At presentation, deep immunodepression and severe depletion of circulating CD4+ lymphocytes or B symptoms are common. They reflect the disease aggressiveness and the frequent preexistent compromise of general conditions of the affected patients. Other symptoms and signs of disease largely depend on distribution of sites of disease. The pattern of clinical presentation translates in heterogeneous aspects in the field of radioimaging (see below) .
PEL may be the primary presentation of KSHV infection. However, KSHV-related diseases such as MCD and Kaposi's sarcoma may precede or may occur contemporary to PEL. In only a minority of cases of PEL reported in the International Extranodal Lymphoma Study Group–IELSG series, a previous or concomitant diagnosis of Kaposi's sarcoma has been reported and MCD was not frequent and, even when present, was exclusively described in the subset of HIV-positive patients . Apart from AIDS, PEL has also been reported in association with other immunodeficiency conditions, namely iatrogenic immunodeficiency following solid organ transplantation, cirrhosis and cancer. Interestingly, PELs developing in the HIV-negative patients selectively affect elder individuals from geographical areas at high prevalence of KSHV infection .
The strongest predictor of poor clinical outcome in the population of HIV-infected individuals is the absence of highly active antiretroviral therapy (HAART) before PEL diagnosis [27,28]. The HAART effect is probably based on the favorable impact of the reversion of severe immunodeficiency. Moreover, the control of HIV replication and the subsequent immunologic recovery induced by HAART could potentially slow down disease progression .
PELs possess an unique constellation of features that distinguish them from other lymphomas occurring in the body cavities. Lymphomas occurring in patients in whom effusions complicate a tissue-based lymphoma, the so-called secondary lymphomatous effusions, closely mimic phenotypic and genotypic features of the corresponding tissue-based lymphoma and are consistently devoid of KSHV infection . The pyothorax-associated lymphomas, now called diffuse large B-cell lymphomas associated with chronic inflammation, consistently present with a tumor mass localized in the body cavities, but only rarely give rise to a lymphomatous effusion and are devoid of KSHV sequences .
Other types of lymphomas involving the serous body cavities include cases of Burkitt lymphoma, mainly occurring in the context of AIDS, which present as a primary lymphomatous effusions without mass formation [3,6]. KSHV infection (assessed by ORF73/LANA immunoreactivity), which clusters with PEL, and translocation of the c-MYC proto-oncogene, which segregates with Burkitt lymphoma , are mutually exclusive molecular events in the development of these distinct malignant effusions .
Other subtypes of lymphomas can present with a primary neoplastic effusion. Many of these cases are KSHV-unrelated large B-cell lymphomas, also termed KSHV-unrelated PEL-like lymphomas . In these lymphomas, the neoplastic cells do not display evidence of KSHV infection, but display morphologic, immunophenotypic and genotypic features related to large B-cell lymphoma .
PEL and KSHV-unrelated PEL-like lymphomas are different in terms of pathogenesis, morphologic–immunophenotypic features, clinical behavior and prognosis. KSHV-unrelated PEL-like lymphoma cases are associated with hepatitis C virus (HCV) (30–40%). The most frequently involved sites are peritoneum and pleura. The lymphoma cells usually show large cell morphology and B-cell immunophenotype. The outcome of patients with KSHV-unrelated PEL-like lymphomas seems to be better than the one for PEL patients in the HIV-positive setting [27,31].
The basic pathologic feature of PEL is a diffuse spreading along the serous membranes without marked infiltrative or destructive growth patterns [3,14,33]. PEL is associated with peculiar imaging features including peritoneal effusion or bilateral/unilateral pleural effusions, usually associated with pericardial effusion, normal mediastinal and parenchymal imaging findings and diffuse slight thickening of the serous membranes at computed tomography (CT) . As seen at autopsy, PEL presents as multiple small tumor foci involving the serous membranes, which appear irregularly thickened [16,24,33]. Furthermore, the lymphomatous infiltration of serosal surfaces is adjacent to the site of primary malignant effusion. Notably, these aspects correlate closely with imaging findings of PEL revealed by CT scan. Overall, these features would indicate a primary serous membrane neoplasm. In the natural history of PEL, the disease initially affects one single serous cavity, usually remains localized to body cavities throughout the clinical course of the lymphoma and occasionally extends into tissues underlying the serous membranes, including the omentum and the outer parts of the gastrointestinal tract wall. Involvement of mediastinal lymph nodes, visceral lymphatics or other superficial and deep lymph nodes, with or without parenchymal infiltration, has been observed in some cases [2,3,16,33].
The exact mechanism by which KSHV promotes oncogenesis in B cells leading is an area of active investigation. In-vitro infection of B cells with KSHV is inefficient and does not lead to transformation of these cells . Therefore, cell lines derived from PEL specimens, where natural infection by KSHV occurred in vivo, have been the only tool available to study the molecular effects of KSHV gene expression in a B-cell background and the molecular mechanisms of lymphomagenesis. KSHV does not replicate in untreated PEL cell lines in culture and only a limited number of viral genes are expressed, which are designated as latent genes. In contrast, when these cell lines are treated with some chemical agents that include phorbol esters and butyrate, viral replication is induced with the production of infectious virion and a large number of lytic viral genes are expressed. It is thought that with expression of latent genes, infected cells can undergo clonal expansion, eventually leading to neoplastic transformation through mechanisms of increased proliferation and impaired apoptosis, although the true role of transient expression of lytic genes during neoplastic transformation in vivo is not known.
Five latent gene products that are thought to play significant roles in PEL pathogenesis are LANA (ORF73), viral cyclin (v-Cyc, ORF72), viral FLICE inhibitory protein (v-FLIP, ORF71), viral interferon regulatory factor 3 (vIRF-3 or LANA-2) and viral interleukin-6 (vIL-6, ORFK2).
LANA, encoded by ORF73, is required for the replication of the latent episomal viral DNA; it binds to the latent origin of replication in the terminal repeat subunits of the viral genome. In addition, it is a multifunctional protein with the potential to significantly alter cellular physiology by recruiting a large variety of cellular proteins linked to transcriptional regulation or proliferation control, including p53, pRB, c-myc, brd2, brd4, CBP, DNAMt1, DNAMt3, GSK3β (reviewed in ). LANA is expressed during latency and represents the most consistently detected viral protein in KSHV-associated tumor cells.
v-Cyc, encoded by ORF72, represents another candidate KSHV oncogene because of its homology to the human cyclin-D/Prad oncogene. In general, cyclin-D proteins (D1, D2, D3) associate with specific cyclin-dependent kinases (CDKs) and these complexes phosphorylate Rb family members. v-Cyc associates with cdk2, cdk4 and cdk6, but appears to promote phosphorylation of its targets mainly in concert with cdk6 [36,37]. Targets of v-Cyc include not only Rb but also other cellular targets including histone H1, Id2, CDC6, cdc25A, Orc-1, the antiapoptotic protein bcl-2 and the cdk inhibitors p27Kip and p21CIP [37,38]. Phosphorylation of p27Kip by the vcyc/cdk6 complex on Ser10 during latency leads to sequestration of p27Kip in the cytoplasm, thereby allowing PEL cells to proliferate in the presence of high p27Kip levels . Likewise, phosphorylation of p21CIP1 on serine 130 by v-Cyc allows this viral protein to bypass the p21CIP1-mediated G1 arrest . v-Cyc has been shown to promote S-phase entry and also to induce apoptosis in cells with high cdk6 expression, which can be counteracted by the action of the viral bcl-2 homologue, vBCL-2, which is expressed during the lytic cycle . v-Cyc can also induce a DNA damage response in endothelial cells . It is likely that some of these biochemical features of v-Cyc play a role in PEL pathogenesis.
v-FLIP, encoded by ORF-K13 (also called ORF71), is transcribed from the LANA promoter in a tricistronic transcript that also contains v-Cyc and is translated from an internal ribosome entry site [41,42]. It is, therefore, thought to be expressed during latency and in all tumor cells. Although the v-FLIP protein expression is very weak in PEL cells, it has been detected by western blot in PEL cell lines  and also in epithelial cells carrying a recombinant KSHV genome. It inhibits CD95/FAS-induced apoptosis in vitro by blocking caspase-3, caspase-8 and caspase-9 . Both CD95/Fas-L and TRAIL/TNF-alpha induce apoptosis through a similar mechanism [45,46]. A more recent line of inquiry found v-FLIP to be involved in NF-κB signaling by binding to IKKγ/NEMO [47,48]. TRAF-2 is also involved in v-FLIP signaling to NF-κB . Eliminating either v-FLIP or NF-κB activity from PEL induces apoptosis [50–52], demonstrating that this pathway is essential for lymphomagenesis.
KSHV encodes four homologues to the cellular interferon regulatory factors, an important family of transcription factors involved in interferon signaling. Only one of these is expressed in latently infected PEL cells, which is vIRF-3, also called LANA-2, encoded by encoded by ORF-K10.6. The functional aspects of this viral protein were recently reviewed . Importantly, when vIRF-3 is knocked down with RNA interference techniques in PEL cell lines, there is a reduction of proliferation and induction of apoptosis, suggesting that this viral protein is indeed involved in the pathogenesis of PEL .
Investigative studies on the pathogenesis of PEL have revealed a peculiar profile of cytokines that is characterized by secretion of large amounts of human interleukin (IL)-6, vIL-6, IL-10 and vascular endothelial growth factor (VEGF) (Fig. 3) that play a role in PEL pathogenesis in vitro and/or in vivo. vIL-6, encoded by ORF-K2, is considered to be a lytic viral protein but is expressed in a variable but significant proportion of PEL cells, both in primary tumors and established cell lines [55,56]. vIL-6 is a multifunctional cytokine that is capable of promoting hematopoiesis, plasmacytosis and angiogenesis . It is a homologue of IL-6 but differs from the cellular protein in that it can signal through gp130 in the absence of the IL-6 receptor α subunit (gp80), making this viral cytokine able to affect a broader range of cell types [56,58]. As vIL-6 is secreted, it can affect neighboring cells in a paracrine fashion and support their growth .
Deregulation of VEGF appears to be a feature common to all KSHV-related disorders. However, in contrast to Kaposi's sarcoma and MCD, the role exerted by VEGF in PEL would be essentially to accelerate vascular permeability .
A number of continuous lymphoma cell lines have been established from different specimens obtained from patients with AIDS-associated and non-AIDS-associated PEL. The names of 22 PEL cell lines can be found in the literature. Two cell lines are sisters of already existing lines, which were established from the same patients but independently in other laboratories; one cell line was published under two different designations. Fourteen PEL-derived cell lines are well characterized and described [61–74] (Table 1). Five additional PEL cell lines have been described in the literature: cell line BC-4 , BC-5 , HH-B2 , PEL-5  and VG-1 .
It is important to point out that the derivation of 13 of the 14 PEL cell lines is authenticated (Table 1), which means that it has been proven that the new cell line was indeed derived from the patients whose primary cells were seeded into culture and was not the result of an inadvertent cross-contamination with cells from an established, faster growing cell line. Overall, in a recent large study, some 15% of human leukemia–lymphoma cell lines were found to be cross-contaminated with other cell lines . With regard to the PEL cell lines, comparisons of primary cells and cell lines using immunoglobulin heavy and light chain gene rearrangement patterns, EBV sequencing, cytogenetic analysis and DNA fingerprinting proved the correct derivation.
Availability of cell lines is another important issue. Ten of the PEL cell lines listed in Table 1 can be obtained from public repositories such as American Type Culture (ATCC), German Collection of Microorganisms and Cell Cultures (DSMZ) or the NIH AIDS Program. One cell line was received directly from the original investigators.
Consistent with their derivation, all 14 PEL cell lines are KSHV-positive. It has been reported that a switch from latent infection to productive (lytic) replication of KSHV could be induced, for example, in cells exposed to the phorbol ester TPA or butyrate [63,69,72]. Eight PEL cell lines are co-infected with EBV, showing a monoclonal pattern, whereas the remaining six cell lines are EBV-negative (Table 1). None of the cell lines were found to be positive for other herpes viruses such as CMV (0/2), HHV6 (0/1) or HSV1/2 (0/1); the cell lines are further negative for HBV (0/4), HCV (0/4), HIV (0/10) and HTLV1/2 (0/6).
Except for two cell lines with limited immunoprofiles, the PEL cell lines were extensively immunophenotyped using large panels of immunomarkers. In general, the cells are devoid of any lineage-specific or lineage-associated markers. None of the cell lines express any markers associated with the T-cell or natural killer cell lineages (CD2, CD3, CD4, CD5, CD7, CD8, CD56). The cells are also generally negative for typical B-cell antigens such as CD10, CD19, CD20, CD21, CD22, CD40, CD72, CD79a/b, CD80, or surface immunoglobulin. Some B-cell markers are occasionally positive: CD23 (56% of the cell lines), CD37 (60%), CD39 (60%), CD74 (80%) and, of particular note, late B-cell/plasma cell marker, CD138 (100%). As expected, all myelomonocytic markers are negative (CD13, CD14, CD15, CD33 and others). There is variable expression of nonlineage markers and activation-associated antigens: HLA-DR (80%), CD30 (83%), CD34 (0%), CD38 (89%), CD45 (92%), CD45RA (25%), CD45RO (40%), CD70 (100%) and CD71 (100%). All cell lines tested express EMA. Finally, expression of adhesion markers is again variable: CD11a (67%), CD11b (20%), CD11c (0%), CD18 (0%), CD43 (67%), CD44 (80%), CD49a (0%), CD49d (80%), CD49f (100%), CD54 (71%) and CD58 (67%). In summary, the cells are generally negative for classical T-cell, NK cell and B-cell immunomarkers, except for CD138, suggesting a preterminal B or plasma cell stage, and are positive at variable levels for some activation and adhesion markers.
Southern blot analysis of the PEL cell lines demonstrated clonal rearrangements of the heavy and/or light chain immunoglobulin genes, indicating their B-cell derivation. No rearrangements of the proto-oncogenes MYC, BCL1, BCL2 or BCL6 were found . Also, the tumor suppressor gene p53 was always seen in its wild-type form without any point mutations. The majority of the PEL cell lines examined were described as carrying mutations of the BCL-6 5 noncoding regions .
Several PEL cell lines could be successfully heterotransplanted into immunocompromised mice (e.g. BNX, NOD/SCID or SCID), in which the malignant cells proliferated developing tumors, lymphomatous effusions and marked angiogenesis [64,74,81].
Several cell lines (BC-1, BC-2, BC-3, BCBL-1, CRO-AP/2, CRO-AP/3, CRO-AP/5) were shown to produce constitutively high levels of the cytokines IL-6, IL-10 and hepatocyte growth factor (HGF) . In the colony formation assay on agar, two cell lines (BCP-1 and HBL-6) had high colony-forming efficiency, attesting their malignant nature.
The STR-428 cell line was derived from a 53-year-old man with HIV− KSHV− ‘malignant effusion lymphoma’, which, according to the authors, corresponds to diffuse large B-cell lymphoma in the WHO classification . The cell line is EBV− KSHV−, carries various surface B-cell immunomarkers (CD19, CD20, CD22, kappa light chain), and finally also contains the other lymphomas characteristic t(14;18)(q32:q21) with the IGH–BCL2 fusion and a MYC rearrangement . Hence, STR-428 represents a unique high-grade B-cell lymphoma-derived cell line and has features that are clearly distinct from those of canonical PEL cell lines.
A concise summary of the most relevant and distinctive features of PEL cell lines is given in Table 2. These cell lines are very well characterized and described. Their profiles in the various areas (virology, immunophenotypes, cytogenetics, molecular genetics, functional aspects) are unique and, taken together, characteristic. The number of cell lines established is limited, but the majority of them are available from public repositories. As they are continuously available model systems, the PEL cell lines represent valuable and indispensable tools for the pathophysiological characterization of the primary effusion lymphomas. Furthermore, these cell lines provide a permanent source for the analysis of KSHV sequences. Notably, PEL-derived cell lines stably retain the KSHV latent viral genome in high copy number (50–100 copies/cell), whereas latent genomes do not persist in latently infected primary endothelial cells derived from Kaposi's sarcoma lesion nor in epithelial, endothelial or fibroblast cell lines. [83,84].
A major instrument toward the definition of PEL phenotype and the cell biology has been gained with characterization by gene expression profiling (GEP) analysis of a number of PEL cell lines representative of EBV-positive and EBV-negative PEL [85–87].
Through GEP, PELs were defined by the overexpression of genes that are involved in inflammation, cell adhesion and invasion, which may be responsible for their presentation in body cavities . Klein et al.  used GEP (of 12 000 genes) of PEL – primary specimens – to further define the phenotype of AIDS-related PEL and to investigate the relationship of lymphoma with normal B cells and with other tumor subtypes, including NHL of immunocompetent hosts and AIDS-NHL. The comparative analysis of the PEL profiles versus those of normal and malignant B cells identified several genes and their products that were not previously associated with PEL. They included aquaporin-3, a water channel protein involved in water transport; the P-selectin glycoprotein ligand PSGL-1/SELPLG, a ligand for P-selectin involved in leukocyte adhesion and upregulated on the cell surface of plasma cells in mice; mucin-1, originally identified as a tumor-associated glycoprotein that is highly upregulated on various tumor types (e.g. adenocarcinoma); and the vitamin D3 receptor, which is expressed on various cell types of the immune system (Fig. 4).
Inoculation of a KSHV-associated PEL cell line into the peritoneal cavity of severe combined immunodeficiency mice resulted in the formation of effusion and solid lymphomas in the peritoneal cavity. Proteomics using two-dimensional difference gel electrophoresis and DNA microarray analyses identified 14 proteins and 105 genes, respectively, whose expression differed significantly between effusion and solid lymphomas. Among these, 49 and 56 genes were identified as showing higher expression in effusion and solid lymphomas, respectively. The group showing predominant expression in effusion lymphoma contained transactivator/cell cycle-associated genes (MAPKAPK2, C/EBPD, G protein-coupled receptor, RRAS, etc.), enzymes and a cell surface antigen (CD68). The group showing predominant expression in solid lymphoma contained structural proteins (collagens, proteoglycans), adhesion molecules (integrins) and cell cycle-associated genes (MAPKs, IRF1, etc.). Moreover, KSHV-encoded lytic proteins, including viral IL-6, were highly expressed in effusion lymphoma compared with solid lymphoma. These data suggested that differences in gene and protein expression between effusion and solid lymphomas may be associated with the formation of effusion lymphoma or invasive features of solid lymphoma .
Taken together, the PEL cell lines display unique features, are clearly distinct from other lymphoma cell lines, represent important model systems for the study of the pathology of PEL and are useful tools for the analysis of KSHV. Notably, PEL have been instrumental in determining the genomic sequence of KSHV as well as in establishing the expression pattern and function of several of the genes harbored by the virus [89–94]. Furthermore, PEL cell lines represent an indispensable tool for the understanding of the impact of KSHV biology on the pathology of PEL. Ongoing research should lead to a deeper understanding of additional questions relevant to the pathobiology of PEL, with the hope of eventual improvement in patient care. Aspects of PEL that merit further investigation include the following:
This work was supported in part by a grant from the Ministero della Salute, Rome, within the framework of the ‘Progetto Integrato Oncologia-Advanced Molecular Diagnostics’ project (RFPS-2006-2-342010.7) (to A.C.) and National Institutes of Health/National Cancer Institute grants R01CA068939 and R01CA103646 (to E.C.).
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