ALBERT

Description: Iron overload is an inevitable consequence of multiple transfusions and occurs in many hematological diseases including sickle cell anemia (SCA) and beta thalassemia (β-thal). Liver biopsy provides quantification of iron content in the liver, but is not without risks such as bleeding, pain, and infection. MRI R2* has the advantage of quantifying liver iron without the risks of invasive procedures, however, this technique has not been fully investigated or validated. Furthermore, the variability introduced by multiple MRI readers has not been investigated to date. Patients with hematological diseases were selected to participate in this prospective IRB-approved study if they received ≥ 18 transfusions or had a serum ferritin ≥ 1000 ng/mL. All study participants completed 1.5 Tesla MRI R2* testing (Siemens Symphony), serum ferritin, and liver biopsy with quantification of liver iron content (LIC) within 30 days. Regions of interest (ROI) were drawn on R2* maps in a homogeneous area of the right hepatic lobe, avoiding blood vessels and obvious bile ducts. Three independent reviewers, blinded to the patients’ clinical status and the other 2 reviewers’ results, performed the ROI analysis. The correlation between LIC and liver R2* was calculated using the Spearman’s Rank-Order Correlation Coefficient. Due to possible outliers in the data, robust simple linear regression methods were used to fit a regression line to scatter plots. All liver biopsy samples were sent to Mayo Laboratories for LIC quantification. The agreement among the 3 raters was assessed using the interclass correlation coefficient (ICC). Forty-seven patients, median age 14 years (range 7 – 37) participated; 24 were female. Thirty-five of them had SCA, 8 had β-thal (major or intermedia), and 4 had bone marrow failure syndromes. Total table time for R2* MRI testing was between 20 to 30 minutes. All patients tolerated the liver biopsy without complications. The mean (±1SD) ferritin was 2917ng/mL (±2239), mean LIC was 12.139 mg/ 100g of dry weight liver (±8.269), and mean liver R2* ranged from 425 to 432 Hz (±257 to 249 Hz). All 3 raters produced R2* values strongly associated with LIC, with correlation coefficients from 0.93 to 0.95 (p

Description: Introduction: Unsuccessful transition from pediatric to adult care among youth with sickle cell disease (SCD) contributes to frequent disease complications and early death. The literature is sparse on longitudinal SCD transition program outcomes. The St. Jude SCD Transition to Adult Care Program started in 2007, is a comprehensive, co-located, and interdisciplinary program embedded in a larger SCD medical home. The objective of this study is to describe transfer rates, time to transfer, and maintenance of adult care over a 9-year period. Methods: With St. Jude IRB approval, we conducted a retrospective review of the rate of matriculation in adult care, the latency time from leaving pediatric care and initiating adult care, and maintenance of engagement in adult care 12 and 24 months later. Eligibility criteria: 1) SCD diagnosis and 2) Completion of pediatric care between 2007 and 2016. Results: Three hundred and sixty participants transferred to adult care over the study period. Mean age at transfer was 18 years. Matriculation in adult care within 6 months from leaving pediatric care substantially increased: 71% in 2007 to 93% in 2016. We observed a progressive reduction in the latency time from pediatric to adult care, from 12 months in 2007 to 1.5 months in 2016. Fifty-five percent (n=198) of youth who transitioned to the partner Methodist Comprehensive Sickle Cell Center (MCSCC) post-pediatric care continued to be followed by our program. Of these, 77% and 57% remained with the MCSCC 12 and 24 months after transfer to adult care, respectively. Conclusions: The St. Jude SCD Transition to Adult Care Program demonstrated a gradual increase in transfer rates and a reduction in length of time-to-adult transfer since its creation. Adult care engagement declined over time and warrants future interventions. Improved health care transition outcomes could potentially translate into improved quality of life and greater longevity. Disclosures Hankins: bluebird bio: Consultancy; NCQA: Consultancy; Novartis: Research Funding; Global Blood Therapeutics: Research Funding.

Description: Background: Up to 40% of children with sickle cell anemia (SCA) will have abnormalities on brain imaging due to their hematologic disorder, much of which is subclinical. Common abnormalities on brain magnetic resonance imaging/magnetic resonance angiography (MRI/MRA) include leukoencephalopathy from microvascular ischemic insult and vascular stenosis from endothelial damage. Silent cerebral ischemic insults are progressive; further neurologic abnormalities, including overt stroke, are more common among children with ischemic findings on MRI compared to children without them. Furthermore, poor performance on neuropsychological testing, lower IQ, and higher rates of grade retention are common in children with SCA and cerebral ischemic disease. The effect of hydroxyurea treatment on the development and progression of vascular stenosis and leukoencephalopathy is unclear. This study aimed to longitudinally evaluate the development of intracerebral abnormalities through serial MRI/MRA in children with SCA (HbSS and HbSβ0-thalassemia) who receive long-term therapy with hydroxyurea. Methods: Children with SCA and no prior history of overt stroke enrolled in the Hydroxyurea Study of Long-Term Effects (HUSTLE NCT00305175) underwent brain MRI/MRA, transcranial doppler (TCD) examinations, and laboratory evaluations immediately before hydroxyurea initiation and after 3 and 6 years of treatment to maximum tolerated dose. MRI/MRAs were reviewed for the presence or absence of vascular stenosis and leukoencephalopathy. Leukoencephalopathy was defined as white matter T2 hyperintensity. Proportions of abnormal MRI/MRA findings were compared between baseline and 3 years and baseline and 6 years using McNemar’s test and the Wilcoxon-Mann-Whitney exact test was used to explore associations with laboratory parameters. Results: Forty-six children with SCA, mean age 9.4 years (range 1-17.3), had an MRI/MRA at baseline and 3 years post-initiation of hydroxyurea. Ten children had an additional MRI/MRA after 6-years of hydroxyurea therapy. Frequencies of leukoencephalopathy and vascular stenosis are shown in the table. Prevalence of leukoencephalopathy before hydroxyurea therapy was higher than that reported in the literature for untreated children with SCA of similar age. There were no significant differences between baseline imaging findings and those at 3 and 6 years. Table: Prevalence of leukoencephalopathy and vascular stenosis at baseline, 3 and 6 years Baseline (n=47) Median age 9.6 years (range 1.0 to 17.3) 3 years (n=46) Median age 13.1 years (range 4.4 to 21.1) 6 years (n=10) Median age 14.5 years (range 8.3 to 18.3) Leukoencephalopathy 27 (57.4%) 28 (60.9%)* 5 (50%)* Stenosis 3 (6.4%) 1 (2.2%)* 0 *all p〉0.05 Children with leukoencephalopathy at baseline and 3 years were older than those without (mean 10.7 vs. 7.7 years, p=0.01; 10.5 vs. 7.5 years, p=0.02 respectively). Lower HbF at baseline was associated with the presence of leukoencephalopathy at year 3 (median HbF 4.6% vs. 12.4%, p=0.008), but there was no association between HbF at 3-years and the presence of leukoencephalopathy at 3-years (median HbF 16.4% vs. 13.2%, p=0.55). When stratified by age, these findings were similar. TCD velocities and other hematologic parameters were not associated with MRI/MRA abnormalities. The small number of vascular stenosis cases precluded further analyses of this outcome. Conclusions: In this longitudinal study, children treated with hydroxyurea for 3-6 years did not demonstrate an increased frequency of vascular stenosis or leukoencephalopathy on brain MRI/MRA during treatment. Older age at hydroxyurea initiation and lower pre-hydroxyurea HbF percentage were associated with the presence of leukoencephalopathy at baseline and 3-years. These findings suggest that hydroxyurea may mitigate the expected progression of vascular stenosis and leukoencephalopathy in children with SCA, and that this therapy should be initiated early on, before the development of cerebrovascular disease, particularly among those with a low HbF percentage. Disclosures Off Label Use: hydroxyurea for children with sickle cell disease.

Description: Background: Central nervous system complications of sickle cell disease (SCD) include stroke, silent cerebral infarct, and neurocognitive deficits, but few studies have examined developmental delays in preschool-age children. Using the Brigance developmental screen in 3 year-old children with SCD we found a high frequency of scores below the “normal” cutoff for age, but control data were lacking (Ped. Blood Cancer 2011;56:620). We hypothesized that children with SCD would be more likely to “fail” this screening test than age-matched children from a similar ethnic/socio-economic background. Methods: This prospective study was approved by the St. Jude Children’s Research Hospital (SJCRH) IRB and informed consent obtained for each subject. 3.0-3.9 year-old SCD subjects were tested in the SJCRH Sickle Cell Clinic. Patients receiving chronic transfusion therapy were excluded. Age-matched and race-matched control subjects with similar socio-economic backgrounds were recruited from 8 Memphis daycare centers. The Brigance Preschool Screen was administered by trained examiners in the clinic or the daycare and required about 15 minutes to complete; the primary caretaker simultaneously completed the demographic form. None of the participants had previous exposure to the test. The proportions of subjects who failed the Brigance test (scoring below the age-related “cutoff”) and test raw scores (higher scores indicating better performance) were compared between SCD and control subjects using the exact Chi-square test and the two-sample t-test, respectively; logistic regression was used to model the associations between the Brigance failure proportions and other covariates for the groups. P-values

Description: Background: Hematopoietic stem cell (HSC) gene therapy is potentially curative for sickle cell disease (SCD) provided that a sufficient quantity of long-term engrafting CD34+ HSCs can be safely collected and infused. In patients with SCD, peripheral blood (PB) mobilization with granulocyte colony stimulating factor is contraindicated, and steady-state bone marrow (BM) harvesting is associated with suboptimal HSC quality and yield. Hence, agents that promote safe and effective peripheral HSC mobilization in SCD are required. Methods: This phase I multicenter study investigated the safety and efficacy of plerixafor mobilization (240 µg/kg) followed by apheresis, processing, and HSC enrichment in 15 adult subjects with SCD (NCT03226691). Hydroxyurea treatment was stopped at least two weeks prior to mobilization and all participants who were not receiving long-term transfusion therapy received prophylactic red blood cell exchange prior to plerixafor administration to achieve a target sickle Hb (HbS)

Description: Background: Cardiac abnormalities in sickle cell anemia (SCA) include pan-chamber dilation, biventricular hypertrophy, ventricular diastolic dysfunction and elevated pulmonary artery pressures. Diastolic dysfunction in SCA has been reported with a variable prevalence, ranging from 11-77%. Its onset is in childhood, and data show an increase in prevalence with age [Pediatr Blood Cancer 2017, 64 (11): e26607]. While its etiology has remained elusive, it has been shown to be associated with early mortality in adults [J Am Coll Cardiol 2007, 49(4): 472-479] and decreased exercise capacity in children and adults [Circulation 2011, 124 (13):1452-1460]. The effect of hydroxyurea (HU), a SCA disease-modifying therapy, on ventricular diastolic dysfunction has not been investigated, and could represent a potential therapeutic strategy. Objective: To determine the longitudinal effect of HU therapy on left ventricular diastolic function and to identify hematological correlates of altered left ventricular diastolic indices. Methods: Children aged 5-18 years with HbSS or HbS/β0 thalassemia enrolled in the Long-Term Effects of Erythrocyte Lysis Trial [Pediatr Blood Cancer 2019, 66(7): e27717.], underwent prospective measurement of diastolic function by 2D-echocardiogram. Echocardiogram was obtained at steady state (≥ 4 weeks from illness, transfusion, or hospitalization), and then repeated 2 years later. Echocardiogram parameters used for clinical evaluation of diastolic function included early diastolic mitral annular velocity (lateral e'), an index of ventricular relaxation and an early marker of abnormal diastolic function, and the ratio between early mitral inflow velocity and early diastolic mitral annular velocity (lateral E/e'). Diastolic function was categorized as normal/abnormal, using normative age‐specific ranges [J Am Soc Echocardiogr 2004,17(3):212-221] and compared between baseline and 2 years. Hemoglobin and hemolysis markers including absolute reticulocyte count (ARC), and lactic dehydrogenase (LDH) were obtained concurrently. Results: 155 children with SCA, mean age 10.3 years (SD 3.54), were followed longitudinally. Of those, 59 (38%) did not receive HU, 61 (39%) were treated with HU both at baseline and at the 2-year evaluation, and 35 (23%) were untreated at baseline but initiated HU during the 2-year follow-up period after the initial echocardiogram. The prevalence of abnormal E/e' ratio did not change over time in the entire cohort (baseline 56% and 2-year 61%, p=0.23) or across the subgroups. However, the proportion of subjects with abnormal e' increased from 32% at baseline to 44% 2 years-later (p=0.01, Figure 1). In the subgroup analysis, participants who were continuously treated with HU were older [mean age 12 years (SD 3.54)] and had an increase in prevalence of abnormal e' at the 2-year time-point from 31% to 51% (p= 0.01, Figure 1). Individuals who were recently initiated on HU (baseline 29% and 2-year 43%, p = 0.27) or untreated (baseline 34% and 2-year 37%, p = 0.79) were comparatively younger [mean age 9.2 years (SD 3.02)] and did not have any significant change in diastolic function over time (Figure 1). A multivariate linear generalized mixed model was used to determine the association between abnormal diastolic and hematological parameters. An increase in hemoglobin concentration or a decrease in ARC and LDH did not affect the probability of having an abnormal e'. However, improvement in anemia among participants recently initiated on HU by 1 gm/dL, appeared to decrease the probability of having abnormal E/e' (Odds ratio, OR: 0.57, 95% CI: 0.32-1.03, p = 0.06). No such association was seen among participants on continuous HU therapy, whose hemoglobin did not significantly increase over the 2-year period from their baseline assessment. Conclusion: Among children with SCA, HU therapy does not appear to significantly alter left ventricular diastolic indices over a follow-up of 2 years. Our findings may indicate HU's inability to affect cardiac remodeling in older children despite stable hematological parameters. Longer follow up is required to investigate clinically significant changes in diastolic indices during HU therapy. Additional therapeutic modalities that mitigate ventricular remodeling may need to be explored, to prevent the progression of diastolic dysfunction into adulthood, where it has been identified as a predictor of early mortality. Disclosures Kang: MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Zhao:MBIO: Other: St. Jude Children's Research Hospital has an existing exclusive license and ongoing partnership with Mustang Bio for the further clinical development and commercialization of this XSCID gene therapy. Ataga:Novartis: Consultancy, Honoraria, 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; Emmaus Life Sciences: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bioverativ: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding; Modus Therapeutics: Honoraria. Hankins:Bluebird Bio: Consultancy; NHLBI: Research Funding; ASPHO: Honoraria; Novartis: Research Funding; LYNKS Foundation: Research Funding; NHLBI: Honoraria; Global Blood Therapeutics: Research Funding; National Committee for Quality Assurance: Consultancy.

Description: Introduction: Gene variants in the apolipoprotein L-1 (APOL1) gene are strong modifiers for the development of chronic kidney disease in individuals of African descent and are associated with progression of renal disease and albuminuria in cross-sectional studies of individuals with sickle cell anemia (SCA). While the association of APOL1 with albuminuria in older SCA patients is established, it is unclear whether participants with APOL1 G1 (rs73885319/ rs6090145) and G2 (rs71785313) variants (Ashley-Koch Br J Hematol 2011; Kormann Br J Haematol 2017) are more likely to develop albuminuria early in life. We hypothesized that individuals with SCA with the APOL1 G1 and G2 variants experience albuminuria at a higher rate and at a younger age than individuals without these APOL1 mutations. Methods: APOL1 G1 (rs73885319/ rs6090145) and G2 (rs71785313) variants were identified from whole genome sequence (WGS) data for individuals with SCA (HbSS or HbSβ0 thalassemia) enrolled in the longitudinal Sickle Cell Clinical Research and Intervention Program (SCCRIP; NCT02098863) (Hankins et al, Pediatr Blood Cancer 2018). WGS data were generated by aligning paired-end 150 bp reads to the GRCh38 human reference using the Burrows-Wheeler Aligner (BWA-ALN v0.7.12) and the GATK best-practices workflow implemented in GATK v3.4.0. We modeled the time to first albuminuria diagnosis defined by abnormal urine albumin:creatinine ratio (〉30mg/g). Covariates included age, sex, and hydroxyurea therapy. We considered two risk models. Model 1 defined high risk APOL1 mutation as either APOL1 G1 homozygotes or G2 homozygotes or G1/G2 double heterozygotes. Model 2 was defined additively wherein G1 or G2 homozygotes were assumed to confer more risk compared to G1/G2 double heterozygotes. Results: In 285 individuals with SCA, 93% (n=266) with HbSS and 7% (n=19) with HbSβ0 thalassemia, 14% (n=41) experienced albuminuria at a mean age of 11.4 (±3.3) years. In total, 11% (n=32) of this SCA cohort had an APOL1 mutation; 6% (n=17) were G1/G2 double heterozygote, 4% (n=11) were G1 homozygotes, and 1% (n=4) were G2 homozygotes. Among the 32 participants with an APOL1 mutation, 40% (n=13) had albuminuria as compared to 11% (n=28) of the 253 participants without an APOL1 mutation (OR: 9.85, 95% CI 3.84-25.25). There was also a significant association with albuminuria based on the additive genetic model (OR: 4.38, 95% CI 2.39 -8.02) (Table). In a survival analysis, participants with an APOL1 mutation had a hazard ratio (HR) of 3.75 (95% CI: 1.93-7.29, p

Description: Introduction RBC alloimmunization is common in patients with sickle cell disease (SCD). Despite serological matching RBCs for major Rh antigens, Rh alloimmunization remains problematic. The Rh blood group is encoded by two genes RHD and RHCE, which exhibit extensive nucleotide polymorphism and chromosome structural changes, resulting in the formation of Rh variant antigens. Rh variants can result in loss of protein epitopes or expression of neo-epitopes, and are common in SCD patients. Hence SCD patients harboring Rh variants can be predisposed to Rh alloimmunization. Given the limitation of traditional serologic antigen typing for detection of Rh variants, molecular genotyping has become required. A DNA microarray-based platform, BioArray RHCE and RHD BeadChip (Immuncor) is available for RH genotyping. However, it detects the most common, but not all, variants. Whole exome sequence data have been used for prediction of Rh variants (Chou, et. al, Blood Adv., 2017), offer some advantages, including detection of rare variants, structural rearrangements and copy number variation. However, whole genome sequence (WGS) analysis of RHD/RHCE is challenging due to difficulties in mapping next generation sequencing (NGS) reads to this duplicated gene family. We developed a computational algorithm to identify RH variants using WGS data. Methods The pipeline included three major components, RH allele database construction, RH variant calling, and classification of Rh blood group according the identified variants. The RH allele database was built based on NCBI Blood Group Antigen Gene Mutation (BGMUT) and International Society of Blood Transfusion (ISBT) database. Since the alleles in the BGMUT and ISBT databases were specified according to conventional RH genes (RHD, L08429; RHCE, DQ322275) that are different from those on reference human genome, we first called the variations based on the reference human genome. The positions of the identified variations were subsequently corrected to match with the BGMUT and ISBT annotation system. Next, the NGS reads with low base quality and/or mapping quality were discarded during the variation calling step. Synonymous and non-synonymous amino acid changes were characterized for each polymorphism. Haplotypes were constructed for the segments with NGS read support. Gene sequencing coverage was calculated to determine gene deletions or amplifications. Lastly, we implemented an algorithm to predict RH genotypes based on a selection of candidate alleles by read-mapping profile which considers both sequence variations and sequence consistency followed by a likelihood-based ranking of all pairwise combinations of the selected alleles. The allele combination with the highest likelihood is considered the most likely pair of alleles at a given locus. Patient specimens used in this study were from participants of the Sickle Cell Clinical Research and Intervention Program (SCCRIP, Hankins et al. Pediatr Blood Cancer. 2018). Results We validated our method in a cohort of 58 SCD patients whose RH genotypes had been determined by BioArray RhCE and RhD BeadChip and supplementary molecular tests that identify the most common variants among individuals of African descent. In this validation cohort including a total of 11 RHD and 13 RHCE alleles, our approach achieved a concordance rate of 85.85% (91 of 106 alleles) for RHD and 83.02% (88 of 106 alleles) for RHCE genotyping. WGS was highly sensitive in distinguishing homozygosity from heterozygosity of genes. By comparing the numbers of NGS reads on RH regions and whole genome average coverage, heterozygous deletion can be determined. Since WGS provides comprehensive genotyping, our analysis identified single nucleotide polymorphisms that were not identified by the BeadChip and supplemental molecular testing. The final source of discordance was likely due to the short read length of NGS such that haplotype phases cannot be correctly predicted if the variations are separated by thousands of base pairs, for which long read DNA sequencing or RNA/cDNA sequencing are required. Evaluation of the identified discrepancies is ongoing. Conclusions We developed and validated a diagnostic method for RH genotyping that leveraged the accuracy and flexibility of RH genotyping based on WGS data. With further optimization of our method, this may be useful for RBC genotype matching sickle cell patients to blood donors in the future. Disclosures Hankins: Novartis: Research Funding; Global Blood Therapeutics: Research Funding; NCQA: Consultancy; bluebird bio: Consultancy.

Description: Introduction Sickle Cell Anemia (SCA) is a rare, devastating, and debilitating disease marked by the pathophysiologic features of hemolytic anemia, vaso-occlusion, and progressive end-organ damage. The most devastating complication of pediatric SCA is the development of central nervous system events such as overt stroke or silent cerebral infarcts (SCIs) which can produce significant physical and neurocognitive deficits. Transcranial Doppler ultrasonography (TCD) measures cerebral artery blood flow velocity in children with SCA. Elevated TCD velocity levels are a reliable predictor of stroke risk, and children with conditional TCD levels (defined as 〉 170 to 〈 200 cm/sec) are at increased risk of stroke compared to those with normal TCD levels (200 cm/sec), but the extent to which hemoglobin (Hb) concentration influences TCD level elevation is unknown. Using recently collected longitudinal, real world data, this study sought to quantify the impact of the rise in Hb obtained during hydroxyurea (HU) therapy on the change observed in TCD levels as an estimate for the magnitude of Hb rise that would be predicted to convert an individual from the conditional TCD level range (moderate risk of stroke) to the normal TCD level (low risk of stroke). Methods Children (age 0 g/dl, ≥0.5 g/dl, ≥1 g/dl, and ≥2 g/dl). A mixed- model repeated measures (MMRM) using longitudinal panel data were employed next to estimate the impact of Hb levels on TCD velocity, controlling for participants' demographic and clinical characteristics. Results A total of 202 children (female: 46.5%, mean age at BL TCD: 7.53 yrs) were analyzed. At BL, 33 patients (16.3%) had conditional TCD level, 162 patients (80.2%) had normal TCD level. Mean (SD) BL Hb level was 8.4 g/dl (0.97 g/dl). In cross-sectional analysis, Hb was negatively associated with corresponding TCD levels at 1 yr, 2 yrs and 4 yrs post HU (Spearman correlation coefficients: -0.20, -0.64 and -0.38, respectively, all p-values