Alessandro Busca

Mesenchymal Stem Cell-Derived Microvesicles Protect Against Acute Tubular Injury

Administration of mesenchymal stem cells (MSCs) improves the recovery from acute kidney injury (AKI). The mechanism may involve paracrine factors promoting proliferation of surviving intrinsic epithelial cells, but these factors remain unknown. In the current study, we found that microvesicles derived from human bone marrow MSCs stimulated proliferation in vitro and conferred resistance of tubular epithelial cells to apoptosis. The biologic action of microvesicles required their CD44- and beta1-integrin-dependent incorporation into tubular cells. In vivo, microvesicles accelerated the morphologic and functional recovery of glycerol-induced AKI in SCID mice by inducing proliferation of tubular cells. The effect of microvesicles on the recovery of AKI was similar to the effect of human MSCs. RNase abolished the aforementioned effects of microvesicles in vitro and in vivo, suggesting RNA-dependent biologic effects. Microarray analysis and quantitative real time PCR of microvesicle-RNA extracts indicate that microvesicles shuttle a specific subset of cellular mRNA, such as mRNAs associated with the mesenchymal phenotype and with control of transcription, proliferation, and immunoregulation. These results suggest that microvesicles derived from MSCs may activate a proliferative program in surviving tubular cells after injury via a horizontal transfer of mRNA.

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.).

Analysis of immune reconstitution in children undergoing cord blood transplantation

OBJECTIVE The aim of this study was to investigate and compare immune reconstitution in allogeneic cord blood transplantation (CBT) and bone marrow transplantation (BMT) recipients. MATERIALS AND METHODS Twenty-three children underwent CBT from either human leukocyte antigen-identical siblings (11 cases) or unrelated donors (12 cases) were enrolled in the study, together with 23 matched children receiving BMT. Patients were analyzed 2-3 and 12-15 months after transplant. Recovery of T-, B-, and NK-lymphocyte subsets, proliferative in vitro response to mitogens, as well as cytotoxic activities, were investigated. RESULTS CBT recipients showed a marked increase in the number of B lymphocytes as compared with patients who underwent BMT (p < 0.001). The absolute number of CD3(+) and CD8(+) T cells, as well as the proliferative response to T-cell mitogens, recovered with time after transplantation, irrespective of the source of stem cells used. Recipients of unrelated CBT had a better recovery of CD4(+) T lymphocytes (p < 0.01). Among patients experiencing acute graft-versus-host disease (GVHD), children given CBT had a much greater production of CD4(+) CD45RA(+) T cells than BMT recipients (p < 0.005). Recovery of NK cell number and innate cytotoxic activities was fast, irrespective of the source of stem cells used. CONCLUSIONS Despite the much lower number of lymphocytes transferred with the graft, recovery of lymphocyte number and function toward normal in CBT recipients was rapid and comparable to that observed after transplantation of bone marrow progenitors. This prompt immune recovery possibly was favored by the reduced incidence and severity of GVHD observed in children who underwent CBT.

Risk factors and severe outcome in thrombotic microangiopathy after allogeneic hematopoietic stem cell transplantation

Background. Thrombotic microangiopathy (TMA) has been described as severe complication after hematopoietic stem cell transplantation (HSCT). The principal aim of this study was to focus the incidence and the outcome of TMA in the era of more complex HSCTs. Methods. We analyzed the role of some predicting factors for the incidence and the outcome of TMA after HSCT. We enrolled 539 consecutive patients (307 males, median age 31 years) undergoing HSCT from match or mismatch human leukocyte antigen family donor (314) or match/mismatch unrelated (195) and haploidentical donor (30) for malignant or nonmalignant diseases. TMA diagnosis was performed by homogeneous clinical and laboratory criteria. Results. Sixty-four of 539 patients presented TMA (11,87%) and the five-year cumulative incidence of TMA was 14% (HR=0.13). Fifty nine of 64 patients were affected by malignant and 5/64 by non-malignant diseases. On multivariate analysis, TMA occurrence was influenced by graft versus host disease >grade II (P=0.0001), donor type (P=0.029), gender (P=0.0233), total body irradiation based conditioning regimen (P=0.0041). Three factors for TMA outcome proved to be statistically significant by multivariate analysis: age (P=0.009), donor type (P=0.0187) and TMA index (P=0.029). The TMA mortality rate was 50%. The outcome was influenced by defibrotide (P=0.02 in univariate analysis). Conclusions. The study underlines the possibility of finding out which patients are more prone to developing post-HSCT TMA, and identifies which risk factors are more frequently associated with a dismal outcome after TMA.

Incidence and outcome of invasive fungal diseases after allogeneic stem cell transplantation: A prospective study of the gruppo italiano trapianto midollo osseo (GITMO)

Epidemiologic investigation of invasive fungal diseases (IFDs) in allogeneic hematopoietic stem cell transplantation (allo-HSCT) may be useful to identify subpopulations who might benefit from targeted treatment strategies. The Gruppo Italiano Trapianto Midollo Osseo (GITMO) prospectively registered data on 1858 consecutive patients undergoing allo-HSCT between 2008 and 2010. Logistic regression analysis was performed to identify risk factors for proven/probable IFD (PP-IFD) during the early (days 0 to 40), late (days 41 to 100), and very late (days 101 to 365) phases after allo-HSCT and to evaluate the impact of PP-IFDs on 1-year overall survival. The cumulative incidence of PP-IFDs was 5.1% at 40 days, 6.7% at 100 days, and 8.8% at 12 months post-transplantation. Multivariate analysis identified the following variables as associated with PP-IFDs: transplant from an unrelated volunteer donor or cord blood, active acute leukemia at the time of transplantation, and an IFD before transplantation in the early phase; transplant from an unrelated volunteer donor or cord blood and grade II-IV acute graft-versus-host disease (GVHD) in the late phase; and grade II-IV acute GVHD and extensive chronic GVHD in the very late phase. The risk for PP-IFD was significantly higher when acute GVHD was followed by chronic GVHD and when acute GVHD occurred in patients undergoing transplantation with grafts from other than matched related donors. The presence of PP-IFD was an independent factor in long-term survival (hazard ratio, 2.90; 95% confidence interval, 2.32 to 3.62; P < .0001). Our findings indicate that tailored prevention strategies may be useful in subpopulations at differing levels of risk for PP-IFDs.

Busulfan plus cyclophosphamide versus busulfan plus fludarabine as a preparative regimen for allogeneic haemopoietic stem-cell transplantation in patients with acute myeloid leukaemia: an open-label, multicentre, randomised, phase 3 trial.

BACKGROUND The standard busulfan-cyclophosphamide myeloablative conditioning regimen is associated with substantial non-relapse mortality in patients older than 40 years with acute myeloid leukaemia who are undergoing allogeneic stem-cell transplantation. Because the combination of busulfan plus fludarabine has been proposed to reduce non-relapse mortality, we aimed to compare this treatment with busulfan plus cyclophosphamide as a preparative regimen in these patients. METHODS We did an open-label, multicentre, randomised, phase 3 trial for patients with acute myeloid leukaemia at 25 hospital transplant centres in Italy and one in Israel. Eligible patients were aged 40-65 years, had an Eastern Cooperative Oncology Group performance status less than 3, and were in complete remission. Patients were randomly assigned 1:1 to receive intravenous busulfan plus cyclophosphamide or busulfan plus fludarabine. Treatment allocations were not masked to investigators or patients. Randomisation was done centrally via a dedicated web-based system using remote data entry, with patients stratified by donor type and complete remission status. Patients allocated to busulfan plus cyclophosphamide received intravenous busulfan 0·8 mg/kg four times per day during 2 h infusions for four consecutive days (16 doses from days -9 through -6; total dose 12·8 mg/kg) and cyclophosphamide at 60 mg/kg per day for two consecutive days (on days -4 and -3; total dose 120 mg/kg). Patients allocated to busulfan plus fludarabine received the same dose of intravenous busulfan (from days -6 through -3) and fludarabine at 40 mg/m(2) per day for four consecutive days (from days -6 through -3; total dose 160 mg/m(2)). The primary endpoint was 1-year non-relapse mortality, which was assessed on an intention-to-treat basis; safety outcomes were assessed in the per-protocol population. This trial has been completed and is registered with ClinicalTrials.gov, number NCT01191957. FINDINGS Between Jan 3, 2008, and Dec 20, 2012, we enrolled and randomly assigned 252 patients to receive busulfan plus cyclophosphamide (n=125) or busulfan plus fludarabine (n=127). Median follow-up was 27·5 months (IQR 9·8-44·3). 1-year non-relapse mortality was 17·2% (95% CI 11·6-25·4) in the busulfan plus cyclophosphamide group and 7·9% (4·3-14·3) in the busulfan plus fludarabine group (Grays test p=0·026). The most frequently reported grade 3 or higher adverse events were gastrointestinal events (28 [23%] of 121 patients in the busulfan plus cyclophosphamide group and 26 [21%] of 124 patients in the busulfan plus fludarabine group) and infections (21 [17%] patients in the busulfan plus cyclophosphamide group and 13 [10%] patients in the busulfan plus fludarabine group had at least one such event). INTERPRETATION In older patients with acute myeloid leukaemia, the myeloablative busulfan plus fludarabine conditioning regimen is associated with lower transplant-related mortality than busulfan plus cyclophosphamide, but retains potent antileukaemic activity. Accordingly, this regimen should be regarded as standard of care during the planning of allogeneic transplants for such patients. FUNDING Agenzia Italiana del Farmaco.

Mycophenolate mofetil (MMF) as therapy for refractory chronic GVHD (cGVHD) in children receiving bone marrow transplantation.

Mycophenolate mofetil (MMF) is an alternative immunosuppressant which inhibits the proliferation of T and B lymphocytes. The purpose of the present study was to evaluate the safety and efficacy of MMF as salvage therapy for chronic GVHD (cGVHD) in children receiving allogeneic bone marrow transplantation. Fifteen children, 3–16 years of age, who had received grafts from HLA-compatible siblings (n = 8), partially matched related donors (n = 2) or matched unrelated donors (n = 5), developed extensive cGVHD which had proved unresponsive to standard immunosuppressive therapy. Patients were treated with MMF at the dose of 15–40 mg/kg/day in combination with other immunosuppressive therapy for a median of 4 months (range 1–15 months). The overall response rate (complete or partial response) was 60%. Thirteen percent had only minor responses, whereas 27% of patients had progressive disease. Best responses were seen in patients with GI tract (60% of complete responses) or mouth (33% of complete responses) cGVHD and skin involvement (43% of complete responses) that did not include sclerodermatous manifestations. Once MMF was started, improvements in the clinical manifestations of cGVHD allowed a significant reduction of steroids in 45% of patients and discontinuation in 27% of cases. Six patients (40%) experienced adverse events, with gastrointestinal symptoms predominating. Five patients experienced opportunistic infections. MMF was discontinued after 35–180 days in six patients for the following reasons: parents choice (n = 2), liver toxicity (n = 1), poor compliance (n = 2), and no response (n = 1). In conclusion, these preliminary results suggest that MMF in combination with other immunosuppressive agents may have a role to play in patients with cGVHD. Prospective clinical trials are needed to establish exact indications for therapy and dosage scheduling. Bone Marrow Transplantation (2000) 25, 1067–1071.

Second allogeneic bone marrow transplantation in acute leukemia : a multicenter study from the Gruppo Italiano Trapianto Di Midollo Osseo (GITMO)

Thirty-eight second allogeneic bone marrow transplants (BMT) for acute leukemia relapsed after first BMT were performed in 13 Italian centers between 1987 and 1994. Twenty-one patients had acute myelogenous leukemia (AML), 17 acute lymphoblastic leukemia (ALL); at second BMT 24 patients were in complete remission (CR) and 14 in relapse. The median time to relapse after first BMT was 10 months (range 1–70). Grade II or greater acute graft-versus-host disease (GVHD) after second transplant occurred in 34.2% of patients and a chronic GVHD in 31.5% of patients. Twenty-four patients died: seven from early transplant-related mortality (TRM), 13 from relapse and four from late toxicity. As of 31 July 1996, at a median follow-up of 47 months (range 22–85), there are 14 survivors. The three-year probability of TRM, relapse and event-free survival (EFS) is 28%, 40% and 42% respectively. In 20 of 27 evaluable patients, remission duration after second BMT was longer than after the first BMT. A diagnosis of AML was correlated with a better outcome. These data support the usefulness of second allograft in selected patients with AML relapsing after a first BMT.

Impact of thrombotic thrombocytopenic purpura on leukemic children undergoing bone marrow transplantation

Thrombotic thrombocytopenic purpura (TTP) has emerged as one of the main transplant-related complications over the last 15 years. The current study defines the incidence and the risk factors for the occurrence of TTP in 131 consecutive leukemic children who were transplanted between January 1994 and December 1997 at four Italian pediatric centers. Patients with ALL (101), AML (21), MDS (9), underwent an HLA-identical sibling BMT (82) or an HLA-identical unrelated BMT (49), receiving a conditioning regimen consisting of high-dose chemotherapy in 24 patients and of F-TBI combined with high-dose chemotherapy in 107 patients. The diagnosis of TTP was retrospectively evaluated on the basis of parallel criteria. TTP treatment varied according to the protocol of each treatment center. Twenty-eight of 131 patients (21.4%) developed TTP at a median of 46 days (range 21–80) after BMT. Multivariate analysis demonstrated that the risk of TTP was higher in patients who underwent unrelated BMT (P value = 0.02). Acute GVHD, stage of disease at BMT, conditioning with TBI, gender, age, did not appear to be associated with the occurrence of TTP. As to the outcome, TTP resolved in 19 patients while in nine it was the principal cause of death (32.1%). In patients with TTP, LDH peak value was the only statistically significant factor (P = 0.001) related to severe TTP. In conclusion, our experience demonstrates that leukemic children undergoing BMT, especially from an unrelated donor, should be carefully assessed for TTP which appears to be a severe and relatively common transplant-related complication when strict diagnostic criteria are applied. Bone Marrow Transplantation (2000) 26, 1005–1009.

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.