ALBERT

Description: Umbilical cord is an extra-embryonic-annex rich of both hematopoietic stem and progenitor cells (HSPC) and mesenchymal stem cells (MSC) and it is easily accessible. The HSPC derived from umbilical cord blood (UCB) are promising as graft for allogeneic bone marrow (BM) transplantation and as source of target cells for autologous HSPC gene correction. UCB-HSPC have several advantages compared to adult ones: a less risk of graft-versus-host disease, a higher frequency of progenitors with a greater clonogenic potential and more susceptibility to be transduced by lentiviral vectors. Nonetheless, the HSPC yield from single cord blood unit is not sufficient for these clinical approaches in adults. Therefore, ex-vivo expansion of HSPC in media supplemented by cytokines and/or in vitro culture systems with feeder layers, is a valid approach to exceed this limit. MSC are a component of BM-microenvironment that play a key role in supporting of hematopoiesis by ability to secrete soluble factors and probably by the direct cell-cell interaction too. In this work, we investigated the ability of umbilical cord extracellular matrix-MSC (Wharton's Jelly-MSC) to support the ex-vivo expansion of UBC- purified CD34+ cells. In particular, we evaluated the fold increase, and the frequency of CD34+ cell and CD34+subtypes during expansion at the following culture conditions: by direct contact with WJ-MSC layer, by exposure to the soluble factors secreted by WJ-MSC layer in transwell system. The fold expansion was compared with the CD34+ cells expanded in a customized serum-free medium. CD34+ cells were isolated by immuneselection from 8 fresh UCB. The WJ-MSC were isolated from UC cut-pieces by non-enzymatic procedure but thanks to their capacity to migrate to plastic substrate. At the confluence of 60-70% the WJ-MSC were treated with mytomicin-C to arrest the cell cycle. After 48h, the immune-selected CD34+ cells were seeded in WJ-MSC at the density of 5-10 x104 in 12 well plates by direct or indirect contact (by transwell system). CD34+cells were grown in absence of feeder layers at the same conditions. Early hematopoietic cytokines (Flt-3, TPO, SCF) were supplemented in all three conditions and freshly replaced every two days of culture. Numbers and frequency of CD34+cells were evaluated according to ISHAGE method and CD34+ subtyping was performed by four color method to investigated the co-expression of the primitive surface antigens (CD38, CD133, CD90). The frequency of CD34+ cells at day 5 of culture decreased only 10% and was about 50% after 8 days of culture in conditions. The expansion of CD34 + cells at direct contact with WJ-MSC was superior (5.5 fold increase) compared to that of the other two conditions (3 fold on average). At day 8of culture, the CD34+ cells expanded 12 fold at direct contact with feeder layer, about 7 fold in a transwell system and 6 fold in basic medium. No substantial differences in the grade of expansion was revealed in heterologous vs homologous co-cultures of HSPC/WJ-MSC. Noteworthy is that in the contact system in addition to the fluctuating CD34+ cells harvested from the medium (floating CD34+ cells), we found approximately 50% of the total CD34+ cells be adherent to WJ-MSC layer, these cells were released only after enzymatic proteolytic treatment. Subtyping the CD34+cell population growing in contact to the WJ-MSC or in the conditioned medium we found that the CD34+/CD133+cell population was maintained high (72% ±12 over the total CD34+ cells) as in unmanipulated CB-HSC. The CD34+CD38- cells decreased by 2,5 fold in both systems, as early as day 5 of culture. However, in the contact system this population was 3 times more represented in the attached CD34+ cell fraction. The CD34+/CD90+ subtype was also expanded (more than 8 fold) particularly in the attached fraction, as early as 5 days of culture and was maintained to the end WJ-MSC supported ex-vivo HSPC expansion with superior effect in a cell contact system. Two phenotypically different populations of HSPC developed in this system with an increased frequency of CD34+ cells that co-expressed markers typical of more early progenitors in the attached CD34+ cell fraction. We are assessing the significance of these differences by performing molecular and functional studies of WJ-MSC-supported HSPC. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F12000150004 Disclosures No relevant conflicts of interest to declare.

Description: In countries with a high prevalence of hemoglobinopathy carriers, the only realistic approach to control the birth of new patients with thalassemia major or sickle cell disease is population screening in combination with invasive prenatal diagnosis[1]. In the early 1990's, molecular definition of the thalassemia defects, development of procedures for their detection by DNA analysis and introduction of amniotic fluid sampling (amniocentesis) or chorionic villus sampling (CVS) led to early prenatal diagnosis at 16 and 11 weeks, respectively [2]. An alternative technique for earlier diagnosis is celocentesis [3]. At the end of week 4 of gestation, the developing exocoelomic cavity (ECC) splits the extra-embryonic mesoderm into two layers, the somatic mesoderm lining the trophoblast and the splanchnic mesoderm covering the secondary yolk sac and the embryo. The ECC is the largest anatomical space inside the gestational sac between 5 and 9 weeks of gestation and is surrounded by celomic fluid (CF), which contains cells of fetal origin [4-5]. This fluid can be sampled by a technique that involves the ultrasound-guided insertion of a needle through the vagina from as early as 7 weeks of gestation. Previous studies utilizing coelocentesis for prenatal determination of single-gene defects reported variable success ranging from 58 to 95% because of presence of maternal cell contamination (MCC) [6]. In this work we demostred as this problem can be overcome through the identification of embryo-fetal erythroid precursors presented in the coelomatic fluid and their specific selection. 302 couples from different regions of Italy, at risk for ß thalassemia or sickle cell disease asked for prenatal diagnosis by coelocentesis that was carried out at between 6+6 and 9+2 weeks' gestation. Coelomic fluid samples with no or very low (5% maternal contamination, two different procedures were used to isolate embryo-fetal cells: the first technique involved positive selection of embryo-fetal erythroid precursors by anti-CD71 MicroBeads (160 samples), the second procedure was through the use of a micromanipulator (42 samples). In 68/300 (22.6%) cases the fetus was affected by ß-thalass/ß-thalassemia and 66 women chosen to terminate the pregnancy. Two families decided to continue the pregnancy for ethical reasons despite the documented presence of an affected fetus. The antenatal diagnosis was confirmed in all 66 cases by molecular analysis of placental tissue after termination (Table 1). In 232/300 (77.4%) cases the fetus was diagnosed as being normal or a carrier for ß-thalassemia or sicke cell disease. The results obtained after coelocentesis were confirmed by amniocentesis or postnatally. In two case (0.66%) was no obtained a reliable diagnosis because no fetal cells were found (Table 1). The findings of this study in a large number of pregnancies investigated by coelocentesis, demonstrate that embryo-fetal erytroid cell selection from coelomatic fluid allows reliable and earlier prenatal diagnosis of hemoglobinopathies. This technique is attractive to parents because it provides prenatal diagnosis of genetic disease at least 4 weeks earlier respect to traditional procedures reducing anxiety of parents and from a clinical point of view this procedure would allow women to undergo medical TOP at 8-10 weeks of gestation which is less traumatic and safer than second trimester surgical TOP. Table 1. Overall results of prenatal diagnosis of hemoglobinopathies by coelocentesis. Number of coelocentesis effectuated 302 Results of Prenatal diagnosis: Affected 68 Carrier of hemoglobinopathies 161 Non Affected 71 diagnosis not reiable 2 Control post coelocentesis: Placental tissue after termination 66 CVS or amniocentesis 153 Postnatal test 61 Awaiting 20 Coelocentesis diagnostic errors 0 Disclosures No relevant conflicts of interest to declare.

Description: Introduction. In the last few decades, the life expectancy of Thalassemia Major (TM) patients has progressively been increasing. The improvement can be due to several factors, including introduction of chelation treatment (Deferoxamine 1965, Deferiprone 1987, Deferasirox 2006), screening of blood for the most common viral agents, aggressive treatment of infection and improved treatment of cardiac complications. However, no comparative survival curves between TM versus Thalassemia Intermedia (TI) have been so far reported. Moreover, no data on life expectancy, after introduction of chelation treatment have been described. Methods. Data coming from several randomized clinical trials, carried ahead by Campus of Hematology Franco and Piera Cutino-A.O.O.R Villa Sofia-V. Cervello, Palermo (Italy), were retrospectively considered for this study. Primary goal of the study was to provide evidence of possible differences in survival curves between TM versus TI. Survival curves in TM versus TI patients were compared using Kaplan-Meier method and the log-rank test before and after the introduction of Deferoxamine (DFO) (1965). Moreover, Cox regression model was even used to explore risk of death between the two diagnoses. Each dead patient was observed from its birth to its death, and each alive patient was observed from its birth to June 30, 2015. Results. Three hundred seventy-nine patients with TM (n=284, dead 40) and TI (n=95, dead 13) entered into the study. Males were 50.7% of this cohort of patients. Among the cohort of dead patients, 15% (6/40) TM and 76.9% (10/13) TI patients were born before introduction of DFO (1965) . The mean age survival was 50.6 (SE 0.9) and 70.6 (SE 1.7) for TM and TI, respectively. Table 1 shows the main causes of death. In TM patients the most common causes of death were heart damage (16 cases, 40%, Tab. 1), followed by cancer (3 cases, 7.5%, Tab. 1), liver cirrhosis (3 cases, 7.5%, Tab. 1) and infections (3 cases, 7.5%, Tab. 1). In TI patients the most common causes of death were cancer (2 cases, 38.5%, Tab. 1), followed by infections (3 cases, 23.1% , Tab. 1), heart damage (2 case, 15.4%, Tab. 1). Kaplan-Meir curves showed statistically significant difference in TM versus TI survival (log-rank test, p- value

Description: Introduction. Congenital dyserythropoietic anemias (CDAs) are hereditary rare erythropoietic disorder characterized by distinct morphological abnormalities of the bone marrow cells, ineffective erythropoiesis and systemic iron overload. This conditions is characterized by a maturation arrest during erythropoiesis with a reduced reticulocyte production in contrast with erythroid hyperplasia in bone marrow. Three types of CDA are known: types 1, 2 and 3.The identification of their causative genes provided evidence that these conditions have different molecular mechanisms that induce abnormal cell maturation and division. We describe the clinical and laboratory manifestations, the diagnosis procedure, the therapeutic approaches and the clinical phenotype in some cases of congenital dyserythropoietic anemia (CDAs) in Italian patients. The molecular analysis allow us to identify several genetic variants some of which have never been described previously. This report highlights the importance of recognizing CDAI even in countries where thalassemia is common. Among the different tools genetic study of CDA-related genes remains the main approach for pursuing the right diagnosis. Methods. Peripheral blood samples from 100 normal adults and 20 patients with different types of anemia were collected. Blood samples were analyzed by EMA binding test and by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate ( SDS-PAGE). SDS PAGE was carried out in a 3.5-17% exponential gradient gel in Fairbanks buffer Genomic DNA was extracted from mononuclear cells of peripheral blood samples, by using a phenol-chloroform method. Polymerase chain reaction (PCR) products were sequenced directly using Big-Dye terminator 3.1 cycle sequencing kit and run on ABI PRISM 3130 DNA analyzer. The bone marrow of all patients was analysed with light or electron microscopy. The diagnosis of CDAs was based on the presence of mild to moderate anemia ineffective erythropoiesis and bone marrow erythroblasts morphological abnormalities. SEC23B, CDAN1, KLF1, BCL11A gene sequencing analysis was performed to highlight the presence of nucleotide variations and their relationship with the clinical presentation. Results and Discussion. We collected blood samples from 20 Italian patients with suspect of CDAs and 100 samples belonging to the healthy control subjects.None mutation in the genes analyzed was found in the samples controls.We identified SEC 23 B mutations in 4 out of 20 patients analysed. In two patients with suspect of CDAII we found two nucleotide variations; SDS PAGE in these patients identified the presence of abnormal band 3 and the EMA binding test resulted pathologic. Bone marrow (BM) light microscopy revealed more than 10% mature binucleated erythroblasts. In the patients suspected of CDA1, several gene variants were found in the CDAN1 gene, some described in the literature as mutations or polymorphisms, others of uncertain significance. In a patient diagnosed with CDA1 two novel mutations was found. EMA binding test resulted normal in all patients with CDA1. Light microscopy of the BM of CDA1 patients showed erythroid hyperplasia, presence of internuclear bridges between intermediate erythroblasts and characteristic pattern of spongy "swiss cheese" hetrochromatin in electron microscopy. None KLF1 and BCL11A mutations correlated with atypical CDAs was identified. In this last patients the EMA binding test resulted normal. CDAs are rare clinical hereditary disorders whose correct diagnosis is often delayed to adolescence or adulthood, when significant iron overload and end organ damage may have been occurred. Patients are often misdiagnosed with other congenital haemolytic anaemias. Inaccurate diagnosis can lead to inappropriate therapies, such as iron supplements, aggressive transfusion or splenectomy. However even if the gold standard for the CDAs diagnosis is the electronic microscopy, the identification of the mutated genes involved in the majority of CDAs patients made in recent years has improved the possibility of detecting these diseases. Disclosures No relevant conflicts of interest to declare.

Description: Introduction. Mesenchymal stem cells (MSCs) are multipotent precursors able to differentiate into bone, cartilage and fat. In humans, the MCS were isolated from bone marrow, cord blood, peripheral blood, adipose tissue and the amniotic fluid (AF) in the second trimester of pregnancy (FA-MSC). The FA-MCS have immunosuppressive properties, extensive proliferative potential and self-renewal, and express the fetal stem transcription factor Oct4. The purpose of this work was to isolate and characterize cells isolated from human FA as alternative source of multipotent MSC for regenerative medicine and transplantation. Materials and Methods. Expansion. The FA cells (extracted from the first three milliliters of FA collected during diagnostic amniocentesis and can not be used for prenatal diagnosis), were cultured in serum free medium specific to the MSC (STEM CELL). Phenotypic analysis. On the FA-expanded MSC the presence of mesenchymal markers CD106, CD146 and CD105, and non-mesenchymal markers CD45 and CD34 have been evaluated by flow cytometry. The fetal origin of the FA-expanded MSC was verified by QF-PCR comparing FA-MSC DNA and maternal mononuclear DNA. Stemness analysis. The stemness, potential before and after expansion, was evaluated by expression analysis(RT-PCR) of Oct4 and Nanog gene. cDNA from adult peripheral blood and cDNA from chorionic villus at 10 weeks of gestation were used as control. The specific primers for the analysis of Oct4 and Nanog genes were designed in the laboratory on the basis of the gene maps (GENE ATLAS). Results and Conclusions. After 8 days of culture FA cells have produced CFU-F colonies. At 15 days of culture started the proliferation of fibroblasts, and at 28 days of culture the fibroblasts have reached 80% confluency. Phenotypic analysis. The flow cytometry assay allowed cultures immunophenotypic characterization. The flow cytometry analysis of the FA-MSC showed the presence of mesenchymal markers CD146, and CD105 and the absence of hematopoietic/endothelial markers CD34 and CD45. The QF-PCR confirmed the fetal origin of the expanded FA-MSC. Using medium serum-free adherence method, it has been developed a reproducible protocol for the isolation and expansion of AF-MSC. Phenotypic analysis results, confirmed the nature of the mesenchymal cells expanded AF. The presence of expression of Nanog (a homeobox playing a key role in embryonic development and in maintaining the pluripotency of stem cells), and Oct4 (a transcription factor involved in stem cells pluripotency and self-renewal maintenance) confirmed the stamness persistence of AF-MSCs after expansion. The expanded AF-MSCs could be used in different therapeutic fields: bone marrow transplantation in adults, transplant "in utero" and gene therapy for congenital blood diseases, treatment of chronic inflammatory bowel disease. The use of AF-MSCs also can open new perspectives for research in regenerative medicine because of their possible de-differentiation by "ex vivo" procedures. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Sickle cell disease (SCD) is a monogenic, yet highly phenotypically variable disease with multisystem pathology. HbS is prone to polymerization upon deoxygenation, and this property underlies all the pathophysiology of SCD. The net result is a chronic hemolytic anemia, with intravascular and extravascular components, and a tendency for microvascular obstruction or vaso-occlusion (VOC). The ability to predict ''severe disease'' depends on the ability to define it specifically and reproducibly. Many genetic, clinical and laboratory modifiers have been suggested as possible predictors of a "severe disease". Despite of this wealth of data the modelling of predictor variable continues to be difficult (Quinn et al, 2016). Considering that the hallmark of SCD is the chronic hemolitic anemia causing increasing of gastro-intestinal iron absorption, our aim was to evaluate if LIC-R2 (Ferriscan) determination may be related with phenotype severity of SCD. Methods This was a retrospective study of patients with SCD attending 7 Italian centres participating in the LICNET-S (Liver Iron Cutino NETwork in Sickle Cell Disease), collected at the Campus of Hematology Franco and Piera Cutino, AOOR Villa Sofia-V. Cervello (Palermo, Italy). The LICNET-S protocol was established on June 2018 by Foundation Franco and Piera Cutino of Palermo and approved by our Ethics Committee on 4, July 2018. This analysis included data from LIC-R2 for those patients presenting between July 2018 and July 2019. The MRI-R2 used protocol was that of St Pierre et al, 2005. Retrieved information included laboratory and clinical findings. These are shown as mean±sd and percentages. A linear regression model (LRM) (Draper & Smith, 1998) was used to study the correlation between LIC-R2 and some potential indicators of phenotype severity. All statistical analyses were performed using Stata 12 (StataCorp, College Station, TX, USA). Results Overall 65 patients (32 (49.3%) females) whom 16 patients with S/S, 15 with S/beta+ tahalseemia and 34 with S/beta0 thalassemia were enrolled in the study. Table 1 shows summary of the main clinical findings included in the study to evaluate the LIC-R2 predictivity based on SCD phenotype severity. Table 2 shows the results of the LRM analysis. The variables statistically significant were the number of the VOC, the Aseptic Avascular Necrosis (AVN) and the Chelation Treatment (CT) (Table 2). However, transfusion regimen was not related with LIC-R2 value, suggesting that chronic hemolitic anemia causing increasing of gastro-intestinal iron absorption rather than transfusion treatment was the main determinant in the LIC-R2 values. This issue is supported by the report of high LIC-R2 in non transfused patients with SCD (Yassim et al, 2017). Table 2 shows even the relationship between the impairment of the single indicator (VOC, AVN, CT) and the LIC-R2 value. All other laboratory and main clinical findings included in Table 2 were not statistically significant. The value of VOC in determining phenotype severity is well known. AVN is a consequence of a chronic ischaemia with inflammatory process evolving at the same time as medullary osteoblastic activity (Mukizi-Musaka et al, 2010). Therefore, it may be related with the chronic hemolitic anemia condition in SCD. Finally, the relationship between CT and LIC-R2 may be explained considering that patients with higher iron body burden are those who received earlier chelation treatment. Conclusions This study suggests, as LIC-R2 value is a strong predictive indicator of phenotype severity in SCD. Probably, this is due because of LIC-R2 is related both with acute and chronic hemolitic anemia of SCD causing an increase, during the years, of gastro-intestinal iron absorption. Therefore, the possibility of preventing VOC, even using new anti-sickling drugs, should be hardly pursued. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 5108 Background Hepatocellular carcinoma (HCC) is the most common primary hepatic cancer, and the fifth and eighth most common malignancy worldwide in men and women, respectively. Hepatitis B and C, primary and secondary hemochromatosis and cirrhosis have been identified as major risk factors. Hepatitis C or B virus infection and secondary hemochromatosis are the major cause of morbidity and mortality. In the last years, HCC on cirrhosis is a common reported complication in patients with thalassemia syndromes1,2,3. In non thalassemia patients HCC is a late complication of cirrhosis and prognosis depends on suitability for treatment. In this group, the last is low (40%) considering that cirrhosis severe stage C, according to Child-Pugh score is generally observed (personal communication). Aim of the study describing, in a homogeneous cohort of thalassemia patients, the main clinical findings of HCC. Patients and Methods All subjects with risk factors for HCC as iron overloading, HCV or HBV chronic infection, histological evidence of cirrhosis were undergone every six months to an ultrasound evaluation associated with alphaphetoprotein determination. Nine new cases, since January 2000 until July 2009, were detected. Table I shows the main clinical findings. Mean value of serum ferritin levels, in the last 2 years before diagnosis, was 1049±721. All patients except one were anti-HCV positive (Table I). Seven of them were HCV-RNA positive (Table I). None of them had previous HBV infection. Results Only one patient had severe stage C, according to Child-Pugh score. None of the patients had significant high levels of alphaphetoprotein (AFP) at diagnosis. However, a slightly increase of AFP levels was shown, during the 6 months before diagnosis, in 50% of cases. Three patients had a delayed diagnosis because of atypical imaging for HCC at TC scan or a negative previous histology for HCC after liver biopsy. Five patients had multifocal HCC at the diagnosis (Table I). Four showed an unifocal HCC, developing two of them multifocal HCC during the follow-up (Table I). Four patients died during the follow-up for decompensated cirrhosis. Five patients are alive after treatment. Main used treatments are shown on Table I. The overall mean survival was 28±25 months (range 3-64). Conclusions 1) almost all talassemia patients with HCC are suitable for treatment ; 2) baseline liver function is the most main factor conditioning survival and suitability for treatment ; 3) periodical serum AFP determnations can reveal a slight increase; 3) iron overloading alone can be related to the development of HCC in a liver with still good functional reserve, giving more opportunity for treatment 4) monitoring for HCC by liver ultrasound evaluation, every six months, in at risk patients with thalassemia syndromes should be mandatory. Disclosures No relevant conflicts of interest to declare.

Description: Background Heart failure (HF) is the most important cause of death in Thalassemia Major (TM) patients, and results from iron overload which determines progressive systolic dysfunction of the left ventricle. T2* Magnetic Resonance Imaging (CMR) is the only non-invasive tool for detecting and quantifying myocardial iron storage.We had observed that a large number of Thalassemia patients recently observed at our Centre develops a different form of HF, with evidence of diastolic dysfunction and often in absence of systolic dysfunction. Methods We evaluated the clinical, electrocardiographic, echocardiographic and Doppler data of 16 adult Thalassemia patients with HF observed at our Centre between 2008 and 2016, together with the data obtained by means of T2* CMR. All statistical analyses were descriptive. Results are provided as means ± standard deviations, medians with interquartile ranges (IQR), and percentages. Results Table 1 describes demographics, T2* and Echo-Doppler data of 16 TM patients. The 31.2% were females and the mean age was 44.2±5.7 years.One patient presented systolic dysfunction of the left ventricle whereas the others had echocardiographic and Doppler evidence of diastolic dysfunction. Systolic dysfunction of the right ventricle was also found in 81.25% of cases. Furthermore, 30.75% of cases had T2* values consistent with significant risk for heart failure (≤14 ms), whereas the others had normal values. In 68.75% of the cases ECG showed inversion of T wave beyond V2 lead, and low voltages. Conclusions Most of the patients with heart failure recently observed at our Centre had diastolic dysfunction of the left ventricle with normal systolic function, and impairment of systolic function of the right ventricle, and normal values of cardiacT2*. In 68.75% of cases ECG showed inversion of T wave beyond V2 lead and low voltages. Limitations of this study can be summarizes in: a) small number of cases (16 pts); b) Evidence of normal values of T2* values in most patients does not exclude an iron overload in precedent years. However patients with HF due to systolic dysfunction usually show low or very low values; c) a possible bias of this study may be linked to the Centre where this study has been performed. Our Centre is the Reference Centre of Sicilian Region for Thalassemia patients. This implies the possibility of a very strict surveillance of chelation therapy with frequent evaluations of the data of T2* in order to improve at best the treatment with chelation therapy. It is possible that this, at least in part, might prevent the onset of the classical form of systolic dysfunction of the left ventricle due to iron overload, and that in these patients, differently than in patients followed up in other centres, different forms of HF noit linked to cardiac iron overload may occur: that is heart failure preserved ejection fraction (HFpEF), with prevalent left ventricular diastolic dysfunction. Table 1. Demographics, Echo-Doppler and T2* data Table 1. Demographics, Echo-Doppler and T2* data Disclosures No relevant conflicts of interest to declare.

Description: 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.

Description: Introduction: Genetic modification of autologous hematopoietic stem and progenitor cells (HSPC) is a promising clinical intervention to cure inherited monogenic diseases. Successful gene therapy trials have already been conducted using CD34+ cells from bone marrow and from mobilized peripheral blood. In this regard, cord blood (CB) represents an attractive source of HSCs due to its high concentration of high proliferative HSPC and increased susceptibility to be transduced by lentiviral vectors. Unfortunately, the major disadvantage is the limited number of HSC in the CB collection. Consequently, ex-vivo expansion of CB-HSC is desirable to extend clinical applications. Purposes: To investigate the ability of UCB-cd34+ cells to be expanded in serum-free media supplemented with the early acting hematopoietic cytokines SCF,TPO and Flt-3 ligant (STF) and to characterize CD34+ cells subtypes, clonogenic capacity and gene expression profile during expansion. We also wanted to investigate the susceptibility of the expanded cd34+ cells to be transduced by a GFP-lentiviral vector (LV-GFP) Material and Methods: CD34+ immunoselected cells from 10 UCB were grown for 8 days in customized serum-free medium formulated for HSC expansion, supplemented with STF cytokines. Numbers end frequency of CD34+cells and co-expression of the primitive surface antigens (CD38, CD133, CD90) was evaluated during expansion. Colonies developed in methylcellulose were scored for enumeration ad typing. LV-GFP transduction efficiency was evaluated in CD34+ cells cultured for 4 days in expansion medium plus STF and for 24 hrs in X-vivo10 medium with STF±IL-3 cytokines; the last condition slightly expands CD34+ cells (1.3 fold) and are currently used for HSPC-lentivector transduction in gene therapy clinical trials. The transduction efficiency was evaluated by measuring the percentage of GFP+ cells in the bulk and in colonies developed in methylcellulose and the VCN/cell by Q-PCR. Gene expression profiles were analyzed by human whole genome Agilent microarray Technology to detect differentially expressed genes between expanded, ex-vivo medium cultured and un-cultured cells. Results: We found an average of 8 fold-increase CD34+cells at day 4 and of 22 fold- increase at day 8 of culture. The frequency of CD34+ was maintained at day 4 and declined of about 50% at day 8. CD34+/CD38- early progenitors doublet as early as day 4, differences in CD34+/CD133+ and CD34+/CD90+cells were not significant. The number of CFU slightly increased during expansion while the relative frequency of colonies type did not significantly changed. Four days expanded CD34+ cells were transduced more efficiently than those grown in ex-vivo medium even in presence of IL-3 added to the STF cytokine cocktail. Comprehensive gene expression profile analysis highlighted about 4000 genes differentially expressed in CD34+ cells expanded for 4 and for 8 days compared to that of the un-cultured cells. Conversely, the expression profiles analysis did not show any clear separation between different cell culture methods (expansion vs ex-vivo medium). Specifically, the number of differentially expressed genes in common between the different culture conditions compared with the un-cultured cells was statistically significant. Unsurprisingly, the common up-regulated genes were related to the cell cycle. The likeness between the gene expression profiles of the different culture conditions was also validated by the identification of a significantly small number of differentially expressed genes between them. Conclusions: UCB-CD34+ cells can be efficiently expanded and transduced in serum free conditions. The expanded cells exhibited phenotypic marchers typical of early progenitors and developed colonies in number and in type similar to the unmanipulated cells and exhibited whole gene expression profile that is consisted with that of CD34+ cells exposed for the short term culture conditions currently used in gene therapy trial mediated by lentiviral vectors. Results from this study open a window on the future possibility of using homologous UCB-HSC as target for gene correction in patients diagnosed for a genetic disorder in prenatal time. The genetically modified cells would be stored and used for gene therapy in the same individual in pediatric age. This work was funded by the F and P Cutino Foundation - Project RiMedRi CUP G73F12000150004 Disclosures No relevant conflicts of interest to declare.