Search

Patients with beta-thalassemia show 5-fold increase in agestandardized lethality due to SARS-CoV-2 infection, representing a high-risk population compared with age- and sex-matched healthy subjects.(1) Vaccination against SARS-CoV-2 is crucial to reduce mortality and morbidity of frail patients.(2) Up to now, limited data have been available on the responses of patients with beta-thalassemia immunized with anti-SARS-CoV-2 mRNA vaccines.(3)

Pulmonary arterial hypertension (PAH) is a life-threatening complication of β-thalassemia, especially in untransfused patients with thalassemia intermedia. Pinto and colleagues analyzed the outcome of 24 patients with PAH documented by right heart catheterization, and they report that with a median follow-up of 4 years, 54% died, most of which deaths were attributable to PAH. Patients who receive treatment that reduce their pulmonary pressures have improved survival, suggesting that improvement in monitoring and treatment are critical imperatives for these patients.

Background: Children with sickle cell disease (SCD) are at increased risk of cerebrovascular events such as stroke, silent infarcts and neurocognitive impairment. The role of Transcranial Doppler ultrasound scanning (TCD) to identify sickle cell anemia (SCA) children at high risk of stroke is well established. Adam et al in 1998 recommended those with abnormal cerebrovascular flow velocities are offered prophylactic blood transfusion therapy to prevent stroke between ages 2 to 16 years. Therefore, TCD screening for stroke prevention in now is a mandatory in all guidelines for the management of children with SCA. However, there is still no uniform implementation of the program globally and in European countries (Rees 2016). Moreover, the information available on the quality of the TCD screening is limited to educational experiences in a few countries (Inusa 2019) but to evaluation of stroke prevention programs has been performed in Europe. As more disease modifying therapies become available for children with SCD, it is mandatory to know TCD availability, screening practices, and real-world data on stroke prevention in Europe. EuroBloodNet is the European Reference Network (ERN) on rare Hematological Disorders, one of the 24 ERN established by the European Union to improve care of patients with rare disorders in Europe. EuroBloodNet's main goal is to improve the healthcare and overall quality of life of patients with a Rare Hematological Disease by: 1) Improving equal access to highly specialized healthcare delivery through a multidisciplinary patient centered approach; 2) Enhancing the best practices in prevention, diagnosis and safe clinical care across Europe based on promotion of evidence based guidelines. We wanted to assess the state of the art of TCD screening and stroke prevention programs in European Expert Centers. Methods: An online survey was developed by SCD experts in 5 European countries and sent to all Representatives of the Health Care Providers (HCP) and the Red Cell Disorder representatives in each HCP within the EuroBloodNet network, as well as to National Representatives of Scientific Societies within European Countries. Items in the survey are listed in Table 1. Results 81 hematologists or pediatricians from 77 centers in 16 European countries responded to the survey (14/16 in Western Europe); 39/77 (51%) were EuroBloodNet Expert centers, 14/77 (18%) were under evaluation as being recognized; 67/77 specified their expertise: 24% were pediatric, 3% adult, 58% both; 12 centers had >200 patients in the age range 1-16 years. 36% Physicians reported not having a dedicated TCD/TCDi service for children with SCD so exams had to be performed by cardiologists (10%), general radiologists (28%), TCD is not performed (31%), or patients have to be sent in another center (31%). 74% reported requesting annual TCD for their patients, but to the question "What percentage of your patients receives annual TCD" only 28% confirmed that all their patients managed to actually receive annual TCD, due to lack of trained staff (43%), lack of TCD instruments (11%), refusal of patients due to logistical difficulties (22%) (i.e TCD in another city), lack of funds for dedicated staff or equipment (11%), or other reasons. Only 74% of hematologists were aware of the protocol in use at their center by the staff performing TCD; the STOP criteria were applied by 64% of the physicians, mainly due non evaluation of the Internal Carotid Artery. The extracranial part of the carotid artery was evaluated only in 30% of the respondents. In case of abnormal/conditional TCD results, the approach varies and is not uniform across centers. Conclusions Our data show that less than 30% of children with SCD followed in European Centers receive annual TCD according to recognized guidelines. This first multinational European survey allowed the identification of issues related to the lack of access to TCD, lack of trained staff, lack of adequate protocols for implementation of TCD and treatment afterwards, which will need to be addressed through dedicated actions. Figure 1 Figure 1. Disclosures Casale: Novartis Farma SpA: Honoraria, Membership on an entity's Board of Directors or advisory committees. Mañú Pereira: Novartis: Research Funding; Agios Pharmaceuticals: Research Funding. de Montalembert: Vertex: Membership on an entity's Board of Directors or advisory committees; BlueBirdBio: Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Colombatti: Global Blood Therapeutics: Research Funding; Addmedica: Consultancy; Forma Therapeutics: Consultancy; Novartis: Consultancy; NovoNordisk: Consultancy; BlueBirdBio: Consultancy; Global Blood Therapeutics: Consultancy; BlueBirdBio: 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 Allogenic CAR-T cell therapies for cancer provide a new option to reduce barriers faced by autologous cell therapies, but several challenges remain. One challenge is the risk of graft versus host disease (GvHD) caused by the infused T cells. A potential solution is the use of a subset of gamma delta (γδ) CAR-T cells whose T cell receptors (TCRs) recognize invariant antigens rather than hypervariable MHC molecules. Here we describe an off-the-shelf, induced pluripotent stem cell (iPSC)-derived γδ CAR-T (γδ CAR-iT) for treatment of cancer and a process for deriving such cells. Methods T cell-derived iPSCs (TiPSC) are generated by reprogramming γδ T cells to yield pluripotent stem cells. For proof-of-concept studies, TiPSC were engineered using CRISPR gene editing to deliver a CD19 CAR transgene. TiPSC are then subjected to a two-stage differentiation process. First, TiPSC are differentiated into CD34-expressing hematopoietic progenitor cells (HPCs). HPCs are then exposed to a feeder-free differentiation process that results in uniform γδ CAR-iT cells. The purity and identity of γδ CAR-iT cells were assessed by flow cytometry and the ability of γδ CAR-iT cells to respond to homeostatic growth factors was determined by intracellular staining of phosphorylated signaling proteins and mRNA transcriptome analysis. Cytokine production by CAR-iT cells was measured by immunoassays following stimulation of the CAR. Tumor cell killing by γδ CAR-iT cells was performed using IncuCyte cytotoxicity assays. In vivo control of tumors by γδ CAR-iT in immunodeficient mice was determined using a NALM-6 B cell lymphoblastic xenograft model. Results A research-grade γδ TiPSC line was used to develop an iT differentiation process. This γδ TiPSC line was engineered to express a CD19 CAR molecule and then subjected to the differentiation process after which >95% of cells were CD3 + γδ TCR + CAR + iT cells. These γδ CAR-iT cells responded to IL-2 and IL-15. STAT5 phosphorylation levels were similar but STAT3 phosphorylation levels were greater in response to IL-15 compared to IL-2 at equimolar concentrations of cytokine. IL-2 and IL-15 elicited qualitatively similar transcriptional responses, but the magnitude of cytokine-induced gene expression was generally greater in IL-15-treated cells. Upon activation, γδ CAR-iT cells released markedly less IFN-γ and other inflammatory cytokines than conventional blood-derived ab CAR-T cells. In an IncuCyte serial killing assay, γδ CAR-iT cells exhibited sustained killing of NALM-6 tumor cells for at least one week in the presence of IL-15. In vivo, γδ CAR-iT cells caused a significant reduction in NALM-6 tumor burden with a single dose of γδ CAR-iT resulting in >95% tumor growth inhibition. To establish an efficient method for derivation of clinical grade γδ TiPSC lines, we investigated methods to isolate, expand, and reprogram human γδ T cells. When γδ T cells were expanded by exposure to the chemical zoledronic acid (zoledronate) and IL-2, we found a large disparity between donors; some donors exhibit robust expansion while others are seemingly resistant to zoledronate. In order to enhance γδ T cell expansion we screened dozens of activation conditions and eventually established a universal activation protocol that can elicit robust expansion of γδ T cells from all donors tested. When expanded γδ T cells were subjected to reprogramming conditions, dozens to hundreds of individual TiPSC colonies were obtained from each donor. The identity of the rearranged γδ TCR locus was confirmed using molecular assays. New γδ TiPSC lines were engineered with a CD19 CAR molecule and killing activity was confirmed in an in vitro serial killing assay. Conclusions γδ CAR-iT cells provide a new opportunity to treat cancers with an off-the-shelf universal T cell platform without the risk for GvHD. γδ CAR-iT cells are readily manufacturable, and we have derived an end-to-end process that enables new TiPSC line reprogramming, genetic modification of TiPSC lines, and feeder-free differentiation. γδ CAR-iT cells exhibit potent antigen-specific tumor killing and they release less inflammatory cytokine than conventional CAR-T cells, potentially reducing the risk for cytokine-mediated toxicities. We believe that this off-the-shelf platform will enable safer and more accessible allogenic cell therapies for hematologic and solid cancers. Disclosures Wallet: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Nishimura: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Del Casale: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Lebid: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Salantes: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Santostefano: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Bucher: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Mendonca: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Beqiri: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Thompson: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Morse: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Millar Quinn: Century Therapeutics: Current Employment, Current holder of stock options in a privately-held company. Borges: Century Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Introduction Asplenic patients are at high risk of potentially fatal invasive infections, such as sepsis, meningitis, and pneumonia. It has been shown that infection from influenza viruses can precede or increase the risk of bacterial infection and of serious complications of the underlying disease. International and national guidelines recommend annual influenza vaccination in asplenic subjects. Following the Covid-19 pandemic, the major government and medical-scientific institutions in the US and in Europe have been planning how to contain infection during the 2020-2021 influenza season. Extending influenza vaccination is the safest and most effective way to reduce the circulation of influenza virus and to promote the correct diagnosis and management of suspected cases of SARS-CoV-2. Influenza vaccination also reduces complications associated with the underlying disease and visits to Emergency Units. Our study aims to evaluate influenza vaccination in a large population of asplenic patients and explore the main causes for non-vaccination to identify critical areas for improvement in the vaccination programme in these at-risk patients for the 2020-2021 influenza season. Methods The Italian Network of Asplenia (INA) is made up of 88 doctors working in 50 clinical centers in 27 cities and 16 of the 20 regions of Italy. It aims to build a large, prospective cohort of asplenic patients throughout Italy through which to study the interaction between asplenia and its associated underlying conditions, collecting precise, accurate data also in cases of rarer diseases. The study also aims to improve the quality of healthcare for this at-risk population. The number of patients enrolled in the Network who had had at least one dose of influenza vaccine at the time of diagnosis of asplenia was retrieved from the INA database. All participating centers were asked to answer a questionnaire to report the main obstacles for influenza vaccination. Results At 1st August 2020, 1,670 patients had been enrolled in the INA (783 females; 887 males). All underlying causes of asplenia are shown in Table 1. Only 466 (28%) patients had had at least one influenza vaccination, while 1,204 (72%) had never been vaccinated since diagnosis of asplenia. Thirty-five (70%) of the 50 centers answered the questionnaire. Main causes of non-vaccination were physicians' ambivalence concerning vaccination and patients' inadequate awareness or logistical problems. Conclusions These data show very low seasonal influenza vaccination cover even though asplenic patients are considered at-risk of complications associated with infection from influenza viruses. Since the 2020-2021 influenza season could see influenza viruses in circulation with SARS-CoV-2, influenza vaccination must be expanded as widely as possible, in particular to subjects of all ages at high risk. These results reveal important areas of concern in the management of asplenic patients and the need to improve the quality of information to physicians and patients alike. The INA co-ordinating center will launch a campaign to provide information and organize ad hoc meetings to widen influenza vaccination coverage in asplenic patients and reduce the pressure on the national health service during the next influenza season. Disclosures Forni: Novartis: Membership on an entity's Board of Directors or advisory committees. Colombatti:Addmedica: 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; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Giona:Sanofi Genzyme: Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Novartis: Research Funding. Ferrero:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Research Funding, Speakers Bureau; Servier: Speakers Bureau. Perrotta:Novartis: Consultancy, Research Funding, Speakers Bureau. Casale:Novartis: Membership on an entity's Board of Directors or advisory committees.

The outcome of high-risk (HR) Acute Lymphoblastic Leukemia (ALL) patients enrolled in AIEOP-BFM ALL 2000 study (NCT00613457) in Italy is described. Overall, 1999 Philadelphia negative ALL patients entered the study. HR criteria were: minimal residual disease (MRD) levels ≥10-3 at day 78 (HR-MRD), no complete remission (no-CR) at day 33, t(4;11) translocation, Prednisone Poor Response (PPR). Treatment (2 years) included protocol I, 3 polychemotherapy blocks, delayed intensification (protocol IIx2 or IIIx3), cranial radiotherapy, maintenance. 312 HR patients (15.6% of the total) had 5-year event-free survival (EFS) and overall survival (OS) of 58.9%(SE 2.8) and 68.9%(2.6). In hierarchical order, EFS was 45.9%(4.4) in 132 HR-MRD patients, 41.2%(11.9) in 17 patients no-CR at day 33, 36.4%(14.5) in 11 patients with t(4;11), 74.0%(3.6) in 152 HR patients only for PPR. No statistically significant difference was found for disease-free survival (DFS) in patients with very high risk features (HR-MRD, no-CR at day 33, t(4;11) translocation), given HSCT (n=66) or chemotherapy only (n=88), after adjusting for waiting time to hematopoietic stem cell transplantation (HSCT) (5.7 months). Patients at HR only for PPR have favorable outcome. High risk MRD is associated with poor outcome despite intensive treatment and/or HSCT and may qualify for innovative therapies. The study is registered at the US National Institutes of Health website http://clinicaltrials.gov as "Combination Chemotherapy Based on Risk of Relapse in Treating Young Patients With Acute Lymphoblastic Leukaemia" with the protocol identification number NCT00613457.

Introduction. A prospective magnetic resonance imaging (MRI) study demonstrated a good control of myocardial iron overload (MIO) in terms of prevention and treatment in children with thalassemia major (TM). The aim of the present study was to evaluate if changes in MIO were related to baseline hepatic iron or changes in hepatic iron overload (HIO). Methods. We considered 68 TM patients enrolled in the MIOT (Myocardial Iron Overload in Thalassemia) project with less than 18 years at the first MRI scan and who performed a follow-up (FU) study at 18±3 months. Myocardial and hepatic iron burdens were quantified by the T2* technique. The value of 20 ms was used as conservative normal value for the global T2* value. Liver T2* values were converted into liver iron concentration (LIC) values. A LIC Results. Thirty-six patients were females and mean age at the time of the baseline MRI was 13.74±3.09 years. Baseline global heart T2* values were 29.72±11.21 ms and 16 (23.5%) patients showed significant baseline MIO. The percentage changes in global heart T2* values per month in the whole patient population were 0.66±1.70 and they resulted significantly higher in the 16 patients with significant baseline MIO versus the patients with no baseline MIO (1.99±2.53% vs 0.25±1.09% ms; P=0.002). Percentage changes in global heart T2* values per month were not influenced by initial MRI LIC values (R=0.048; P=0.695) (Figure 1A) and were comparable among the 4 groups of patients identified on the basis of baseline MRI LIC values (14 no HIO: 0.29±1.12% vs 21 mild HIO: 0.75±1.56% vs 15 moderate HIO: 0.82±2.03% vs 18 severe HIO: 0.71±2.00%; P=0.876). Percentage changes in global heart T2* values per month were not associated to final MRI LIC values (R=-0.134; P=0.277) (Figure 1B). The correlation between % changes in global heart T2* and MRI LIC values did not reach the statistical significance (R=-0.244; P=0.067) (Figure 1C). In patients with baseline MIO no correlation was found between % changes in global heart T2* values per month and initial MRI LIC values (R=-0.325; P=0.219) or % changes in MRI LIC values per month (R=-0.353; P=0.180). Conclusion. In pediatric TM patients changes in cardiac iron are not correlated to baseline MRI LIC values and changes in hepatic iron. So, our data seem not supporting the hypothesis for which it is necessary to clean the liver before removing iron from the heart. Figure 1 Disclosures Pepe: Chiesi Farmaceutici S.p.A., ApoPharma Inc., and Bayer: Other: No profit support.

Introduction and Aim Till today, the only curative and most widely used therapy for β-thalassemia (β-TDT) is allogeneic HSCT: the European Society for Bone Marrow Transplantation Hemoglobinopathy Registry reported over 90% overall survival in under-14 patients transplanted from an HLA identical family donor. However, with the new therapeutic scenario opened up by Gene Therapy (GT), it has become essential to identify and prioritize patient profiles for which GT could be applied. The "Pesaro Patients Risk Classification" showed the importance of risk stratification in order to achieve the best results. The Italian Scientific Society for Thalassemias and Hemoglobinopathies (SITE) closely involved in the cure and overall approach to these pathologies, decided to carry out this project of analysis and assessment to establish the possible inclusion and exclusion criteria for access to GT of patients with β-TDT and to collect the outcomes. The aim of this study is to identify which patients with β-TDT could benefit from GT, based on evidence and consensus. Methods The decision-making algorithm was developed within the scientific activity of SITE, which defined the feasibility of the project and selected the multidisciplinary panel of experts in hemoglobinopaties for the elaboration of clinical issues. Published literature (Medline, PubMed, Embase, Cochrane Library) was searched in order to get solid evidences regarding best candidates to allogeneic SCT, who should not therefore undergo GT. Evaluation of literature and scientific evidences were reported and discussed through call conferences and mails communications. The final review has been performed by a pool of external reviewers who evaluated the clinical relevance, the applicability, the legibility of the document and the consistency between the recommendations and the summaries of the tests produced to test the algorithm. This step is in progress. The final version of the document will be made available on the web site of SITE (www.site-italia.org). The outcomes of the procedures will be collected in the electronic clinic database used by the Centers (i.e Webthal©) to perform a risk stratification. Results and Conclusions The patient selection procedure must be performed by the joint evaluation of the Center of the "Hemoglobinopathies Italian Network" together with the qualified treatment Center for HCS transplantation. The document is proposed as a dynamic and up-datable tool. It is structured such as to allow two levels of consultation: an interactive flow chart (Figure) presenting the different clinical issues discussed with conclusive practical indications linked to chapters providing detailed information on each aspect of the subject. Applying the appropriate cautionary considerations, patients are considered non-eligible in the case of: exclusion criteria indicated by the Regulatory Authorities (i.e. EMA/CHMP/166977/2019);Severe iron overload and/or organ damage (i.e. pulmonary hypertension). Manageable iron overload is a condition of re-evaluation. Caution is mandatory in the evaluation of patients with complications or comorbidities. The access to GT needs to be re-evaluated on the basis of scientific and regulatory updates. Figure Disclosures Longo: Blue Bird Bio: Consultancy. Pinto:Novartis: Consultancy; Bluebird Bio.: Consultancy. Angelucci:Novatis: Honoraria, Other: Chair Steering Committee TELESTO protocol; Celgene: Honoraria, Other: Participation in DMC; BlueBirdBio: Other: Local advisory board; Jazz Pharmaceuticals: Other: Local advisory board; Roche: Other: Local advisory board; Vertex Pharmaceuticals Incorp., and CRISPR Therapeutics: Other: Participation in DMC. Cappellini:Novartis: Membership on an entity's Board of Directors or advisory committees; Vifor Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Honoraria; CRISPR Therapeutics: Membership on an entity's Board of Directors or advisory committees; Genzyme/Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees. Piga:Acceleron Pharma: Research Funding; Novartis: Consultancy, Research Funding; Celgene Corporation: Consultancy, Research Funding. Forni:Roche, Erithropoiesis Stimulation: Research Funding; Celgene, Erithropoiesis Stimulation: Research Funding; Novartis, Iron chelation: Research Funding; BlueBirdBio: Consultancy.

Background: Recent studies on the impact of migration on the geographical distribution of the HbS allele have highlighted sickle cell disease (SCD) as a global public health issue. Although considered a "rare disease" due to its global frequency in the 28 countries of the European Union, SCD is the most common genetic disease in France and the United Kingdom and its frequency is steadily rising in many other countries of central and southern Europe. At present, less than 50% of children with SCD have access to TCD screening in the USA and Europe. Most centers use the non-imaging approach, as described in the STOP trials, which is a "blind technique" where there is no guiding anatomical information and thus relies heavily on operator experience. Some centers now use imaging TCD which provides anatomical information enabling the Circle of Willis to be visualized and so facilitates identification of the basal cerebral arteries and orientation of the Doppler beam when acquiring blood velocities. The primary study objective was to determine the effectiveness of the modular training program in achieving the high level of scanning competency described in the STOP trial, irrespective of practitioner background and when using either non-imaging or imaging TCD. Methodology. The modular TCD training program was developed at the training center in London and delivered to trainees at all three centers (London-UK, Padova-Italy and Dublin, Ireland). The program comprised of a 2-day instructional course covering theory and practical aspects of TCD and incorporated significant hands-on instruction. This was followed by trainees scanning at their own hospital until they had collected a log book of at least 40 scans (within a one year period), after which a scan review and competency evaluation was performed. Results.Modular training program.Nine training courses were held (six in England, one in Ireland and two in Italy); these were attended by a total of 51 trainees (Table 1). Approximately half the trainees (45%) successfully completed the competency evaluation, 20 were still in training, two of whom had failed the assessment and eight withdrew from the program due to problems with local funding for staff or equipment. The ten trainees with an ultrasound background (clinical scientists) were able to acquire TCD skills rapidly as demonstrated by the high pass rate. The findings were more variable in the clinician group (pediatricians and nurses) with five requiring refresher courses and twelve failing to complete the minimum annual scan number (forty) due to small local sickle populations. Comparative analysis of TCD data obtained before and after training.A total of 555 patients were included in this study; 181 patients at Center 1 (52 males, mean age 7.9±3.8 (range 2-15.4 years), genotypes: 134 HbSS, 39 HbSC, 8 HbSβ thalassemia), 194 patients. Center 2 (53 males, mean age 7.4±3·2 (range 2-15.1 years), genotypes: 158 HbSS, 32 HbSC, 4 HbSβ) and 154 patients at Center 3 (50 males, mean age 6.4±3.5 (range 2-15.1 years), genotypes: 154 HbSS, 10HbSC, 16 HbSβ thalassemia). There was no significant difference in gender distribution (Chi-Square=0.313, p=0.85), but more young patients were recruited in Center 3 (ANOVA, F=8.9, p Disclosures Colombatti: AddMedica: Consultancy; Novartis: Consultancy; Global Blood Therapeutics: Consultancy.

We conducted a long-term prospective study of 89 cancer survivor children who had acquired hepatitis B virus (HBV) and/or hepatitis C virus (HCV) during treatment for neoplasia, the aim being to evaluate the natural history of the diseases and the effect of interferon (IFN) treatment. Patients were followed up for a median period of 13 years (range, 8 to 20); 46 were infected by HBV, 11 by HCV, and 32 coinfected by HBV and HCV. A spontaneous clearance of hepatitis B surface antigen (HBsAg) occurred more frequently in coinfected patients (19%) than in the HBV-infected (2%; P = .004), with an annual seroconversion rate of 2.1% and 0.2%, respectively (P= .008). Loss of hepatitis Be antigen (HBeAg) occurred in 44% of coinfected and in 28% of HBV-infected patients. Clearance of serum HCV-RNA was observed in 34% and 9%, respectively, of coinfected and HCV-infected patients. Seventeen HBV-infected, 4 HCV-infected, and 16 coinfected patients received -IFN treatment. In the HBV group, 6 patients (35%) cleared serum HBV DNA and seroconverted to anti-HBe; in the HCV-group, none cleared HCV-RNA. In the coinfected group, 1 patient cleared both HBV DNA and HCV-RNA, 6 patients cleared serum HCV-RNA alone, and 1 only HBV DNA and HBeAg. Overall, the diseases showed a mild histological course with no evidence of liver cirrhosis. A reciprocal interference on viral replication between HBV and HCV may occur in coinfected patients. Treatment seems to be effective for selected cases and is justified in view of the uncertain prognosis of the disease in these patients.

Although the heart and liver have traditionally been the organs of interest for iron-related complications in Thalassemia Major (TM), endocrine disorders are common and carry significant morbidity. This is thought to result from iron accumulation in multiple organs functioning as part of endocrine systems. Deferasirox chelation therapy is well tolerated in TM and reduces iron burden effectively in the heart and liver. The impact of deferasirox on endocrine parameters remains unclear. The aim of this study was to examine the long-term efficacy of deferasirox for both the prevention of endocrine dysfunction in patients free of endocrine complications, and in improving endocrine parameters in those with pre-existing endocrine disorders. This was a multicentre retrospective cohort study of TM patients over 2 years of age receiving chelation treatment with once daily oral deferasirox monotherapy. Data on relevant clinical and laboratory parameters, including glycaemia, bone mineral density (BMD) z-scores, and testing of the pituitary, gonadal, thyroid and adrenal axes were collected at baseline and follow-up. Dosing regimens were titrated according to standard practice and only patients treated for at least 3 years were included in the present analysis. Data are presented as mean with standard deviation unless otherwise stated. Statistical comparisons were made using paired t-tests for continuous data, and chi-square test or Fisher's exact test to compare categorical data distributions. A total of 72 patients (69% female) with a mean age 26 ± 15 years were included. Mean duration of exposure to deferasirox was 6.9 ± 2.7 years with a mean daily dose of 25 ± 5 mg/kg. Myocardial T2* significantly increased from baseline to follow up (29.30 ± 10.29ms vs. 33.5 ± 13.06ms, P=0.01), signifying effective removal of iron from the heart. Among patients free of thyroid dysfunction at baseline, we observed no significant difference in mean TSH (2.10 ± 1.00 mi/u/ml vs. 2.28 ± 1.06 mi/u/ml, P=0.23 ), FT4 (6.89 ± 4.50 ng/ml vs. 7.10 ± 4.71 ng/ml, P=0.34) or FT3 (2.87 ± 1.16 pg/ml vs. 3.00 ± 0.58 pg/ml, P=0.35) during the study period. Of 9 patients with biochemical hypothyroidism at baseline, including 4 receiving thyroxine, we observed no significant change in thyroid function tests or thyroxine requirements. Two new incidences of subclinical hypothyroidism were noted. Among patients free of diabetes mellitus at baseline, we observed no significant variation in blood glucose measurements (pooled over 3 consecutive readings) at the end of treatment (88.62 ± 10.53 mg/dl vs. 88.36 ± 9.35 mg/dl, P=0.83). There was 1 incidence of type 2 diabetes during the study period. No significant difference was seen in blood sugar control or insulin requirements between baseline and end of treatment in patients with type 1 diabetes. Excluding normal pre-pubertal paediatric patients, there was no significant increase in the prevalence of hypogonadism between baseline and the end of study, 21 patients (38%) vs. 24 (44%) were diagnosed with hypogonadism respectively (P=0.56). Biochemical indices of bone metabolism showed no significant difference in calcium levels or vitamin D from baseline to follow up. BMD was improved from baseline to follow-up, with normal Z scores in 7 (20%) patients, 20 (57%) osteopenic, and 8 (23%) osteoporotic, compared with 7 (20%), 24 (69%), and 4 (11%) at end of treatment, respectively. Adverse effects were infrequent and of low grade, effecting 7 (9.7%) patients. Endocrine disorders are common sequelae of iron overload in patients with TM, and follow an insidious course of progressive dysfunction. We report infrequent development and progression of endocrine abnormalities, and significant improvement in bone density in TM patients treated with deferasirox over a 7-year follow-up period. In comparison with the incidence of complications from other studies of TM, deferasirox appears effective in reducing risk of developing endocrine disorders. Increasing myocardial T2* values confirmed effective removal of iron from the heart. Although multiple biological pathways are likely responsible, reduction in global iron alleviating siderosis of endocrine organs may be responsible for preserved function and bone mineral metabolism. Our study suggests that chelation therapy with deferasirox may prevent endocrine complications in patients with TM and prevent progression in those with established abnormalities Disclosures: Silverio: Novartis: Consultancy, Research Funding.

Introduction: Recently, the ability of LIC (liver iron concentration) and serum ferritin in predicting myocardial iron overload (MIO) has been challenged by magnetic Resonance Imaging (MRI) monitoring which demonstrated no or weak correlation between serum ferritin or LIC and MIO. Anyway, the role of this traditional markers could result particularly useful in pediatric population, where MRI assessment is difficult to carry out, because of early age, scarce collaboration or limited availability. So, we derived objective thresholds for these markers for predicting cardiac T2* Methods: From the 2171 patients with hemoglobinopathies enrolled in the MIOT (Myocardial Iron Overload in Thalassemia) network, we retrospectively selected 107 paediatric patients with thalassemia major (TM) (61 boys, median age 14.4 years). MIO was assessed using a multislice multiecho T2* approach. Hepatic T2* values were assessed in a homogeneous tissue area and converted into LIC. Results: Twenty-three patients (21.5%) showed an abnormal global heart T2* value and none of them was under 7.9 years of age. Serum ferritin was negatively correlated with global heart T2* values (r=-0.425; P There was a significant negative correlation between global heart and MRI LIC values (P=-0.436; P Conclusion: A weak connection between serum ferritin levels or hepatic iron and cardiac iron was demonstrated in our pediatric population. Anyway, MRI LIC≥14 mg/g/dw and serum ferritin levels≥2000 ng/ml were found to be significant risk factors for a global heart T2* value Figure 1. Figure 1. Disclosures Pepe: Novartis: Speakers Bureau; ApoPharma Inc: Speakers Bureau; Chiesi: Speakers Bureau.

BACKGROUND Sickle Cell Disease (SCD) is the most frequent severe genetic disease worldwide. Its frequency is rising in European countries, including Italy and Ireland. In Europe Sickle SC (HbSC) is the second most common form of SCD after sickle cell anaemia (HbSS/HbSB°) and accounts for 25-30% of cases. Neurological events are among the most frequent and disabling complications in children with SCD with an important impact on quality of life, health and educational system costs (DeBaun et al., 2012). Overt and silent stroke are reported in in HbSC disease, although to a lesser extent. Studies suggest that the life-time risk of stroke in HbSC is 2-3% (Deane et al., 2008). Stroke Prevention is limited to only for HbSS/HbSB° but not HbSC. CBF-V as measured by TCD ranges of velocities in the Middle Cerebral Artery (MCA) and in the distal Internal Carotid Artery (dICA) used to stratify patients with HbSS/HbSB° in risk categories might be inappropriate for HbSC patients. Unlike North America, in Europe HbSC phenotype is more common; we therefore set out in this three country European study (SCATES) to describe the pattern of CBF-velocity and also compare the findings with HbSS patients attending the same facilities:. Hypothesis: Aims is to determine mean reference values of TCD velocities in MCA and dICA in a European prospective cohort of children with HbSC in comparison with a cohort of HbSS. Main objectives To assess the pattern of cerebral flow velocity distribution in HbSC and compare with HbSS in three European countries To test if the impact of clinical and hematological factors on Cerebral blood flow velocity in HbSC and HbSS in order to make recommendation for screening HbSC. METHODS Following a formal evaluation and validation of the competency of the screening centres to perform TCD in sickle cell disease; consecutive patients were recruited into the prospective observational study. TCD was performed with imaging TCD (Philps, other makes) by certified TCD operators. The data were entered on-line (Study Trax) and downloaded for statistical analysis using STATA 10.0 (Stata-Corp LP, College Station, Texas). Before regression analyses were performed, all variables that did not have a normal distribution (BP diastolic, albumin creatinine ratio (ACR), lactate dehydrogenase (LDH) and bilirubin) were transformed to achieve a normal distribution. All variables were standardised to allow for clear interpretation. Univariable regression analyses were performed to examine the influence of laboratory and haematological parameters on maximum TCD velocity from the MCA or TICA, in each SCD type subgroup. As TCD velocity had a non-parametric distribution the 95% confidence intervals of TCD velocity for HbSC subgroup was determined using the bootstrap method (resampling with replacement from our data set), with 5,000 replications(Singh & Xie, 2010) RESULTS At recruitment, the participants' age ranged from 2 to 16 years with a mean age of 8.11 (sd ±4.07). Overall, 224 (76.12%) children had HbSS phenotype, and 61 (18.21%) children had HbSC phenotype. Mean values for haematological parameters for HbSS and HbSC subgroups: Mean Hb g/dl (std) was 8.32(1.16) for HbSS and 10.93(0.91) for HbSC; Albumin: creatinine ratio as 12.39 (3.92) and 8.32 (2.68) for HbSS and HbSC respectively. HbSS as expected show inverse relationship between CBF-V and hemoglobin (n = 224; beta = -7.90; 95%CI = -11.9 to -3.89; p = 0.00014); positive correlation with systolic blood pressure increase (beta=11.03; 95% 3.10 to 18.995 and P=0.008); and total leukocyte count (n-120; beta 1.50; 95% CI 0.38-2.63; P=0.009). However, there was no correlation between TCD and any parameter in the HbSC group. Discussion and Conclusion From this pan-European patient cohort with a substantial proportion of patients we show that the CBF-V in HbSC does not follow a normal distribution pattern and appears entirely unrelated to clinical or hemolytic markers as observed with HbSS. Higher systolic blood pressure has been reported as risk factor for the development of silent cerebral infarct in HbSS (DeBaun et al., 2014). To the best of our knowledge this is the first time systolic and increased leukocyte as a marker of thrombosis (Marchetti & Falanga, 2007) is a risk factor in SCD. CBF-V. The fact there is relationship with these markers in HbSC suggests the lack of benefit for this measurement. Disclosures No relevant conflicts of interest to declare.

Introduction: Although risk-adapted therapy improved the prognosis of pediatric T-ALL in the last decades, patients with T-ALL still have a worse outcome compared to B Cell Precursor (BCP)-ALL, and therefore they would benefit from the identification of new prognostic markers for a better treatment stratification and novel strategies to complement current chemotherapy regimens. Among newly reported genomic abnormalities, a subset of BCP-ALL patients is characterized by the over-expression of the Cytokine Receptor-like Factor 2 (CRLF2) gene, due to either an intra-chromosomal deletion causing the P2RY8-CRLF2 fusion or the IGH@-CRLF2 translocation. These two CRLF2 rearrangements were shown to correlate with poor outcome in BCP-ALL patients. In T-ALL, alterations of CRLF2 were not reported yet, but recently, mutations in its partner IL7Rα have been identified in about 10% of T-ALL patients. Aim: To estimate the incidence of CRLF2 aberrations and their prognostic value in pediatric T-ALL. Methods: We analyzed CRLF2 gene expression in 120 T-ALL patients, consecutively enrolled in the AIEOP-BFM ALL2000 study in Italian centers (AIEOP) from September 2000 to July 2005, and, as a validation cohort, in 92 consecutive patients treated with the same protocol in German centers (BFM-G), from July 1999 to December 2004. CRLF2 transcript levels were analyzed by RQ-PCR. Relative gene expression of CRLF2 was quantified by the 2-DDCt method. The DDCt for AIEOP and BFM-G samples was referred to the median DCt of their respective cohort. Results: An heterogeneous expression of CRLF2 was observed among AIEOP T-ALL patients (range: 0.06 to 82 fold change). Seventeen patients (14.2%) presented an expression 5 times higher than the median (‘CRLF2-high’). Interestingly, none of the CRLF2-high cases resulted to be positive for P2RY8-CRLF2 fusion, 1/5 was positive for the IGH@ translocation and 1/7 showed a supernumerary X chromosome. JAK2 and CRLF2 mutations were absent in all 120 cases, while IL7R mutations were detected in 5/107 patients (4.7%), but unexpectedly they were not associated to CRLF2 over-expression. CRLF2-high patients had a significantly inferior 5-y EFS (41.2%±11.9 vs. 68.9%±4.6, p=0.006) and an increased, although not statistically significant, cumulative incidence of relapse (CIR) compared to CRLF2-low patients (41.2%±11.9 vs. 26.3%±4.3, p=0.17). The prognostic value of CRLF2 over-expression was confirmed in the BFM-G cohort (5-y EFS in 12 CRLF2-high patients was 50.0%±14.4 vs 83.8%±4.1, p-=0.008; CIR: 33.3%±13.6 vs. 11.3%±3.5, p= 0.06). Interestingly, CRLF2-high patients were more frequently allocated to the high risk (HR) T-ALL subgroup (20.9% in HR vs.8.3% in no-HR in the two cohorts analyzed together). In this subgroup, CRLF2 over-expression was significantly associated to a poor prognosis (5-y EFS: 31.6%±10.7 vs. 62.5%±5.7, p-value=0.008; CIR: 47.4%±11.5 vs. 29.2%±5.4, p=0.14). When analyzed according to prednisone (PDN) response, 17/28 (61%) were prednisone poor responders (PPR), and in the ‘PPR-only’ subgroup (non-HR by other features) 4/9 (44%) CRLF2-high patients relapsed versus 4/36 (11%) CRLF2-low. Cox model analysis adjusted by risk group showed that CRLF2-high expression had a relevant prognostic impact with a 2-fold increased risk of relapse (Hazard ratio 2.12; 95% CI 1.07-4.21; p=0.03). The mechanisms responsible for CRLF2 over-expression in T-ALL and its contribution to the pathogenesis of the disease is still being investigated. Notably, gene set enrichment analysis (GSEA) showed an inverse correlation between the expression of CRLF2 and positive cell cycle regulators, thus suggesting that CRLF2-high blasts have a low proliferating activity and may therefore be less sensitive to conventional chemotherapy. Conclusions: CRLF2 over-expression is a poor prognostic marker not only in BCP-ALL patients, but also in T-ALL, identifying a subset of HR T-ALL patients with extremely severe outcome. Specifically, this marker would allow identifying T-ALL patients that could benefit from alternative therapy, potentially targetting the CRLF2 pathway. Disclosures No relevant conflicts of interest to declare.

Aim: To evaluate the outcome of the Italian Association of Pediatric Hematology and Oncology (AIEOP) patients diagnosed with Early T-cell Precursor (ETP) Acute Lymphoblastic Leukemia (ALL) in the period September 2000-December 2011 and treated in the context of the AIEOP-BFM ALL 2000 Study and the subsequent - very similar – AIEOP ALL R2006 Study. Patients and methods: ETP-ALL was defined by staining negative for CD1a and CD8, mildly positive or negative for CD5 and positive for at least one of CD34, CD117, HLADR, CD13, CD33, CD11b, or CD65 antigens. Treatment consisted of BFM protocol I either with dexamethasone or prednisone in phase IA, 4 HDMTX courses (5 g/sqm over 24 hrs) in non-high risk patients or 3 poly-chemotherapy blocks in high risk patients, delayed intensification based on protocol(s) II or III, protracted intrathecal therapy or cranial radiotherapy, maintenance therapy for a total of 2 years of treatment. Hematopoietic stem cell transplantation (HSCT) was indicated for patients with poor treatment response assessed either morphologically at day +8 (Prednisone Poor Response, PPR) or day +33 (no Complete Remission, CR) or by PCR at day +78 (high-level Minimal Residual Disease, HR-MRD; ≥10-3). Results: Of the 439 T-ALL eligible patients, 34 had ETP ALL. The incidence (7.7%) may be underestimated since the full set of the data needed was not available for all patients. Out of the 34 patients with ETP ALL 14 were at high risk due to PPR; of them, 3 were HR-MRD and 5 did not achieve CR after Phase IA of induction therapy (including one with HR-MRD). Of the remaining 20 patients (all prednisone good responders), 3 patients were at high risk due to resistance to Phase IA and HR-MRD (n=1) or HR-MRD only (n=2). 17/30 patients could not be monitored by MRD due to death in Induction (n=1), or absent (n=10) or inadequate PCR markers (n=6). Of the17 patients monitored by MRD, 13 had HR-MRD at day 33 and 6 of them also at day +78. One patient died during induction therapy. The remaining 33 achieved morphological CR: 27 after phase IA and 6 (those resistant to phase IA) after phase IB of protocol I. Of 12 patients who underwent HSCT, 3 died of HSCT-related complications and 3 relapsed. With a median follow-up of 6.6 years, the 5-year event-free survival (EFS) and Survival in the 34 ETP ALL patients were of 60.9%(SE 8.5) and 66.6%(8.3), versus 71.2%(2.3) and 77.1%(2.1) respectively in the non-ETP patients. The overall cumulative incidence of relapse was 27.3%(7.8) and 22.2%(2.1) in ETP and non-ETP T-ALL patients, respectively (p=0.52). EFS in ETP vs non-ETP patients was respectively 81.9%(9.5) vs 83.8%(2.4) in non high risk patients (p=0.87) and 41.2%(11.9) vs 53.2%(4.0) in high risk patients (p=0.24). Conclusions: The outcome of T-ALL patients treated with BFM-type therapy is comparable in ETP and non-ETP for those with good response to initial chemotherapy; it was slightly, but not significantly, inferior in ETP-ALL patients with poor initial response. Phase IB treatment element is very effective in ETP-ALL, suggesting that intensification with antimetabolite and alkylating agents may be beneficial, while the benefit of HSCT in first CR needs to be further investigated. Disclosures No relevant conflicts of interest to declare.

Abstract 2214 Bernard-Soulier syndrome (BSS) has been historically described as an autosomal recessive disease, and more than 50 different mutations have been identified in homozygous or double-heterozygous patients. However, a few reports have suggested that BSS may be occasionally transmitted in an autosomal-dominant fashion. In 2001, we described six Italian families that were previously diagnosed with Mediterranean macrothrombocytopenia and possessed the monoallelic c.515C>T transition in the GPIBA gene, which results in a p.Ala156Val substitution (Bolzano mutation) in GPIb alpha (Savoia et al, Blood 2001;97:1330–5) Since 2001, we have searched for this mutation in all patients referred to us for a non-syndromic, autosomal dominant form of inherited macrothrombocytopenia of unknown origin. Screening for the c.515C>T mutation identified 42 carriers and genetic molecular testing in all of the available relatives indicated that this mutation was present in 61 additional individuals. When the 42 families described here are added to the 6 families previously described, our database of 210 pedigrees of inherited thrombocytopenia includes 48 families with monoallelic, dominant BSS caused by the Bolzano mutation; 22 of the pedigrees have MYH9-related disease, 21 have thrombocytopenia 2 caused by ANKRD26 mutations (Noris et al, Blood 2011;117:6673–80), and 10 pedigrees have the classic biallelic, recessive form of BSS (Savoia et al, Haematologica 2011;96:417-23). Our experience therefore indicates that the Bolzano mutation is the most frequent cause of inherited thrombocytopenia in Italy. Analyses of the geographic origin of affected pedigrees and haplotypes indicated that this mutation originated in southern Italy. The bleeding tendency was variable, with 42% of the cases presenting recurrent, spontaneous hemorrhages that only rarely were severe (5 patients required medical intervention and platelet transfusions). 19 women delivered 32 children (vaginal and caesarean births) with no platelet transfusions, and excessive bleeding was reported in one case only. None of the patients experienced abnormal bleeding associated with dental extractions or surgery. Thrombocytopenia was usually mild (a few subjects even had normal platelet counts), and electronic counters overestimated the degree of platelet deficiency with respect to manual counting with optical microscopy in a Neubauer chamber (Table 1). Platelets were larger than normal in most patients, and their GPIb/IX/V content was 50% of controls. However, in vitro platelet aggregation in response to ristocetin 1.5 mg/mL was defective only in 22% of cases. The serum thrombopoietin level was two-fold greater than that of healthy subjects and similar to that observed in a case series of patients with ITP. Beta 1-tubulin polymorphisms, which have been associated with platelet size and function (Freson et al, Blood 2005;106:2356–62), did not explain the variation in clinical and laboratory features of our patients. In conclusion, our study indicates that the monoallelic Bolzano mutation is a frequent cause of macrothrombocytopenia in Italy and that this mutation induces a mild form of BSS that is often misdiagnosed with ITP. Further analyses in other countries will reveal whether this form of the disease is exclusive to Italy or if it also affects other populations.Table 1.Platelet count and size in patients with the Bolzano mutation in GPIb alpha.PatientsControlsmean±SD (range)Manual platelet count, x10e9/L114±36† (42–184)276±50 (170–420)Impedance platelet count, x10e9/L89±33**† (21–147)255±53 (141–396)Optical platelet count, x10e9/L103±32*† (21–162)280±54 (164–412)MPV, fL, by optical counter15±1.7† (9.7–17.8)8.2±0.8 (7.1–11.1)MPD, um, by optical microscopy3.6±0.5† (2.6–5.1)2.4±0.3 (1.9–3.4)*: p Disclosures: Cattaneo: Eli Lilly Daiichi Sankyo: Honoraria; AstraZeneca: Honoraria, Research Funding.

Abstract 2643 Poster Board II-619 C/EBP is a transcription factor that regulates terminal granulocytic differentiation. CEBPA mutations have been associated with improved outcome in both adult and pediatric patients with acute myeloid leukemia (AML). However, the impact in different treatment protocols, as well as the different outcome between single and double mutants, is still to be definitively established. We evaluated the prevalence and prognostic significance of CEBPA mutations in children with de novo non promyelocitic AML treated in Italy with the AIEOP-LAM 2002/01 pediatric protocol. Among 205 patients enrolled in the protocol between December 2002 and December 2007, 103 were successfully analyzed for CEBPA mutations by PCR amplification of TAD and bZIP domains of the gene, and through sequencing of positive cases after DHPLC analysis. Characteristics of analyzed and non analyzed patients were not statistically different. Two types of CEBPA mutations, N-terminal (TAD) truncating mutations and in-frame bZip domain mutations, were detected in 13/103 (12.6%) patients tested; 8 of them (61.5%) harboured both mutation types. Laboratory, clinical characteristics and outcomes for patients with CEBPA mutations were compared to those of patients with wild-type CEBPA. CEBPA mutations were significantly more common in older patients (8/13 vs 30/90 children were older than 10 years), in patients with FAB M1 (7/13 vs 4/90), and in patients with normal cytogenetics (13/13). None of the CEBPA mutated cases carried either FLT3 or NPM1 mutations. Only 1 mutated case was found in Standard Risk patients (defined as children carrying isolated CBF abnormality and achieving complete remission after 1 cycle of induction therapy), while the other 12 patients belonged to the High Risk group. Although the values did not reach statistical significance because of the low prevalence of CEBPA mutations, with a median follow up of 39 months (range 4–86) the event-free survival probability at 5 years was 76.9% vs. 59.6% for children with or without CEBPA mutations, respectively. The values for Disease-Free Survival were 83.3% vs. 65.4% and those for Overall Survival were 90.0%. 66.4%, respectively. No difference in terms of outcome was found between patient with a single and those with double mutants, neither between those with TAD- and bZIP- mutations. Therefore, patients in the AIEOP-LAM 2002/01 pediatric trial carrying CEBPA mutations seem to have a lower relapse rate and improved outcome, their overall survival approaching that of children belonging to the Standard Risk group (90% vs. 97%). If confirmed in a larger cohort of patients and with longer follow-up, these data suggest that CEBPA mutation analysis could be usefully employed to identify patients at lower risk of treatment failure and for allocating them into different classes of risk. Disclosures: No relevant conflicts of interest to declare.

Radioimmunotherapy (RIT) with 90Y-Zevalin® combined with high dose therapy and autologous stem cell transplantation (ASCT) is gaing increasing importance for the treatment of relapsed or refractory non Hodgkin Lymphoma (nHL). We evaluated the feasibility and the clinical results of the addition of 90Y-Zevalin® at standard dose to BEAM regimen (Z-BEAM) in nHL pts who failed to achieve complete remission (CR) after previous chemoimmunotherapy. Methods. Between October 2005 and June 2008, 53 patients were enrolled in 11 italian centers. The treatment strategy is shown in figure 1. Salvage treatment consisted of 2 courses of R-DHAP. PBSCs were collected after mobilization with DHAP and G-CSF plus in vivo purging with Rituximab. Patients’ characteristics are shown in table 1. Results. The median CD34+ cells infused was 5.5 x10^6/Kilograms (range 2.55–34). All patients engrafted. The median number of red blood cell and platelet transfusion were 4 (1–7) and 6 (1–8), respectively. The median time to platelet and neutrophil counts higher than 20x10^9/L and 0.5x10^9/L were 14 (range, 9–60 days) and 10 days (range, 8–20), respectively. Mucosites occurred in all pts (grade III in 20 and grade IV in 5 patients). Febrile neutropenia occurred in 39 pts (74%). Eight pneumonitis and 12 blood stream infections, mainly by Gram+, were documented. One patient developed an atrial fibrillation. Five pts were not evaluable for response because too early. The 90-day overall response rate was 86% with 74% of CR. Three relapses (relapse rate 9%) and four progression were documented at a median follow-up of 247 days post Z-BEAM (range, 125–818). The potential factor to predict CR was: at last PR before Z-BEAM (p=0.06). Fourthy patients are alive at a median follow-up of 175 days post HST (range, 6–590): thirty pts in CR (57%), three pts in PR (5.5%), three pts in progressive disease (PD, 6%)(fig. 2). Fourtheen pts died (26%): 5 deaths due to TRM before day 90, 1 for ARDS (+230), 1 TRM post a subsequent RIC allotransplant (+95) and 6 due to PD (median follow-up 110 days, range 97–150). The Kaplan-Meyer estimated 3y-EFS is 64%. Five early deaths before day-90 occurred: 2 due to septic shock (day +6 and +39), 1 to pneumonitis (+22), 1 for BK viral encephalites (+61) and 1 to MOF (+14). The Kaplan-Meyer estimated Treatment Related Mortality (TRM) is 9.3%. Two statistically risk factors for 90-day TRM (p Conclusion. In pts with different histology nHL, who failed to achieve CR after previous immuno-chemotherapy, RIT integrated with high-dose chemotherapy (Z-BEAM) is capable to induce 86% of ORR, 74% of CR and 3 ys EFS of 64%, with sustained engraftment and an acceptable extra-haematological toxicity, mainly restricted to pts older then 65 ys. The power of this program needs to be assessed in a larger series of patients and in a randomized fashion. Table 1: Patient Characteristics and 90-day response post HST Figure. 1 Treatment Plan Figure. 1. Treatment Plan