Jude Children's Research Research Articles

Neurocognitive Impairment Predicts Poor Transition Outcomes Among Patients with Sickle Cell Disease

Introduction: In the United States, most children with sickle cell disease (SCD) survive into adulthood and transfer from pediatric to adult-centered care. Cognitive deficits begin during childhood and are highly prevalent among individuals with SCD, potentially affecting their functional ability to establish adult care and navigate the new adult care environment. Lack of engagement in adult care can place youth with SCD at higher risk for care discontinuity and higher disease morbidity and mortality. The relationship between cognition and transition to adult care has not been examined. We hypothesized that better performance on measures of neurocognition were associated with decreased latency in initiating adult care, greater retention in adult care, and increased utilization of adult ambulatory services. As a secondary objective, we examined the relationship of environmental outcomes to transition outcomes. Methods: We included participants enrolled in the Sickle Cell Research and Intervention Program (SCCRIP; Hankins J. et al, Pediatric Blood and Cancer 2018), a longitudinal lifetime cohort study of individuals with SCD that monitors neurocognition. Participants were included if they underwent neurocognitive screening assessment in adolescence, prior to their transfer to adult care and if they satisfied their first appointment in adult care. The neurocognitive screening battery included measures of estimated global intelligence (Wechsler Abbreviated Scales of Intelligence, 2nd Ed; WASI-2) and sustained attention (Continuous Performance Test, 2nd Ed; CPT-2). Environmental factors included the Economic Hardship Index (EHI), guardian employment status while in pediatric care, and the number of persons living in the household. Use of adult ambulatory services was measured by the number of outpatient visits per patient-year. The association between cognitive performance and the latency from pediatric to adult care, adult care retention and environmental variables was examined using the 2-sample t test if the data were normally distributed or the Wilcoxon rank-sum test otherwise. Categorical variables were analyzed with the Chi-square test or Fisher's exact test. Transition outcomes were also analyzed as continuous variables using univariate linear regression. All reported p-values are two-sided. Results: Eighty adolescents with SCD ages 15-18 years at the time of their cognitive assessment (58% male, 63% HbSS/HbSβ0-thalassemia) were included; most transferred <6 months from the last pediatric visit Table 1). Of these 80 patients, 61 and 43 had sufficient follow-up time to examine their retention in adult care 12 and 24 months after transfer, respectively. Fifty out of the 61 patients (82%) remained in adult care > 12 months, and 31 of the 43 (72%) remained in adult care >24 months after their first adult visit. Higher Full-Scale IQ was associated with establishing adult care ≤2 months from last pediatric visit (Table 1; Figure 1A, 1B). Belonging to families with fewer children, smaller households and a higher WASI-2 Verbal Comprehension Index were associated with establishing adult care ≤6 months from last pediatric visit. Better CPT-2 Commissions performance (less attention deficit) was associated with increased adult care retention at 12 and 24 months (Table 2; Figure 1C,1D). Having a working guardian was associated with less retention at 12 months (p=0.01), whereas having an unemployed primary guardian was associated with greater retention at 24 months (p=0.02). Further, an employed guardian was associated with greater utilization of adult ambulatory services (p=0.01). EHI was not significantly related to transition outcomes. No relationship was found between adult ambulatory services and neurocognitive assessment. Conclusion: Neurocognitive deficit (lower IQ and attention deficits) may decrease short and long-term engagement in adult care among youth with SCD as demonstrated by longer latency periods between pediatric and adult care and shorter adult care retention. Socio-economic factors may also play a role in transition outcomes but require further investigation. Investigation of disease modifying therapies that preserve cognitive function should be prioritized. Interventions that account for patients' cognitive level and their environment should be considered in the individualization of transition plans. Disclosures King: Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novimmune: Research Funding; Amphivena Therapeutics: Research Funding; Incyte: Consultancy; Tioma Therapeutics (formerly Vasculox, Inc.):: Consultancy; Cell Works: Consultancy; Bioline: Consultancy; Celgene: Consultancy; RiverVest: Consultancy; WUGEN: Equity Ownership. Wang:Agios Pharmaceuticals: Consultancy; Novartis: Consultancy. 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. 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. Hankins:National Committee for Quality Assurance: Consultancy; NHLBI: Research Funding; Global Blood Therapeutics: Research Funding; Novartis: Research Funding; LYNKS Foundation: Research Funding; NHLBI: Honoraria; ASPHO: Honoraria; Bluebird Bio: Consultancy.

Longitudinal Effect of Hydroxyurea Therapy on Left Ventricular Diastolic Function in Sickle Cell Anemia

Longitudinal Effect of Hydroxyurea Therapy on Left Ventricular Diastolic Function in Sickle Cell Anemia

The Genomic Landscape of Childhood Acute Lymphoblastic Leukemia

Introduction: Although recent studies have refined the classification of B-progenitor and T-lineage acute lymphoblastic leukemia into gene-expression based subgroups, a comprehensive integration of significantly mutated genes and pathways for each subgroup is needed to understand disease etiology. Methods: We studied 2789 children, adolescents and young adults (AYA) with newly diagnosed B-ALL (n=2,322 cases) or T-ALL (n=467) treated on Children's Oncology Group (n=1,872) and St. Jude Children's Research Hospital trials (n=917). The cohort comprised childhood NCI standard-risk (41.8%; age range 1-9.99 yrs, WBC ≤ 50,000/ml), childhood NCI high-risk (44.5%; age range ≥10 to 15.99 yrs) and AYA (9.9%; age range 16-30.7 yrs). Genomic analysis was performed on tumor and matched-remission samples using whole transcriptome sequencing (RNA-seq; tumor only; n=1,922), whole exome sequencing (n=1,659), whole genome sequencing (n=757), and single nucleotide polymorphism array (n=1,909). Results: For B-ALL, 2104 cases (90.6%) were classified into 26 subgroups based on RNA-seq gene expression data and aneuploidy or other gross chromosomal abnormalities (iAMP21, Down syndrome, dicentric), deregulation of known transcription factors by rearrangement or mutation (PAX5 P80R, IKZF1 N159Y), or activation of kinase alterations (Ph+, Ph-like). For T-ALL, cases were classified into 9 previously described subtypes based on dysregulation of transcription factor genes and gene expression. In 1,659 cases subject to exome sequencing (1259 B-ALL, 405 T-ALL) we identified 18,954 nonsynonymous single nucleotide variants (SNV) and 2,329 insertion-deletion mutations (indels) in 8,985 genes. Overall, 161 potential driver genes were identified by the mutation-significance detection tool MutSigCV or by presence of pathogenic variants in known cancer genes. Integration of sequence mutations and DNA copy number alteration data in B-ALL identified 7 recurrently mutated pathways: transcriptional regulation (40.6%), cell cycle and tumor suppression (38.0%), B-cell development (34.5%), epigenetic regulation (24.7%), Ras signaling (33.0%), JAK-STAT signaling (12.0%) and protein modification (ubiquitination or SUMOylation, 5.0%). The top 10 genes altered by deletion or mutation in B-ALL were CDKN2A/B (30.1%), ETV6 (27.0%), PAX5 (24.6%), CDKN1B (20.3%), IKZF1 (17.6%), KRAS (16.5%), NRAS (14.6%), BTG1 (7.5%) histone genes on chromosome 6 (6.9%) and FLT3 (6.1%), and for T-ALL, CDKN2A/B (74.7%), NOTCH1 (68.2%), FBXW7 (21.3%), PTEN (20.5%) and PHF6 (18.2%) (Figure 1A). We identified 17 putative novel driver genes involved in ubiquitination (UBE2D3, UBE2A, UHRF1, and USP1), SUMOylation (SAE1, UBE2I), transcriptional regulation (ZMYM2, HMGB1), immune function (B2M), migration (CXCR4), epigenetic regulation (DOT1L) and mitochondrial function (LETM1). We also observed variation in the frequency of genes and pathways altered across B-ALL subtypes (Figure 1B). Interestingly, alteration of SAE1 and UBA2, novel genes that form a heterodimeric complex important for SUMOylation, and UHRF1 were enriched in ETV6-RUNX1 cases. Deletions of LETM1, ZMYM2 and CHD4 were associated with near haploid and low hypodiploid cases. Deletion of histone genes on chromosome 6 and alterations of HDAC7 were enriched in Ph+ and Ph-like ALL. Mutations in the RNA-binding protein ZFP36L2 were observed in PAX5alt, DUX4 and MEF2D subgroups. Genomic subtypes were prognostic. ETV6-RUNX1, hyperdiploid, DUX4 and ZNF384 ALL were associated with good outcome (5-yr EFS 91.1%, 87.2%, 91.9% and 85.7%, respectively), ETV6-RUNX1-like, iAMP21, low hyperdiploid, PAX5 P80R and PAX5alt were associated with intermediate outcome (5-yr EFS 68.6%, 72.2%, 70.8%, 77.0% and 70.9%, respectively), whilst KMT2A, MEF2D, Ph-like CRLF2 and Ph-like other conferred a poor prognosis (55.5%, 67.1%, 51.5% and 62.1%, respectively). TCF3-HLF and near haploid had the worst outcome with 5-yr EFS rates of 27.3% and 47.2%, respectively. Conclusions: These findings provide a comprehensive landscape of genomic alterations in childhood ALL. The associations of mutations with ALL subtypes highlights the need for specific patterns of cooperating mutations in the development of leukemia, which may help identify vulnerabilities for therapy intervention. Disclosures Gastier-Foster: Bristol Myers Squibb (BMS): Other: Commercial Research; Incyte Corporation: Other: Commercial Research. Willman:to come: Patents & Royalties; to come: Membership on an entity's Board of Directors or advisory committees; to come: Research Funding. Raetz:Pfizer: Research Funding. Borowitz:Beckman Coulter: Honoraria. Zweidler-McKay:ImmunoGen: Employment. Angiolillo:Servier Pharmaceuticals: Consultancy. Relling:Servier Pharmaceuticals: Research Funding. Hunger:Jazz: Honoraria; Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees. Mullighan:Amgen: Honoraria, Other: speaker, sponsored travel; Loxo Oncology: Research Funding; AbbVie: Research Funding; Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel.

Transcranial Doppler Velocities Conversion Rate Based on Increasing Hemoglobin Concentration: Analysies from the SCCRIP Cohort Study

Transcranial Doppler Velocities Conversion Rate Based on Increasing Hemoglobin Concentration: Analysies from the SCCRIP Cohort Study

Comprehensive Genomic Profiling of Pediatric Therapy-Related Myeloid Neoplasms Identifies Mecom Dysregulation to be Associated with Poor Outcome

Therapy-related myeloid neoplasms (tMN) occur in children secondary to cytotoxic therapies used to treat pediatric malignancies, are typically resistant to conventional chemotherapy, require hematopoietic cell transplantation as the only curative option, and have a dismal prognosis. The genomic alterations that drive tMN in children have yet to be comprehensively described, and it is unclear if particular genomic lesions hold prognostic value. We have characterized the genomic profile of 62 pediatric tMN cases (tMDS: n=23, tAML: n=39) obtained from the St. Jude Children's Research Hospital Tissue Bank from patients diagnosed between 1987 and 2018. These cases arose following treatment for a variety of primary tumors (hematological (74%), bone and soft tissue (23%), and brain (3%)). Acute lymphoblastic leukemia was the most frequent primary tumor (n=39, 63%). Conventional cytogenetics (n=60) showed a complex karyotype (≥3 structural alterations) in 19 (32%) cases, and 7 of these cases contained a deletion involving chromosome 7 (del(7)). Eleven (18%) other cases without complex karyotypes had del(7). Deletions of chromosome 5 were present in 9 (15%) cases, but only in the context of a complex karyotype. We hypothesized that the patients' younger age and the different spectrum of primary tumor types and chemotherapy would give rise to a mutational spectrum distinct from adult tMN. We used whole exome (WES), whole genome (WGS), and RNA sequencing (RNA Seq) to describe the mutational profile of our pediatric tMN cohort. WES was completed for 58 tumor/normal pairs using Nextera Rapid Capture Expanded Exome (Illumina). Fifteen cases were analyzed by WGS (11 also had WES). Normal comparator genomic DNA was obtained from flow-sorted lymphocytes. An average of 21 coding variants/patient (range: 1-131) was observed from the gene-coding region, and these include synonymous, non-synonymous, and splice site variants. Ras/MAPK pathway mutations were present in 44% of the cases (43 mutations in 27 cases). Canonical KRAS (n = 16), NF1 (n = 10), and NRAS (n = 7) mutations were the most frequent coding mutations. Eleven (18%) patients had either heterozygous deletion or a copy neutral loss of heterozygosity event involving chromosome 17p and the TP53 locus; 5 of these cases had concurrent TP53 missense mutations identified at allele frequencies near 100%. Unlike tMN in adults, mutations in PPM1D were not identified. RNA-Seq completed on 56 evaluable cases identified 28 (50%) cases with KMT2A rearrangement (KMT2Ar). MLLT3 was the most common fusion partner (n=13, 46%). In addition to KMT2A rearrangements, RNA-Seq also identified a RUNX1-MECOM fusion. Alterations involving the MECOM locus have been described in some myeloid neoplasms like tMN, and its overexpression is associated with a poor prognosis and some AMLs with KMT2Ar. MECOM expression levels were variable in this cohort (FPKM range: 0.004 - 38.4) with 24 cases (43%) having an FPKM>5 (MECOMHigh). In addition to the RUNX1-MECOM event, these 24 MECOMHigh cases included 18 with KMT2Ar (64% of KMT2Ar group) and 1 with a NUP98 fusion (NUP98-HHEX). The remaining 4 MECOMHigh cases demonstrate allele-specific MECOM expression, suggesting a cis-regulatory element is driving this expression. Two of these 4 cases have WGS and were found to contain a t(2;3)(p21;q26.2) involving MECOM on chromosome 3 and noncoding regions of chromosome 2 adjacent to ZFP36L2, a gene highly expressed in hematopoietic cells. ENCODE data supports that this region of the genome is an active enhancer in hematopoietic cells, suggesting a proximity effect in which this enhancer has been hijacked to drive high levels of MECOM expression. In our cohort, MECOM expression levels are predictive of a worse outcome (overall survival (OS) at 2 years: High=14.6% vs. Low=46.3%; log rank p<0.01). Although KMT2Ar was frequently present in our cohort and enriched in the MECOMHigh group (High=75% (18/24) vs. Low=31% (10/32); p<0.01), high MECOM expression did not confer a significant survival difference within the KMT2Ar group (OS at 2 years: High=16.7% vs Low=40%; log rank p=0.33). Further, the presence of a KMT2Ar or a complex karyotype did not significantly affect the OS in this cohort. In conclusion, we report the genomic profile of a large cohort of pediatric tMN cases and show that high levels of MECOM expression, a portion of which is driven by enhancer hijacking, predicts a worse outcome. Disclosures Gruber: Bristol-Myers Squibb: Consultancy.

Identification of New Risk Loci and Regulatory Mechanisms Influencing Genetic Susceptibility to Acute Lymphoblastic Leukaemia

There is increasing evidence for an inherited genetic basis of susceptibility to acute lymphoblastic leukaemia (ALL) in children. In particular, genome-wide association studies (GWAS) have identified nine genomic regions with common polymorphisms associated with ALL risk. However, these loci cumulatively only explain a minority of the genetic risk of childhood ALL, and differences in the etiology amongst molelcular subtypes of ALL remain poorly understood. In this study, we performed one of the largest ALL susceptibility GWAS, totalling 5,321 cases and 16,666 controls of European descent, to comprehensively map ALL risk loci. With this large sample size, we also sought to identify risk genes for two major subtypes of ALL, namely those with ETV6-RUNX1 fusion and hyperdiploid ALL. This meta-analysis included four previously published GWAS datasets with European cases from the UK ALL cohorts and the German Berlin-Frankfurt-Münster ALL trials, and the US cases enrolled on ALL trials in Children's Oncology Group and at St. Jude Children's Research Hospital (Nat Genet2009, Blood 2012, Nat Genet 2013, Blood 2013, Nat Commun 2018). The replication series included 2,237 ALL cases of non-European ancestry from the COG and St. Jude GWAS cohorts. For each GWAS dataset, the genotypes of ~10 million SNPs in each study were imputed. After filtering out SNPs on the basis of minor allele frequency and imputation quality, we assessed associations between ALL status and SNP genotype in each study using logistic regression. Risk estimates were combined through an inverse variance weighted fixed-effects meta-analysis. The meta-analysis identified 16 risk loci above genome-wide significance (P < 5 x 10-8), of which ten confirmed previously published associations. Of the six new candidate risk loci, four were validated in the replication series (P < 0.05): for all B-ALL at 9q21.3 (nearest gene TLE1), for hyperdiploid ALL at 5q31.1 (C5orf56) and 6p21.31 (BAK1), and for ETV6-RUNX1 positive ALL at 17q21.32 (IGF2BP1). As well as providing further evidence for the 21q22.3 (ERG) association for all B-ALL, we also identified novel risk variants with the hyperdiploid subtype-specific association. To gain insight into the biological basis of association signals, we examined the epigenetic landscape of risk regions in B-cells. Of particular note, hyperdiploid ALL-specific risk variant at 6p21 is located within an intron 1kb downstream of the BAK1 transcription start site and possess histone marks characteristic of active promoter activity and open chromatin accessibility. The top SNP at this locus falls within a transcription factor binding cluster, and the C-risk allele is associated with reduced BAK1 expression (P<2×10-16). Mendelian randomization analysis confirmed a significant association with BAK1 expression and ALL consistent with a causal relationship. Similarly, the ETV6-RUNX1-specific risk variant at 17q12 is located in the second intron of IGF2BP1 and is predicted to influence topological associated domain structure in cis. Interestingly, ETV6-RUNX1 positive ALL leukemia cells showed a dramatic overexpression of IGF2BP1 compared to those without this fusion gene (P=3.7×10-23), suggesting its potential role in the pathogenesis of this ALL subtype. Analysis of disruption of transcription factor binding sites at ALL risk loci genome wide identified over-representation of PBX1 (P=0.007), TCF3 (P=0.007), ETS1 (P=0.009), RUNX1 (P=0.012) and ERG (P=0.030), BRD4 (P=0.007), and NR3C1 (P=0.009). Many of these transcription factors are also somatically mutated in ALL, pointing to multiple mechanisms by which they can contribute to leukemogenesis. Using the Linkage Disequilibrium Adjusted Kinships method, we estimated that ALL risk loci identified so far accounted for 31% of the total variance in genetic risk of this cancer. Polygenic risk score analysis indicates that an individual in the top 1% of genetic risk would have a 4.7-fold increased risk of ALL when compared to an individual with median genetic risk. In summary, our study provides further evidence for inherited susceptibility to ALL and support for subtype specificity at risk loci. Our findings also highlight a model of ALL pathogenesis relying on transcriptional deregulation, particularly of genes involved in B cell differentiation. Disclosures Raetz: Pfizer: Research Funding. Mullighan:Pfizer: Honoraria, Other: speaker, sponsored travel, Research Funding; AbbVie: Research Funding; Loxo Oncology: Research Funding; Amgen: Honoraria, Other: speaker, sponsored travel; Illumina: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: sponsored travel. Hunger:Amgen: Consultancy, Equity Ownership; Bristol Myers Squibb: Consultancy; Jazz: Honoraria; Novartis: Consultancy. Schrappe:Together with study group from SHIRE, JazzPharma, Servier, SigmaTau, Amgen, and Novartis.: Research Funding; SHIRE, Servier, and JazzPharma: Honoraria. Relling:Servier Pharmaceuticals: Research Funding. Loh:Medisix Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees.

Progression of Central Nervous System Vasculopathy in Young Adults with Sickle Cell Anemia

Progression of Central Nervous System Vasculopathy in Young Adults with Sickle Cell Anemia

CRISPR-Cas9 Genome Editing of γ-Globin Promoters in Human Hematopoietic Stem Cells to Induce Erythrocyte Fetal Hemoglobin for Treatment of β-Hemoglobinopathies

Induction of fetal hemoglobin (HbF, α2γ2) via genome editing-mediated disruption of DNA regulatory elements that repress expression of γ-globin genes (HBG1 and HBG2) is a promising therapeutic strategy for b-hemoglobinopathies including sickle cell disease (SCD) and β-thalassemia. Optimal technical approaches and safety profiles are yet to be fully defined. We used CRISPR/Cas9 to target a DNA repressor element near the distal CCAAT box of the HBG1/HBG2 promoters. This region contains a "TGACC" motif recognized by BCL11A, a transcriptional repressor protein that regulates γ-to-β globin switch after birth. Rare germline variants at or near this motif are associated with hereditary persistence of fetal hemoglobin, a benign genetic condition that alleviates the clinical manifestations of co-inherited b-hemoglobinopathies. Previously, we showed that transduction of human CD34+ cells with lentiviral vector encoding Cas9 and guideRNA (gRNA) targeting the HBG1/HBG2 promoter caused induction of HbF in red blood cell (RBC) progeny generated in vitro (Traxler et. al, Nature Medicine v22,2016). Here we present a clinically tractable approach for disrupting the HBG1/HBG2 BCL11A binding site in human hematopoietic stem cells (HSCs). Electroporation of Cas9:gRNA ribonucleoprotein (RNP) complex into healthy or SCD donor CD34+ cells resulted in up to 80% on-target insertion-deletion (indel) mutations and 35% HbF in erythroid progeny generated in vitro. Sixteen to 17 weeks after transplantation of gene edited CD34+ cells into immunodeficient NBSGW mice, up to 75% of donor CD34+ cells in recipient bone marrow contained on-target indels, demonstrating efficient modification of repopulating human HSCs. No differences in CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages were observed between edited and control cells. Moreover, up to 78% of gene edited erythroid cells stained with anti-HbF antibody ("F-cells") compared to 15% in control erythroid cells, suggesting a "pan-cellular" pattern of HbF expression after editing. Strikingly, human donor-derived erythroid cells in recipient bone marrow expressed up to 40% HbF compared to 3% HbF in controls. Although the editing frequencies of HBG1 and HBG2 promoters varied between different donor CD34+ cells, an engineered variant of Cas9 containing 3 nuclear localization sequences (Wu et. al,Nature Medicine v25, 2019) edited repopulating HSCs more efficiently and consistently than conventional Cas9 with two nuclear localization signals. Simultaneous on-target RNP-induced DSBs at both HBG1 and HBG2 can result in the deletion of the intervening 4.9-kb region, leaving a single hybrid gene with HBG2 promoter sequences fused to the downstream HBG1 gene. We detected this deletion in approximately 30% of edited cells, with no associated decline in HbF expression determined by clonal analysis of erythroid colonies. No off-target mutations were detected by targeted sequencing of the 26 top candidate sites identified by CIRCLE-seq, an in vitro genome-scale method for detecting Cas9 activity. Analysis of gene edited human donor cells purified from mouse bone marrow showed no chromosomal rearrangements by G-banding (n=20) or fluorescence in situ hybridization with a probe located distal to the HBG1/HBG2 loci (n=225). Taken together, our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a CRISPR-Cas9 genome editing approach to induce HbF for treating hemoglobinopathies. Figure. Gene editing of the HBG1/HBG2 promoters in HSCs and HbF induction of erythroid progeny in vivo. Plerixafor-mobilized CD34+ cells from an individual with SCD were edited with RNP and transplanted into NBSGW mice, which were analyzed 16-17 weeks later. A. On-target indel frequency before (Pre) and after bone marrow transplantation (BM). The black bars represent a 13-nucleotide deletion associated with human hereditary persistence of fetal hemoglobin. B. Human erythroblasts and reticulocytes derived from RNP-edited and non-edited Control CD34+ donor cells. Scale bar = 10 mm. C. HbF immunostaining control and RNP edited erythroid cells in recipient bone marrow assessed by flow cytometry. D. %HbF protein in hemolysates of control (C) and RNP edited erythroid cells assessed by ion-exchange HPLC. n= 3 biological replicates. **** P < 0.0001. Figure Disclosures Metais: 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. Sharma:Doris Duke Foundation: Research Funding; Vertex Pharmaceuticals: Other: Study PI. Weiss:Beam Therapeutics: Consultancy; Rubius INC: Consultancy; GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy.

Pipeline for Discovering Neoepitopes Generated By Alternative Splicing in B-ALL

Pediatric B-cell acute lymphoblastic leukemias (B-ALL) are striking for their low mutational burdens. Ostensibly, this paucity of mutation-associated neoantigens could limit the development of novel immunotherapies. However, our lab has previously shown that globally aberrant splicing is a hallmark of B-ALL, as compared to normal pro-B cells. Here we hypothesize that alternative splicing represents a new source of targetable neo-epitopes, distinct from missense mutations. To test this hypothesis, we constructed a pipeline for neo-epitope discovery, validation, and therapeutic development. First, we obtained a very large RNA-seq dataset covering over 400 pediatric B-ALL samples from St. Jude Children's Research Hospital. For normal controls, RNA-seq was performed on 4 pro-B cell fractions from bone marrow donors. We then applied a bioinformatic splicing analysis, MAJIQ 2.0, to deeply mine the dataset for local splicing variants (LSVs) unique to B-ALL. To bypass MHC presentation barriers, we focused on LSVs mapping to exons encoding extracellular protein domains (ectoLSVs). This filtering yielded a list of 914 ectoLSVs in 430 genes. These ectoLSVs were further filtered for those that preserved opening reading frames, occurred with high prevalence, and demonstrated a large differential expression between B-ALL and normal controls. One such prominent event was the increased skipping in B-ALL of CD22 of exon 5-6 (Δex5-6). We validated this event at the RNA level with RT-PCR in 18 out of 18 primary B-ALL samples from CHOP Biobank. Using Nanopore-based long read RNA sequencing, we confirmed the Δex5-6 event exists within a larger translatable transcript. BALB/c mice were then immunized against a peptide containing the junction site of exon 4 and 7 to generate hybridomas; several monoclonal antibody (mAb) against CD22 Δex5-6 were successfully obtained. To validate the specificity of our mAbs, we used CRISPR to delete endogenous CD22 in OCI-Ly10 cells and reconstituted the cells with either CD22 Δex5-6 or full length CD22. The mAb demonstrated remarkable specificity for CD22 Δex5-6 and did not bind to the full-length isoform. We also demonstrated that at least one of these mAbs (clone 3A3) specifically binds to endogenously expressed CD22 Δex5-6 in multiple B-ALL cell lines by immunoprecipitation and Western blot analysis. Our next steps are to develop 3A3-based antibody-drug conjugates and chimeric antigen receptors for further therapeutic testing. In summary, our results validate that 1) our current discovery pipeline is able to identify targetable splicing-derived neo-epitopes, and that 2) antibodies with impressive specificity can be generated against such neo-epitopes. Furthermore, this new paradigm has the promise of increasing the repertoire of highly specific immunotherapy targets in B-ALL, despite its low mutation burden. Of note, this strategy could also be carried forward into therapeutic development for many other cancers beyond B-ALL. Disclosures No relevant conflicts of interest to declare.

Asparaginase formulation impacts hypertriglyceridemia during therapy for acute lymphoblastic leukemia.

Glucocorticoids and asparaginase, used to treat acute lymphoblastic leukemia (ALL), can cause hypertriglyceridemia. We compared triglyceride levels, risk factors, and associated toxicities in two ALL trials at St. Jude Children's Research Hospital with identical glucocorticoid regimens, but different asparaginase formulations. In Total XV (TXV), native Escherichia coli l-asparaginase was front-line therapy versus the pegylated formulation (PEG-asparaginase) in Total XVI (TXVI). Patients enrolled on TXV (n=498) and TXVI (n=598) were assigned to low-risk (LR) or standard/high-risk (SHR) treatment arms (ClinicalTrials.gov identifiers: NCT00137111 and NCT00549848). Triglycerides were measured four times and were evaluable in 925 patients (TXV: n=362; TXVI: n=563). The genetic contribution was assessed using a triglyceride polygenic risk score (triglyceride-PRS). Osteonecrosis, thrombosis, and pancreatitis were prospectively graded. The largest increase in triglycerides occurred in TXVI SHR patients treated with dexamethasone and PEG-asparaginase (4.5-fold increase; P<1×10-15 ). SHR patients treated with PEG-asparaginase (TXVI) had more severe hypertriglyceridemia (>1000mg/dL) compared to native l-asparaginase (TXV): 10.5% versus 5.5%, respectively (P=.007). At week 7, triglycerides did not increase with dexamethasone treatment alone (LR patients) but did increase with dexamethasone plus asparaginase (SHR patients). The variability in triglycerides explained by the triglyceride-PRS was highest at baseline and declined with therapy. Hypertriglyceridemia was associated with osteonecrosis (P=.0006) and thrombosis (P=.005), but not pancreatitis (P=.4). Triglycerides were affected more by PEG-asparaginase than native l-asparaginase, by asparaginase more than dexamethasone, and by drug effects more than genetics. It is not clear whether triglycerides contribute to thrombosis and osteonecrosis or are biomarkers of the toxicities.

Enrichment of heterozygous germline RECQL4 loss-of-function variants in pediatric osteosarcoma

Patients harboring germline pathogenic biallelic variants in genes involved in the recognition and repair of DNA damage are known to have a substantially increased cancer risk. Emerging evidence suggests that individuals harboring heterozygous variants in these same genes may also be at heightened, albeit lesser, risk for cancer. Herein, we sought to determine whether heterozygous variants in RECQL4, the gene encoding an essential DNA helicase that is defective in children with the autosomal recessive cancer-predisposing condition Rothmund–Thomson syndrome (RTS), are associated with increased risk for childhood cancer. To address this question, we interrogated germline sequence data from 4435 pediatric cancer patients at St. Jude Children's Research Hospital and 1127 from the National Cancer Institute Therapeutically Applicable Research to Generate Effective Treatment (TARGET) database and identified 24 (0.43%) who harbored loss-of-function (LOF) RECQL4 variants, including five of 249 (2.0%) with osteosarcoma (OS). These RECQL4 variants were significantly overrepresented in children with OS, the cancer most frequently observed in patients with RTS, as compared to 134,187 noncancer controls in the Genome Aggregation Database (gnomAD v2.1; P = 0.00087, odds ratio [OR] = 7.1, 95% CI, 2.9–17). Nine of the 24 (38%) individuals possessed the same c.1573delT (p.Cys525Alafs) variant located in the highly conserved DNA helicase domain, suggesting that disruption of this domain is central to oncogenesis. Altogether these data expand our understanding of the genetic factors predisposing to childhood cancer and reveal a novel association between heterozygous RECQL4 LOF variants and development of pediatric OS.

PF740 CEREBRAL BLOOD FLOW IN ADOLESCENTS WITH SICKLE CELL ANEMIA RECEIVING VOXELOTOR

Background:Oxygenated sickle hemoglobin (HbS) does not polymerize; therefore, increasing oxygen affinity to HbS is a therapeutic strategy for sickle cell anemia (SCA; HbSS and HbSβ0thalassemia), and voxelotor (GBT440) is a first‐in‐class, small molecule that increases the affinity of hemoglobin for oxygen. It has been postulated that increasing hemoglobin‐oxygen affinity could limit oxygen offloading from hemoglobin, thus impairing tissue oxygenation which could lead to an increase in cerebral blood flow and potential risk of stroke. This study used MRI techniques to evaluate cerebrovascular hemodynamics to inform on cerebral oxygenation with voxelotor therapy.Aims:To assess effects of voxelotor on grey matter cerebral blood flow (CBF) in children with SCA.Methods:This was a St. Jude Children's Research Hospital (St. Jude) investigator‐initiated, ancillary study of children enrolled in the HOPE‐KIDS1 (NCT02850406) trial. Study activities were approved by the St. Jude IRB and all participants provided documentation of informed consent. Participants underwent two non‐sedated functional magnetic resonance imaging with angiography (MRI/MRA) evaluations, once prior to receiving voxelotor and again following multiple doses (Table). Time resolved phase‐contrast (PC) MR imaging was used to measure CBF during multiple phases of the cardiac cycle at several anatomic locations, including the internal carotid arteries and anterior, middle and posterior cerebral arteries, and arterial spin labeling (ASL) sequences utilizing a multi‐channel head coil in a 3T scanner were performed.Results:Preliminary data on three patients are available. All three participants (Table) were being treated with a stable dose of hydroxyurea and had normal transcranial doppler velocities upon study entry. As per the HOPE‐KIDS1 study design, two received voxelotor at 900 mg per day and one received 1500 mg per day. Baseline MRI/MRA exams were normal in two participants and one had silent cerebral infarcts without vasculopathy. Generally, participants had higher hemoglobin levels and decreased hemolysis while receiving voxelotor. Two patients had lower global CBF while receiving voxelotor and the third showed essentially no change in CBF. Repeat MRI/MRAs showed no new or evolving areas of ischemia and EPO levels did not increase over time.Summary/Conclusion:Vasodilatory autoregulation of the cerebral vasculature enables a rapid and effective mechanism to titrate blood flow based on metabolic demand; increasing cerebral blood flow to offset impaired tissue oxygenation (Guilliams, K. Stroke 2019). In our small cohort of adolescents with SCA, the lack of increase in CBF after treatment with voxelotor suggests that there is no impairment in oxygen unloading to brain tissue. In fact, these preliminary findings of decreasing CBF with rising hemoglobin levels in two patients suggest improved oxygen delivery to the brain. Further study on the effects of voxelotor on cerebral hemodynamics and oxygen delivery is warranted.image

Global childhood cancer survival estimates and priority-setting: a simulation-based analysis.

Accurate childhood cancer survival estimates are crucial for policy makers and clinicians for priority-setting and planning decisions. However, observed survival estimates are lacking for many countries, and when available, wide variation in outcomes is reported. Understanding the barriers to optimising survival can help improve childhood cancer outcomes. We aimed to provide estimates of global childhood cancer survival, accounting for the impact of multiple factors that affect cancer outcomes in children. We developed a microsimulation model to simulate childhood cancer survival for 200 countries and territories worldwide, accounting for clinical and epidemiologic factors, including country-specific treatment variables, such as availability of chemotherapy, radiation, and surgery. To ensure model results were consistent with reported survival data, we calibrated the model to estimates from the CONCORD-2 and CONCORD-3 studies using an Approximate Bayesian Computation approach. We estimated 5-year net survival for diagnosed cases of childhood cancer in each country and territory and estimated potential survival gains of seven policy interventions focused on improving treatment availability and delivery (ie, increasing the availability of chemotherapy, radiation, general surgery, neurosurgery, or ophthalmic surgery, reducing treatment abandonment, and improving the quality of care to the mean of high-income countries) implemented in isolation or as packages. Our model estimated that, for diagnosed cases, global 5-year net childhood cancer survival is currently 37·4% (95% uncertainty interval 34·7-39·8), with large variation by region, ranging from 8·1% (4·4-13·7) in eastern Africa to 83·0% (81·6-84·4) in North America. Among the seven policy interventions modelled, each individually provided small gains, increasing global 5-year net survival to between 38·4% (35·8-40·9) and 44·6% (41·7-47·4). 5-year net survival increased more substantially when policy interventions were bundled into packages that improved service delivery (5-year net survival 50·2% [47·3-53·0]) or that expanded treatment access (54·1% [50·1-58·5]). A comprehensive systems approach consisting of all policy interventions yielded superadditive gains with a global 5-year net survival of 53·6% (51·5-55·6) at 50% scale-up and 80·8% (79·5-82·1) at full implementation. Childhood cancer survival varies widely by region, with especially poor survival in Africa. Although expanding access to treatment (chemotherapy, radiation, and surgery) and addressing financial toxicity are essential, investments that improve the quality of care, at both the health-system and facility level, are needed to improve childhood cancer outcomes globally. Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard TH Chan School of Public Health, Harvard Medical School, National Cancer Institute, SickKids, St Jude Children's Research Hospital, Union for International Cancer Control, Children with Cancer UK Davidson and O'Gorman Fellowship.

The changing burden of long-term health outcomes in survivors of childhood acute lymphoblastic leukaemia: a retrospective analysis of the St Jude Lifetime Cohort Study

Treatment for childhood acute lymphoblastic leukemia has evolved over the past five decades, with moderation of traditional chemotherapy and radiotherapy and the introduction of targeted immune-based and cellular-based therapies. The affect of these changes on late health outcomes has not been assessed. Using data from the The St Jude Lifetime (SJLIFE) Cohort, we aimed to characterise the magnitude of morbidity and patterns of late health outcomes among survivors of childhood acute lymphoblastic leukaemia treated over time. The St Jude Lifetime (SJLIFE) Cohort is a retrospective cohort study with prospective follow-up and ongoing data accrual designed to facilitate longitudinal, clinically-based assessment of health outcomes among survivors of paediatric malignancies. 980 survivors included in this analysis were diagnosed with paediatric acute lymphoblastic leukaemia at St Jude Children's Research Hospital (SJCRH) between Aug 28, 1963, and July 19, 2003, were aged 18 years old and older at enrolment, had a minimum follow-up of 10 years after diagnosis, and completed an initial on-campus SJLIFE assessment as of data cutoff (June 30, 2015). 272 community control participants, matched to survivors on 5-year age blocks in each sex, were recruited for comparison. Cumulative chemotherapy and radiation dose exposures and major medical events during and after therapy were retrieved from the medical records of the survivors. History or physical examination, laboratory analysis, physical fitness, and neurocognitive testing were done. Health conditions were graded according to a modified version of the Common Terminology Criteria for Adverse Events. Neurocognitive domains of attention (Trial Making Test Part A and Conner's Continuous Performance Test-II) and executive function (Trail Making Test Part B, Controlled Oral Word Association Test, and Wechsler Adult Intelligence Scale-III Digit Span Test Backward) were measured and age-adjusted Z scores were calculated. Mean cumulative count was used to calculate the age-standardised cumulative burden of health conditions over time. This cohort study is registered at ClinicalTrials.gov, number NCT00760656. 980 survivors of acute lymphoblastic leukaemia (50% women, median age at diagnosis 5 years [IQR 3·1-9·1 years], and median time from diagnosis of 30·0 years [22·7-36·3]) had a median age of 35·8 years (29·4-42·9) at assessment compared with 35·1 years (28·7-42·6) for 272 controls. Survivors had significantly more growth hormone deficiency, hypogonadism, and neuropathy than controls. By age 30 years, survivors of acute lymphoblastic leukaemia had, on average, 5·4 (95% CI 5·1-5·8) grade 1-4 health conditions, including 3·2 (2·9-3·4) grade 2-4 health conditions, compared with 2·0 (CI 1·7-2·2) grade 1-4 and 1·2 (1·03-1·4) grade 2-4 health conditions among controls. The cumulative burden of grade 2-4 health conditions involved multiple organ systems for survivors treated on protocols between 1962-91, but after elimination of cranial radiotherapy for children with acute lymphoblastic leukaemia, conditions now predominately include musculoskeletal and endocrine disorders for survivors on protocols between 1991-2007. Although changes in paediatric acute lymphoblastic leukaemia treatment protocols have improved overall survival, the burden of late morbidity remains high for these patients. We show that the pattern of late toxic effects has markedly changed over time, with survivors having a reduction in health conditions that are immediately life-threatening, however, maintaining health status and quality of life for survivors of paediatric acute lymphoblastic leukaemia requires continued medical surveillance, counselling, and lifestyle modifications. US National Cancer Institute and the American Lebanese Syrian Associated Charities.

Reducing Compassion Fatigue in Inpatient Pediatric Oncology Nurses.

To develop an evidence-based compassion fatigue program and evaluate its impact on nurse-reported burnout, secondary traumatic stress, and compassion satisfaction, as well as correlated factors of resilience and coping behaviors. The quality improvement pilot program was conducted with 59 nurses on a 20-bed subspecialty pediatric oncology unit at the St. Jude Children's Research Hospital in Memphis, Tennessee. Validated measures of compassion fatigue and satisfaction (Professional Quality of Life Scale V [ProQOLV]), coping (Brief COPE), and resilience (Connor-Davidson Resilience Scale-2) were evaluated preprogram and at two, four, and six months postprogram, with resilience and coping style measured at baseline and at six months postprogram. Secondary traumatic stress scores significantly improved from baseline to four months. Select coping characteristics were significantly correlated with ProQOLV subscale scores. Ongoing organizational support and intervention can reduce compassion fatigue and foster compassion satisfaction among pediatric oncology nurses.

Screening for Early Childhood Intervention in Oncology

Infants and toddlers (birth to 3 years of age) diagnosed with cancer are at a critical phase of development and are at greater risk for developmental delays due to this early adversity. Because of this risk, St. Jude Children's Research Hospital (SJCRH) developed an Early Childhood Clinic (ECC). Psychologists screen children using the Battelle Developmental Inventory, Second Edition (BDI-2). If physical and occupational therapy services are needed on the basis of the results of the BDI-2, the physical therapists and occupational therapists administer the Peabody Developmental Motor Scales, Second Edition (PDMS-2). If scores from the PDMS-2 display a delay in gross and/or fine motor skills, physical therapy and/or occupational therapy are initiated. At SJCRH, the psychologists in the ECC have screened 107 patients. Eighty-eight of the 107 patients (82%) were referred to physical therapy. Because 82% of patients who received a developmental screen are then referred to physical therapy, it appears beneficial to screen children younger than 3 years who have been diagnosed with a malignancy. The ECC at SJCRH is a program dedicated to improving quality of life for children and their families through hospital-based early intervention for chronically ill children. The ECC developed at SJCRH may be a model of care for pediatric oncology settings to encourage the development of age-appropriate motor skills while providing families with resources in home communities to further this development.

Catherine Lam

Global childhood cancer survival rates show a stark contrast between high-income countries, where survival rates are as high as 80%, and low-income and middle-income countries, where they are about 20%. For more than two decades, St Jude Children's Research Hospital (Memphis, TN, USA) has been working to tackle this imbalance. In March, 2018, St Jude became the first WHO Collaborating Centre for Childhood Cancer, with the ambitious aim of curing at least 60% of children with six common types of cancer by 2030. Co-director of the WHO Collaborating Centre is Catherine Lam, a St Jude paediatric oncologist and Director of the Health Systems Unit and of the Asia-Pacific Regional Program at St Jude Global. “There are still lots of disparities within countries and across countries that we are tackling”, Lam says. She hopes that the WHO collaboration will draw the attention of policy makers and donors so that they will dedicate sustainable resources to the challenges.

No evidence that G6PD deficiency affects the efficacy or safety of daunorubicin in acute lymphoblastic leukemia induction therapy.

Anthracyclines are used in induction therapy of pediatric acute lymphoblastic leukemia (ALL) and are known to generate oxidative stress; whether this translates into enhanced antileukemic activity or hemolytic effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency is unknown. Among 726 pediatric patients with newly diagnosed ALL treated at St. Jude Children's Research Hospital, 22 had deficient G6PD activity. We compared the prevalence of positive minimal residual disease (MRD) ≥1% at Day 15/Day 19 of induction or ≥0.01% at Day 42/Day 46 (end of induction) and the number of red blood cell (RBC) transfusions after daunorubicin in induction between patients with or without G6PD deficiency, adjusting for ALL risk group, treatment protocol, age, and gender. There was no difference in Day 15/19 (P=1) or end of induction MRD (P=0.76) nor in the number of RBC transfusions (P=0.73); the lack of association with MRD was confirmed in a dataset of 1192 newly diagnosed male patients enrolled in a Children's Oncology Group trial (P=0.78). We found no evidence that G6PD deficiency affects daunorubicin activity during induction treatment for ALL.

Quality Improvement Interventions across a Network of Pediatric Hematology-Oncology Clinics.

Achieving improvement in quality among different institutions is challenging. Immunocompromised children with febrile neutropenia are at high risk of severe infection. Pediatric hematology-oncology patients frequently experience central line-associated bloodstream infections (CLABSIs) associated with implanted catheters. A network of 8 pediatric hematology-oncology clinics affiliated with St. Jude Children's Research Hospital launched 2 initiatives designed to reduce the incidence of infections and improve infection treatment. We reviewed the timing of antibiotic administration for immunocompromised patients with a fever before and after a quality improvement intervention tailored to each affiliate clinic. We also reviewed the frequency of CLABSIs before and after implementing a central line care bundle for implanted catheters in ambulatory patients. Across the affiliate clinic network, the timing of antibiotic administration improved from the preintervention period (23% of patients received antibiotics within 60 min of registration) to 53% and 73%, in successive postintervention periods. Implementing a central line bundle for implanted catheters was associated with increased compliance and a trend toward increased time between CLABSIs. We describe an approach to quality improvement utilizing a system of monitoring with annual clinical audits, development of joint quality improvement initiatives, ongoing education, and focused training of staff for effecting change that improves patient healthcare across multiple institutions.

Estimating the total incidence of global childhood cancer: a simulation-based analysis.

Accurate estimates of childhood cancer incidence are important for policy makers to inform priority setting and planning decisions. However, many countries do not have cancer registries that quantify the incidence of childhood cancer. Moreover, even when registries do exist, they might substantially underestimate the true incidence, since children with cancer might not be diagnosed. We therefore aimed to provide estimates of total childhood cancer incidence accounting for underdiagnosis. We developed a microsimulation model to simulate childhood cancer incidence for 200 countries and territories worldwide, taking into account trends in population growth and urbanicity, geographical variation in cancer incidence, and health system barriers to access and referral that contribute to underdiagnosis. To ensure model results were consistent with epidemiological data, we calibrated the model to publicly available cancer registry data using a Bayesian approach in which the observed data are fixed and the model parameters (cancer incidence and probabilities of health system access and referral) are random variables. We estimated the total incidence of childhood cancer (diagnosed and undiagnosed) in each country in 2015 and projected the number of cases from 2015 to 2030. Our model estimated that there were 397 000 (95% uncertainty interval [UI] 377 000-426 000) incident cases of childhood cancer worldwide in 2015, of which only 224 000 (95% UI 216 000-237 000) were diagnosed. This finding suggests that 43% (172 000 of 397 000) of childhood cancer cases were undiagnosed globally, with substantial variation by region, ranging from 3% in western Europe (120 of 4300) and North America (300 of 10 900) to 57% (43 000 of 76 000) in western Africa. In south Asia (including southeastern Asia and south-central Asia), the overall proportion of undiagnosed cases was estimated to be 49% (67 000 of 137 000). Taking into account population projections, we estimated that there will be 6·7 million (95% UI 6·3-7·2) cases of childhood cancer worldwide from 2015 to 2030. At current levels of health system performance, we estimated that 2·9 million (95% UI 2·7-3·3) cases of childhood cancer will be missed between 2015 and 2030. Childhood cancer is substantially underdiagnosed, especially in south Asia and sub-Saharan Africa (including western, eastern, and southern Africa). In addition to improving treatment for childhood cancer, health systems must be strengthened to accurately diagnose and effectively care for all children with cancer. As countries expand universal health coverage, these estimates of total incidence will hopefully help guide efforts to appropriately increase health system capacity to ensure access to effective childhood cancer care. Boston Children's Hospital, Dana-Farber Cancer Institute, Harvard T H Chan School of Public Health, Harvard Medical School, National Cancer Institute, SickKids, St Jude Children's Research Hospital, and Union for International Cancer Control.