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PV, NR and MMP contributed equally Introduction Patients with red blood cell disorders (RBCD), chronic life threating multisystemic disorders in their severe forms, are likely to be at increased risk of complications from SARS-Cov-2 (Covid-19), but evidence in this population is scarce due to its low frequency and heterogeneous distribution. ERN-EuroBloodNet, the European Reference Network in rare hematological disorders, established a European registry to determine the impact of COVID-19 on RBCD patients and identify risk factors predicting severe outcomes. Methods The ERN-EuroBloodNet registry was established in March 2020 by Vall d'Hebron Research Institute based on REDcap software in accordance with the Regulation (EU) 2016/679 on personal data. The local Research Ethics Committee confirmed that the exceptional case of the pandemic justifies the waiver of informed consent. The ERN-EuroBloodNet registry on RBCD and COVID-19 is endorsed by the European Hematology Association (EHA). Eligible patients had confirmed RBCD and COVID-19. Data collected included demographics, diagnosis, comorbidities, treatments, and COVID-19 (severity grade, clinical manifestations, acute events, treatments, hospitalization, intensive care unit, death). For analysis of COVID-19 severity, two groups were established 1) Mild: asymptomatic or mild symptoms without clinical pneumonia and 2) Severe: pneumonia requiring oxygen/respiratory support and/or admission to intensive care unit. Continuous variables were compared using the Wilcoxon rank-sum test or Kruskall Wallis test, while categorical variables were analyzed using the Chi-square test or Fisher's Exact test. Relevant factors influencing disease or severity were examined by the logistic regression adjusted for age. Results As of June 2021, 42 medical centers from 10 EU countries had registered 373 patients: 191 Sickle cell disease (SCD), 156 Thalassemia major and intermedia (THAL) and 26 other RBCD. 84% of the SCD patients were reported by Spain, Belgium, Italy and The Netherlands and 92% of the THAL patients by Italy and Greece. The mean age of SCD was lower (22.5y) than of THAL (39.6y) with pediatric population accounting for 50.5% in SCD and 9% in THAL (p Age and BMI correlated with COVID-19 severity, as described in the general population (p=0.002, p Potential risk factors such as elevated ferritin, current chelation or history of splenectomy did not confer additional risk for developing severe COVID-19 in any patient group. Only diabetes as a comorbidity correlated with severity grade in SCD (p=0.011) and hypertension in THAL (p=0.014). While severe COVID-19 infection in SCD was associated with both ACS (p Overall, 14.8% RBC patients needed oxygen/respiratory support, 4.4% were admitted to ICU with an overall mortality rate of 0.8% (no deaths were registered in pediatric age), much lower than reported in other similar cohorts. Discussion Results obtained so far show that severe COVID-19 occurs at younger ages in more aggressive forms of SCD and THAL. Current preventive approaches (shielding, vaccinations) focus on age over disease severity. Our data highlights the risk of severe COVID-19 infection in some young patients, particularly those with SS/SB0 SCD, suggesting that immunization should be considered in this pediatric group as well. Results between similar sized cohorts of RBCD patients vary between each other and those presented here, highlighting the importance of collecting all of these small cohorts together to ensure adequate statistical power so that definitive risk factors (eg. age, genotype, comorbidities) can be reliably identified and used to guide management of patients with these rare disorders in the light of the ongoing pandemic. Figure 1 Figure 1. Disclosures Longo: Bristol Myers Squibb: Honoraria; BlueBird Bio: Honoraria. Bardón-Cancho: Novartis Oncology Spain: Research Funding. Flevari: PROTAGONIST COMPANY: Research Funding; ADDMEDICA: Consultancy, Research Funding; BMS: Research Funding; IMARA COMPANY: Research Funding; NOVARTIS COMPANY: Research Funding. Voskaridou: BMS: Consultancy, Research Funding; IMARA: Research Funding; NOVARTIS: Research Funding; ADDMEDICA: Consultancy, Research Funding; GENESIS: Consultancy, Research Funding; PROTAGONIST: Research Funding. Biemond: GBT: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau; Novo Nordisk: Honoraria; Celgene: Honoraria; Sanquin: Research Funding. Nur: Celgene: Speakers Bureau; Roche: Speakers Bureau; Novartis: Research Funding, Speakers Bureau. Beneitez-Pastor: Agios: Honoraria; Alexion: Honoraria; Novartis: Honoraria; Forma Therapeutics: Honoraria. Pepe: Chiesi Farmaceutici S.p.A: Other: no profit support; Bayer S.p.A.: Other: no profit support. de Montalembert: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Membership on an entity's Board of Directors or advisory committees; Vertex: Membership on an entity's Board of Directors or advisory committees. Glenthøj: Agios: Consultancy; Novo Nordisk: Honoraria; Novartis: Consultancy; Alexion: Research Funding; Bluebird Bio: Consultancy; Bristol Myers Squibb: Consultancy; Saniona: Research Funding; Sanofi: Research Funding. Benghiat: Novartis: Consultancy; BMS: Consultancy. Labarque: Novartis: Consultancy; Bayer: Consultancy; Sobi: Consultancy; NovoNordisk: Consultancy; Octapharma: Consultancy. Diamantidis: Genesis Pharma: Honoraria; Uni-Pharma: Honoraria; Bristol Myers Squibb: Consultancy; IONIS Pharmaceuticals: Research Funding; NOVARTIS, Genesis Pharma SA: Research Funding. Kerkhoffs: Sanofi: Research Funding; Terumo BCT: Research Funding. Iolascon: Celgene: Other: Advisory Board; Bluebird Bio: Other: Advisory Board. Taher: Vifor Pharma: Consultancy, Research Funding; Agios Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy, Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Colombatti: Novartis: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novonordisk: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Research Funding. Mañú Pereira: Novartis: Research Funding; Agios Pharmaceuticals: Research Funding.

Langerhans cell histiocytosis (LCH) is a rare disease with an unknown etiology characterized by heterogeneous lesions containing CD207+CD1a+ cells that can arise in almost any tissue and cause significant morbidity and mortality. Precursors of pathological Langerhans cells have yet to be defined. Our aim was to identify circulating CD207+CD1a+ cells and their inducers in LCH. Expression of CD207 and CD1a in the blood myeloid compartment as well as thymic stromal lymphopoietin (TSLP) and transforming growth factor β (TGF-β) plasma levels were measured in 22 pediatric patients with active disease (AD) or nonactive disease (NAD). In patients with AD vs those with NAD, the myeloid compartment showed an increased CD11b (CD11bhigh plus CD11b+) fraction (39.7 ± 3.6 vs 18.6 ± 1.9), a higher percentage of circulating CD11bhighCD11c+CD207+ cells (44.5 ± 11.3 vs 3.2 ± 0.5), and the presence of CD11chighCD207+CD1a+ cells (25.0 ± 9.1 vs 2.3 ± 0.5). Blood CD207+CD1a+ cells were not observed in adult controls or umbilical cord. Increased TSLP and TGF-β levels were detected in patients with AD. Interestingly, plasma from patients with AD induces CD207 expression on CD14+ monocytes. We conclude that CD207+CD1a+ cells are circulating in patients with active LCH, and TSLP and TGF-β are potential drivers of Langerhans-like cells in vivo. Fil: Carrera Silva, Eugenio Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Nowak, Wanda. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Tessone, Licina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Olexen, Cinthia Mariel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Ortiz Wilczyñski, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentina Fil: Estecho, Ivana Gisele. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Elena, Graciela. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños Pedro Elizalde (ex Casa Cuna); Argentina Fil: Errasti, Andrea Emilse. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Rosso, Diego. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños Pedro Elizalde (ex Casa Cuna); Argentina

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome comprising familial/genetic HLH (FHL) and secondary HLH. In the HLH-94 study, with an estimated 5-year probability of survival (pSu) of 54% (95% confidence interval, 48%-60%), systemic therapy included etoposide, dexamethasone, and, from week 9, cyclosporine A (CSA). Hematopoietic stem cell transplantation (HSCT) was indicated in patients with familial/genetic, relapsing, or severe/persistent disease. In HLH-2004, CSA was instead administered upfront, aiming to reduce pre-HSCT mortality and morbidity. From 2004 to 2011, 369 children aged

Langerhans cell histiocytosis (LCH) is a rare disease with an unknown etiology characterized by heterogeneous lesions containing CD207

Introduction. The MIOT (Myocardial Iron Overload in Thalassemia) Network was a national Italian network constituted by thalassemia and magnetic resonance imaging (MRI) centers built in 2006. The main aim was to assure available, accessible homogeneous and standardized T2* MRI cardiac and liver iron overload assessments for a significant number of patients with emoglobinopathies. Moreover, the creation of a solid clinical-instrumental web based database allowed data exchange between centers and constituted a means of monitoring health care processes and outcomes. We describe the impact of this ten-year Network on cardiac complications in patients with thalassemia major (TM). Methods. Among the 2497 emoglobinopathies patients consecutively enrolled in the MIOT Network we considered the 1401 TM patients who performed an end-of-study MRI. Per protocol the MRI follow up was scheduled every 18±3 months. Myocardial iron overload (MIO) was quantified by the multislice multiecho T2* technique. Biventricular function was quantified by cine images. Results. At the last MRI significantly higher global heart T2* values (35.5±10.7 ms vs 29.2±12.0 ms; P In patients with significant baseline MIO (global heart T2* Based on MRI results the 75% of the patients changed the chelation therapy. At the last MRI the percentage of patients with an excellent/good compliance was significantly higher (94.7% vs 92.7%%; P=0.034). The 13.1% of the patients had a cardiac complication (heart failure, arrhythmias, pulmonary hypertension, myocardial infarction, angina, myo/pericarditis, and peripheral vascular disease) before the enrolment in the project. During the study, the frequency of cardiac complications was 7.9 %, significantly lower (P Conclusion. Over a period of 10 years, the continuous monitoring of cardiac iron levels and a tailored chelation therapy allowed a reduction of MIO in the 70% of patients and a consequent improvement of cardiac function and reduction of heart failure. So, a national networking as the MIOT project was effective in improving the care and reducing cardiac outcomes of TM patients. Figure 1 Figure 1. Disclosures Pepe: Chiesi Farmaceutici S.p.A., ApoPharma Inc., and Bayer: Other: No profit support.

Introduction. Chronic iron overload may represent a serious complication of potentially lifesaving blood transfusions in different haematological diseases. Excess iron deposits in various tissues of the body, particularly the liver, heart, and endocrine organs (Cappellini. Thalassaemia International Federation, 2014). This process leads to tissue damage and ultimately to significant morbidity and mortality (Musallam. Haematologica 2013). Indeed, organ failure due to chronic iron overload may represent the major cause of death in patients with different haematological diseases who receive blood transfusions regularly without appropriate chelation therapy (Inati. Pediatr Blood Cancer 2011; N Engl J Med 2000; Cleve Clin J Med 2005). The main aim of this study was to look for the relationships between changes in LIC and changes in serum ferritin (SF) level, during real life experience with larger setting of patients with haematological diseases and different chelation regimens, previously described at baseline, according to the LICNET protocol (Vitrano et al., Eur J Haematol 2016). Methods. This was a cross-sectional study of patients with haematological disorders attending 9 Italian centres participating in the LICNET. The LICNET protocol was approved on December 4, 2012 by our Ethical Committee. The underlying diagnoses were regularly transfused Thalassemia Major (TM), Thalassemia Intermedia (TI), Sickle Cell Disease (SCD), Myelodysplastic Syndrome (MDS), Diamond-Blackfan Anemia (DBA). Transfused status was defined as receipt of ≥7 mL/kg/month of packed red blood cells. The inclusion criteria for the cross-sectional analysis were: 1) underlying diagnosis above described; 2) determination of two R2-MRI scans performed as part of the network, for those patients presenting between February 2013 and December 2016; 3) transfusion dependence; 4) same chelation treatment at T0 (MRI1) and T1 (MRI2) . The settled R2-MRI protocol at the Centre follows that of St Pierre et al. (St Pierre et al. Magn Reson Med 2014). Descriptive analysis was provided as means, standard deviations, medians or percentages. Three classes of risk (low, intermediate and high), on the basis of LIC values ( 15 mg Fe/g dw ) were considered to evaluate the control of iron body burden during the study period. LIC comparisons at MRI1 and MRI2 were made using t -test and/or Wilcoxon test. All p -values are two sided with the level of significance set at Results. A total of 130 patients were evaluated, with a median age of 35 years (range: 6-78). The median duration (range) between MRI1 and MRI2 days was 483 (184-1076). Patients' characteristics for the considered chelation regimens are summarized in Table 1. Overall patients, across all chelation regimens, showed a statistically significant difference in variation of LIC between MRI1 and MRI2 (p=0.011, Table 2). Overall variation of LIC, during a period of 483 (184-1076) days, was -0.8 (-29.0-33.0) mg Fe/g dw. Median changes in LIC (range), mg Fe/g dw for single chelation regimen are reported on Tab. 2. Changes in LIC determinations, during the period of the study, according to the baseline values, are shown on Fig. 1: Overall 7.7% of patients, across different chelation regimens and during a period of 483 (184-1076) days, moved from high risk group (LIC >15 mg Fe/g dw) to intermediate risk group (LIC 7-15 mg Fe/g dw) with stabilization of iron overloading in patients in low risk group at baseline; all chelation regimens were able to move LIC from high risk group to intermediate risk group. In the other combination group, the patients moved from the intermediate risk group to the low risk group. Median changes in SF level (range), ng/ml for overall patients and for single chelation regimen are reported on Tab. 2: Overall patients, across all chelation regimens, did not show any statistically significant difference in variation of SF between MRI1 and MRI2 (p= 0.566, Table 2). Conclusions. In conclusion, SF level trends are unable to predict changes in LIC, even in well chelated patients with haematological disorders. Therefore, variations of SF level must be interpreted with caution and confirmed, when it is possible, by direct measurement of LIC for more correct management of chelation treatment. Disclosures Oliva: Celgene: Consultancy; Amgen Inc.: Consultancy; La Jolla: Consultancy; Novartis: Consultancy; Janssen: Consultancy.

VEGF165, the major angiogenic growth factor, is known to activate various steps in proangiogenic endothelial cell behavior, such as endothelial cell migration and invasion, or endothelial cell survival. Thereby, the urokinase-type plasminogen activator (uPA) system has been shown to play an essential role not only by its proteolytic capacities, but also by induction of intracellular signal transduction. Therefore, expression of its cell surface receptor uPAR is thought to be an essential regulatory mechanism in angiogenesis. We found that uPAR expression on the surface of confluent endothelial cells was down-regulated compared with subconfluent proliferating endothelial cells. Regulation of uPAR expression was most probably affected by extracellular signal-regulated kinase 1/2 (ERK1/2) activation, a downstream signaling event of the VEGF/VEGF-receptor system. Consistently, the receptor-like protein tyrosine phosphatase DEP-1 (density enhanced phosphatase-1/CD148), which is abundantly expressed in confluent endothelial cells, inhibited the VEGF-dependent activation of ERK1/2, leading to down-regulation of uPAR expression. Overexpression of active ERK1 rescued the DEP-1 effect on uPAR. That DEP-1 plays a biologic role in angiogenic endothelial cell behavior was demonstrated in endothelial cell migration, proliferation, and capillary-like tube formation assays in vitro.

Background: No prospective data are available about the efficacy of deferasirox versus deferiprone and desferrioxamine in monotherapy. Our study aimed to prospectively assess the efficacy of deferasirox versus deferiprone and desferrioxamine in monotherapy in a large cohort of thalassemia major (TM) patients by quantitative Magnetic Resonance (MR). Methods: Among the 2551 TM patients enrolled in the MIOT (Myocardial Iron Overload in Thalassemia) network we selected those with an MR follow up study at 18±3 months who had been received one chelator alone between the 2 MR scans. We identified three groups of patients: 235 treated with DFX, 142 with DFP and 162 with DFO. Iron overload was measured by T2* multiecho technique. Liver T2* values were converted into liver iron concentration (LIC) values. Biventricular function parameters were quantitatively evaluated by cine images. Results: Excellent/good levels of compliance were similar in the DFX (98.7%) vs DFP (96.3%) and DFO (97.5%) groups. Among the patients with myocardial iron overload at baseline, in all three groups there was a significant improvement in the global heart T2* value (DFX: +4.58±5.91ms P Among the patients with hepatic iron at baseline (LIC≥3mg/g dw) the changes were not significantly different in DFX versus the other groups. Conclusions: Prospectively in a large clinical setting of TM patients, DFX monotherapy was significantly less effective than DFP in improving myocardial siderosis and biventricular function and it was significantly less effective than DFO in improving the LVEF. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Pepe: Chiesi Farmaceutici and ApoPharma Inc.: Other: Alessia Pepe is the PI of the MIOT project, that receives no profit support from Chiesi Farmaceutici S.p.A. and ApoPharma Inc..

The expression and function of a new cytokine-induced endothelial cell adhesion protein, vascular cell adhesion molecule-1 (VCAM-1), was characterized in vitro by using a monoclonal antibody, MoAb 4B9, which recognizes a functional epitope on this protein. As determined by enzyme-linked immunosorbent assay and radioimmunoprecipitation of metabolically labeled cells, VCAM-1 was minimally expressed on unstimulated human umbilical vein endothelium (HUVE), but was rapidly induced by recombinant human tumor necrosis factor-alpha (rhTNF-alpha), rh interleukin-1, and lipopolysaccharide. In contrast to intercellular adhesion molecule-1, VCAM-1 was not induced on dermal fibroblasts or arterial smooth muscle cells after stimulation with rhTNF, or on keratinocytes after stimulation with rh interferon-gamma. MoAb 4B9 significantly inhibited the adherence of peripheral blood lymphocytes (PBL) and lymphocytic cell lines, but not neutrophils, to rhTNF- activated HUVE. The inhibitory effect of MoAb 4B9 on PBL adherence to HUVE was additive to that produced by the CD18 MoAb 60.3. These results show that VCAM-1 mediates a CD18-independent pathway of peripheral blood lymphocyte adherence to cytokine-stimulated HUVE. We propose that lymphocyte binding to VCAM-1, induced on endothelium by cytokines, may be an important component of lymphocyte emigration at sites of inflammation or immune reaction.

Purpose: The aim of this multi-centre study was to retrospectively assess in thalassemia major (TM) if deferiprone (DFP) had a dose-dependent effect on liver iron concentration (LIC) assessed by quantitative magnetic resonance imaging (MRI). Methods: Among the 958 TM patients enrolled in the MIOT (Myocardial Iron Overload in Thalassemia) network, we identified hose with an MRI follow up study at 18±3 months who had been received DFP monotherapy and had no changes in dose of DFP between the 2 MRI scans. Patients were divided into two groups according to the DFP dose: 79 patients with ≤ 75 mg/kg/d (group 1) and 39 with > 75 mg/kg/d (group 2). Hepatic iron overload was measured by the T2* multiecho technique and T2* values were converted into LIC values using the calibration curve introduced by Wood et al. Results: The two groups had comparable baseline MRI LIC values. The table shows the evolution of different iron overload risk classes between the baseline and the FU MRI. The percentage of patients that worsened their status was significantly higher in group 1 than in group 2 (26.6% vs 7.7%; P=0.016). Subgroup analysis in patients with hepatic iron overload at baseline (MRI LIC > 3mg/g/dw) was conducted: 48 patients from group 1 (DFP dose: mean 70.6±11.2 mg/kg/d, median 75 mg/kg/d) and 30 from group 2 (DFP dose: mean 85.2±6.6 mg/kg/d, median 84 mg/kg/d). The two subgroups had comparable baseline MRI LIC values (10.2±8.1 mg/g dw vs 11.1±8.7 mg/g dw (P=0.314). While the mean change in subgroup 2 ( -1.8±6.3mg/g/dw, P=0.131) was more favourable than in subgroup 1 (+0.1±7.7 mg/g/dw, P=0.903), the change in MRI LIC values did not reach statistical significance between the two subgroups (P=0.579) (Figure 1), which may be due to small cohort evaluated. Conclusions: In TM patients the worsening in MRI LIC can be prevented by increasing the dose of deferiprone above the widely used regimen of 75 mg/kg body weight. Our results are consistent with the iron balance studies performed by Grady RW et al. Table 1. Evolution of different iron overload risk classes between the baseline and the FU MRI. The underlined numbers represent the patients who remained in the same risk class. DFP dose ≤ 75 mg/kg/d (N=79) FU LIC 75 mg/kg/d (N=39) FU LIC Figure 1. Changes of MRI LIC values in patients with basal MRI LIC > 3 mg/g/dw. Figure 1. Changes of MRI LIC values in patients with basal MRI LIC > 3 mg/g/dw. Disclosures Pepe: Chiesi: Speakers Bureau; ApoPharma Inc: Speakers Bureau; Novartis: Speakers Bureau.

Introduction: Cardiovascular Magnetic Resonance (CMR) has an established role in managing and predicting prognosis of patients with Thalassemia Major (TM). Thalassemia Intermedia (TI) is a milder variant of beta-thalassemia showing a different clinical and prognostic profile; pulmonary hypertension (PH) is a more common complication in TI patients. We prospectively determined the predictive value of CMR parameters, including measurement of right ventricular mass, for cardiac complications in TI. Methods: We considered 342 TI patients enrolled in the Myocardial Iron Overload in Thalassemia network; about half of them (178/302, 58.9%) were transfusion-dependent. Myocardial and liver iron overload were measured by T2* multiecho technique. Atrial dimensions, left and right ventricular mass and systolic function were quantified by cine images. Late gadolinium enhancement (LGE) images were acquired to detect myocardial fibrosis. Results: Twenty-three patients were excluded because a cardiac complication was present at the time of first CMR, so we prospectively followed 319 patients. All 319 patients were white, with a mean age at time of their first scan of 38.02±11.69 years and 165 (51.7%) of them were females. Mean follow-up time was 52.24±24.87 months (median 54.64 months). Cardiac events were recorded in 22 patients (6.9%): heart failure (HF) in 1 patient, arrhythmias in 12 patients, pulmonary hypertension (PH) in 7 patients and myocardial infarction (MI) in 2 patients. Due to the low number of events, only arrhythmias, PH and cardiac complications globally considered were taken as cardiac outcomes for univariate and multivariate analysis. In the multivariate analysis RV hypertrophy was the only independent predictive factor for arrhythmias (HR=33.83, 95% CI=6.07-188.74, P The Figures display the Kaplan-Meier curves showing the impact of the independent predictive factors on each outcome. Conclusions: For the first time we studied the prognostic value of right ventricular mass as part of multiparametric CMR imaging in a population of TI patients. RV hypertrophy identified patients at high risk for arrhythmias and PH. Both RV hypertrophy and fibrosis detected by LGE were independent predictive factor for cardiac complications. Measurement of RV mass should be part of the multi-parametric CMR study of patient with thalassemia intermedia. Figure 1. Figure 1. Disclosures Pepe: ApoPharma Inc: Speakers Bureau; Novartis: Speakers Bureau; Chiesi: Speakers Bureau.

Introduction. This was a cross-sectional study of patients with hemoglobinopathies attending 13 Italian centers participating in the LICNET (Liver Iron Cutino Network) network promoted from Piera Cutino partnership and instituted by our center (Campus of Haematology Franco e Piera Cutino-A.O.O.R. Villa Sofia Cervello, Italy) on February 2013. LICNET is addressed to the diagnostics of iron overload in liver by R2 MRI (Ferriscan®) in patients with hemoglobinopathies. Ferriscan is a rapid scan method now available (10 minutes). This tool is crucial to have accurate and reliable measures for iron body burden control in hemoglobinopathies. Methods. Data included patients with β-thalassemia major (TM) (regularly transfused) (TX), β-thalassemia intermedia (TI) (both TX and non-transfused) (non-TX), and sickle cell disease (SCD) (both TX and non-TX). The main aim of the study was to evaluate how serum ferritin levels (SFLs) predict liver iron concentration (LIC) in different hemoglobinopathies, and to have valuable information about prognosis and response to therapy. In particular, to identify SFLs that best predict LIC thresholds of clinical significance (7 and 15 mg Fe/g dw) by identifying levels with highest sum of sensitivity and specificity was used the receiver operating characteristic (ROC) curve analysis. Results. A total of 363 patients were evaluated in this analysis, with a mean age of 35.6 ± 13.0 years (range: 6-76) and including 160 (44.1%) males. The underlying diagnosis were β-TM (n=204, 56.2), β-TI (n=102, 28.1%), and SCD (n=57, 15.7%). Among β-TI patients, 60 (58.8%) were on transfusion therapy. Similarly, in patients with SCD 34 (59.6%) were on transfusion therapy. The mean LIC in the study population was 7.8 ± 9.6 mg Fe/g dw and the median was 4.0 mg Fe/g dw. Across underlying diseases, LIC distribution was as follows: β-TM (mean: 9.0 ± 10.7, median: 4.9 mg Fe/g dw), TX β-TI (mean: 7.1 ± 7.3, median: 5.0 mg Fe/g dw), non-TX β-TI (mean: 5.1 ± 6.0, median: 3.2 mg Fe/g dw), TX SCD (mean: 8.5 ± 11.0, median: 4.5 mg Fe/g dw), and non-TX SCD (mean: 3.1 ± 1.9, median: 2.4 mg Fe/ g dw). It was apparent that TX patients irrespective of underlying diagnosis have a comparable proportion of patients with high LIC risk categories (>7 mg Fe/g dw) (p=0.627). Among chelated patients, LIC distribution was as follows: Deferoxamine (DFO) (mean: 7.3 ± 8.5, median: 4.7 mg Fe/g dw), Deferiprone (DFP) (mean: 11.6 ± 11.4, median: 8.4 mg Fe/g dw), Deferasirox (DFX) (mean: 7.8 ± 10.3, median: 3.2 mg Fe/g dw), DFO+DFP (mean: 8.2 ± 10.6, median: 4.5 mg/ Fe g dw), and other combinations (mean: 6.5 ± 4.0, median: 5.1 mg Fe/ g dw), with a statistically significant difference noted between groups (p =0.009) with the highest median among chelated patients noted in DFP treated patients and lowest median noted in DFX treated patients. For underlying disease groups, ROC curve analysis showed that SFLs that best predict LIC thresholds of 7 and 15 mg Fe/g dw varied, although patients with β-TI showed lowest SFLs to predict these thresholds especially non-TX patients (Fig. 1, Fig.2). Discussion. This study suggest as high values of LIC are present even in patients with TI or SCD, confirming that gastro-intestinal iron absorption is one of the main mechanism for secondary iron overloading. Moreover, close to 20% of patients with non-TX β-TI continue to have high LIC thresholds, while none of non-TX patients with SCD have LIC values > 7 mg Fe/g dw. The evidence that SFLs of 900 ng/mL are related in β-TI with LIC > 15 mg Fe/g dw (Fig. 2) suggests as chelation treatment could be reconsidered earlier in this cohort of patients. Finally, these findings suggest as LIC is predicted by different SFLs according to the different types of hemoglobinopathy. Figure 1. Receiver operating characteristic curve analysis of serum ferritin level for predicting LIC>7 mg Fe/g dw in Thalassemia Major, Thalassemia Intermedia and Sickle Cell Disease patients. Figure 1. Receiver operating characteristic curve analysis of serum ferritin level for predicting LIC>7 mg Fe/g dw in Thalassemia Major, Thalassemia Intermedia and Sickle Cell Disease patients. Figure 2. Receiver operating characteristic curve analysis of serum ferritin levels for predicting LIC>15 mg Fe/g dw in Thalassemia Major, Thalassemia Intermedia and Sickle Cell Disease patients. Figure 2. Receiver operating characteristic curve analysis of serum ferritin levels for predicting LIC>15 mg Fe/g dw in Thalassemia Major, Thalassemia Intermedia and Sickle Cell Disease patients. Disclosures No relevant conflicts of interest to declare.

Endothelial urokinase-type plasminogen activator receptor (uPAR) is thought to provide a regulatory mechanism in angiogenesis. Here we studied the proangiogenic role of uPAR in endothelial colony-forming cells (ECFCs), a cell population identified in human umbilical blood that embodies all of the properties of an endothelial progenitor cell matched with a high proliferative rate. By using caveolae-disrupting agents and by caveolin-1 silencing, we have shown that the angiogenic properties of ECFCs depend on caveolae integrity and on the presence of full-length uPAR in such specialized membrane invaginations. Inhibition of uPAR expression by antisense oligonucleotides promoted caveolae disruption, suggesting that uPAR is an inducer of caveolae organization. Vascular endothelial growth factor (VEGF) promoted accumulation of uPAR in ECFC caveolae in its undegraded form. We also demonstrated that VEGF-dependent ERK phosphorylation required integrity of caveolae as well as caveolar uPAR expression. VEGF activity depends on inhibition of ECFC MMP12 production, which results in impairment of MMP12-dependent uPAR truncation. Further, MMP12 overexpression in ECFC inhibited vascularization in vitro and in vivo. Our data suggest that intratumor homing of ECFCs suitably engineered to overexpress MMP12 could have the chance to control uPAR-dependent activities required for tumor angiogenesis and malignant cells spreading.

A new and rare type of Bcr/Abl junction between exon C3 of the 3′ portion of the Bcr gene and Abl exon 2 has been identified in the leukemic cells of two Ph1-positive chronic myelogenous leukemia patients in chronic phase. This is the fourth type of Bcr/Abl junction so far identified in Ph1-positive hematologic malignancies and is a consequence of an unusual breakpoint position on chromosome 22 that falls approximately 20 kb downstream of the major breakpoint cluster region (bcr) of the Bcr gene. The new hybrid mRNA is 540 base pairs (bp) longer than that expressed by the K562 cell line and could codify for a Bcr/Abl protein carrying 180 additional aminoacids with respect to the larger P210 protein so far identified. The hematologic phenotype expressed by the two patients carrying this unusual type of Bcr/Abl rearrangement does not significantly differ from that commonly seen in chronic myelogenous leukemia.

We investigated the role of c-Kit and the membrane-bound ligand (mbKitL) in endothelial progenitor cell (EPC) recruitment by microvascular endothelial cells (ECs). We demonstrated that inflammatory activation induced the expression of the mbKitL on ECs both in vitro and in vivo, and that recruitment of EPCs depended on c-Kit/mbKitL interaction. Depletion of endogenous c-Kit or inhibition of c-Kit enzymatic activity by imatinib mesylate prevented adhesion of EPCs to activated ECs both in vitro and in vivo, indicating that a functional c-Kit on EPCs is essential. We also demonstrate that Akt was the downstream molecule regulating cell adhesion. A potential role of the c-Kit/mbKitL interaction in pathological settings is sustained by the expression of the mbKitL on ECs lining intraplaque neovessels. Thus, our results provide new insights into the mechanisms underlying EPC recruitment and the bases for novel strategies to hinder pathological angiogenesis.

BACKGROUND: The multiple myeloma (MM) represents a process in which an asymptomatic stage of monoclonal gammopathy of undetermined significance (MGUS) precedes virtually all cases of MM. It is known that tumor progression are determined by a favorable tumor microenvironment (TME) and in this scenario fibroblasts represent the principal cellular component in the TME. A particular subpopulation of fibroblasts, cancer associated fibroblasts (CAFs), has recently raised the interest of many researchers due to their active participation in tumor growth and invasion and their association with higher malignancy grade, and poor prognosis. Recent findings indicate that the urokinase plasminogen activator (u-PA), and the urokinase receptor (u-PAR) are critical in cell invasion and degradation processes. Degradation and remodeling of the surrounding tissues are crucial in the early steps of tumor progression by facilitating expansion of the tumor mass, tumor cell proliferation, migration, and invasion. uPAR is expressed by multiple tumor associated cell types found in tumors. Targeting uPAR expressed on tumor-associated cells may be as important as targeting uPAR expressed on tumor cells and may lead to enhanced antitumor activity especially in those tumor types expressing uPAR on both types of cells. METHODS: Cell purification and cultures: BM mononuclear cells (BMMCs) were isolated by Ficoll-Hypaque gradient from heparinized bone marrow (BM) aspirates from 10 patients with relapse/refractory MM, 7 patients with asymptomatic MM, 7 with remission MM, 10 with MGUS. Fibroblasts were purified from BM stromal cells BMSCs through anti-fibroblasts-microbeads, and culture in DMEM medium with 10% FBS. Cell phenotype analysis: CAFs were analyzed on heparinized bone marrow aspirates and they were identified by FSP1 and α-smooth muscle actin (α-SMA) expression on gated CD45-population. Expression of alpha-SMA in BM purified fibroblasts was also demonstrated by immunofluorescence staining. Immunofluorescence: CAFs were cultured in DMEM medium, fixed in paraformaldehyde, and permeabilized according to routine methods. The primary antibodies were anti–uPAR, and anti–α-SMA. Fibroblast nuclei were stained with DAPI. Quantitative PCR analysis: Complementary DNA was prepared from 1 ug total RNA using a GoScript reverse transcription system. The relative quantity of uPA, uPAR, MMP-2, α-SMA and vimentin messenger RNA were measured using the Applied Biosystems 7500 Fast Real-Time PCR System and determined by the comparative Ct method using 18S ribosomal RNA as the normalization gene. The study was approved by the local Ethics Committee and all patients provided their informed consent in accordance with the Declaration of Helsinki. RESULTS: Cell phenotype analysis:Flow cytometry analysis showed that CAFs were increased in patients with relapse-MM compared to patients with asymptomatic, remission MM and MGUS suggesting that CAFs expansion is involved in MM progression. CAFs activation: The increased frequency of alpha-SMA in CAFs of relapsed MM patients was demonstrated by the immunofluorescence analysis. Overall, these results suggest that MM activation is associated with the overexpression of uPAR. (Fig.1) CAFs activation was also demonstrated by Real Time PCR and the figure 2 shows the overexpression of activation molecules as well as proinvasive systems in CAF of relapsed MM in comparison of MGUS and asymptomatic MM. CONCLUSIONS: In MM developement and progression the BM niche appears to play an important role in differentiation, migration, proliferation, and drug resistance of the malignant PCs. The main goal of this proposal was to globally approach the expression of CAFs’ activation and proinvasive systems in the initiation and progression of MM. Our results highlight an important mile stone on the phisiopatology of MM progression demonstrating that the CAF-activated phenotype is associated with an over-expression of the most important pro-invasive systems, and in particular the relapsed-MM CAFs seem to overexpress the fibrinolytic pattern of invasive tumor-like cells. On the basis of these results we aim to develop a biological model which can become a potential therapeutic target. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Endothelial progenitor cell (EPC) mobilization has been reported following tissue damage, whereas no data are available regarding the mobilization of hematopoietic progenitor cells (HPCs). We performed the phenotypic and functional analysis of circulating CD34+ progenitor cells in patients with acute myocardial infarction (AMI), assessed from admission up to 60 days, in patients with stable angina pectoris (SA), and in healthy controls (CTRLs). In patients with AMI at admission (T0), the number of circulating CD34+ cells was higher (P < .001) than in CTRLs and became comparable with CTRLs within 60 days. Both the number of CD34+ cells coexpressing CD33, CD38, or CD117 and the number of HPCs was higher (P < .02 for all) in patients with AMI at T0 than in CTRLs, as was the number of hematopoietic colonies (P < .03). Patients with AMI (T0) had a significantly increased number of CD34+ vascular endothelial growth factor receptor 2–positive (VEGFR-2+) cells (P < .002) with respect to CTRLs, including CD34+ CD133+VEGFR-2+ and CD34+ CD117+VEGFR-2+ EPCs. The number of endothelial colonies was higher in patients with AMI (T0) than in CTRLs (P < .05). No significant difference was documented between patients with SA and CTRLs. Spontaneous mobilization of both HPCs and EPCs occurs within a few hours from the onset of AMI and is detectable until 2 months.

The expression and function of a new cytokine-induced endothelial cell adhesion protein, vascular cell adhesion molecule-1 (VCAM-1), was characterized in vitro by using a monoclonal antibody, MoAb 4B9, which recognizes a functional epitope on this protein. As determined by enzyme-linked immunosorbent assay and radioimmunoprecipitation of metabolically labeled cells, VCAM-1 was minimally expressed on unstimulated human umbilical vein endothelium (HUVE), but was rapidly induced by recombinant human tumor necrosis factor-alpha (rhTNF-alpha), rh interleukin-1, and lipopolysaccharide. In contrast to intercellular adhesion molecule-1, VCAM-1 was not induced on dermal fibroblasts or arterial smooth muscle cells after stimulation with rhTNF, or on keratinocytes after stimulation with rh interferon-gamma. MoAb 4B9 significantly inhibited the adherence of peripheral blood lymphocytes (PBL) and lymphocytic cell lines, but not neutrophils, to rhTNF-activated HUVE. The inhibitory effect of MoAb 4B9 on PBL adherence to HUVE was additive to that produced by the CD18 MoAb 60.3. These results show that VCAM-1 mediates a CD18-independent pathway of peripheral blood lymphocyte adherence to cytokine-stimulated HUVE. We propose that lymphocyte binding to VCAM-1, induced on endothelium by cytokines, may be an important component of lymphocyte emigration at sites of inflammation or immune reaction.