Somatic, hematologic phenotype, long-term outcome, and effect of hematopoietic stem cell transplantation. An analysis of 97 Fanconi anemia patients from the Italian national database on behalf of the Marrow Failure Study Group of the AIEOP (Italian Association of Pediatric Hematology-Oncology).

We analyzed 97 Fanconi anemia patients from a clinic/biological database for genotype, somatic, and hematologic phenotype, adverse hematological events, solid tumors, and treatment. Seventy-two patients belonged to complementation group A. Eighty percent of patients presented with mild/moderate somatic phenotype and most with cytopenia. No correlation was seen between somatic/hematologic phenotype and number of missense mutations of FANCA alleles. Over follow-up, 33% of patients improved or maintained mild/moderate cytopenia or normal blood count, whereas remaining worsened cytopenia. Eleven patients developed a hematological adverse event (MDS, AML, pathological cytogenetics) and three developed solid tumors. 10 years cumulative risk of death of the whole cohort was 25.6% with median follow-up 5.8 years. In patients eligible to hematopoietic stem cell transplantation because of moderate cytopenia, mortality was significantly higher in subjects transplanted from matched unrelated donor over nontransplanted subjects, whereas there was no significant difference between matched sibling donor transplants and nontransplanted patients. In patients eligible to transplant because of severe cytopenia and clonal disease, mortality risk was not significantly different in transplanted from matched unrelated versus matched sibling donor versus nontransplanted subjects. The decision to transplant should rely on various elements including, type of donor, HLA matching, patient comorbidities, impairment, and clonal evolution of hematopoiesis. Am. J. Hematol. 91:666-671, 2016. © 2016 Wiley Periodicals, Inc.

Diagnosis and management of acquired aplastic anemia in childhood. Guidelines from the Marrow Failure Study Group of the Pediatric Haemato-Oncology Italian Association (AIEOP).

Acquired aplastic anemia (AA) is a rare heterogeneous disease characterized by pancytopenia and hypoplastic bone marrow. The incidence is 2-3/million inhabitants/year, in Europe, but higher in East Asia. Survival in severe aplastic anemia (SAA) has markedly improved in the past 2 decades because of advances in hematopoietic stem cell transplantation, immunosuppressive and biologic drugs, and supportive care. In SAA hematopoietic stem cell transplant (HSCT) from a matched sibling donor (MSD) is the treatment of choice. If a MSD is not available, the options include immunosuppressive therapy (IST) or unrelated donor HSCT. The objective of this guideline is to provide healthcare professionals with clear guidance on the diagnosis and management of pediatric patients with AA. A preliminary, evidence-based document issued by a group of pediatric hematologists was discussed, modified and approved during a series of "Consensus Conferences" according to procedures previously validated by the AIEOP Board. The guidelines highlight the importance of referring pediatric patients with AA to pediatric centers with long experience in diagnosis, differential diagnosis, management, supportive care and follow-up of AA.

p38 Mitogen-activated protein kinase inhibition enhances in vitro erythropoiesis of Fanconi anemia, complementation group A-deficient bone marrow cells.

Bone marrow failure in Fanconi anemia (FA) has been linked in part to overproduction of inflammatory cytokines, to which FA stem and progenitor cells are hypersensitive. In cell lines and murine models p38 mitogen-activated protein kinase (MAPK)-dependent tumor necrosis factor α (TNF-α) overexpression can be induced by the Toll-like receptors (TLRs) 4 and 7/8 ligands Lipopolysaccharide (LPS) and R848. Ex vivo exposure of FA stem cells to TNF-α suppresses their replication and selects preleukemic clones. Here we show that inhibition of p38 MAPK also reduces TLR4 and 7/8-mediated TNF-α production in primary human FA complementation group A (FANCA)-deficient monocytes from nine patients and demonstrate that, while p38 MAPK inhibition also enhances clonal growth of FANCA-deficient erythroid progenitors, the effect was mediated indirectly by the influence of the inhibitor on auxiliary cells, not erythroid colony-forming units themselves. Taken together, these results support the view that inhibition of the p38 MAPK pathway in monocytes may improve hematopoiesis in FANCA patients.

Molecular analysis of Fanconi anemia: the experience of the Bone Marrow Failure Study Group of the Italian Association of Pediatric Onco-Hematology.

Fanconi anemia is an inherited disease characterized by congenital malformations, pancytopenia, cancer predisposition, and sensitivity to cross-linking agents. The molecular diagnosis of Fanconi anemia is relatively complex for several aspects including genetic heterogeneity with mutations in at least 16 different genes. In this paper, we report the mutations identified in 100 unrelated probands enrolled into the National Network of the Italian Association of Pediatric Hematoly and Oncology. In approximately half of these cases, mutational screening was carried out after retroviral complementation analyses or protein analysis. In the other half, the analysis was performed on the most frequently mutated genes or using a next generation sequencing approach. We identified 108 distinct variants of the FANCA, FANCG, FANCC, FANCD2, and FANCB genes in 85, 9, 3, 2, and 1 families, respectively. Despite the relatively high number of private mutations, 45 of which are novel Fanconi anemia alleles, 26% of the FANCA alleles are due to 5 distinct mutations. Most of the mutations are large genomic deletions and nonsense or frameshift mutations, although we identified a series of missense mutations, whose pathogenetic role was not always certain. The molecular diagnosis of Fanconi anemia is still a tiered procedure that requires identifying candidate genes to avoid useless sequencing. Introduction of next generation sequencing strategies will greatly improve the diagnostic process, allowing a rapid analysis of all the genes.

Risk of seizures in children receiving busulphan-containing regimens for stem cell transplantation.

Busulphan (BU) is associated with neurotoxicity and risk of seizures. Hence, seizure prophylaxis is routinely utilized during BU administration for stem cell transplantation (SCT). We collected data on the incidence of seizures among children undergoing SCT in Italy. Fourteen pediatric transplantation centers agreed to report unselected data on children receiving BU as part of the conditioning regimen for SCT between 2005 and 2012. Data on 954 pediatric transplantation procedures were collected; of them, 66% of the patients received BU orally, and the remaining 34%, i.v. All the patients received prophylaxis of seizures, according to local protocols, consisting of different schedules and drugs. A total of 13 patients (1.3%) developed seizures; of them, 3 had a history of epilepsy (or other seizure-related pre-existing condition); 3 had documented brain lesions potentially causing seizures per se; 1 had febrile seizures, 1 severe hypo-osmolality. In the remaining 5 patients, seizures were considered not explained and, thus, potentially related to BU administration. The incidence of seizures in children receiving BU-containing regimen was very low (1.3%); furthermore, most of them had at least 1-either pre-existing or concurrent-associated risk factor for seizures.

Treatment of graft versus host disease with mesenchymal stromal cells: a phase I study on 40 adult and pediatric patients.

This phase I multicenter study was aimed at assessing the feasibility and safety of intravenous administration of third party bone marrow-derived mesenchymal stromal cells (MSC) expanded in platelet lysate in 40 patients (15 children and 25 adults), experiencing steroid-resistant grade II to IV graft-versus-host disease (GVHD). Patients received a median of 3 MSC infusions after having failed conventional immunosuppressive therapy. A median cell dose of 1.5 × 10(6)/kg per infusion was administered. No acute toxicity was reported. Overall, 86 adverse events and serious adverse events were reported in the study, most of which (72.1%) were of infectious nature. Overall response rate, measured at 28 days after the last MSC injection, was 67.5%, with 27.5% complete response. The latter was significantly more frequent in patients exhibiting grade II GVHD as compared with higher grades (61.5% versus 11.1%, P = .002) and was borderline significant in children as compared with adults (46.7 versus 16.0%, P = .065). Overall survival at 1 and 2 years from the first MSC administration was 50.0% and 38.6%, with a median survival time of 1.1 years. In conclusion, MSC can be safely administered on top of conventional immunosuppression for steroid resistant GVHD treatment. Eudract Number 2008-007869-23, NCT01764100.

Epidemiology of infections in children with acquired aplastic anaemia: a retrospective multicenter study in Italy.

Infection is a significant cause of death in patients with aplastic anaemia (AA). However, few studies have examined the characteristics of infections in patients with AA, especially in children. The aim of this retrospective study was to evaluate the incidence and types of infections in a large cohort of paediatric patients with AA referred to eight AIEOP (Italian Association of Paediatric Oncology and Haematology) centres in Italy. The study included 78 patients, 45 boys and 33 girls, median age 9.29 yrs (1st-3rd quartile 3.59-13.09) diagnosed with AA. During the study period, 111 infectious episodes were observed in 42 (54%) patients. Fifty-one (46%) episodes were fever of unknown origin and 60 (54%) were documented infections (DI). In this group, microbiologically documented infection (MDI) with bacteremia accounted for 23 (38%) episodes, MDI without bacteremia for 7 (12%), clinically documented infection for 25 (42%) and invasive fungal diseases for 5 (8%). The rate (episodes/1000 d at risk) was similar in severe aplastic anemia and very severe aplastic anemia both before and after day 120. During the first 120 d from diagnosis, the cumulative risk of a DI was 21% (95% CI 12-29) with the last episode at day 117, but the 50% of episodes were observed in the first 24 d. After day 120, the cumulative risk of DI was again 21% (95% CI 12-29), with the last episode at day 445 of follow-up, with 50% of episodes observed in the first 120 d of observation (240 d from the diagnosis of AA). We found a statistically significant association between the grade of aplasia at diagnosis and the incidence of IEs (P = 0.0002). No association was found between gender, age at diagnosis, response at day +120 and at day +180, use of G-CSF and occurrence of IEs. The actuarial overall survival at 5 yrs was 90% ± 3.6. The mortality rate attributable to infection complication was 9%. This is a large paediatric cohort study reporting the epidemiology of infectious complications in children with AA and that allow us to compare the epidemiological data in this diseases with that of the most recent studies in neutropenic children with cancer. Our findings confirm that infections represent the main cause of death in patients with AA and they are important for the design of management strategies of febrile neutropenia in these patients.

Ubiquitin-proteasome-rich cytoplasmic structures in neutrophils of patients with Shwachman-Diamond syndrome.

BACKGROUND: Shwachman-Diamond syndrome is an autosomal recessive disorder in which severe bone marrow dysfunction causes neutropenia and an increased risk of leukemia. Recently, novel particulate cytoplasmic structures, rich in ubiquitinated and proteasomal proteins, have been detected in epithelial cells and neutrophils from patients with Helicobacter pylori gastritis and several epithelial neoplasms. DESIGN AND METHODS: Blood neutrophils from 13 cases of Shwachman-Diamond syndrome - ten with and three without SBDS gene mutation - and ten controls were investigated by confocal microscopy and ultrastructural immunocytochemistry using antibodies against ubiquitinated proteins, proteasomes, p62 protein, and Helicobacter pylori VacA, urease and outer membrane proteins. RESULTS: Many extensively disseminated particulate cytoplasmic structures, accounting for 22.78 ± 5.57% (mean ± standard deviation) of the total cytoplasm, were found in blood neutrophils from mutated Shwachman-Diamond syndrome patients. The particulate cytoplasmic structures showed immunoreactivity for polyubiquitinated proteins and proteasomes, but no reactivity for Helicobacter pylori products, which are present in particulate cytoplasmic structures of Helicobacter pylori-positive gastritis. Neutrophils from patients with Shwachman-Diamond syndrome frequently showed p62-positive autophagic vacuoles and apoptotic changes in 5% of cells. No particulate cytoplasmic structures were observed in most control neutrophils; however, in a few cells from two cases we noted focal development of minute particulate cytoplasmic structures, accounting for 0.74 ± 0.56% of the total cytoplasm (P<0.001 versus particulate cytoplasmic structures from mutated Shwachman-Diamond syndrome patients). Neutrophils from non-mutated Shwachman-Diamond-syndrome-like patients resembled controls in two cases, and a third case showed particulate cytoplasmic structure patterns intermediate between those in controls and those in mutated Shwachman-Diamond syndrome patients. CONCLUSIONS: Particulate cytoplasmic structures are a prominent feature of neutrophils from patients with Shwachman-Diamond syndrome. They may help us to understand the mechanism of granulocyte dysfunction and the neoplastic risk of the disease.

Endothelial fate and angiogenic properties of human CD34+ progenitor cells in zebrafish.

OBJECTIVE: The vascular competence of human-derived hematopoietic progenitors for postnatal vascularization is still poorly characterized. It is unclear whether, in the absence of ischemia, hematopoietic progenitors participate in neovascularization and whether they play a role in new blood vessel formation by incorporating into developing vessels or by a paracrine action. METHODS AND RESULTS: In the present study, human cord blood-derived CD34(+) (hCD34(+)) cells were transplanted into pre- and postgastrulation zebrafish embryos and in an adult vascular regeneration model induced by caudal fin amputation. When injected before gastrulation, hCD34(+) cells cosegregated with the presumptive zebrafish hemangioblasts, characterized by Scl and Gata2 expression, in the anterior and posterior lateral mesoderm and were involved in early development of the embryonic vasculature. These morphogenetic events occurred without apparent lineage reprogramming, as shown by CD45 expression. When transplanted postgastrulation, hCD34(+) cells were recruited into developing vessels, where they exhibited a potent paracrine proangiogenic action. Finally, hCD34(+) cells rescued vascular defects induced by Vegf-c in vivo targeting and enhanced vascular repair in the zebrafish fin amputation model. CONCLUSIONS: These results indicate an unexpected developmental ability of human-derived hematopoietic progenitors and support the hypothesis of an evolutionary conservation of molecular pathways involved in endothelial progenitor differentiation in vivo.

Diamond-Blackfan anemia: genotype-phenotype correlations in Italian patients with RPL5 and RPL11 mutations.

BACKGROUND: Diamond-Blackfan anemia is a rare, pure red blood cell aplasia of childhood due to an intrinsic defect in erythropoietic progenitors. About 40% of patients display various malformations. Anemia is corrected by steroid treatment in more than 50% of cases; non-responders need chronic transfusions or stem cell transplantation. Defects in the RPS19 gene, encoding the ribosomal protein S19, are the main known cause of Diamond-Blackfan anemia and account for more than 25% of cases. Mutations in RPS24, RPS17, and RPL35A described in a minority of patients show that Diamond-Blackfan anemia is a disorder of ribosome biogenesis. Two new genes (RPL5, RPL11), encoding for ribosomal proteins of the large subunit, have been reported to be involved in a considerable percentage of patients. DESIGN AND METHODS: In this genotype-phenotype analysis we screened the coding sequence and intron-exon boundaries of RPS14, RPS16, RPS24, RPL5, RPL11, and RPL35A in 92 Italian patients with Diamond-Blackfan anemia who were negative for RPS19 mutations. RESULTS: About 20% of the patients screened had mutations in RPL5 or RPL11, and only 1.6% in RPS24. All but three mutations that we report here are new mutations. No mutations were found in RPS14, RPS16, or RPL35A. Remarkably, we observed a higher percentage of somatic malformations in patients with RPL5 and RPL11 mutations. A close association was evident between RPL5 mutations and craniofacial malformations, and between hand malformations and RPL11 mutations. CONCLUSIONS: Mutations in four ribosomal proteins account for around 50% of all cases of Diamond-Blackfan anemia in Italian patients. Genotype-phenotype data suggest that mutation screening should begin with RPL5 and RPL11 in patients with Diamond-Blackfan anemia with malformations.

Interleukin-17-producing T-helper cells as new potential player mediating graft-versus-host disease in patients undergoing allogeneic stem-cell transplantation.

OBJECTIVES: Graft-versus-host disease (GVHD) is a major obstacle to safe allogeneic hematopoietic stem-cell transplantation, leading to significant mortality. Recently, T-helper (TH)-17 cells have been shown to play a central role in mediating several autoimmune diseases. The aim of our study was to investigate the relationship between TH-17 cells and GVHD occurring in transplanted patients. METHODS: Blood samples were collected from 51 hematopoietic stem-cell transplantation patients and 15 healthy donors. Patients with GVHD were monitored for the presence of TH-17 cells by ELISPOT or flow cytometry in the peripheral blood and by confocal microscopy in GVHD lesions. Cytokine plasma levels were detected by ELISA. RESULTS: An increased TH-17 population (up to 4.8% of peripheral blood CD4+T lymphocytes) was observed in patients with acute GVHD and (up to 2.4%) in patients with active chronic GVHD along with an inflammatory process. In contrast, the percentage of TH-17 cells drastically decreased in patients with inactive chronic GVHD. TH-17 cells consisted of both interleukin (IL)-17+/interferon (IFN)-gamma- and IL-17+/IFN-gamma+ subsets and expressed IL-23 receptor. Interestingly, IFN-gamma+ TH-17 cells were able to infiltrate GVHD lesions as observed in liver and skin sections. Moreover, the proportion of TH-17 was inversely correlated with the proportion of regulatory T cells observed in the peripheral blood and tissues affected by GVHD. Finally, we demonstrated a strong correlation between TH-17 levels and the clinical status of patients with GVHD. CONCLUSIONS: These findings support the hypothesis that TH-17 are involved in the active phases of GVHD and may represent a novel cellular target for developing new strategies for GVHD treatment.

Changes in cytokine profile pre- and post-immunosuppression in acquired aplastic anemia.

Cytokine expression assessed by flow cytometry in 53 acquired aplastic anemia patients before and after combined immunosuppression (EBMT WPSAA protocols) showed that CD3(+) marrow cells containing TNF-alpha, IFN-gamma and IL4 were similar in subjects with disease at onset (DO) and responsive to treatment who had more CD3(+)/TNF-alpha(+) and CD 3(+)/IFN-gamma(+) cells than normal controls. In vitro block of TNF-alpha and/or IFN-gamma significantly increased BFU-e over baseline in 28 patients. In responsive to treatment patients only TNF-alpha block significantly incremented colonies over normal controls. Absolute marrow CD3(+)/TNF-alpha(+) and CD3(+)/IFN-gamma(+) cells prospectively tested in a group of 21 subjects declined significantly more in Responders than in Non Responders to immunosuppression at Response Evaluation Time respect to Diagnosis. Both in Responders and in Non Responders these cells remained higher than in normal controls. This study suggests that immunosuppression does not fully clear excess TNF-alpha and IFN-gamma from marrow of patients with good outcome and raises the hypothesis that additional cytokine blockade might be useful in immunosuppression for acquired aplastic anemia.

NOLA1 gene mutations in acquired aplastic anemia.

BACKGROUND: Telomerase complex genes mutations (DKC1, TERC, TERT, and NOP10) lead to premature telomere shortening and are responsible for different forms of dyskeratosis congenita. TERC and TERT mutations were also found in patients with aplastic anemia. The aim of this work is to analyze the possible involvement of the telomerase complex gene NOLA1, in a population of Italian AA patients. PROCEDURE: DNA of 108 AA patients and 170 normal controls was amplified by PCR and analyzed by DHPLC. For each abnormal elution profile PCR products was directly sequenced using ABI prism 3100 Genetic Analyzer. RESULTS: We identified, in two patients and two control, the new c.390A > T variation, which is not reported in GenBank, and leads to p.H28L amino acidic change. Telomere analysis shows that the subjects carrying the change have a telomere length comparable to that of healthy controls thus suggesting that this variation has no effect on telomerase complex activity. CONCLUSIONS: We did not find any clear disruptive mutation in NOLA1 gene. The non-conservative variation identified in our sample has no effect on telomeres length. This result suggests that heterozygous point mutations in NOLA1 gene are not responsible for AA in our patients at least acting via telomere. However, in our experience, molecular analysis of other telomerase complex gene (TERC, TERT) is important for AA patients and family members in order to set up an adequate therapeutic or surveillance program and identify carriers or exclude them as potential bone marrow donors.

Advanced pediatric myelodysplastic syndromes: can immunophenotypic characterization of blast cells be a diagnostic and prognostic tool?

BACKGROUND: The diagnosis of myelodysplastic syndromes (MDS) is mainly based on morphology and cytogenetic analysis. Several efforts to analyze MDS by flow cytometry have been reported in adults. These studies have focused on the identification of abnormalities in the maturation pathway of antigen expression of myelo-monocytic cells, and characterization of blast populations. Therefore, phenotype has been proposed as a diagnostic and prognostic criterion tool for adult MDS. The current article provides data concerning the blast phenotype in pediatric MDS. PROCEDURE: We evaluated by multiparameter flow cytometry 26 MDS pediatric patients with more than 2% of blast cells at bone marrow morphological examination (17 de novo MDS and 9 secondary MDS) and 145 pediatric de novo acute myeloid leukemia (AML) cases (M3 excluded). As control group, 12 healthy age-matched donors for allogenic bone marrow transplantation (BMD) and 6 regenerating bone marrow samples, collected from children with acute lymphoblastic leukemia (ALL) in remission after induction chemotherapy, were studied. RESULTS: We identified a blast immunophenotype typically expressed in most MDS cases and a strong correlation between CD7 expression and poor outcome. CD34+ compartment in MDS bone marrow was also analyzed: a significant decrease of B-cell precursors was detected in MDS patients independent of age. CONCLUSIONS: Our data suggest that the blasts phenotypic features can constitute a diagnostic and prognostic tool also for pediatric MDS.

Cyclosporin A response and dependence in children with acquired aplastic anaemia: a multicentre retrospective study with long-term observation follow-up.

Immunosuppressive therapy (IST) with antithymocyte globulin and cyclosporin A (CyA) is the standard treatment for children with acquired aplastic anaemia (AAA) lacking a matched donor. Survival rates of more than 80% at 5 years are achieved, but the response is drug-dependent in 15-25% of cases. This study, of 42 consecutive children with AAA treated with IST, assessed the incidence of CyA-dependence, CyA and granulocyte colony-stimulating factor (G-CSF) tapering schedules and the impact of drug accumulation on progression to myelodysplasia/acute myeloid leukaemia (MDS/AML). Overall survival was 83% at 10 years. CyA-dependence without a predictive marker was observed in 18% of responders. Probability of discontinuing CyA was 60.5% at 10 years; a slow CyA tapering schedule was performed in 84% of patients; the cumulative incidence of relapse was 16% at 10 years. Relapse risk was significantly associated with rapid CyA discontinuation: 60% compared to 7.6% in the slow tapering group (P = 0.001). Cumulative incidence of MDS/AML was 8% at 10 years, with a significant correlation with both G-CSF cumulative dose and second IST. This long-term follow-up of children with AAA shows that IST with a slow CyA tapering course is an effective treatment with a low-relapse rate in these cases.

The outcome of children with Fanconi anemia given hematopoietic stem cell transplantation and the influence of fludarabine in the conditioning regimen: a report from the Italian pediatric group.

BACKGROUND AND OBJECTIVES: Hematopoietic stem cell transplantation (HSCT) still represents the only treatment potentially able to prevent/rescue the development of marrow failure and myeloid malignancies in patients with Fanconi anemia (FA). While in the past HSCT from an HLA-identical sibling was proven to cure many patients, a higher incidence of treatment failure has been reported in recipients of an unrelated donor (UD) or HLA-partially matched related allograft. DESIGN AND METHODS: We analyzed the outcome of 64 FA patients (age range, 2-20 years) who underwent HSCT between January 1989 and December 2005. Patients were transplanted from either an HLA-identical sibling (n=31), an UD (n=26), or an HLA-partially matched relative (n=7). T-cell depletion of the graft was performed in patients transplanted from an HLA-disparate relative. RESULTS: The 8-year estimate of overall survival (OS) for the whole cohort was 67%; it was 87%, 40% and 69% when the donor was an HLA-identical sibling, an UD and a mismatched relative, respectively (p<0.01). The outcome of recipients of grafts from an UD improved over time, the probability of survival being 10% and 72% for patients transplanted before and after 1998, respectively (p<0.05). The OS probability of children who did or did not receive fludarabine in preparation for the allograft was 86% and 59%, respectively (p<0.05). INTERPRETATION AND CONCLUSIONS: These data, useful for counselling, provide support to the concept that a relevant proportion of FA patients undergoing HSCT can now be successfully cured, even in the absence of an HLA-identical sibling, especially if the conditioning regimen includes fludarabine.

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.