Raffaella Trovato

Treatment of herpesvirus associated primary effusion lymphoma with intracavity cidofovir.

Primary effusion lymphoma (PEL) is a B-cell non-Hodgkin lymphoma, involving the serous cavities, which is invariably associated with Kaposi sarcoma-associated herpesvirus (KSHV)/ human herpesvirus-8 (HHV-8) and often with Epstein–Barr virus (EBV) infections. The disease accounts for less than 3% of AIDS-associated lymphomas, as recognized by the WHO, but a few cases have also been reported in transplant patients as well as in elderly human immunodeficiency virus (HIV)-negative men originating from areas endemic for HHV-8 infection, and at high risk for classic Kaposi sarcoma (KS). PEL has a poor prognosis, with a median survival of few months, in the majority of affected patients. In AIDS patients, combined chemotherapy, including high-dose methotrexate, may be attempted with the possibility to achieve complete remission, at least in some cases. On the contrary, in elderly and transplant patients, the use of chemotherapeutic regimens has been generally hampered by severe toxicity, and has revealed unsuccessful in the vast majority of the treated patients, raising the need for alternative treatment approaches. Cidofovir is an antiviral with a broad spectrum of activity against DNA viruses, and has been reported to be one of the most effective agents to inhibit HHV-8 replication in vitro. In addition to its antiviral activity, a potent antitumor activity has been recently attributed to cidofovir. Topical application of cidofovir resulted effective to treat both neoplastic genital lesions and recurrent laryngeal papillomatous lesions caused by human papillomavirus (HPV). In animal models, cidofovir has been found to be effective on virus-associated tumors, such as in nude mice with EBV-associated nasopharyngeal carcinoma, in which intratumoral injection of cidofovir induced in vivo regression of the tumors. The antitumor activity of cidofovir is not specifically related to the antiviral action of the compound. Consistent with this, cidofovir has also been shown to inhibit the growth of tumors, such as hemangiosarcomas, which are not associated with oncogenic viruses. In the current study, we tested whether cidofovir exerts an antitumor effect against two PEL cell lines in vitro, namely the HHV-8-positive BCBL-1 and the HHV-8-positive and EBVpositive HBL-6 cell lines. The EBV-negative and HHV-8negative RAMOS cell line was tested as control. Then, we investigated the effect of intracavity injections of cidofovir for the treatment of three elderly, HIV negative, patients with PEL. The BCBL-1, HBL-6 and the RAMOS cell lines were cultured in RPMI 1640 medium, supplemented with 15% of heat inactivated fetal calf serum (FBS), glutamine 1 mM and antibiotics, at 371C in a 5% CO2 humidified incubator. Cells were treated with cidofovir at 0.01, 0.1, 0.5, and 1 mM, for 3 and 6 days. At the end of the incubation period, cell count and viability were evaluated in triplicate, with Trypan blue dye exclusion assay. Apoptosis was studied by a combination of methods, including, morphology, propidium iodide staining (50 mg/ml) and analysis by flow cytometry (Becton and Dickinson Italia, Milano, Italy), the DNA fragmentation assay, and the in situ cell death detection kit (Boheringer Mannheim, Mannheim, Germany), which relies on the use of terminal deoxynucleotidyl transferase (TdT) that catalyses the polymerization of fluorescein-labeled nucleotides to free 30-hydroxyl residues of DNA fragments generated by endonucleases during apoptosis (TUNEL). Cidofovir caused a dose-dependent inhibition of BCBL-1 and HBL-6 cell proliferation and viability (Figure 1a–d), while in RAMOS cells, cidofovir inhibited proliferation but had no effect on viability (Figure 1e and f). Cidofovir induced a dose-dependent apoptosis in both the BCBL-1 and HBL-6 cell lines, after 3 and 6 days, as detected by flow cytometry analysis (Figure 2a). Apoptosis induced by cidofovir was confirmed by the observation of typical apoptotic features of cell morphology, by the characteristic ladder of fragmented genomic DNA on the DNA fragmentation assay, as well by the detection of fluorescein-labeled DNA strand breaks in apoptotic cells, by TUNEL assay (Figure 2b–d in supplementary information). On the contrary, RAMOS cells did not show any significant apoptosis up to 6-day treatment with cidofovir (Figure 2a and analysis of cell cycle distribution of RAMOS cells in supplementary information). In three patients, (pt. 1, pt. 2 and pt. 3) a diagnosis of HHV-8positive PEL, in Ann Arbor stage IV, was made, on the basis of morphologic, immunophenotypic and molecular analysis, as described. EBV coinfection was documented in pt. 1, while serology for HIV, hepatitis B and C viruses was negative in all three patients. In detail, pt. 1, a 96-year-old Italian man, was hospitalized for bilateral pleural effusion. No clinical evidence of KS was found. The patient was subjected to pleural drainage every 2 weeks for 4 months, without receiving any chemotherapy. Then, he was treated with two doses of intrapleura cidofovir, at 2.5 mg/kg, every 1 week. No recurrence of pleural effusion was observed after the second injection. PEL relapsed at the same site, as documented on standard radiographic examination, 10 months later. The patient refused cidofovir therapy and was subjected to pleural drainage. The patient died for heart failure. Pt. 2, a 70-year-old Italian man, was hospitalized for recurrent peritoneal effusions. The patient had a history of KS, for which he had received chemotherapy and radiotherapy 10 years before, with complete tumor regression. The patient was subjected to peritoneal drainages every 10 days for 2 months. Then, the patient received three doses of intraperitoneal cidofovir, at 5 mg/kg, every 1 week. No recurrence of peritoneal effusion was observed after the last cidofovir injection. PEL relapse was documented in the pleura 5 months later. The patient died for a cerebrovascular accident, with no peritoneal relapse. Pt. 3, a 77-year-old Italian man, was hospitalized for a 5-month history of bilateral pleural effusion requiring repeated pleural drainages. Chemotherapy was avoided because the patient had been suffering from heart failure. He was treated with three doses of intrapleura cidofovir, Received 31 May 2004; accepted 19 November 2004; Published online 20 January 2005 Correspondence: Dr M Luppi, Department of Oncology and Hematology, University of Modena and Reggio Emilia, Policlinico, Via del Pozzo 71, 41100 Modena, Italy; Fax: þ 39 059 4224549; E-mail: mluppi@unimore.it

Angiogenesis in multiple myeloma: correlation between in vitro endothelial colonies growth (CFU-En) and clinical–biological features

Mouse models and studies performed on fixed bone marrow (BM) specimens obtained from patients with multiple myeloma (MM) suggest that plasma cell growth is dependent on endothelial cell (EC) proliferation within the BM microenvironment. In order to assess whether EC overgrowth in MM reflects a spontaneous in vitro angiogenesis, BM mononucleated cells from 13 untreated (UT) MM, 20 treated (11 with melphalan and nine with DAV schedule) MM, eight patients with monoclonal gammopathy of uncertain significance (MGUS) and eight controls were seeded in an unselective medium to assess EC proliferation. Furthermore, the influence of IL6 on the EC growth was investigated. Endothelial colonies (CFU-En) appeared as small clusters, formed by at least 100 slightly elongated and sometimes bi-nucleated cells expressing factor VIII, CD31 and CD105 (endoglin). The CFU-En mean number/106 BM mononucleated cells in untreated MM samples (2.07 s.d. ± 1.3) was significantly higher than in normal BM (0.28 ± 0.48), while no difference was seen between normal BM and MGUS (0.28 ± 0.54). Interestingly, the mean number of CFU-En in the DAV group (1.88 ± 1.6) did not differ from the UT, while it was found to be lower in the melphalan group (0.31 ± 0.63). The addition of anti-IL6 monoclonal antibody induced a reduction of both the plasma cells in the supernatant and the CFU-En number. This study describes a rapid and feasible assay providing support for the association between EC and plasma cells further suggesting that the in vitro angiogenesis process may parallel that observed in vivo.

Deletion of the p16INK4A gene in ex vivo acute adult T cell lymphoma/leukemia cells and methylation of the p16INK4A promoter in HTLV type I-infected T cell lines.

The stoichiometry of the p16INK4A and p15INK4B proteins bound to the cyclin D-CDK4/6 complex regulates the entry of cells into the G1 phase of the cell cycle. Thus, their level of expression is essential in maintaining regulated cell growth. In several tumors, deletion of these genes has been reported and, more recently, promoter methylation has been suggested as an alternative mechanism to decrease the expression of these cell cycle inhibitor proteins. Here, we studied the methylation status and the integrity of the p16INK4A and p15INK4B genes in 8 chronically HTLV-I-infected T cell lines and in ex vivo cells from 14 ATLL patients. Deletion of the locus carrying both genes was not found in the HTLV-I-infected T cell lines but was found in seven of eight acute ATLL cases and in none of the PBMCs from the chronic cases or the affected lymph nodes of the lymphoma type. In contrast, partial or complete methylation of one or both genes was found only in chronically HTLV-I T cells. Thus, HTLV-I infection targets the p16INK4A and p15INK4B loci both in vitro and in vivo, although the mechanisms may differ.

Human herpesvirus‐6 reactivation in a longitudinal study of two HIV‐1 infected patients

After primary infection, human herpesvirus‐6 (HHV‐6) persists in latent form and can be reactivated in immunocompromised subjects. A longitudinal study of HHV‐6 infection was carried out in two HIV‐1 seropositive patients to provide in vivo evidence of HHV‐6 reactivation. Concomitant with a significant rise of anti‐HHV‐6 IgG detected by IFA, a transient increase of HHV‐6 viral load was shown in PBLs by PCR. During HHV‐6 reactivation it was also identified either cell‐free HHV‐6 by PCR in plasma or IgM antibody titers. HHV‐6 reactivation was followed by a temporary decrease in CD4+ count and by a progressive dramatic loss of CD4+ during the following 18 months. HHV‐6 strain characterization by PCR demonstrated that first patient (MM) initially showed the B variant, followed by reactivation and persistence of the A variant, while in the second (SG) only the A variant was detected. The evidence of HHV‐6 reactivation suggests its involvement in immunologic damage underlying the disease. J. Med. Virol. 51:259–264, 1997.

PCR with degenerate primers for highly conserved DNA polymerase gene of the herpesvirus family shows neither human herpesvirus 8 nor a related variant in bone marrow stromal cells from multiple myeloma patients

The possibility has been raised that either a human herpesvirus‐8 (HHV‐8) variant or a novel, unidentified, γ‐herpesvirus related to HHV‐8 is frequently associated with multiple myeloma (MM), which could explain the lack of antibodies to HHV‐8 antigens and the discordant results from polymerase chain reaction (PCR) studies of HHV‐8‐specific sequences in MM patients. Thus, we used a sensitive PCR assay with degenerate primers targeting the highly conserved DNA polymerase gene of the herpesvirus family to examine the long‐term cultures of bone marrow stromal cells (BMSCs) from 19 MM, 3 monoclonal gammopathies of undetermined significance and 6 control patients. Both the culture supernatant and the adherent stromal layer were examined from the 2nd until the 8th week of culture to assess the immunophenotype of the various cell types harvested for the molecular analysis. BMSCs consisted of a mixed population of fibroblast, macrophage, dendritic and endothelial cells. An amplified product of the expected size was obtained only in 3 MM cases, both in the adherent and nonadherent fractions. Direct sequencing and alignment of the nucleotide and amino acid sequences showed that the DNA sequences were 100% identical to Epstein‐Barr virus (EBV) DNA. The PCR positivity was due to the presence of EBV‐infected lymphoblastoid cells with plasmacytoid features, expressing the EBV‐encoded latent membrane protein‐1 and detectable either in the stromal cells or in the culture supernatant. Our data do not support a causal role of either HHV‐8 or a novel herpesviral variant related to HHV‐8 in MM. Int. J. Cancer 86:76–82, 2000.

Quantitation of HCV viraemia by branched DNA signal amplification in patients treated with α-interferon — A longitudinal study

SummaryUsing bDNA, the plasma viral load trend of HCV-infected patients undergoing IFN therapy was analyzed. Nine patients were enrolled, each assigned to one of three groups, based on IFN response as determined by ALT and AST level trend. HCV was genotyped using DEIA. Each patients clinical stage was determined by liver biopsy analysis. In nonresponding patients elevated viral loads and biochemical parameters were observed. These values were not influenced by IFN treatment. In relapsed patients the cessation of IFN treatment increased viral load; this was associated with a rise in ALT and AST values. In responders ALT and AST levels remained normal; viral load was low. Patients with elevated HCV viral load showed a worsening in their liver histology during the follow-up period. These results confirm that plasma viral load is a good marker of biochemical change and disease progression.ZusammenfassungDer Verlauf der Virämie bei mit Hepatitis C Virus (HCV) infizierten Patienten unter Therapie mit Interferon-α wurde analysiert. Neun Patienten wurden nach dem Trend der AST- und ALT-Spiegel drei Gruppen zugeordnet: Patienten, die auf eine Therapie mit Interferon α ansprachen, Patienten, die nicht ansprachen und Patienten, die ein Rezidiv entwickelten. Der HCV-Genotyp wurde mittels DEIA bestimmt. Das klinische Stadium wurde durch Leberbiopsien ermittelt. Bei Patienten, die nicht ansprachen, war die Virämie hoch, die biochemischen Parameter wiesen hohe Spiegel auf. Die Werte wurden durch die Interferon α-Therapie nicht verändert. Bei Patienten, die nach Absetzen von Interferon ein Rezidiv entwickelten, stiegen Viruslast, ALT- und AST-Werte erneut an. Bei erfolgreich Therapierten blieben die ALT- und AST-Spiegel in einem normalen Bereich. Hohe Virustiter waren mit einer Verschlechterung der Leberhistologie während der Verlaufsbeobachtung verbunden. Die Ergebnisse belegen, daß die Virustiter im Plasma ein guter Marker für die biochemischen Parameter und die weitere Krankheitsentwicklung sind.