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Ba ckground : Lysosomal storage diseases (LSDs) are a heterogeneous group of rare chronic genetic conditions. The standard-of-care treatment for LSDs is hospital-based infusion of enzyme replacement therapy (ERT), however, over time this can be stressful and inconvenient. The Italian TuTor program, established in 2011 by Sanofi Genzyme, is a professional nursing service providing home-based ERT to patients with LSDs. Objectives: The current questionnaire-based study was conducted to investigate the level of patient satisfaction with the TuTor program and to shed light on disease perception. Methods: Patients were enrolled in the TuTor program from 2011 onwards. The first 100 patients enrolled were interviewed at baseline with follow-up interviews conducted at 6, 12 and 18 months. R esults: Overall, 52 patients were female; 46 had Gaucher’s disease, 46 had Fabry disease and 8 had mucopolysaccharidosis type 1. Patients took on average >2 hours to receive hospital-based ERT, plus time associated with the infusion; 2 out of 3 patients needed a caregiver to travel to the hospital. After receiving home-based ERT for 6 months, 37% of patients considered their quality of life ‘greatly improved’ (60% at 18 months). Overall, 99% to 100% of patients rated the home-based nursing service as ‘positive’ or ‘very positive’ and reported that they would recommend the service to other patients with their condition. Conclusions: For patients with LSDs eligible for ERT, a disease-specific home-based nursing service increased their perception of quality of life over a hospital-based service and was advantageous in terms of their time and expenditure.

Transient elastography (TE) is a valuable noninvasive technique of measuring liver stiffness and a reliable tool for predicting hepatic fibrosis in patients with chronic liver disease. The role of TE in patients with beta-thalassemia has not been extensively investigated. The present study aimed to evaluate the role of TE in the assessment of hepatic fibrosis in 115 adult patients with beta-thalassemia major (TM) (#59) or intermedia (TI) (#56). TE was performed according to current practice. Histologic data were obtained in 14 cases. Liver iron concentration was assessed by atomic absorption spectrometry and T2 magnetic resonance. In patients with TM, the proportion of anti-HCV positive viremic patients, median serum ferritin levels, and TE values were significantly higher than in TI. In the group of 14 patients who underwent liver biopsy, a significant positive correlation was observed between liver stiffness and fibrosis stage (r = 0.73, P = 0.003). Severe fibrosis is diagnosed with a sensitivity of 60% and a specificity of 89%, whereas cirrhosis is detected with a sensitivity of 100% and a specificity of 92%. At multivariate analysis, the variables independently associated with TE were ALT, GGT, and bilirubin levels in both groups and, in patients with TM, HCV RNA positivity. In beta-thalassemia patients, TE is a reliable tool for assessing liver fibrosis even if the influence of iron overload has to be clarified.

Beta-thalassemia major (TM), a chronic, genetically determined hematological disorder, has received little investigation on the psychological aspects of the disease and the psychosocial adjustment of patients with this anemia. Unfortunately, the few psychological studies referred on the literature are generally limited to the investigation of the only children with thalassemia major. The study was planned to assess the self-image, the quality of life, the way of coping and to investigate the existence of psychiatric disorders in young adults with thalassemia major. 147 patients were included in the study. Patients were psychologically investigated by three interviews: the first connects some psychosocial information, the second submits patients to the psychological test and the third gives back the results obtained by the test. The psychological test consists of the ways of coping questionnaire (WCQ), the Machover's test, The short form 36-health survey questionnaire (SF-36) and symptom-check-list-90 revised (SCL-90-R) were performed on all patients. Vis-à-vis identity and self-image were found to be low with feeling of insufficiency and being exposed to vulnerability in 80% of patients with TM. Evaluation of mean values of symptomatological dimensions in these patients showed a personality characterized by somatization (SOM), depression (DEP) and obsessive-compulsive traits. The principal coping strategy used is escape-avoidance. No statistically significant differences occurred to relation to gender, age, level education and SCL-90-R and WCQ scores. Estimation of the SF-36 scores showed that the emotional role and social function values were considerably lower than in all of the domain. As a result, the study showed that most of the patients with TM had severe psychosocial problems. Relying on these data, it was concluded that medical therapy of these patients should be supported with psychological aid and psychiatric treatment.

The study aimed to evaluate, over a 3-year period, the progression towards sustained hypertension and left ventricular (LV) changes in patients with isolated office (IO) hypertension (office BP140 and/or 90 mmHg, daytime BP130/80 mmHg). After 3 years from the basal evaluation, 38 subjects with basal normal BP and 42 subjects with basal IO hypertension underwent a second 24-h BP monitoring and echocardiography; 19 patients of the basal IO hypertension group were not revaluated because they had already developed ambulatory hypertension and were on antihypertensive treatment. At the second evaluation, the 38 normotensive subjects had unchanged BP and LV parameters; 25 IO hypertensives have developed sustained hypertension. Considering them together with the 19 patients already treated, 72% of 61 IO hypertensives developed ambulatory hypertension over a 3-year period. The patients who subsequently developed hypertension differed from the group who did not only for lower basal values of LV diastolic parameters; all the patients with basal LV hypertrophy and/or preclinical diastolic impairment subsequently developed sustained hypertension. In conclusion, IO hypertensive patients show a high rate of progression towards sustained hypertension. Basal LV hypertrophy and/or preclinical diastolic dysfunction were the only markers of a greater risk of becoming hypertensives.

Objectives : Thrombocytopenia and splenomegaly are common features in several haematological disorders. Gaucher disease (GD) is a rare lysosomal storage disorder frequently characterized by thrombocytopenia and splenomegaly, which represents a clinical challenge for haematologists and internists. Case : We describe the case of a 37-year-old patient with a diagnosis of spherocytosis since childhood, who developed hepatic failure and presented striking features of GD including hepatosplenomegaly, bone fractures and post-partum bleeding. We reconsidered the diagnosis of spherocytosis and investigated Gaucher disease. Conclusion : GD should be considered in the differential diagnosis of thrombocytopenia and splenomegaly.

Due to the increasing survival of thalassemic patients, osteopathy is a mounting clinical problem. Low bone mass alone cannot account for the high fracture risk described; impaired bone quality has been speculated but so far it cannot be demonstrated noninvasively. We studied bone quality in thalassemia major using trabecular bone score (TBS), a novel texture measurement extracted from spine dual-energy X-ray absorptiometry (DXA), proposed in postmenopausal and secondary osteoporosis as an indirect index of microarchitecture. TBS was evaluated in 124 adult thalassemics (age range 19–56 years), followed-up with optimal transfusional and therapeutical regimens, and in 65 non-thalassemic patients (22–52 years) undergoing DXA for different bone diseases. TBS was lower in thalassemic patients (1.04 ± 0.12 [range 0.80–1.30]) versus controls (1.34 ± 0.11 [1.06–1.52]) (p

Summary The risk of developing hepatocellular carcinoma (HCC) in patients with thalassaemia is increased by transfusion-transmitted infections and haemosiderosis. All Italian Thalassaemia Centres use an ad hoc form to report all diagnoses of HCC to the Italian Registry. Since our last report, in 2002, up to December 2012, 62 new cases were identified, 52% of whom were affected by thalassaemia major (TM) and 45% by thalassaemia intermedia (TI). Two had sickle-thalassaemia (ST). The incidence of the tumour is increasing, possibly because of the longer survival of patients and consequent longer exposure to the noxious effects of the hepatotropic viruses and iron. Three patients were hepatitis B surface antigen-positive, 36 patients showed evidence of past infection with hepatitis B virus (HBV). Fifty-four patients had antibodies against hepatitis C virus (HCV), 43 of whom were HCV RNA positive. Only 4 had no evidence of exposure either to HCV or HBV. The mean liver iron concentration was 8 mg/g dry weight. Therapy included chemoembolization, thermoablation with radiofrequency and surgical excision. Three patients underwent liver transplant, 21 received palliative therapy. As of December 2012, 41 patients had died. The average survival time from HCC detection to death was 11·5 months (1·4–107·2 months). Ultrasonography is recommended every 6 months to enable early diagnosis of HCC, which is crucial to decrease mortality.

Objectives Gaucher disease (GD) diagnosis relies on the demonstration of deficient β- d -glucosidase (GBA) activity in cellular homogenates. Diagnosis process, however, can be delayed as (i) some GD symptoms are non-specific; and (ii) diagnostic tests are performed in specialized laboratories. These difficulties negatively impact on timely access of patients to therapy. GBA assay in dried blood spots (DBS) represents a method facilitating early identification of patients who will be finally diagnosed with gold standard assay of nucleated cells. Aim of this study is to investigate the DBS analytical performance compared with gold standard method. Design & methods A cross-sectional study started by comparing data of 50 DBS and 50 homogenate samples from the same subjects (25 known-GD and 25 controls). The subsequent phase examined 443 DBS samples. Along with these, 73 blood samples were sent for leukocyte separation and/or EBV-lymphoblast cell lines, and 1 skin biopsy for fibroblast cell lines. Overall the study included a total of 493 subjects. Results While the results from this first validation group did not yield false positive/negative values, when the analysis was extended to 443 DBS, 14.4% (64 samples) of positive results was yielded. Among these, only 15 were confirmed as GD values with gold standard test. In addition, a thorough examination of some clinical data also revealed 2 false negative results which were confirmed by both enzymatic and molecular analyses. Conclusions DBS test could be useful as screening method although with cautions, whereas the standardized GBA assay should remain the gold standard for laboratory diagnosis of Gaucher disease.

INTRODUCTION In thalassemia patients transfusion therapy and inappropriately increased iron absorption due to ineffective erythropoiesis cause iron overload, which account for most morbidity in these patients, with involvement of liver, heart and endocrine glands. The most frequent endocrine defect is hypogonadotropic hypogonadism, followed by diabetes mellitus, hypothyroidism and hypoparathyroidism. The hypothalamic-pituitary-adrenal axis is rarely affected, but the identification of asymptomatic patients is crucial, as adrenal insufficiency is a potentially life-threatening condition. Hypoadrenalism can play a dramatic role in stressful situations like acute cardiac failure, sepsis or surgery. A better understanding of emerging complications, including adrenal insufficiency, is essential in the thalassemia population, considering their increased survival and life activities. We describe three cases of thalassemia major patients regularly monitored at our Rare disease Center. The annual endocrinological follow-up at our Clinic includes the evaluation of basal adrenal function (cortisol and corticotrophin serum levels), with completely normal results in the patients described. In all of them there were no symptoms nor clinical signs of hypoadrenalism, which dramatically emerged only on occasion of acute events and was confirmed by the finding of inappropriately low serum cortisol levels. CASE REPORT 1. A 52-year-old male was admitted to the Intensive Care Unit for severe sepsis due to bacterial pneumonia and heart failure associated with atrial fibrillation in November 2015. He was regularly transfused every three weeks, on oral iron chelation therapy with deferasirox with no liver nor cardiac iron overload. Conversely, endocrinopathy was present with type 2 diabetes, hypogonadotropic hypogonadism and hypoparathyroidism. Adrenal crisis was suspected because of persistent hypotension despite intensive treatment for septic shock, including intravenous norepinephrine. Intravenous replacement therapy with hydrocortisone rapidly reverted the hypotension. CASE REPORT 2. A 44-year-old male patient was admitted to the Intensive Care Unit in February 2015 for severe sepsis, hyponatremia, hypoglycemia and mental confusion. He was regularly transfused with two/three units of packed red cells every three weeks, on iron chelation therapy with deferasirox. He had not liver nor heart iron overload, and the only endocrine defect was hypogonadotropic hypogonadism. Hypoadrenalism was suspected as severe hypoglycemia did not respond to continuous ev glucose administration. Glucose blood levels normalized only after hydrocortisone ev. CASE REPORT 3. A 40-year-old male patient had a right abdominal mass known since 2002, and ascribed to extramedullary erythropoiesis. The mass volume had been progressively increasing up to 19 x 15 x 25 cm³. In 2015 a computed tomography scan of the abdomen showed a huge well circumscribed retroperitoneal mass in the right abdomen, originating from the right adrenal gland, displacing the liver medially and the right kidney caudally and anteriorly; moreover, it compressed the inferior vena cava and displaced the mesenteric vessels. In view of surgery, though adrenal basal function was normal an ACTH test was performed, disclosing adrenal insufficiency. Replacement therapy with hydrocortisone was set to make the patient able to cope with the stressful event. Laparotomy was performed and a 3.5 kg encapsulated tumour attached to the right adrenal gland was removed; histology revealed a giant myelolipoma. CONCLUSIONS The three cases presented show that adrenal insufficiency requires early detection, that it may be underdiagnosed and it can present dramatically in acute and even life-threatening conditions. Pituitary-adrenal axis should be screened in asymptomatic thalassemic patients, and once adrenal insufficiency has been identified, it should be treated to prevent potentially lethal consequences. Currently, our patients are asymptomatic on chronic oral replacement therapy with cortisone acetate. Increased awareness of the risks of hypoadrenocorticism in patients with iron overload is needed, because symptoms are usually not clinically evident and often neglected because of their mildness, especially in non-stressful situations. Disclosures Cappellini: Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Genzyme-Sanofi: Membership on an entity's Board of Directors or advisory committees.

BACKGROUND: Ruxolitinib (RUX) is approved in adult patients (pts) with myelofibrosis (MF), and in pts with polycythemia vera (PV) who are resistant/intolerant to hydroxyurea. Splenomegaly, a key clinical feature in advanced MF/PV, is also common in pts with transfusion-dependent thalassemia (TDT). In pts with TDT, splenomegaly worsens anemia, leading to increased transfusion requirement (TR). Similar to murine models of MF (Ostojic A, 2012), JAK2 inhibition led to a decrease in spleen size in murine models of thalassemia (Musallam KM, 2013). Additionally, thalassemia-related ineffective erythropoiesis was associated with hyperactivation of JAK-STAT pathway in preclinical studies. These findings indicate that RUX treatment (Tx) might benefit pts with TDT and splenomegaly. Present exploratory study aims to evaluate the effect of RUX Tx on TR, spleen volume (SV), and pre-transfusion hemoglobin (Hgb) levels. METHODS: TRUTH is a single-arm, multicenter, phase 2a study exploring the efficacy and safety of RUX in regularly transfused adult pts (N = 30) with thalassemia and splenomegaly, for 30 weeks (core study). Starting dose of RUX was 10 mg twice daily (maximum dose of 25 mg in 5 mg/10 mg increments). Pts were required to receive iron chelation (deferoxamine/deferasirox) for at least 4 week prior to screening and throughout the study. Primary end point was the percent change of red blood cells (RBCs) transfused between week 6 to 30 vs baseline period (BL; defined as period between 24 weeks prior to start of Tx and week 0). Change of SV from BL (by MRI/CT) at week 12 and week 30 was a secondary end point. Other secondary end points included safety (N = 30, safety set) and change of pre-transfusion Hgb level from BL. RESULTS: Of the 30 pts enrolled (median age, 24 years; 60% male), 26 completed the core phase at week 30 and 4 discontinued before week 30 (adverse event [AE], N = 2; withdrew consent, N = 1; subject/guardian decision, N = 1). Of those 26 who completed core Tx, 20 pts continue to receive RUX beyond the core study via other mechanisms. The median duration of exposure during the core phase was 30.2 weeks and median actual dose intensity of RUX was 27.2 mg/day (range, 13.3-39.0 mg/day). Mean hematocrit (HCT) adjusted volume of transfused RBC per 4 weeks was 605 mL for the BL period and 560 mL for the on-Tx period (between week 6-30; N = 27, per protocol set; 3 pts received < 18 weeks of Tx). Mean percent change of transfusion rate was −5.9 (95% CI: −14.7, 2.83). Change of HCT adjusted transfused volume per 4 weeks for on-Tx period vs BL is shown in Figure 1A. The percent change from BL in SV at week 30 is represented in Figure 1B. The mean SV reduction from BL at week 12 (N = 26) and week 30 (N = 25) was −19.7% and −26.8%, respectively. A slight trend for improvement was observed in the median pre-transfusion Hgb levels over time (pre-Tx = 8.4 g/L; end of study [week 24-30] = 8.9 g/L). At BL, 77% (23/30) of pts had Hgb levels below LLN but ≥ 8 g/dL and 20% (6/30) of pts had Hgb levels < 8 g/dL. At BL, 20% (6/30) of pts had a platelet (PLT) count below LLN but > 50 × 109/L, while no pt had PLT counts < 50 × 109/L. Worst post-BL hematologic abnormalities were Hgb (< 8g/dL, [hypo] = 17 pts [57%]), and PLT counts (< 50 × 109/L [hypo] = 1 pt [3%]). The most common AEs (all grade [G], ≥ 5%, regardless of study drug relationship) were upper respiratory tract infection (27%), nausea (20%), and upper abdominal pain/anemia/diarrhea/weight increased [each = 17%]). Overall, 25 pts experienced AEs, 11 pts had G 3 or 4 AEs, and 6 pts had serious AEs (regardless of study drug relationship); while, 13 pts experienced AEs, 5 pts had G 3 or 4 AEs, and 3 pts had serious AEs that were suspected to be related to the study drug. No deaths were reported during the study. AEs led to dose reduction/study Tx interruption in 9 pts (regardless of study drug relationship [≥ 5%]: nausea [all G = 2 pts (7%); G 3 or 4 = 1 pt (3%)] and vomiting [all G = 2 pts (7%); G 3 or 4 = 1 pt (3%)]). CONCLUSION: RUX Tx showed a trend for improvement in transfused red cells and a slight improvement in pre-transfusion Hgb; while, there was a noticeable reduction in SV over time. As per investigator assessment of clinical benefit, a majority of pts continued Tx beyond the core study. RUX was well tolerated in the study population with modest incidences of G 3 or 4 and serious AEs, with no new safety findings. Given the sustained decrease in SV, further studies could be valuable to determine if RUX Tx may be an alternative to splenectomy in pts with TDT. Disclosures Aydinok: Shire: Research Funding; Cerus: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Karakas:Novartis: Research Funding. Siritanaratkul:Jansen-Cilag: Research Funding; Novartis: Research Funding; Roche: Research Funding; Pfizer: Research Funding. Kattamis:Novartis: Honoraria, Research Funding; ApoPharma: Honoraria. Hollaender:Novartis: Employment. Mahuzier:Novartis: Employment. Gadbaw:Novartis: Employment. Taher:Novartis: Honoraria, Research Funding; Celgene: Research Funding.

Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a clear relationship between the sources of hematopoietic stem cells (HSCs) infused and their differential homing and engraftment properties. For a long time, bone marrow (BM) harvest has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution following myeloablative conditioning regimen. At present, mobilized peripheral blood (PB) is commonly used for hematopoietic cells transplantation in both adults and children, particularly in the autologous setting, and it has progressively replaced BM as the source of HSCs.HSCs are maintained in their niche by binding to cellular determinants through adhesion molecules and diverse strategies are currently used to promote their egress from BM to PB. Traditionally, the growth factor granulocyte-colony stimulating factor (G-CSF) represents the gold standard agent to mobilize HSPCs for transplantation. Nevertheless, other compounds have been recently tested. One of the most successful mobilizing agents is Plerixafor (AMD3100, Mozobil™), a bicyclam molecule that selectively and reversibly antagonizes the binding of stromal cell derived factor-1 (SDF-1), located on the surface of BM stromal cells and osteoclasts, to chemokine CXC-receptor-4 (CXCR4), located on the surface of HSPCs, with the subsequent mobilization in the blood. The use of this drug is currently approved by FDA and EMA in combination with G-CSF, in patients affected by lymphoma or multiple myeloma whose cells mobilize poorly with G-CSF alone. Clinical trials demonstrated that Plerixafor alone safely and rapidly mobilizes HSCs also in healthy donors, beta-thalassemia patients and pediatric patients affected by malignancies. Previous characterization studies on non-human primates and human samples of Plerixafor mobilized cells in comparison to cells mobilized by G-CSF alone or in combination with Plerixafor showed a different expression profile, cell composition and engrafting potential in a xenotransplant model. From these studies remains unsolved whether Plerixafor, G-CSF, or their combination mobilizes different primitive HSC populations, defined both by multimarker immunophenotype and in vivo functional analysis. In the present study we investigated by controlled comparative analysis the functional and molecular hallmarks of human HSCs collected from BM, G-CSF and/or Plerixafor mobilized peripheral blood. We show that Plerixafor alone mobilizes preferentially long-term hematopoietic stem cells (LT-HSCs), defined as CD34+CD38/lowCD90+CD45RA-CD49f+ cells and primitive populations of HSCs. These cells possess higher ability to home to hematopoietic niches and engraft in NOD/SCID/IL2rγnull (NSG) mice, resulting in enriched scid-repopulating cell frequency, in comparison to other sources. The higher content of CXCR4+ and CD49f+ cells correlates with this feature. Furthermore, global gene expression profiling highlights the superior in vivo reconstitution activity of Plerixafor mobilized cells. The "stemness" signature of cells dislodged from their niche by the drug is attenuated by the combined use with G-CSF, which emphasizes the gene expression profile induced by G-CSF treatment. These data indicate that a qualitative advantage accounts for the superior performance of Plerixafor mobilized cells. These findings provide the rationale for using a suboptimal dose of more primitive HSCs when target cell number for transplantation is limited, or when G-CSF mobilization is too risky like in sickle cell anemia patients. Moreover, CD34+ cells mobilized by Plerixafor alone or with the combination of G-CSF are efficiently transduced by a lentiviral vector encoding for human ß-globin gene (GLOBE LV) and are able to engraft and differentiate in vivo, supporting their use for gene therapy applications. Disclosures Ciceri: MolMed SpA: Consultancy.

BACKGROUND. A substantial proportion of patients with transfusion-dependent beta-thalassemia major suffer from chronic liver disease. Iron overload resulting from repeated transfusions and HCV infection have been implicated in the development of liver fibrosis. The objective of iron chelation is to avoid the complications of siderosis, such as hepatocellular injury. As a matter of fact, adequate chelation therapy is mandatory in order to prevent liver disease progression. In the last few years the availability of different chelating agents (coupled with anti-HCV therapy) allowed control of liver disease due to secondary iron overload. Transient elastography (TE, Fibroscan®) is a non-invasive and rapid diagnostic tool that enables accurate prediction of hepatic fibrosis by measuring liver stiffness (LSM). Patients with TDT could benefit from regular non-invasive assessment of liver fibrosis as an indirect indicator of treatment adequacy and therapeutic compliance. AIM. We investigated the efficacy of adequate iron chelation to prevent the progression of liver fibrosis in TDT patients over a time period of approximately 4 years. MATERIALS AND METHODS. We analysed data in a retrospective cohort study over a 4±1.5-year time period. Ninety-nine patients with beta-thalassemia major (41 M, 58 F, aged 36±6 years), followed at Rare Disease Center, Ca' Granda Hospital in Milan, were enrolled in the study. All patients received regular transfusions every 2-4 weeks. Chelation therapy was registered using deferasirox (DFX), deferoxamine (DFO), deferiprone (DFP) or a combination of the three. All the participants underwent TE and T2-weighted magnetic resonance imaging (T2*MRI) twice (at baseline,T0 and after approximately 4 years, T1). Liver iron concentration (LIC) was assessed by T2*MRI using the appropriate formula at T0 and T1. LSM was measured according to the following cut-off: 7.9 kPa moderate fibrosis (F2), >10.3 advanced fibrosis (F3), >11.9 kPa cirrhosis (F4). We divided our cohort into groups depending on the administered chelating agent and the presence of iron overload defined by a LIC > 4.23 mg Fe/g dry weight (corresponding to a T2* value > 6.3 ms). RESULTS. At baseline the mean pre-transfusion Hb levels was 9.74 ± 1.17 g/dL, the mean iron intake 0.33 ± 0.07 mg Fe/kg/d and median serum ferritin 669.5 ng/ml (range 113-5912). HCV-RNA positivity was found in 33 patients (33%) at T0: 20 patients were treated for hepatitis C during the observation period. The overall mean LSM was 7.4±3.2 kPa, the mean hepatic T2* was 9.92±7.01 ms and the mean LIC was 4.81±3.82 mg/g dw (n=99). Data available at 4±1.5 years showed a significant reduction in liver stiffness (6.6±3.2 kPa, p 0.017), iron overload measured by hepatic T2* (12.84±7.28 ms, p < 0.001) and LIC (3.65±3.45 mg/g dw, p 0.001). This result was confirmed when considering patients with iron overload at the time of the first measurment (n=41). The number of patients with moderate to advanced fibrosis (LSM ≥ F2) decreased, yet not significantly (30% vs 19%, p 0.07). Subjects treated with a stable dose of DFX over the entire period of observation (n=39) showed a reduction of LSM (6.9±2.3 vs 6.06±2.4, p 0.04) and a concomitant improvement of hepatic T2* and LIC values (respectively 11.02±6.57 vs 15.04±6.42 and 3.84±2.81 vs 2.49±1.89, p < 0.001). In this group we observed a substantial decrease in the number of patients with LIC > 4.23 mg Fe/g dw (33% vs 13%, p 0.01). A reduction of LSM, yet not statistically significant due to the limited size of the cohort, was achieved in patients on combined DFO+DFP at T0 and T1 (n=6, 8.6±5.5 vs 7.3±3.8). The group of patients on DFO (n=11) remained stable over time. Patients who underwent anti-HCV therapy showed an even more evident reduction in LSM (9±3 vs 7±3.1, p 0.016). CONCLUSION. These data support evidence on the efficacy of adequate iron chelation therapy and, if necessary, anti-HCV treatment in decreasing or at least controlling the progression of liver fibrosis measured by TE in transfusion-dependent BTM patients. Therapeutic compliance and tailoring of drugs is essential to prevent hepatic injury resulting from siderosis. Moreover, proper chelation and treatment of HCV infection act sinergistically to limit progression of liver disease in TDT patients with active hepatitis C, so that development of cirrhosis could be either prevented or greatly delayed. Disclosures Cappellini: Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Genzyme-Sanofi: Membership on an entity's Board of Directors or advisory committees.

Heart failure due to myocardial iron overload remains the leading cause of morbidity and mortality in adult thalassemia major (TM) patients. We evaluated the removal of cardiac iron and the changes of cardiac function by different iron chelation in TM patients by T2* cardiac magnetic resonance (CMR). Sixty-seven TM patients (27 males/40 females; mean age, 35 ± 6 years) on different chelation regimens underwent T2* CMR at baseline (t 0), after 6–14 months (t 1) and after 32 ± 7 months (t 2). Patients were divided in four groups according to chelation treatment: group A (deferasirox), group B (deferoxamine), group C (combined treatment, deferoxamine plus deferiprone) and group D (deferiprone alone). Myocardial T2* at t 0 was

A 45-year-old white female with thalassaemia major, diabetes mellitus and hypogonadism underwent routine cardiac magnetic resonance (CMR) imaging to evaluate T2*, a myocardial and hepatic iron load indicator useful in the management of iron chelating therapy. At cardiac cine imaging, left ventricular antero-apical mild hypokinesia and pericardial effusion were evident. T2 weighted STIR …

Extramedullary haematopoiesis (EH) is the production of blood cell precursors outside the bone marrow that occurs in various disorders, such as thalassaemia, sickle cell anaemia, hereditary spherocytosis, polycythaemia vera, myelofibrosis and other haematological diseases. In chronic anaemia, it is a physiological response to increased erythropoietin. In some other conditions, such as myeloid metaplasia, polycythaemia vera or chronic myeloid leukaemia, EH is due to a clonal disorder of haematopoiesis that enables progenitor cells to escape from the marrow and lodge in other organs. EH usually involves the liver, spleen and lymph nodes or it can be paravertebral, intrathoracic, pelvic. It is often asymptomatic but can sometimes lead to symptomatic tumour-like masses. Treatment options are still controversial and limited, including hypertransfusion regimen, surgical treatment, radiotherapy and hydroxyurea (HU). We describe intrathoracic and symptomatic pelvic EH masses in a 48-year-old woman and intrathoracic bilateral masses causing respiratory insufficiency with pleural effusion in a 42-year-old male, both affected by thalassaemia intermedia. Both patients showed a clinical improvement with hydroxyurea therapy and occasional blood transfusions.

Heparin induced thrombocytopenia (HIT) in addition to bleeding complications are the most serious and dangerous side effects of heparin treatment. HIT remains the most common antibody-mediated, drug-induced thrombocytopenic disorder and a leading cause of morbidity and mortality. Two types of HIT are described: Type I is a transitory, slight and asymptomatic reduction of platelet count occurring during 1-2 days of therapy. HIT type II, which has an immunologic origin, is characterized by a thrombocytopenia that generally onset after the fifth day of therapy. Despite thrombocytopenia, haemorrhagic complications are very rare and HIT type II is characterized by thromboembolic complications consisting in venous and arterial thrombosis. The aim of this paper is to review new aspects of epidemiology, pathophysiology, clinical features, diagnosis and therapy of HIT type II. There is increasing evidence that platelet factor 4 (PF4) displaced from endothelial cells, heparan sulphate or directly from the platelets, binds to heparin molecule to form an immunogenic complex. The anti-heparin/PF4 IgG immune-complexes activates platelets through binding with the Fcgamma RIIa (CD32) receptor inducing endothelial lesions with thrombocytopenia and thrombosis. Cytokines are generated during this process and inflammation could play an additional role in the pathogenesis of thromboembolic manifestations. The onset of HIT type II is independent from dosage, schedule, and route of administration of heparin. A platelet count must be carried out prior to heparin therapy. Starting from the fourth day, platelet count must be carried out daily or every two days for at least 20 days of any heparin therapy regardless of the route of the drug administration. Patients undergoing orthopaedic or cardiac surgery are at higher risk for HIT type II. The diagnosis of HIT type II should be formulated on basis of clinical criteria and confirmed by in vitro demonstration of heparin-dependent antibodies detected by functional and antigen methods. However, the introduction of sensitive ELISA tests to measure anti-heparin/PF4 antibodies has showed the immuno-conversion in an higher number of patients treated with heparin such as the incidence of anti-heparin/PF4 exceeds the incidence of the disease. If HIT type II is likely, heparin must be immediately discontinued, even in absence of certain diagnosis of HIT type II, and an alternative anticoagulant therapy must be started followed by oral dicumaroids, preferably after resolution of thrombocytopenia. Further studies are required in order to elucidate the pathogenetic mechanism of thrombosis and its relation with inflammation; on the other hand large clinical trials are needed to confirm the best therapeutic strategies for HIT Type II.

We describe two cases of simple heterozygosity for the common beta degrees -thalassemia mutation beta 39 (C-->T), both presenting with a thalassemia intermedia phenotype. In both cases synergic effect deriving from membrane defects or red cell enzyme deficiencies were excluded. In one case a triplication of the alpha-globin genes was found which did not justify the severity of the transfusion-dependent phenotype. Multiplex ligation-dependent probe amplification (MLPA) analysis of the alpha-globin gene cluster revealed two new rearrangements, consisting of a full duplication of the alpha-globin genes locus including the upstream regulatory element. In one case the duplication was in the presence of the common anti-alpha(3.7) triplication in trans, resulting in a total of 7 active alpha-globin genes. In the other case the duplicated allele and the normal allele in trans resulted into a total of 6 active alpha-globin genes. We report the clinical and hematological data and the molecular analysis and discuss the occurrence of alpha-globin genes duplication defects in cases of beta-thalassemia heterozygotes with thalassemia intermedia phenotypes.

Over the past decades outcomes of clinical hematopoietic stem cell transplants have established a clear relationship between the sources of hematopoietic stem cells (HSCs) infused and their differential homing and engraftment properties. For a long time, bone marrow (BM) harvest has been the preferred source of hematopoietic stem and progenitor cells (HSPCs) for hematopoietic reconstitution following myeloablative conditioning regimen. At present, mobilized peripheral blood (PB) is commonly used for hematopoietic cells transplantation in both adults and children, particularly in the autologous setting, and it has progressively replaced BM as the source of HSCs. So far, the intrinsic molecular features of human primitive HSCs from different sources have not been investigated in comparative studies to unravel their variable reconstitution potential. Diverse strategies are currently used to disengage HSCs from the niche, promoting egress from BM to PB. Traditionally the growth factor granulocyte-colony stimulating factor (G-CSF) represents the gold standard agent to mobilize HSPCs for transplantation. Nevertheless, many other compounds have been tested to this regard. One of the most successful mobilizing agents is Plerixafor (AMD3100, Mozobil™), a bicyclam molecule that selectively and reversibly antagonizes the binding of stromal cell derived factor-1 (SDF-1), located on the surface of BM stromal cells and osteoclasts, to chemokine CXC-receptor-4 (CXCR4), located on the surface of HSPCs, with the subsequent mobilization in the PB. This drug, which was shown in preclinical combination studies with G-CSF to enhance mobilization compared to G-CSF alone, is currently approved by FDA and EMA "in combination with G-CSF to enhance the mobilization of HSCs into the peripheral blood for collection and autologous transplantation of patients affected by lymphoma or multiple myeloma whose cells mobilize poorly" We investigated functional and molecular hallmarks of human HSCs from different sources, i.e. BM and PB following mobilization by G-CSF and/or Plerixafor. We show that Plerixafor alone mobilizes preferentially long-term hematopoietic stem cells (LT-HSCs), defined as CD34+ CD38/low CD90+ CD45RA- CD49f+ cells and primitive populations of HSCs. These cells are able to provide stable long-term hematopoietic engraftment in NOD/SCID/IL2rγnull (NSG) mice, resulting in enriched scid-repopulating cell frequency, in comparison to other sources. The quiescence status of these cells correlates with the enriched scid-repopulating cell frequency. Noteworthy, the combined use of G-CSF and Plerixafor mobilizes a CD34+ population enriched in immature cells and with a lower engraftment capacity respect to cells mobilized by Plerixafor alone. Since the signaling provided by the interaction of SDF-1 with CXCR4, plays an essential role in maintaining HSC quiescence and regulating homing, we analyzed the CXCR4 expression. Interestingly, this analysis reveals that the proportion of CXCR4+ primitive cells was lower when using G-CSF combined to Plerixafor in respect to Plerixafor alone. These data indicate that the combination of the two mobilizing agents induce a higher amount of circulating CD34+ cells but containing a lower proportion of cells capable of homing to BM in NSG mice. . As a result, at a defined dose of transplanted CD34+ cells, less SRCs are observed when G-CSF is added to Plerixafor. Indeed, it is expected to observe also a rapid rescue of hematopoiesis in myeloablated subjects conferred by high amount of short-term progenitors. Insights into the transcriptional program reveal the molecular machinery underlying stemness features of cells derived from different sources, defining their specific functional properties. Noteworthy, CD34+ cells exposed to Plerixafor but still resident in the BM acquire an intermediate signature between steady-state and circulating cells, suggesting an effect of this agent on HSC function. From preliminary data, genes of Prostaglandin signaling are up-regulated in HSCs mobilized by Plerixafor, suggesting a role of this pathway. These data uncover unique HSCs properties shaped by their origin and illuminate the choice of different transplantation strategies accordingly to the clinical need. Disclosures No relevant conflicts of interest to declare.

Venous thromboembolic events, such as pulmonary embolism, deep venous thrombosis, and portal vein thrombosis, have been observed in adult thalassemia patients, mainly in beta-thalassemia intermedia. The clinical findings are consistent with the observation of several alterations that indicate a state of activation of the hemostatic mechanisms in thalassemias. These alterations have usually been related to high platelet counts due to splenectomy and/or liver dysfunction. In a retrospective study of a large cohort of adults with thalassemia, we found a larger prevalence of venous thromboembolic events in transfusion-independent patients with thalassemia intermedia (29%) than in regularly transfused patients with thalassemia major (2%); moreover, the higher prevalence occurred particularly in splenectomized thalassemia intermedia patients. More recently, a multicenter study involving 56 tertiary referral centers in 7 countries was planned to assess the magnitude of thrombotic risk in thalassemia patients. The total number of patients who had thrombotic events was 146 (1.65%) out of 8860, with a prevalence of 0.9% in thalassemia major and 4% in thalassemia intermedia. The highest prevalence was confirmed in splenectomized patients. The observation that thrombotic events are more frequent in beta-thalassemia patients who are not receiving regular transfusions (thalassemia intermedia or thalassemia major patients in less developed countries with limited transfusion resources) or in thalassemic patients who have undergone splenectomy strongly supports the procoagulant activity of circulating damaged red blood cells.

The clinical significance of the extent of a decrease in nocturnal blood pressure (BP) and the resulting classification of hypertensives as "dipper" (decrease in BP10% day BP) or "nondipper" (decrease in BP10% day BP) has been questioned recently. The aim of our study was to evaluate if the extent of a nocturnal BP decrease, established on the basis of a single 24-hour BP monitoring, is related to cardiovascular remodeling in essential hypertension. We enrolled 253 never-treated essential hypertensives (24-hour BPor = 140 and/or 90 mm Hg); for each patient we recorded 24-hour BP, left ventricular (LV) echocardiogram, Doppler transmitral flow velocities, and carotid-femoral pulse-wave velocities. A dipper BP profile was found in 161 patients, whereas 92 patients were nondippers. The 2 groups did not differ with regard to age, gender, body mass index, 24-hour and daytime BP, and 24-hour, daytime, and nighttime heart rate. All LV morphologic characteristics LV systolic and diastolic functional parameters, mitral Doppler-derived diastolic indexes, as well as carotid-femoral pulse-wave velocity, and aortic index distensibility were not significantly different between dippers and nondippers. The prevalence of LV hypertrophy and diastolic dysfunction was also similar between the 2 groups. The extent of a decrease in nocturnal BP did not correlate with any cardiovascular parameter. In conclusion, in never-treated hypertensives, the extent of a nocturnal BP decrease is not related to LV morpho-functional characteristics and aortic distensibility; therefore, the nondipping status established on the basis of a single 24-hour BP monitoring does not identify hypertensive patients with greater cardiovascular damage.

Introduction. During the last 10 years the cardiac deaths in adult thalassemia major (TM) patients are significantly reduced because of the availability of new oral iron chelators and the introduction of T2* cardiovascular magnetic resonance (CMR), which allows to evaluate the cardiac iron load. Deferasirox (DFX) is an oral iron chelator with established efficacy and safety. Based on myocardial T2* values, the risk of cardiac heart failure can be estimated prompting to intensify chelation therapy, if necessary. We would evaluate by T2* CMR the efficacy of DFX in preventing and removing cardiac and hepatic iron load and cardiac volume changes, along 5 years in adult TM patients. Methods. Twenty-three TM patients (9 males/14 women, mean age 36 ± 4 yrs) were included in this study. They repeated CMR to assess myocardial and liver iron load. Cardiac and hepatic iron load was assessed measuring myocardial T2* at 3 different time-points: basal value (t0), after a period of 2.5 years ± 6 months (t1) and after a period of five years (t2). During the follow-up period two patients became pregnant and they stopped chelation therapy with deferasirox for the duration of the pregnancy: they restarted oral iron chelation therapy with DFX after the delivery without any adverse event. Six patients underwent treatment with interferon and ribavirin to treat their hepatitis C: the dose of DFX was modulated based on increased iron intake during the treatment period. Results. Overall, the mean iron intake was 0.34±0.10 mg/Kg/day and the pre-transfusional mean hemoglobin (Hb) was 9.7±0.5 g/dl. The median ferritin value was 575 ng/ml (range 252-2530 ng/ml). The mean dosage of deferasirox at the beginning of the observation period was 24.4 ± 5.3 mg/kg/day; the mean dose of DFX along the 5-years follow-up was 25.3 ± 1.2 mg/kg/day. An initial reduction of mean ferritin levels was observed in the first 2.5 years of observation reaching a statistical significance only after 5 years (1055±563 ng/ml at baseline vs 960±568 ng/ml at t1 vs 751±627 ng/ml at t2, p=0.009 between t1 and t2). Myocardial T2* values changed progressively and increased significantly between t0 and t2 (t0: 27.15 ± 9.58 vs t2: 36.64 ± 6.68, p=0.0001). At baseline evaluation, a cardiac T2* value < 20 ms was detected in 6 patients (26 %): they showed an improvement of cardiac T2* values between t0 and t1, with normal T2* levels reached in all patients at t2. In the overall population a significant reduction of both end-diastolic and end-systolic left ventricular volumes (EDV, ESV) were detected between t0 and t2 (EDV, t0: 132±31 ml vs t2: 124±22 ml, p=0.033; ESV, t0: 48±14 ml vs t2: 41±10 ml, p=0.0007) (Table 1). A significant reduction in Liver Iron Concentration (LIC) was detected at t1 (5.36±3.58 mg/g dw at baseline vs 3.35±2.68 mg/g dw at t1, p=0.004). In patients with cardiac iron overload at baseline (n.6), mean cardiac T2* values doubled at t2 and mean LIC value is reduced of 29 %. Conclusions. Wecan conclude that DFX, at a personalized dose, progressively and significantly reduces myocardial and liver iron overload and it prevented the iron deposition along 5-years of continuous treatment. | | Overall patients | Patients with baseline T2* 10–20 ms | || | | t0 | t2 | P -value | t0 | t2 | P -value | | LVEF (%, mean ± SD) | 64 ± 6 | 66 ± 6 | ns | 61 ± 7 | 63 ± 6 | Ns | | EDV (ml, mean ± SD) (nv: 52-141 ml) | 132 ± 31 | 124 ± 22 | 0,033 | 139 ± 33 | 121 ± 22 | 0.03 | | ESV (ml, mean ± SD) (nv: 13-51 ml) | 48 ± 14 | 41 ± 10 | 0,0007 | 54 ± 14 | 44 ± 10 | 0.02 | Table 1. Left ventricular ejection fraction and cardiac volumes during the period of follow-up. LVEF: Left Ventricular Ejection Fraction; EDV: End-Diastolic left ventricular Volume; nv: normal value; ESV End-Systolic left ventricular Volume; SD: Standard Deviation; ns: not significative. Disclosures Cappellini: Novartis, Shire, Cellgene, Sanofi: Advisory board Other.

Background and aim. Transient elastography (TE) is a new, non-invasive and reproducible technique that measures liver stiffness (LSM). It has been demonstrated to be a reliable tool for assessing hepatic fibrosis and cirrhosis in patients with chronic liver disease (CLD). However, its role in patients with b- thalassemia has not been extensively investigated. The aim of the present study was to assess LSM and its possible correlation with iron overload in HCV positive patients with b- thalassemia major and intermedia. Methods. During a six-month period (from January to June 2007) 46 consecutive adults patients with b- thalassemia afferring to a single Italian Thalassemia Care Center in Milan, Italy, were enrolled in the study. Twenty-nine patients (Group I: 7 M and 23 F; mean age 31±SD 7.1 yrs; mean BMI 23.4±SD 3 Kg/m2) had b- thalassemia major and 17 intermedia (Group II: 10 M and 7 F; mean age 43±SD12.4 yrs; BMI 22 ±SD 3 Kg/m2). Sixteen patients (55%) in group I and two (12%) in group II were HCV RNA positive. All patients were examined by TE (FibroScan®; Echosens, Paris, France) and only the examinations with at least 10 validated measurements and a success rate of at least 60% were considered adequate. According to a previous study in CLD patients the considered TE cut off to diagnosing different stages of hepatic fibrosis were: >7.9 kPa for F≥2; >10.3 for ≥F3 and >11.9 for F=4. Twelve patients (all in group I) also had undergone liver biopsy. Necroinflammation and fibrosis were scored by METAVIR classification; liver iron concentration (LIC, mg/gr of liver dry weight) was measured on fresh tissue cores by atomic absorption spectrometry. Twenty-five patients underwent liver iron determination by T2* Magnetic Resonance Imaging (MRI) assessment. Results. In patients who underwent liver biopsy, LSM increased proportionally to the METAVIR stage and a significant positive correlation was observed between LSM and fibrosis stage (r=0.57, p= 0.039). Patients in group I had significantly higher values of mean LSM values (10.6± SD 9.3 kPa) and serum ferritin (SF) (1367±SD 1169 ng/mL) than those in group II (6.0± SD 3.3 kPa and 716±SD 472 ng/mL, respectively) (p=2) or severe fibrosis (F>=3) did not differ significantly according to HCV viremic status. Conclusion TE is a reliable non invasive technique to stage liver fibrosis in patients with b- thalassemia major. In these patients with concomitant HCV infection a significant or severe fibrosis was observed in about one third of the cases. Apart from fibrosis also serum necroinflammatory activity, GGT levels and SF levels may influence LSM values. The reliability of liver iron overload by T2* MRI evaluation remains still to be validated.

Successful gene therapy of inherited blood diseases relies on transplantation and engraftment of autologous genetically engineered hematopoietic stem/progenitor cells (HSPCs) in myeloablated patients. Hematopoietic reconstitution and clinical benefit are related to cell dose, although single disease features might play a role favoring selection of relevant progenitor populations. Gene therapy trials in young pediatric patients are performed isolating CD34+ cells from bone marrow (BM), while in adults mobilized peripheral blood stem cells (PBSC) should represent the favorite target. In the context of gene therapy for thalassemia, the choice of HSPC source is crucial since intrinsic characteristics of patients (splenomegaly and thrombophilia) dictate caution in the use of G-CSF as mobilization agent and prompt investigation of new agents. Moreover, adult thalassemic patients may possibly have a decreased BM stem cell reservoir, due to the BM suppression in response to multiple transfusions. A phase II clinical protocol exploring the use of Plerixafor as a single mobilizing agent in adult patients affected by transfusion dependent beta-thalassemia (EudraCT 2011-000973-30) started in 2012 at our hospital. Plerixafor selectively and reversibly antagonizes the binding of SDF-1 to its receptor CXCR4 with subsequent egress of HSCs to the peripheral blood. The availability of a new source of HSPCs, potentially superior in terms of CD34+ cell yield, transduction efficiency and biological features to steady-state BM, would have a significant impact on the feasibility and efficacy of gene therapy. Four subjects were enrolled and treated by subcutaneously administration of Plerixafor at the single dose of 0.24 mg/kg followed by leukoapheresis. Mobilization of CD34+ cells occurred very rapidly with a peak between 7 to 9 hrs. Three out of four patients achieved the minimal target cell dose (2 x 106 cells/kg) and no severe adverse event occurred. To the aim of engineering Plerixafor-mobilized CD34+ cells for gene therapy, we performed a comprehensive characterization of their biological, molecular and functional properties. In vivo reconstitution potential and lympho-myeloid differentiation were tested following transplantation in NSG mice and compared to those of PBSCs mobilized by G-CSF. Percentages of engrafted human cells in NSG mice transplanted with Plerixafor -PBSCs were about 2- to 5-fold higher than those found in mice transplanted with G-CSF PBSCs. On the same line, the SRC frequency, obtained by pooled engraftment data, was significantly higher (1 SRC out of 47.875 CD34+ cells vs.1 SRC out of 141.203 CD34+ cells). The phenotypic analysis of the frequency of primitive hematopoietic sub-populations revealed that Plerixafor mobilizes preferentially HSPCs and LT-HSPCs, with a percentage of CD34+ CD38-/low CD90+ CD45RA- CD49f+ cells higher than that found in G-CSF PBSCs. This result mirrors the enhanced number of SRCs found in the CD34+ cell population mobilized by Plerixafor. In order to further define the molecular features of HSPCs from different sources, we are studying signalling networks in response to specific cytokines by phospho-proteins analysis and gene expression by microarrays analysis. Our studies are focused on self-renewal, homing, engraftment and multilineage differentiation processes and bioinformatic analysis will reveal the molecular machinery underlying 'stemness' properties of Plerixafor mobilized cells. Disclosures: No relevant conflicts of interest to declare.

Abstract 586 Gene therapy of inherited blood diseases requires harvest of hematopoietic stem cells (HSCs) from patients and autologous transplantation of genetically modified cells. In order to achieve correction of the disease, high number of HSCs and previous conditioning of the host bone marrow (BM) are necessary. In the clinical application of gene therapy for thalassemic patients the choice of the HSC source is a crucial issue. On one side, the minimal target dose poses a challenge for the use of steady state BM since reinfusion of high numbers of beta globin gene modified CD34+ cells is probably necessary to gain sufficient correction of the genetic defect in order to achieve transfusion independency; on the other side, the disease related features and complications of thalassemic patients (i.e. splenomegaly and thrombophilia) dictate caution in the use of G-CSF as mobilizing agent. In April 2011 a clinical protocol exploring the use of Plerixafor (AMD3100) as single agent was started (“Plerixafor mobilized stem cells as source for gene therapy of beta-thalassemia”, acronym AMD-THAL, EudraCT2011-000973-30). Aims of the trial were to explore the ability of Plerixafor in inducing safe and effective stem cells mobilization in adult patients affected by beta-thalassemia, to characterize stem/progenitor cells mobilized from the BM and peripheral blood of treated subjects and to achieve gene transfer efficiency of mobilized CD34+ cells at a level comparable to that obtained using steady state BM. Four patients (01, 02, 03 and 04) were enrolled and already mobilized to date (August 2012). All patients are affected by transfusion dependent beta-thalassemia and aged 28 (01), 41 (02), 39 (03), 33 (04). Two are splenectomized (02 and 03); all subjects are regularly iron chelated with adequate organ function. Administration of Plerixafor subcutaneously as single agent and at the single dose of 0.24 mg/kg resulted in mobilization of CD34+ cells/mcl with a peak of 78 cells at 9 hrs (01), 70 cells at 7 hrs (02) and 69 cells at 8 hrs (03); suboptimal mobilization was observed in patient 04 (peak 18 at 8 hrs). Patient 03 received a second dose at 0.40 mg/kg 24 hrs after the first dose and underwent a second leucoapheretic procedure. Harvest by leukoapharesis resulted in procurement of the following CD34+ cells/kg: 1.84 × 106 (01) and 4.43 × 106 (02) with a unique leukoapheretic procedure, and 3.57 × 106 (03) with two leukoapheresis. No apheresis was performed for patient 04 because the minimum target of 20 CD34+ cells/mcl in peripheral blood was not reached. CD34+ cells selection through Clinimacs Miltenyi resulted in the following yield: 1.2 × 106 CD34+ cells/Kg, 65% recovery (01), 2.66 × 106 CD34+ cells/Kg, 60% recovery (02), 1.78 × 106 CD34+ cells/Kg, 50% recovery (03). No severe adverse event occurred. Recorded side effects were: grade 3 hypotension related to the apheretic procedure (01), mild grade 1 facial disestesia (02 and 04) and hyperleukocytosis (02: WBC from 13.6 to 42.6 × 103/mcl). In addition, steady state and Plerixafor primed BM aspirates were performed to analyze any modification in CD34+ concentration in the BM following Plerixafor administration. In fact, Plerixafor administration resulted in enrichment of CD34+ cells concentration in the BM. Purified CD34+ cells from leukoapheresis of the 4 treated patients were analyzed for their biological and functional properties, subpopulations composition and expression profile. In vivo reconstitution potential and lymphomyeloid differentiation of CD34+ cells were tested following transplantation in NSG mice. Experiments are ongoing but preliminary results indicate that cells mobilized by Plerixafor have a primitive phenotype with a high reconstitution potential and are efficiently transduced with a lentiviral based vector, named GLOBE, encoding for the human beta-globin (Roselli et al., 2010), thus being a suitable source of target cells for gene therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract 1155 Introduction. Anterior spinal artery thrombosis (AST) results in sudden, often irreversible sensory and motor loss, with partial or complete paraplegia. This is recognized as a complication of: prothrombotic states; right to left shunting; trauma and spinal surgery. Both venous and arterial thrombosis, including the central nervous system, are well-recognized risks in sickle cell disease (SCD) but AST has not been described. In thalassemia disorders (Thal), cerebral infarction, often silent, is increasingly recognized (Mussalam et al, Thromb Res, Aug 2012). However AST is not a recognized complication. Here we describe four cases of acute onset AST in adults (3 Thal major, 1 SCD), leading to severe neurological irreversible or partially reversible deficits. Patients and Methods. Patients described were attending outpatients for monitoring drawn from two large adults clinics in the UK (UCLH, Whittington, over 1500 hemoglobinopathy patients) and Italy (Ospedale Maggiore Policlinico, Milan, 400 patients). All events occurred within a 3 year period. Results. Presenting symptoms and Magnetic Resonance Imaging (MRI) findings are shown in the Table. All cases presented acutely with a sensory level on examination and with bladder dysfunction. Three presented with motor weakness of both lower limbs (1 case initially in one limb). Case 3 presented with a sensory deficit affecting the sacral region but no motor deficit. Partial reversibility occurred in cases 1 and 3. In Thal cases, no prodromal syndrome and no prior history of thrombosis were seen. MRI showed changes consistent with acute cord ischemia (delayed in case 4 until 5 days). Extra-medullary hematopoiesis was demonstrated by MRI only in case 2, but was not sufficient to cause cord compression. Cerebrospinal fluid analysis was normal in all cases. Concomitant risk factors such as autoimmune markers, active hepatitis, trauma, or demonstrable prothrombotic markers other than those expected in SCD or Thal were not detected. In none of the Thal cases was a thrombotic history elicited but the SCD patient had a history of retinal artery and renal artery thrombosis (1 year previously). Discussion. A case series of this serious complication has not been previously reported. The known prothrombotic tendency in SCD and Thal is the most likely risk factor as other risk factors were absent. Thal cases were transfusion dependent, where thrombosis risk is generally about a quarter of that in non transfusion dependent Thal (Cappellini, Blood Reviews, 265, 2012, S20–23). In the SCD case, the prior history of arterial thrombosis, consistent with an embolic etiology, led us to examine whether a patent foramen ovale (PFO) was present, which was confirmed by bubble jet studies. This was subsequently closed. In patients with a history of embolic thrombosis, the presence of PFO should be sought and closure considered. Two cases were treated with Methylprednisolone soon after presentation. Two cases were commenced on Aspirin 75mg once a day to limit extension and as secondary prevention. The use of thrombolytic agents such as tissue thromboplastin activator have not been described in AST. In conclusion, spinal cord ischemia should be considered when facing a Thal or SCD patient with acute neurological symptoms affecting legs or bladder. This may be more common in hemoglobin disorders than is apparent from the literature. Table . Case 1 Case 2 Case 3 Case 4 Age at presentation 24 37 32 27 Gender Male Male Female Female Diagnosis SCD Thal Major Thal Major Thal Major Presentation symptoms Bilateral leg weakness Paraparesis Paraplegia Urine retention Sacral paraesthesia Urine retention Lower limb pain paraesthesia, & weakness Urine retention MRI findings Fusiform cord swelling (T9, T10) Secondary to ischemia High T2 signal (T6 to conus) Swollen cord from T9 Low cord signal(T11 - T12) Intramedullary syrinx (T4/5 - T7/8) 1stScan: normal Repeat: High T2 posterior dorsal signal to T9 Conus medullary edema Extramedullary haematopoiesis No Yes but no cord compression No No Hb (g/L) 80 132 113 89 Plt (× 109/L) 451 191 474 438 WBC (× 109/L) 12.7 5.1 12.7 7.7 NRBCs (%) 0.39 ND ND 0.5 Transfusion No Yes Yes Yes Last transfusion None − 2 days − 19 days − 20 days Splenectomy No No Yes No Prior thrombosis Yes No No No Other risk factors * No No No No ND = not detected. * Autoimmune markers, active hepatitis C, prothrombotic screen - non contributory. Disclosures: Cappellini: Novartis Pharmaceuticals: Research Funding.

Abstract 2159 INTRODUCTION. The iron chelation therapy in thalassemia major (TM) patients has been demonstrated to reduce cardiac morbidity and mortality. Deferoxamine (DFO) significantly improved survival but its use has several limitations due to reduced compliance. The once-daily oral chelator deferasirox (DFX) has been shown to remove iron from the liver and from the heart, with improved compliance. The efficacy of DFX in removing cardiac iron has been shown and significant improvements in myocardial T2* in patients with b-thalassemia with T2* levels < 20 ms have been demonstrated. The introduction in the clinical practice of the T2* Cardiac Magnetic Resonance (CMR) evaluation permitted a rapid, direct and highly reproducible method to assess myocardial and hepatic iron overload; moreover this technique allows to evaluate cardiac morphology and function. We evaluated the removal of cardiac iron and the cardiac function parameters in TM patients treated with DFX, undergoing CMR,followed at Hereditary Anemia Centre, Department of Internal Medicine in Milan. METHODS. Forthy-one TM patients (22 females, 19 males, mean age 32 ± 6 yrs), treated with DFX, followed in a single centre, underwent a CMR, performed at baseline (T0), after a variable period of exposure to DFX (median: 12 months, range 4–48 months) and then after at least 6 months of treatment (T1) (median 12 months, range 6–19 months). CMR was performed at Cardiology and CMR Department “A. De Gasperis” at Niguarda Ca' Granda Hospital in Milan, using a 1.5 Tesla MR scanner (Avanto Siemens, Erlangen). The signal intensity of this region was measured for each image with the use of commercial software (CMRtools, Cardiovascular Imaging Solutions, London, UK). All T2* analyses were performed blinded to patient details. Ventricular volumes were analyzed with the same software and stroke volume and ejection fraction calculated from end diastolic and end systolic ventricular volumes. Patients were divided in two groups: group A (28 patients) with baseline T2* values > 20 ms and group B (12 patients) with baseline T2* between 10 and 20 ms. RESULTS. In the overall population, the mean deferasirox dose was 26±7 mg/kg/day; dose adjustment was based on iron intake and on liver and cardiac iron overload at CMR. Despite different durations of deferasirox exposure and different levels of myocardial iron overload, an average improvement of myocardial T2* from 27.4 ms to 29.8 ms was observed at T1 (P=0.004). A significant improvement in LVEF from 63.6 % to 66.5 % (P=0.013), indicative of improved cardiac function, was also observed. Similarly, both end-systolic and end-diastolic ventricular volume assessments (EDV, EDVI, ESV, ESVI) showed significant improvement at T1. No difference in median serum ferritin levels were found between group A and B. Myocardial T2* increased in both groups with DFX treatment, with a significant improvement observed in Group B patients (T2* at T0 was 15.7 ± 2.7 ms and at T1 19.6 ± 3.8 ms, P=0.003) (Table 1). LVEF significantly increased in both groups, from 65.7% to 68.0 % in Group A (P=0.01) and from 59.7% to 64.3% in Group B (P=0.04) (Table 1). In Group A, improvements in EDV, ESV and ESVI were also significant with deferasirox treatment; in Group B significant improvements were observed in ESV and ESVI (Table 1). CONCLUSION. These data confirm the effects of iron chelation with deferasirox in removing cardiac iron in beta TM patients with mild-to-moderate myocardial iron overload and preventing accumulation of myocardial iron in patients with normal baseline cardiac iron levels. Interestingly, improvements in cardiac function were observed both in patients with and without myocardial iron overload at baseline, however it was more significant in patients with normal T2* at baseline. Disclosures: No relevant conflicts of interest to declare.

Abstract 4275 Introduction. Although iron chelation therapy has markedly improved the survival, heart failure due to myocardial iron overload still remains the leading cause of morbidity and mortality in adult thalassemia major (TM) patients. T2* cardiovascular magnetic resonance (CMR) is a non invasive technique that provides rapid and direct assessment of myocardial iron content and its usefulness in monitoring iron chelation has been proved (Wood et al, Circulation 2009). AIM OF THE STUDY. To evaluate the efficacy of different iron-chelation therapies on removal of cardiac iron content assessed by CMR in adult TM patients. PATIENTS AND METHODS. Sixty-seven TM patients (27 males/40 women, mean age 35 ± 6 yrs) treated with different iron chelators underwent repeated cardiac CMR to assess myocardial iron load. Myocardial T2* was assessed at baseline (t0), after 6–14 months (t1) (according to clinical conditions and to T2* at baseline), and as second control (t2) after a mean of 32±7 months from baseline. CMR was performed at Cardiology and CMR Department “A. De Gasperis” at Niguarda Ca' Granda Hospital in Milan, using a 1.5 Tesla MR scanner (Avanto Siemens, Erlangen). Normal cardiac T2* was defined > 20 ms; T2* < 10 ms indicated severe cardiac siderosis and T2* between 10 and 20 ms moderate-to-mild cardiac siderosis. Each patient has to be chelated with the same iron chelation therapy at least for 1 year before the baseline CMR evaluation. Patients were divided based on chelation therapy in 4 groups: group A (n=36, 53.7 %) patients chelated with deferasirox (DFX, mean actual dose 27±7 mg/Kg/die), group B (n=15, 22.4 %) deferoxamine (DFO, actual mean dose 48±9 mg/kg for a median of 6 days/week), group C (n=12, 17,9 %) DFO (mean actual dose 46±7 mg/kg for a median of 4 days/week) plus deferiprone (DFP, mean actual dose 73±7 mg/kg/day) and group D (n=4,6 %) only DFP (mean actual dose 75±0 mg/kg/day). Statistical analysis was performed using a paired Student's t-test. RESULTS. Overall, the pre-transfusional mean hemoglobin (Hb) was 9.7±0.5 g/dl, the median ferritin value was 913 ng/ml (range 229–5934 ng/ml) and the mean iron intake was 0.41±0.12 mg/Kg/day. In the overall population, the baseline myocardial T2* was < 10 ms in 8 patients (11.9 %), between 10 and 20 ms in 22 patients (32.8 %) and ≥ 20 ms in 37 patients (55.2 %). At baseline evaluation, T2* < 10 ms was detected only in 1 patient (1/36: 2.77 %) in group A, in 4 patients (4/15: 26.6 %) in group B and in 3 patients (3/12: 25 %) in group C. In group D all patients showed a myocardial T2* above 20 ms at baseline. Progressive changes in T2* values were observed at t1 and t2 for all the groups. Ten patients (10/36: 27.8 %) in group A, 3 patients (3/15: 20 %) in group B, 3 patients (3/12: 25%) in group C, respectively, moved from an abnormal T2* (< 20 ms) to normal values, in 32±7 months (Table 1). T2* values at t2 improved significantly compared to baseline (p=0.0006) in patients treated with DFX (group A). In patients treated with combination therapy (DFO and DFP), T2* increased more rapidly in those with severe siderosis (T2* < 10 ms) (p=0.006). No significant changes in left ventricular ejection fraction (LVEF) values were observed in all groups of patients: only patients in group A with baseline cardiac T2* between 10 and 20 ms showed a slight improvement in LVEF (p=0.049). No statistically significant reduction in ferritin levels were associated with ameliorating myocardial T2* values. DISCUSSION AND CONCLUSIONS. Our data showed that compliance to chelation therapy at proper doses significantly improve myocardial T2* over 3-year treatment period. Continued treatment with deferasirox significantly increase myocardial T2* over time, showing its efficacy to remove iron from the heart. DFO and DFP combination therapy seems to ameliorate cardiac T2* more rapidly only in patients with T2* < 10 ms at baseline. Disclosures: No relevant conflicts of interest to declare.

Abstract 2011 Poster Board I-1033 Introduction The clinical severity of thalassemia intermedia depends on the degree of a/non-a-chains iimbalance. Among the molecular mechanisms responsible for thalassemia intermedia is the coinheritance of excessive a-globin gene production with a defective beta-globin gene. Materials and Methods: we describe an Italian family where thalassemia intermedia apparently segregates as a dominant form but it tourned out to be due to the coinheritance of a beta-globin mutation and a duplication of the alpha-globin gene cluster. The father (aged 51yrs) showed a well tolerated severe chronic hemolytic anemia (Hb 7.5-8.5 g/dL) not transfusion dependent, jaundice, splenomegaly and leg ulcers:The mother (aged 46yrs) has a completely normal hematological and hemoglobin pattern. Two sons (19 and 14 yrs) showed more severe clinical manifestations than the father. They underwent splenectomy at 12 and 13 years respectively without any benefit and afterwards they become transfusion dependent. Results: The hemoglobin analysis revealed that the father and the sons were heterozygotes for the beta mutation IVSI-110 G>A. MLPA analysis of the alpha-globin gene cluster disclosed a full duplication of the alpha-globin locus, spanning a 175 kb from the telomere to the 3'HVR downstream of the alpha-globin gene and including the upstream regulatory element HS-40. This rearrangement increases the number of the active alpha-globin genes in cis from 2 to 4.Surprisengly it was found in heterozygosis in both parents and in homozygosis in both sons. The hematological and molecular data of the family are reported in the table. In the father the 6 alpha-globin genes led to increased synthesis of alpha-chains; the coinheritance with a beta-thalassemia mutation causes a moderate/severe thalassemia intermedia phenotype. The presence of 8 alpha-globin genes in the sons raises further the degree of globin-chains imbalance and exacerbates the clinical phenotype. It is important to note that splenectomy worsened the clinical course.in the 2 homozygotes for the alpha duplication. Conclusions: Based also on previous experience we suggest that splenectomy in patients with a real excess of alphaa chain production is unconvenient since a large amount of circulating red cells with precipitated alpha chains may be responsible for increased hemolysis as well as increased risk of thrombosis This family moreover raises concerns regarding genetic counselling, suggesting that whenever one of the partner is affected by TI it is advisable a complete molecular screening of the couple in order to exclude any possible alpha gene defects interaction Disclosures: No relevant conflicts of interest to declare.

Concomitant HCV infection and iron overload are responsible for increased risk of chronic liver disease (CLD) in thalassemic patients. Liver biopsy remains so far the gold standard to assess histological activity and iron burden, although it is an invasive method poorly accepted by patients and it can not be repeated regularly. MRI ferriscan is a good tool recently introduced to estimate hepatic and cardiac iron load but it does not allow to evaluate the tissue liver damage. Transient Elastography (TE) is a new non-invasive device that measures liver stiffness (LSM) and assesses liver damage, namely fibrosis and cirrhosis. TE use in thalassemic patients is still limited, thus the aim of this study was to evaluate LSM by TE in a cohort of adults affected by Thalassemia Intermedia (TI) in order to assess the liver damage. Ninety consecutive TI patients followed at a single Italian tertiary Thalassemia Care Center in Milan were enrolled in this study. Eighteen patients (20%) were regularly transfused, 28 (31%) occasionally transfused, 44 (49%) never transfused. No one patient was on regular chelation treatment. Table 1 summarizes the demographic, clinical and laboratory features at time of TE evaluation. Males n° (%) 43 (47.8%) LDH U/l * 570±296 *Mean ± SD TE (FibroScan®) was performed according to Fraquelli et al. and was expressed in KPa. Only the examination with at least 10 validated measurements, a success rate greater than 60% and the interquartile range of all validated measurements lower than 30% of the mean value were considered reliable. TE cut-off to diagnose different stages of hepatic fibrosis was >7.9 kPa for F>2, >10.3 for F>3 and >11.9 for F>4 (cirrhosis). Forty-eight patients underwent T2* Magnetic Resonance Imaging (MRI) in order to estimate liver iron concentration (LIC). Forty-three patients (48%) had normal TE values (TE≤5.0 KPa), 35 (39%) had F>1 (5.02, 2 (2%) had F>3, 5 (6%) had F>4. Mean ± SD TE value was 6.0±2.8 KPa. A significant correlation (p Table 2 describes TE results based on transfusion regimen, ferritin levels and HCV-RNA positivity. TE (mean ± SD) Transfusion regimen Never 5.4±2.2 Occasionally 6.4±3.5 Regular 7.2±2.9 Ferritin levels ng/ml 1000 8.2±3.4 HCV-RNA Positive 7.7±2.9 Negative 5.9±2.8 Moreover, splenectomy (p=0.014) and cholecystectomy (p

Introduction: cardiac failure due to secondary iron overload remains the main cause of death in patients with b-Thalassemia Major (TM). Cardiovascular Magnetic Resonance Imaging (CMR) T2* technique is a new tool to assess myocardial iron concentration that allows to tailor the optimal iron chelation treatment for each patient. Aim of the study: to assess left ventricular function and myocardial iron overload in a cohort of TM patients, cared at Hereditary Anemia Center in Milan, Italy. Methods and Results: In 91 TM patients (33 males/58 females, mean age 32 ± 6 yrs) myocardial iron loading was assessed with the use of CMR T2* measurements (CMR Tools, Cardiovascular Imaging Solutions, London, UK). Left ventricular ejection fraction (LVEF) was also assessed with CMR. In the overall group hemoglobin levels were 9.0 ± 1.0 g/dl; the mean serum ferritin levels and iron intake during the six months before CMR evaluation were 1507 ± 1884 ng/ml and 0.34 ± 0.08 mg/kg/die respectively. T2* was significantly different between females and males (24 ± 11 and 32 ± 12 ms, respectively; p < 0.0001), with significant differences in diabetes mellitus prevalence (17% vs 8%, p 20 ms). LVEF was significantly different between females and males (35% vs 57%, p Conclusions: CMR cardiac function and T2* assessment allow to detect pre-symptomatic cardiac iron overload. Females are more at risk for severe iron overload and left ventricular impairment. The prevalence of diabetes mellitus and compliance to chelation therapy could be relevant in explaining the gender differences. Clinical parameters and T2* values in men and women with thalassemia major Men p Women SD: standard deviation Number of patients (n. of pts) 33 - 58 Age ± SD (years) 33 ± 6 ns 32 ± 6 Hemoglobin levels ± SD (g/dl) 9.0 ± 1.7 ns 9.0 ± 0.8 Ferritin levels ± SD (ng/ml) 964 ± 891 ns 1821 ± 2216 Iron intake ± SD (mg/Kg/die) 0.30 ± 0.07 ns 0.36 ± 0.09 Mean T2* value ± SD (ms) 32 ± 12 20 ms (n. of pts) 25 - 35 T2*< 10 ms (n. of pts) plus LVEF≤ 57 % 0/0 (0%) - 6/7 (85.7%) T2* between 10.1 and 14 ms (n. of pts) plus LVEF≤ 57 % 1/1 (100%) - 3/8 (37.5%) T2* between 14.1 and 20 ms (n. of pts) plus LVEF≤ 57 % 3/7 (42.8%) - 1/8 (12.5%) T2* > 20 ms (n. of pts) plus LVEF≤ 57 % 7/25 (28%) - 5/35 (14.3%)

Gaucher disease (GD) type 1 is the most common lysosomal storage disease and the most common genetic disorder among Ashkenazi Jews. The majority of patients with GD present with unexplained splenomegaly and/or thrombocytopenia, and the disorder often affects children; consequently, haematologists and paediatricians are ideally placed to diagnose this condition. Prompt management of GD type 1 using enzyme-replacement therapy or substrate reduction therapy can reduce the risk of developing long-term GD complications and reverse many of the initial signs/symptoms, thereby improving both quality and duration of life. Treatment is most effective when initiated early; consequently, a prompt diagnosis is essential. Despite this, the average time to diagnosis following the onset of clinical symptoms is 4 years. Reasons for the delay include the heterogeneous nature of the disease, together with a lack of awareness of rare haematological disorders and the benefits of early treatment. Indeed, studies show that only 20% of haematologists consider GD type 1 in their differential diagnosis for patients presenting with splenomegaly and/or thrombocytopenia. To help raise awareness of GD, reduce the diagnostic delay and prevent unnecessary tissue biopsies, simple diagnostic algorithms and screening tools have been developed and validated, both in adults and in children.