-cell lymphomas, which lacked the clear fea- may transform human B-lymphoblastoid cells in vitro, tures of Burkitt's lymphoma and contained normal while transgenic mice carrying Ig-myc chimeric conkaryotypes. The transfected B-lymphoblastoid cells structs have an increased incidence of lymphoma [2]. also demonstrated an altered growth pattern in semi- FLTV-l infection of immortalized B-cell lines can lead solid media, typical of that seen in Burkitf s lymphoma to up-regulation of c-myc transcripts, and HTV may lines, and also demonstrated decreased dependency on also directly effect cellular c-myc expression [7]. Infection of Cynomolgus monkeys by SIV can lead to spegrowth factors. cific T-cell depletion and eventual development of This work has suggested that the expression of EBV-positive B-cell lymphomas [4]. c-myc may normally be controlled by negative feedIt is clear from these data that altered expression of back loops, which, if altered, could eventuate in maligcertain EBV proteins, along with dysregulated expresnant transformation. sion of c-myc, may be sufficient for the development of malignant lymphoma in various animal models. The Cynomolgus monkeys (Macaca fascicularis) inoculated presence of underlying states of abnormal immunity withSW may provide the circumstances necessary for altered expression of these potentially oncogenic genes and Feichtinger and colleagues [4] inoculated 26 Cynomol- proteins. gus monkeys with 10 x 105 infectious doses of simian immunodeficiency virus (SIV^). All animals became infected, and all developed immunodeficiency, as evi- Naturally occurring immunodeficiency syndromes denced by CD4 cell depletion. By day 464, 9 (38%) developed lymphoma, consisting of high-grade tumors The overall risk of cancer is increased approximately in 8 (Burkitt-like in 6; immunoblastic in 2). All lympho- 100 times in patients with various congenital immune mas were of B-cell phenotype, and all exhibited im- deficiency diseases (NOLDS), and lymphoma communophenotypic monoclonality. The tumors were prises approximately half of all cancers which develop
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Using transgenic mice (C57 BL x SJL), various constructs of the c-myc oncogene with different enhancers were implanted by Adams and colleagues [2]. Specific immunoglobulin enhancers (E^-myc or EkSV myc) were most likely to result in a malignant phenotype, with the E^-myc construct leading to disseminated lymphoma in 13 of 15 animals. The lymphomas were of B-lymphoid origin in all and were genorypically monoclonal, or occasionally oligoclonal. Tumor masses at different sites were found to have the same immunoglobulin gene rearrangement. Transplantation of these lymphomas into syngeneic mice resulted in frank tumors in all, extending over four generations. The B-cell lymphomas which occurred were not stage-specific, and both pre-B and mature B phenotypes were encountered. Of note, high expression of c-myc RNA was found within lymphoma tissues.
negative for SFV proteins or genome, although EBV sequences were present in some. Of interest, an additional 26 Cynomolgus monkeys were inoculated with HTV-2. While all became infected, all remained healthy, with no evidence of CD4 depletion or immune deficiency. No lymphoma developed in these animals.
31 [8]. Of interest, full immune reconstitution by bone marrow transplantation may prevent the subsequent development of lymphoma in these individuals. The biologic characteristics of the lymphomas which develop in the setting of NOIDS are not well characterized, although certain differences between them are already apparent. X-linked lymphoproliferative syndrome
Wiskott-Aldrich syndrome Wiskott-Aldrich syndrome is an X-linked recessive disorder, characterized by immunodeficiency, eczema, and thrombocytopenia. Various immune abnormalities have been described, including occasional reports of defective anti-EBNA response to EBV, as well as NK cell defects after EBV infection. Lymphoma develops in approximately 14% of children with Wiskott-Aldrich syndrome, consisting primarily of large-cell immunoblastic lymphoma. These are of B-lymphoid origin, and are primarily extranodal at the time of initial presentation. The central nervous system is involved in approximately 25% [8,11]. The lymphomas which occur are uniformly associated with EBV. Both polyclonal and monoclonal lymphomas have been described [10,11]. Ataxia telangiectasia
Ataxia telangiectasia (AT) is an autosomal recessive disorder, characterized by cerebellar ataxia, oculocuta-
Lymphomas complicating allogeneic organ transplantation
The relative risk of lymphoma in the setting of organ transplantation and iatrogenic immunosuppression is approximately 25-49 times greater than expected [12]. The incidence of lymphoma is approximately 2% in renal transplant recipients, and 5% in cardiac recipients, in whom the most common underlying cardiac disorder is idiopathic cardiomyopathy [13]. Increasing degrees of immunosuppression are associated with increased risk of lymphoma. Thus, history of multiple transplants, use of cyclosporin A, and use of monoclonal antibodies for T-cell depletion have all resulted in an increased risk of post-transplant lymphoproliferative disease [14]. Of interest, decrease or withdrawal of immunosuppressive therapy has resulted in spontaneous regression of these lymphomas in approximately 25%-50%ofcases[15]. Post-transplant lymphomas comprise three groups of lymphoproliferative disorders [10,16]. Polymorphic diffuse B-cell hyperplasia is associated with polyclonal phenotype, a clinical syndrome associated with significant systemic B symptoms, and a polymorphic pathologic picture, consisting of plasma cells, small cleaved cells, typical large cells, and single-cell necrosis. Polymorphic diffuse B-cell lymphoma would be classified as diffuse mixed lymphoma pathologically, characterized by atypical immunoblasts, small cleaved cells, and extensive 'geographic' necrosis. The allograft may be involved in 20% of cases, GI tract in 25%, and CNS in approximately 14%. Polyclonal tumors have been described in approximately 36% of cases, with monoclonal lymphomas in 29%. True monomorphic largecell immunoblastic lymphomas may also evolve in the setting of organ transplantation [17]. Phenotypically, these lymphomas may be nonmarking, poly-, or monoclonal. By Southern blot analysis, monoclonality has been confirmed, with different clones in different anatomic sites of disease [18]. Extranodal disease is expected in the majority, with CNS involvement in up to 77%. Post-transplant lymphomas are uniformly associated
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X-linked lymphoproliferative syndrome (XLP) is an X-linked recessive disorder characterized by defective immune response to EBV. When infected by EBV, affected boys may develop chronic or fatal infectious mononucleosis; aplastic anemia; hypogammaglobulinemia; or malignant lymphoma [9]. Numerous immune defects have been described in XLP including incomplete EBV-specific antibody production, with low or absent anti-EBNA response, poor or decreased activity of EBV-specific memory T cells, abnormal levels and ratios of CD4 and CD8 cells, loss of NK activity after EBV infection, and thymic epithelial destruction after EBV infection [9,10]. Approximately 35% of boys with XLP develop lymphoma. These lymphomas are often extranodal, with prominent involvement of the ileocecal area and the central nervous system. Microscopically, the lymphomas are high-grade tumors of B-cell origin, consisting of either small noncleaved or immunoblastic types [10]. The EBV genome is present in virtually all cases, with presence of EBV within tumor cells. Abnormal karyotypes have been described, with the few cases studied demonstrating t(8;14) or t(8;22). Evolution from polyclonal to monoclonal phenotype has been observed, although careful study of immunophenotype or genotype has not yet been accomplished.
neous telangiectasia, and several immune defects. The immunologic abnormalities described in AT include the occurrence of multiple chromosome breaks with a decreased ability for DNA repair, as well as multiple defects in cell mediated immunity. Chronic, active EBV infection has been described following primary EBV infection, and defective anti-EBNA antibody response may be seen, in addition to NK cell defects [11]. Approximately 10% of children with AT develop lymphoma over time. These lymphomas are large cell in type and usually present in nodal sites. EBV genome is usually not present, although several EBV-associated cases have been described. Both polyclonal and monoclonal lymphomas have been seen [8,11].
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with EBV [10, 12, 16]. Abnormal c-myc expression is not seen. Lymphoma complicating autoimmune disease(s)
AIDS-related lymphoma Clinicopathologic characteristics
The AIDS-related lymphomas are high-grade B-cell tumors, consisting of large-cell immunoblastic or small noncleaved-cell (Burkitt's or Burkitt-like) lymphomas in approximately 80%-90% of cases [27]. Intermediate-grade, large noncleaved-cell lymphomas have also been described, and are considered AIDS-defining. While low-grade B-cell lymphomas, various T-cell lymphomas, and Hodgkin's disease have been diagnosed in HTV-infected patients, their incidence has not increased with the AIDS epidemic, and they are not considered AIDS-defining conditions. Clinically, the AIDS lymphomas present in extranodal sites, occurring in approximately 80%-90% of Endemic Burkitt's lymphoma cases, in distinction to 'usual' lymphomas, in which Endemic African Burkitt's lymphoma has been de- extranodal presentation is expected in approximately scribed in areas of the world which are endemic for 40% of cases [27]. Common sites of involvement inmalaria. While not classically considered an example of clude the central nervous system (33%), gastrointesimmunodeficiency-related lymphoma, several aspects tinal tract (25%), bone marrow (25%), and other diverse sites. of lymphomatous disease do suggest a relationship. Lymphoma primary to the central nervous system Immune deficiency has been demonstrated secondary to chronic malarial infection. Thus, Whittle and col- (P-CNS lymphoma) has a particularly poor prognosis, leagues demonstrated a decrease in CD4 cells, with a and far-advanced underlying HTV-related immunodefidecrease in CD4:CD8 ratios during acute malarial in- ciency is expected. Thus, the median CD4 cell count at 3 fection in Africa [23]. Decreased CD4 cells, with de- diagnosis is 30 mm , and approximately 75% of pacreased CD4:CD8 ratios have been described in Swe- tients have been diagnosed with AIDS prior to the dedish patients with primary P. falciparum infection [24]. velopment of P-CNS lymphoma [28]. Median survival Further, impaired EBV-specific T-cell immunity has is approximately two to three months despite therapy, been described in residents of areas which are holo- and death is often due to opportunistic infection. All such lymphomas are of large-cell or immunoblastic endemic for malaria [25]. type, and EBV genome is uniformly present within Prospective studies of large numbers of children livlymphoma cells. Expression of certain latent EBV proing in the malarial belt of Africa have demonstrated teins, such as EBER, has been demonstrated in 21/21 that EBV infection, with high titers of anti-VCA antisuch cases, while LMP has been found in 45% [29]. body, may predict for the development of Burkitt's lymphoma [26]. Thus, EBV infection precedes the occurrence of lymphoma in such children. Moreover, BL is Pathogenesis of disease uniformly associated with EBV, with EBV genome The mechanisms of lymphomagenesis in AIDS lymdemonstrated within tumor cells in all cases [26]. Pathologically, the lymphomas which develop are phoma are not yet fully understood, although ongoing small noncleaved-cell lymphomas, of B-cell origin. B-cell proliferation and stimulation are integral com-
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Malignant lymphoma has been described with increased frequency in various types of autoimmune disorders, including rheumatoid arthritis (RA), and others. Employing a nationwide tumor registry in Finland, and linking this with data from patients with RA, Hakulinen and colleagues found a two-fold increase in lymphoma among patients with RA [19]. In a retrospective casecontrol study of 238 patients with RA, half of whom received cyclophosphamide, an excess of lymphoma was seen among the patients taking this drug [20]. In studying patients with miscellaneous autoimmune disorders, given various forms of immunosuppressive therapy (azathioprine in 68%, cyclophosphamide in 28%, chlorambucil in 4%), the relative risk of lymphoma was found to be 11 times greater than that expected in the general population. The lymphomas which develop in patients with autoimmune disease tend to be large-cell or immunoblastic lymphomas, of B-cell origin [21]. Extranodal disease is seen in many, and lymphoma confined to the site(s) of prior immune disease has been described [21]. Association with EBV has been confirmed in one case of lymphoma developing in a patient with RA, treated with cyclosporine [22].
Extranodal involvement is common, with lymphoma found prominently in the jaw, ileocecal area, ovaries, and other sites. Central nervous system involvement is rather unusual, occurring in 2%-3% of cases. Endemic Burkitt's lymphoma is a monoclonal tumor. Specific chromosomal translocations have been well described, including t(8;14), t(8;22), and t(8;2), ah1 involving the immunoglobulin heavy- or light-chain genes, as well as the myc oncogene, present on chromosome 8. In this regard, c-myc dysregulation has been described in all cases. Prognosis depends upon the extent of disease, and cure has been possible with single-agent cyclophosphamide, given in high dose.
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ponents of chronic HTV infection. While HTV per se is not present within lymphoma-cell DNA, HIV does induce ongoing B-cell expansion and activation, resulting in the development of reactive B-cell hyperplasia in lymphoid tissues, known as persistent generalized lymphadenopathy (PGL) [30,31]. These reactive lymphoid tissues harbor large quantities of HTV during the course of asymptomatic HIV infection [32]. The relative risk of lymphoma in patients with PGL is increased 850 times over expected [33]. Cytokine networks
EBV In addition to cytokine pathways, EBV may be implicated in the pathogenesis of AIDS lymphoma, perhaps related to the impaired immunosurveillance of EBVinfected cells in patients with AIDS [38,39]. The precise role of EBV in the pathogenesis of AIDS-related lymphoma is somewhat controversial. Shibata and colleagues studied the reactive lymphadenopathy tissues from 35 HTV-infected patients by PCR and in situ hybridization, and found EBV DNA in 13 of 35 (35%); the presence of detectable amounts of EBV DNA in these 13 reactive biopsies was associated with an increased incidence of concurrent lymphoma at another site or with development of lymphoma over time [40]. While it is apparent that AIDS-related primary CNS lymphoma is uniformly associated with latent EBV proteins, this has not been the case in AIDS-related systemic lymphoma, where EBV expression has been reported in 3 of 7 cases (42%) in one study [41], and in only 6 of 16 (38%) cases in another study [42]. Of interest, however, in analyzing 10 of these EBV-positive systemic ATDS-lymphoma cases, Neri and colleagues found evidence of clonal EBV infection in all.
Abnormal DNA rearrangements The DNA rearrangements which occur normally during B-cell differentiation may provide vulnerable, abnormal recombination-prone sites, leading to chromosomal translocations involving the Ig heavy- or lightchain genes. These recombinase errors could lead to the specific chromosomal translocations which have been described in AIDS lymphoma, including t(8; 14), t(8;22), or t(8;2) [45]. Consistent with this hypothesis, Pelicci and colleagues [46] described the development of multiple clonal rearrangements of immunoglobulin genes within reactive PGL tissues; the subsequent selection and growth advantage of one such clone could explain the eventual development of monoclonal B-cell malignancy from ongoing polyclonal B-cell response. Most series have clearly documented the genotypic and/or immunophenotypic monoclonality of AIDSrelated lymphoma [27]. Recently, however, Shiramizu and colleagues employed Southern blot analysis on DNA, as well as frozen-section immunohistochemical analysis from 40 cases of AIDS-related lymphoma, noting that approximately 40% were polyclonal in genotype [47]. While multiple oligoclonal expansions have been documented prior to the development of monoclonal lymphoma [46], the report of polyclonal lymphomas, especially comprising such a large proportion, is most unusual, and its significance awaits confirmation.
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The activation of various cytokine networks may contribute to the chronic B-cell proliferation which characterizes HTV disease. B cells from HTV-infected patients with hypergammaglobulinemia constitutively express TNF-a and IL-6 [34]. High levels of IL-6 gene expression have been demonstrated in both HIV-positive and HTV-negative cases of large-cell lymphoma, independent of EBV status [35]. The number of IL-6expressing cells has been shown to be 17 times higher in lymphoma cases versus control nodal tissues; and seven times higher in large-cell lymphomas containing a high proportion of immunoblasts [35]. Of interest, elevated serum levels of IL-6 have been documented prior to the development of large-cell lymphoma in a group of patients with symptomatic HTV infection [36]. While not unique to AIDS lymphoma, then, IL-6 may play a role in the pathogenesis of diverse types of B-cell neoplasia, in which terminally differentiated cells predominate. Aside from IL-6, IL-10 may also be operative in the development of HTV-related lymphoma [37].
Thus, EBV integration occurred before clonal B-cell expansion, which would be consistent with an etiologic role of EBV [42]. The differences between EBV expression in primary CNS versus systemic lymphoma may be a function of the site of disease, or may be related to differing histologic types, with large-cell or immunoblastic lymphoma more likely to be associated with EBV, as opposed to small noncleaved-cell lymphoma. Thus, EBV genome has been detected by in situ hybridization in 11 of 17 cases of systemic immunoblastic lymphoma (65%), versus only 1 in 5 cases of Burkitt's lymphoma [43]. However, employing Southern blot analysis, PCR and in situ hybridization in 59 cases of systemic AIDS lymphoma, Shibata and colleagues have demonstrated presence of EBV in the majority of all cases, including 85% of immunoblastic, 55% of large-cell, and 57% of small noncleaved-cell lymphomas [44]. Similarly, Emillie noted EBV genome in 66% of small noncleaved-cell, 75% of immunoblastic, and 60% of systemic large-cell lymphomas [35].While these discrepancies may be related to differing interpretations of pathologic material, it is clear that EBV cannot be the 'sole' cause of lymphoma in the setting of AIDS. Still, in those cases which are EBV-positive, mechanisms are in place which could eventuate in malignant transformation [5].
34
C-myc deregulation and other genetic abnormalities
Pathogenesis The lymphomas arising in the setting of AIDS thus involve multiple factors, including underlying immunosuppression; chronic B-cell proliferation (due to HTV, cytokine release, and/or EBV); expression of certain latent EBV viral proteins associated with cellular transformation; occurrence of recombinase errors, leading to chromosomal translocations; and subsequent dysregulation of c-myc and/or other oncogenes, as well as the inactivation of tumor-suppressor gene(s). While some of these factors may occur in the majority of cases, heterogeneity has already been encountered, indicating that distinct mechanisms of lymphomagenesis may occur in different pathologic types or sites of disease.
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The chromosomal translocations described in AIDSrelated lymphoma could result in dysregulation of the c-myc oncogene present on chromosome 8 [48]. Of note, however, c-myc dysregulation has not been uniformly documented in AIDS lymphoma, occurring in 100% of small noncleaved-cell lymphomas in one series [41], and in only a minority of large-cell or immunoblastic lymphomas in another study [49]. Moreover, the specific mechanisms leading to c-myc dysregulation are diverse in the AIDS lymphomas studied to date, with c-myc translocation, similar at the molecular level to sporadic Burkitt's lymphoma, in some series [42], while point mutations were documented in another well-studied case [50]. Furthermore, HTV-1 infection of immortalized B-cell lines, can itself result in up-regulation of c-myc transcripts [5], while HTV may directly affect cellular c-myc gene expression [7]. Regardless of the mechanism of dysregulation, myc protein is a potent regulator of cell proliferation. Dysregulation of c-myc has been shown to contribute to transformation of human B cells in vitro, and may cause B-cell lymphoma in transgenic animals carrying Ig- myc chimeric constructs [2,3]. It is apparent, then, that c-myc dysregulation may provide another mechanism for the development of lymphoma in the setting of AIDS. Mutations or losses of p53 have also been reported in approximately 60% of AIDS lymphoma cases, again restricted to the small noncleaved-cell subtype [49]. Other genetic abnormalities, including 6q deletions [51], and Ras gene mutations [49] have been described as well.
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