Survival In Sickle Cell Disease Research Articles

Myeloid Neoplasia in Patients with Hereditary Hemoglobinopathies and Hemolytic Anemias (HHAs)

Background: Studies have shown an increased incidence of myeloid neoplasia (MN) in patients with sickle cell disease (SCD). It is hypothesized that increased erythropoietic stress may influence acquisition of molecular drivers of MN in SCD. However, present understanding of MN presentation and outcomes in SCD and other hereditary hemoglobinopathies and hemolytic anemias (HHAs) is limited to case reports. Systematic evaluation of MNs in patients with HHAs will better contextualize the risk of MN in this population relative to non-HHA controls. Methods: Patients with HHAs were identified from the Mass General Brigham Research Patient Data Repository using ICD-10 codes. Evaluated HHAs included sickle cell disease (SCD), sickle cell trait (SCT), thalassemia major, thalassemia trait, other hemoglobinopathy traits, and hereditary spherocytosis or elliptocytosis. A final HHA-MN cohort included patients with laboratory confirmed HHA diagnoses as well as incident myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), Ph+ chronic myelogenous leukemia (Ph+ CML), Ph negative myeloproliferative neoplasms (Ph- MPNs) or acute myeloid leukemia (AML). A control cohort of non-HHA MN patients was obtained from the Dana-Farber Hematologic Malignancies Data Repository. Clinical, molecular, and survival data were summarized and compared between HHA-MN and controls. Results: The HHA-MN cohort included 62 patients with HHAs and incident MN (Table 1). HHAs included SCD (n = 6, 9.7%), SCT (n = 9, 14.5%), thalassemia trait (n = 36, 58.1%), thalassemia major (n = 2, 3.2%), hereditary spherocytosis or elliptocytosis (n = 4, 6.5%), and other hemoglobinopathy traits (n = 5, 8.1%). MN was diagnosed at a median age of 61 years [IQR: 48, 70] in the HHA-MN cohort and incident MNs included AML (n = 12, 19.4%), MDS/CMML (n = 22, 35.4%), Ph+ CML (n = 6, 9.7%), and Ph- MPNs (n = 22, 35.4%). A control population of 3388 MN patients without HHA included cases of AML (n = 754, 22.2%), MDS/CMML (n= 1272, 37.5%), Ph+ CML (n = 354, 10.4%), and Ph- MPNs (n = 1008, 29.7%). Compared to HHA patients, control patients were older at MN diagnosis (median age 65 years [IQR: 56, 73], p = 0.0071), driven by significantly younger ages at AML diagnosis for HHA-MN patients (median age 50 years [IQR: 33, 63] vs 65 years [IQR: 57, 73], p =0.0044). AML was the most common MN in SCD (n = 4, 66.7%) whereas incident MDS/CMML was the predominant MN among individuals with thalassemia trait (n = 12, 33.3%) or hereditary spherocytosis and elliptocytosis (n = 2, 50%). Diagnostic cytogenetic data were available for 45 patients in the HHA-MN cohort (72.6% of cohort). Complex karyotype, defined as 3 or more chromosomal aberrations, was observed in 3 of 11 (27.3%) AML, 3 of 19 (15.8%) MDS/CMML and 1 of 17 (5.9%) Ph- MPN cases. Of the 33 HHA-MN patients with available diagnostic next generation sequencing, 60.6% had 2 or more pathogenic mutations at MN diagnosis. Including 2 cases of TP53-mutant AML in patients with SCD, the majority (90%) of AML and 38.9% of MDS/CMML diagnosed in HHA-MN patients were intermediate to adverse risk by ELN criteria or intermediate to very high risk by IPSS-R, respectively. High risk disease was also common in our control MN cohort, where 88.3% of AML cases were intermediate or adverse risk by ELN criteria and 55.9% of MDS/CMML cases were intermediate to very high risk by IPSS-R. Survival varied by subtype of HHA and MN (Figure 1). Survival was lowest for SCD (33.3% at 60-months) and highest for thalassemia traits (67.1% at 60 months). In HHA patients compared to controls, 60-month survival was 65.0% overall (vs 56.4%), 14.3% for AML (vs 23.8%), 51.0% for MDS/CMML (vs 43.9%), 90.5% in Ph- MPN (vs 80.6%), and 100% in Ph+ CML (vs 89.4%). Within the limitations of our sample size, survival differences were not statistically significant. Conclusion: In this retrospective analysis, we observe younger age of diagnosis of myeloid malignancy for patients with HHAs compared to non-HHA controls. Inferior survival in SCD and thalassemia major is largely explained by a higher proportion of AML diagnoses in patients with SCD. AML and MDS/CMML diagnosed in HHA patients tended to be intermediate or high-risk disease, with survival similar to the non-HHA control population that was also enriched for higher risk patients. Validation of our findings in an expanded cohort of HHA patients is currently underway.

Evaluating the implementation of a multi-level mHealth study to improve hydroxyurea utilization in sickle cell disease.

Sickle Cell Disease (SCD) is a progressive genetic disease that causes organ damage and reduces longevity. Hydroxyurea is an underutilized evidence-based medication that reduces complications and improves survival in SCD. In a multi-site clinical trial, part of the NIH-funded Sickle Cell Disease Implementation Consortium (SCDIC), we evaluate the implementation of a multi-level and multi-component mobile health (mHealth) patient and provider intervention to target the determinants and context of low hydroxyurea use. Given the complexity of the intervention and contextual variability in its implementation, we combined different behavioral and implementation theories, models, and frameworks to facilitate the evaluation of the intervention implementation. In this report, we describe engagement with stakeholders, planning of the implementation process, and final analytical plan to evaluate the implementation outcomes. During 19 meetings, a 16-member multidisciplinary SCDIC implementation team created, conceived, and implemented a project that utilized Intervention Mapping to guide designing an intervention and its evaluation plan. The process included five steps: (1) needs assessment of low hydroxyurea utilization, (2) conceptual framework development, (3) intervention design process, (4) selection of models and frameworks, and (5) designing evaluation of the intervention implementation. Behavioral theories guided the needs assessment and the design of the multi-level mHealth intervention. In designing the evaluation approach, we combined two implementation frameworks to best account for the contextual complexity at the organizational, provider, and patient levels: (1) the Consolidated Framework for Implementation Research (CFIR) that details barriers and facilitators to implementing the mHealth intervention at multiple levels (users, organization, intervention characteristics, broader community), and (2) the Technology Acceptance Model (TAM), a conceptual model specific for explaining the intent to use new information technology (including mHealth). The Reach Effectiveness Adoption Implementation and Maintenance (RE-AIM) framework was used to measure the outcomes. Our research project can serve as a case study of a potential approach to combining different models/frameworks to help organize and plan the evaluation of interventions to increase medication adherence. The description of our process may serve as a blueprint for future studies developing and testing new strategies to foster evidence-based treatments for individuals living with SCD.

The Benefits Trial of PB-04 to Evaluate Safety, PK, and Preliminary Efficacy in Inducing Fetal Globin Expression in Beta Thalassemia Intermedia and Sickle Cell Disease

Increased expression of fetal globin (HBG) has been shown to reduce clinical severity of beta-globin disorders and increase survival in sickle cell disease (SCD), through improved globin chain balance in beta thalassemia and reduced HbS polymerization. Pharmacologic approaches are considered most feasible for a majority of patients. Both proportions of F-cells and quantity of hemoglobin F (HbF)/cell are important for therapeutic effects. PB-04 was identified in a high-throughput screen of US and EU-approved drugs to activate HBG gene transcription, without cytotoxicity, to induce fetal globin expression (gamma globin mRNA, F-cells, HbF/cell, HbF), and to suppress or displace 4 repressors of the fetal globin gene promoter (BCL11A, LSD-1, KLF-1, and HDAC-3) in hemoglobinopathy patients' erythroid progenitors. PB-04 enhanced HbF in progenitors from hydroxyurea (HU)-treated patients with sickle cell disease. In in vivo studies, PB-04 induced fetal globin expression >20-fold with oral dosing in anemic baboons, and by 3.5-fold in mice transgenic for 2 copies of the human β-globin gene locus. This agent has been approved and in broad use for Parkinson's Disease treatment for 5 decades in Europe and Canada, to enhance the PK of an active Parkinson's agent, solely in a combination formulation. Because of its safety with chronic dosing up to 1300 mg/day, PB-04 is therefore of interest for repurposing in the treatment of thalassemia and SCD.This Ph1b trial (NCT004432623) will evaluate safety, tolerability, PK, and preliminary efficacy (fetal globin expression) with at least 3 escalating dose cohorts in up to 24 beta thalassemia intermedia (BTI) patients and 12 patients with sickle cell disease, > 18 years, both genders, with 12 weeks of treatment and 4 weeks of follow-up. Doses to be explored were selected from safe and active human equivalent doses in preclinical toxicology and efficacy studies in 2 species. Primary inclusion criteria include clinical diagnosis of BTI, including HbE beta thalassemia, with at least one beta globin gene mutation, or diagnosis of HbSS or HbSb thalassemia (after dose selection); total Hb levels 6-10 gm/dL. Exclusion criteria include red blood cell (RBC) transfusion within 2 months prior to administration of study medication, receiving regular transfusions, and hepatic or renal function > 3 x institutional ULN.Primary endpoints include occurrence, severity, and duration of adverse events by CTCAE v5.0 criteria and PK parameters. Secondary endpoints include change from baseline in assays of HbF expression, total Hb, markers of hemolysis . Exploratory endpoints include select polymorphisms associated with basal fetal globin expression and in vitro responses to the study drug. Statistical analysis will assess the rate of adverse events, PK parameters by dose, and changes in F-cells, F-reticulocytes, HbF/cell, HbF (% and total), compared to 2 averaged baseline values using the Wilcoxon signed rank test. Changes will be analyzed by mixed effect models during the 12-week dosing and 4-week follow-up period. Currently 3 dose cohorts with BTI are enrolled. This study, if successful, will provide a rational basis for definitive trials of an established safe oral therapeutic, with no overlapping toxicities with other experimental or approved agents, for combined use with HU and other therapeutics which enhance red cell viability in the globin disorders.The expertise and contributions of Jim Cradock of NCATS TRND to this work before his passing are gratefully acknowledged. DisclosuresKuo: Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Apellis: Consultancy; Bluebird Bio: Consultancy; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Honoraria; Alexion: Consultancy, Honoraria. Sheth: CRISPR: Consultancy; Bluebird bio: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; Chiesi: Consultancy; Imara: Research Funding; Agios: Consultancy; Dispersol: Research Funding. Al-Samkari: Moderna: Consultancy; Amgen: Research Funding; Rigel: Consultancy; Argenx: Consultancy; Novartis: Consultancy; Dova/Sobi: Consultancy, Research Funding; Agios: Consultancy, Research Funding. Pace: Imara Inc.: Consultancy. Kuypers: Forma Therapeutics, Inc.: Research Funding. Nouraie: Phoenicia BioScience Inc.: Consultancy. Perrine: Agios Pharmaceuticals: Consultancy; Emmaus Medical: Consultancy.

Selenium supplementation may improve COVID-19 survival in sickle cell disease.

Sickle cell disease is associated with lower selenium levels, and the serum selenium level is inversely associated with haemolysis in SCD. The SCD population is more vulnerable to adverse COVID-19 outcomes. SARS-CoV-2 infection lowers the serum selenium level and this is associated with severity of COVID-19. Selenium supplementation is proposed to improve COVID-19 outcomes in the sickle cell disease population.

A Systematic Review of the Effect of Iron Chelation Therapy on Survival in Patients with Sickle Cell Disease and β-Thalassemia

Background: Patients with sickle cell disease (SCD) and beta (β)-thalassemia often require regular blood transfusions; however, each unit of blood contains approximately 200 mg of iron, which can rapidly lead to iron overload (IO). This can result in iron deposition in the heart, liver, and endocrine organs, ultimately leading to organ failure, including cardiac disease, liver fibrosis, and metabolic disorders. The introduction of iron chelation therapy (ICT) with deferoxamine (DFO) and more recently deferiprone (DFP) and deferasirox (DFX) has had a significant impact on IO, demonstrating reductions in serum ferritin level and liver iron content. Although ICT has become the standard of care over the past few decades, there are limited data on the effects of these agents on overall survival. The objective of this systematic review is to evaluate the impact of ICT on survival in patients with transfusion-dependent SCD or β-thalassemia.Methods: This systematic review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols(PRISMA-P) guidelines. Electronic databases, including Embase, PubMed, Cochrane Library, and the World Health Organization (WHO) Clinical Trials Search Portal, along with American Society of Hematology annual meeting abstracts from 2010-2015 were searched to identify English language articles that were relevant to the topic. Search terms included “sickle cell anemia” or “SCA,” “sickle cell disease” or “SCD,” “beta thalassemia” or “thalassemia,” “iron chelation,” “iron overload,” “survival,” “death,” “study,” and “trial.” Abstracts identified by the literature were independently reviewed and only articles with unanimous support were included; when the eligibility of an abstract was disputed, a full copy of the article was retrieved for review. References were excluded for lack of evidence of survival analysis, comparison between regimens, or if trials had fewer than 25 patients. Details of the included studies were collected for categorization. Although a meta-analysis of the data was initially planned, there were too few studies with similar study parameters to complete the analysis.Results: For SCD, the literature search yielded 203 articles of which 6 were deemed relevant (Figure 1). Of these, 3 were excluded and 3 (published between 2001 and 2014) were included. Reasons for exclusion included small patient number, no comparator, and being a theoretical paper. Only 1 of the 3 was a randomized clinical trial (RCT). For β-thalassemia, theliterature analyses yielded 398 articles of which 40 were deemed relevant (Figure 2). After reviewing the articles, 22 were excluded while 18 (published between 1991 and 2015) were included. Reasons for exclusion included small patient number, no comparator, no survival data, no chelation data, or a focus on hematopoietic stem cell or bone marrow transplantation. Only 3 of the 18 were RCTs; the remainder were retrospective and/or longitudinal studies. Most data on effectiveness of ICT were from longitudinal studies that suggest treatment outcomes have improved significantly over time mainly due to the introduction of new and improved iron chelation agents, including oral treatments and combination therapy. Effectiveness was also investigated in studies evaluating treatment compliance that suggest survival is improved in patients compliant to ICT. Compliance has been enhanced with the introduction of oral agents such as DFP and DFX compared with DFO, which requires daily subcutaneous injections.Conclusions: This systematic review identified few RCTs of ICT reporting survival in SCD and β-thalassemia. Despite limited data, the available literature suggests that ICT improves overall survival in SCD and β-thalassemia. Survival may also have been affected by factors such as hydroxyurea usage and the number of blood transfusions. Newer oral treatments have improved compliance, shown to be an important factor in survival, although there have not been any prospective long-term RCTs adequately powered to compare the efficacy among available iron chelation agents in either SCD or β-thalassemia. [Display omitted] DisclosuresBallas:Novartis: Honoraria, Speakers Bureau. Zeidan:Takeda: Speakers Bureau; AbbVie, Otsuka, Pfizer, Gilead, Celgene, Ariad, Incyte: Consultancy, Honoraria; Otsuka: Consultancy.

Diet and Gender Influence Survival in Sickle Cell Disease

Increased childhood mortality and reduced longevity persist in the resource-limited settings with the highest populations of sickle cell disease (SCD) (Piel et al., New Engl J Med 2017). A high catabolic rate caused by multiple phenomena including compensatory erythropoiesis suggest increased calorie and nutritional requirements for individuals with SCD; however, a vast majority of individuals with SCD live under malnourished conditions. Also, it is unknown if the survival of offspring is influenced by the parents' diet and if the diet of individuals has a gender-specific effect on their survival.To address this critical unmet need for information under controlled conditions, we performed a randomized double-blind trial on transgenic Berkley mice with SCD comparing an enriched 'sickle diet' (SD) with a standard 'rodent diet' (RD). The study was performed following protocols approved by the Institutional Animal Care and Use Committee. Breeder pairs comprised of hemizygous (HbAS) females crossbred with homozygous (HbSS) male mice were divided into two dietary groups: [i] SD (59M3, TestDiet; St. Louis, MO) and [ii] RD (9F, 5020, Harlan Laboratories; Madison, WI). SD contained 26.4% protein, 11.1% fat, 27.5% kcal from protein, and 26% kcal from fat. RD contained 18.6% protein, 6.2% fat, 24% kcal from protein, and 18% kcal from fat. SD had a higher mineral, vitamin, amino acid, and omega-3 fatty acid content than RD. Pups were weaned 3 weeks after birth and were then genotyped and phenotyped to select HbSS pups. Pups were then fed SD for 10 days and randomly assigned to either [i] SD or [ii] RD for the remainder of the study. Their survival was followed until the pups were 150 days of age. Analysis was performed on the relationship between rates of survival and the following factors of interest: pup gender, pup diet, and parental diet. The outcome variable of interest, survival in days, was examined using (1) Kaplan-Meier plots of estimated survival and (2) Cox regression to obtain hazard ratios (HR). For Cox regression, the significance of individual factors adjusted for multivariate data and the significance of overall differences were based on Wald tests and log-rank tests respectively. Data were analyzed using the package 'survival' in ' R 3.4.0 ' (R Foundation for Statistical Computing; Vienna, Austria). P <.05 was considered significant.Of the 601 pups, 482 were on SD and 119 were on RD. After 150 days, the overall survival percentages of pups on SD and RD were 77% and 51% respectively. Pups fed SD had significantly prolonged survival rates compared to pups on RD when adjusted for multivariate data (p = 6.03*10-8, HR: .3988, 95% confidence interval (CI): .2860 - .5561). Cumulatively, there were 338 female pups and 263 male pups. After 150 days, the overall survival percentages of female and male pups were 76% and 68% respectively. Gender significantly independently impacted survival with females showing greater survival than males when adjusted for multivariate data (p = .0125, HR: 1.4797, 95% CI: 1.0879 - 2.0126). After 150 days, the overall survival percentages of the 488 pups with parents on SD and the 113 pups with parents on RD were 73% and 68% respectively. Pups born to parents fed SD showed a trend towards increased survival; however, parental diet did not have a statistically significant impact on the survival of pups adjusted for multivariate data (p = .5392, HR: .8878, 95% CI: .6071 - 1.2982). Notably, male pups on RD born to parents on RD showed the poorest survival compared to all other groups, and male pups on SD born to parents on RD showed a significantly improved survival in comparison (p=0.0228), suggesting that improving the individual's diet can overcome the effect of the parents' diet.A diet rich in protein, fat, and micronutrients significantly prolonged the survival of HbSS mice overall. HbSS females survived significantly longer than HbSS males. HbSS mice born to parents fed SD showed a trend towards increased survival; however, parental diet did not have a statistically significant impact on the survival of female pups. Overall, the data emphasized the importance of parents' diet, individual's diet, and gender in SCD. We speculate that an improvement of diet may reduce manifestations of SCD including pain, infections, inflammation, and organ damage and result in increased survival. These data also suggest the consideration of gender and diet in studies using mice with SCD to improve translational outcomes. DisclosuresGupta:Fera Pharmaceuticals LLC: Consultancy, Honoraria; Tautona Group: Consultancy, Honoraria.

Phase 1 Multiple Ascending Dose Study of Safety, Tolerability, and Pharmacokinetics/Pharmacodynamics of Mitapivat (AG-348) in Subjects with Sickle Cell Disease

Phase 1 Multiple Ascending Dose Study of Safety, Tolerability, and Pharmacokinetics/Pharmacodynamics of Mitapivat (AG-348) in Subjects with Sickle Cell Disease

Variation in hospital admission of sickle cell patients from the emergency department using the Pediatric Health Information System.

Universal newborn screening and improved treatment options have led to increased survival in sickle cell disease (SCD). However, patients with SCD still rely heavily on acute care services. To determine the variation seen in hospitalizations for the top complaints for ED visits for children with SCD nationally. We performed a retrospective review of the Pediatric Health Information Systems (PHIS) Database between October 2011 and September 2015. Emergency department (ED) encounters were selected by using International Classification of Diseases, Ninth Edition, Clinical Modification (ICD-9-CM) codes for SCD with and without crisis, fever, and pain. Univariate analyses were performed, as well as index of dispersion (ID) to assess variation by day of the week and region. ANOVA and t-test were used to determine statistical significance. A total of 68661 ED encounters at 36 hospitals met the criteria for inclusion. Of those encounters, 50.1% were admitted to the hospital. Pain and fever were the most common primary diagnoses among this population. Although variation in hospitalization was seen overall, as well as for a primary diagnosis of pain or fever, this variation was not explained by weekday/weekend designation. The results of our study confirm pain and fever as the most common primary diagnoses for children with SCD who seek acute care, as well as demonstrate that while significant variation in hospitalization exists, it is not associated with day of the week. Further studies to elucidate patient- and hospital-level factors that influence admission variation are necessary.

The effect of iron chelation therapy on overall survival in sickle cell disease and β-thalassemia: A systematic review.

Red blood cell transfusions have become standard of care for the prevention of life-threatening anemia in patients with β-thalassemia and sickle cell disease (SCD). However, frequent transfusions can lead to accumulation of iron that can result in liver cirrhosis, diabetes mellitus, arthritis, arrhythmias, cardiomyopathy, heart failure, and hypogonadotropic hypogonadism. Iron chelation therapy has been shown to reduce serum ferritin levels and liver iron content, but limitations of trial design have prevented any demonstration of improved survival. The objective of this systematic review was to investigate the impact of iron chelation therapy on overall and event-free survival in patients with β-thalassemia and SCD. Eighteen articles discussing survival in β-thalassemia and 3 in SCD were identified. Overall iron chelation therapy resulted in better overall survival, especially if it is instituted early and compliance is maintained. Comparative studies did not show any significant differences between available iron chelation agents, although there is evidence that deferiprone is better tolerated than deferoxamine and that compliance is more readily maintained with the newer oral drugs, deferiprone and deferasirox. Iron chelation therapy, particularly the second-generation oral agents, appears to be associated with improved overall and event-free survival in transfusion-dependent patients with β-thalassemia and patients with SCD.

Studies of novel variants associated with Hb F in Sardinians and Tanzanians in sickle cell disease patients from Cameroon

High level of Hb F has been shown to improve survival in sickle cell disease. Among 453 Cameroonians with sickle cell disease, we have investigated 18 selected single-nucleotide polymorphisms (SNPs) in novel and suggestive loci associated with Hb F level identified through a genomewide association study in sickle cell disease patients in Tanzania, and whole-genome sequencing of a population from Sardinia. Seven of 10 variants reported in Sardinians were either monomorphic or very rare in the Cameroonians. No associations were observed with any SNPs and Hb F levels in Cameroonians affected by sickle cell disease. The present study illustrates the complexity of replicating Hb F-promoting variants association results across populations.

Activation of the erythroid K-Cl cotransporter Kcc1 enhances sickle cell disease pathology in a humanized mouse model

AbstractWe used an N-ethyl-N-nitrosurea-based forward genetic screen in mice to identify new genes and alleles that regulate erythropoiesis. Here, we describe a mouse line expressing an activated form of the K-Cl cotransporter Slc12a4 (Kcc1), which results in a semi-dominant microcytosis of red cells. A missense mutation from methionine to lysine in the cytoplasmic tail of Kcc1 impairs phosphorylation of adjacent threonines required for inhibiting cotransporter activity. We bred Kcc1M935K mutant mice with a humanized mouse model of sickle cell disease to directly explore the relevance of the reported increase in KCC activity in disease pathogenesis. We show that a single mutant allele of Kcc1 induces widespread sickling and tissue damage, leading to premature death. This mouse model reveals important new insights into the regulation of K-Cl cotransporters and provides in vivo evidence that increased KCC activity worsened end-organ damage and diminished survival in sickle cell disease.

Nano-Formulated Hydroxyurea Enhances Drug Delivery in K562 Cells

The vasculopathy of sickle cell disease (SCD), characterized by a chronic hemolytic anemia, endothelial dysfunction and recurrent vaso-occlusive events, underlies most of the clinical complications. Hydroxyurea (HU), the only FDA approved medication to treat patients with SCD, acts via the induction of fetal hemoglobin (HbF) production. Recent studies suggest that HU increases endothelial nitric oxide (NO) production and reduces adhesion molecule expression as another potential mechanism for the observed reduction in vaso-occlusive events and improved survival in SCD. Despite its benefits, HU is currently under-utilized and we hypothesize that improved targeted drug delivery may increase its usage. The use of nano-emulsions, a class of stable emulsions formed by a monolayer of phospholipids and/or biodegradable material, is a mechanism by which to enhance targeted cellular delivery of therapeutics. We hypothesized that a nano-formulation of HU would enhance drug delivery to the bone marrow and this may be a direct way of impacting HbF expression in SCD. Nano-formulated HU (NHU) was developed using a phospholipid approach and this compound was found to be biologically stable for > 6 months.Chronic myelogenous leukemia bone marrow (K562) cells were treated with a NHU or HU suspension in concentrations ranging from 25µM to 250µM over a course of 4, 5 and 6 days. Cells were harvested for RNA isolation and qPCR was performed using primers for HbF and basal cell adhesion molecule (BCAM, a cellular adhesion molecule on red and white blood cells. On day 5, when HbF expression in K562 cells peaks, there was a five-fold increase in HbF expression treated with lower concentrations of NHU compared to untreated cells and a two-fold increase in expression compared with HU treated cells. With increasing concentrations of NHU and HU, HbF expression became similar. This suggests that the use of NHU can provide increased HbF expression at a lower concentration with likely less toxicity. Treatment of K562 cells with NHU and HU resulted in decreased BCAM expression on day 6 of treatment. Treatment with NHU produced a four-fold decrease in BCAM expression compared to the same concentration of a HU suspension which supports the concept that NHU can impact gene expression at lower doses than unformulated HU. Nanoformulated HU produces a molecule which is highly stable, and able to achieve higher intracellular concentrations with increased efficacy in K562 cells. This suggests a potential novel therapeutic for patients with sickle cell disease. DisclosuresKlings:Pfizer: Consultancy; Actelion Pharmaceuticals: Research Funding.

Survival in Sickle Cell Disease: Data from a Well-Resourced, National Health System Setting

Background: In well-resourced countries, early mortality in sickle cell disease (SCD) is no longer a major feature, thus the burden of SCD mortality has shifted to adults. Recent data on patients with SCD in USA confirmed continuing improvement in adult survival, attributed to better preventative care. Health economics may play a key role in clinical outcome in SCD; we hypothesise that survival would be longer in a system which is not insurance based. King's College Hospital, London, UK has a large SCD cohort in a well-resourced setting within a national health service framework.Methods: 712 adult (≥18 years) SCD patients were observed over 10 years (2004-13 inclusive) to identify mortality outcome (total 5268 patient-years of observation with median 8 years of observation per patient, over median 9 total hospital visits). Demographic data, sickle and alpha genotype were documented. Laboratory data were also collected (list in table): outpatient laboratory values were averaged over the 10 year period to create a "steady state" value for each patient. Mean number of hospital admissions with a sickle pain crisis was calculated (total admissions/number of observed years per patient).IBM SPSS Statistics 22 was used for Kaplan Meier survival analysis and then regression analysis was undertaken, based on the Cox proportional hazards model, to identify variables which were associated with mortality, and produce hazard ratios for putative predictors (demographic, laboratory and admission data).Results: 43 patients (6.0%) died during the study at a median age of 42 years (IQR: 31-48). Kaplan Meier analysis showed an estimated median survival of 77 years (95% CI: 68-85) for all sickle genotypes. Sub-analysis by sickle genotype showed: HbSS/HbSβ0 thalassaemia together (33 deaths in 450 patients) with estimated median survival 67 years; and HbSC (8 deaths in 229 patients) with significantly higher estimated median survival 82 years (p<0.001), see figure 1. For both genotypes, there was no gender difference on sub-analysis. For HbSS/HbSβ0, there was a significant difference by hospitalisation rate with survival: median survival in those with ≤0.5 admissions/year was 67 years, significantly higher than 60 years (CI: 43-77) for high admission rates (p=0.001), see figure 2.Cox regression analysis was conducted for HbSS/HbSβ0 patients (demographic, laboratory and admission data), see table. Risk of death is increased: 2.5-fold if ferritin is high (>1000ug/L), 6% for every extra 10umol/L total bilirubin, 20% for every extra 100 IU/L LDH, 18% for every extra 1 x109/L WBC (all steady state values). Hospitalisation rates were analysed two ways: using the continuous variable, risk of death is increased by 37% for every admission accrued in an individual's mean admission/year statistic; and using a dichotomous variable (high versus low admissions), those with high admission rates have a hazard ratio over 3-fold that of those with low admission rates.Conclusions: These data indicate that adult survival in SCD in our institution is high; estimated median survival of 67 years for HbSS/Sβ0 and 82 years for HbSC. This is markedly longer than those recently quoted from the USA, which may be due to disparities in health economics. Cox regression sub-analysis for the HbSS/Sβ0 group reveals multiple "predictors" of increased mortality risk including classical laboratory markers of SCD severity - iron loading (likely correlated with disease severity via transfusion rate), inflammatory and haemolytic parameters - as well as confirming hospitalisation rate as a simple but strong predictor of survival in SCD.TableCox regression analysis for HbSS/HbSβ0 thalassaemiaHbSS/HbSβ0 thalassaemia (n=450, 33 deaths)Hazard ratio (CI)p valueDemographicsAlpha Thalassaemia*1.342 (0.666-2.706)0.411Sex* (M=default)0.674 (0.339-1.340)0.261LaboratoryTotal bilirubin umol/L1.006 (1.004-1.008)<0.000001C-reactive protein (CRP) mg/L1.019 (0.998-1.042)0.079High Ferritin >1000ug/L*2.518 (1.212-5.232)0.013Lactate dehydrogenase (LDH) IU/L1.002 (1.000-1.004)0.038WBC x109/L1.181 (1.037-1.345)0.012Hb g/L0.979 (0.956-1.002)0.073Reticulocytes x109/L1.002 (0.999-1.005)0.285AdmissionsMean number admissions / year1.374 (1.159-1.630)<0.001High admission rate (>0.5/year)3.131 (1.567-6.259)0.001*Dichotomous variables [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Use of Nano-Formulated Hydroxyurea Enhances Drug Delivery to Pulmonary Artery Endothelial Cells

The vasculopathy of sickle cell disease (SCD), characterized by a chronic hemolytic anemia, endothelial dysfunction and recurrent vasoocclusive events, underlies most of the clinical complications. Recent work has suggested that hydroxyurea (HU) enhances endothelial nitric oxide (NO) production and reduces adhesion molecule expression which may contribute to its associated observed reduction in vasoocclusive events and improved survival in SCD. However, clinically HU is under-utilized, at least in part related to medication related toxicity, stressing a need for enhanced drug delivery. The use of nano-emulsions, a class of stable emulsions formed by a monolayer of phospholipids and/or biodegradable material such as chitosan, is a mechanism by which to enhance targeted cellular delivery of therapeutics. We hypothesized that a nano-formulation of HU would enhance drug delivery to the endothelium and may be a direct way of targeting endothelial dysfunction in SCD. Nano-formulated HU (nano-HU) was developed using a phospholipid approach and this compound was found to be biologically stable for > 6 months. Human pulmonary arterial endothelial cells (HPAECs) were co-incubated with 25µM, 125µM and 250µM HU, nano-HU, or the empty nano-emulsification preparation for 48 hours. HPAEC were isolated, and stained with propidium iodide (PI) for cell cycle analysis by FACS. HPAEC treated with 25µM nano-HU demonstrated the highest level of cell viability (55%) and similar levels of S phase inhibition as HPAEC treated with 125µM HU (where 10-20% cell viability was observed). RNA isolation of treated HPAECs was performed and qPCR for VCAM1 and SLX4, a regulator of DNA damage which promotes S phase inhibition was performed. 25µM nano-HU significantly decreased VCAM1 and increased SLX4 expression compared with 25µM HU suggesting increased efficacy at lower levels of toxicity. The impact of nano-HU on endothelial gene expression is currently being evaluated by RNA-seq. Nano-formulation of HU produces a molecule which is highly stable, and able to achieve higher intracellular concentrations with lower rates of toxicity in HPAECs .which provides the opportunity to gain greater understanding of the impact of this medication on the vascular endothelium and the potential for a more targeted and better tolerated delivery of this therapeutic to patients with sickle cell disease. DisclosuresKlings:Pfizer: Consultancy.

Factors associated with survival in a contemporary adult sickle cell disease cohort: Factors Associated with Survival in Sickle Cell Disease

Factors associated with survival in a contemporary adult sickle cell disease cohort: Factors Associated with Survival in Sickle Cell Disease

Sudden Death in Adult Sickle Cell Patients: A Single Institutional Experience

Abstract 1061▪▪This icon denotes a clinically relevant abstractIn 1994 Platt et al published the Cooperative Study of Sickle cell Disease (CSSCD) which evaluated the course of nearly 4,000 children and adults with sickle cell disease. This study was one of the largest studies to look at survival in sickle cell disease and suggested a median survival of 42 years for males and 48 years for females with homozygous SS disease. In the past 20 years numerous supportive care measures have been implemented and the impact of these on survival of our adult sickle cell patients is unclear. We undertook a retrospective review of charts and autopsies for all patients in our adult sickle cell clinic that died from the time of inception until 2010 to determine if there was any impact on cause of deathWe performed a retrospective chart review study of all deaths of our adult sickle cell patients since inception of the clinic in 1998. Of the 260 patients actively enrolled in our clinic, 36 died during the time period of 1998 to 2010. Thirty four patients had sickle cell disease, 2 patients had sickle beta thalassemia, and no patient had Hemoglobin SC disease. Data regarding cause of death was determined from autopsy reports and from hospital charts at the time of death. The median age at death was 40 yrs. Despite most patients dying at or before age 40, only 9 of the 36 (25%) patients had autopsies. The cause of death in the autopsies obtained included liver infarct(1), drug overdose (2), cardiomyopathy (3), pulmonary emboli(1), coronary artery dissection(1) and chest syndrome(1). Of the patients who did not undergo autopsy, cause of death was evident in the hospital chart in 9 of the remaining 27 patients. In this group of patients cause of death was noted to be intracranial hemorrhage (2), sepsis (3), small bowel obstruction (1), cancer (1), chest syndrome (1) and heart failure (1). The remainder of the 18 patients died unexpectantly of sudden cardio respiratory arrest of undetermined etiology. Five of these patients died at home and 13 patients died within 48 hours of being hospitalized for acute pain crisis. In chart review of these 18 patients 7 patients had hypertension and 9 patients had renal failure. Of these 18 patients prior echocardiographic data was available in 14 patients. Interestingly, 7 of the 14 patients had TR jet velocity >2.5 m/second. The incidence of TR jet velocity >2.5 m/sec is higher than the 25–30% cited by Gladwin et al in a general sickle cell population.In summary despite numerous advances in supportive care over the past 15 years since the CSSCD the mean age of death in our small group of patients was still in the fourth decade. As in the CSSCD the majority of patients still died of cardiac and pulmonary complications many of which were not evident at the time of hospitalization. This retrospective data suggests close monitoring of these patients during the first two days of hospitalization given the high incidence of sudden cardiac death in this population. This retrospective review also suggests the necessity for improved screening and treatment modalities to prevent cardiopulmonary complications such as pulmonary hypertension in this population. Disclosures:No relevant conflicts of interest to declare.

Genomic Assessment of Mortality in Sickle Cell Disease- A Novel Association with Cytokine Imbalance and T Cell Dysregulation

Abstract 1081 Rationale:Sickle cell disease (SCD) is characterized by systemic complications and significant clinical variability. While some patients never experience severe clinical events, others suffer lifelong morbidity and accelerated mortality. To address this variability, we prospectively followed patients with SCD at steady-state to assess outcomes and leveraged microarray analysis of RNA isolated from peripheral blood mononuclear cells (PBMCs) to identify novel gene expression patterns and signaling pathways that could be associated with survival in SCD. Methods:Forty-two adult patients with SCD at steady-state (no VOC or ACS episodes for >3 weeks) were followed for 5 years (median follow-up 1154 ± 317 days). At baseline enrollment, patients underwent phlebotomy for plasma and serum collection. The plasma was utilized to extract mRNA from PBMCs. The serum was used to assess targeted cytokine levels using high-sensitive cytokine kits. Microarray expression analysis of PBMC mRNA utilized an FDR<10% and a fold-change >1.2. Student t-tests were performed for statistical significance. Results:Five out of forty-two patients (∼12%) died over the course of the study (median follow-up 1093 ± 320 days for surviving patients vs 1246 ± 325 days for patients who died, p=0.42). Although there were trends towards older age, higher tricuspid regurgitation jet velocity, NT-pro-BNP levels, AST levels, and lifetime number of transfusions in the patients that died, there were no statistically significant differences between groups (Table 1). Patients who died (n=5) exhibited 278 differentially regulated genes in comparison to those who survived (n=37). Gene ontology analysis revealed significantly represented T-cell receptor and immune mediated pathways, comprising seven out of the top ten pathways (p<0.05). Using a fold-change of 1.5 threshold on the transcripts, we identified a 14 gene signature which discriminated the survival of patients with 100% accuracy. Multiple genes within this molecular signature for survival also included immune-mediate pathways including CD160, nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (NFKBIA), tumor necrosis factor, alpha-induced protein 3 (TNFAIP3), interleukin 1, beta (Il1B), killer cell lectin-like receptor subfamily G, member 1 (KLRG1), CD3d molecule, delta (CD3D), and killer cell lectin-like receptor subfamily F, member 1 (KLRF1). Given this robust representation of immune-mediate pathways in the signature and ontology analysis, we measured targeted plasma cytokine levels (based on candidates known to be involved in SCD) in all patients. IL-6 (5.7 ± 4.8 vs 11.7 ± 4.8 pg/mL, p=0.006) and IL-8 levels (3.5 ± 2.3 vs 6.2 ± 2.3 pg/mL, p=0.009) were significantly higher during steady-state in patients who later died than in those who survived. Interestingly, the anti-inflammatory cytokine IL-10 (35.2 ± 109.6 vs 16.1 ± 15.6 pg/mL) trended higher in the survival group while tumor necrosis factor levels trended lower (38.2 ± 28.0 vs 48.8 ± 38.5 pg/mL) but this did not reach statistical significance.Table 1Clinical characteristics of study patientsClinical CharacteristicsPatients who survived (n=37)Patients who died (n=5)P valueAge (years)38 ± 1247 ± 100.07Gender (M/F)22/153/20.65Systolic/Diastolic Blood Pressure (mm Hg)122/70 ± 11/9123/69 ± 12/90.37TRV (m/s)2.5 ± 0.302.6 ± 0.350.27Transfusions (#)3.1 ± 1.34.0 ± 1.20.08History of ACS (#)13 (35%)2 (40%)0.56# of ACS episodes1.0 ± 1.91.0 ± 2.00.47WBC Count (10−3/mm3)9.6 ± 3.39.6 ± 5.30.49Hemoglobin (g/dl)9.3 ± 1.99.4 ± 2.10.44Platelet Count (10−3/mm3)321 ± 143342 ± 1440.37Creatinine (mg/dl)1.1 ± 1.60.7 ± 0.20.30AST (U/L)36 ± 1446 ± 340.10Total Bilirubin (mg/dL)2.7 ± 1.32.0 ± 1.00.13Ferritin (mg/L)1209 ± 1860782 ± 4140.30Pro-NT-BNP (ng/L)112 ± 125125 ± 2100.42 Conclusion:These preliminary data suggest that a PBMC genomic signature is a potential prognostic biomarker in SCD. Patients who subsequently died demonstrated a unique PBMC-gene expression profile with significant representation of specific T-cell and immune-mediated pathways. Concurrently, these at-risk patients also demonstrated higher serum IL-6 and IL-8 levels. Given that high circulating levels of these type 2 cytokines suppress both humoral and cell-mediated immune functions, we speculate that T-cells may play novel role in the variable morbidity and mortality observed in SCD. Disclosures:No relevant conflicts of interest to declare.

Relationship of erythropoietin, fetal hemoglobin, and hydroxyurea treatment to tricuspid regurgitation velocity in children with sickle cell disease

AbstractHydroxyurea and higher hemoglobin F improve the clinical course and survival in sickle cell disease, but their roles in protecting from pulmonary hypertension are not clear. We studied 399 children and adolescents with sickle cell disease at steady state; 38% were being treated with hydroxyurea. Patients on hydroxyurea had higher hemoglobin concentration and lower values for a hemolytic component derived from 4 markers of hemolysis (P ≤ .002) but no difference in tricuspid regurgitation velocity compared with those not receiving hydroxyurea; they also had higher hemoglobin F (P < .001) and erythropoietin (P = .012) levels. Hemoglobin F correlated positively with erythropoietin even after adjustment for hemoglobin concentration (P < .001). Greater hemoglobin F and erythropoietin each independently predicted higher regurgitation velocity in addition to the hemolytic component (P ≤ .023). In conclusion, increase in hemoglobin F in sickle cell disease may be associated with relatively lower tissue oxygen delivery as reflected in higher erythropoietin concentration. Greater levels of erythropoietin or hemoglobin F were independently associated with higher tricuspid regurgitation velocity after adjustment for degree of hemolysis, suggesting an independent relationship of hypoxia with higher systolic pulmonary artery pressure. The hemolysis-lowering and hemoglobin F–augmenting effects of hydroxyurea may exert countervailing influences on pulmonary blood pressure in sickle cell disease.

Improved Survival of Children and Adolescents with Sickle Cell Disease.

The survival of children with sickle cell disease (SCD) has improved over the past several decades, especially for very young children. However, we know less about mortality during adolescence, and we do not accurately know the current proportion of children born with SCD who survive to adulthood. The first report from the Dallas Newborn Cohort (DNC), which included follow-up through June 2002, estimated overall survival at 18 years of age to be 85.6% (95% C.I.: 73.4 – 97.8) for individuals with sickle cell anemia (SS) or sickle-β0-thalassemia (Sβ0) (Blood 2004; 103:4023–7). The confidence interval of this estimate was wide because only 8 cohort subjects were 18 years old at the time. Here we update the survival analysis with 5 more years of accrual and follow-up to provide an accurate, contemporary estimate of mortality for patients with SCD through 18 years of age. The DNC includes all individuals with SS, Sβ0, sickle-hemoglobin C disease (SC), or sickle-β+-thalassemia (Sβ+) who were diagnosed by the newborn screening program of Texas (initiated November 1, 1983) and seen at least once in our center. Subjects were analyzed in two separate groups because of clinical similarity: SS/Sβ0 and SC/Sβ+. Overall survival was analyzed by the Kaplan-Meier method. Subjects were censored at the time of their last clinical encounter. Between July 2002 and July 2007 we identified 229 new members of the DNC and added 3,201 additional patient-years of follow-up. The cohort now includes 940 subjects (572 SS, 284 SC, 63 Sβ+, 21 Sβ0; 52.8% male), and it provides a total of 8,857 patient-years of follow-up (5,819 SS/Sβ0 patient-years, 3,039 SC/Sβ+ patient-years). Mean follow-up is 9.4 years (range 0.1– 20.6 years), and 97 subjects are now at least 18 years of age at last follow-up. To date, 92 subjects (9.8%) have been lost to follow-up (not seen for >5 years), and 33 subjects have died (30 SS/Sβ0, 3 SC/Sβ+). There were 7 new deaths in DNC patients since 2002, all of which occurred in patients who were 18 years of age or older. Of all deaths, 23 were SCD-related (5 acute chest syndrome, 5 sepsis, 4 multi-organ failure syndrome, 9 other), and 10 were apparently unrelated to SCD (4 trauma or accidental death, 6 unrelated medical conditions). All SC/Sβ+ deaths were apparently unrelated to SCD. Overall survival at 18 years was 93.9% (95% C.I. 90.3 – 96.2; 81 patients > 18 years of age) for SS/Sβ0 subjects and 98.4% (95% C.I. 94.4 – 99.5; 16 patients > 18 years) for SC/Sβ+ subjects. The overall incidence of death in the SS/Sβ0 and SC/Sβ+ subgroups was 0.52 and 0.10 per 100 patient-years, decreased from 0.59 and 0.24 in the original DNC analysis. Survival also appears to be improving across cohort eras (Figure). In conclusion, approximately 6% of children with SS or Sβ0 die during childhood, but almost all children with SC or Sβ+ live to become adults. Although early childhood mortality has greatly decreased, we show that many adolescents and young adults still die from SCD. Notably, acute chest syndrome and multi-organ failure have now surpassed sepsis as the leading causes of death. These data provide the accurate, contemporary foundation for the counseling of parents of newborns with SCD and for genetic counseling for prospective parents. Finally, given the marked decrease in early mortality we show here, new efforts to improve survival in SCD should focus on adolescents and young adults. [Display omitted]

Predictors of In-Hospital Mortality and Charges in Sickle Cell Disease: Results from the California Discharge Databases 1998–2005.

Most studies of survival in sickle cell disease (SCD) include only patients followed at referral centers. We used the public set of the California Patient Discharge Databases (1998–2005) to compare in-hospital mortality and charges by patient characteristics. We included discharges with any diagnostic code for SCD (282.41–2, 282.60–69). We used the Charlson index (adapted for administrative data) to adjust for comorbid conditions. Hospital charges were adjusted to 1998 levels. Statistical methods included ANOVA, Wilcoxon rank-sum, and multivariate linear and logistic regression. We identified 57,887 admissions for SCD and 376 in-hospital deaths (0.65%) from 1998–2005. The mean age of death was 40 years (95% CI 38–42) for sickle cell anemia (HbSS) and 48.3 years (95% CI 44.1–52.5, p<.0005) for all other SCD. Median length of stay was 4 days (IQR 2–7 days) and charges were $10,027 (IQR 5547–18,302) for HbSS and 4 days (IQR 2–6 days) and $8045 (IQR 4375–16,253) for all other SCD (p<.0001). Women with SCD were older at the time of death (43.6 years) than men (41.4 years, p=.29) and age of death increased from 40.6 years in 1998–2000 to 44.2 in 2004–2005 (p=.17) but these differences were not significant. Mortality was increased with a Charlson Index of 1 or 2, older age, private insurance, a diagnosis of HbSS, and transfusion during the admission (Table 1). Women had a lower odds of death than men (OR 0.7, p<.05). Average annual charges for hospitalization in patients with SCD, adjusted to 1998 values, were $117,000,000 and decreased $1,960,000/year (−4.1%, p<.0001) for children and increased $1,150,000/year (1.4% p<0.05) for adults. Adjusted charges for all children were stable and increased 1.5% per year for adults. Charges per admission were $3167 higher for adults than children, $5608 higher per comorbid diagnosis, $6506 higher in transfused patients, and $3595 higher with a diagnosis of crisis (p<.0001 for all). Compared to MediCal, charges were $1186 higher for Medicare (p<.05), $2651 lower for other government insurance (p<.0001), and $3521 lower for self-pay (p<.05). Government (77%) and private insurance (21%) paid for most admissions. We identified a greatly increased risk of in-hospital morality with comorbid diagnoses and older age and moderately increased risk with private insurance, HbSS genotype, and transfusion during the admission. The increased risk with private insurance was surprising, as higher socioeconomic status is often associated with better health outcomes. This may reflect more stringent requirements for admission or financial barriers to outpatient and preventive services for SCD. Total charges decreased dramatically in children, possibly reflecting increased use of hydroxyurea for frequent pain and chronic transfusions for primary stroke prevention.Table 1:Odds Ratio for In-Hospital Death Based on Multivariate Logistic RegressionVariableOdds Ratio (95% CI)P-ValueCharlson Index=13.3 (2.4–4.6)<.0001Charlson Index=212.9 (9.3–18)<.0001Age (per year)1.04 (1.03–1.05)<.0001Private Insurance vs. MediCal1.6 (1.2–2.2)<.0005Medicare vs. MediCal0.9 (0.7–1.3)NSOther SCD vs. HbSS0.7 (0.5–0.9)<.0005Transfusion1.6 (1.2–2.0)<.0001NS indicates not significant