Characterization of a novel HHV-8-positive cell line reveals implications for the pathogenesis and cell cycle control of primary effusion lymphoma.

Primary effusion lymphoma (PEL) represents a peculiar type of B cell lymphoma which associates with HHV-8 infection and preferentially grows in liquid phase in the serous body cavities. In this report, we provide the detailed characterization of a newly established PEL cell line, termed CRO-AP/6. The cell line was obtained from the pleural effusion of a HIV-positive patient with PEL. Its derivation from the tumor clone was established by immunogenotypic analysis. Detailed phenotypic investigations defined that CRO-AP/6 reflects pre-terminally differentiated B cells expressing the CD138/syndecan-1 antigen. Karyotypic studies of CRO-AP/6 identified several chromosomal abnormalities, whereas genotypic studies ruled out the involvement of molecular lesions associated with other types of B cell lymphoma. Both CRO-AP/6 and the parental tumor sample harbored infection by HHV-8. Conversely, EBV infection was present in the parental tumor sample although not in CROAP/6, indicating that CRO-AP/6 originated from the selection of an EBV-negative tumor subclone. The pattern of viral (HHV-8 v-cyclin) and cellular (p27Kip1) regulators of cell cycle expressed by CRO-AP/6, together with the results of growth fraction analysis, point to abrogation of the physiological inverse relationship between proliferation and p27Kip1 expression. Also, both CRO-AP/6 and the parental tumor sample display biallelic inactivation of the DNA repair enzyme gene O6-methylguanine-DNA methyltransferase (MGMT) by promoter methylation. Overall, the CRO-AP/6 cell line may help understand cell cycle control of PEL cells, may clarify the relative contribution of HHV-8 and EBV to the disease growth and development and may facilitate the identification of recurrent cytogenetic abnormalities highlighting putative novel cancer related loci relevant to PEL.

Molecular characterization of HHV-8 positive primary effusion lymphoma reveals pathogenetic and histogenetic features of the disease.

BACKGROUND: Primary effusion lymphoma (PEL) associates with HHV-8 infection, preferentially develops in immunodeficient patients and grows in the serous body cavities. PEL derives from post-germinal center, pre-terminally differentiated B-cells. The pathogenesis of PEL is unclear and the sole identified genetic lesions are human herpesvirus type-8 (HHV-8) infection in all cases and EBV infection in 70% of cases. Epstein-Barr virus (EBV) infection in PEL displays a latency I phenotype. OBJECTIVES: To clarify the pathogenesis and histogenesis of PEL by investigating (1) the lymphoma karyotype; (2) the expression status of the Met tyrosine kinase receptor and of its ligand hepatocyte growth factor (HGF); (3) the molecular profile of EBV, with particular focus on mutations of EBNA-1 genes, which are thought to affect viral tumorigenicity in EBV-infected neoplasms displaying the latency I phenotype. STUDY DESIGN: Twenty-four PEL (nine cell lines and 15 primary specimens) formed the basis of the study. Karyotypes were investigated by conventional cytogenetics and fluorescent in situ hybridization (FISH) in selected cases. The expression status of Met and HGF was defined by multiple techniques, including RT-PCR, FACS analysis, immunocytochemistry, Western blot studies and ELISA. The molecular profile of EBNA-1 genes of EBV were investigated by DNA direct sequencing. RESULTS: Trisomy 7, trisomy 12 and breaks at 1q21-q25 are recurrently associated with PEL. PEL consistently co-express Met and HGF both at the mRNA and protein level. Among aggressive B-cell lymphomas, Met/HGF co-expression appears to be relatively specific for PEL. The EBNA-1 gene of EBV displays a high degree of genetic heterogeneity in PEL, with no preferential association with one specific variant. CONCLUSIONS: PEL associates with recurrent chromosomal alterations, suggesting that viral infection is not sufficient for tumor development and that lesions of cellular genes may be required. The expression of Met/HGF by PEL cells may bear implications for the lymphoma proliferation and growth pattern, since Met/HGF interactions influence cell mitogenesis and motogenesis. EBV infection in PEL displays a latency I phenotype and fails to associate with specific EBNA-1 variants, suggesting that the role of EBV in PEL is not mediated by the major transforming pathways currently known in EBV positive lymphomas.

AIDS-related Burkitt's lymphoma. Morphologic and immunophenotypic study of biopsy specimens.

In patients infected with HIV, high-grade B-cell non-Hodgkin's lymphomas (NHL), including the small noncleaved cell (SNCC) category, exhibit pleomorphic features, which makes precise definition difficult. Sixty-nine pathologic specimens with HIV-related systemic lymphomas, including 42 SNCC, 20 immunoblastic lymphomas (IBL), and 7 cases with features "intermediate" between SNCC and IBL were morphologically and immunophenotypically investigated. The host immune status was also analyzed in 57 of 69 patients. In 29 representative SNCC lymphomas, in 9 IBL cases, and an additional 3 intermediate lymphomas, both p53 protein overexpression and the association of Epstein-Barr virus (EBV) genetic information were assessed. Small noncleaved cell lymphomas included tumors exhibiting features of the 2 established subtypes (27 Burkitt's and 15 non-Burkitt's). In the seven intermediate cases, cells showed features intermediate between SNCC with plasmablastic differentiation and immunoblasts plasmacytoid. Immunoblast-like cells were also present. p53 protein overexpression and EBV association were found in a proportion of SNCC (14 of 29; 7 of 29) and intermediate (3 of 3; 2 of 3) lymphomas. Conversely, IBL cases were consistently p53 negative, but showed a high EBV association (7 of 9). All the evaluated patients with intermediate lymphomas had a considerably lower mean (76.6 per mm3 +/- 77.4 SD) and median (54 per mm3) number of CD4+ lymphocyte count than SNCC patients (mean 227.9 per mm3 +/- 186.9 SD, median 193 per mm3), thus mirroring IBL patients (mean 95.3 per mm3 +/- 82.8 SD, median 81 per mm3). All data provide evidence that lymphomas showing intermediate features constitute a distinct subgroup from either SNCC or IBL.

Primary effusion lymphoma cell lines harbouring human herpesvirus type-8.

Primary effusion lymphoma (PEL) is a novel lymphoma entity consistently infected by HHV-8 that occurs predominantly in immunodeficient patients and is characterized by liquid growth in the serous body cavities. In order to facilitate the understanding of PEL pathogenesis and histogenesis, we have established three PEL cell lines termed CRO-AP/2, CRO-AP/3 and CRO-AP/5. All cell lines have been derived from HIV positive homosexual men affected by PEL with (in the case of CRO-AP/2 and CRO-AP/5) or without (in the case of CRO-AP/3) a previous history of Kaposi's sarcoma. The cell lines are representative of both virologic variants of PEL, i.e. HHV-8+ EBV+ PEL (CRO-AP/2 and CRO-AP/5) and HHV-8+ EBV- PEL (CRO-AP/3). Morphologic and phenotypic features of CRO-AP/2, CRO-AP/3 and CRO-AP/5 are typical of PEL, and include morphology bridging immunoblastic and anaplastic features as well as an indeterminate (non B- non T-cell) phenotype. The B-cell nature of the cell lines is documented by the presence of rearranged immunoglobulin genes. The detailed analysis of the molecular and phenotypic features of CRO-AP/2, CRO-AP/3 and CRO-AP/5 has allowed the identification of recurrent chromosomal abnormalities of PEL and has contributed to the definition of PEL as a lymphoma of post-germinal center, pre-terminally differentiated B-cells.

The role of cytokines in the pathogenesis and management of AIDS-related lymphomas.

AIDS-related non-Hodgkin lymphomas (AIDS-NHL) consistently derive from B-cells and are characterized by extreme clinical aggressiveness. At histological level, AIDS-NHL are classified as AIDS-related Burkitt's lymphoma (AIDS-BL), AIDS-related diffuse large cell lymphoma (AIDS-DLCL) and AIDS-related primary effusion lymphoma (AIDS-PEL). The role of cytokines in the pathogenesis and management of AIDS-NHL has been studied to a certain extent. Production of large quantities of human IL-10 occurs frequently in AIDS-BL and correlates with latent EBV infection of the tumor clone. Lesser amounts of the cytokine are released in EBV negative cases. The pathogenetic role of IL-10 in AIDS-BL is suggested by the observation that IL-10 antisense oligonucleotides inhibit proliferation of the lymphoma. A significant fraction of AIDS-BL cell lines produce TNFbeta. Among AIDS-NHL, the release of TNFbeta appears to be specific for AIDS-BL. The pathogenetic relevance of TNFbeta in lymphomagenesis is suggested by the observation that some BL cell lines use TNFbeta as an autocrine growth factor. Some cases of AIDS-BL, particularly those carrying EBV infection, also secrete IL-6, IL-7 and IL-12. With respect to AIDS-DLCL, many cases express the IL-6R, rendering these cells responsive to the paracrine stimulation by the IL-6 produced by nearby T-cells, macrophages and endothelial cells which are frequently abundant in these tumor samples. The tumor clone itself, however, generally fails to release IL-6. AIDS-PEL is characterized by secretion of large amounts of IL-6 and IL-10. Some PEL cases also release oncostatin M. Apart from human IL-6, PEL also express viral IL-6, which is encoded by the HHV-8 genome. The biological relevance of both IL-6 and IL-10 in PEL proliferation and growth has been recently clarified in vitro and in vivo. Overall, these data suggest that activation of different cytokine loops clusters with different clinico-pathologic categories of AIDS-NHL and may represent the potential target of novel therapeutic strategies.

BCL-6 in aids-related lymphomas: pathogenetic and histogenetic implications.

AIDS-related non-Hodgkin's lymphomas (AIDS-NHL) are classified into Burkitt's lymphoma, diffuse large cell lymphoma (DLCL), and body cavity based lymphoma. The molecular pathogenesis of AIDS-NHL is complex and involves the genetic alteration of several cancer related genes, including the BCL-6 proto-oncogene. BCL-6 encodes a zinc finger transcription factor which is selectively expressed by germinal center (GC) B-cells, but not by pre-GC or post-GC B-cells. Genetic alterations of BCL-6 occur frequently among B-cell NHL and comprise gross rearrangements as well as small mutations of the 5' noncoding region of the gene. Gross rearrangements of BCL-6 among AIDS-NHL cluster with 20% AIDS-DLCL. Conversely, mutations of the 5' noncoding region of BCL-6 occur at sustained frequency throughout the clinico-pathologic spectrum of AIDS-NHL and represent the most common genetic alteration presently detectable in these lymphomas. The frequency of BCL-6 mutations, as well as their location in the proximity of the BCL-6 regulatory regions, suggest that they may play a pathogenetic role in AIDS-related lymphomagenesis. Beside their pathogenetic implications, the occurrence of BCL-6 mutations among AIDS-NHL bears histogenetic relevance because BCL-6 mutations are regarded as a marker of B-cell transition through the GC. Thus, it is conceivable that a large fraction of AIDS-NHL is histogenetically related to GC or post-GC B-cells. This notion is further confirmed by the observation that AIDS-NHL frequently express the BCL-6 protein, which stains selectively GC B-cells throughout B-cell differentiation.

High frequency of EBV association with non-random abnormalities of the chromosome region 1q21-25 in AIDS-related Burkitt's lymphoma-derived cell lines.

Chromosome 1q abnormalities represent the second most frequent cytogenetic lesion of Burkitt lymphoma (BL) and acute lymphoblastic leukemia (ALL)-L3. The most frequent change is partial duplication of the long arm of chromosome 1, involving variable bands but consistently including 1q23. Among AIDS-related BL similar chromosome 1q abnormalities have also been found. We have now characterized in detail the chromosome 1q abnormalities of 4 AIDS-BL cell lines and compared them to other molecular features of the tumor clone, namely infection by Epstein Barr virus (EBV). Immunophenotypic characteristics were also assessed by conventional in situ immunocytochemical and flow cytometric procedures. The B-cell origin of all cell lines was demonstrated by the expression of B-cell-restricted markers (e.g., CD19). Analysis of Ig light chains confirmed their monoclonal nature. The t(8;14) was present in 3 of the 4 lines, whereas variant translocation t(8;22) was detected in the remaining cell line. Additional chromosomal changes were found in all cases, with chromosome 1 being involved in all. Structural changes encompassed in each case the 1q21-25 bands, in either duplication or partial trisomy. EBER ISH studies identified EBV association in 3 of the 4 AIDS-BL cell lines in contrast to previous studies of BL of immunocompetent individuals. Our findings of a high frequency of chromosome 1q abnormalities in EBV-infected AIDS-related BL cell lines demonstrate that such chromosomal abnormality and EBV positivity are not mutually exclusive and are possibly independent factors, whereas their close association in AIDS may be related to the immunodeficiency.

Establishment and characterization of EBV-positive and EBV-negative primary effusion lymphoma cell lines harbouring human herpesvirus type-8.

In this study we report on the establishment and characterization of two novel lymphoma cell lines (CRO-AP/3 and CRO-AP/5) which carry infection by human herpesvirus type-8 (HHV-8) and have derived from AIDS-related primary effusion lymphoma (PEL). These two cell lines are representative of different virologic subtypes of PEL, i.e. HHV-8+/EBV- PEL in the case of CRO-AP/3 and HHV-8+/EBV+ PEL in the case of CRO-AP/5. Consistent with the diagnosis of PEL, both CRO-AP/3 and CRO-AP/5 expressed indeterminate (i.e. non-B, non-T) phenotypes although immunogenotypic studies documented their B-cell origin. Both cell lines are devoid of genetic lesions of c-MYC, BCL-2 and p53 as well as gross rearrangements of BCL-6. Detailed histogenetic characterization of these novel PEL cell lines suggests that PEL may derive from a post-germinal centre B cell which has undergone pre-terminal differentiation. The CRO-AP/3 and CRO-AP/5 cell lines may provide a valuable model for clarifying the pathogenesis of PEL. In particular, these cell lines may help understand the relative contribution of HHV-8 and EBV to PEL growth and development and may facilitate the identification of recurrent cytogenetic abnormalities highlighting putative novel cancer related loci relevant to PEL.

Borreliosis risk after tick bite in north-eastern Italy.

Lyme Borreliosis is an infectious disease caused by the Borrelia burgdorferi spirochete transmitted to man by a tick bite. The aim of this study was to assess the predictive value of tick bites for the diagnosis of Borreliosis in a sample of 266 subjects residing in an endemic area of north-eastern Italy. In the serological diagnosis of Borreliosis, positive and negative predictive values of tick bites were found to be 24% and 88% respectively.

Establishment of HHV-8-positive and HHV-8-negative lymphoma cell lines from primary lymphomatous effusions.

Primary lymphomatous effusions are represented by cases of non-Hodgkin's lymphoma (NHL) which grow in liquid phase in the serous body cavities in the absence of solid tumour masses. Based on morphologic, immunophenotypic, virologic and genotypic features, primary lymphomatous effusions are distinguished into body cavity-based lymphoma (BCBL), Burkitt's lymphoma (BL) and immunoblastic large-cell lymphoma. The histogenesis and pathogenesis of primary lymphomatous effusions are virtually unclarified. In this study, we have established 2 cell lines (CRO-AP/1 and CRO-AP/2) representative of 2 distinct categories of primary lymphomatous effusion, BCBL (CRO-AP/2) and BL (CRO-AP/1). Both CRO-AP/1 and CRO-AP/2 carry monoclonal re-arrangements of the immunoglobulin genes which are identical to those of the respective parental tumours. Consistent with its BCBL origin, CRO-AP/2 is characterised by a non-B, non-T phenotype and harbours infection by HHV-8 (approx. 100 viral copies/cell) and Epstein-Barr virus. Conversely, CRO-AP/1 expresses several B cell-associated antigens, lacks HHV-8 infection and carries the genetic hallmark of BL, i.e., a chromosomal breakpoint of band 8q24. CRO-AP/1 and CRO-AP/2 may be valuable for the biologic characterization of primary lymphomatous effusions, particularly since the number of available cell lines derived from such lymphomatous effusions is extremely limited.

Genetic characterization of HHV-8/KSHV-positive primary effusion lymphoma reveals frequent mutations of BCL6: implications for disease pathogenesis and histogenesis.

Human herpesvirus-8/Kaposi sarcoma-associated herpesvirus-positive primary effusion lymphoma (PEL) is a recently identified B-cell non-Hodgkin lymphoma category characterized by liquid growth in the serous body cavities. Apart from viral infection, no genetic alteration is known to be associated with PEL and no recurrent cytogenetic abnormality has been identified in these lymphomas. Yet the consistent monoclonality of PEL indicates that the disease is not solely a virus-driven proliferation. Here we report that PEL is associated with a high frequency of mutations of BCL6 5' noncoding regions, and we identify karyotypic abnormalities that may be recurrently involved in these lymphomas. Mutations of BCL-6 5' noncoding regions occurred in 8/13 PEL. Mutations occurred in the absence of BCL6 gross rearrangements were often multiple in the same patient (7/8 mutated cases), and occurred in both HIV-positive and HIV-negative individuals. Since BCL6 mutations are regarded as a genetic marker of B-cell transition through the germinal center (GC), these data are consistent with histogenetic derivation of PEL from GC or post-GC B-cells. Cytogenetic and FISH analysis of seven PEL cell lines showed frequent occurrence of complete or partial trisomy 12 (7/7 cases), trisomy 7 (4/7 cases), and abnormalities of bands Iq21-25 (5/7 cases).

Biologically relevant phenotypic changes and enhanced growth properties induced in B lymphocytes by an EBV strain derived from a histologically aggressive Hodgkin's disease.

Epstein-Barr virus (EBV) isolates show a wide genomic heterogeneity, and a key issue is whether distinct strain variations may contribute to the development and/or malignancy of EBV-related disorders. Herein, we report on the virologic and biologic characterization of an EBV strain derived from a cyto-histologically aggressive EBV-related Hodgkin's disease (HD) (case HD-3) showing a high number of "anaplastic" Reed-Sternberg cells expressing markedly high levels of CD30, CD40 and LMP-1. The HD-3-derived EBV showed strong in vitro immortalizing properties, as suggested by the unusually high number of spontaneous lymphoblastoid cell lines (LCLs) obtained from the patient. Immunofluorescence and immuno-cytochemical analyses showed that HD-3 LCLs expressed significantly higher levels of CD23, CD30, CD38, CD39, CD40 and CD71 antigens and CD54 and CD58 adhesion molecules than B95.8 LCLs. In contrast, the expression of CD11a, CD24, CD95, bcl-2, LMP-1 and EBNA-2 was similar in both groups of LCLs. These phenotypic changes are consistent with the induction of a pronounced activation status and are not dependent on the cellular background, having been closely reproduced by the same virus in LCLs from an unrelated donor (DEN-HD-3 LCLs). HD-3 LCLs were able to grow in vitro at low serum concentrations (up to 0.1%) and were significantly more clonogenic in soft agarose than B95.8 LCLs. Moreover, although no evidence of tumor formation was observed in nude mice injected with B95.8 LCLs, all 5 spontaneous LCLs of patient HD-3 and the 2 DEN-HD-3 LCLs grew in transplanted animals as lymphoproliferations composed of EBER+, LMP-1+ cells. Our findings indicate that the biologic properties of the HD-3 EBV strain are significantly different from those of the B95.8 virus and may have contributed to the cytologic and histo-pathologic malignancy of this HD case. Moreover, molecular characterization of the HD-3 EBV genome identified a 63-bp deletion within the 3' end of the LMP-1 gene as a likely significant change that may be responsible, at least in part, for the biologically relevant phenotypic modifications and enhanced in vitro and in vivo growth potential induced in B lymphocytes by this virus strain.

Patterns of cytokine expression in AIDS-related non-Hodgkin's lymphoma.

The pathogenesis of AIDS-related non-Hodgkin's lymphomas (AIDS-NHL) involves accumulation of genetic lesions, stimulation and selection by antigen, as well as infection by viruses. Deregulation of cytokine loops has also been proposed to contribute to AIDS-NHL development, although data are available only for a limited number of cytokines. In this study we have utilized a panel of AIDS-NHL cell lines to investigate in detail the pattern of tumour expression and production of a wide spectrum of cytokines. The cytokines investigated included interleukin (IL)-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, TNF alpha, TNF beta, IFN gamma, TGF beta2, G-CSF, GM-CSF and SCF. The AIDS-NHL cell lines utilized were representative of both AIDS-related Burkitt lymphoma (AIDS-BL) and AIDS-related body cavity-based lymphoma (AIDS-BCBL). Overall, AIDS-NHL were found to produce IL-6, IL-10 and TNF beta, although with different patterns depending upon the biological features of the tumour. Production of high levels of IL10 preferentially associated with Epstein-Barr virus (EBV) positive AIDS-BL and AIDS-BCBL, although lower levels of the cytokine were also detectable among EBV-negative AIDS-BL. Production of IL-6 was restricted to EBV-positive AIDS-BL and AIDS-BCBL, whereas it was absent among EBV-negative AIDS-BL. Production of TNF beta clustered with AIDS-BL, whereas this was absent among AIDS-BCBL. These results define that the pattern of cytokine expression of AIDS-NHL depends upon the biological features of the tumour and may have implications for the pathogenesis of these disorders.

High CD40 membrane expression in AIDS-related lymphoma B cell lines is associated with the CD45RA+, CD45RO+, CD95+ phenotype and high levels of its soluble form in culture supernatants.

CD40 is a membrane glycoprotein expressed on normal and neoplastic B lymphocytes. Stimulation through CD40 regulates important cellular functions, but the effects depend on its membrane density. While extensive studies have characterized CD40 in non-Hodgkin lymphomas of immunocompetent individuals, little is known on the characteristics of this molecule in lymphomas arising in immunocompromised hosts. The aim of this study was to characterize the pattern of CD40 expression in an in vitro model constituted by AIDS small non-cleaved lymphoma (SNCCL) cell lines. The analysis of CD45 isoforms, a group of molecules alternatively spliced during B cell differentiation, has been chosen to correlate this process to the number of CD40 molecules per cell in these cell lines. Since Apo 1/Fas expression is upregulated on B lymphocytes after CD40 ligation and this expression is functionally relevant, we wanted to know whether a different CD40 pattern in AIDS-SNCCL cell lines could influence CD95 expression. We have shown that 3 of these cell lines (PA 682, Es III, and HBL-2) have high membrane CD40 expression (> 100,000 molecules/cell); they release large amounts of soluble CD40 (sCD40) in culture supernatants (>500 pg/ml), are CD45RA/RO double labelled, and express the Apo 1/Fas (CD95) antigen. On the contrary, low CD40 membrane antigen cell lines (BRGIgA, HBL-2, NC 71, AS 283A, and LAM C3+, < 50,000 molecules/cell) release low amounts of sCD40 (<300 pg/ml), are CD45RA+ but CD45RO-, and do not express CD95. EBV has no role in CD40 and CD45 isoform behaviour, because EBV superinfection of the EBV negative, low membrane CD40 HBL-2 cell line does not modify CD40 membrane expression, sCD40 production, or CD45 isoform and CD95 expression. Our data suggest that membrane CD40 in AIDS-SNCCL cell lines might be a key element in the regulation of their pathophysiology by influencing the expression of CD45 isoforms and of CD95, and by the release of its soluble form.

Association of Kaposi's sarcoma-associated herpesvirus-positive primary effusion lymphoma with expression of the CD138/syndecan-1 antigen.

Primary effusion lymphoma (PEL) represents a novel B-cell non-Hodgkin's lymphoma (NHL) type associated with Kaposi's sarcoma-associated herpesvirus infection and typically growing as lymphomatous effusions in the body cavities. The precise B-cell subset from which PEL originates as well as the biologic mechanisms responsible for its peculiar growth pattern are unclear. In this study, we have analyzed PEL for the expression status of CD138/syndecan-1, a molecule selectively associated with late stages of B-cell differentiation and implicated in cell-to-cell and cell-to-extracellular matrix interactions. PEL patient samples (n = 7) and cell lines (n = 5) were investigated by multiple approaches, including immunocytochemistry, flow cytometry, RNA analysis, and Western blot studies. For comparison, lymphomatous effusions other than PEL (n = 13) and tissue-based NHL (n = 103) were also tested. Expression of CD138/syndecan-1 associates at high frequency with PEL (5 of 7 patient samples and 5 of 5 cell lines), whereas it is consistently absent among other lymphomatous effusions (n = 13). The CD138/syndecan-1 isoform expressed by PEL has an average molecular weight of 420 kD, which is substantially different from that of CD138/syndecan-1 molecules generally expressed by plasma cells. These data, along with previous immunophenotypic evidence, unequivocally define that PEL cells represent a preterminal stage of B-cell differentiation and may bear implications for the peculiar growth pattern of this lymphoma.