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HHV-8) in lymphoproliferative diseases
seminars in C A N C E R B I OLOG Y, Vol 9, 1999: pp. 165]174 Article No. scbi.1998.0118, available online at http:rrwww.idealibrary.com on
The role of Kaposi’s sarcoma-associated herpesvirus (KSHVr r HHV-8) in lymphoproliferative diseases Ethel CesarmanU and Daniel M. Knowles
The Kaposi’s sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), has been found to be present in a limited subset of lymphoproliferative disorders. Among these are the primary effusion lymphomas, formerly designated body cavity-based lymphomas, a rare type of malignant lymphoma which possesses an unusual set of clinical and biologic features, suggesting that they represent a distinct disease entity. This virus is also present in a large proportion of cases of multicentric Castleman’s disease, particularly those associated with HIV-infection. In addition, KSHV has been implicated in the pathogenesis of multiple myeloma, where it has been identified in bone marrow adherent cells but not in the neoplastic myeloma plasma cell population. However, the latter finding remains controversial. The discovery of KSHV in a subset of malignant lymphomas has allowed the development of lymphoma cell lines which now serve as biological reagents for propagating the virus, as a substrate for serologic assays, and as a model system for pathobiologic studies. This review discusses the features of KSHV-associated lymphoproliferative disorders and the evidence supporting its role in the pathogenesis of these diseases.
less of its presentation in immunocompromised or immunocompetent hosts.2 ] 6 This association appears to be quite specific, since this virus was not identified in nearly any other diseases or tissues, including a variety of vascular lesions.7] 10 Early sequencing data suggested that KSHV was a novel herpesvirus, most homologous with two g-herpesviruses, the EpsteinBarr virus ŽEBV. and herpesvirus saimiri ŽHVS..1 It is well known that EBV immortalizes B cells in vitro and is present in various types of malignant lymphoma.11 HVS is a virus of squirrel monkeys Ž Saimiri sciureus. that can be isolated from peripheral blood mononuclear cells of healthy animals but causes fulminant T-cell lymphomas in New World primates other than its natural hosts.12 HVS is also capable of transforming human T lymphocytes to continuous growth in vitro.13 Therefore, the identification of this virus in a subset of malignant lymphomas was not surprising. What was unanticipated, was the specificity of this association with a small subset of malignant lymphomas that possess distinctive and unusual clinicopathologic features, including their presentation as lymphomatous effusions in body cavities, therefore being initially called body cavity-based lymphomas ŽBCBL., and subsequently primary effusion lymphomas ŽPEL.. The finding of KSHV in this subset of lymphomas not only turned out to be important for the recognition of a distinct disease entity, but also allowed the development of cell lines as a tool to propagate this virus, serving as the basis for serologic assays and providing a reagent to study its pathobiology. We will review our current knowledge of primary effusion lymphomas in the following section. KSHV has also been identified in two other lymphoproliferative disorders: multicentric Castleman’s disease and multiple myeloma. While the presence of this virus in the latter remains controversial, the information regarding the possible role of KSHV in both of these disorders will be reviewed in sections that follow. Finally, the development of lymphoma cell lines and their uses will be discussed.
Key words: Kaposi’s sarcoma r KSHV r HHV-8 r lymphoproliferative disease Q1999 Academic Press
Introduction FOLLOWING THE IDENTIFICATION OF KSHV,1 it immediately became evident that this virus was practically invariably present in Kaposi’s sarcoma tissues, regardU
From the Department of Pathology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY, 10021, USA Q1999 Academic Press 1044-579Xr 99r 030165q 10 $30.00r 0
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Primary effusion lymphomas
immunoglobulin gene rearrangements Ž97%.; Ž5. coinfection with Epstein-Barr virus Ž86%.; Ž6. lack of c-myc gene rearrangements Ž97%.; and Ž7. lack bcl-2, ras, and p53 gene alterations Ž87%.. AIDS-related lymphomas displaying several of these features should be evaluated for the presence of KSHVrHHV-8 to confirm the diagnosis of PEL. Patients with this type of lymphoma have a very poor clinical outcome, with a median survival of 5 months. PELs are extremely rare tumors, estimated to account for approximately 3% of AIDS-related lymphomas and 0.4% of all AIDS unrelated diffuse large cell non-Hodgkin’s lymphomas.27 Nevertheless, small series and isolated cases reported by other investigators include a similar set of characteristics.20,22,27,28 An additional complication to the understanding of PELs is the finding that lymphomas containing KSHV can present as solid tissue masses, usually extranodaly, similar to other AIDS-related non-Hodgkin’s lymphomas. While some of these lymphomas subsequently develop an effusion, others apparently do not. In fact, when screening 76 cases of systemic AIDS-related lymphomas for KSHV, we identified four cases in which the presence of high viral copy numbers was confirmed by Southern blot analysis of genomic DNA and which contained this virus in practically all the lymphoma cells as demonstrated by in situ hybridisation ŽNador et al, manuscript in preparation.. In support of the notion that these fall within the spectrum of PEL are the following features: Ž1. all four patients were male homosexuals; Ž2. all four cases were classified as large cell immunoblastic lymphomas; Ž3. three of the four cases lacked the expression of lineage restricted antigens and the fourth case only expressed surface immunoglobulin; Ž4. clonal EBV infection was detected in all four cases; and Ž5. all four cases lacked oncogene and tumor suppressor gene alterations. Therefore, it appears that KSHV-associated lymphomas represent a distinct pathobiologic category which is frequently, but not exclusively, associated with a lymphomatous effusion, comprising approximately 5% or less of all AIDS-related lymphomas. These cases could be referred to as ‘extracavitary’ PELs or secondary effusion lymphomas, but regardless of nomenclature, additional studies are necessary to confirm whether all KSHV- associated lymphomas have clinical and pathobiological features, as well as response to therapy, which are similar enough to determine whether all of these should be considered to belong to the same disease entity. Cases of AIDS-related lymphoma which have been
The identification of a novel human herpesvirus in KS lesions, and sequence data showing homology to the lymphotropic g-herpesviruses ŽEBV and HVS. prompted us to assess its presence in a diverse panel of lymphoid malignancies, representing every major clinicopathologic category. We found that all eight AIDS-related PELs, but none of the remaining 185 cases of AIDS and non-AIDS related NHL, Hodgkin’s disease and lymphoid leukemia contained KSHV.14 These lymphomas contained very high amounts of viral DNA, ranging between 40 and 80 copies per cell. Therefore, this virus was easily identifiable by Southern blot analysis, in contrast to KS tissues, which may contain less that one copy per cell, making it necessary to perform PCR for its detection in some cases. The first reported cases of malignant lymphoma occurring as body cavity effusions were described as AIDS-associated lymphohematopoietic neoplasms displaying an indeterminate immunophenotype.15 In this report of three cases, two of which were lymphomatous effusions, a B cell lineage and the presence of EBV were demonstrated using DNA-based assays. Subsequent studies recognized these lymphomatous effusions as a distinct subset of AIDS-related lymphomas.16 ] 18 The specific association between KSHV and PEL has been confirmed by several investigators.19 ] 23 In addition, cases of PEL occurring in HIV-negative men as well as women have been identified, and these cases also contain KSHV.24 ] 27 However, lymphomas other than PELs can involve body cavities as lymphomatous effusions, even in the absence of a tumor mass. Effusions are particularly common in Burkitt’s lymphomas, but can also be seen in other non-Hodgkin’s lymphomas. Therefore, certain criteria should be used for the diagnosis of PEL.25 In our experience, which includes a total of 33 cases, lymphomas containing KSHV have the following features: Ž1. presentation as a lymphomatous effusion in the pleural, peritoneal andror pericardial cavity without a contiguous tumor mass Ž86%., frequently remaining localized to the body cavity of origin Ž81%.; Ž2. morphology bridging large cell immunoblastic lymphoma and anaplastic large cell lymphoma Ž100%. ŽFigure 1, Panel A.; Ž3. expression of CD45 and one or more activation-associated antigens Ž95%. in the frequent absence of B cell-associated antigens Ž95%. and immunoglobulin expression Ž76%.; Ž4. B cell origin as demonstrated by the presence of clonal 166
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Figure 1. KSHV in primary effusion lymphomas. ŽA. Wright-Giemsa-stained air dried cytocentrifuge preparation of a KSHV-positive primary effusion lymphoma. The cells are considerably larger than normal benign lymphocytes and exhibit cytomorphologic features that appear to bridge large cell immunoblastic lymphoma and anaplastic large-cell lymphoma. The cells display significant polymorphism and possess moderately abundant amphophilic to deeply basophilic cytoplasm. A prominent clear perinuclear Golgi zone is frequently present. The nuclei vary from large and round to highly irregular, multilobated and pleomorphic and often contain one or more prominent nucleoli Žoriginal magnification= 1000.. ŽB. In situ hybridisation for KSHV using a viral cyclin probe. A case of primary effusion lymphoma with solid tissue involvement shows cytoplasmic and nuclear hybridisation signals in practically all lymphoma cells. A spindle-
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positive for KSHV by PCR but not by Southern blot analysis have been identified by us as well as by others,22,23 suggesting low viral copy numbers in the tumor tissue. In situ hybridisation studies of these cases have shown that the virus is present only in scattered atypical cells and some reactive appearing cells in these cases ŽNador et al, manuscript in preparation.. The cases we identified were not reminiscent of PEL, since they exhibited a polymorphic morphology and expression of B cell associated antigens CD19 and CD20. While KSHV may be playing an indirect role in these lymphomas as well, cases containing low copy numbers of KSHV should not be considered part of the spectrum of PEL. Further studies are necessary to determine whether KSHV is acting as an antigenic stimulus or using paracrine mechanisms to stimulate a proliferation of B cells and therefore is involved in the pathogenesis of these lesions, or whether its presence in these lymphomas is merely the result of disseminated viremia in KSHV-positive patients. Closer examination of PELs has provided information about the biology of this type of disease, and its place in the spectrum of non-Hodgkin’s lymphomas. Most cases have been B cell lymphomas, as determined by the presence of clonal immunoglobulin gene rearrangements. However, two biphenotypic cases expressing CD3 have been identified, both of which contained B and T cell antigen receptor gene rearrangements.22,29 PELs usually lack expression of B-cell associated antigens, although most of them express monotypic k or l mRNA30 and some cases have been reported to express surface or cytoplasmic immunoglobulin.22,25 In our experience, faint but distinct cytoplasmic staining using antisera to k or l can be seen in a subset of cells in some PEL cell lines Žunpublished observation.. Most PELs are thought to originate from post-germinal center B cells, since they have hypermutation of the immunoglobulin genes,31 although this association is not strict as one case that is not mutated and a second case with ongoing mutations were identified, suggesting they can be of pre-germinal center origin, or can be arrested in the germinal center stage. While most cases have functional rearrangements, many do not express immunoglobulin proteins. The reason for this discrepancy is unclear.
In addition to an immunoblastic morphology, PELs have a set of immunophenotypic features suggesting that they are at a preterminal stage of B cell differentiation. Loss of expression of B cell antigens occurs in plasma cells, and this is a frequent finding in multiple myeloma as well as in immunoblastic lymphom as. Furtherm ore, m ost PELs express CD138rSyndecan-1, an adhesion molecule which is selectively expressed by a subset of pre-B cells and by plasma cells, including myeloma plasma cells. The expression of CD138 by PELs seems to be quite specific, as it is not expressed by other lymphomatous effusions, primary or secondary, or by most other solid lymphomas.32 The almost invariable presence of KSHV in lymphomas having the features described above suggests that this virus is necessary for the development of PELs. However, since PELs are so uncommon, even in populations where the seroprevalence of KSHV is relatively high, it is evident that infection by this virus represents only one of several genetic events involved in their development. One such other factor appears to be EBV infection, as the vast majority of PELs, especially in immunocompromised hosts, contain both viral genomes. The specific role of each of these viruses and their interaction is still poorly understood, but analysis of the genes expressed by both of them has shed some light into their possible roles. Both herpesviruses can be lytic or latent, expressing distinct subsets of genes. PELs in vivo, as well as in culture Žsee below. express mostly latent genes, but there is always a small proportion of cells in which EBV and KSHV lytic gene expression occurs. However, most cells have a latent pattern of gene expression. It is known that EBV can establish different types of latency. Latency type I Žrestricted latent gene expression. is seen in Burkitt’s lymphomas, while latency type III Žfull pattern of latent gene expression. is seen in lymphoblastoid cell lines and large cell lymphomas in immunocompromised patients, particularly those with immunoblastic features. While the EBNA1 gene, necessary for EBV replication, is expressed in all latency types, other latent genes, including the EBV transforming genes LMP1 and EBNA2, are only expressed in latencies type II ŽLMP1. and type III ŽLMP1 and all EBNAs.. It is thought that expression of these transforming genes in Burkitt’s
shaped cell which is negative for KSHV hybridisation signals is seen in the lower left corner of this panel Žoriginal magnification= 630.. ŽC. Abundant viral IL-6 protein expression is seen in numerous lymphoma cells by immunohistochemistry using a rabbit polyclonal antiserum raised against a vIL-6-specific peptide Žoriginal magnification= 400..
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lymphomas is not ‘necessary’, since these carry a translocated c-myc oncogene, and the lack of expression of these immunogenic proteins further provides this type of lymphoma with an advantage to evade the immune system. Analysis of the pattern of EBV gene expression in PELs revealed that only EBNA1 was expressed, corresponding to type I latency.33,34 This was an unexpected finding, given the resemblance of PEL cells to immunoblastic lymphoma cells. This observation suggests that KSHV is playing a transforming role in PELs, as the major EBV oncogenes are not expressed. Also consistent with this hypothesis is the lack of structural alterations in the cellular transforming genes frequently involved in malignant lymphomas. In particular, c-myc gene rearrangements have only been identified in one case Žpersonal unpublished observation., being extremely uncommon in contrast to other lymphomatous effusions and EBV-associated lymphomas, where alterations in this gene are a frequent finding. While mutations of the p53 gene have only been identified in one case of PEL,25 mutations in the non-coding, presumably regulatory region of the BCL-6 gene have been reported in a significant proportion of cases.35 The BCL-6 gene is frequently mutated in diffuse large B cell lymophomas, although the functional significance of these mutations remains unclear. Cytogenetic analyses have not provided concrete additional evidence for the presence of a single specific genetic alteration. Of nine PEL cell lines examined, it appears that numerous abnormalities are present, with complex hyperdiploid karyotypes. Frequent abnormalities are trisomy 7, trisomy 12 and aberrations of chromosomal bands 1q21-q25. 36 Translocations specific to other lymphoma types have not been identified. Since there has been no demonstration of expression of EBV transforming genes, or alterations of cellular oncogenes, with the possible exception of mutations in the BCL-6 gene, expression of KSHVencoded transforming genes may be particularly relevant. The genome of KSHV has been found to contain at least 11 open reading frames ŽORFs. that encode homologues to cellular proteins involved in signal transduction, cell cycle regulation andror inhibition of apoptosis.37 To this date, two of these genes have been found to be transforming in transfected cells, andror to cause tumors when these are injected into immunodeficient mice, namely the viral interferon regulatory factor ŽK9, vIRF.,38 ] 40 and the viral G protein-coupled receptor ŽORF 74, KSHV GPCR..41,42 Two additional genes with no known
cellular homolog have been found to be transforming in cell culture systems: K143 and K12 Žkaposin..44 At least two chemokines ŽvMIP I and II, K6 and K4. are encoded by KSHV.45 This virus also contains ORFs homologous to cellular oncogenes involved in lymphomagenesis, and are thus potentially involved in the pathogenesis of PELs. These are the viral cyclin D ŽORF 72., which is homologous to the bcl-1 gene, and the viral bcl-2 ŽORF 16.. KSHV also encodes a homolog of interleukin 6 ŽK2, vIL6., an important growth and differentiation factor for B cells, and a vFLIP ŽK13., an inhibitor of apoptosis.46 While all of these genes appear to be transcribed in PELs, the levels are highly variable. Latent expression in PELs has been well documented for the vIL-6 using specific antisera45 ŽFigure 1, Panel C.. The viral cyclin D ŽORF72. gene is also transcribed during latency in PELs within the neoplastic cell population, as determined by in situ hybridisation ŽFigure 1, Panel B. ŽNador, unpublished observations.. The kaposin ŽK12., vIRF ŽK9., vMIP-II and vFLIP ŽK13. genes have been found to be transcribed during latency in PEL cell lines,47,48 so they are likely to be expressed in PELs in vivo. Lytic genes with ‘transforming’ potential are the K1 protein, vMIP-I, vBCL-2, and GPCR. Although in situ localisation for each of these genes has not been performed, there is indirect evidence to suggest that these lytic genes are expressed only by a subset of PEL cells in vivo. While a viral transforming gene is expected to be expressed by most, if not all, tumor cells, paracrine effects may be relevant to the pathogenesis of KS,42,49,50 and have been postulated to play a role in multiple myeloma.51 Therefore, some of these lytic genes may also be involved in the pathogenesis of PEL.
Multicentric Castleman’s disease KSHV has also been found to be present in multicentric Castleman’s disease ŽMCD..52 ] 54 This virus is present in almost all cases of MCD occurring in patients with AIDS. In fact, in HIV positive patients MCD has been found to be frequently associated with KS, and is usually observed in men infected with HIV by sexual contact.55 Notably, MCD, also called multicentric angiofollicular hyperplasia, is characterized by a vascular proliferation in the germinal centers which is reminiscent of KS. However, this virus is present in MCD in patients with AIDS whether accompanied by KS or not. Furthermore, KSHV has also been identified in approximately half of the 169
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cases of MCD occurring in HIV-negative individuals, suggesting that it is not a coincidental association occurring in HIV positive individuals. KSHV sequences have also been detected in peripheral blood mononuclear cells of patients with MCD.52 Multicentric Castleman’s disease is an atypical lymphoproliferative disorder thought to be related to immune dysregulation.56 This disease occurs more frequently in older individuals, and is more common in men. The patients usually present with multiple lymphadenopathies and a variety of constitutional symptoms. They may develop auto-immune phenomena, cytopenias, skin rashes and intercurrent infections. Patients with MCD frequently develop malignancies, most commonly KS and NHL.52 The identification of KSHV in AIDS and non-AIDS-associated MCD supports an even closer relationship between MCD and KS than previously hypothesized, as in both KSHV may be playing a crucial role. The role of KSHV in the pathobiology of MCD remains poorly understood, and it is not clear whether the KSHV-positive cases differ clinically from KSHV-negative ones in individuals without AIDS. It has been shown that the germinal centers of hyperplastic lymph nodes of patients with Castleman’s disease produce large quantities of interleukin 6 ŽIL-6 .. This may explain the large proportion of plasma cells in these lymph nodes, since IL-6 induces B-cell differentiation. Notably, KSHV encodes for a viral IL-6 homolog, which is also expressed in MCD in scattered cells surrounding the lymphoid follicles.45,57
stimulate the proliferation of the myeloma plasma cells. Multiple studies have been published since, with conflicting results. Two additional independent laboratories confirmed the specific presence of KSHV DNA sequences in multiple myeloma biopsies.60,61 However, other investigators have been unable to confirm this association when looking for KSHV DNA by PCR in bone marrow biopsies andror dendritic cell cultures from bone marrow or peripheral blood.62 ] 71 Serologic studies have been almost uniform in their findings, demonstrating that patients with multiple myeloma lack antibodies to KSHV.62,63,65,68,72 ] 75 This lack of reactivity is not due to generalized immunosuppression, since antibodies to other more ubiquitous herpesviruses, such as EBV and CMV, are easily detectable in these patients using comparable methodologies. Our own group has also been unable to confirm the association between KSHV infection and multiple myeloma, using a variety of methods Žmanuscript in preparation.. In one paper, very weak seroreactivity to KSHV antigens was detected using immunoblotting.76 While this may be confirmatory of the association between KSHV infection and multiple myeloma, it is possible that this low reactivity represents higher levels of non-specific cross-reactivity than in control patients because of the gammopathy in multiple myeloma patients. Furthermore, the epidemiologic patterns of Kaposi’s sarcoma and multiple myeloma are distinct, suggesting different etiologies.77,78 Three explanations can be proposed to explain this controversy: Ž1. KSHV is not present in multiple myeloma or MGUS, and the identification of viral sequences by a fraction of investigators is due to technical artifacts, such as PCR contamination; Ž2. KSHV is present in such low copy numbers in bone marrow and dendritic cell preparations in patients with multiple myeloma that the technique utilized by most laboratories is not sufficiently sensitive to detect them, although in several studies sensitivity controls suggest detection of 1 to 10 copies of KSHV. In this instance, the absent or very low serologic reactivity remains to be explained; it has been hypothesized that HHV-8 localisation within the bone marrow-based dendritic cells may prevent the maturation of an antigenic response due to B-cell tolerance of antigens presented in the bone marrow.79 This reasoning would not explain, however, the discrepant epidemiology of both diseases; and Ž3. a different virus is present in multiple myeloma, similar enough to be recognized by some PCR primers when certain protocols with low stringency are used. Antibodies to this novel virus may not recognize most
Multiple myeloma Initial studies evaluating the presence of KSHV in large series of lymphoid proliferations failed to identify this virus in multiple myeloma or other plasma cell malignancies.14,20,22,23 However, an association between this virus and multiple myeloma was subsequently reported.58 In that study, KSHV DNA and RNA sequences were detected in bone marrow stromal cell cultures from patients with multiple myeloma, as well as two of eight patients with monoclonal gammopathy of undetermined significance ŽMGUS.. This result, which was based on PCR and RT-PCR analyses, was subsequently confirmed by the same team using in situ hybridisation and PCR analysis of DNA extracted from fresh bone marrow core biopsies.59 Since transcripts KSHV vIL-6 were detected, it was hypothesized that KSHV uses a paracrine mechanism, through production of this cytokine, to 170
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KSHV antigens. This explanation may be supported by sequence analysis of PCR products from specimens obtained from patients with multiple myeloma, where significant interpatient variation has been noted, and the reported higher homology between different myeloma patients than when these sequences are compared with KSHV for KS specimens.79 One study has suggested that sequences homologous to one of the regions of KSHV is widely disseminated in a variety of tissues, including multiple myeloma.66 While this last possibility appears to be the most conciliatory explanation, much additional evidence needs to be provided to confirm the presence of a distinct KSHV-like virus and then to confirm its specific association with multiple myeloma and MGUS.
the number of potential transforming genes in KSHV, and its clear association with a subset of malignant lymphomas, it is surprising that this virus is not obviously transforming when lymphoid cells are infected in culture. This observation is consistent with the requirement of other transforming events for KSHV to play a role in the development of PELs. It is also likely that having a high viral copy number in each cell, as well as a particular program of gene expression, are necessary for transformation by this virus, neither of which has been achieved so far in cell culture experiments.
Concluding remarks The identification of KSHV in PEL has led to the recognition of a rare but distinct type of malignancy. Subsequent studies have revealed the possible existence of an ‘extracavitary’ form of this disease and the realisation that perhaps some of the systemic lymphomas, in particular a fraction of those with immunoblastic morphology occurring in HIV-positive individuals, may also belong to the spectrum of KSHV-associated lymphomas. In addition, this virus is unquestionably associated with MCD in the setting of AIDS, as well as in half of cases occurring in HIVnegative individuals. The recognition of the presence of this virus in PEL and MCD, and the eventual understanding of the mechanisms utilized by KSHV in the pathobiology of these diseases, should lead to the identification of rational therapeutic approaches. As both diseases are incurable and deadly at the present time, this is an important ongoing line of investigation. A third disease in which a possible association with KSHV infection has been reported is multiple myeloma. However, this association remains highly controversial, and subsequent studies are necessary to determine whether an as yet undescribed KSHV-like virus is present in this type of lymphoid malignancy. The recognition of KSHV in PEL has resulted in the development of cell lines that have been crucial for the rapid characterisation of this virus, and for the large number of seroepidemiologic studies that have provided strong evidence supporting a causal role for KSHV in the development of KS.
Primary effusion lymphoma cell lines The recognition of the presence of KSHV in PEL allowed the establishment of cell lines to be used as a biological tool for subsequent studies. These cell lines still represent the most efficient method for propagating KSHV in culture, and for obtaining relatively large quantities of this virus. We derived the first KSHV-positive cell lines from two patients with PEL.80 Both of these cell lines also contained the EBV genome, but nevertheless one of them, called BC-1, served as a source of virus for genomic sequencing,81,82 including the first almost full length sequence,37 and for the first serologic assays.83,84 Subsequently, several cell lines containing KSHV but not EBV were established, called BC-3 85 and KS-1 Žestablished independently from the same patient specimen.,86 BCBL-1, 87 BCP-1, 88 and CRO-APr3. 36 A comprehensive review of the features of all these cell lines has been recently published.36 Serologic assays for the presence of KSHV antibodies are now frequently done by immunofluorescence using one of the PEL cell lines as a substrate. Detection of antibodies to latent proteins can be done using untreated cultured cells, while for detection of antibodies to lytic antigens these cells can be treated to induce lytic viral replication using either TPA or butyrate. Another recently developed method for screening for the presence of antibodies to this virus is a whole virus ELISA,89 which also utilizes viral particles purified from a PEL cell line. PEL cell lines have also been used to purify viral particles to infect lymphoid82,90,91 and endothelial cells.50,92 While both cell types can be infected, only endothelial cells seem to be altered by KSHV. Given
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19. Karcher DS, Alkan S Ž1995. Herpes-like DNA sequences, AIDS-related tumors, and Castleman’s disease ŽLetter to the Editor.. N Engl J Med 333:797]798 20. Pastore C, Gloghini A, Volpe G, Nomdedeu J, Leonardo E, Mazza U, Saglio G, Carbone A, Gaidano G Ž1995. Distribution of Kaposi’s sarcoma herpesvirus sequences among lymphoid malignancies in Italy and Spain. Br J Haematol 91:918]920 21. Carbone A, Tirelli U, Gloghini A, Pastore C, Vaccher E, Gaidano G Ž1996. Herpesvirus-like, DNA sequences selectively cluster with body cavity-based lymphomas throughout the spectrum of AIDS-related lymphomatous effusions ŽLetter to the Editor.. Eur J Cancer 32A:555]556 22. Otsuki T, Kumar S, Ensoli B, Kingma DW, Yano T, StetlerStevenson M, Jaffe ES, Raffeld M Ž1996. Detection of HHV8rKSHV DNA sequences in AIDS-associated extranodal lymphoid malignancies. Leukemia 10:1358]1362 23. Gessain A, Briere ` J, Angelin-Duclos C, Valensi F, Merle Beral ´ H, Davi F, Nicola M-A, Sudaka A, Fouchard N, Gabarre J, Troussard X, Dulmet E, Audouin J, Diebold J, de The ´ G Ž1997. Human herpes virus 8 ŽKapsoi’s sarcoma herpes virus. and malignant lymphoproliferations in France: a molecular study of 250 cases including two AIDS-associated body cavity based lymphomas. Leukemia 11:266]272 24. Nador RG, Cesarman E, Knowles DM, Said JW Ž1995. Herpes-like DNA sequences in a body cavity-based lymphoma in an HIV-negative patient ŽLetter to the Editor.. N Engl J Med 333:943 25. Nador RG, Cesarman E, Chadbum A, Dawson DB, Ansari MQ, Said J, Knowles DM Ž1996. Primary effusion lymphoma: a distinct clinicopathologic entity associated with the Kaposi’s sarcoma-associated herpesvirus. Blood 88:645]656 26. Said JW, Tasaka T, Takeuchi S, Asou H, de Vos S, Cesarman E, Knowles DM, Koeffler HP Ž1996. Primary effusion lymphoma in women: Report of two cases of Kaposi’s sarcomaherpes virus-associated effusion-based lymphoma in human immunodeficiency virus- negative women. Blood 88: 3124]3128 27. Carbone A, Gloghini A, Vaccher E, Zagonel V, Pastore C, Dalla Palma P, Branz F, Saglio G, Volpe R, Tirelli U, Gaidano G Ž1996. Kaposi’s sarcoma-associated herpesvirus DNA sequences in AIDS-related and AIDS-unrelated lymphomatous effusions. Br J Haematol 94:533]543 28. Karcher DS, Alkan S Ž1997. Human herpesvirus-6 associated body cavity based lymphoma in human immunodeficiency virus-infected patients: a unique B-cell neoplasm. Hum Pathol 28:801]808 29. Said JW, Shintaku IP, Asou H, de Vos S, Baker J, Hanson G, Cesarman E, Nador R, Koeffler HP ŽIn press. Herpesvirus 8 inclusions in primary effusion lymphoma: Report of a unique case with T-cell phenotype. Arch Pathol Lab Med 30. Gaidano G, Pastore C, Gloghini A, Volpe G, Capello D, Polito P, Vaccher E, Tirelli U, Saglio G, Carbone A Ž1997. Human herpesvirus type-8 ŽHHV-8 . in haematopoietic neoplasia. Leuk Lymphoma 24:257]266 31. Matolcsy A, Nador RG, Cesarman E, Knowles DM Ž1998. Immunoglobulin VH gene mutational analysis suggests that primary effusion lymphomas derive from different stages of B cell maturation. Am J Pathol 153:1609]1614 32. Gaidano G, Gloghini A, Gattei V, Rossi MT, Cilia AM, Godeas C, Degan M, Perin T, Canzonieri V, Aldinucci D, Saglio G, Carbone A, Pinto A Ž1997. Association of Kaposi’s sarcomaassociated herpesvirus-positive primary effusion lymphoma with expression of the CD138rsyndecan-1 antigen. Blood 90:4894]4900 33. Horenstein MG, Nador RG, Chadburn A, Hyjek EM, Inghirami G, Knowles DM, Cesarman E Ž1997. Epstein-Barr virus latent gene expression in primary effusion lymphomas con-
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Blood dendritic cells from myeloma patients are not infected with Kaposi’s sarcoma-associated herpesvirus ŽKSHVrHHV-8 .. Blood 92:402]404 Cathomas G, Stalder A, Kurrer MO, Regamey N, Erb P, Joller-Jemelka HI Ž1998. Multiple myeloma and HHV8 infection. Blood 91:4391]4393 Tisdale JF, Stewart AK, Dickstein B, Little RF, Dube I, Cappe D, Dunbar CE, Brown KE Ž1998. Molecular and serological examination of the relationship of human herpesvirus 8 to multiple myeloma: orf 26 sequences in bone marrow stroma are not restricted to myeloma patients and other regions of the genome are not detected. Blood 92:2681]2687 Tarte K, Olsen SJ, Yang Lu Z, Legouffe E, Rossi JF, Chang Y, Klein B Ž1998. Clinical-grade functional dendritic cells from patients with multiple myeloma are not infected with Kaposi’s sarcoma-associated herpesvirus. Blood 91:1852]1857 Mitterer M, Mair W, Gatti D, Sheldon J, Vachula M, Coser P, Schultz TF Ž1998. Dendritic cells derived from bone marrow and CD34q selected blood progenitor cells of myeloma patients, cultured in serum-free media, do not contain the Kaposi sarcoma herpesvirus genome. Br J Haematol 102:1338]1340 Perna AM, Viviano E, Iannitto E, Marceno R, Romano N Ž1998. No association between human herpesvirus type 8 infection and multiple myeloma. J Natl Cancer Inst 90: 1013]1014 Bouscary D, Dupin N, Fichelson S, Grandadam M, FontenayRoupie M, Marcelin AG, Blanche P, Picard F, Freyssinier JM, Ravaud P, Dreyfus F, Calvez V Ž1998. Lack of evidence of an association between HHV-8 and multiple myeloma. Leukemia 12:1840]1841 Tarte K, Olsen SJ, Rossi JF, Legouffe E, Lu ZY, Jourdan M, Chang Y, Klein B Ž1998. Kaposi’s sarcoma-associated herpesvirus is not detected with immunosuppression in multiple myeloma. Blood 92:2186]2188 Whitby D, Boshoff C, Luppi M, Torelli G Ž1997. Kaposi’s sarcoma- associated herpesvirus infection and multiple myeloma. Science 278:1971]1972 MacKenzie J, Sheldon J, Morgan G, Cook G, Schulz TF, Jarrett RF Ž1997. HHV-8 and multiple myeloma in the UK wletterx. Lancet 350:1144]1145 Marcelin AG, Dupin N, Bouscary D, Bossi P, Cacoub P, Ravaud P, Calvez V Ž1997. HHV-8 and multiple myeloma in France wletterx. Lancet 350:1144]1144 Santarelli R, Angeloni A, Farina A, Gonnella R, Gentile G, Martino P, Petrucci MT, Mandelli F, Frati L, Faggioni A Ž1998. Lack of serologic association between human herpesvirus-8 infection and multiple myeloma and monoclonal gammopathies of undetermined significance. J Natl Cancer Inst 90:781]782 Gao SJ, Alsina M, Deng JH, Harrison CR, Montalvo EA, Leach CT, Roodman GD, Jenson HB Ž1998. Antibodies to Kaposi’s sarcoma-associated herpesvirus Žhuman herpesvirus 8. in patients with multiple myeloma. J Inf Dis 178:846]849 Hjalgrim H, Frisch M, Melbye M Ž1998. Incidence rates of classical Kaposi’s sarcoma and multiple myeloma do not correlate. Br J Cancer 78:419]420 Cottoni F, Uccini S Ž1997. Kaposi’s sarcoma-associated herpesvirus infection and multiple myeloma. Science 278:1972 Said JW, Heppner K, Shintaku IP, Schrage M, Green E, Rettig MR, Vescio RA, Schiller G, Ma HJ, Belson D, Savage A, Berenson JR, Koeffler HP, Asou H, Pinkus G, Pinkus J Ž1998. Multiple myeloma and HHV8 infection. Response. Blood 91:4392]4393 Cesarman E, Moore PS, Rao P, Inghirami G, Knowles DM,
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The role of Kaposi’s sarcoma-associated herpesvirus (KSHVr r HHV-8) in lymphoproliferative diseases Ethel CesarmanU and Daniel M. Knowles
The Kaposi’s sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV-8), has been found to be present in a limited subset of lymphoproliferative disorders. Among these are the primary effusion lymphomas, formerly designated body cavity-based lymphomas, a rare type of malignant lymphoma which possesses an unusual set of clinical and biologic features, suggesting that they represent a distinct disease entity. This virus is also present in a large proportion of cases of multicentric Castleman’s disease, particularly those associated with HIV-infection. In addition, KSHV has been implicated in the pathogenesis of multiple myeloma, where it has been identified in bone marrow adherent cells but not in the neoplastic myeloma plasma cell population. However, the latter finding remains controversial. The discovery of KSHV in a subset of malignant lymphomas has allowed the development of lymphoma cell lines which now serve as biological reagents for propagating the virus, as a substrate for serologic assays, and as a model system for pathobiologic studies. This review discusses the features of KSHV-associated lymphoproliferative disorders and the evidence supporting its role in the pathogenesis of these diseases.
less of its presentation in immunocompromised or immunocompetent hosts.2 ] 6 This association appears to be quite specific, since this virus was not identified in nearly any other diseases or tissues, including a variety of vascular lesions.7] 10 Early sequencing data suggested that KSHV was a novel herpesvirus, most homologous with two g-herpesviruses, the EpsteinBarr virus ŽEBV. and herpesvirus saimiri ŽHVS..1 It is well known that EBV immortalizes B cells in vitro and is present in various types of malignant lymphoma.11 HVS is a virus of squirrel monkeys Ž Saimiri sciureus. that can be isolated from peripheral blood mononuclear cells of healthy animals but causes fulminant T-cell lymphomas in New World primates other than its natural hosts.12 HVS is also capable of transforming human T lymphocytes to continuous growth in vitro.13 Therefore, the identification of this virus in a subset of malignant lymphomas was not surprising. What was unanticipated, was the specificity of this association with a small subset of malignant lymphomas that possess distinctive and unusual clinicopathologic features, including their presentation as lymphomatous effusions in body cavities, therefore being initially called body cavity-based lymphomas ŽBCBL., and subsequently primary effusion lymphomas ŽPEL.. The finding of KSHV in this subset of lymphomas not only turned out to be important for the recognition of a distinct disease entity, but also allowed the development of cell lines as a tool to propagate this virus, serving as the basis for serologic assays and providing a reagent to study its pathobiology. We will review our current knowledge of primary effusion lymphomas in the following section. KSHV has also been identified in two other lymphoproliferative disorders: multicentric Castleman’s disease and multiple myeloma. While the presence of this virus in the latter remains controversial, the information regarding the possible role of KSHV in both of these disorders will be reviewed in sections that follow. Finally, the development of lymphoma cell lines and their uses will be discussed.
Key words: Kaposi’s sarcoma r KSHV r HHV-8 r lymphoproliferative disease Q1999 Academic Press
Introduction FOLLOWING THE IDENTIFICATION OF KSHV,1 it immediately became evident that this virus was practically invariably present in Kaposi’s sarcoma tissues, regardU
From the Department of Pathology, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY, 10021, USA Q1999 Academic Press 1044-579Xr 99r 030165q 10 $30.00r 0
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immunoglobulin gene rearrangements Ž97%.; Ž5. coinfection with Epstein-Barr virus Ž86%.; Ž6. lack of c-myc gene rearrangements Ž97%.; and Ž7. lack bcl-2, ras, and p53 gene alterations Ž87%.. AIDS-related lymphomas displaying several of these features should be evaluated for the presence of KSHVrHHV-8 to confirm the diagnosis of PEL. Patients with this type of lymphoma have a very poor clinical outcome, with a median survival of 5 months. PELs are extremely rare tumors, estimated to account for approximately 3% of AIDS-related lymphomas and 0.4% of all AIDS unrelated diffuse large cell non-Hodgkin’s lymphomas.27 Nevertheless, small series and isolated cases reported by other investigators include a similar set of characteristics.20,22,27,28 An additional complication to the understanding of PELs is the finding that lymphomas containing KSHV can present as solid tissue masses, usually extranodaly, similar to other AIDS-related non-Hodgkin’s lymphomas. While some of these lymphomas subsequently develop an effusion, others apparently do not. In fact, when screening 76 cases of systemic AIDS-related lymphomas for KSHV, we identified four cases in which the presence of high viral copy numbers was confirmed by Southern blot analysis of genomic DNA and which contained this virus in practically all the lymphoma cells as demonstrated by in situ hybridisation ŽNador et al, manuscript in preparation.. In support of the notion that these fall within the spectrum of PEL are the following features: Ž1. all four patients were male homosexuals; Ž2. all four cases were classified as large cell immunoblastic lymphomas; Ž3. three of the four cases lacked the expression of lineage restricted antigens and the fourth case only expressed surface immunoglobulin; Ž4. clonal EBV infection was detected in all four cases; and Ž5. all four cases lacked oncogene and tumor suppressor gene alterations. Therefore, it appears that KSHV-associated lymphomas represent a distinct pathobiologic category which is frequently, but not exclusively, associated with a lymphomatous effusion, comprising approximately 5% or less of all AIDS-related lymphomas. These cases could be referred to as ‘extracavitary’ PELs or secondary effusion lymphomas, but regardless of nomenclature, additional studies are necessary to confirm whether all KSHV- associated lymphomas have clinical and pathobiological features, as well as response to therapy, which are similar enough to determine whether all of these should be considered to belong to the same disease entity. Cases of AIDS-related lymphoma which have been
The identification of a novel human herpesvirus in KS lesions, and sequence data showing homology to the lymphotropic g-herpesviruses ŽEBV and HVS. prompted us to assess its presence in a diverse panel of lymphoid malignancies, representing every major clinicopathologic category. We found that all eight AIDS-related PELs, but none of the remaining 185 cases of AIDS and non-AIDS related NHL, Hodgkin’s disease and lymphoid leukemia contained KSHV.14 These lymphomas contained very high amounts of viral DNA, ranging between 40 and 80 copies per cell. Therefore, this virus was easily identifiable by Southern blot analysis, in contrast to KS tissues, which may contain less that one copy per cell, making it necessary to perform PCR for its detection in some cases. The first reported cases of malignant lymphoma occurring as body cavity effusions were described as AIDS-associated lymphohematopoietic neoplasms displaying an indeterminate immunophenotype.15 In this report of three cases, two of which were lymphomatous effusions, a B cell lineage and the presence of EBV were demonstrated using DNA-based assays. Subsequent studies recognized these lymphomatous effusions as a distinct subset of AIDS-related lymphomas.16 ] 18 The specific association between KSHV and PEL has been confirmed by several investigators.19 ] 23 In addition, cases of PEL occurring in HIV-negative men as well as women have been identified, and these cases also contain KSHV.24 ] 27 However, lymphomas other than PELs can involve body cavities as lymphomatous effusions, even in the absence of a tumor mass. Effusions are particularly common in Burkitt’s lymphomas, but can also be seen in other non-Hodgkin’s lymphomas. Therefore, certain criteria should be used for the diagnosis of PEL.25 In our experience, which includes a total of 33 cases, lymphomas containing KSHV have the following features: Ž1. presentation as a lymphomatous effusion in the pleural, peritoneal andror pericardial cavity without a contiguous tumor mass Ž86%., frequently remaining localized to the body cavity of origin Ž81%.; Ž2. morphology bridging large cell immunoblastic lymphoma and anaplastic large cell lymphoma Ž100%. ŽFigure 1, Panel A.; Ž3. expression of CD45 and one or more activation-associated antigens Ž95%. in the frequent absence of B cell-associated antigens Ž95%. and immunoglobulin expression Ž76%.; Ž4. B cell origin as demonstrated by the presence of clonal 166
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Figure 1. KSHV in primary effusion lymphomas. ŽA. Wright-Giemsa-stained air dried cytocentrifuge preparation of a KSHV-positive primary effusion lymphoma. The cells are considerably larger than normal benign lymphocytes and exhibit cytomorphologic features that appear to bridge large cell immunoblastic lymphoma and anaplastic large-cell lymphoma. The cells display significant polymorphism and possess moderately abundant amphophilic to deeply basophilic cytoplasm. A prominent clear perinuclear Golgi zone is frequently present. The nuclei vary from large and round to highly irregular, multilobated and pleomorphic and often contain one or more prominent nucleoli Žoriginal magnification= 1000.. ŽB. In situ hybridisation for KSHV using a viral cyclin probe. A case of primary effusion lymphoma with solid tissue involvement shows cytoplasmic and nuclear hybridisation signals in practically all lymphoma cells. A spindle-
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positive for KSHV by PCR but not by Southern blot analysis have been identified by us as well as by others,22,23 suggesting low viral copy numbers in the tumor tissue. In situ hybridisation studies of these cases have shown that the virus is present only in scattered atypical cells and some reactive appearing cells in these cases ŽNador et al, manuscript in preparation.. The cases we identified were not reminiscent of PEL, since they exhibited a polymorphic morphology and expression of B cell associated antigens CD19 and CD20. While KSHV may be playing an indirect role in these lymphomas as well, cases containing low copy numbers of KSHV should not be considered part of the spectrum of PEL. Further studies are necessary to determine whether KSHV is acting as an antigenic stimulus or using paracrine mechanisms to stimulate a proliferation of B cells and therefore is involved in the pathogenesis of these lesions, or whether its presence in these lymphomas is merely the result of disseminated viremia in KSHV-positive patients. Closer examination of PELs has provided information about the biology of this type of disease, and its place in the spectrum of non-Hodgkin’s lymphomas. Most cases have been B cell lymphomas, as determined by the presence of clonal immunoglobulin gene rearrangements. However, two biphenotypic cases expressing CD3 have been identified, both of which contained B and T cell antigen receptor gene rearrangements.22,29 PELs usually lack expression of B-cell associated antigens, although most of them express monotypic k or l mRNA30 and some cases have been reported to express surface or cytoplasmic immunoglobulin.22,25 In our experience, faint but distinct cytoplasmic staining using antisera to k or l can be seen in a subset of cells in some PEL cell lines Žunpublished observation.. Most PELs are thought to originate from post-germinal center B cells, since they have hypermutation of the immunoglobulin genes,31 although this association is not strict as one case that is not mutated and a second case with ongoing mutations were identified, suggesting they can be of pre-germinal center origin, or can be arrested in the germinal center stage. While most cases have functional rearrangements, many do not express immunoglobulin proteins. The reason for this discrepancy is unclear.
In addition to an immunoblastic morphology, PELs have a set of immunophenotypic features suggesting that they are at a preterminal stage of B cell differentiation. Loss of expression of B cell antigens occurs in plasma cells, and this is a frequent finding in multiple myeloma as well as in immunoblastic lymphom as. Furtherm ore, m ost PELs express CD138rSyndecan-1, an adhesion molecule which is selectively expressed by a subset of pre-B cells and by plasma cells, including myeloma plasma cells. The expression of CD138 by PELs seems to be quite specific, as it is not expressed by other lymphomatous effusions, primary or secondary, or by most other solid lymphomas.32 The almost invariable presence of KSHV in lymphomas having the features described above suggests that this virus is necessary for the development of PELs. However, since PELs are so uncommon, even in populations where the seroprevalence of KSHV is relatively high, it is evident that infection by this virus represents only one of several genetic events involved in their development. One such other factor appears to be EBV infection, as the vast majority of PELs, especially in immunocompromised hosts, contain both viral genomes. The specific role of each of these viruses and their interaction is still poorly understood, but analysis of the genes expressed by both of them has shed some light into their possible roles. Both herpesviruses can be lytic or latent, expressing distinct subsets of genes. PELs in vivo, as well as in culture Žsee below. express mostly latent genes, but there is always a small proportion of cells in which EBV and KSHV lytic gene expression occurs. However, most cells have a latent pattern of gene expression. It is known that EBV can establish different types of latency. Latency type I Žrestricted latent gene expression. is seen in Burkitt’s lymphomas, while latency type III Žfull pattern of latent gene expression. is seen in lymphoblastoid cell lines and large cell lymphomas in immunocompromised patients, particularly those with immunoblastic features. While the EBNA1 gene, necessary for EBV replication, is expressed in all latency types, other latent genes, including the EBV transforming genes LMP1 and EBNA2, are only expressed in latencies type II ŽLMP1. and type III ŽLMP1 and all EBNAs.. It is thought that expression of these transforming genes in Burkitt’s
shaped cell which is negative for KSHV hybridisation signals is seen in the lower left corner of this panel Žoriginal magnification= 630.. ŽC. Abundant viral IL-6 protein expression is seen in numerous lymphoma cells by immunohistochemistry using a rabbit polyclonal antiserum raised against a vIL-6-specific peptide Žoriginal magnification= 400..
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lymphomas is not ‘necessary’, since these carry a translocated c-myc oncogene, and the lack of expression of these immunogenic proteins further provides this type of lymphoma with an advantage to evade the immune system. Analysis of the pattern of EBV gene expression in PELs revealed that only EBNA1 was expressed, corresponding to type I latency.33,34 This was an unexpected finding, given the resemblance of PEL cells to immunoblastic lymphoma cells. This observation suggests that KSHV is playing a transforming role in PELs, as the major EBV oncogenes are not expressed. Also consistent with this hypothesis is the lack of structural alterations in the cellular transforming genes frequently involved in malignant lymphomas. In particular, c-myc gene rearrangements have only been identified in one case Žpersonal unpublished observation., being extremely uncommon in contrast to other lymphomatous effusions and EBV-associated lymphomas, where alterations in this gene are a frequent finding. While mutations of the p53 gene have only been identified in one case of PEL,25 mutations in the non-coding, presumably regulatory region of the BCL-6 gene have been reported in a significant proportion of cases.35 The BCL-6 gene is frequently mutated in diffuse large B cell lymophomas, although the functional significance of these mutations remains unclear. Cytogenetic analyses have not provided concrete additional evidence for the presence of a single specific genetic alteration. Of nine PEL cell lines examined, it appears that numerous abnormalities are present, with complex hyperdiploid karyotypes. Frequent abnormalities are trisomy 7, trisomy 12 and aberrations of chromosomal bands 1q21-q25. 36 Translocations specific to other lymphoma types have not been identified. Since there has been no demonstration of expression of EBV transforming genes, or alterations of cellular oncogenes, with the possible exception of mutations in the BCL-6 gene, expression of KSHVencoded transforming genes may be particularly relevant. The genome of KSHV has been found to contain at least 11 open reading frames ŽORFs. that encode homologues to cellular proteins involved in signal transduction, cell cycle regulation andror inhibition of apoptosis.37 To this date, two of these genes have been found to be transforming in transfected cells, andror to cause tumors when these are injected into immunodeficient mice, namely the viral interferon regulatory factor ŽK9, vIRF.,38 ] 40 and the viral G protein-coupled receptor ŽORF 74, KSHV GPCR..41,42 Two additional genes with no known
cellular homolog have been found to be transforming in cell culture systems: K143 and K12 Žkaposin..44 At least two chemokines ŽvMIP I and II, K6 and K4. are encoded by KSHV.45 This virus also contains ORFs homologous to cellular oncogenes involved in lymphomagenesis, and are thus potentially involved in the pathogenesis of PELs. These are the viral cyclin D ŽORF 72., which is homologous to the bcl-1 gene, and the viral bcl-2 ŽORF 16.. KSHV also encodes a homolog of interleukin 6 ŽK2, vIL6., an important growth and differentiation factor for B cells, and a vFLIP ŽK13., an inhibitor of apoptosis.46 While all of these genes appear to be transcribed in PELs, the levels are highly variable. Latent expression in PELs has been well documented for the vIL-6 using specific antisera45 ŽFigure 1, Panel C.. The viral cyclin D ŽORF72. gene is also transcribed during latency in PELs within the neoplastic cell population, as determined by in situ hybridisation ŽFigure 1, Panel B. ŽNador, unpublished observations.. The kaposin ŽK12., vIRF ŽK9., vMIP-II and vFLIP ŽK13. genes have been found to be transcribed during latency in PEL cell lines,47,48 so they are likely to be expressed in PELs in vivo. Lytic genes with ‘transforming’ potential are the K1 protein, vMIP-I, vBCL-2, and GPCR. Although in situ localisation for each of these genes has not been performed, there is indirect evidence to suggest that these lytic genes are expressed only by a subset of PEL cells in vivo. While a viral transforming gene is expected to be expressed by most, if not all, tumor cells, paracrine effects may be relevant to the pathogenesis of KS,42,49,50 and have been postulated to play a role in multiple myeloma.51 Therefore, some of these lytic genes may also be involved in the pathogenesis of PEL.
Multicentric Castleman’s disease KSHV has also been found to be present in multicentric Castleman’s disease ŽMCD..52 ] 54 This virus is present in almost all cases of MCD occurring in patients with AIDS. In fact, in HIV positive patients MCD has been found to be frequently associated with KS, and is usually observed in men infected with HIV by sexual contact.55 Notably, MCD, also called multicentric angiofollicular hyperplasia, is characterized by a vascular proliferation in the germinal centers which is reminiscent of KS. However, this virus is present in MCD in patients with AIDS whether accompanied by KS or not. Furthermore, KSHV has also been identified in approximately half of the 169
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cases of MCD occurring in HIV-negative individuals, suggesting that it is not a coincidental association occurring in HIV positive individuals. KSHV sequences have also been detected in peripheral blood mononuclear cells of patients with MCD.52 Multicentric Castleman’s disease is an atypical lymphoproliferative disorder thought to be related to immune dysregulation.56 This disease occurs more frequently in older individuals, and is more common in men. The patients usually present with multiple lymphadenopathies and a variety of constitutional symptoms. They may develop auto-immune phenomena, cytopenias, skin rashes and intercurrent infections. Patients with MCD frequently develop malignancies, most commonly KS and NHL.52 The identification of KSHV in AIDS and non-AIDS-associated MCD supports an even closer relationship between MCD and KS than previously hypothesized, as in both KSHV may be playing a crucial role. The role of KSHV in the pathobiology of MCD remains poorly understood, and it is not clear whether the KSHV-positive cases differ clinically from KSHV-negative ones in individuals without AIDS. It has been shown that the germinal centers of hyperplastic lymph nodes of patients with Castleman’s disease produce large quantities of interleukin 6 ŽIL-6 .. This may explain the large proportion of plasma cells in these lymph nodes, since IL-6 induces B-cell differentiation. Notably, KSHV encodes for a viral IL-6 homolog, which is also expressed in MCD in scattered cells surrounding the lymphoid follicles.45,57
stimulate the proliferation of the myeloma plasma cells. Multiple studies have been published since, with conflicting results. Two additional independent laboratories confirmed the specific presence of KSHV DNA sequences in multiple myeloma biopsies.60,61 However, other investigators have been unable to confirm this association when looking for KSHV DNA by PCR in bone marrow biopsies andror dendritic cell cultures from bone marrow or peripheral blood.62 ] 71 Serologic studies have been almost uniform in their findings, demonstrating that patients with multiple myeloma lack antibodies to KSHV.62,63,65,68,72 ] 75 This lack of reactivity is not due to generalized immunosuppression, since antibodies to other more ubiquitous herpesviruses, such as EBV and CMV, are easily detectable in these patients using comparable methodologies. Our own group has also been unable to confirm the association between KSHV infection and multiple myeloma, using a variety of methods Žmanuscript in preparation.. In one paper, very weak seroreactivity to KSHV antigens was detected using immunoblotting.76 While this may be confirmatory of the association between KSHV infection and multiple myeloma, it is possible that this low reactivity represents higher levels of non-specific cross-reactivity than in control patients because of the gammopathy in multiple myeloma patients. Furthermore, the epidemiologic patterns of Kaposi’s sarcoma and multiple myeloma are distinct, suggesting different etiologies.77,78 Three explanations can be proposed to explain this controversy: Ž1. KSHV is not present in multiple myeloma or MGUS, and the identification of viral sequences by a fraction of investigators is due to technical artifacts, such as PCR contamination; Ž2. KSHV is present in such low copy numbers in bone marrow and dendritic cell preparations in patients with multiple myeloma that the technique utilized by most laboratories is not sufficiently sensitive to detect them, although in several studies sensitivity controls suggest detection of 1 to 10 copies of KSHV. In this instance, the absent or very low serologic reactivity remains to be explained; it has been hypothesized that HHV-8 localisation within the bone marrow-based dendritic cells may prevent the maturation of an antigenic response due to B-cell tolerance of antigens presented in the bone marrow.79 This reasoning would not explain, however, the discrepant epidemiology of both diseases; and Ž3. a different virus is present in multiple myeloma, similar enough to be recognized by some PCR primers when certain protocols with low stringency are used. Antibodies to this novel virus may not recognize most
Multiple myeloma Initial studies evaluating the presence of KSHV in large series of lymphoid proliferations failed to identify this virus in multiple myeloma or other plasma cell malignancies.14,20,22,23 However, an association between this virus and multiple myeloma was subsequently reported.58 In that study, KSHV DNA and RNA sequences were detected in bone marrow stromal cell cultures from patients with multiple myeloma, as well as two of eight patients with monoclonal gammopathy of undetermined significance ŽMGUS.. This result, which was based on PCR and RT-PCR analyses, was subsequently confirmed by the same team using in situ hybridisation and PCR analysis of DNA extracted from fresh bone marrow core biopsies.59 Since transcripts KSHV vIL-6 were detected, it was hypothesized that KSHV uses a paracrine mechanism, through production of this cytokine, to 170
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KSHV antigens. This explanation may be supported by sequence analysis of PCR products from specimens obtained from patients with multiple myeloma, where significant interpatient variation has been noted, and the reported higher homology between different myeloma patients than when these sequences are compared with KSHV for KS specimens.79 One study has suggested that sequences homologous to one of the regions of KSHV is widely disseminated in a variety of tissues, including multiple myeloma.66 While this last possibility appears to be the most conciliatory explanation, much additional evidence needs to be provided to confirm the presence of a distinct KSHV-like virus and then to confirm its specific association with multiple myeloma and MGUS.
the number of potential transforming genes in KSHV, and its clear association with a subset of malignant lymphomas, it is surprising that this virus is not obviously transforming when lymphoid cells are infected in culture. This observation is consistent with the requirement of other transforming events for KSHV to play a role in the development of PELs. It is also likely that having a high viral copy number in each cell, as well as a particular program of gene expression, are necessary for transformation by this virus, neither of which has been achieved so far in cell culture experiments.
Concluding remarks The identification of KSHV in PEL has led to the recognition of a rare but distinct type of malignancy. Subsequent studies have revealed the possible existence of an ‘extracavitary’ form of this disease and the realisation that perhaps some of the systemic lymphomas, in particular a fraction of those with immunoblastic morphology occurring in HIV-positive individuals, may also belong to the spectrum of KSHV-associated lymphomas. In addition, this virus is unquestionably associated with MCD in the setting of AIDS, as well as in half of cases occurring in HIVnegative individuals. The recognition of the presence of this virus in PEL and MCD, and the eventual understanding of the mechanisms utilized by KSHV in the pathobiology of these diseases, should lead to the identification of rational therapeutic approaches. As both diseases are incurable and deadly at the present time, this is an important ongoing line of investigation. A third disease in which a possible association with KSHV infection has been reported is multiple myeloma. However, this association remains highly controversial, and subsequent studies are necessary to determine whether an as yet undescribed KSHV-like virus is present in this type of lymphoid malignancy. The recognition of KSHV in PEL has resulted in the development of cell lines that have been crucial for the rapid characterisation of this virus, and for the large number of seroepidemiologic studies that have provided strong evidence supporting a causal role for KSHV in the development of KS.
Primary effusion lymphoma cell lines The recognition of the presence of KSHV in PEL allowed the establishment of cell lines to be used as a biological tool for subsequent studies. These cell lines still represent the most efficient method for propagating KSHV in culture, and for obtaining relatively large quantities of this virus. We derived the first KSHV-positive cell lines from two patients with PEL.80 Both of these cell lines also contained the EBV genome, but nevertheless one of them, called BC-1, served as a source of virus for genomic sequencing,81,82 including the first almost full length sequence,37 and for the first serologic assays.83,84 Subsequently, several cell lines containing KSHV but not EBV were established, called BC-3 85 and KS-1 Žestablished independently from the same patient specimen.,86 BCBL-1, 87 BCP-1, 88 and CRO-APr3. 36 A comprehensive review of the features of all these cell lines has been recently published.36 Serologic assays for the presence of KSHV antibodies are now frequently done by immunofluorescence using one of the PEL cell lines as a substrate. Detection of antibodies to latent proteins can be done using untreated cultured cells, while for detection of antibodies to lytic antigens these cells can be treated to induce lytic viral replication using either TPA or butyrate. Another recently developed method for screening for the presence of antibodies to this virus is a whole virus ELISA,89 which also utilizes viral particles purified from a PEL cell line. PEL cell lines have also been used to purify viral particles to infect lymphoid82,90,91 and endothelial cells.50,92 While both cell types can be infected, only endothelial cells seem to be altered by KSHV. Given
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