Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) DNA sequences are absent in leukapheresis products and ex vivo expanded CD34+ cells from multiple myeloma patients

Recently it was reported that Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8) infects bone marrow (BM) dendritic cells (DC) in multiple myeloma (MM) patients and therefore might play a role in MM development. Because of the use of myeloid growth factors like GM-CSF and G-CSF for the mobilization of peripheral blood progenitor cells (PBPC), the subsequent increase of DC precursors might imply a risk for KSHV contamination in PBPC grafts. Therefore, in this study leukapheresis products and ex vivo cultured CD34+ cell suspensions were analysed. KSHV DNA could not be amplified in any of them.     

Low incidence of human herpesvirus 8 in stem cell collections from myeloma patients

Human herpesvirus 8 is a gammaherpesvirus which may be implicated in the pathogenesis of multiple myeloma. Viral DNA sequences have been found in the bone marrow, peripheral blood and leukapheresis products of myeloma patients. These findings have significant implications for the use of leukapheresed cells in the transplantation and immunotherapy of myeloma. The studies suggest the cell which harbours the virus may be dendritic in origin. We have previously reported that dendritic cells cultured for use in the clinical setting do not harbour HHV-8. In this study, we examined the leukapheresis products of a larger cohort of myeloma patients for the presence of HHV-8 using a highly sensitive PCR technique. A strong association between HHV-8 and myeloma was not confirmed, with only 4% of the patient samples positive for viral sequences. While further study is needed, the current use of apheresis cells and their cultured progeny in the treatment of myeloma should not be compromised.     

Absence of herpesvirus DNA sequences in the 5T murine model of human multiple myeloma

Kaposi's sarcoma-associated herpesvirus (KSHV, also known as HHV-8) has been found in patients with multiple myeloma (MM) and postulated to be aetiologically associated with the development of this common plasma cell malignancy. A murine model of MM was previously established in which intravenous transfer of 5T myeloma cells into C57BL/KaLwRij mice resulted in characteristic features of human MM. In the present study, we sought to identify herpesvirus DNA sequences in this murine model of MM through polymerase chain reaction (PCR) analysis using primers specific for KSHV, murine herpesvirus 68 (MHV68) and murine cytomegalovirus (MCMV) as well as consensus primers designed from the highly conserved DNA polymerase genes of the Herpesviridae family. None of the DNA samples from whole bone marrow (n = 6) or dendritic cells enriched by long-term culture (n = 8) of 5T myeloma-bearing mice as well as the 5T myeloma cell lines (n = 3) maintained in long-term culture yielded specific amplification products in any of the PCR assays. Two KSHV-specific serological assays measuring antibodies to KSHV latent and lytic antigens also failed to detect the presence of anti-KSHV antibodies in mice that developed MM. These results suggest that the development of 5T murine MM is unlikely to be involved with KSHV or a KSHV-like murine herpesvirus.     

Leukapheresis cells of patients with multiple myeloma collected after mobilization with chemotherapy and G-CSF do not bear Kaposi's sarcoma associated herpesvirus DNA

The presence of Kaposi's sarcoma associated herpesvirus (KSHV) in bone marrow dendritic cells, in bone marrow biopsies and in dendritic cells of peripheral blood from patients with multiple myeloma (MM) has been reported. These data suggested an association between infection with KSHV and the development of MM. The mobilization of infected cells into leukapheresis products (LP) has also been described. We assessed the LP of 35 patients with MM for the presence of KSHV using a sensitive and specific nested PCR assay, capable of detecting one copy of the virus genome. None of the samples tested revealed positivity for KSHV after amplification and subsequent hybridization with a KSHV-specific probe. Amplification products of approximately the same size as the positive control seen in eight samples did not hybridize with the specific oligonucleotide. No homologies of these products to the KSHV genome could be discovered after sequencing. Therefore we have no evidence that LP of patients with MM bear KSHV, and they can therefore be used as a source for dendritic cells for immunotherapy.     

Immunohistochemical study association between human herpesvirus 8 and multiple myeloma

Multiple myeloma (MM) is a plasma cell neoplasm characterized by a clonal proliferation of plasma cells that produce a monoclonal protein and involve the skeleton at multiple sites. Although human herpesvirus-8(HHV-8) has been implicated in pathogenesis of disease, but this role is not clear. The aim of this study was to show direct evidence of HHV-8 in bone marrow tissue of MM patients with use of immunohistochemical method. Standard immunohistochemstry was performed with HHV-8 marker on formalin fixed paraffin embedded bone marrow tissue in 30 MM cases and 30 subjects with normal bone marrow tissue. Slides were examined for nuclear immunoreactivity by two pathologists. The data were analyzed with 2 × 2 contingency tables and Fischer’s exact test, also differences with P-value under 0.05 (P ≤ 0.05) was considered statistically significant. Nuclear immunoreactivity for HHV-8 was detected in four patients (13.3%) with MM but was not detectable in any normal bone marrow cells. Fisher exact test showed no difference for HHV-8 immunoreactivity between two groups of case and control (P = 0.11). Our finding demonstrated that HHV-8 infection did not seem influence on pathogenesis of multiple myeloma.     

Danish patients with untreated multiple myeloma do not harbour human herpesvirus 8

The role of human herpesvirus 8 (HHV-8) in multiple myeloma (MM) remains controversial. We examined 15 Danish MM patients before cytoreductive therapy. Mononuclear cells isolated from peripheral blood and bone marrow aspirates, as well as long-term cultured bone marrow stromal cells, were assayed for the presence of HHV-8 DNA. All material was tested by three simple unnested polymerase chain reaction (PCR) assays (amplifying regions of ORF26, ORFK1 and ORF75) and two nested PCR assays (amplifying regions of ORF26). HHV-8 was not demonstrated in any of the samples. Our findings do not suggest an association between HHV-8 and MM in the Danish population.     

Human herpesvirus 8 enhances human immunodeficiency virus replication in acutely infected cells and induces reactivation in latently infected cells

Human herpesvirus 8 (HHV-8) is etiologically associated with Kaposi sarcoma (KS), the most common AIDS-associated malignancy. Previous results indicate that the HHV-8 viral transactivator ORF50 interacts synergistically with Tat protein in the transactivation of human immunodeficiency virus (HIV) long terminal repeat (LTR), leading to increased cell susceptibility to HIV infection. Here, we analyze the effect of HHV-8 infection on HIV replication in monocyte-macrophage and endothelial cells, as potential targets of coinfection. Primary or transformed monocytic and endothelial cells were infected with a cell-free HHV-8 inoculum and subsequently infected with lymphotropic or monocytotropic strains of HIV. The results show that HHV-8 coinfection markedly increases HIV replication in both cell types. HHV-8 infection induces also HIV reactivation in chronically infected cell lines and in peripheral blood mononuclear cells (PBMCs) from patients with asymptomatic HIV, suggesting the possibility that similar interactions might take place also in vivo. Furthermore, coinfection is not an essential condition, since contiguity of differently infected cells is sufficient for HIV reactivation. The results suggest that HHV-8 might be a cofactor for HIV progression and that HHV-8-infected endothelial cells might play a relevant role in transendothelial HIV spread.     

Human herpesvirus 8 genome is not found in whole bone marrow core biopsy specimens of patients with plasma cell dyscrasias

Recently, molecular evidence of the gamma herpesvirus, human herpesvirus 8 (HHV-8), was found in the nonmalignant bone marrow stromal cells of patients with multiple myeloma using a polymerase chain reaction (PCR)-based assay. Other investigators have been unable to confirm either the presence of HHV-8 using molecular techniques or serologic evidence of prior infection with HHV-8. In order to maximize the likelihood of detection of small quantities of the virus and minimize the risk of potential nucleic acid contamination, we used entire bone marrow biopsy core specimens for DNA extraction and amplification. These specimens included both malignant plasma cells and bone marrow stromal cells and were subjected to minimal manipulation prior to DNA extraction and PCR. We tested eight patients with various plasma cell dyscrasias and compared them to negative controls with non-Hodgkin's lymphoma using standard PCR assays utilizing the KS330(233)primers and probe for HHV-8. This assay is reproducibly positive in Kaposi's sarcoma tissue. We found no evidence of HHV-8 DNA in either the lymphoma controls or the samples from patients with the plasma cell dyscrasias using these methods. We conclude that HHV-8 is unlikely to play a major role in the pathogenesis of the plasma cell dyscrasias in the majority of patients with these diseases. This report adds to the body of evidence that HHV-8 is not associated with plasma cell dyscrasias like multiple myeloma.     

Dendritic cells cultured from mononuclear cells and CD34 cells in myeloma do not harbour human herpesvirus 8

Dendritic cells (DC) are antigen-presenting cells with the potential to be a powerful adjuvant in the immunotherapy of haematological malignancy, including myeloma. Recently, human herpesvirus 8 (HHV-8) infection of dendritic cells in the long-term bone marrow stromal cultures of patients with myeloma has been reported. This finding is of great potential importance regarding oncogenesis in myeloma in addition to having significant implications for the use of DC in the immunotherapy of this disease. Therefore DC generated from mobilized blood mononuclear cells (MO-DC) and purified CD34⁺ cells (CD34-DC) of myeloma patients were examined for the presence of HHV-8 using a sensitive PCR technique. HHV-8 was not demonstrated in MO-DC or CD34-DC and we conclude that these cells remain a suitable vehicle for investigation in the immunotherapy of myeloma.     

Clearance of human herpesvirus 8 from blood and regression of leukopenia-associated aggressive classic Kaposi's sarcoma during interferon-alpha therapy: a case report.

A human immunodeficiency virus-negative woman with severe classic Kaposi's sarcoma, idiopathic leukopenia, and massive spread of human herpesvirus 8 (HHV-8) in circulating cells showed stable disease remission in response to systemic interferon-alpha treatment that was accompanied by increased CD3(+) and CD4(+) T cell numbers and complete clearance of HHV-8 from the circulation. These results suggest a direct relationship between HHV-8 clearance from blood and regression of Kaposi's sarcoma and are consistent with the in vitro inhibitory effects of interferon-alpha on HHV-8 infection.     

Identification of an HLA A*0201-restricted CD8(+) T-cell epitope for the glycoprotein B homolog of human herpesvirus 8

Human herpesvirus 8 (HHV-8; Kaposi sarcoma-associated herpesvirus)-specific cytotoxic T-lymphocyte (CTL) and interferon-gamma (IFN-gamma) responses to proteins produced during the lytic cycle of HHV-8 replication are mediated by HLA class I-restricted, CD8(+) T cells. We have characterized the fine specificity of the CD8(+) T-cell response to 25 peptides derived from 5 HHV-8 lytic cycle proteins based on a prediction model for HLA A*0201 binding motifs. One of the 25 HLA A*0201 peptides derived from the glycoprotein B (gB) homolog of Epstein-Barr virus (gB(492-500); LMWYELSKI; single-letter amino acid codes) bound to HLA A*0201 and stimulated IFN-gamma responses in CD8(+) T cells from HHV-8(+), HLA A*0201 persons, but not HHV-8-seronegative or non-HLA A*0201 persons. The peptide also induced IFN-gamma and CTL reactivity to naturally processed gB protein. The peptide was a major immunogenic epitope of HHV-8 as indicated by induction of IFN-gamma responses in peripheral blood mononuclear cells from 5 of 5 HHV-8 seropositive, HLA A*0201 persons when gB(492-500) was presented by autologous dendritic cells. T-cell reactivity to gB(492-500) was not related to detectable HHV-8 DNA in the blood. These data show that CD8(+) T cells recognize an HLA A*0201-restricted epitope for HHV-8 lytic cycle protein gB, particularly when presented by dendritic cells. This epitope may be important in control of HHV-8 infection by CD8(+) T cells.     

Lack of serologic association of human herpesvirus-8 (KSHV) in patients with monoclonal gammopathy of undetermined significance with and without progression to multiple myeloma

Because human herpesvirus-8 (HHV-8) DNA has been found in multiple myeloma (MM) patients by polymerase chain reaction, it was suggested that HHV-8 may play a role in the transformation of monoclonal gammopathy of undetermined significance (MGUS) to MM. Therefore, 362 MGUS sera with and without progression to MM were tested for IgG antibody to HHV-8. Only 7.8% of the MGUS sera contained HHV-8 antibody to lytic proteins, and IgG antibody to HHV-8 latent antigen was even lower than lytic antibody (2.9%). No differences were observed in the distribution of antibody to HHV-8 in sera from MGUS patients who progressed to MM. The seroprevalences of HHV-8 in MGUS (7.8%), MM (5.4%), and healthy donors (5.9%) were similar, thus arguing for the lack of epidemiologic evidence of HHV-8 participation in the pathogenesis of MM. MGUS patients were immune competent in response to Epstein-Barr virus (EBV) infection because 97% contained antibody to EBV virus capsid antigen.     

Autocrine nerve growth factor is essential for cell survival and viral maturation in HHV-8-infected primary effusion lymphoma cells

High levels of nerve growth factor (NGF) are found in sera from individuals infected with human herpesvirus 8 (HHV-8). BC-1 and BCBL-1 cells are primary effusion lymphoma-derived B-cell lines; BC-1 cells are infected by HHV-8 and the Epstein-Barr virus (EBV), and BCBL-1 cells are infected only by HHV-8. Both cells express NGF receptors and produce NGF, whereas RAMOS cells (a B-cell line that is negative for HHV-8 and EBV) express NGF receptors but do not produce detectable NGF. Neutralization of endogenous NGF results in cell growth inhibition and apoptosis in BCBL-1 cells and, to a minor extent, in BC-1 cells. When the HHV-8 lytic cycle is induced in BCBL-1 cells by tetradecanoyl phorbol acetate (TPA), an initial reduction of endogenous NGF production is observed, and many cells undergo apoptosis. However, at 48 hours, TPA-treated cells produce significantly more NGF than untreated controls, and a subsequent recovery of cell viability is observed. Consistent with this finding, the addition of exogenous NGF or anti-NGF antibodies to TPA-treated cells reduces or increases, respectively, the rate of apoptosis in response to TPA. Finally, electron microscopy of TPA-treated BCBL-1 cells shows that the addition of exogenous NGF increases the number of cells producing and releasing complete virions as compared with the controls (25% versus 5%). On the contrary, NGF neutralization leads to the production of defective viral progeny in about 2% of cells. These data indicate that NGF is essential for both cell survival and virus maturation in HHV-8-infected cell lines. (Blood. 2000;95:2905-2912)     

Distribution of human herpesvirus-8 latently infected cells in Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma

Human herpesvirus 8 (HHV-8, also called KSHV) is linked to the etiopathogenesis of Kaposi's sarcoma (KS), multicentric Castleman's disease (MCD), and primary effusion lymphoma (PEL). The universal presence of HHV-8 in early KS has not yet been shown. We used a mAb (LN53) against latent nuclear antigen-1 (LNA-1) of HHV-8 encoded by ORF73 to study the distribution of the cell types latently infected by HHV-8 in patch, plaque, and nodular KS, MCD, and PEL. In early KS, HHV-8 is present in <10% of cells forming the walls of ectatic vessels. In nodular KS, HHV-8 is present in cells surrounding slit-like vessels and in >90% of spindle cells, but not in normal vascular endothelium. In addition, HHV-8 colocalizes with vascular endothelial growth factor receptor-3 (VEGFR-3), a marker of lymphatic and precursor endothelium. In early KS lesions, VEGFR-3 is more extensively expressed than LNA-1, indicating that HHV-8 is not inducing the proliferation of VEGFR-3-positive endothelium directly. In MCD, HHV-8 is present in mantle zone large immunoblastic B cells. No staining for LNA-1 is seen in samples from multiple myeloma, prostate cancer, and angiosarcoma, supporting the absence of any etiological link between these diseases and HHV-8.     

Concomitant mobilization of plasma cells and hematopoietic progenitors into peripheral blood of multiple myeloma patients: positive selection and transplantation of enriched CD34+ cells to remove circulating tumor cells

One advantage of the use of peripheral blood stem cells (PBSCs) over autologous bone marrow would be a reduced risk of tumor cell contamination. However, the level of neoplastic cells in the PB of multiple myeloma (MM) patients after mobilization protocols is poorly investigated. In this study, we evaluated PB samples from 27 pretreated MM patients after the administration of high dose cyclophosphamide (7 g/m2 or 4 g/m2) and granulocyte-colony stimulating factor for the detection of myeloma cells as well as hematopoietic progenitors. Plasma cells containing intracytoplasmic lg were counted by microscope immunofluorescence after incubation with appropriate antisera directed against light- and heavy-chain lg. Moreover, flow cytometry studies were performed to determine the presence of malignant B-lineage elements by using monoclonal antibodies against the CD19 antigen and the monotypic light chain. Before initiation of PBSC mobilization, circulating plasma cells were detected in all MM patients in a percentage ranging from 0.1% to 1.8% of the mononuclear cell fraction (mean value, 0.7% +/- 0.4% SD). In these patients, a higher absolute number of PB neoplastic cells was detected after chemotherapy and granulocyte colony-stimulating factor. Kinetic analysis showed a pattern of tumor cell mobilization similar to that of normal hematopoietic progenitors with a maximum peak falling within the optimal time period for the collection of PBSCs. The absolute number of plasma cells showed a 10 to 50-fold increase as compared with the baseline value. Apheresis products contained 0.7% +/- 0.2% SD of myeloma cells (range, 0.2% to 2.7%). Twenty-three MM patients were submitted to PBSC collection. In 10 patients, circulating hematopoietic CD34+ cells were highly enriched by avidin-biotin immunoabsorption, were cryopreserved, and used to reconstitute bone marrow function after myeloablative therapy. The median purity of the enriched CD34+ cell population was 89.5% (range, 51% to 94%), with a 75-fold increase as compared with the pretreatment samples. The median overall recovery of CD34+ cells and colony-forming unit-granulocyte-macrophage was 58% (range, 33% to 95%) and 45% (range, 7% to 100%), respectively. Positive selection of CD34+ cells resulted in 2.5- to 3-log depletion of plasma cells and CD19+ B-lineage cells as determined by immunofluorescence studies, although DNA analysis of CDR III region of IgH gene showed the persistence of minimal residual disease in 5 of 6 patient samples studied. Myeloma patients were reinfused with enriched CD34+ cells after myeloablative therapy consisting of total body irradiation (1,000 cGy) and highdose melphalan (140 mg/m2). They received a median of 4 x 10(6) CD34+ cells/kg and showed a rapid reconstitution of hematopoiesis; the median time to 0.5 x 10(9) neutrophils and to 20 and 50 x 10(9) platelets per liter of PB was 10, 11, and 12 days, respectively. These results, as well as other clinically significant parameters, did not significantly differ from those of patients (n = 13) receiving unmanipulated PBSCs after the same pretransplant conditioning regimen. In summary, our data show the concomitant mobilization of tumor cells and hematopoietic progenitors in the PB of MM patients. Positive selection of CD34+ cells reduces the contamination of myeloma cells from the apheresis products up to 3-log and provides a cell suspension capable of restoring a normal hematopoiesis after a total body irradiation-containing conditioning regimen.     

Clinical and molecular follow-up by amplification of the CDR-III IgH region in multiple myeloma patients after autologous transplantation of hematopoietic CD34+ stem cells.

BACKGROUND AND OBJECTIVE: Autologous blood stem cell transplantation (ABSCT) using chemotherapy-induced mobilization of peripheral blood stem cells (PBSC) is being increasingly used in the treatment of multiple myeloma (MM). We report the clinical and molecular follow-up of 10 MM patients who underwent autologous stem cell transplantation with peripheral blood selected CD34+ cells, as support therapy following a myeloablative conditioning regimen. DESIGN AND METHODS: The CDR-III coding region of the IgH gene was studied by a) consensus PCR applied to 8 MM patients, or b) by direct sequencing of PCR product generated by family-specific primers in the remaining two patients (who became immunofixation analysis (IF) negative). In this case, two patient-specific primers were generated, thus obtaining a high PCR assay sensitivity and specificity (ASO PCR). RESULTS: Seven patients are alive: 4 of them have serum M protein assessable by IF, while 1 was not a secretor and 2 converted from serum IF positivity to negativity 6 and 12 months after ABSCT. Three patients died: 1 from disease progression and 2 from infective complications during clinical remission. The molecular analysis during the follow-up showed that the bone marrow samples from the two patients who obtained IF negativity were persistently PCR positive for the presence of rearranged CDR-III region. Moreover, despite the remarkable reduction of myeloma burden, a minimal level of residual myeloma cells was still detectable by molecular analysis. INTERPRETATION AND CONCLUSIONS: These results confirm that although positive selection of CD34+ cells markedly reduces the contamination of myeloma cells from apheresis products by up to 3 log, and provides a cell suspension capable of restoring normal hematopoiesis after ablative conditioning regimen, it does not abrogate myeloma cell contamination in most of the apheresis products.     

The restricted cellular host range of human herpesvirus 8

DESIGN: A selection of primary and transformed cell types were evaluated for their susceptibility to infection with human herpesvirus 8 (HHV-8)/Kaposi's sarcoma-associated herpesvirus. METHODS: Sources of HHV-8 included Kaposi's sarcoma lesion punch biopsies that were either cocultured directly with target cells or that were first cocultured with human lymphocytes to derive HHV-8-containing fluids that were inoculated onto target cells. HHV-8 was also obtained from primary effusion lymphoma-derived cell lines. Techniques to detect infection included the PCR, immunofluorescence assays and in situ hybridization. RESULTS: Susceptible cells included human umbilical cord blood mononuclear cells (UCMC), adult CD19 B cells, macrophages and certain endothelial cells of human and animal origin, including some that are transformed with human papilloma virus type 16 E6 and E7 genes. The infection of lymphocytes did not yield established lymphoblastoid cell lines (LCL) and virus infection persisted for only 4-7 days. However, long-term HHV-8 infection of UCMC could be achieved by coinfection with Epstein-Barr virus. HHV-8 could also infect UCMC LCL recently derived by Epstein-Barr virus transformation, but long-established LCL could not be infected with HHV-8. CONCLUSIONS: These data provide further biological evidence in cell culture for the limited cellular host range of HHV-8 to CD19 B cells, macrophages, and certain endothelial cells.     

Remission of HHV-8 and HIV-associated multicentric Castleman disease with ganciclovir treatment

Multicentric Castleman disease (MCD) is a lymphoproliferative disorder associated with human herpesvirus 8 (HHV-8) infection among persons with human immunodeficiency virus (HIV) infection. Treatment often includes chemotherapy, and progression to non-Hodgkin lymphoma frequently occurs. MCD is characterized in part by active HHV-8 replication, and many of the symptoms of MCD may be attributable to viral gene products. We describe the effect of ganciclovir on the clinical and virologic course of MCD in a series of 3 case reports. Two patients experienced a reduction in the frequency of episodic flares of MCD and detectable HHV-8 DNA with intravenous or oral ganciclovir, whereas the third patient recovered from an acute episode of renal and respiratory failure with intravenous ganciclovir therapy. These data provide in vivo evidence for the utility of antiviral agents against HHV-8 in the management of MCD.     

Retrospective analysis of HHV-8 viremia and cellular viral load in HIV-seropositive patients receiving interleukin 2 in combination with antiretroviral therapy

The combination of interleukin 2 (IL-2) and antiretroviral therapy (ART) represents an emerging strategy in the treatment of patients infected with HIV. Aside from its immunomodulatory role, however, IL-2 may induce replication of human herpesvirus 8 (HHV-8)/Kaposi sarcoma (KS)-associated herpesvirus. We retrospectively evaluated HHV-8 plasma viremia and cellular load, as well as anti-HHV-8 antibody titers, in sequential samples from 84 patients receiving ART alone or in combination with IL-2. At baseline, HHV-8 plasma viremia was present only in 2 HHV-8-seropositive patients in whom KS subsequently developed during or immediately after termination of IL-2 therapy. The level of viremia increased during follow-up and peaked at the time of the clinical manifestation of KS. Moreover, transient peaks of HHV-8 viremia were temporally associated with administration of IL-2. HHV-8 plasma viremia was never detected in the other 47 patients receiving IL-2 nor in 35 controls treated only with ART. Thus, IL-2 therapy seems safe in most patients infected with both HIV and HHV-8, except for those with detectable HHV-8 viremia, who may not be eligible for IL-2 treatment.