Lucia Fassone

Epstein-Barr Virus Infection Is Predictive of CNS Involvement in Systemic AIDS-Related Non-Hodgkin’s Lymphomas

PURPOSE This study aimed at correlating Epstein-Barr virus (EBV) infection of systemic AIDS-related non-Hodgkin lymphomas (AIDS-NHL) with the development of a CNS localization of the tumor. PATIENTS AND METHODS Demographic, epidemiologic, clinical, histologic, and virologic features were collected for all systemic AIDS-NHL patients included in the study (n = 50). Pathologic specimens were classified according to the working formulation for NHL and the Revised European-American Lymphoma classification. EBV infection in tumor tissue samples was studied by EBV small encoded RNA in situ hybridization; EBV-DNA detection in CSF was carried out by nested polymerase chain reaction using Epstein-Barr nuclear antigen-1-specific primers. In addition, selected EBV-positive lymphomas were subjected to a detailed characterization of EBV molecular heterogeneity. RESULTS Eleven patients had a CNS involvement at some point during their clinical history (four at diagnosis and seven at relapse). Thirty patients (11 with CNS involvement and 19 without) harbored EBV infection of the tumor. Sensitivity, specificity, and positive and negative predictive values of EBV-DNA detection in CSF for CNS involvement by lymphoma were 90%, 100%, 100%, and 97.6%, respectively. Factors significantly predictive of CNS involvement were EBV infection of the tumor (P=.003), an extranodal disease at diagnosis other than CNS (P=.006), and a non-CNS relapse (P=.01). In four cases of CNS involvement, EBV-DNA in CSF preceded any other sign of disease by a mean of 35 days. CONCLUSION These results show that EBV infection of the tumor clone significantly increases the risk of CNS involvement by systemic AIDS-NHL, without regard of specific molecular features. The detection of EBV-DNA in the CSF of AIDS-NHL patients may select cases with higher risk of CNS involvement and, therefore, may prove useful in the therapeutic stratification of these tumors.

Molecular profile of Epstein-Barr virus infection in HHV-8-positive primary effusion lymphoma

Primary effusion lymphoma (PEL) selectively involves the serous body cavities, occurs predominantly in immunodeficient patients and is infected consistently by human herpesvirus type-8. PEL is also frequently infected by Epstein–Barr virus (EBV). The precise pathogenetic role of EBV coinfection in PEL is not fully understood. The lymphoma fails to express the EBV transforming proteins EBNA-2 and LMP-1, whereas it expresses EBNA-1 (latency I phenotype). Some studies have hypothesized that other EBV-positive lymphomas expressing the latency I phenotype may associate with specific molecular variants of EBNA-1, although this issue has not been addressed in PEL. On this basis, this study is aimed at a detailed molecular characterization of EBV in PEL. Fifteen EBV positive PEL (12 AIDS-related, one post-transplant, two arising in immunocompetent hosts) were subjected to molecular characterization of the viral genes EBNA-1 and LMP-1, as well as definition of EBV type-1/type-2. The EBNA-1 gene displayed a high degree of heterogeneity in different cases of PEL, with seven distinct recognizable variants and subvariants. A wild-type LMP-1 gene was detected in 10/15 cases, whereas in 5/15 cases the LMP-1 gene harbored a deletion spanning codons 346–355. EBV type-1 occurred in 11/15 PEL whereas EBV type-2 occurred in 4/15 cases. Despite a high degree of genetic variability of the virus in different PEL cases, each single PEL harbored only one EBV variant, consistent with monoclonality of infection and suggesting that infection preceded clonal expansion. Overall, our results indicate that: (1) individual PEL cases consistently harbor a single EBV strain; (2) EBNA-1 displays a high degree of heterogeneity in different PEL cases; (3) no specific EBV genotype preferentially associates with PEL.

The tyrosine kinase receptor Met and its ligand HGF are co-expressed and functionally active in HHV-8 positive primary effusion lymphoma

Primary effusion lymphoma (PEL) harbors consistent infection by human herpesvirus-8, preferentially develops in immunodeficient patients and selectively localizes to the serous body cavities. Histogenetic analysis has suggested that PEL originates from post-germinal center, pre-terminally differentiated B cells sharing phenotypic features with plasma cells. Here we have investigated the expression status and functional integrity of the Met tyrosine kinase receptor and of its ligand hepatocyte growth factor (HGF). Thirteen PEL (nine cell lines and four primary specimens) were analyzed for Met and HGF expression and function by multiple assays. For comparison, a panel of 34 high grade B cell non-Hodgkin lymphomas (NHL) other than PEL was also investigated. Co-expression of Met and HGF was found in all PEL analyzed, whereas it was restricted to 1/34 B cell NHL other than PEL (P < 0.001; χ2 test). The Met protein expressed by PEL displays biochemical characteristics typical of Met expressed by other cell types and is capable of tyrosine autophosphorylation. By using a combination of immunological and biological assays, production and secretion of a functional HGF species was identified in all PEL cell lines analyzed. HGF stimulation of PEL cells rapidly induces Met tyrosine phosphorylation, demonstrating the functional integrity of the Met/HGF loop. Because of the well known mitogenic and motogenic properties of Met/HGF interactions, these data may bear implications for PEL growth and dissemination. Among B cell neoplasms, Met/HGF co-expression selectively clusters with PEL and, as demonstrated by previous studies, with multiple myeloma plasma cells, thus reinforcing the notion that PEL displays biologic similarities with tumors derived from late stages of B cell differentiation.

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 CRO-AP/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.

HHV‐8/KSHV is Not Associated with AIDS‐Related Primary Central Nervous System Lymphoma

Primary central nervous system lymphoma (PCNSL) is a major complication of the late stages of human immunodeficiency virus (HIV) disease. Epstein Barr virus (EBV) infection is the only genetic lesion consistently associated with this neoplasia. Recently, it has been proposed that the pathogenesis of AIDS‐related PCNSL (AIDS‐PCNSL) may be associated with infection by human herpesvirus‐8/Kaposis sarcoma associated herpesvirus (HHV‐8/KSHV), although at present such association remains controversial. In order to conclusively assess the link between HHV‐8/KSHV infection and AIDS‐PCNSL, we performed a comprehensive study based on multiple molecular assays on cerebral tissues and cerebrospinal fluid (CSF) as well as specific immunologic assays on patients’ sera. A well characterized panel of 33 Italian patients with AIDS‐PCNSL and 13 controls with other HIV‐related brain focal diseases from the same geographical area was analyzed. No signs of HHV‐8/KSHV infection were detected in cerebral tissues by single‐step PCR. Cerebral tissues of all AIDS‐PCNSL scored negative for HHV‐8/KSHV DNA sequences also by nested PCR, with the exception of one single patient who was simultaneously affected by Kaposis sarcoma. All CSF samples analyzed were consistently devoid of HHV‐8/KSHV sequences by molecular assays. By serologic assays, detecting both latent and lytic HHV‐8/KSHV antigens, a specific immunoreactivity was observed in 16/33 (48%) AIDS‐PCNSL and in 6/13 (46%) controls (P=0.88). A significant correlation with HHV‐8/KSHV serum reactivity was seen with a homosexual route of HIV transmission (P=0.018), but not with the presence of AIDS‐PCNSL. The results of our analysis conclusively assess that HHV‐8/KSHV infection is not a feature of AIDS‐PCNSL.

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.

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 Burkitts 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 significan fraction of AIDS-BL cell lines produce TNFβ Among AIDS-NHL, the release of TNFβ appears to be specific for AIDS-BL. The pathogenetic relevance of TNFβ in lymphomagenesis is suggested by the observation that some BL cell lines use TNFβ 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.

Characterization of Epstein–Barr Virus Genotype in AIDS-Related Non-Hodgkin's Lymphoma

In the present study sequence variations at the C terminus of the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1), EBV-encoded latent membrane protein 1 (LMP-1), and EBNA-2 and EBNA-3C genes were investigated in 64 cases of EBV-positive AIDS-related diffuse large cell lymphoma (AIDS-DLCL), both systemic (12) and localized primarily to the central nervous system (52), and in 12 cases of EBV-positive AIDS-related Burkitts lymphoma (AIDS-BL). Sequence analysis of the EBNA-1 C-terminal region led to the distinction of two major unrelated EBV strains, termed strain P (prototype) and strain V (variant), and their related subtypes, namely P-ala, P-thr, V-leu, V-val, and V-pro. Analysis of the LMP-1 gene was performed to assess the frequency of the C-terminus deletion variant, whereas analysis of EBNA-2 and EBNA-3C genes led to the identification of the distribution of the EBV type 1 and type 2 strains. The frequency of EBNA-1 subtypes was assessed in 49 cases of AIDS-NHL, including 37 cases of AIDS-DLCL and 12 cases of AIDS-BL. The P strain was detected in 45 of 49 cases (91.8%) whereas the V strain was found in 4 of 49 samples (8.1%). A significant difference in the distribution of the P and V strains was found between AIDS-DLCL and AIDS-BL (p < 0.01), because of the exclusive infection by the P strain of the AIDS-DLCL samples analyzed. The frequency of LMP-1 deletion variants and of EBV type 1 and type 2 strains in AIDS-DLCL overlapped with that of the general population, and no correlation was found with the evaluated clinicoepidemiological data of patients, that is, disease site, tumor histology, CD4(+) cell counts, and HIV transmission route. In conclusion, we found that the distribution of the EBV genotype in all of the AIDS-NHL samples analyzed is similar to the viral representation found in control individuals of both immunocompetent and immunocompromised populations.

Mutation of BAX occurs infrequently in acquired immunodeficiency syndrome-related non-Hodgkin's lymphomas

Acquired immunodeficiency syndrome (AIDS)‐related non‐Hodgkins lymphomas (AIDS‐NHLs) consistently derive from B cells, are histologically heterogeneous, and are associated with distinct molecular pathways depending upon histology. Recently, it has been proposed that inactivating mutations of the bax death agonist may contribute to the pathogenesis of human tumors. In particular, among B‐cell malignancies, BAX mutations have been detected at a certain frequency in Burkitt lymphomas. This study is aimed at defining the status of the BAX gene throughout the clinicopathologic spectrum of AIDS‐NHL (n = 54), including AIDS‐related Burkitt lymphoma (n = 14), AIDS‐related Burkitt‐like lymphoma (n = 8), AIDS‐related diffuse large cell lymphoma (n = 15), AIDS‐related primary central nervous system lymphoma (n = 6), and AIDS‐related primary effusion lymphoma (n = 11). All 6 BAX exons and flanking sequences were subjected to mutational analysis by polymerase chain reaction‐single strand conformation polymorphism followed by DNA direct sequencing of positive cases. Mutations of BAX among AIDS‐NHL were restricted to a cell line of AIDS‐related primary effusion lymphoma, which harbored a frameshift mutation causing the introduction of a proximal stop codon. All other AIDS‐NHL displayed wild‐type BAX alleles. In order to investigate whether BAX inactivation in AIDS‐NHL may occur through mechanisms other than gene mutation, bax protein expression was investigated by Western blot analysis or immunohistochemistry in selected cases. All AIDS‐NHL analyzed expressed normal bax proteins. Overall, this study indicates that deregulation of apoptotic control in AIDS‐NHL is not caused by BAX alterations. Genes Chromosomes Cancer 27:177–182, 2000.