Kaposi's sarcoma-associated herpesvirus induction of chromosome instability in primary human endothelial cells

Chromosome instability contributes to the multistep oncogenesis of cancer cells. Kaposi's sarcoma (KS), an angiogenic vascular spindle cancer of endothelial cells, displays stage advancement with lesions at early stage being hyperproliferative, whereas lesions at late stage are clonal or multiclonal and can exhibit a neoplastic nature and chromosome instability. Although infection with KS-associated herpesvirus (KSHV) has been associated with the initiation and promotion of KS, the mechanism of KS neoplastic transformation remains unclear. We show that KSHV infection of primary human umbilical vein endothelial cells induces abnormal mitotic spindles and centrosome duplication. As a result, KSHV-infected cells manifest chromosome instability, including chromosomal misalignments and laggings, mitotic bridges, and formation of micronuclei and multinucleation. Our results indicate that KSHV infection could predispose cells to malignant transformation through induction of genomic instability and contributes to the development of KS.     

HIV-1 infection of primary effusion lymphoma cell line triggers Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gammaherpesvirus etiologically linked to primary effusion lymphoma (PEL), to a subset of multicentric Castleman's disease and to Kaposi's sarcoma (KS), the most common neoplasm associated with AIDS. Among KSHV-infected individuals, the risk of KS is much higher in those with human immunodeficiency-1 (HIV-1) infection than among those with other types of immunosuppression, suggesting a direct action of HIV-1 on KSHV replication. We show in our report that BC-3 cells, a chronically KSHV-infected B-cell line of a PEL origin, are permissive to HIV-1, offering a new tool for studying the interactions between these 2 viruses. In these cells, HIV-1 infection leads to reactivation of latent KSHV genomes, as demonstrated by the expression of KSHV lytic viral mRNAs. Although recombinant HIV-1 gp120 fails to enhance herpesvirus expression, transient transfection of the HIV-1 trans-activator Tat suffices to reactivate latent KSHV. By showing that HIV-1 infection directly reactivates latent KSHV, our results suggest a direct role of HIV-1 in the onset of KS in coinfected individuals.     

Prognostic factors and outcome of human herpesvirus 8-associated primary effusion lymphoma in patients with AIDS.

PURPOSE: Primary effusion lymphoma (PEL) is a rare high-grade B-cell non-Hodgkin's lymphoma associated with Kaposi sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/HHV-8) infection, and is mostly observed in the course of HIV infection. The prognosis is poor, with reported median survival time shorter than 6 months. To date, no prognostic factor has been identified in this subset of lymphoma. PATIENTS AND METHODS: We describe here a large series of HIV-infected patients with PEL, including 28 cases diagnosed in six centers during an 11-year time period. Prognosis analysis was performed using a Cox proportional hazard regression model. Statistically significant covariates were further analyzed in a forward, stepwise multivariate model. RESULTS: After a median follow-up of 3.8 years (range, 10 months to 10.8 years), nine patients (32%) were still alive, and eight of them remained progression free. The median survival was 6.2 months, and the 1-year overall survival rate was 39.3%. Fourteen patients (50%) achieved complete remission, with a 1-year disease-free survival rate at 78.6%. In a multivariate analysis, only a performance status more than 2 (hazard ratio, 5.84; 95% CI, 1.76 to 19.33) and the absence of highly active antiretroviral therapy (HAART) before PEL diagnosis (hazard ratio, 3.26; 95% CI, 1.14 to 9.34) were found to be independent predictors for shorter survival. CONCLUSION: Based on a retrospective series of 28 patients, two prognostic factors were identified as being independently associated with impaired clinical outcome in HIV-related PEL--(1) a poor performance status and (2) the absence of HAART before PEL diagnosis.     

Prevalence and transmission of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in Ugandan children and adolescents

We studied the seroprevalence and transmission of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8), among 215 Ugandan children, adolescents and young adults. We measured antibodies to a latent nuclear antigen (LANA) and a lytic cycle protein encoded by open reading frame (orf) 65. Infection with KSHV/HHV8 occurred during early childhood and reached adult levels (approx. 50%) before the age of puberty. In children younger than 12 years of age, antibodies to LANA and the orf65 protein were independently associated with hepatitis B infection (p < 0.005). KSHV/HHV8 infection was not associated with antibodies to hepatitis A virus and hepatitis C virus, nor with the quality of the water supply, household size, previous blood transfusions, number of boy/girl friends or marital status. Antibodies to the orf65 protein, but not LANA, were weakly associated with a history of i.v. injections. Our results show that, in contrast to its sexual mode of transmission among homo/bisexual men and sexually transmitted diseases clinic attendees of Northern Europe and the US, transmission of KSHV in Uganda occurs largely before puberty. Among Ugandan children, KSHV transmission follows a horizontal pattern similar to other herpesviruses, in particular the related γ herpesvirus, Epstein-Barr virus. Transmission of KSHV may be facilitated by living conditions that also promote infection with hepatitis B virus. Int. J. Cancer 77:817–820, 1998.© 1998 Wiley-Liss, Inc.     

The role of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in lymphoproliferative diseases.

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.     

Targeting human herpesvirus-8 for treatment of Kaposi's sarcoma and primary effusion lymphoma

PURPOSE OF REVIEW: Human herpesvirus-8, also called the Kaposi's sarcoma herpesvirus, is present in all cases of Kaposi's sarcoma and primary effusion lymphoma and in some cases of multicentric Castleman's disease. This review discusses mechanisms by which human herpesvirus-8 contributes to tumorigenesis and how this knowledge can be used to target the virus for the treatment of these tumors. RECENT FINDINGS: Most primary effusion lymphomas and Kaposi's sarcoma tumor cells are latently infected with human herpesvirus-8 and hence resistant to antiherpesvirus drugs that are dependent on lytic replication. In contrast, many of the cells infected with human herpesvirus-8 in multicentric Castleman's disease support lytic replication, so that clinical improvement frequently occurs in response to treatment with antiherpesvirus drugs. The resistance of latently-infected tumor cells to antiherpesvirus drugs can be overcome by inducing human herpesvirus-8 to reenter the lytic cascade in the presence of antiherpesvirus drugs. This leads to apoptosis of virally infected cells without increasing production of infectious virus. Alternatively, the replication and maintenance of the human herpesvirus-8 episome during latency can be disrupted by glycyrrhizic acid or hydroxyurea so that the virus no longer contributes to tumorigenesis. Both the innate and acquired immune systems can also be augmented to help prevent or treat human herpesvirus-8-associated tumors. SUMMARY: Novel strategies targeting human herpesvirus-8, which is present in all cases of Kaposi's sarcoma and primary effusion lymphoma, provide opportunities for selectively killing tumor cells.     

Immortalization of primary endothelial cells by the K1 protein of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to three different human cancers: Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. The Kaposi's sarcoma lesion expresses high levels of angiogenic factors and is comprised of a mixed cell population, including endothelial cells that are infected with KSHV. We find that the KSHV K1 protein is expressed in Kaposi's sarcoma lesions and can immortalize and extend the life span of primary human umbilical vein endothelial cells in culture. Vascular endothelial growth factor (VEGF) is critical for the survival of endothelial cells, and we show that expression of K1 in endothelial cells resulted in increased levels of secreted VEGF and the activation of key signaling pathways, including the VEGF/VEGF receptor and the phosphatidylinositol-3'-OH-kinase (PI3K) pathway. The SH2 binding motifs present in the cytoplasmic tail of K1 were critical for K1's ability to activate these pathways. Activation of PI3K by K1 results in activation of Akt kinase and mammalian target of rapamycin and inactivation of the proapoptotic proteins FKHR, glycogen synthase kinase-3, and Bad, which are events indicative of cell survival. Because activation of the PI3K pathway is critical for transformation of many human cells, we suggest that PI3K activation by K1 is involved in endothelial cell immortalization and contributes to KSHV-associated tumorigenesis. We also report that K1 enhances angiogenesis in vivo and increases tumor vasculature and tumor size.     

The sero-epidemiology of Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in adults with cancer in Uganda

The association between the prevalence of antibodies against Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8 [HHV-8]) and sociodemographic, sexual, reproductive and lifestyle factors was investigated in a study of adults presenting with cancer at hospitals in Kampala, Uganda. Patients were interviewed and tested for antibodies against KSHV (using an indirect immunofluorescent assay). Data are presented for 607 patients who were not infected with the human immunodeficiency virus-1 (HIV) and who did not have Kaposi's sarcoma (these included people with cancers of the uterine cervix [140], breast [58], liver [41], oesophagus [36], lymphoma [47], other cancers [285] and benign tumours [63]). The prevalence of anti-KSHV antibodies was 50% overall (302/607) and did not differ significantly by cancer site (p = 0.4) or sex (p = 0.2), but increased linearly with age from 35% in those under 25 years to 55% in those 45 years and over (chi(2) trend [1 df] = 9.1; p < 0.001). After adjusting for age and sex, anti-KSHV antibodies were more common in tribal groups other than the Baganda tribe (54% vs. 45% among Baganda; p = 0.02), but there was no significant (p > 0.05) variation in seroprevalence by district of birth, region of residence prior to becoming ill or various measures of wealth. The prevalence of anti-KSHV antibodies decreased with increasing number of older siblings, although this may be due to chance (p = 0.05) and was higher among people who had ever been married (p = 0.03). There was no significant association (p > 0.05) between the presence of antibodies against KSHV and other sexual and reproductive factors. Among the 302 patients with anti-KSHV antibodies, the proportion with high titres increased linearly with increasing age (p = 0.03) and was higher among those reporting having had a blood transfusion (p = 0.03). In conclusion, in this population in Uganda, where KSHV is relatively common, the prevalence of anti-KSHV antibodies increased with age but showed little association with nearly 50 other factors studied.     

Induction of human herpesvirus 8 gene expression in a posttransplantation primary effusion lymphoma cell line

Human herpesvirus 8 (HHV-8 or Kaposi's sarcoma herpesvirus) is a gamma herpesvirus that is most likely the etiologic agent of both Kaposi's sarcoma and primary effusion lymphoma (PEL), a rare HIV-associated lymphoma. The role of HHV-8 in post-transplant lymphoma is less well characterized. We demonstrate that HHV-8 is constitutively present in LH5-21 cells, an atypical patient derived posttransplant PEL cell line. LH5-21 cells lack detectable Epstein-Barr virus, express T cell-associated surface markers and have undergone immunoglobulin heavy chain gene rearrangement. Incubation with 12-O-tetradecanoyl-phorbol- 13-acetate or butyrate induces high levels of several HHV-8 encoded genes that are associated with lytic replication. The patient from whom this cell line was derived demonstrated a dramatic clinical response to withdrawal of immunosuppressive therapy. While HHV-8 associated PELs in the post-transplant setting are rare, this study suggests that improvement in the host immunologic function might help in the management of some PELs.     

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.     

Human-immunodeficiency-virus-negative, human-herpes-virus-8-negative abdominal cavity primary effusion lymphoma

Primary effusion lymphoma (PEL) is an unusual class of non-Hodgkin's lymphoma, which is characterised by lymphomatous effusions in body cavities, but no associated mass lesions. It is usually associated with an immunodeficient state most often with the human immunodeficiency virus (HIV). We describe a case of a man with HIV-negative, human-herpes-virus-8 (HHV8)-negative PEL, with a history of heavy alcohol intake. The abdominal cavity was the only area involved; no solid tumour masses were observed on scanning, and the bone-marrow investigations were normal. The ascites contained numerous pleomorphic lymphoid, lymphoplasmacytoid cells of B-cell origin. The immunophenotyping was moderately positive for CD 38 and 138, and strongly positive for Ki 67. It is postulated that the immunosuppressed state in this patient may have been caused by the long history of heavy alcohol intake.     

Cisplatin disrupts the latency of human herpesvirus 8 and induces apoptosis in primary effusion lymphoma cells

Human herpesvirus 8 (HHV8) is the etiologic agent for primary effusion lymphoma (PEL). The aim of this study is to investigate the effects of cisplatin on the PEL cells. Cisplatin treatment induced apoptosis and inhibited the growth of PEL cells, and the effect was more profound in the HHV8-positive lymphoma cells compared with the EBV-positive lymphoma cells. Cisplatin treatment decreased the expression of HHV8 latent genes and activated p53 at serine 15 in PEL cells. Our results indicate that cisplatin can disrupt HHV8 latency and induce reactivation of p53 and highly selective treatment modality for this virally induced lymphoma.     

A prospective study of Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus in adults with human immunodeficiency virus-1

Antibody titres against Kaposi's sarcoma associated herpesvirus (KSHV or human herpesvirus 8 (HHV-8)) and Epstein-Barr virus (EBV) were examined in people who subsequently developed Kaposi's sarcoma and non-Hodgkin's lymphoma, within randomised controlled trials of antiretroviral therapy in adults infected with the human immunodeficiency virus-1 (HIV). For each case of Kaposi's sarcoma (n=189) and each case of non-Hodgkin's lymphoma (n=67), which developed after randomisation, one control was randomly selected from other trial participants, after matching for age, sex, ethnicity, mode of HIV transmission, type of treatment received and period of follow-up. Using sera taken an average of two and a half years before the diagnosis of cancer, titres of antibodies against KSHV latent (LANA) and lytic (K8.1) antigens and against EBV (VCA) antigens were investigated in relation to subsequent risks of cancer by calculating odds ratios (OR) using conditional logistic regression. Latent antibodies against KSHV were detectable among 38% (72 out of 189) of Kaposi's sarcoma cases and 12% (23 out of 189) of their controls (OR=4.4, 95% confidence intervals (CI) 2.3-8.3, P<0.001). The OR for Kaposi's sarcoma increased with increasing antilatent KSHV antibody titre (chi(2)(1) for trend=32.2, P<0.001). Lytic antibodies against KSHV were detectable among 33% (61 out of 187) of Kaposi's sarcoma cases and 19% (36 out of 187) of their controls (OR=2.0, 95% CI 1.2-3.4, P=0.003) and the OR for Kaposi's sarcoma increased with increasing antilytic KSHV antibody titre (chi(2)(1) for trend=6.2, P=0.02). Virtually, all cases and controls had anti-EBV antibodies detected and the OR for non-Hodgkin's lymphoma associated with a doubling of the anti-EBV antibody titre was estimated to increase by a multiplicative factor of 1.3 (95% CI 0.9-1.7, P=0.1). Kaposi's sarcoma was not associated with antibody levels against EBV (P=0.4) and non-Hodgkin's lymphoma was not associated with antibodies against KSHV (latent P=0.3; lytic P=0.5). Adjustment for CD4 count at the time of sample collection made no material difference to any of the results. In conclusion, among human immunodeficiency virus infected people, high levels of antibodies against KSHV latent and lytic antigens are strongly associated with subsequent risk of Kaposi's sarcoma but not non-Hodgkin's lymphoma. Antibody titre to EBV does not appear to be strongly associated with subsequent risk of Kaposi's sarcoma or non-Hodgkin's lymphoma in HIV infected people.     

Inhibition of HHV-8/KSHV infected primary effusion lymphomas in NOD/SCID mice by azidothymidine and interferon-alpha

Kaposi's sarcoma-associated herpesvirus/human herpesvirus type-8 (KSHV/HHV-8) is associated with primary effusion lymphomas (PEL), a rare form of B-cell lymphoma. PEL cell lines infected with HHV-8, but negative for Epstein-Barr virus (EBV), were analyzed for their tumorigenic potential in a small animal model system. Inoculation of PEL cell lines into non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice results in efficient engraftment and tumorigenesis in vivo. PEL-engrafted NOD/SCID (PEL/SCID) mice displayed malignant ascites development with notable abdominal distension, consistent with the clinical manifestations of PEL in humans. Azidothymidine (AZT, zidovudine) and interferon-alpha (IFN-alpha) induce apoptosis in HHV-8+/EBV- PEL cells in culture, by induction of a tumor necrosis factor-related apoptosis inducing ligand (TRAIL) mediated suicide program and has been proposed as a therapy for herpesvirus-associated lymphomas. Daily injection of AZT and IFN-alpha significantly increased mean survival time (MST) of PEL/SCID mice suggesting that induction of apoptosis in PEL cells in vivo may be exploited as an effective relatively non-toxic therapy targeting HHV-8 infected PEL. These data demonstrate that the PEL/SCID mouse is an important preclinical model to characterize efficacy and anti-tumor mechanisms of new therapeutic targets in vivo and will be useful in the design and testing of agents in viral lymphoproliferative diseases.     

Mono/oligoclonal pattern of Kaposi Sarcoma-associated herpesvirus (KSHV/HHV-8) episomes in primary effusion lymphoma cells

Primary effusion lymphoma (PEL) is a rare lymphoma of B-cell origin, developed in serous cavities. PEL tumor cells are latently infected with Kaposi sarcoma-associated herpesvirus (KSHV) and in most cases co-infected with Epstein-Barr virus (EBV). In 15 primary PEL tumors including 10 EBV-positive cases, we analyzed the fused terminal repeat (TR) regions of KSHV episomes using pulsed-field gel electrophoresis and Southern blot. On the same genomic DNA samples, the cellular clonality was assessed by Southern blot and PCR detection of monoclonal immunoglobulin heavy chain (IGH) VDJ gene rearrangements, associated in the EBV-infected cases, with Southern blot analysis of the fused termini of EBV episomes. Monoclonal IGH gene rearrangements were detected in 13 tumors using Southern blot, in 11 cases using PCR, and in all cases considering both methods. EBV infection was monoclonal in all EBV-positive cases. However, only 5 PEL tumors were found to be monoclonally infected with KSHV. In the 10 other cases, we found a biclonal (2 bands; n = 4) or an oligoclonal pattern (3-6 bands; n = 6) of KSHV episomes. We hypothesized that the apparent discrepancy between viral and cellular clonalities in PEL might be due to several phenomena including complex mechanisms of genomic recircularization, insertion of duplicated sequences into the TR region and simultaneous infection of tumor cells with defective KSHV variants. KSHV infection of contaminating nontumoral cells, superinfection from lytically infected cells or viral integration events might also explain the oligoclonal pattern of KSHV infection. Several of these mechanisms, not mutually exclusive, might coexist in a single tumor.