Leonardo Potenza

Invasive aspergillosis in patients with acute myeloid leukemia: a SEIFEM-2008 registry study.

Background The aim of this study was to evaluate prognostic factors, treatments and outcome of invasive aspergillosis in patients with acute myeloid leukemia based on data collected in a registry. Design and Methods The registry, which was activated in 2004 and closed in 2007, collected data on patients with acute myeloid leukemia, admitted to 21 hematologic divisions in tertiary care centers or university hospitals in Italy, who developed proven or probable invasive aspergillosis. Results One hundred and forty cases of invasive aspergillosis were collected, with most cases occurring during the period of post-induction aplasia, the highest risk phase in acute myeloid leukemia. The mortality rate attributable to invasive aspergillosis was 27%, confirming previous reports of a downward trend in this rate. Univariate and multivariate analyses revealed that the stage of acute myeloid leukemia and the duration of, and recovery from, neutropenia were independent prognostic factors. We analyzed outcomes after treatment with the three most frequently used drugs (liposomal amphotericin B, caspofungin, voriconazole). No differences emerged in survival at day 120 or in the overall response rate which was 71%, ranging from 61% with caspofungin to 84% with voriconazole. Conclusions Our series confirms the downward trend in mortality rates reported in previous series, with all new drugs providing similar survival and response rates. Recovery from neutropenia and disease stage are crucial prognostic factors. Efficacious antifungal drugs bridge the period of maximum risk due to poor hematologic and immunological reconstitution.

Genetic PTX3 Deficiency and Aspergillosis in Stem-Cell Transplantation

BACKGROUND The soluble pattern-recognition receptor known as long pentraxin 3 (PTX3) has a nonredundant role in antifungal immunity. The contribution of single-nucleotide polymorphisms (SNPs) in PTX3 to the development of invasive aspergillosis is unknown. METHODS We screened an initial cohort of 268 patients undergoing hematopoietic stem-cell transplantation (HSCT) and their donors for PTX3 SNPs modifying the risk of invasive aspergillosis. The analysis was also performed in a multicenter study involving 107 patients with invasive aspergillosis and 223 matched controls. The functional consequences of PTX3 SNPs were investigated in vitro and in lung specimens from transplant recipients. RESULTS Receipt of a transplant from a donor with a homozygous haplotype (h2/h2) in PTX3 was associated with an increased risk of infection, in both the discovery study (cumulative incidence, 37% vs. 15%; adjusted hazard ratio, 3.08; P=0.003) and the confirmation study (adjusted odds ratio, 2.78; P=0.03), as well as with defective expression of PTX3. Functionally, PTX3 deficiency in h2/h2 neutrophils, presumably due to messenger RNA instability, led to impaired phagocytosis and clearance of the fungus. CONCLUSIONS Genetic deficiency of PTX3 affects the antifungal capacity of neutrophils and may contribute to the risk of invasive aspergillosis in patients treated with HSCT. (Funded by the European Society of Clinical Microbiology and Infectious Diseases and others.).

HHV-6A in Syncytial Giant-Cell Hepatitis

Syncytial giant-cell hepatitis is a rare but severe form of hepatitis that is associated with autoimmune diseases, drug reactions, and viral infections. We used serologic, molecular, and immunohistochemical methods to search for an infectious cause in a case of syncytial giant-cell hepatitis that developed in a liver-transplant recipient who had latent infection with variant B of human herpesvirus 6 (HHV-6B) and who had received the organ from a donor with variant A latent infection (HHV-6A). At the onset of the disease, the detection of HHV-6A (but not HHV-6B) DNA in plasma, in affected liver tissue, and in single micromanipulated syncytial giant cells with the use of two different polymerase-chain-reaction (PCR) assays indicated the presence of active HHV-6A infection in the patient. Expression of the HHV-6A-specific early protein, p41/38, but not of the HHV-6B-specific late protein, p101, was demonstrated only in liver syncytial giant cells in the absence of other infectious pathogens. The same markers of HHV-6A active infection were documented in serial follow-up samples from the patient and disappeared only at the resolution of syncytial giant-cell hepatitis. Neither HHV-6B DNA nor late protein was identified in the same follow-up samples from the patient. Thus, HHV-6A may be a cause of syncytial giant-cell hepatitis.

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

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-γ, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, α1–3glucan, β1–3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-γ) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with IA.

Changes in the immune responses against human herpesvirus-8 in the disease course of posttransplant kaposi sarcoma

In nine patients with posttransplant Kaposi sarcoma (KS) T-cell responses to human herpesvirus (HHV)-8 latent and lytic antigens, as detected by enzyme-linked-immunospot (Elispot) assay, were absent at disease onset. Virus-specific T-cell responses were detected in six renal recipients at remission after a reduction of calcineurin inhibitors (CIs), and in two HHV-8 seropositive renal recipients without KS. In two liver recipients undergoing switch from CIs to sirolimus (SRL), normalization of the T-cell repertoire and recovery of both HHV-8-specific effector and memory T lymphocytes were associated with complete KS remission. In a renal recipient undergoing SRL conversion, the early recovery of HHV-8-specific effector but not of memory T lymphocytes, was associated only with partial remission. Neither rejection nor changes in graft function were observed after SRL conversion. HHV-8-specific T-cell responses are required to achieve posttransplant KS remission, and may be restored under SRL, while maintaining effective immunosuppression.

Evaluation of the Practice of Antifungal Prophylaxis Use in Patients With Newly Diagnosed Acute Myeloid Leukemia: Results From the SEIFEM 2010-B Registry

BACKGROUND To analyze the efficacy of antifungal prophylaxis (AFP) with posaconazole and itraconazole in a real-life setting of patients with acute myeloid leukemia (AML) during the first induction of remission. METHODS From January 2010 to June 2011, all patients with newly diagnosed AML were consecutively registered and prospectively monitored at 30 Italian hematological centers. Our analysis focused on adult patients who received intensive chemotherapy and a mold-active AFP for at least 5 days. To determine the efficacy of prophylaxis, invasive fungal disease (IFD) incidence, IFD-attributable mortality, and overall survival were evaluated. RESULTS In total, 515 patients were included in the present analysis. Posaconazole was the most frequently prescribed drug (260 patients [50%]) followed by fluconazole (148 [29%]) and itraconazole (93 [18%]). When comparing the groups taking posaconazole and itraconazole, there were no significant differences in the baseline clinical characteristics, whereas there were significant differences in the percentage of breakthrough IFDs (18.9% with posaconazole and 38.7% with itraconazole, P< .001). The same trend was observed when only proven/probable mold infections were considered (posaconazole, 2.7% vs itraconazole, 10.7%, P= .02). There were no significant differences in the IFD-associated mortality rate, while posaconazole prophylaxis had a significant impact on overall survival at day 90 (P= .002). CONCLUSIONS During the last years, the use of posaconazole prophylaxis in high-risk patients has significantly increased. Although our study was not randomized, it demonstrates in a real-life setting that posaconazole prophylaxis confers an advantage in terms of both breakthrough IFDs and overall survival compared to itraconazole prophylaxis. CLINICAL TRIALS REGISTRATION NCT01315925.

Combined antifungal approach for the treatment of invasive mucormycosis in patients with hematologic diseases: a report from the SEIFEM and FUNGISCOPE registries.

Invasive mucormycosis (IM) in patients with acute leukemia and allogeneic stem cell transplant (allo-SCT) recipients treated with antifungal monotherapy is associated with high mortality rates of 44–49%.[1][1]–[3][2] Among the available antifungals, amphotericin B (AmB) formulations and

How I treat HHV8/KSHV-related diseases in posttransplant patients

Posttransplantation human herpesvirus-8 (HHV8)/Kaposi sarcoma herpesvirus (KSHV) primary infection and/or reactivations are associated with uncommon and sometimes fatal, neoplastic, and non-neoplastic diseases. HHV8-related clinical manifestations notably range from Kaposi sarcoma (KS) to either primary effusion lymphoma or multicentric Castleman disease B-cell malignancies, and from polyclonal HHV8-positive plasmacytic lymphoproliferative disorders to bone marrow failure and peripheral cytopenias, associated or not with hemophagocytic syndromes, and to acute hepatitis syndromes. We reviewed the patient series reported in the literature and summarized clinical management aspects, in terms of diagnosis, follow-up, and treatment. We described typical clinical presentations and histopathologic diagnostic features of these diseases, and we discussed the role of HHV8-specific serologic, molecular, and immunologic assays, particularly focusing on recent data from HHV8-specific T-cell monitoring in posttransplantation KS patients. We finally discussed actual therapeutic options, namely, the reduction or discontinuation of immunosuppressive therapy or the switch from calcineurin inhibitors to mTOR inhibitors, as alternatives to antineoplastic chemotherapy, along with the use of antiherpesvirus agents as prophylactic or therapeutic measures, and treatment with rituximab in posttrans-plantation multicentric Castleman disease patients and non-neoplastic HHV8-associated syndromes.

CDKN2A/B Alterations Impair Prognosis in Adult BCR-ABL1–Positive Acute Lymphoblastic Leukemia Patients

Purpose: The 9p21 locus, encoding three important tumor suppressors (p16/CDKN2A, p14/ARF, and p15/CDKN2B), is a major target of inactivation in the pathogenesis of many human tumors. Patients and Methods: To explore, at high resolution, the frequency and size of alterations affecting this locus in adult BCR-ABL1–positive acute lymphoblastic leukemia (ALL) and to investigate their prognostic value, 112 patients (101 de novo and 11 relapsed cases) were analyzed by genome-wide single-nucleotide polymorphism arrays and gene candidate deep exon sequencing. Paired diagnosis–relapse samples were further available and analyzed for 19 (19%) cases. Results: CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 25% of newly diagnosed patients, respectively. Deletions were monoallelic in 72% of cases, and in 43% of them, the minimal overlapping region of the lost area spanned only the CDKN2A/B gene locus. An analysis conducted at relapse showed an increase in the detection rate of CDKN2A/ARF loss (47%) compared with the time of diagnosis (P = 0.06). Point mutations within the 9p21 locus were found at very low levels, with only a nonsynonymous substitution in the exon 2 of CDKN2A. Of note, deletions of CDKN2A/B were significantly associated with poor outcomes in terms of overall survival (P = 0.0206), disease free-survival (P = 0.0010), and cumulative incidence of relapse (P = 0.0014). Conclusions: Inactivation of the 9p21 locus by genomic deletion is a frequent event in BCR-ABL1–positive ALL. Deletions are frequently acquired during leukemia progression and are a poor prognostic marker of long-term outcomes. Clin Cancer Res; 17(23); 7413–23. ©2011 AACR.