P. Barozzi

Clinico-pathological characterization of hepatitis C virus-related B-cell non-Hodgkin's lymphomas without symptomatic cryoglobulinemia

BACKGROUNDnEpidemiological evidence has suggested an association between hepatitis C virus (HCV) infection and B-cell lymphoproliferation. We studied the prevalence of HCV infection in a series of de novo B-cell non-Hodgkins lymphoma (B-NHL) cases and correlated virological findings with clinico-histological features.nnnPATIENTS AND METHODSnOne hundred fifty-seven patients with de novo B-NHL were included in the study. Their serum was examined by ELISA and RIBA for the presence of anti-HCV antibodies, and either the peripheral blood mononuclear cells or the pathology tissues of all of the patients were examined by reverse transcriptase polymerase chain reaction for the presence of HCV RNA sequences.nnnRESULTSnHCV infection occurred in 22.3% of B-NHL patients and was documented before the diagnosis in about half of the positive cases. Of interest, HCV infection was more frequently found in follicular center, marginal zone and diffuse large-cell lymphoma types, but was not associated with symptomatic cryoglobulinemia. The median survival time was 48 months in HCV-positive and 52 months in HCV-negative B-NHL patients.nnnCONCLUSIONSnOur findings strengthen the pathogenetic link between HCV and B-NHL and show that HCV infection may be associated with the malignant proliferation of defined B-cell subsets other than the immunoglobulin Mk B-cell subset involved in the pathogenesis of mixed cryoglobulinemia type II and associated lymphoplasmacytoid lymphoma type. HCV-related liver disease did not affect the survival of our B-NHL patients.

Detection of antibodies to human herpesvirus 8 in Italian children: evidence for horizontal transmission.

Human herpesvirus 8 (HHV-8), also known as Kaposis sarcoma associated herpesvirus (KSHV), has been shown to be the causative agent for Kaposis sarcoma (KS) and to be more prevalent in populations or risk groups at increased risk for KS. HHV-8 infection is rare in children from the US and the UK, but has been reported in African children. In this study we examine HHV-8 infection in children from Italy, a country with an elevated prevalence of HHV-8 in adults and high socio-economic conditions.

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

Prevalence of Human Herpesvirus-6 Chromosomal Integration (CIHHV-6) in Italian Solid Organ and Allogeneic Stem Cell Transplant Patients

The unique phenomenon of human herpesvirus‐6 (HHV‐6) chromosomal integration (CIHHV‐6) may account for clinical drawbacks in transplant setting, being misinterpreted as active infection and leading to unnecessary and potentially harmful treatments. We have investigated the prevalence of CIHHV‐6 in 205 consecutive solid organ (SO) and allogeneic stem cell transplant (alloSCT) Italian patients. Fifty‐two (38.5%) of 135 solid organ transplant (SOT) and 16 (22.8%) of 70 alloSCT patients resulted positive for plasma HHV‐6 DNA by real‐time polymerase chain reaction. Seven SOT and three alloSCT patients presented HHV‐6‐related diseases, requiring antivirals. Two further patients (0.9%) were identified, presenting high HHV‐6 loads. The quantification of HHV‐6 on hair follicles disclosed the integrated state, allowing the discontinuation of antivirals. Before starting specific treatments, CIHHV‐6 should be excluded in transplant patients with HHV‐6 viremia by the comparison of HHV‐6 loads on different fluids and tissues. Pretransplantation screening of donors and recipients may further prevent the misdiagnosis of CIHHV‐6.

Feedbacks and adaptive capabilities of the PI3K/Akt/mTOR axis in acute myeloid leukemia revealed by pathway selective inhibition and phosphoproteome analysis.

Acute myeloid leukemia (AML) primary cells express high levels of phosphorylated Akt, a master regulator of cellular functions regarded as a promising drug target. By means of reverse phase protein arrays, we examined the response of 80 samples of primary cells from AML patients to selective inhibitors of the phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) axis. We confirm that >60% of the samples analyzed are characterized by high pathway phosphorylation. Unexpectedly, however, we show here that targeting Akt and mTOR with the specific inhibitors Akti 1/2 and Torin1, alone or in combination, result in paradoxical Akt phosphorylation and activation of downstream signaling in 70% of the samples. Indeed, we demonstrate that cropping Akt or mTOR activity can stabilize the Akt/mTOR downstream effectors Forkhead box O and insulin receptor substrate-1, which in turn potentiate signaling through upregulation of the expression/phosphorylation of selected growth factor receptor tyrosine kinases (RTKs). Activation of RTKs in turn reactivates PI3K and downstream signaling, thus overruling the action of the drugs. We finally demonstrate that dual inhibition of Akt and RTKs displays strong synergistic cytotoxic effects in AML cells and downmodulates Akt signaling to a much greater extent than either drug alone, and should therefore be explored in AML clinical setting.

Trends in Immune Cell Function Assay and Donor‐Specific HLA Antibodies in Kidney Transplantation: A 3‐Year Prospective Study

The immune cell function assay (ICFA) and de novo anti‐donor‐specific HLA antibodies (DSA) have been proposed as assays for immune monitoring in renal transplantation, but longitudinal studies examining the modification of both parameters over time and their relation with clinical events are lacking. We prospectively measured longitudinal changes in ICFA and DSA levels in 55 kidney transplant recipients over 3‐year follow‐up (534 visits) and analyzed their relation with the risk of developing acute rejections or infections. Seven patients (12.7%) developed biopsy‐proven acute rejection, and 20 (36.4%) developed viral infections. At 3 years posttransplant, 28% of the patients had developed de novo DSA. ICFA levels peaked at 1–2 months posttransplant (pu2009=u20090.005) and leveled off thereafter. They were not associated with the risk of acute rejections, viral infections or development of de novo DSA. Instead, the incidence of de novo DSA was higher in patients who previously had viral infections (adjusted‐odds ratio of de novo DSA associated with prior infections: 6.03 [95% CI, 1.64–22.06; pu2009=u20090.007]). Our prospective, longitudinal study does not support using ICFA to quantify the immune risk in kidney transplantation. Further studies are needed to confirm the relationship between viral infections and the subsequent development of de novo DSA.

The oncogenic 30 and 69 bp deletion variants of the EBV LMP-1 gene are common in HIV-negative lymphoproliferations, both malignant and benign

BACKGROUNDnIn vitro studies have shown that the 30 and 69 base pair (bp) deletion variants of the latent membrane protein (LMP)-1 gene of the Epstein-Barr virus (EBV) have a higher transforming capacity than the wild-type variant. In recent years these studies have triggered an in vivo search for such potentially oncogenic variants in lymphoid tissues.nnnPATIENTS AND METHODSnWe used polymerase chain reaction (PCR) to investigate the prevalence of LMP-1 gene variants in EBV-positive lymph nodes from 60 HIV-negative Italian patients with benign and malignant lymphoid disorders.nnnRESULTSnThe 30 bp variant was detected in 10 of 39 (25.6%) malignant lymphomas but also in 4 of 13 (30%) reactive lymphadenitis with follicular hyperplasia. Of note is the fact that the 69 bp variant was detected in three cases of malignant lymphoproliferation but also in two cases of localized Castlemans disease of hyalin vascular type.nnnCONCLUSIONSnThe molecular detection of the oncogenic variants of the LMP-1 gene in a lymph node biopsy as an indicator of the aggressiveness of the EBV-associated lymphoproliferative disease must be considered with caution. The relatively high frequency of the 69 bp variant in our series compared with that reported in the literature probably reflects a different incidence of LMP-1 variants in healthy populations from different geographical areas.

β-HHVs and HHV-8 in Lymphoproliferative Disorders.

Similarly to Epstein-Barr virus (EBV), the human herpesvirus-8 (HHV-8) is a γ-herpesvirus, recently recognized to be associated with the occurrence of rare B cell lymphomas and atypical lymphoproliferations, especially in the human immunodeficiency virus (HIV) infected subjects. Moreover, the human herpesvirus-6 (HHV-6), a β-herpesvirus, has been shown to be implicated in some non-malignant lymph node proliferations, such as the Rosai Dorfman disease, and in a proportion of Hodgkin’s lymphoma cases. HHV-6 has a wide cellular tropism and it might play a role in the pathogenesis of a wide variety of human diseases, but given its ubiquity, disease associations are difficult to prove and its role in hematological malignancies is still controversial. The involvement of another β-herpesvirus, the human cytomegalovirus (HCMV), has not yet been proven in human cancer, even though recent findings have suggested its potential role in the development of CD4+ large granular lymphocyte (LGL) lymphocytosis. Here, we review the current knowledge on the pathogenetic role of HHV-8 and human β-herpesviruses in human lymphoproliferative disorders.