Combined chemotherapy including high-dose methotrexate in KSHV/HHV8-associated primary effusion lymphoma

Primary effusion lymphoma (PEL) is a rare KSHV/HHV8-associated high-grade non-Hodgkin's lymphoma (NHL) of B-cell origin, characterized by serous effusions in body cavities. Most patients are HIV-infected homosexual men with severe immunosuppression and other KSHV/HHV8-associated diseases such as Kaposi's sarcoma (KS). The prognosis is poor with a median survival of less than 6 months in most cohorts. The achievement of a sustained complete remission is rare. High-dose chemotherapy regimens are warranted to improve complete remission rate and survival. Seven patients with AIDS-associated PEL were treated with a combined chemotherapy including high-dose methotrexate followed by leucovorin rescue. In all cases, KSHV/HHV8 sequences were detected in the effusion samples using quantitative PCR assays. Five patients had a pre-existing KS, associated in three cases with multicentric Castleman's disease (MCD). Upon diagnosis, 6 patients received antiretroviral therapy, which was maintained during chemotherapy in 5 of them. At time of analysis, 3 out of 7 patients were in complete remission 18, 26, and 78 months after PEL diagnosis. Three patients died with a progressive PEL at 22, 67, and 153 days after diagnosis, and 1 patient died 9 months after PEL diagnosis with a MCD-associated plasmablastic NHL. Complete remission was obtained in 3 out of 7 patients treated for AIDS-associated PEL with combined chemotherapy containing high-dose methotrexate.     

Distinct distribution of rare US genotypes of Kaposi's sarcoma-associated herpesvirus (KSHV) in South Texas: implications for KSHV epidemiology

Genotypes of Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) from patients with KS in South Texas were examined. Open-reading frame (ORF)-K1 and ORF-K15 DNA segments from 16 KSHV isolates were amplified by polymerase chain reaction, and KSHV subtypes were assigned on the basis of sequence variations. K1 genotyping showed that 75% exhibited C subtype and 25% exhibited A subtype. K15 genotyping showed that 56% exhibited M form, of which 89% exhibited C3 K1 subtype and 44% exhibited P form. A unique isolate was found and was classified as C6 clade. All of the M KSHV isolates had been obtained from human immunodeficiency virus-negative classic KS patients >50 years of age, of whom 78% were Hispanic. Conversely, all KS patients with AIDS were <36 years of age and exhibited P form KSHV. These findings indicate that C3/M KSHV genotypes are more prevalent in South Texas (50%) than in other US regions (3%) and that M form KSHV likely existed in this region long before the AIDS epidemic.     

Novel association of haemophagocytic syndrome with Kaposi's sarcoma-associated herpesvirus-related primary effusion lymphoma

Haemophagocytic syndrome (HPS) is a fulminant, often fatal, systemic illness that occurs in association with infection and malignancy. We provide the first report of HPS that heralded a primary effusion lymphoma (PEL), a rare neoplasm linked to Kaposi's sarcoma-associated herpesvirus. The patient was a 38-year-old man with acquired immunodeficiency syndrome who presented with fever, sweats, lymphadenopathy, splenomegaly and refractory anaemia. Examination of the spleen demonstrated haemophagocytosis; analysis of ascites revealed PEL. Treatment with chemotherapy and ganciclovir resulted in complete remission of both conditions. This case illustrates the diagnostic challenges posed by HPS and supports the trial of antiviral agents in combination with chemotherapy in patients with PEL.     

Establishment and characterization of a primary effusion (body cavity- based) lymphoma cell line (BC-3) harboring kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) in the absence of Epstein-Barr virus

The recently identified Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus 8 (HHV-8), has been found to be consistently associated with an unusual subset of acquired immunodeficiency syndrome-related lymphomas, the so-called body cavity- based lymphomas (BCBL) or primary effusion lymphomas (PEL). These lymphomas are characterized by a unique spectrum of morphologic and molecular characteristics, and grow as lymphomatous effusions without an identifiable contiguous tumor mass. Until now, efforts to delineate the role of KSHV in the pathogenesis of PELs have been hampered by the lack of appropriate model systems and the concomitant presence of Epstein-Barr virus (EBV) in nearly all cases examined, and in all previously established cell lines. We now report the establishment and characterization of a novel PEL cell line, BC-3, which is KSHV+ by polymerase chain reaction (PCR) but EBV- as assessed by a variety of methods including PCR for EBER, EBNA-2, and EBNA-3C. This cell line was established from a lymphomatous effusion obtained from an HIV- patient, and has immunophenotypic and molecular features consistent with the diagnosis of PEL, including an indeterminate immunophenotype with a B- cell immunogenotype and lack of c-myc proto-oncogene rearrangements. Pulsed-field gel electrophoresis shows an intact KSHV genome of about 170 kb both in the cell line and in the viral isolate, whereas herpesvirus-like capsids are visible by electron microscopy. Consequently, the BC-3 cell line represents an invaluable tool as a source of KSHV, for both the evaluation of the pathogenic potential of this virus and the mechanistic characterization of its role in the development of Kaposi's sarcoma and malignant lymphoma.     

Kaposi's sarcoma-associated herpesvirus-infected primary effusion lymphoma has a plasma cell gene expression profile

Kaposi's sarcoma-associated herpesvirus is associated with three human tumors: Kaposi's sarcoma, and the B cell lymphomas, plasmablastic lymphoma associated with multicentric Castleman's disease, and primary effusion lymphoma (PEL). Epstein-Barr virus, the closest human relative of Kaposi's sarcoma-associated herpesvirus, mimics host B cell signaling pathways to direct B cell development toward a memory B cell phenotype. Epstein-Barr virus-associated B cell tumors are presumed to arise as a consequence of this virus-mediated B cell activation. The stage of B cell development represented by PEL, how this stage relates to tumor pathology, and how this information may be used to treat the disease are largely unknown. In this study we used gene expression profiling to order a range of B cell tumors by stage of development. PEL gene expression closely resembles that of malignant plasma cells, including the low expression of mature B cell genes. The unfolded protein response is partially activated in PEL, but is fully activated in plasma cell tumors, linking endoplasmic reticulum stress to plasma cell development through XBP-1. PEL cells can be defined by the overexpression of genes involved in inflammation, cell adhesion, and invasion, which may be responsible for their presentation in body cavities. Similar to malignant plasma cells, all PEL samples tested express the vitamin D receptor and are sensitive to the vitamin D analogue drug EB 1089 (Seocalcitol).     

Quantitative analysis of Kaposi sarcoma-associated herpesvirus (KSHV) in KSHV-associated diseases

BACKGROUND: Accurate numbers of copies of Kaposi sarcoma-associated herpesvirus (KSHV) and numbers of virus-infected cells in lesions caused by KSHV-associated diseases are unknown. METHODS: Quantitative polymerase chain reaction (PCR) and computerized imaging of immunohistochemical analysis were performed on pathologic sections of samples from persons with KSHV-associated diseases. RESULTS: Real-time PCR and semiquantitative PCR-Southern blotting demonstrated that DNA extracted from biopsy samples of KS lesions contained approximately 1-2 viral copies/cell. KSHV-associated lymphoma contained 10-50 viral copies/cell. Computerized-image analysis demonstrated that approximately 49% of cells expressed KSHV-encoded latency-associated nuclear antigen in KS biopsy samples. On the basis of results of real-time PCR and computerized-image analysis, the predicted number of viral copies was 3.2 viral copies/cell in KS lesions. Computerized-image analysis also revealed that the expression of open-reading frame (ORF)-50 protein, an immediate early protein of KSHV, was very rare in KS lesions, which implies that they were mainly composed of proliferating cells latently infected with KSHV. In multicentric Castleman disease lesions, 25% of virus-infected cells expressed ORF50 protein, which suggests the frequent lytic replication of KSHV. CONCLUSIONS: Numbers of viral copies and of virus-positive cells vary among KSHV-associated diseases, which suggests different mechanisms of viral pathogenesis. The combination of real-time PCR and computerized-image analysis provides a useful tool for the assessment of the number of viral copies in KSHV-associated diseases.     

HIV-1 Tat enhances Kaposi sarcoma-associated herpesvirus (KSHV) infectivity

The high frequency of Kaposi sarcoma (KS) in immunodeficiency states, particularly in patients with AIDS, has been attributed to increased replication of KS-associated herpesvirus (KSHV), a necessary cofactor for KS development. However, experimental KSHV infection of endothelial lineage cells that compose KS lesions has been difficult even in the absence of immune cells. Here we show that HIV-1 Tat protein can directly promote KSHV transmission. Full-length HIV-1 Tat and a 13-amino-acid peptide corresponding to the basic region of Tat specifically enhances the entry of KSHV into endothelial and other cells, presenting evidence for an active role of HIV-1 in the development of KSHV-associated diseases. These results can explain why AIDS-KS is more frequent and clinically more aggressive than KS in other immunodeficiency states.     

T-cell immunity to Kaposi sarcoma-associated herpesvirus: recognition of primary effusion lymphoma by LANA-specific CD4+ T cells

T-cell immunity is important for controlling Kaposi sarcoma-associated herpesvirus (KSHV) diseases such as the endothelial cell malignancy Kaposi sarcoma, or the B-cell malignancy, primary effusion lymphoma (PEL). However, little is known about KSHV-specific T-cell immunity in healthy donors and immune control of disease. Using PBMCs from healthy KSHV-infected donors, we found weak ex vivo responses to the KSHV latent antigens LANA, vFLIP, vCyclin, and Kaposin, with LANA most frequently recognized. CD4(+) T-cell clones specific to LANA, a protein expressed in all KSHV-infected cells and malignancies, were established to determine whether they could recognize LANA-expressing cells. B-cell targets expressing or fed LANA protein were consistently recognized by the clones; however, most PEL cell lines were not. PELs express the KSHV protein vIRF3 that inhibits promoter function of the HLA class II transactivator, decreasing expression of genes controlled by this transactivator. Re-expressing the class II transactivator in the PELs increased expression of downstream targets such as HLA class II and restored recognition but not killing by the LANA-specific clones. We suggest that PELs are poorly controlled in vivo because of inefficient recognition and killing by T cells.     

Persistence of Kaposi sarcoma-associated herpesvirus (KSHV)-infected cells in KSHV/HIV-1-coinfected subjects without KSHV-associated diseases

Inguinal lymph nodes from 24 human immunodeficiency virus (HIV) type 1-infected subjects without Kaposi sarcoma-associated herpesvirus (KSHV)-associated diseases were examined for KSHV infection. KSHV-infected cells were detected at a very low frequency in the lymph nodes of 7 subjects (median frequency, 2 infected cells/10(7) lymph node cells). Latent, but not lytic, KSHV gene expression was detected and KSHV-infected cells were located in B cell-rich areas of lymph node follicles. These findings provide evidence that, in the absence of KSHV-associated diseases, latent infection of lymph node cells provides a mechanism for the persistence of KSHV in KSHV/HIV-1-coinfected persons.     

Human herpesvirus 8 (HHV-8)-associated peritoneal primary effusion lymphoma (PEL) in two HIV-negative elderly patients

Human herpesvirus 8 (KSHV/HHV-8) is associated with all forms of Kaposi sarcoma (KS), with a rare high-grade B-cell non-Hodgkin lymphoma characterized by serous effusions in body cavities called primary effusion lymphoma (PEL) and with some forms of multicentric Castleman disease (MCD). Although mostly observed during AIDS, such disorders have also been described with a lower incidence in human immunodeficiency virus-negative patients. We describe here the features of two novel cases of AIDS-unrelated PEL. Two patients, a 78-year-old man (case 1) and a 86-year-old woman (case 2), both of French origin, presented exudative ascitic effusion containing numerous KSHV/HHV-8(+) EBV(-) large lymphomatous cells of B-cell clonal origin, characterized by a CD45(+) CD30(+) CD19(-) CD20(-) immunophenotype. The PEL tumor cells harbored a homogenous and isolated trisomy 12 in case 1 and an aberrant expression of the T-cell lineage antigen CD7 in case 2. Both patients were lymphopenic at the time of PEL diagnosis and rapidly died with progressive lymphoma. Moreover, patient 2 had a previous history of classic KS and MCD clinically improved after treatment with all-trans-retinoid acid and a concomitant metastatic breast adenocarcinoma. Compared to AIDS-related PEL, these two cases displayed distinct features in particular the advanced age of patients, as observed for Mediterranean KS, and the absence of EBV coinfection.     

Establishment and characterization of a Kaposi's sarcoma-associated herpesvirus- and Epstein-Barr virus-negative malignant lymphoma cell line (OHK) with primary effusion lymphoma immunophenotype

A novel cell line, designated OHK, was established from ascites of a 59-year-old Japanese woman with diffuse large B-cell lymphoma showing a peculiar serosal tropism, as seen in primary effusion lymphomas (PEL). OHK exhibited a large pleomorphic morphology with irregular nuclei and distinct nucleoli, and included immunoblastic and Reed-Sternberg-like giant cells. On ultrastructural examination, rich intermediate filaments, and well-developed Golgi apparati and rough endoplasmic reticulum, were seen. Immunophenotypically, OHK lacked T and B cell-associated antigens, and had CD10, CD30, CD33 and CD138 antigens. Although OHK cells did not express immunoglobulin (Ig) protein, Southern blot analysis demonstrated clonal rearrangements of Ig heavy and light chain genes. These observations suggest that OHK cells are derived from preterminally differentiated B cells, and that they have features of PEL. Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus were not detected. OHK displayed hyperploid karyotypes with multiple structural abnormalities, and produced some cytokines such as macrophage-colony-stimulating factor (M-CSF), granulocyte-CSF, interleukin 6 and transforming growth factor beta 1. In particular, vascular endothelial growth factor (VEGF), whose stimulation of vascular permeability is thought to be critical to the pathogenesis of PEL, was also produced in large quantities. These results indicate that OHK may be a useful tool for the investigation of PEL.     

Kaposi sarcoma-associated herpesvirus (KSHV) induces heme oxygenase-1 expression and activity in KSHV-infected endothelial cells

Kaposi sarcoma (KS) is the most common AIDS-associated malignancy and is characterized by angiogenesis and the presence of spindle cells. Kaposi sarcoma-associated herpesvirus (KSHV) is consistently associated with all clinical forms of KS, and in vitro infection of dermal microvascular endothelial cells (DMVECs) with KSHV recapitulates many of the features of KS, including transformation, spindle cell proliferation, and angiogenesis. To study the molecular mechanisms of KSHV pathogenesis, we compared the protein expression profiles of KSHV-infected and uninfected DMVECs. This comparison revealed that heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in heme catabolism, was up-regulated in infected endothelial cells. Recent evidence suggests that the products of heme catabolism have important roles in endothelial cell biology, including apoptosis and angiogenesis. Here we show that HO-1 mRNA and protein are up-regulated in KSHV-infected cultures. Comparison of oral and cutaneous AIDS-KS tissues with normal tissues revealed that HO-1 mRNA and protein were also up-regulated in vivo. Increased HO-1 enzymatic activity in vitro enhanced proliferation of KSHV-infected DMVECs in the presence of free heme. Treatment with the HO-1 inhibitor chromium mesoporphyrin IX abolished heme-induced proliferation. These data suggest that HO-1 is a potential therapeutic target for KS that warrants further study. (Blood. 2004;103: 3465-3473)      

Relationship between the quantity of Kaposi sarcoma-associated herpesvirus (KSHV) in peripheral blood and effusion fluid samples and KSHV-associated disease

The Kaposi sarcoma-associated herpesvirus (KSHV)-DNA level was determined in samples from 71 patients with Kaposi sarcoma (KS), 28 patients with multicentric Castleman disease (MCD), and 8 patients with primary effusion lymphoma (PEL). KSHV-DNA levels were higher in patients with active KS or MCD than in those with KS or MCD in remission. Among patients with active disease, the highest KSHV-DNA levels were observed in effusion fluid samples from patients with PEL (7.2 log(10) copies/150,000 cells), followed by blood samples from patients with MCD and PEL (4.86 and 3.83 log(10) copies/150,000 cells, respectively), and the lowest levels were observed in blood samples from patients with KS (2.63 log(10) copies/150,000 cells). Determining the KSHV-DNA level may be useful in diagnosing KSHV-associated disease and for following up patients with KS when the development of MCD or PEL is suspected.     

Human herpesvirus-8/Kaposi's sarcoma-associated herpesvirus in organ transplant patients with immunosupression

Several recent studies have demonstrated Kaposi's sarcoma-associated herpes virus (KSHV), also known as putative human herpes virus-8 (HHV-8), DNA in various epidemiologic forms of Kaposi's sarcoma (KS), including AIDS-associated, classic, and endemic types. Risk of developing KS in non-HIV-infected immunosuppressed hosts, such as patients following solid organ transplantation, is also significantly higher compared to normal individuals. We have retrospectively evaluated 28 organ transplant patients with KS (23 cutaneous and five visceral) for the presence of KSHV genome by polymerase chain reaction (PCR) amplification of DNA isolated from formalin-fixed, paraffin-embedded archival tissue samples. 27/28 KS patients were positive for the presence of KSHV. In four KS patients, tissue samples with no histologic evidence of KS were also analysed for KSHV. No evidence of positivity in three samples was noted, but one patient had weak positive amplification products on DNA samples isolated from a gastric biopsy with chronic gastritis and lymph node with sinus histiocytosis. These data support the association of KSHV with KS developing in non-HIV-infected immunosuppressed patients, similar to other forms of KS, and suggest that KSHV may play a significant role in the development of all forms of KS.     

An unusual case of primary effusion lymphoma in a HIV-negative patient not pathogenetically associated with HHV8

The development of an unusual case of primary pleural effusion in a 90-year-old human immunodeficiency virus (HIV)-negative Japanese woman with no identifiable tumor mass has been described. Pleural effusion specimens contained large diffuse lymphoma cells, with the phenotype and genotype of a B-cell lineage (positive for CD20, CD79a and clonal rearrangement of Ig heavy chain) and the c-myc gene rearrangement, but were negative for T-cell markers (CD45RO and CD3). The patient was negative for human herpes virus 8 (HHV8), Epstein-Barr virus (EBV) and hepatitis C virus (HCV), as well as human T-cell lymphotropic virus type-1 (HTLV-1). The patient died of respiratory failure 5 months after the diagnosis of primary effusion lymphoma (PEL), and an autopsy was performed. Autopsy findings revealed no evidence of tumor mass or bone marrow involvement of lymphoma cells. This case has been considered as a PEL in a HIV-, HHV8-, EBV- and HCV-negative patient. Although cytomorphology of lymphoma cells was classified as large-cell lymphoma in this case, it is interesting that the present case may represent an unusual subset of Burkitt-like disease because of clear B-cell phenotype and c-myc gene rearrangement.