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Luspatercept has recently been approved for the treatment of beta-thalassemia and its use in clinical practice has been increasing. As it is the first erythroid maturation drug available for this diagnosis, the expertise about its use is still limited. To address this point, and to promote awareness and guide the clinical use of luspatercept in beta-thalassemia, this paper was developed as a consensus by experts from the Italian Society of Thalassemia and Hemoglobinopathies (SITE). After a brief presentation of the core features of luspatercept, a comprehensive set of questions is addressed, covering relevant aspects for the practical management of this new therapeutic option.

β-thalassemia is an inherited disease causing an impaired hemoglobin production, eventually leading to a severe chronic anemia. Resolutive treatments are still limited to a small number of patients and blood transfusions still represent the standard of care. In this scenario, luspatercept is the first approved drug able to significantly modify the disease phenotype. Developed as a fusion protein, it binds TGF-β ligands, contributing to a reduction of ineffective erythropoiesis. As shown by clinical trials in thalassemia, this effect determines an increase in mean hemoglobin levels and/or a decrease in transfusion burden. While some potential indications are still being evaluated in trials, luspatercept has recently entered the clinical practice for transfusion-dependent thalassemia patients.

β-thalassaemia is a rare genetic condition caused by mutations in the β-globin gene that result in severe iron-loading anaemia, maintained by a detrimental state of ineffective erythropoiesis (IE). The role of multiple mechanisms involved in the pathophysiology of the disease has been recently unravelled. The unbalanced production of α-globin is a major source of oxidative stress and membrane damage in red blood cells (RBC). In addition, IE is tightly linked to iron metabolism dysregulation, and the relevance of new players of this pathway, i.e., hepcidin, erythroferrone, matriptase-2, among others, has emerged. Advances have been made in understanding the balance between proliferation and maturation of erythroid precursors and the role of specific factors in this process, such as members of the TGF-β superfamily, and their downstream effectors, or the transcription factor GATA1. The increasing understanding of IE allowed for the development of a broad set of potential therapeutic options beyond the current standard of care. Many candidates of disease-modifying drugs are currently under clinical investigation, targeting the regulation of iron metabolism, the production of foetal haemoglobin, the maturation process, or the energetic balance and membrane stability of RBC. Overall, they provide tools and evidence for multiple and synergistic approaches that are effectively moving clinical research in β-thalassaemia from bench to bedside.

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

Iron overload and chronic viral hepatitis underlie the increased incidence of hepatocellular carcinoma in patients with Beta thalassemia, as recently emerged due to the prolonged survival. Conversely, there are very few studies examining the relationship between thalassemia and other malignancies. Although some Authors have indicated an increase in incidence of hematologic and solid tumours such as thyroid cancer and renal cell carcinoma, there are not enough data to make a conclusion. Moreover, there is even less data on a possible relationship between other Hemoglobinopathies and tumors risk. Seven Italian Specialized Centres for the treatment of Hemoglobinopathies, with the patronage of the "Società Italiana Talassemie ed Emoglobinopatie" (SITE), evaluated the incidence of malignant neoplasms in Hemoglobinopathies, their site and features. Each Center considered all consecutive patients of its own historical series; the cohort included 4104 patients (48% males), followed between 1970 and 2021 (Figure 1A), for a total of 143255 person-years of observation. The most common diagnosis was Transfusion-Dependent Beta Thalassemia (TDT) with 2122 subjects (51.7%), followed by Non-Transfusion-Dependent Beta Thalassemia (NTDT, 789 subjects, 19.2%), Sickle Cell Disease (SCD, 787, 19.2%), Hemoglobin H Disease (HbH, 375, 9.1%) and Other (34, 0.8%). Two-hundred-sixty-two patients had undergone bone marrow transplantation (BMT). A total of 157 diagnoses of cancer (153 patients, 52% males) was reported during the period of observation, corresponding to a prevalence of 3.8%, with the mean age at the diagnosis of 47±13 years. Hepatocellular carcinoma (HCC) was the most frequent site (62 cases) in both sexes (males 50%, females 32%) contributing to 41% of total number of cases, excluding non-melanoma skin cancer, and in all type of hemoglobinopathies (except HbH). No significant differences were found in the age of diagnosis of HCC stratifying for gender (males 48±14 years, females 51±9 years) and types of Hemoglobinopathies (TD 48±9 years, NTDT 51±10 years, SCD 50±14 years). After HCC, hematologic tumours (11), kidney (6), and lung (6) were the most frequent sites in men, accounting for 29% of all male cancers. For women, the most common incident cancers were breast (9), thyroid (8) and kidney (7), these representing 33% of all female cancer cases (Figure 1B). The first diagnoses of cancer dated back to the 1980s and their number sharply increased after the 2000s with a peak in the five-year period 2008-2012 and a (not significant) reduction in recent years. A similar trend may be noted considering HCC alone but in this specific case, the reduction reaches the level of significance (Figure 1C). The overall age-adjusted incidence rate of cancer in Hemoglobinopathies was estimated to be 391 cases/100000 person-years (95% C.I. 290-650) which was not statistically different from that of the Italian general population (632 cases/100000 person-years), both considering Hemoglobinopathies in total and each subgroup of patients. However, age-adjusted incidence rate of HCC was more than 7 times lower in general population (22 cases/100000 person-years) than in patients with Hemoglobinopathies (153 cases/100000 person-years, 95% C.I. 96-222, p Although preliminary, these findings provide novel insights into the relationship between cancer and Hemoglobinopathies, suggesting that the overall cancer risk is not increased in these patients. HCC is confirmed as the most frequent tumor, especially in patients with Beta Thalassemia (TD and NTDT), but advances in chelation, with renewed attention to hepatic iron, and the new drugs that have led to the eradication of hepatitis C even in these patients, may explain the recent reduction in the number of diagnoses that we have reported in this study. Acknowledgment. We would like to thank ALT (Associazione per la Lotta alla Talassemia R.Vullo - Ferrara). Figure 1 Figure 1. Disclosures Origa: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; BlueBird Bio: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria. Longo: Bristol Myers Squibb: Honoraria; BlueBird Bio: Honoraria. Pinto: BlueBird Bio: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Quarta: Sanofi - Genzyme: Membership on an entity's Board of Directors or advisory committees, Other: collaboration relationships for Advisory boards, Webinar events, editorial projects; Speaker at conferences; Blue Bird Bio: Other: collaboration relationships for Advisory boards, Webinar events, editorial projects; Celgene: Other: collaboration relationships for Advisory boards, Webinar events, editorial projects; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: collaboration relationships for Advisory boards, Webinar events, editorial projects; Speaker at conferences; Takeda: Other: collaboration relationships for Advisory boards, Webinar events, editorial projects; speaker at conferences; Bristol Meyer Squibb: Membership on an entity's Board of Directors or advisory committees, Other: Speaker at conferences. Casale: Novartis Farma SpA: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maggio: Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Perrotta: Novartis Farma SpA: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Blue Bird Bio: Honoraria, Membership on an entity's Board of Directors or advisory committees. Forni: Celgene: Membership on an entity's Board of Directors or advisory committees; Bluebirdbio: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Introduction: Ineffective erythropoiesis (IE) is a crucial hallmark of β-Thalassemia (β-Thal) and sets the goal for treatment of both Transfusion-Dependent (TDT) and Non Transfusion-Dependent Thalassemia (NTDT) patients. The Growth Differentiation Factor (GDF) group has a relevant role in the molecular regulation of IE. Specifically, GDF11 contributes to the inhibition of RBC maturation and it is targeted by activin traps, such as luspatercept. However, its role is still debated; i.e., in a mouse model of β-Thal, the absence of GDF11 alone is not sufficient to mitigate IE (Guerra A et al., Blood, 2019). GDF15 increases from early until late phases of erythroid differentiation and negatively regulates erythroid cell development in-vitro, modulating maturation and apoptosis. (Ranjbaran R et a.l, Exp Cell Res, 2020). A few clinical studies found elevated serum GDF15 levels in β-Thal (Tanno T et al., Nat Med, 2007; Huang Y et al., Int J Med Sci, 2019), but data are sparse and a clear correlation with the severity of the pathology is still missing. Methods: We run an observational study at our institution. At routine checks, patients were asked to consent to a specific blood sample for GDF15, whereas hemoglobin (Hb), serum erythropoietin (Epo), ferritin (Ftn), iron, and transferrin saturation (TSat) were measured as part of clinical practice. In a small subset of patients, a consent for serial sampling was added. Serum GDF15 was measured by ELISA (DuoSet DY957, R&D Systems). Demographics were collected from clinical records. Statistical analysis was performed using Statistica 10 (Statsoft). Results: GDF15 levels were measured in 458 individuals: 267 TDT, 77 NTDT, 45 β-Thal trait carriers (BTC), and 69 healthy (H) subjects. Median (IQR) levels of GDF15 were significantly different among diagnoses (P Discussion: GDF15 levels correlated with the severity of β-Thal phenotype, showing a 26-fold (TDT), a 6-fold (NTDT) and a 2-fold (BTC) increase compared to controls. In TDT patients, higher GDF15 levels correlated with lower Hb and higher Epo, which are typically observed as a result of IE in thalassemia. In addition, GDF15 correlated with markers of altered iron metabolism, such as TSat and serum iron. In individual patients, GDF15 showed strong and consistent variation with treatment. GDF15 was also associated with quantitative markers of disease in NTDT and BTC patients. This is to our knowledge the larger sample of patients carrying β-thal mutations in which GDF15 levels were measured and correlated with the severity of the disease. These results show that GDF15 may be a suitable and useful quantitative marker of IE. Figure 1 Figure 1. Disclosures Piga: Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Acceleron: Research Funding. Longo: Bristol Myers Squibb: Honoraria; BlueBird Bio: Honoraria.

Introduction: The majority of published data about Deferasirox (DFX) are obtained on the dispersible formulation (DFX DT). A film-coated formulation (DFX FCT) with higher bioavailability is now approved and used in most countries, in tablets containing 30% less active principle. However, only few reports exist about long-term effects of DFX FCT and no independent comparison of the two formulations is available. Hypothesizing a bioavailability even higher than described, we conducted a study comparing PK/PD and safety of both formulations in a cohort of patients with congenital anemias. Methods: Clinical records of patients treated at our referral Centre in the period from 2011 to March 2019 were reviewed, identifying patients that performed a 24-hours PK profile for both DFX formulations and completed at least 1 year of continuous treatment for each one. Data about PK (drug dose, maximum plasma concentration (Cmax), Area Under Curve (AUC)), PD (serum ferritin (FTN), Liver Iron Concentration (LIC) by biosusceptometry) and safety (ALT, AST, creatinine, eGFR, protein/creatinine ratio) were collected. Acute Kidney Injury (AKI) was defined according to guidelines as previously described (Bird, 2018). Estimated compliance was also considered. Serum DFX concentrations were measured by HPLC (De Francia et al, 2012). For PD and safety analysis, study time was defined as the period between two subsequent LIC measures; timeframes closest to the beginning of the therapy were selected. Wilcoxon matched-pairs test, Chi-square test and Spearman r index were used to analyze differences and correlations between DFX DT and DFX FCT treatments, with a significance threshold of 0.01. Results: Seventy-four patients diagnosed of β-thalassemia major (61), β-thalassemia intermedia (11), Diamond-Blackfan anemia (1) and Stomatocytosis (1) met inclusion criteria for PK analysis. DFX was tested at a mean dose of 26.1 (DT) and 15.5 mg/kg (FCT; -41%), with a Cmax of 71.0 and 101.5 μMol/L (+43%) and an AUC of 855.0 and 1301.3 (+52%), respectively. Cmax and AUC correlated significantly with dose for both formulation, but highly for FCT (r(dose-Cmax) = 0.41 and 0.58, r(dose-AUC) = 0.37 and 0.52 for DT and FCT, respectively), suggesting a stronger association between administered dose and systemic bioavailability (Fig.1). Forty-nine out of 74 patients, with at least 1-year follow-up for each formulation, were further analyzed for PD and safety. Actual administered dose was adjusted if indicated, but no significant differences were observed from doses tested at PK. LIC and FTN levels did not change significantly during treatments; as expected, the rate of iron overload variation (ΔLIC) was proportional to the iron overload at baseline for both formulations, but this correlation was stronger for FCT (Tab.1). Mean compliance was high with both formulations but improved significantly with DFX FCT (DT=89.3%; FCT=91.6%; p=0.001). Liver enzymes, creatinine, eGFR and protein/creatinine ratio at the end of treatments did not differ from baseline. AKI episodes were experienced in both formulations at rates comparable or higher than described (Diaz-Garcia, 2014 and Bird, 2018), but significantly lower with DFX FCT (DT=1.47; FCT=0.49 episodes/patient/year; p=0.0083). Overall kidney function was maintained at the end of the study for both treatments. Discussion: This study presents a direct and extended comparison of DFX formulations from real-word experience. DFX FCT, even at doses lower than recommended (-41% instead of -30%) was superior to DT in terms of PK and comparable in overall PD after 1 year of observation. Here described for the first time, DFX FCT also resulted to be superior in terms of scalability, i.e. it showed better correspondence between administered dose and serum concentration and between the baseline iron burden and the rate of iron elimination during treatment. Acute renal impairment was confirmed to be a major complication of DFX treatment, however AKI prevalence was significantly lower for FCT and, consistently, a significant dose-dependent association was observed for FCT, but not DT. Altogether, our results suggest that FCT formulation is more bioavailable than expected and could improve chronic treatment with DFX, underlining the critical role of personalized dose tailoring and monitoring to maximize the delicate balance between effectiveness and safety . Disclosures Longo: Blue Bird Bio: Consultancy. Piga:Celgene Corporation: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Acceleron Pharma: Research Funding.

Background Medulloblastoma (MB) is the most common malignant childhood brain tumor with the propensity to disseminate at an early stage, and is associated with high morbidity. New treatment strategies are needed to improve cure rates and to reduce life-long cognitive and functional deficits associated with current therapies. Extracellular Vesicles (EVs) are important players in cell-to-cell communication in health and diseases. A clearer understanding of cell-to-cell communication in tumors can be achieved by studying EV secretion in medullospheres. This can reveal subtle modifications induced by the passage from adherent to non-adherent growth, as spheres may account for the adaptation of tumor cells to the mutated environment. Methods Formation of medullospheres from MB cell lines stabilized in adherent conditions was obtained through culture conditioning based on low attachment flasks and specialized medium. EVs collected by ultracentrifugation, in adherent conditions and as spheres, were subjected to electron microscopy, NanoSight measurements and proteomics. Results Interestingly, iron carrier proteins were only found in EVs shed by CSC-enriched tumor cell population of spheres. We used iron chelators when culturing MB cell lines as spheres. Iron chelators induced a decrease in number/size of spheres and in stem cell populations able to initiate in vitro spheres formation. Conclusions This work suggests a not yet identified role of iron metabolism in MB progression and invasion and opens the possibility to use chelators as adjuvants in anti-tumoral chemotherapy.