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The fusion gene MLL-AF4 defines a high-risk subtype of pro-B acute lymphoblastic leukaemia. However, relapse can be associated with a switch from acute lymphoblastic to acute myeloid leukaemia. Here we show that these myeloid relapses share oncogene fusion breakpoints with their matched lymphoid presentations and can originate in either early, multipotent progenitors or committed B-cell precursors. Lineage switching is linked to substantial changes in chromatin accessibility and rewiring of transcriptional programmes indicating that the execution and maintenance of lymphoid lineage differentiation is impaired. We show that this subversion is recurrently associated with the dysregulation of repressive chromatin modifiers, notably the nucleosome remodelling and deacetylation complex, NuRD. In addition to mutations, we show differential expression or alternative splicing of NuRD members and other genes is able to reprogram the B lymphoid into a myeloid gene regulatory network. Lineage switching in MLL-AF4 leukaemia is therefore driven and maintained by defunct epigenetic regulation.Statement of SignificanceWe demonstrate diverse cellular origins of lineage switched relapse within MLL-AF4 pro-B acute leukaemia. Irrespective of the developmental origin of relapse, dysregulation of NuRD and/or other epigenetic machinery underpins fundamental lineage reprogramming with profound implications for the increasing use of epitope directed therapies in this high-risk leukaemia.

TO THE EDITOR: Factor XI (FXI) is a key element for the amplification of thrombin generation, which participates in the contact pathway. This explains why FXI deficiency has minor physiological relevance but protects against thrombotic events, supporting FXI as an excellent target for

Management of immune thrombocytopenia (ITP) during pregnancy can be challenging because treatment choices are limited. Thrombopoietin receptor agonists (Tpo-RAs), which likely cross the placenta, are not recommended during pregnancy. To better assess the safety and efficacy of off-label use of Tpo-RAs during pregnancy, a multicenter observational and retrospective study was conducted. Results from 15 pregnant women with ITP (pregnancies, n = 17; neonates, n = 18) treated with either eltrombopag (n = 8) or romiplostim (n = 7) during pregnancy, including 2 patients with secondary ITP, were analyzed. Median time of Tpo-RA exposure during pregnancy was 4.4 weeks (range, 1-39 weeks); the indication for starting Tpo-RAs was preparation for delivery in 10 (58%) of 17 pregnancies, whereas 4 had chronic refractory symptomatic ITP and 3 were receiving eltrombopag when pregnancy started. Regarding safety, neither thromboembolic events among mothers nor Tpo-RA–related fetal or neonatal complications were observed, except for 1 case of neonatal thrombocytosis. Response to Tpo-RAs was achieved in 77% of cases, mostly in combination with concomitant ITP therapy (70% of responders). On the basis of these preliminary findings, temporary off-label use of Tpo-RAs for severe and/or refractory ITP during pregnancy seems safe for both mother and neonate and is likely to be helpful, especially before delivery.

Background: Sickle cell disease (SCD) is a lifelong, inherited blood disorder, resulting from a mutation in the hemoglobin (Hb) subunit β gene, that leads to sickle hemoglobin (HbS) formation. Polymerization of HbS causes red blood cell sickling and damage, leading to hemolysis, chronic anemia, and vaso-occlusive crises (VOCs). Patients with SCD are at higher risk of end-organ damage, increased morbidity, and early mortality as a result of low Hb and increased hemolysis. Voxelotor (Oxbryta ®) tablets, a HbS polymerization inhibitor, are approved in the US for treatment of SCD in adults and adolescent patients aged ≥12 years. The randomized, placebo-controlled HOPE trial showed that significantly more patients treated with voxelotor 1500 mg achieved a >1 g/dL Hb increase compared with placebo (89% vs 25%, respectively) at any time up to week 72. These Hb increases were associated with reductions in markers of hemolysis (eg, indirect bilirubin, reticulocyte percentage). To confirm the safety and efficacy of long-term voxelotor use, we report an interim analysis of an ongoing open-label extension (OLE) of the HOPE trial. Methods: Patients who completed the phase 3 HOPE trial were eligible to enroll in the multicenter OLE study and receive treatment as long as they continued to receive clinical benefit and/or until they had access to voxelotor through commercialization or a managed access program. All patients, including those who previously received placebo or voxelotor 900 mg, received voxelotor 1500 mg as ongoing treatment. Adverse event data were collected beginning on the date of informed consent through 28 days after discontinuation of voxelotor. Measurements of Hb and clinical markers of hemolysis are ongoing and summarized here for 48 weeks of treatment in the OLE. Central laboratory assessments were used for the US and Europe, and local laboratory assessments were used for the rest of the world. Data presented are based on an interim data cut (December 31, 2020). Results: Of the 199 patients who completed the HOPE trial, 178 (89.4%) were enrolled and dosed in the OLE. Median age at enrollment was 25 years (15.7% adolescents, 84.3% adults). At the cutoff date, the median duration of voxelotor exposure the OLE was 69.9 weeks (range: 1.9-102.0 weeks), with 78 patients treated for ≥72 weeks. Of these 78 patients, 52 had previously received voxelotor in the randomized part of the study, for a combined exposure duration of ≥144 weeks. Among those who previously received placebo, the mean (SD) change in Hb from baseline (ie, the start of the OLE) to week 48 was 1.3 (1.51) g/dL, consistent with the HOPE trial results. Mean (SD) Hb changes for patients who previously received voxelotor 900 mg and 1500 mg were 0.7 (1.48) g/dL and 0.2 (1.15) g/dL, respectively, indicating durability of response. Markers of hemolysis improved from baseline to week 48 in patients who received placebo in the HOPE trial (-39.5% indirect bilirubin; -28.6% reticulocyte percentage). Patients who received voxelotor during the HOPE trial showed a stable response at week 48 (-2.0% and 1.1% indirect bilirubin, -14.6% and -21.0% reticulocytes for voxelotor 900 mg and 1500 mg, respectively). The annualized incidence rate of VOCs was 1.3 (95% CI: 1.1-1.4) events per year across all patients. A total of 83.7% of patients (149/178) experienced a non-SCD-related treatment-emergent adverse event (TEAE), with the most commonly reported being arthralgia, headache, pain, nausea, and pain in extremity (Table). Most non-SCD-related TEAEs were grade 1 or 2 in severity. Eleven patients (6.2%) had an adverse event that led to treatment discontinuation. No TEAEs consistent with lack of tissue oxygenation were observed. Conclusions: In this OLE study, treatment with voxelotor 1500 mg resulted in improvements in Hb and clinical measures of hemolysis at 48 weeks in patients who received placebo in the HOPE trial. Treatment with voxelotor 1500 mg showed durability of response in patients previously treated with voxelotor of any dosage in the HOPE trial. The safety profile in the OLE was consistent with the findings from the HOPE trial, and no new safety signals were identified with exposure through a combined 144 weeks of treatment. Based on these results, long-term use of voxelotor is safe, well tolerated, and effective in reducing anemia and hemolysis, with a low rate of VOCs, in patients with SCD. Funding: This study was supported by Global Blood Therapeutics. Figure 1 Figure 1. Disclosures Achebe: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pharmacosmos: Membership on an entity's Board of Directors or advisory committees; Fulcrum Therapeutics: Consultancy. Hassab: Global Blood Therapeutics: Research Funding. Alkindi: Global Blood Therapeutics: Speakers Bureau; Emmaus: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Brown: Global Blood Therapeutics: Consultancy, Research Funding; Imara: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Novo Nordisk: Consultancy; Forma Therapeutics: Research Funding; Pfizer: Research Funding. Telfer: BlueBirdBio: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Addmedica: Membership on an entity's Board of Directors or advisory committees; Emmaus: Membership on an entity's Board of Directors or advisory committees; Terumo: Honoraria; ApoPharma: Membership on an entity's Board of Directors or advisory committees. Biemond: Sanquin: Research Funding; Global Blood Therapeutics: Honoraria, Research Funding, Speakers Bureau; Celgene: Honoraria; Novo Nordisk: Honoraria; Novartis: Honoraria, Research Funding, Speakers Bureau; CSL Behring: Honoraria. Gordeuk: Modus Therapeutics: Consultancy; Global Blood Therapeutics: Consultancy, Research Funding; Emmaus: Consultancy, Research Funding; Incyte: Research Funding; Novartis: Research Funding; CSL Behring: Consultancy. Lipato: Global Blood Therapeutics: Speakers Bureau. Tonda: Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gray: Global Blood Therapeutics: Current Employment, Current equity holder in publicly-traded company. Howard: Resonance Health: Honoraria; Bluebird Bio: Research Funding; Agios Pharmaceuticals: Consultancy; Forma Therapeutics: Consultancy; Novo Nordisk: Consultancy; Novartis: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy; Imara: Consultancy, Honoraria.

Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day.

Introduction: The chemotherapeutic agents are effective in destroying cancerous cells for patient with hematological malignancies, but can damage the germinal cells. Hence, the infertility is the therapy-induced complication, which can be transient or permanent, depending on type of treatment protocol used and other factors. The aim of the study was to assess the fertility status among patients treated for Hodgkin lymphoma (HL) and Non-Hodgkin lymphoma (NHL). Methods: A chart review was done for patients followed up in hematology clinic. All male patients treated for hematological malignancies during the period from 2004-2014 were assessed. The infertility status was assessed using WHO infertility criteria. The fertility status was defined based on semen analysis or having a child post chemotherapy. The data was captured for type of protocol, number of cycles, fertility status, having a child pre and post chemotherapy, and sperm bank utilization. Results were summarized as frequency and percentages. Data was analyzed using SAS. Results: 400 medical charts were reviewed. 208/400 were included based on age inclusion criteria (18-60 years). 59/208 included based on available fertility data, of which 31 were diagnosed with HL, 21 NHL, 4 acute lymphocytic leukemia, and 3 acute myeloid leukemia. Only HL and NHL were considered for further analysis. 9/31 (29%) with HL and 1/21 (4.7%) with NHL had infertility prior to starting chemotherapy based on semen analysis with no follow-up fertility assessment done post-treatment. The final sample consisted of 42 patients who had data available on their fertility status post-treatment (22 HL and 20 NHL) (Figure 1). The mean age was 38 ± 8 years. 25(68%) had advanced disease. Among HL, 8/22 (36.36%) were fertile (7 conceived a child and 1 with normal semen analysis) (Figure 2). This includes 6 out of 9 (66.7%) patients who received ABVD protocol alone, 0 out of 6 who received ABVD + BEACOPP protocol, and 2 (28.6%) out of 7 who received salvage chemotherapy. Among the NHL, 11/20 (55%) were fertile (8 conceived a child and 3 with normal semen analysis) (Figure 2). This includes 7 out of 11 (63.6%) who received CHOP +/-Rituximab protocol, 3 out of 5 (60%) who received CHOP + other chemotherapy (MTX/CVP), 1 out of 3 (33.3%) who received only other chemotherapies (Hyper CVAD/MTX) (Figure 2). 9 out of 22( 41%) HL and 6 out of 20 NHL patients had sperm banking done, and only two utilized their sperms with one successful conception in a patient with NHL (Figure 3). Conclusion: Although the sample size is very small, we found a trend that may suggest HL to affect fertility and that ABVD protocol may not be as safe as it thought to be on fertility. The advanced stage of the disease being the majority may have influenced this finding. We found the documentation of fertility status among patients receiving chemotherapy to be scarce. Hence, it is important to educate physicians and dedicate a protocol for assessment of fertility pre- and post-treatment with encouragement to utilize sperm banking. Disclosures No relevant conflicts of interest to declare.

Chromosomal translocations found in acute myeloid leukemia (AML) can generate oncogenic fusions with aberrant epigenetic and transcriptional functions. However, direct therapeutic targeting of leukemia fusion proteins has not been accomplished so far. Although high remission rates can be induced in patients diagnosed with AML1-ETO/t(8;21)-positive AML only half of them achieve long-term disease-free survival (Papaemmanuiel et al., NEJM, 2016). In the other half of these patients, the disease maintaining leukemia stem cell (LSC) clone is not eliminated by chemotherapy. A functional characteristic of LSCs is unlimited self-renewal capacity and several signaling pathways have been identified that maintain stem cell self-renewal. Targeting the oncogene induced self-renewal capacity of LSCs has great potential to eliminate the malignant clone and prevent relapse. To identify oncogenic cellular functions with relevance for LSC self-renewal, we performed global proteome profiling in murine AML1-ETO9a (AE) compared to MLL-AF9 (MA9) driven LSCs. Gene set enrichment analyses revealed a significant enrichment of calcium-dependent cellular functions and Phospholipase C (PLC)-signaling in AE LSCs. These data could be confirmed in sorted CD34+ blasts from AE-positive AML when compared to non-AE-AML. All PLC family members are regulators of Ca2+ homeostasis. However, when analyzing published AML gene expression datasets we found exclusively PLCG1 to be highly expressed in t(8;21) AML. Conditional activation of AE in embryonic stem cells resulted in induction of PLCG1 expression and PLCG1 was identified as a direct target of the AE fusion by ChIP-sequencing in AE-positive Kasumi-1 cells.Here, PLCG1 depletion resulted in reduced Ca2+ release, impaired proliferation and reduced colony formation in vitro. In a xenograft model, inactivation of PLCG1 resulted not only in delayed disease development (median survival shNT vs. shPLCG1: 135 days vs. not reached, p=0.02) but also in reduction of disease penetrance by 87%. Consistent with these results, transcriptome analysis revealed strong induction of gene sets related to myeloid differentiation and down-regulation of gene sets linked to proliferation, stemness and c-Myc targets. To confirm the functional role of PLCG1-signaling in AE-driven LSCs, we generated a new conditional knockout mouse model for Plcg1 and induced leukemia using the oncogenes AE and KRAS-G12D (AE/K). Genetic inactivation of Plcg1in vivo after engraftment of leukemic cells resulted in significant reduction of LSC numbers (p=0.04) and a reduction of disease penetrance by 67% in primary recipients. Isolated LSCs revealed induction of differentiation, loss of cell cycle activity and failed to re-establish disease in secondary recipients (Plcg1+/+ vs. Plcg1-/-: median survival 12 days vs. not reached; p=0.0001). In contrast, genetic deletion of Plcg1 appeared to be dispensable for normal murine HSC function during primary and secondary transplantation. Primary human t(8;21) AML cells (derived from 4 different donors) showed impaired colony forming capacity following PLCG1 inactivation in vitro irrespective of co-occurring mutations while colony formation of human CD34+ BM cells was not affected to a major extent. As Ca2+ signaling appeared deregulated in t(8;21) AML, we aimed to investigate the effects of pharmacologic Ca2+ inhibition as a tractable target downstream of PLCG1. To assess specifically for LSC function, we treated primary recipient mice with established AE/K-driven leukemia with the clinically approved calcineurin inhibitor ciclosporin (CsA), a compound that blocks intracellular Ca2+ release. CsA-treated animals showed reduction in total leukemic burden (spleen weight diluent vs. CsA, p=0.01) and LSC numbers (p=0.02). This resulted in increased survival of secondary recipient hosts (diluent vs. CsA: median 15 vs. 29 days, p=0.0002). These effects could not be observed for other oncogenes (e.g. MA9), confirming its specificity for AE-induced disease. Consistently, CsA treated primary human t(8;21)-positive AML blasts failed to form colonies in methylcellulose. In summary, our findings identified PLCG1-dependent Ca2+ signaling as a critical pathway for t(8;21) LSC maintenance and self-renewal. Most importantly, as PLCG1 is dispensable for maintenance of normal HSPCs, PLCG1 could serve as a novel therapeutic target in t(8;21) AML. Disclosures Döhner: Daiichi: Honoraria; Jazz: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Janssen: Honoraria; CTI Biopharma: Consultancy, Honoraria. Bullinger:Novartis: Honoraria; Menarini: Honoraria; Jazz Pharmaceuticals: Honoraria; Abbvie: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Seattle Genetics: Honoraria; Sanofi: Honoraria; Janssen: Honoraria; Hexal: Honoraria; Gilead: Honoraria; Daiichi Sankyo: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Bayer: Other: Financing of scientific research; Pfizer: Honoraria.

Objectives: Sickle Cell Disease (SCD) is an inherited hemoglobinopathy with multiple complications including stroke. It is observed that siblings with SCD have an increased risk of stroke if there was a sibling in the family with SCD and stroke (Driscoll M C, Blood, 2003). Human Leukocyte Antigen (HLA) is extremely variable among individuals and certain HLA alleles have previously been associated with a number of diseases. It has been shown that HLA-DRB1*0301, HLA-DRB1*0302 and HLA-DQB1*0201 increase the risk of stroke and HLA-DRB1*1501 and HLA-DQB1*0602 lower the risk of stroke in patients with SCD (Styles L, Blood, 2000). These alleles are not common in all populations. We therefore planned to assess the impact of other HLA alleles on the risk of stroke in patients with SCD. Methods: This is a retrospective case-control study conducted at Sultan Qaboos University Hospital. We included all adult and pediatric patients with SCD and diagnosed with stroke using CT/MRI of the brain. The study included patients diagnosed during the period of 1995 to 2015. HLA typing was performed using molecular techniques. HLA alleles with a frequency of at least 5% were selected for analysis. Association studies were performed using Fisher Exact test. All statistical tests were performed using R program (version 3.1.2). Results: A total of 244 patients were included (66 patients with SCD and stroke [cases] and 178 patients with SCD with no stroke. The median age was 26 years (Interquartile Range [IQR]: 19-32) and 23 (IQR: 17-29 ) for cases and controls respectively. The median hemoglobin, mean corpuscular volume, hemoglobin S level and lactate dehydrogenase level was 9 g/dL, 70 fL, 70% and 400 U/L respectively for the cases, and 9 g/dL, 60 fL, 70% and 400 U/L respectively for the control. The most frequent HLA-A allele was HLA-A*02 (24%) which did not impact the risk of stroke (Odds Ratio [OR]: 1.59, 95% Confidence Interval [CI]: 0.84-3.02) as were the other tested HLA-A alleles (all CIs crossed OR of 1.00). The most frequent HLA-B allele was HLA-B*51 (15%) which did not impact the risk of stroke (OR: 0.89, 95% CI: 0.43-1.85) as were the other tested HLA-B alleles (all CIs crossed OR of 1.00). The most frequent HLA-C allele was HLA-C*04 (17%) which did not impact the risk of stroke (OR: 1.34, 95% CI: 0.67-2.65); however, HLA-C*06 (OR: 2.37, 95% CI: 1.01-5.54) increased the risk of stroke while HLA-C*07 protected from stroke (OR: 0.35, 95% CI: 0.13-0.93). None of the other HLA-C alleles were statistically significant (all CIs crossed OR of 1.00). The most frequent HLA-DRB1 allele was HLA-DRB1*16 (18%) which did not impact the risk of stroke (OR: 1.02, 95% CI:0.51-2.03) as were the other tested HLA-DRB1 alleles (all CIs crossed OR of 1.00). Conclusions: HLA-C*06 is associated with an increased risk of stroke in patients with SCD while HLA-C*07 is protective from stroke in these patients. Early identification of high-risk patients would be beneficial for preventive intervention, such as chronic transfusion or bone marrow transplantation. The impact of these HLA alleles should be validated in other populations. Disclosures Al-Khabori: Shire (Takeda): Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; SOBI: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Honoraria; NovoNardisk: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background: Sickle cell disease is a common hemaglopinapthy worldwide and in Oman. Avascular necrosis (AVN) of the hip causes significant morbidity to patients with sickle cell disease and has a profound impact on their quality of life. This study aims to identify the prevalence and outcome of AVN among young Omani patients with sickle cell disease. Methods: The is a cross sectional study done in the main tertiary care and referral facility in Oman, Sultan Qaboos University Hospital. Out of 3000 registered patients 85 patients found to have clinical and radiological proven AVN, between June 2017-January 2019. Data obtained included demographics, the affected joint, uni or bilateral), radiological staging by FICAT (MRI), hydroxyurea use, dose and duration as well as laboratory data at time of diagnosis. Results: The prevalence of AVN among SCD Omani children and young adults is 2.8 %. Their age ranges between 5-25 (Mean 14.6 +3.4). Male to female ratio was 1.6:1. Based on FICAT system score, most of the cases (82%) are AVN grade 3 and above at diagnosis. Thirty-six patients (42%) were diagnosed to have stage IV AVN. Regarding the hydroxyurea use, 43 patients (50.6%) were already started on HU before the development of AVN. Their mean duration of HU use was 5.3 years, with an average dose of 15.7 mg/kg/day. Fifteen patients developed AVN and were started on HU as part of their management. The follow up MRI of 4 of them showed improvement of their AVN stage (from 4 to 2). Fifteen patients (17.6%) underwent joint replacement because of chronic pain and disability and almost half of them (7 patients) were on hydroxyurea. Conclusion: Prevalence of AVN in young patients with SCD in the main tertiary care referral facility in Oman is 2.8% which is less than the internationally reported. Patients are diagnosed at later stages (3 and above) indicating a possibility of underdiagnosis of asymptomatic stage one and 2 patients. Hydroxyurea use has improved the severity of the AVN in few patients. Disclosures Al-Khabori: SOBI: Honoraria; AstraZeneca: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Shire (Takeda): Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; NovoNardisk: Membership on an entity's Board of Directors or advisory committees. Wali:Sultan Qaboos University Hospital: Employment.

Background: Hematopoietic stem cell transplantation (HSCT) is still the only curative therapy for β-thalassemia major (BTM). Several new approaches have been applied to reduce the toxicity of conditioning regimens, improve strategies for the prevention of graft-versus-host disease (GVHD), and optimize supportive care. Objectives: This study aimed to evaluate the thalassemia-free survival and outcome of HSCT in the Egyptian experience and to analyze patients and donors' characteristics as well transplantation related factors that could affect the graft success rate. Methods: We report the results of 174 consecutive first related allogeneic stem cell transplants for children with β‐thalassaemia major performed between January 1997 and December 2014. Patients and methods: the study included 174 consecutive patients with β-thalassaemia major received allogenic hematopoietic stem cell transplant (HSCT) from HLA fully matched sibling (n=153) or parent (n=10) donners. Patients mean age was 6.1±4.2 (range: 0.7 to 23.7 yrs.), the median follow up was 34 months (range 1 to 134 months). The Pesaro risk class I, II, and III categories were 21.8, 63.2% and 14.9%, respectively. Bone marrow was the source of stem cells in 26 (14.9%) and peripheral blood(PBSC) in 148 (85.1%)cases. One hundred fifty-seven patients (90.2%) received conditioning with Busulphan(BU) followed by cyclophosphamide. ATG was added in 155/157 patients at a total dose of 110 mg/kg divided into 10 doses (5 pre and 5 post-transplant). Whereas 17 (9.8%) patients received long duration conditioning with Fludarabine, Busulphan followed by cyclophosphamide in 5 patients and ATG was added in 12/17 patients at a total dose of 60 mg/kg. Regarding Graft versus Host Disease GVHD prophylaxis; all patients received cyclosporine (CsA) at a dose of 3 mg/kg/day IV from day -1 until oral intake was possible then shifted to oral dose 5 mg/kg/day divided to 2 daily doses and maintained till day 270, then gradually tapered off. CSA was combined with methotrexate (MTX) in 79 patients (51.3%); at a dose of 15 mg/m2 IV on day +1, then 10 mg/m2 on days +3, +6, and +11. While 75 patients (48.7%) received CSA and methylprednisone 2 mg/kg starting from day -7 till day +4 then tapered gradually over two weeks. Results: The median time to achieve neutrophil recovery was 18 days (range 8-60 days) and for platelet recovery was 20.5 days (range 7-60 days). Graft failure rate was 12.6% (n=22); 12 (6.9%) as primary and 10 (5.7%) as secondary. The risk of GF was investigated in relation to risk class, serum ferritin level, stem cell source and use of ATG in conditioning regimen. Bone marrow as a source of stem cell was related to a higher graft failure rate (p=0.018) Five-year. Probabilities of overall-, disease-free and event-free survival in all patients were 74.9%, 61% and 55.8%, respectively. Five years overall survival was higher in patients with Pesaro risk class II (p=0.001). Moreover, DFS and EFS were significantly higher in patients with serum ferritin ≤2000 ng/ml (p =0.012, and 0.021 respectively). Transplant related mortality (TRM) was 18.4%, the leading causes of death were infection ( 40.5%), GVHD (16.7%)and respiratory failure(14.3%). Conclusion: Patients with low serum ferritin , Pesaro Risk Class I &I I and PBSC transplant have a more favorable outcome .Infection was the leading cause of mortality among our patients. Figure Disclosures No relevant conflicts of interest to declare.

The gene encoding Wilms Tumor 1 (WT1) is recurrently upregulated in Acute Myeloid Leukemia (AML). WT1 transcript burden is associated with primary refractory disease and with relapse when there is detectable WT1 minimal residual disease (Lambert et al., Oncotarget, 2014). We have recently described the gene regulatory networks in purified blasts from AML patients with various driver mutations (Assi et al., Nature Genetics, 2019) and found that WT1 was a key transcription factor node in all networks examined. Furthermore, in knockdown experiments as part of a shRNA depletion screen against transcription factors in vitro and in murine xenotransplantation experiments, we showed that WT1 was essential for leukemic maintenance (Martinez-Soria et al., Cancer Cell, 2018). WT1 produces 8 primary isoforms, all of which are expressed in hematopoietic cells by employing one of two transcription start sites and alternative splicing in exon 5 and exon 9. To further understand how WT1 maintains leukemia, we cloned each of the 8 isoforms into a doxycycline-inducible vector and transduced them into a t(8;21) AML cell line, Kasumi-1, which well recapitulates primary t(8;21) AML (Ptasinska et al., Leukemia, 2014). We carefully overexpressed each of the 8 WT1 isoforms to levels seen in AML patient samples and functionally and molecularly characterised them. Overexpression of isoforms lacking 3 amino acids in the zinc finger DNA binding domain (WT1 -KTS) significantly reduced cell growth and colony formation, increased apoptosis, caused a G1 cell cycle arrest and caused myeloid differentiation of leukemic blasts. However, overexpression of isoforms with the 3 amino acids (WT1 +KTS) in the DNA-binding domain had the opposite effects, thus having a major role in the maintenance of a leukemic phenotype. Also, we found that the expression of WT1 +KTS isoforms in purified blasts from patients was higher than WT1 -KTS isoforms and may therefore permit leukemic maintenance. We did not find any functional differences between isoforms employing alternate transcriptional start sites or between exon 5 splice variants. We then performed the first ever WT1 chromatin immunoprecipitation (ChIP-seq) in AML and show that WT1 -KTS shows increased binding as compared to WT1 +KTS with 9451 unique WT1 binding sites in WT1 -KTS expressing cells and only 1355 sites in WT1 +KTS expressing cells with 2391 sites overlapping between the isoforms. This finding suggests that WT1 isoforms compete for binding at multiple sites and may explain why the ratio of expression between WT1 isoforms is critical (Calabrese et al., Blood, 2012). We also correlated WT1 ChIP-seq peaks to transcription as assayed by RNA-seq. We found that differentially bound cis-regulatory elements are associated with altered expression of genes involved in proliferation, cell cycle, apoptosis and differentiation pathways, explaining many of the functional alterations in cellular behaviour that we see. Several vaccine studies and more recently a T cell receptor study targeted against WT1 have been undertaken but they have shown mixed efficacy in AML (Chapuis et al, Nature Medicine, 2019). Since we find that different isoforms of WT1 have antagonistic effects, we hypothesise that a more effective therapeutic strategy would be to selectively target only WT1 +KTS isoforms. Disclosures No relevant conflicts of interest to declare.

It is known that cells within the inflammatory background in classical Hodgkin lymphoma (cHL) provide signals essential for the continual survival of the neoplastic Hodgkin and Reed-Sternberg (HRS) cells. However, the mechanisms underlying the recruitment of this inflammatory infiltrate into the involved lymph nodes are less well understood. In this study, we show in vitro that HRS cells secrete lymphotoxin-α (LTα) which acts on endothelial cells to upregulate the expression of adhesion molecules that are important for T cell recruitment. LTα also enhances the expression of hyaluronan which preferentially contributes to the recruitment of CD4(+) CD45RA(+) naïve T cells under in vitro defined flow conditions. Enhanced expression of LTα in HRS cells and tissue stroma; and hyaluronan on endothelial cells are readily detected in involved lymph nodes from cHL patients. Our study also shows that although NF-κB and AP-1 are involved, the cyclooxygenase (COX) pathway is the dominant regulator of LTα production in HRS cells. Using pharmacological inhibitors, our data suggest that activity of COX1, but not of COX2, directly regulates the expression of nuclear c-Fos in HRS cells. Our findings suggest that HRS cell-derived LTα is an important mediator that contributes to T cell recruitment into lesional lymph nodes in cHL.

Understanding and blocking the self-renewal pathway of preleukemia stem cells could prevent acute myeloid leukemia (AML) relapse. In this study, we show that increased FOXO1 represents a critical mechanism driving aberrant self-renewal in preleukemic cells expressing the t(8;21)-associated oncogene AML1-ETO (AE). Although generally considered as a tumor suppressor, FOXO1 is consistently upregulated in t(8;21) AML. Expression of FOXO1 in human CD34+ cells promotes a preleukemic state with enhanced self-renewal and dysregulated differentiation. The DNA binding domain of FOXO1 is essential for these functions. FOXO1 activates a stem cell molecular signature that is also present in AE preleukemia cells and preserved in t(8;21) patient samples. Genome-wide binding studies show that AE and FOXO1 share the majority of their binding sites, whereby FOXO1 binds to multiple crucial self-renewal genes and is required for their activation. In agreement with this observation, genetic and pharmacological ablation of FOXO1 inhibited the long-term proliferation and clonogenicity of AE cells and t(8;21) AML cell lines. Targeting of FOXO1 therefore provides a potential therapeutic strategy for elimination of stem cells at both preleukemic and leukemic stages.

Background: Transient elastography (Fibroscan®) is an ultrasound technique used to measure liver stiffness (LS), and thus assess for liver fibrosis, in patients with various chronic hepatic disorders. It can also be used to predict severity in multiple other diseases that might affect LS such as amyloidosis and possibly conditions associated with iron overload. Objectives: To assess the frequency of liver fibrosis in patients with chronic hemolytic anemia using Transient elastography (Fibroscan®), and to determine the reliability of this tool as a non-invasive method to predict hepatic iron content as compared to liver iron concentration (LIC) measured by magnetic resonance imaging (MRI). Patients and methods: Seventy-five transfusion dependent patients (50 β-thalassemia major;25 sickle cell disease) with a mean age of 13.4±5.2 years in addition to 75 -age and sex matched- healthy children were recruited. All subjects underwent assessment of LS in kilopascals (kPa), by Transient elastography measurement using FibroScan (Echosens, Paris, France І). Steady state serum ferritin (SF), and hepatitis B serologies (HBsAg and antiHB core antibodies) were assessed by enzyme linked immunoassay (ELISA). LIC values, within 6 months' duration, as identified by quantitative MRI of hepatic iron stores as a signal intensity ratio method based on T1 and T2* contrast imaging without gadolinium were retrieved. Informed consent was obtained from patients' legal guardians prior to enrollment in the study. Results: The median SF was 2280 ng/ml (84% had values exceeding 1000 ng/ml). The median LIC was 13.86 mg/g dw (78.7% patients showed LIC above 7 mg/g dw). The median cardiac T2* was 30.8 ms (3 patients had values below 20). Fifty-two (69.3%) patients were categorized as F0-1 and 21 (28%) were stage F2, 2 (1.3%) were stage F3, and 2 patients had severe fibrosis. The mean and median fibroscan (FS) values were 6.19 ±1.76 kPa and 5.9 kPa (range 3 to 14.1) respectively. Patients had significantly higher mean FS compared to control group (p ˂0.001). Patients with no or mild fibrosis (F0-1) had lower FS values (5.3kPa) compared to patients with fibrosis grades 2-4 (p ˂0.001). FS values were not affected by disease type (thalassemia or sickle cell disease), age (above 12 years), or HCV sero-positivity. FS values correlated with SF (r=0.410, p˂ 0.001). Simple regression analysis of the two variables suggested that changes in SF were associated with minimal but significant changes in FS values (p=0.04) with good agreement (kappa =0.324, p=0.003). LIC did not differ in relation to grade of fibrosis (p>0.05), did not correlate with FS values (r= 0.014, p=0.908), and no changes in FS were expected with LIC changes on regression analysis (p=0.466) with low agreement between LIC and FS at cutoff value 5.3 kPa (kappa = 0.015, p=0.9). Sensitivity and specificity of FS values to predict LIC were high at cutoff values ranging between 3.2 to 3.75 kPa but decreased markedly at higher cutoff values. On comparing sensitivity and specificity of FS values in prediction of iron overload at different cutoff values by ROC curve, it could not significantly predict iron overload (p=0.7). No correlations were found between LIC and other variables including SF (r=0.2), and changes in SF were not significantly associated with changes in LIC values (p =0.089). However, sensitivity and specificity of SF in predicting LIC were good at cutoff 1003.85 ng/ml but decreased markedly at higher cutoff values. Comparing its sensitivity and specificity to that of SF in the prediction of iron overload at different cutoff values by ROC curve, FS could not predict iron overload accurately (p=0.9) and the degree of agreement between these two variables as indicators of iron overload was low (kappa=0.063, p=0.478). Conclusion: Fibroscan could be a valuable tool to assess the degree of liver fibrosis in patients with elevated SF, but it does not appear to reliably predict LIC in such group of patients especially with severe iron overload. FS values were not affected by disease type, age above 12 years, or HCV sero-positivity. Figure Figure. Disclosures No relevant conflicts of interest to declare.

Background and Purpose: Venous thromboembolism (VTE) is common in patients with sickle cell disease (SCD). Traditional risk factors such as central venous catheters, frequent hospitalization, orthopedic surgeries for avascular necrosis, and pregnancy often leads to an increased incidence of VTE in the SCD. In addition, SCD itself appears to be a hypercoagulable state with many SCD-specific factors such as genotype, splenectomy and thrombophilia modifying the risk of VTE. This study aims to assess the clinical and pathological characteristics of VTE amongst a cohort of patients with SCD at the Sultan Qaboos University Hospital and determine its relation to morbidity and mortality. Methodology: In this retrospective case control study, medical details of all patients with SCD who developed thromboembolic complications over the past decade were retrieved from the hospital information system. SCD patients matched for age and gender (2:1 ratio) who did not have thromboembolic complications but had a thrombophilia screen performed served as controls. The study was approved by the local Medical Research and Ethics Committee. Results & Discussion: A total of 53 SCD patients were enrolled [34 cases, 19 controls] in this study. Amongst the 34 cases (mean age-30 yrs.), 18 had pulmonary embolism, eight had deep venous thrombosis, whereas, three each had cerebral venous thrombosis and portal venous thrombosis and one each had cerebral arterial thrombosis and VTE. A higher incidence of autosplenectomy(69.7% v/s 52.6%) and central venous catheters(42.4% v/s 5.3%) were significantly associated with thrombosis (p Conclusions: The study shows that thromboembolic complications in SCD has a high impact on the morbidity and mortality. It confirms PE as the leading cause for VTE in SCD with asplenia, central venous catheter, high LDH, WBC and Platelet counts being significant risk factors. Disclosures No relevant conflicts of interest to declare.

Introduction: Sickle cell disease (SCD) is a major public health problem in Oman with high morbidity and mortality. The ability to identify the risk factors that are associated with mortality among SCD patients would permit accurate prognostication and provide an opportunity to use effective prophylactic management. Objective: The study aimed at identifying the risk factors associated with mortality in SCD patients by studying the clinical course of 86 patients who died at two tertiary care hospitals in Oman. Methods: We analyzed the electronic records of 86 SCD patients who died between 2006 to 2016. These data included cardiac parameters, SCD manifestations like frequency of painful crisis, acute chest syndrome, splenic and hepatic sequestration, dactylitis, and stroke. Furthermore, we also analyzed the haematological, biochemical and radiological parameters as well as use of antibiotics and exchange blood transfusion. Data were transcribed to Excel and later analyzed by IBM SPSS Ver23. Results±13.7. Previous complications prior to death included acute chest syndrome in 66.3% cases, followed by stroke in 20.9%, hepatic sequestration in 6.9%, splenic sequestration in 3.5% and dactylitis in 2.3% cases. At the terminal event, patients presented with fever, cough and crepitation in 47%, 29% and 36% respectively, while abnormal pulse, blood pressure, respiratory rate and O2 saturation were seen in 55%, 64%, 46% and 55% respectively. Further abnormal chest x-ray, CT scan and abdominal ultrasound were seen in 57.5%, 88.5% and 50% respectively. Amongst the haematology parameters, there was a significant drop in the mean hemoglobin and platelet counts from baseline, whereas there was a significant rise in the WBC counts (p Conclusion: Multi-organ failure was mostly associated with the terminal events relating to mortality. Although sepsis played a major role as a cause of death in association with multiorgan failure, low hemoglobin, and low platelet counts with increased WBC counts along with elevated LDH and CRP played a significant role in the terminal event in this cohort of SCD patients. Disclosures No relevant conflicts of interest to declare.

Introduction: Mutations in the epigenetic regulators are commonly found in myeloid disorders including Myeloproliferative Neoplasm (MPN). Primary myelofibrosis, dysplastic changes and severity of the disease were associated with the mutation load. Most of the studies had a limited number of targeted genes and included a mixture of JAK2 positive and negative disease. The objective of this study is to assess the impact of mutations in the epigenetic regulators on the presentation of patients with JAK2 V617F positive MPN. Methods: We retrieved the clinical and laboratory information on 61 consecutive eligible patients. Mutation analysis of the entire coding regions of ASXL1, ASXL2, CBL, CEBPA, CSF3R, DNMT3A, EZH2, IDH2, TET1 and TET2 genes was performed using next generation sequencing (NGS; Ion PGM Sequencer; Thermo Fisher ScientificÒ). The library was constructed and the templates were prepared using the PGM tool. The variants were annotated using the ClinVar database and the prediction from the Scale-Invariant Feature Transform (SIFT) and or Polymorphism Phenotyping (Polyphen) algorithms. Alignment, variant filtering and annotation were performed using Ion Torrent Suite. Standard descriptive and analytical statistics were used as appropriate to describe and compare different groups. The MPN subtype, bleeding, thrombosis, hemoglobin, platelet count, White Cell Count (WBC), Lactate Dehydrogenase (LDH) and erythropoietin level were compared for each candidate variant. An alpha threshold of 0.05 was used with no adjustment for multiple comparisons as the analyses were considered exploratory. All statistics were performed using R program. Variants were selected for further experimentation based on their frequency and association with the clinical information at diagnosis. Results: Sixty-one patients were included (Polycythemia Vera: 29, Essential Thrombocythemia: 21, Primary Myelofibrosis: 9, MPN unclassifiable: 2) with a median age of 62 years (Interquartile Range [IQR]: 44 - 70). Male to female ratio was 35:26. The median hemoglobin, WBC, platelet count, LDH and erythropoietin were 14.6 g/dL (IQR: 12.8 - 16.8), 11.5 *109/L (IQR: 11.5 - 14.4), 507 *109/L (IQR: 391 - 779), 265 mU/mL (IQR: 231 - 409) and 1.2 mU/mL (IQR: 1.0 - 4.8) respectively. At presentation, 54% had splenomegaly, 23% had an arterial or venous thrombosis, and 5% had bleeding. Sixty-three variants were found in the samples tested. The median mutation load was 13 variants (Range: 11-14). Patients with higher than the median mutation load had higher mean erythropoietin (7.8 vs. 0.9 g/dL; p = 0.02016). ASXL1 p.Leu815Pro variant was found in all patients. Only three variants were found in the ClinVar database. Seven variants were predicted to be pathogenic (ASXL1: 1, EZH2: 1, IHD1: 1, TET1: 1 and TET2: 3). Patients with TET2: p.Leu1721Trp variant had 6.4 higher odds of bleeding (p = 0.04345). Patients with TET2: p.His1778Arg variant had a lower WBC (9.1 vs. 13.9 *109/L; p = 0.01699) and LDH (213 vs. 348 mU/mL; p = 0.0006528) while those with TET1: p.Lle1123Met variant had a higher WBC (13.5 vs. 8.3 *109/L; p = 0.02756). None of the remaining comparisons were statistically significant. Conclusions: Incorporation of clinical information facilitates the prioritization of variants from DNA sequencing experiments. In MPN, we recommend ASXL1: p.Leu815Pro (expressed in all patients), TET2: p.Leu1721Trp (associated with bleeding), TET2: p.His1778Arg (correlated with the WBC and LDH) and TET1: p.Lle1123Met (correlated with LDH) for further functional experimentation. Disclosures No relevant conflicts of interest to declare.