Pediatric ALL Research Articles

A Retrospective Cytogenetic Abnormality in Pediatric Acute Lymphoblastic Leukemia: Report of 11 Years.

Acute lymphoid leukemia (ALL) is the largest subset of hematologic malignancies, accounting for approximately 70%-80% of childhood leukemia, and is most common at age 4 years. The aim of this study was to define the frequency of chromosomal abnormalities in pediatric ALL. In this 11-year retrospective study, we investigated 99 patients which referred to our department due to ALL from 2010 to 2020. The age group of the patients ranged from 6 months to 14 years with a mean of 6.71 ± 4.09 years. Clinical and diagnostic findings were extracted from patients' medical records. We showed cytogenetic abnormalities of 99 pediatric ALL patients, including 78 pre-B-ALL, 9 common B-ALL, and 12 T-ALL cases. The 5-year overall survival rate (OSR) and event-free survival (EFS) of all cytogenetic abnormalities (n = 99) were 48% and 43%, respectively. There was a significant relationship between the two cytogenetic abnormalities, hypodiploidy and t(9;22), with death (P < 0.05). On comparing the subjects with normal cytogenetics to the other cytogenetic abnormalities, EFS was significantly low for hypodiploidy (P = 0.0163, hazard ratio = 0.5308) and t(9;22) (P = 0.0131, hazard ratio = 0.4908), while other cytogenetic abnormalities did not have a statistically significant difference in EFS. Our results emphasized the importance of the cytogenetic findings in evaluating the survival outcomes, which allows identifying a variety of OSR and EFS, because some of the cytogenetic abnormalities may interfere with the death and prognosis.

Health-related quality of life in adults treated for paediatric acute lymphoblastic leukaemia: a cross-sectional and longitudinal cohort study

IntroductionAcute lymphoblastic leukaemia (ALL) is the most common form of cancer in children. Although treatment methods have improved and resulted in significant improvement of survival and reduction in late effects...

The treatment development for T cell acute lymphoblastic leukemia in children, adolescents, and young adults

T-cell acute lymphoblastic leukemia (T-ALL) accounts for 10%-15% of pediatric ALL, and its frequency increases during the adolescent and young adult (AYA) periods. The improvements in pediatric T-ALL outcomes have been achieved through the treatment intensification strategies addressing the cranial radiotherapy omission issue. Furthermore, pediatric-inspired regimens have been adopted for AYA patients with T-ALL, improving outcomes. In Japan, the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG) ALL-T11/JALSG T-ALL-211-U trial has been conducted for newly diagnosed patients with T-ALL aged <25 years and has been shown to have excellent outcomes. While early T cell precursor ALL is not an obvious poor prognostic factor in recent treatment strategies for pediatric T-ALL, the minimal residual disease (MRD) has been recognized as the most reliable prognostic indicator. Therefore, MRD is now widely used in T-ALL clinical trials for risk stratification and stem cell transplantation indications. Further progress in the novel therapeutic approaches can potentially improve the outcomes of pediatric and AYA patients with T-ALL.

Electronic pill box: An easy and effective strategy to strengthen adherence to 6MP during maintenance chemotherapy for pediatric ALL

Electronic pill box: An easy and effective strategy to strengthen adherence to 6MP during maintenance chemotherapy for pediatric ALL

Expression of Heat Shock Protein 70 (Hsp-70) in Children’s Acute Lymphoblastic Leukemia

Heat shock protein (Hsp) 70 is one of the most common Hsp proteins found in humans. Like other types of Hsp, Hsp-70 acts as a chaperone protein in the process of protein synthesis. Its important role in protein synthesis means that these proteins are supposed to be inside the cell and cannot be examined outside the cell. In its development, Hsp-70 turned out to be examined outside of cells due to an increase in Hsp-70 caused by stress on cells which were caused by environmental stress conditions (heat shock, cold shock, heavy metal, oxygen-free radicals), inflammatory and infectious processes, ischemia, tissue damage, aging, and oncogenesis. Cancer studies have found high expression of Hsp-70 in solid tumors and hematological malignancies including ALL. High expression of Hsp-70 is said to have adverse implications on the outcome of ALL and therefore Hsp-70 is auspicious as a therapeutic target in the pediatric treatment of ALL. This review aims to elucidate the role of Hsp-70 expression in pediatric ALL patients.

Impact of Race/Ethnicity and Language Preferences on Pediatric ALL Survival Outcomes

Impact of Race/Ethnicity and Language Preferences on Pediatric ALL Survival Outcomes

Prognostic Value and Outcome for ETV6/RUNX1-Positive Pediatric Acute Lymphoblastic Leukemia: A Report From the South China Children's Leukemia Group.

PurposeTo analyzed the outcome of ETV6/RUNX1-positive pediatric acute B lymphoblastic leukemia (B-ALL) with the aim of identifying prognostic value.MethodA total of 2,530 pediatric patients who were diagnosed with B-ALL were classified into two groups based on the ETV6/RUNX1 status by using a retrospective cohort study method from February 28, 2008, to June 30, 2020, at 22 participating ALL centers.ResultsIn total, 461 (18.2%) cases were ETV6/RUNX1-positive. The proportion of patients with risk factors (age <1 year or ≥10 years, WB≥50×109/L) in ETV6/RUNX1-positive group was significantly lower than that in negative group (P<0.001), while the proportion of patients with good early response (good response to prednisone, D15 MRD < 0.1%, and D33 MRD < 0.01%) in ETV6/RUNX1-positive group was higher than that in the negative group (P<0.001, 0.788 and 0.004, respectively). Multivariate analysis of 2,530 patients found that age <1 or ≥10 years, SCCLG-ALL-2016 protocol, and MLL were independent predictor of outcome but not ETV6/RUNX1. The EFS and OS of the ETV6/RUNX1-positive group were significantly higher than those of the negative group (3-year EFS: 90.11 ± 4.21% vs 82 ± 2.36%, P<0.0001, 3-year OS: 91.99 ± 3.92% vs 88.79 ± 1.87%, P=0.017). Subgroup analysis showed that chemotherapy protocol, age, prednisone response, and D15 MRD were important factors affecting the prognosis of ETV6/RUNX1-positive children.ConclusionsETV6/RUNX1-positive pediatric ALL showed an excellent outcome but lack of independent prognostic significance in South China. However, for older patients who have the ETV6/RUNX1 fusion and slow response to therapy, to opt for more intensive treatment.

Genetic Variants in METTL14 are Associated with the Risk of Acute Lymphoblastic Leukemia in Southern Chinese Children: A Five-Center Case-Control Study.

Background/AimAcute lymphoblastic leukemia (ALL) is the most common form of pediatric cancer. METTL14, an N6-methyladenosine (m6A) modification protein, plays several roles in cancer development and is involved in the pathogenesis of various types of cancers. However, the role of METTL14 gene single nucleotide polymorphisms (SNPs) in pediatric ALL susceptibility remains to be investigated.MethodsA case-control design and multinomial logistic regression were used to develop models to estimate the overall risk for pediatric ALL and three METTL14 gene SNPs (rs298982 G/A, rs298981 A/G and rs1064034 T/A) in 808 cases and 1340 controls, which were genotyped using a TaqMan assay. The associations were estimated by odds ratios (ORs) with their 95% confidence intervals (CIs). Furthermore, stratified analysis was performed to explore associations of rs298982 and rs1064034 with pediatric ALL susceptibility in terms of age, sex, immunophenotype, minimal residual disease (MRD), and other clinical characteristics.ResultsAmong the three analyzed SNPs, rs298982 G/A and rs1064034 T/A exhibited a significant association with decreased childhood ALL risk, while rs298981 A/G exhibited no difference. In stratified analysis, rs298982 GA/AA and rs1064034 TA/AA had a protective effect in children <120 months of age and males, common B ALL, TEL-AML, non gene fusion, normal diploid, and high WBC. However, the rs1064034 TA/AA genotype was associated with an increased risk of mixed immunophenotyping. Compared with the reference haplotype GAT, haplotypes CAA, CGT and CGA were significantly associated with elevated ALL risk, while haplotype GGT was significantly associated decreased ALL risk. Moreover, subjects carrying rs298982 A or rs1064034 A exhibited less minimal MRD after induced chemotherapy. Functional annotations revealed that METTL14 gene SNPs rs298982 G/A and rs1064034 T/A could be potential functional variants.ConclusionIn conclusion, METTL14 gene polymorphisms influence the risk of ALL in southern Chinese children and might be potential biomarkers for pediatric ALL susceptibility and chemotherapeutics.

Transcriptional and Mutational Profiling of B-Other Acute Lymphoblastic Leukemia for Improved Diagnostics.

Simple SummaryPediatric acute lymphoblastic leukemia is the most common malignancy in children. Based on the genetic characteristics of the tumor, patients are risk-stratified and treated with different treatment intensities. However, in a proportion of cases, known as B-other, no genetic alterations relevant for risk stratification are found with routine diagnostic procedures. In this study, we performed RNA sequencing, a comprehensive and cutting-edge method, of 185 children with B-other leukemia and analyzed gene fusions, expression profiles and mutations. Furthermore, we validated our findings using commonly used diagnostic techniques. Our results identified a subgroup of cases clustering with known leukemia subtypes, e.g., DUX4-positive, and subgroups characterized by mutations in PAX5 and IKZF1, resulting in more cases being assigned to a defined subgroup. Moreover, we identified new fusion partners of ETV6, IKZF1 and PAX5. Our data demonstrate the applicability and technical considerations for the use of RNA sequencing to personalize genetic diagnostics in pediatric leukemia. B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common cancer in children, and significant progress has been made in diagnostics and the treatment of this disease based on the subtypes of BCP-ALL. However, in a large proportion of cases (B-other), recurrent BCP-ALL-associated genomic alterations remain unidentifiable by current diagnostic procedures. In this study, we performed RNA sequencing and analyzed gene fusions, expression profiles, and mutations in diagnostic samples of 185 children with BCP-ALL. Gene expression clustering showed that a subset of B-other samples partially clusters with some of the known subgroups, particularly DUX4-positive. Mutation analysis coupled with gene expression profiling revealed the presence of distinctive BCP-ALL subgroups, characterized by the presence of mutations in known ALL driver genes, e.g., PAX5 and IKZF1. Moreover, we identified novel fusion partners of lymphoid lineage transcriptional factors ETV6, IKZF1 and PAX5. In addition, we report on low blast count detection thresholds and show that the use of EDTA tubes for sample collection does not have adverse effects on sequencing and downstream analysis. Taken together, our findings demonstrate the applicability of whole-transcriptome sequencing for personalized diagnostics in pediatric ALL, including tentative classification of the B-other cases that are difficult to diagnose using conventional methods.

Therapeutic Targeting of CD74 with STRO-001 Antibody-Drug Conjugate in AML and ALL

Introduction: Despite advances in cytotoxic and targeted therapies, recurrent disease remains the most significant obstacle to long-term survival in patients with childhood and adult leukemias. As part of our efforts to identify therapies that can be repurposed for immediate use in patients with leukemia, we interrogated a library of all available antibody-drug conjugates (ADCs) whose targets are expressed in leukemias (AML or ALL). A list of targets with available ADCs was merged with transcriptome data from over 3000 pediatric and adult leukemias (AML and ALL) to identify targets that are expressed in a large cohort of leukemias with immediate therapies for repurposing. The transcriptome data set included nearly 2000 pediatric AML cases sequenced as part of Target Pediatric AML (TpAML), 419 adult AML cases from TCGA LAML and the Beat AML program, as well as 853 ALL cases from COG and St. Jude trials. In this repurposing endeavor, CD74 emerged as the most expressed transcript in AML and ALL. CD74 encodes for a cell surface protein that associates with the class II major histocompatibility complex and is involved in the regulation of antigen presentation for immune response and B-cell differentiation. STRO-001 (Sutro Biopharma) is a CD74-directed, site-specific ADC developed for the treatment of multiple myeloma and lymphomas. Given broad expression of CD74 in leukemias, we evaluated the efficacy of STRO-001 in AML and ALL preclinical models.Methods: To characterize CD74 expression, RNA-seq data obtained from pediatric and adult AML and ALL patients was examined. Cell surface expression of CD74 was determined by flow cytometry using PE labeled anti-human CD74 antibody. The CD74-targeting ADC (STRO-001) was obtained from Sutro Biopharma.The preclinical efficacy of STRO-001 was evaluated against AML and ALL cell lines and patient samples expressing various levels of CD74 in vitro and in vivo. For in vivo studies, AML and ALL cell lines were transduced with GFP/Luciferase construct, and GFP+ cells were injected intravenously into NSG mice. Leukemia burden was measured by bioluminescence (IVIS) imaging weekly.Results: Transcriptomics analysis showed CD74 expression in a majority of adult and pediatric AML (>99% of cases) and at a much higher level compared CD33 and CD123 (targets currently developed for AML, Fig. 1A). CD74 is also broadly expressed in pediatric ALL, with a significant increase in expression observed compared to CD19 and CD22 (known targets in ALL, Fig. 1B). We confirmed that CD74 is expressed on the cell surface of AML blasts in primary patient samples (Fig. 1C) as well as AML and ALL cell lines (Fig. 1D). Given confirmation of cell surface expression of CD74, we investigated whether targeting CD74 can effectively eliminate leukemia cells. We evaluated the in vitro cytotoxicity of STRO-001 against K562 (a CML cell line that does not express CD74), AML cell lines (MV4;11 and NOMO-1), and ALL cell lines (REH1 and RS4;11) with varied CD74 expression. STRO-001 demonstrates target-specific cytotoxicity against CD74-expressing AML and ALL cell lines, but not K562 cells (Fig. 1E). STRO-001 exhibited high potency in CD74 expressing cells, with IC-50s of 41nM (MV4;11), 1.3nM (NOMO-1), 0.7nM (REH-1) and 3nM (RS4;11). In vivo studies in NSG mice transplanted with AML and ALL cell lines showed high potency. Treatment with STRO-001 at 3mg/kg once a week for 3 weeks effectively eradicated the leukemia in NOMO-1, REH-1, and RS4;11-bearing xenograft mice, while disease progression was observed in untreated control mice (Fig. 1F). We further evaluated the efficacy of STRO-001 in primary patient samples. Primary leukemia specimens from 3 patients with varied CD74 expression (Fig. 1G) were incubated with increasing concentrations of STRO-001 for 3 days. STRO-001 exhibited potent anti-leukemia activity against primary AML cells with IC-50s of 17.4nM, 12.8nM, and 4.07nM, respectively (Fig. 1H).Conclusion: Through transcriptomics profiling and validation of the cell surface expression by flow cytometry, we have identified CD74 as a viable therapeutic target for AML and ALL in children and adults. We further demonstrate that targeting CD74 with STRO-001 effectively eliminates leukemia cells both in vitro and in vivo, providing the preclinical data to compel evaluation of STRO-001 in clinical trials for childhood and adult leukemia. [Display omitted] DisclosuresHylkema: Moderna: Current equity holder in publicly-traded company; Quest Diagnostics Inc: Current equity holder in publicly-traded company. Pardo: Hematologics, Inc.: Current Employment. Abrahams: Sutro Biopharma: Current Employment. Bedard: Sutro Biopharma: Current Employment. Molina: Sutro Biopharma: Current Employment. Eidenschink Brodersen: Hematologics, Inc.: Current Employment, Other: Equity Ownership. Loken: Hematologics, Inc.: Current Employment, Other: current equity holder in a privately owned company.

The Effect of Cytomegalovirus on Pediatric Acute Lymphoblastic Leukemia

Background: Recent evidence supports the role of cytomegalovirus (CMV) in the development of childhood ALL. The underlying mechanism and CMV's role in the leukemic cell phenotype is unknown, but CMV typically interacts with the host immune system allowing the virus to survive in a latent state; it may be that this immune dysregulation affects the risk of ALL. This study aims to explore the association of CMV and ALL at the time of leukemia diagnosis, using AML cases as a control and further, to determine whether CMV affects certain subgroups of ALL patients such as specific ethnicities, age groups, or cytogenetic subtypes.Methods: Pediatric diagnostic leukemia bone marrow samples obtained from the California Childhood Leukemia Study were screened for the presence of CMV DNA using a custom-designed droplet digital PCR assay. A total of 869 cases were analyzed including 125 AML cases and 744 ALL cases. Demographic and clinical features were compared between patients found to be CMV positive (cases with any detectable CMV positive droplets) and those who were CMV negative (cases with no detectable CMV positive droplets). The effect of the level of CMV viral DNA load was also assessed. For a subset of cases (n=61), Affymetrix Array gene expression data were available and differential gene expression performed to compare CMV positive cases with high viral load to CMV negative cases. Odds ratios and confidence intervals were estimated using logistic regression.Results: ALL cases were more likely to be CMV positive compared to AML (OR: 2.50; CI 1.00, 5.47, p = 0.039 for CMV highest quintile vs. CMV negative). Within ALL cases, B-cell ALL (B-ALL) was significantly associated with CMV positivity compared to T-cell ALL (T-ALL) (OR: 2.93; CI: 1.01, 8.52, p = 0.048 for CMV highest tertile vs. CMV negative). Further subtype analysis of B-ALL cases revealed CMV positivity to be significantly associated with high hyperdiploidy, one of the most common ALL subtypes, when compared to ETV6-RUNX1 (OR: 2.52; CI:1.34, 4.73, p = 0.004 for highest CMV tertile vs. CMV negative). CMV positive B-ALL cases were also more likely to harbor deletions of EBF1, a B-cell development gene, compared to CMV negative cases (OR: 6.10; CI: 1.09, 34.06, p = 0.04 for CMV 2 nd tertile vs. CMV negative; OR: 5.54; CI: 1.13, 27.18, p = 0.03 for CMV highest tertile vs. CMV negative). Differential gene expression analysis revealed 830 genes to be significantly differentially expressed between the highest quintile of CMV positive cases and CMV negative cases, and gene ontology analysis revealed upregulation of processes involved in viral infection and replication. Specifically, cytokine signaling pathways including IL-1, IL-8, and IL-7 were upregulated in CMV positive cases while Th1 and the pathway facilitating crosstalk between dendritic cells and natural killer cells were downregulated. Interestingly, B-cell receptor signaling was also upregulated in CMV positive cases.Conclusion: Our results support the hypothesis that CMV plays an enhanced role in leukemia development in specific subtypes of ALL, and not in AML development. The ability of CMV to interact with the host immune system, highlights immune dysregulation as a potential mechanism by which CMV contributes to risk of ALL. Gene expression analysis on a subset of cases revealed differentially expressed genes to be enriched in pathways involved in immune response, suggesting a potential role for active CMV infection in the leukemic phenotype. The patterns of up- and downregulation in these pathways were consistent with the host response to CMV. Additionally, acute CMV infection has been shown to promote B-cell activation and proliferation. Further, CMV seropositive individuals have been reported to have altered immune responses even with the virus in a latent state highlighting the virus's effect on B-cells. In our study we also found B-cell receptor signaling to be upregulated in CMV positive cases. This is consistent with the known effects of CMV in a typical host, but in patients with leukemia it provides an interesting potential link between CMV infection and development of pediatric ALL. These intriguing results require validation and warrant continued investigation of the role of CMV in pediatric leukemia development. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phase 1b Study of Carfilzomib in Combination with Induction Chemotherapy in Children with Relapsed or Refractory Acute Lymphoblastic Leukemia (ALL)

Phase 1b Study of Carfilzomib in Combination with Induction Chemotherapy in Children with Relapsed or Refractory Acute Lymphoblastic Leukemia (ALL)

Histone Hypoacetylation-Driven CD9 Repression Arrests Differentiation and Evades Immunosurveillance in Pediatric Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a genetically heterogeneous hematologic malignancy characterized by uncontrolled proliferation of myeloid progenitor cells accompanied by impaired terminal differentiation. Despite intensive treatment regimes, the clinical outcomes remain poor, underscoring the need to decipher the underlying pathology and implement therapeutic interventions. Emerging evidence suggest myeloblasts could evolve machineries to evade T cell patrol and hinder immunotherapies. Here, we present a new mechanism driving immune escape in the context of pediatric AML, based on our discoveries of CD9 in hematopoietic stem cells (HSC; Leung et al, Blood, 2011) and acute lymphoblastic leukemia (ALL; Leung et al, Leukemia, 2020).We first examined CD9 expression and its prognostic impact in patient cohorts of childhood leukemia. The expression of cell surface CD9 on blasts of pediatric AML patients (13.2%, n=81) was significantly lower than that of pediatric ALL patients (90.4%, n=181, P<0.001) or that on CD34+ HSC of normal bone marrow donors (48.4%, n=22, P=0.014). Among pediatric AML cases, the blasts of 32 patients (39.5%) were CD9+. The 5-year RFS rate of CD9- patients was significantly lower than CD9+ patients (34.1% vs. 61.2%, P=0.018).Enforced CD9 expression in MV4-11 cells significantly suppressed proliferation (P<0.01), Ki-67 expression (P=0.041) and colony formation (P=0.002). NOD/SCID mice transplanted with CD9+ cells exhibited a drastic reduction of leukemic load in the bone marrow, spleen, blood and liver by 70.7-91.8% (P<0.05), a significantly prolonged survival duration (P<0.001), and a marked regression of extramedullary myeloid sarcoma when compared with animals transplanted with CD9- MV4-11 cells.A marked decrease of H3K9/27Ac occupancy in the CD9 locus was observed in AML than in ALL cells (4.8-14.2-fold, P<0.05), and strongly correlated with CD9 repression (r=0.585-0.719, P<0.01). Exposure of CD9- AML cell lines (n=8) and samples (n=9) with a histone deacetylase inhibitor panobinostat significantly elevated CD9 mRNA and protein expression (3.1-32.2-fold, P<0.05), restored activating histone acetylation marks (4.1-41.6-fold, P<0.05) and suppressed myeloblast proliferation ex vivo (median IC50: 21.4 nM).Mechanistically, global transcriptome profiling of pediatric AML (n=31) revealed decreased stemness (NES: -1.7, P=0.01) and increased monocyte (NES: 1.8, P=0.034) gene signatures in CD9+ patient samples. Moreover, single-cell transcriptomic analyses of total bone marrow cells from MV4-11-tranplanted mice detected a significant enrichment of differentially regulated genes functioning in the antigen processing and presentation pathway. Concordantly, we observed a profound up-regulation of CD9 (9.4-51.1-fold, P<0.001) in PMA-mediated monocyte/macrophage-like AML differentiation cultures preceding the appearance of lineage markers CD14, CD36 and iCD68 as well as the antigen presentation molecule MHC-I. In the overexpression system, CD9 not only elevated the expression of monocytic markers, but also promoted basal and IFNγ-induced MHC-I/II expression (P<0.01) through the JAK/STAT axis. Inter-patient comparisons of bone marrow samples (n=27) revealed a higher MHC-I expression in CD9+ than CD9- AML (MFI: 61820 vs. 18601, P<0.001). Interestingly, tetraspanin CD9 physically bound to MHC-I/II and formed an immune complex as revealed by co-immunoprecipitation. In NSG mice, co-transplantation of human PBMCs mounted an effective immunity against CD9+ but not CD9- AML (MV4-11 and MOLM-13), concomitant with a robust bone marrow infiltration of cytotoxic T cells. Syngeneic transplantation in immunocompetent mice, AML/T cell co-cultures, antigen-specific assays and panobinostat priming/T cell adoptive transfer are currently underway to fully dissect the role of CD9 in leukemia immunity.Taken together, our data provided molecular, cellular and clinical evidence showing the plausible function of CD9 as a key driver intertwining differentiation and immunosurveillance in pediatric AML, and inspired a new combinatorial epigenetic/immunotherapy for this rare but aggressive malignancy. DisclosuresNo relevant conflicts of interest to declare.

MTHFR and SLCO1B1 Gene Polymorphism and Methotrexate Induced Toxicity in Children with Acute Lymphoblastic Leukemia in the Northwest of Mexico

Introduction.Acute lymphoblastic leukemia (ALL) represents approximately 52% of pediatric cancer diagnoses in Mexico and is the leading cause of death by disease among children of 5 to 14 years. The use of pharmacogenomics for the detection of germline genetic variations, which are associated with sensitivity or toxicity to chemotherapy, is one of the recent strategies to improve survival. Some mutations present in genes related to the metabolism or transport of methotrexate (MTX) have been associated with the occurrence of toxicities during treatment in pediatric acute lymphoblastic leukemia. The effect of the SNP´s rs1801131, rs1901133 in the MTHFR and rs4149058, rs4149081 in the SLCO1B1 gene have not been studied in Northwest Mexican children with acute lymphoblastic leukemia.Patients and methods.Eligible patients were infants less than 1 year to adolescents less than 18 years of age, diagnosed with B-ALL at the Specialties Children's Hospital of Chihuahua, Mexico. Patients were treated according to the locally protocols based on the St. Jude Children's Research Hospital Total XIIIB and Total XV protocols. We followed-up for two to four weeks after 24 hours high dose MTX (1 to 5 g/m 2) administration. Toxicity data were collected objectively from the patient's medical files and adverse effects were classified as a dichotomous variable yes/no. Genomic DNA was extracted with the Master Pure DNA purification kit (Epicentre Illumina® Company) from peripheral blood. Genotyping assays were performed by real-time polymerase chain reaction, using rhAMp® IDT® genotyping probes (Iowa, USA) and Quant Studio 3 Applied Biosystems Real-Time System Thermal Cycler (Thermo Fisher Scientific®). For statistical analysis association between MTX dose, presence of toxicity, and genetic polymorphisms was evaluated by the chi-square or Fisher's exact test. The effect sizes of the associations were estimated by the OR's from univariate logistic regressions and multivariate logistic regressions to account for the possible confounding effect of sex and age. Analyses were performed by using SAS System and IBM SPSS Statistics Base 22.0 software.Results.The study population demographics and clinical characteristics are summarized in Table 1. MTHFR and SLCO1B1 genotype in our patients and controls obtained from the 1000 Genomes Project database are shown in table 2. In general, a predominance of toxicity events is observed in patients heterozygous for both polymorphisms in the MTHFR gene, but not in the SLCO1B1 gene (Figure 1). Through a logistic regression analysis, we observed an association of 8% between the polymorphisms rs1801131, rs1901133, rs4149058, rs4149081 and the presence of anemia. The AC heterozygous of the rs1801131 has the strongest association with a positive coefficient (+1.1355) and when comparing AC heterozygotes with CC mutated homozygotes, an Odds Ratio (OR) of 4.64 (CI: 95%, 0.719) was observed, identifying it as a risk factor. In contrast, the homozygous AA of the same gene, presented a negative coefficient (-0.7354), thus decreasing the probability of presenting anemia in this population. The presence of neutropenia was associated in 25% with the polymorphisms rs1801131, rs1901133, rs4149058, rs4149081. The AA homozygous of rs1801131 decreased the probability of developing neutropenia (coefficient -0.7643, p=0.04) and the TC heterozygous of rs4149056 increased the probability when comparing with the wild TT homozygotes (OR of 2.91, CI: 95%, 0.496). Liver enzymes elevation was associated in 84% with the polymorphisms rs1801131, rs1901133, rs4149058, rs4149081. We found that the presence of TC heterozygote of rs4149056 decreased the probability of hepatotoxicity (coefficient -1.5718, p=0.03) and GA heterozygote of rs41419081 increased it (coefficient +3.2056, p=0.004) (Table 3). According to this statistical model, we could not analyze the association between the polymorphisms rs1801131, rs1901133, rs4149058, rs4149081 and thrombocytopenia, mucositis, febrile neutropenia or creatinine elevation.ConclusionToxicity related to treatment is one of the most important causes of death among pediatric ALL patients in Mexico, the development of precision medicine through the identification of SNPs associated with the variability in our patients' response is an alternative to minimize methotrexate toxicity and maximize its benefit during its use. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Decoding Clonal Evolution in Relapsed T Cell Acute Lymphoblastic Leukemia at Single Cell Resolution

T-lineage acute lymphoblastic leukemia (T-ALL) comprises approximately 10-15% of pediatric ALL cases with distinct feature in biology and largely inferior outcome compared to B-ALL. Growing evidence has reflected pivotal roles of clonal evolution in T-ALL recurrence, but bulk sequencing may not serve as the perfect model to reliably infer clonal heterogeneities and their immunomodulatory milieu during leukemia development. In this study, single-cell sequencing was applied to uncover leukemic clonal relationships with relapse throughout chemotherapy in T-ALL at a more accurate resolution.We performed bulk whole-exome sequencing for sorted CD7 + BMMCs from 5 pairs of diagnosis-relapse (Dx_Rel) samples, revealing a series of well-reported hotspot mutations in T-ALL. Among those, we observed diagnosis-specific variations and relapse-emerged variations, suggesting the putative correlations with chemo-resistance. Transcriptomic sequencing highlighted additional stemness and metabolic abnormalities underlying leukemic re-occurrence. Incorporated Dx_Rel paired ATAC-seq depicted relapse-specific activated chromatin regions, such as ELK1, ELK4, RUNX1.To dissect clonal diversities within and across the 5 Dx_Rel T-ALL pairs, we carried out high-throughput droplet-based 5'-single-cell RNA-seq (scRNA-seq) and paired T cell receptor sequencing (scTCR-seq). By performing unsupervised clustering of scRNA-seq profiles encompassing 10 samples, we identified 23 distinct T-lineage clusters (Cluster 0-22) based on the two-dimensional UMAP visualization. In 2 out of 5 patients (T593 and T788), diffusion map of T-lineage sub-clusters between diagnostic and relapsed samples appeared to be almost identical, while distinct shifts from diagnosis to relapse in the compositions have been observed in the other 3 out of 5 patients (T956, T723 and T856). Besides, it was noteworthy that two T-cell sub-clusters were concluded as “normal” T cells (Cluster 9 and 12) uniformly presented in both diagnostic and relapsed diffusion of T-cell sub-clusters across 5 Dx_Rel, from which TCR repertoires and expression profiles could well discriminate leukemic cells.Next, we sought to further deconvolute the clonal evolution patterns for T-ALL Dx_Rel pairs. We observed that except in T788 lacking of clonal TCRs, dominant diagnostic clones of the other 4 patients diminished (T593) or vanished (T956, T723, T856) at relapse, sparing newly emerged subclones predominantly substituted at relapse. We clearly depicted two distinct patterns of evolutionary trajectories in these 4 Dx_Rel pairs by comprehensively mapping hierarchical TCR clonotypes onto leukemic clonotypes at single cell levels. Specifically, in T956 and T723, we observed significant outgrowth of incidental diagnostic sub-clones at relapse, whereby surrogate TCR repertoires correspondingly enumerated, suggestive of dynamic shifts in dominant clone over continuous chemo-exposure. Whereas in T593 and T856, expanding clones at relapse were showed up with completely different gene signatures from the diagnostic ones, but dominant clones at diagnosis and relapse were surprisingly presented with identical TCR repertoires. This was undoubtedly informative of leukemic “clonal drift” within which hypothetical intrinsic transformation happened to the same subclones over persistent chemotherapy.Besides, we took advantage of our well-discriminated model to fully delineated the involvement of “normal” T subclusters in leukemic latency and chemo-responsiveness. By analyzing TCR repertoires in combined with expression profiles, we noted that “normal” T cells infiltrated by T-ALL were majorly distributed in CD8-effector sub-clusters compared to those from healthy donor, suggesting a robust leukemic stimulation on effector CD8 signaling in T-ALL microenvironment.Collectively, our presented study accurately distinguished leukemic cells from normal T cells in T-ALL at a single-cell resolution. By tracking transcriptomic profiles within and across Dx_Rel T-ALL pairs, we further identified distinct clonal evolutionary patterns, which may determine diversified fates of leukemic clones in response to therapeutic pressures. In the meantime, we provided a comprehensive phenotypic view on “normal” T cells under leukemic prevalence and re-occurrence, extending significant implications for future precise immunotherapies. DisclosuresNo relevant conflicts of interest to declare.

Genome-Wide Analysis of Allele-Specific Expression Genes in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction. Pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) is the most common pediatric hematological malignancy and it remains an important cause of morbidity and mortality in children. In this study, we performed an allele-specific expression (ASE) analysis of pediatric BCP ALL with the aim to investigate the relationship between cis-regulatory mutations and gene expression patterns.Materials and methods. Twenty-two high hyperdiploid ALL, twenty ETV6/RUNX1-positive ALL, seven TCF3/PBX1-positive ALL and twenty-eight genetically unclassified BCP ALL (“B-other”) were subjected to whole genome sequencing, SNP array analysis and RNA sequencing. The binomial test was applied to estimate the allelic bias of heterozygous exonic single nucleotide variants (SNVs) in the RNA sequencing data against the genomic data. Allelic ratios >2 or <0.5, and P values <0.05 were used to identify allele-specific expression protein-coding genes.Results. We identified 12,693 expressed genes, of which 9,672 (76%) had heterozygous exonic SNVs (informative genes), in multiple BCP ALL samples (n>2) in 77 of the investigated samples. Genes with ASE were distributed evenly across the autosomal chromosomes in the different subtypes with a range of 30 - 165 ASE genes per case (median number, 86). We found that 630 (6.5%) genes displayed ASE in multiple BCP ALL samples (n>2), of which only eight autosomal genes had monoallelic expression in more than two investigated samples. This suggests that ASE and monoallelic expression are relatively rare in BCP ALL. Gene enrichment analyses showed that pathways involving negative regulation of natural killer cell-mediated cytotoxicity and cell proliferation were enriched, indicating that ASE events possibly were associated with the cell proliferation and leukemia progression in BCP ALL. Furthermore, the hematopoiesis pathway was also enriched in ASE genes that showed high allelic expression bias (allelic ratios >2.5), suggesting that ASE genes might be associated with leukemia development. Somatic genomic aberrations that could cause ASE were also investigated in this study. All informative cases with TCF3/PBX1 rearrangement (n=4) showed monoallelic expression of the PBX1 gene, likely associated with the PBX1 truncation caused by the fusion. Additionally, CHP1, located in 15q15.1, displayed ASE in one case with an inversion involving that chromosome band, indicating a potential cis-acting element in the inversion region that regulated the CHP1 gene expression. Notably, PAX5 displayed various patterns of ASE in BCP ALL. One of three cases with PAX5/ZCCHC7 gene rearrangements displayed PAX5 ASE while the other two did not, indicating a potential uncovered cis-regulatory element around the PAX5/ZCCHC7 breakpoints. Furthermore, two cases with no PAX5 gene rearrangement displayed monoallelic expression of the PAX5 gene, suggesting that there are additional epigenetic alterations were also involved in the regulation of PAX5 gene expression in BCP ALL.Conclusions. In this study, we have characterized genes displaying ASE in childhood BCP ALL. Our data provide new insight into pathogenesis of BCP ALL and may be used to identify novel targets for treatment. DisclosuresNo relevant conflicts of interest to declare.

Access to CAR-T Cell Therapy in Underrepresented Populations: A Multicenter Cohort Study of Pediatric and Young Adult ALL Patients

Introduction: Social determinants of health such as race, ethnicity, and socioeconomic status (SES) are associated with inferior outcomes in patients with B-cell acute lymphoblastic leukemia (B-ALL). Latinx patients have worse event-free survival and are at increased risk of relapse when compared to non-Latinx peers; given that low SES is also an independent predictor of relapse, some of this disparity is likely the result of structural racism. Chimeric antigen receptor T-cell therapy (CAR-T) is a promising approach to improve survival for patients with relapsed/refractory (r/r) B-ALL. However, given the limited number of cellular therapy centers, these therapies may not be equally accessible to patients of low SES or patients from historically disadvantaged populations. The sociodemographic characteristics of pediatric and young adult patients referred for CAR-T have not been well-described. We aimed to evaluate how sociodemographic characteristics of patients referred for CAR-T from outside institutions (referred CAR-T) differed from r/r patients referred for CAR-T at their home institution (local CAR-T), r/r patients not referred for CAR-T at their home institution (other relapse), and patients without r/r disease (without relapse). We hypothesized that there would be a higher proportion of non-Latinx White patients and patients with higher SES among referred CAR-T patients.Methods: We conducted a multicenter, retrospective cohort study of children and young adults with B-ALL treated at five large pediatric hospitals between 2012-2018. Patients diagnosed with B-ALL between age 0 and 29 were included. Clinical and demographic data (including race/ethnicity, insurance, and primary language) were collected from the electronic health record and analyzed using descriptive statistics. For patients residing within the United States, ArcGIS NSES Index software was used to assign SES score by census tract (0-100, with 50 as the national average).Results: Our cohort included 1374 patients with B-ALL, of which 372 (27%) had r/r disease (Table 1). Among 372 r/r patients, 142 were referred CAR-T patients and 230 were treated for r/r leukemia at their home institution (35% local CAR-T and 65% other relapse). Median distance traveled to CAR-T therapy was 824 miles among referred patients and 20.5 miles among local CAR-T patients. The majority of patients (76% of the overall cohort and 79% of those receiving CAR-T) were either White or Latinx.A higher proportion of local CAR-T patients were Latinx compared to referred CAR-T patients (56% vs. 29%; p<0.01). Conversely, fewer local CAR-T patients were non-Latinx White compared to referred CAR-T patients (28% vs. 47%; p<0.01). A higher proportion of local CAR-T patients had public insurance compared to referred CAR-T patients (65% vs. 31%; p<0.001). A higher proportion of local CAR-T patients identified Spanish as their primary language compared to referred CAR-T patients (24% vs. 6%; p<0.001). Mean SES scores were similar across all groups.Conclusions: In a large multicenter cohort of B-ALL pediatric and young adult patients, the demographics of referred CAR-T patients were notably different than local CAR-T patients. Latinx patients are at increased risk of relapse, making access to CAR-T all the more essential; however, we found less than one-third of patients referred from outside institutions for CAR-T cell therapy were Latinx, while a majority of patients receiving CAR-T cell therapy locally were Latinx. This association suggests that barriers to access such as distance and need for travel may differentially impact Latinx patients due to structural racism. Spanish-speaking patients and patients with public insurance were also underrepresented in referrals from outside institutions. However, these observational differences may be in part due to regional variations in the demographics of the population. Preliminary analysis by site is underway and indicates that center level demographic variation may be driving some of the observed differences. Very few Black and Asian patients received CAR-T within our cohort making any assessments of patterns of care difficult. Future work will involve additional analysis to identify non-clinical factors that may influence referral and enrollment rates on CAR-T clinical trials as well as qualitative work to understand these factors from the patient and provider perspectives. [Display omitted] DisclosuresWalters: AllCells, Inc: Consultancy; BioLabs, Inc: Consultancy; Vertex pharmaceuticals: Consultancy; Ensoma, Inc.: Consultancy. Ramsey: Vertex Pharmaceuticals: Consultancy; Cystetics Medicines: Consultancy.

Novel Associations Between METTL3 Gene Polymorphisms and Pediatric Acute Lymphoblastic Leukemia: A Five-Center Case-Control Study.

ObjectiveTo reveal the contributing role of METTL3 gene SNPs in pediatric ALL risk.Patients and MethodsA total of 808 pediatric ALL cases and 1,340 cancer-free controls from five hospitals in South China were recruited. A case-control study by genotyping three SNPs in the METTL3 gene was conducted. Genomic DNA was abstracted from peripheral blood. Three SNPs (rs1263801 C>G, rs1139130 A>G, and rs1061027 A>C) in the METTL3 gene were chosen to be detected by taqman real-time polymerase chain reaction assay.ResultsThat rs1263801 C>G, rs1139130 A>G, and rs1061027 A>C polymorphisms were significantly associated with increased pediatric ALL risk was identified. In stratification analyses, it was discovered that rs1263801 CC, rs1061027 AA, and rs1139130 GG carriers were more likely to develop ALL in subgroups of common B-ALL, MLL gene fusion. Rs1263801 CC and rs10610257 AA carriers were more possible to increase the risk of ALL in subgroups of low hyperdiploid, and all of these three SNPs exhibited a trend toward the risk of ALL. All of these three polymorphisms were associated with the primitive/naïve lymphocytes and MRD in marrow after chemotherapy in ALL children. Rs1263801 CC and rs1139130 AA alleles provided a protective effect on MRD ≥0.01% among CCCG-treated children. As for rs1139130, AA alleles provided a protective effect on MRD in marrow ≥0.01% on 33 days and 12 weeks among CCCG-treated children, but provided a risk effect on MRD in the marrow ≥0.01% among SCCLG-treated children. As for rs1263801 CC and rs1139130 AA, these two alleles provided a protective effect on MRD in the marrow ≥0.01% among CCCG-treated children.ConclusionIn this study, we revealed that METTL3 gene polymorphisms were associated with increased pediatric ALL risk and indicated that METTL3 gene polymorphisms might be a potential biomarker for choosing ALL chemotherapeutics.

CAR T for Pediatric ALL: What’s Next?

Introduction With intensified chemotherapy having long since reached its limits in ALL, patients who do not respond to such chemotherapy previously had very poor outcomes. Immunotherapies have improved these outcomes, with CAR T-cells being the most powerful of these immunotherapies. Chimeric antigen receptors (CARs) combine a binding fragment (scFv) of an antibody with intracellular signaling domains, using synthetic biology to create a molecule which combines binding and activation functions to bring T-cells to leukemia cells, kill leukemia cells, and activate T-cells for significant in vivo proliferation. In the current products, the T-cells are collected from the patient, making this an autologous immunocellular therapy.

Treatment of childhood acute lymphoblastic leukemia in Iraq: a 17-year experience from a single center

We performed a retrospective analysis of 1415 acute lymphoblastic leukemia children diagnosed between January 2000 and December 2016 at Children Welfare Teaching Hospital, Baghdad, Iraq. Patients were divided into three cohorts according to treatment period (2000–2005; 2006–2011; 2012–2016). Treatments were based on modified-UKALL protocols; a steroid-pre-phase was introduced from September 2008. The overall complete remission was 86%, increased from 80% to 91% in the last period. Early deaths occurred in 10%, decreasing to 6%, overtime. Relapses were 23%; toxic deaths and abandonment 8% and 13%, respectively. At a median follow-up of 65.3 months, with abandonment considered as an event, the 5-year overall survival (OS) and event-free survival were 62.2% and 46.3%, statistically influenced by treatment period (5-year OS 62.6%, 59.1%, 66.3%; p=.057, respectively). Though pediatric ALL survival in Iraq is still below that observed in high income countries, survival rates progressively improved. Toxic deaths remain an important cause of failure.