Acute Lymphoblastic Leukemia Subtypes Research Articles

Down-Regulated FOXO1 in Refractory/Relapse Childhood B-Cell Acute Lymphoblastic Leukemia.

Background: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer, with an overall prevalence of 4/100,000, accounting for 25–30% of all childhood cancers. With advances in childhood ALL treatment, the cure rate for childhood ALL has exceeded 80% in most countries. However, refractory/relapsed ALL remains a leading cause of treatment failure and subsequent death. Forkhead box O1 (FOXO1) belongs to the forkhead family of transcription factors, but its role in B-cell ALL (B-ALL) has not been determined yet.Procedures: RNA sequencing was applied to an ALL case with induction failure (IF) to identify the possible genetic events. A cytokine-dependent growth assay in Ba/F3 cells was used to test the leukemic transformation capacity of MEIS1–FOXO1. The propidium iodide (PI) staining method was used to evaluate the effect of MEIS1–FOXO1 on cycle distribution. FOXO1 transactivity was examined using a luciferase reporter assay. FOXO1 mRNA expression levels were examined using real-time quantitative PCR among 40 children with B-ALL treated with the CCCG-ALL-2015 protocol. Association analysis was performed to test the correlation of FOXO1 transcription with childhood B-ALL prognosis and relapse in a series of GEO datasets. An MTT assay was performed to test the drug sensitivity.Results: In this ALL case with IF, we identified a novel MEIS1–FOXO1 fusion gene. The transactivity of MEIS1–FOXO1 was significantly lower than that of wild-type FOXO1. MEIS1–FOXO1 potentiated leukemia transformation and promoted Ba/F3 cell cycle S-phase entry. Low FOXO1 transcription levels were found to be strongly associated with unfavorable ALL subtype, minimal residual disease (MRD) positivity, and relapse. Lower FOXO1 expression was associated with prednisone and cyclophosphamide resistance.Conclusions: Low FOXO1 transcription was associated with high-risk stratification and relapse in children with B-ALL, probably due to multi-drug resistance.

Pharmacogenomics of intracellular methotrexate polyglutamates in patients' leukemia cells in vivo.

BACKGROUNDInterpatient differences in the accumulation of methotrexate's active polyglutamylated metabolites (MTXPGs) in leukemia cells influence its antileukemic effects.METHODSTo identify genomic and epigenomic and patient variables determining the intracellular accumulation of MTXPGs, we measured intracellular MTXPG levels in acute lymphoblastic leukemia (ALL) cells from 388 newly diagnosed patients after in vivo high-dose methotrexate (HDMTX) (1 g/m2) treatment, defined ALL subtypes, and assessed genomic and epigenomic variants influencing folate pathway genes (mRNA, miRNA, copy number alterations [CNAs], SNPs, single nucleotide variants [SNVs], CpG methylation).RESULTSWe documented greater than 100-fold differences in MTXPG levels, which influenced its antileukemic effects (P = 4 × 10-5). Three ALL subtypes had lower MTXPG levels (T cell ALL [T-ALL] and B cell ALL [B-ALL] with the TCF3-PBX1 or ETV6-RUNX1 fusions), and 2 subtypes had higher MTXPG levels (hyperdiploid and BCR-ABL like). The folate pathway genes SLC19A1, ABCC1, ABCC4, FPGS, and MTHFD1 significantly influenced intracellular MTXPG levels (P = 2.9 × 10-3 to 3.7 × 10-8). A multivariable model including the ALL subtype (P = 1.1 × 10-14), the SLC19A1/(ABCC1 + ABCC4) transporter ratio (P = 3.6 × 10-4), the MTX infusion time (P = 1.5 × 10-3), FPGS mRNA expression (P = 2.1 × 10-3), and MTX systemic clearance (P = 4.4 × 10-2) explained 42% of the variation in MTXPG accumulation (P = 1.1 × 10-38). Model simulations indicated that a longer infusion time (24 h vs. 4 h) was superior in achieving higher intracellular MTXPG levels across all subtypes if ALL.CONCLUSIONSThese findings provide insights into mechanisms underlying interpatient differences in intracellular accumulation of MTXPG in leukemia cells and its antileukemic effectsFUNDINGTHE National Cancer Institute (NCI) and the Institute of General Medical Sciences of the NIH, the Basque Government Programa Posdoctoral de Perfeccionamiento de Personal Investigador doctor, and the American Lebanese Syrian Associated Charities (ALSAC).

Response Adapted Therapy in ETV6/RUNX1-Positive Childhood Acute Lymphoblastic Leukemia Treated per Modified St Jude Total XV Study

Introduction: ETV6-RUNX1 is the most common genetic aberration in childhood acute lymphoblastic leukemia (ALL), occurring in approximately 25% of cases with a precursor-B phenotype. The presence of ETV6-RUNX1 has been associated with a relatively low rate of relapse in multiple studies. Relapses tend to occur late and have a better salvage rate than other ALL subtypes. Moreover, conflicting data in literature regarding the prognostic significance of ETV6-RUNX1 after accounting for age, initial count and treatment intensity. We sought to study the clinical features, therapy response in newly diagnosed ETV6-RUNX1-positive treated at King Hussein Cancer Center. Methods: Patients were treated per modified St Jude Total (XV) study, risk stratification was further refined by including minimal residual disease (MRD)measurements on day 15 and day 46 of remission induction therapy. Patients with the ETV6- RUNX1 fusion or hyperdiploidy without CNS or testicular disease and a satisfactory early MRD decline (<1% on day 15 and <0.01% on day 46) were classified as being low-risk for relapse and were treated on lower -risk arm regardless of age and leukocyte count. Results: Seventy patients (n=70) with ETV6-RUNX1-positive treated at our institution between May 2006 to October 2017. The median age at diagnosis, 5.8 years (range, 1.5-10.8). ETV6-RUNX1 was associated with favorable age between 1- and 6-years in 55 patients (79%), male gender in 41 patients (59%), initial leukocyte count <10 in 39 patients (56%), CNS status 1 in 100% of patients. The majority, n= 58 (83%) of patients had NCI standard risk (SR) and n=12 (17%) had NCI high risk (HR). Sixty-six patients (94%) had MRD <1% at day 15 and all patients achieved remission with MRD<0.01% at day 46 of remission induction. All patients were treated as LR ALL per modified St Jude total XV protocol. At median follow up of (36 months; range, 26 to 154), in the subgroups of ETV6-RUNX1-positive patients classified as NCI standard risk (SR)and NCI high risk (HR) the 5-year EFS estimates were 94.1% ± 3.2 and 82%±1(P=.13), 5-year OS estimates were 100% and 92% ± 8 (p=0.31) respectively. Conclusion: MRD treatment schema in ETV6/RUNX1 -positive patients have excellent outcomes if they achieved favorable response at day 15 and end of remission induction regardless of the age and Initial leukocyte count. Treatment reduction is feasible and declared safe for children with NCI-HR who are predicted to have a low risk of relapse on the basis of rapid clearance of MRD post remission induction therapy. Disclosures No relevant conflicts of interest to declare.

Genetic Determinants of Blood Cell Traits Play a Role in Susceptibility to Acute Lymphoblastic Leukemia

Background: Genome-wide association studies (GWAS) have identified several genes associated with childhood acute lymphoblastic leukemia (ALL) that are also implicated in variation in hematological traits. We performed a comprehensive study of the shared heritability of blood cell traits and ALL, and investigated whether genetic variation in blood cell traits may underlie ALL risk. Methods: We leveraged genome-wide single nucleotide polymorphism (SNP) data from: 1) 335,332 European ancestry, cancer-free subjects without immune deficiencies or hematopoietic disorders in the UK Biobank (UKB); and 2) a childhood ALL GWAS meta-analysis including 2,121 cases and 59,965 controls of European ancestry, to investigate the shared genetic basis of blood cell trait variation and ALL susceptibility. Co-heritability between blood cell profiles and ALL susceptibility was evaluated by LD score regression. To formally test variation in blood cell traits as potential causal pathways in ALL development, we conducted Mendelian randomization (MR) analyses, which use genetic predictors of blood cell phenotypes to overcome potential confounding and reverse causation in directly measured blood counts. Genetic instruments for MR were developed using a two-stage GWAS of 6 cell types (lymphocytes, monocytes, neutrophils, eosinophils, basophils, platelets), and their ratios (lymphocyte/monocyte - LMR; neutrophil/monocyte - NLR; platelet/lymphocyte - PLR) in the UKB. Next we applied multi-trait analysis of GWAS (MTAG) to improve power for identifying trait-specific loci by exploiting the correlated nature of blood cell traits. Causal odds ratios (OR) for ALL were estimated per 1 standard deviation increase in normalized cell counts (109cells/L) or 1-unit increase in cell type ratios. Results: Using genome-wide SNP data, we found that ALL has a heritability of hg=0.235 (95% confidence intervals, CI:0.203-0.268) in European ancestry individuals. In LD score regression, ALL susceptibility was correlated at the genetic level with overall blood cell counts (rg=0.070, P=0.003), lymphocyte counts (rg=0.088, P=0.004), LMR (rg=0.065, P=0.012), and PLR (rg= -0.072, P=0.001). Genetic instruments for MR were selected from independent (LD r2<0.05) variants with P<5×10-8 in the discovery blood cell trait GWAS/MTAG (N=234,778) and P<0.05 in the replication analysis (N=100,554). The resulting genetic predictors explained between 4.0% (basophils, NSNP=144) and 23.9% (platelets, NSNP=661) of trait variation. Our findings lend support to a modest but significant causal effect of variation in lymphocyte counts (OR=1.16, 95% CI:1.01-1.33), LMR (OR=1.23, 95% CI:1.07-1.41), NLR (OR=0.67, 95% CI:0.54-0.83), and PLR (OR=0.80, 95% CI:0.70-0.92) on ALL risk. There was no evidence of directional pleiotropy based on the MR Egger intercept test (P>0.05). However, significant heterogeneity among SNP-specific causal effects (Cochran's Q P<0.05) indicated potential for confounding due to balanced pleiotropy. In sensitivity analyses using MR-PRESSO framework, which corrects for distortion in causal effects due to pleiotropy, the associations with ALL risk became attenuated for lymphocytes (OR=1.14, 95% CI:0.98-1.32), but persisted for LMR (OR=1.18, 95% CI:1.01-1.38), NLR (OR=0.76, 95% CI:0.60-0.97), and PLR (OR=0.82, 95% CI:0.70-0.95). Next, we searched for individual variants that may underlie the effects on ALL risk using heterogeneity-based clustering of genetic instruments. This analysis confirmed that regulation of blood cell traits partially mediates the effects of known ALL risk variants: rs4948492, rs4245597 (ARID5B), rs2239630 (CEBPE), and rs78697948 (IKZF1). We also identified putative novel ALL risk loci at chromosome 2q22 (OR=1.28, P=2.5×10-6), chr6q23 (OR=1.21, P=9.3×10-6), and chr13q12 (OR=1.79, P=3.2×10-5), though further analyses are required to confirm these associations and identify likely causal variants and genes at these loci. Conclusions: Our findings suggest that dysregulation of blood cell profiles characterized by a genetic predisposition to increased lymphocyte counts, particularly in relation to neutrophils, monocytes, and platelets, confers a modest but significant increase in ALL risk. Additional research is required to determine whether a genetic propensity to higher lymphocyte levels is associated with ALL subtypes or clinical outcomes in ALL patients. Disclosures Ma: Celgene/BMS: Research Funding; BMS: Consultancy.

Comparative Analysis for Differential Drug Response between Early T-Cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL) and T-Cell Acute Lymphoblastic Leukemia (T-ALL) Patients Using the Cellworks Omics Biology Model (CBM): Mycare-021-03

Background:Early T-cell Precursor Acute Lymphoblastic Leukemia (ETP-ALL), an orphan disease, is a sub-type of T-Cell Acute Lymphoblastic Leukemia (T-ALL) with very poor prognosis and limited therapy options. ETP-ALL is a heterogeneous disease with many distinct genomic profiles, often with more myeloid than lymphoid characteristics. However, standard of care (SOC) drugs for acute myeloid leukemia (AML) have shown limited efficacy for ETP-ALL (PMID: 32733662, 25435716). The genomic profiles of ETP-ALL patients have more complex cytogenetics and larger numbers of genomic aberrations when compared to non-ETP-ALL (T-ALL) profiles (PMID: 22237106, 30641417). We present an alternative multi-gene analysis approach using the Cellworks Omics Biology Model (CBM) workflow to identify unique, intersecting protein pathways in patient-specific disease profiles. The CBM predictive workflow was used to design novel personalized therapy options for an ETP-ALL representative PEER human lymphoid cell line in comparison to a T-ALL JURKAT cell line. The predicted combination therapies were then validated in a lab model. Methods:A PEER cell line was selected to represent ETP-ALL and a JURKAT cell line was selected as a representative for non-ETP T-ALL. Next Generation Sequencing (NGS) was performed for the PEER cell line. For the JURKAT cell line, publicly available NGS whole exome sequencing from cBioPortal and Sanger, along with array CGH from Agilent, were used. The genomic data for the PEER and JURKAT cell lines were used as inputs to the CBM to generate dynamic patient-specific disease protein network maps. Biomarkers and pathway characteristics unique to the PEER and JURKAT cell lines were identified. A digital drug library of targeted FDA-approved agents was simulated on the disease models using both single drug agents and drug combinations at varying doses. The treatment impact was assessed by quantitatively measuring drug effect on a cell growth score, which is a composite of the quantified values of cell proliferation, survival and apoptosis along with impact on the patient-specific disease biomarker score. Comparative dose response studies were run to assess IC50 differences for both cell lines. Cellworks VenturaTM predicted novel therapy combinations for the ETP-ALL representative PEER cell line, which were then prospectively validated by in vitro experiments. The same therapy options were predicted to be less effective in the T-ALL representative JURKAT cell line, which was also confirmed by in vitro studies. Results:The CBM predicted three novel combination therapies for the ETP-ALL representative PEER cell line: nilotinib + cytarabine, bortezomib + cytarabine and bortezomib + idarubicin. All three therapies were predicted to be less effective in JURKAT cells. In vitro, PEER cells were sensitive to all 3 combinations, as predicted by the CBM; whereas, JURKAT cell lines were not sensitive to the first 2 combinations (as predicted), but were sensitive to bortezomib + idarubicin. The CBM analysis is supported by scientific rationales for these combinations based on the genomics-driven disease characteristics of the cell-line. The reasons for drug sensitivity and resistance were determined. These combinations were then prospectively validated in vitro on both cell lines and the experimental responses matched the predicted outcomes. Conclusion:The Cellworks Omics Biology Model integrates the multiple genomic abnormalities in a patient to identify disease network characteristics unlike other NGS analytic tools that attempt to interpret the impact of each genomic alteration in isolation. CBM identified 3 novel therapy options for ETP-ALL that were validated in vitro, similar to anecdotal experience in vivo. This predictive technology can improve clinical decision-making and identify novel treatment options. Disclosures Howard: Cellworks:Consultancy;Servier:Consultancy, Other: Speaker;EUSA Pharma:Consultancy;Sanofi:Consultancy, Other: Speaker;Boston Scientific:Consultancy.Kumar:Cellworks Research India Private Limited:Current Employment.Pampana:Cellworks Research India Private Limited:Current Employment.Ullal:Cellworks Research India Private Limited:Current Employment.Tyagi:Cellworks Research India Private Limited:Current Employment.Lala:Cellworks Research India Private Limited:Current Employment.Kumari:Cellworks Research India Private Limited:Current Employment.Joseph:Cellworks Research India Private Limited:Current Employment.Raju:Cellworks Research India Private Limited:Current Employment.Balakrishnan:Cellworks Research India Private Limited:Current Employment.Mundkur:Cellworks Group Inc.:Current Employment.Macpherson:Cellworks Group Inc.:Current Employment.Nair:Cellworks Research India Private Limited:Current Employment.Kapoor:Cellworks Research India Private Limited:Current Employment.

Incidence and Therapeutic Implications of Germline TP53 Mutations in Hypodiploid Childhood Acute Lymphoblastic Leukemia: A Retrospective Analysis of the Italian Cohort

Background. Hypodiploid Acute Lymphoblastic Leukemia (ALL) is a rare subtype of childhood ALL known to be associated with a poor prognosis. Currently, intensive chemotherapy and haematopoietic stem cell transplantation are recommended as the main therapeutic strategy for these patients. Previous studies showed that low-hypodiploid ALL frequently carry TP53 variants, the majority of which are germline mutations. This finding suggests that hypodiploid ALL may be a manifestation of underlying Li-Fraumeni syndrome, with an associated high risk for secondary tumors. We analyzed TP53 variants in the Italian cohort of hypodiploid pediatric ALL patients diagnosed between 2000 and 2019, to explore frequency and characteristics, as well as the potential role of germline variants in the therapeutic strategy. Patients were treated according to the AIEOP-BFM ALL2000 (n=16), ALL2009 (n=16) and ALL2017 (n=8) protocols. Methods. We performed a targeted Next Generation Sequencing (NGS) Nextera Flex DNA panel of 40 genes, including TP53, in a retrospective series of hypodiploid paediatric ALL Italian patients enrolled in four nationwide frontline protocols. Ploidy was defined based on DNA index (DNAi) and/or cytogenetics/FISH, and cases with DNAi<0.8 were included in this study. Only TP53 variants with VF >5% and coverage 500X were considered. Bioinformatics analysis has been carried out by Sophia DDM software. NGS has been performed both in disease (hematopoietic) and germline (remission or buccal brush) tissues samples. Results. Nineteen TP53 variants were observed in 20/40 (50%) hypodiploid ALL patients, with 19/20 being low-hypodiploid ALL cases (DNAi between 0,6 and 0,8); 13/19 TP53 variants were known to be pathogenic whereas 6/19 were classified as Variant of Unknown Significance (VUS). Considering the VUS variants, 3/6 were missense, 1/6 frameshift and 2/6 in-frame ins/del. Notably, 13 patients out of 20 (65%) were found to carry a germline variant, while 5 patients presented a somatic variant; in 2 cases the remission sample was not available, but the VF<20% was indicative of a somatic variant. Among the germline variants, 1 was found to be a germline mosaicism, presenting a nonsense pathogenic TP53 variant both in remission (VF 10%) and buccal brush sample at remission (VF 8.5%). 16/19 variants reside in the p53 core DNA binding domain, known to be a fundamental site that mediates the transcription of p53 regulated genes, where most of the pathogenic mutations in cancer cells occur. The 3 variants outside the DNA binding domain were 1 nonsense, 1 frameshift and 1 missense, and were localized in the nuclear localization signal region (NLS) or in the oligomerization domain (OD). Considering the 32 patients with at least 5 years of follow-up, EFS was not significantly different between TP53 wild type (47.1% ± 12.1) vs TP53 variant (51.9% ± 17,8) subgroups. According to MRD stratification criteria, the patients carrying a TP53 variant were classified as medium risk in 14/20, standard risk in 5/20 and HR in 1 case, not statistically different from TP53 wild type. Collection of data on secondary neoplasms in patients and their relatives in ongoing. Conclusion. Our data represent the largest low hypodiploid patient cohort tested so far, confirming the high frequency of deleterious TP53 mutations. Mutational testing of TP53 in patients with hypodiploid ALL is warranted, in order to ensure a proper genetic counseling for patients with germline mutations and their families, with tailored clinical surveillance (according to Li-Fraumeni Syndrome guidelines). The best therapeutic regimen for hypodiploid patients is still a matter of debate, with MRD at the end of induction and TP53 germline mutations potentially indicating a highest relevance in the indication to hematopoietic stem cell transplantation. Chemotherapy-only treatment could be indicated for MRD-low patients and HSCT for MRD-High, potentially considering a reduced conditioning regimen for TP53 mutated cases. Disclosures Locatelli: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharmaceeutical: Speakers Bureau. Rizzari:Sobi: Consultancy, Other: Advisory Board.

Role of rs10406069 in miR-5196 in hyperdiploid childhood acute lymphoblastic leukemia.

Aim: To determine the role of single nucleotide polymorphisms (SNPs)in noncoding RNAs in childhood acute lymphoblastic leukemia (ALL) subtypes. Materials & methods: We screened all SNPs in 130 pre-miRNA genes to assess their role in the susceptibility of the most common subtypes of ALL: hyperdiploid and ETV6-RUNX1. Results: In two independent cohorts, we found a significant association between rs10406069 in miR-5196 and the risk of developing hyperdiploid ALL. This observation could be explained by the impact of the SNP on miR-5196 expression and in turn, in its target genes. Indeed, rs10406069 was associated with expression changes in SMC1A, a gene involved in sister chromatin cohesion. Conclusion: rs10406069 in miR-5196 may have a relevant role in hyperdiploid ALL risk.

Expression Profile of LncRNAs in Childhood Acute Lymphoblastic Leukemia: A Pilot Study

Background: Childhood acute lymphoblastic leukemia (ALL) explains 26% of pediatricmalignancies and is one of the leading causes of disease-related deaths in children. A novelmolecular class of non-coding genes, long non-coding RNAs (lncRNAs) having over 200nucleotides, have been defined as regulators of different cellular processes including pluripotency,oncogenesis, and transcription. It has been demonstrated that lncRNA transcription profilescan distinguish pre B-cell subtype of ALL accurately and act as early diagnostic and prognosticbiomarkers. Hence, the aim of this pilot study was the prior evaluation of expression profileof several lncRNA candidates including RP11-68I18.10, RP11-624C23.1, RP11-446E9, RP11-137H2.4, and RP11-203E8 in patients with ALL. Methods: In this study, 80 blood samples were obtained from patients, definitely diagnosed bypathologists with ALL, and from healthy subjects. Total RNA was extracted from blood samples,and cDNA was synthesized. Real-time PCR was applied to determine the expression of lncRNAs.A P-value of 0.010 was considered statistically significant. Results: Our findings revealed that the expression levels of lncRNAs RP11-624C23.1, RP11-446E9, RP11-137H2.4, RP11-68I18.10, and RP11-203E8 were significantly decreased in ALLsamples compared to those of healthy samples (P<0.0001, P =0.0616, P =0.0292, P<0.0001, andP = 0.0007). Moreover, the relationship between these five lncRNA expression changes and theimmunophenotype in ALL patients was not significant. Conclusion: The dysregulation of lncRNAs in ALL samples could provide a novel and interestingpossibility for early diagnosis and prognosis, as well as mastering the treatment of ALL.

New Approaches to Treating Challenging Subtypes of ALL in AYA Patients.

The treatment of acute lymphoblastic leukemia (ALL) in adolescent and young adult (AYA) patients has markedly improved with the adoption of pediatric-inspired protocols. However, there remain several subtypes of ALL that represent significant therapeutic challenges. Here, we review the current evidence guiding treatment of Philadelphia chromosome-positive (Ph+), Philadelphia chromosome-like (Ph-L), and early T-precursor (ETP) ALL in the AYA population. Clinical trials in Ph + ALL have demonstrated the superior efficacy of second- and third-generation tyrosine kinase inhibitors (TKIs) to induce and maintain remission. Current efforts now focus on determining the durability of these remissions and which patients will benefit from transplant. For Ph-like and ETP ALL, recent studies are investigating the addition of novel agents to standard treatment. The treatment of Ph + ALL has significantly improved with the addition of potent TKIs. However, the treatment of Ph-like and ETP ALL remains a challenge. At this time, the judicious use of allogenic transplant is the only current approach to modify this increased risk.

Identification of Acute Lymphoblastic Leukemia Subtypes in Touching Cells Based on Enhanced Edge Detection

Identification of Acute Lymphoblastic Leukemia Subtypes in Touching Cells Based on Enhanced Edge Detection

Decitabine mildly attenuates MLL-rearranged acute lymphoblastic leukemia in vivo, and represents a poor chemo-sensitizer.

MLL‐rearranged acute lymphoblastic leukemia (ALL) represents a highly aggressive ALL subtype, characterized by aberrant DNA methylation patterns. DNA methyltransferase inhibitors, such as decitabine have previously been demonstrated to be effective in eradicating MLL‐rearranged ALL cells in vitro.Here, we assessed the in vivo anti‐leukemic potential of low‐dose DNA methyltransferase inhibitor decitabine using a xenograft mouse model of human MLL‐rearranged ALL. Furthermore, we explored whether prolonged exposure to low‐dose decitabine could chemo‐sensitize MLL‐rearranged ALL cells toward conventional chemotherapy as well as other known epigenetic‐based and anti‐neoplastic compounds.Our data reveal that decitabine prolonged survival in xenograft mice of MLL‐rearranged ALL by 8.5 days (P = .0181), but eventually was insufficient to prevent leukemia out‐growth, based on the examination of the MLLAF4 cell line SEM. Furthermore, we observe that prolonged pretreatment of low‐dose decitabine mildly sensitized toward the conventional drugs prednisolone, vincristine, daunorubicin, asparaginase, and cytarabine in a panel of MLL‐rearranged cell lines. Additionally, we assessed synergistic effects of decitabine with other epigenetic‐based or anticancer drugs using high‐throughput drug library screens. Validation of the top hits, including histone deacetylase inhibitor panobinostat, BCL2 inhibitor Venetoclax, MEK inhibitor pimasertib, and receptor tyrosine kinase foretinib, revealed additive and moderate synergistic effects for the combination of each drug together with decitabine in a cell line‐dependent manner.

Targeting lactate dehydrogenase A (LDHA) exerts antileukemic effects on T-cell acute lymphoblastic leukemia.

BackgroundT‐cell acute lymphoblastic leukemia (T‐ALL) is an uncommon and aggressive subtype of acute lymphoblastic leukemia (ALL). In the serum of T‐ALL patients, the activity of lactate dehydrogenase A (LDHA) is increased. We proposed that targeting LDHA may be a potential strategy to improve T‐ALL outcomes. The current study was conducted to investigate the antileukemic effect of LDHA gene‐targeting treatment on T‐ALL and the underlying molecular mechanism.MethodsPrimary T‐ALL cell lines Jurkat and DU528 were treated with the LDH inhibitor oxamate. MTT, colony formation, apoptosis, and cell cycle assays were performed to investigate the effects of oxamate on T‐ALL cells. Quantitative real‐time PCR (qPCR) and Western blotting analyses were applied to determine the related signaling pathways. A mitochondrial reactive oxygen species (ROS) assay was performed to evaluate ROS production after T‐ALL cells were treated with oxamate. A T‐ALL transgenic zebrafish model with LDHA gene knockdown was established using CRISPR/Cas9 gene‐editing technology, and then TUNEL, Western blotting, and T‐ALL tumor progression analyses were conducted to investigate the effects of LDHA gene knockdown on T‐ALL transgenic zebrafish.ResultsOxamate significantly inhibited proliferation and induced apoptosis of Jurkat and DU528 cells. It also arrested Jurkat and DU528 cells in G0/G1 phase and stimulated ROS production (all P < 0.001). Blocking LDHA significantly decreased the gene and protein expression of c‐Myc, as well as the levels of phosphorylated serine/threonine kinase (AKT) and glycogen synthase kinase 3 beta (GSK‐3β) in the phosphatidylinositol 3′‐kinase (PI3K) signaling pathway. LDHA gene knockdown delayed disease progression and down‐regulated c‐Myc mRNA and protein expression in T‐ALL transgenic zebrafish.ConclusionTargeting LDHA exerted an antileukemic effect on T‐ALL, representing a potential strategy for T‐ALL treatment.

13q12.2 deletions in acute lymphoblastic leukemia lead to upregulation of FLT3 through enhancer hijacking

AbstractMutations in the FMS-like tyrosine kinase 3 (FLT3) gene in 13q12.2 are among the most common driver events in acute leukemia, leading to increased cell proliferation and survival through activation of the phosphatidylinositol 3-kinase/AKT-, RAS/MAPK-, and STAT5-signaling pathways. In this study, we examine the pathogenetic impact of somatic hemizygous 13q12.2 microdeletions in B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) using 5 different patient cohorts (in total including 1418 cases). The 13q12.2 deletions occur immediately 5′ of FLT3 and involve the PAN3 locus. By detailed analysis of the 13q12.2 segment, we show that the deletions lead to loss of a topologically associating domain border and an enhancer of FLT3. This results in increased cis interactions between the FLT3 promoter and another enhancer located distally to the deletion breakpoints, with subsequent allele-specific upregulation of FLT3 expression, expected to lead to ligand-independent activation of the receptor and downstream signaling. The 13q12.2 deletions are highly enriched in the high-hyperdiploid BCP ALL subtype (frequency 3.9% vs 0.5% in other BCP ALL) and in cases that subsequently relapsed. Taken together, our study describes a novel mechanism of FLT3 involvement in leukemogenesis by upregulation via chromatin remodeling and enhancer hijacking. These data further emphasize the role of FLT3 as a driver gene in BCP ALL.

Abstract 1936: Immunoproteasome inhibitors for the treatment of ALL

Abstract The t(4;11)(q21;q23) chromosomal translocation that creates MLL-AF4 fusion protein confers a poor prognosis protein in infant acute lymphoblastic leukemia (ALL). This translocation also sensitizes cells to proteasome inhibitors bortezomib and carfilzomib, which are approved by the FDA for the treatment of multiple myeloma. Clinical activity of bortezomib in combination with standard chemotherapy has been documented in several clinical trials of ALL patients, and a case of a single-agent activity against relapsed leukemia driven by MLL-AF4 translocation has been described. However, toxicities of bortezomib and carfilzomib may be unacceptable to pediatric patients. We found that the overwhelming majority of proteasomes in this subtype of ALL are lymphoid tissue specific immunoproteasomes. Cells with MLL-AF4 translocations were sensitive to pharmacologically relevant concentrations of specific immunoproteasome inhibitor ONX-0914. Furthermore, ONX-0914 dramatically delayed growth of orthotopic xenograft tumors in mice. Thus, immunoproteasomes are therapeutic targets in ALL; replacing bortezomib and carfilzomib with immunoproteasome inhibitors in ALL should reduce toxicities associated with inhibition of the proteasomes in non-lymphoid tissues. Citation Format: Tyler W. Jenkins, Sondra Downey-Kopyscinski, William C. Colley, Jennifer L. Fields, Steven N. Fiering, Alexei F. Kisselev. Immunoproteasome inhibitors for the treatment of ALL [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1936.

Abstract 4181: The XPO1 inhibitior, eltanexor, exhibits potent in vivo activity against a broad range of pediatric acute lymphoblastic leukemia subtypes

Abstract Introduction: Exportin 1 (XPO1/CRM1) plays a central role in the export of proteins from the nucleus including those with tumor suppressor and growth regulatory activity. Eltanexor (KPT-8602) is a second-generation Selective Inhibitor of Nuclear Export (SINE) that specifically blocks XPO1 cargo interactions and is under clinical evaluation for adults with cancer (NCT02649790). In previous Pediatric Preclinical Testing Consortium (PPTC) studies, eltanexor showed potent in vivo activity against acute lymphoblastic leukemia (ALL) patient-derived xenografts (PDXs). In an effort to understand whether eltanexor exhibited subtype-specific activity in pediatric ALL we carried out a single-mouse trial (SMT) against an intended 90 pediatric ALL PDXs. Methods: Pediatric ALL PDXs, an orthotopic model of the disease, were inoculated into NSG mice in an SMT format (a single mouse inoculated with a single PDX was treated with a single drug). Engraftment and drug responses were assessed by weekly enumeration of the % human leukemic blasts in the peripheral blood (%huCD45+/HLA-ABC+). Treatment commenced when the median %huCD45+/HLA-ABC+ exceeded 1%, and mice received eltanexor at 12.5 mg/kg per oral daily x 5 for 4 weeks. The baseline level of the %huCD45+/HLA-ABC+ in each mouse served as its own control. PDX responses to treatment were assessed by time to event, the maximum decrease in %huCD45+/ABC+ at any point after treatment initiation, and by stringent Objective Response Measures (ORMs) modeled after the clinical setting (Houghton et al, Pediatr Blood Cancer, 2007, 49:928-40). Results: Eltanexor was well tolerated with a mean maximum weight loss of 5.5% across all mice. SMT testing was successful in all 90 pediatric ALL PDXs representative of B-ALL (typical B-ALL, Philadelphia chromosome-positive ALL [Ph+-ALL] and Ph-like ALL), T-ALL (typical T-ALL and early T-cell precursor ALL [ETP-ALL]), and MLL-rearranged ALL (MLLr-ALL). Event-free survival (EFS) of mice ranged from 2.6-137 days, and 61 (68%) of mice experienced EFS that extended beyond the treatment window. Regressions of ≥50% from baseline were observed in 56 (62%) of mice, and these were most pronounced in T-ALL (14/18, 78%) and Ph-like ALL (11/19, 58%). ORMs classified as Complete Response (CR) or Maintained CR (MCR) were elicited in 43 (48%) of PDXs, and again these were more common in T-ALL (12/18, 67%). The ALL subtypes that exhibited the poorest responses to eltanexor were Ph+-ALL and ETP-ALL, although with relatively small sample size (n=3 and 6, respectively). ORMs in the SMT correlated highly with historical conventional drug testing (R=0.94, P&amp;lt;0.001, n=12). Conclusions: Eltanexor exhibits potent single-agent in vivo activity against PDXs derived from a broad range of ALL subtypes including T-ALL and Ph-like ALL and warrants further investigation in pediatric ALL. (Supported by NCI Grants CA199222 &amp; CA199000) Citation Format: Richard B. Lock, Kathryn Evans, Connor D. Jones, Stephen W. Erickson, Beverly A. Teicher, TJ Unger, Yosef Landesman, Malcolm A. Smith. The XPO1 inhibitior, eltanexor, exhibits potent in vivo activity against a broad range of pediatric acute lymphoblastic leukemia subtypes [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4181.

DUX4r, ZNF384r and PAX5-P80R mutated B-cell precursor acute lymphoblastic leukemia frequently undergo monocytic switch.

Recently, we described B-cell precursor acute lymphoblastic leukemia (BCP-ALL) subtype with an early switch to the monocytic lineage and the loss of the B-cell immunophenotype, including CD19 expression. Thus far, the genetic background has remained unknown. Among 726 children consecutively diagnosed with BCP-ALL, 8% patients experienced a switch detectable by flow cytometry (FC). Using exome and RNA sequencing, the switch was found to positively correlate with three different genetic subtypes: PAX5-P80R mutation (five cases with switch of five), rearranged (DUX4r) (30 cases of 41) and rearranged (ZNF384r) (four cases of ten). Expression profiles or phenotypic patterns correlated with genotypes, but within each genotype no cases who subsequently switched could be indentified. If switching was not taken into account, the B-cell-oriented FC assessment underestimated the minimal residual disease level. For patients with PAX5-P80R, a discordance between FC-determined and polymerase chain reactiondetermined minimal residual disease was found on day 15, resulting from a rapid loss of the B-cell phenotype. Discordance on day 33 was observed in all the DUX4r, PAX5-P80R and ZNF384r subtypes. Importantly, despite the substantial phenotypic changes, possibly even challenging the appropriateness of BCP-ALL therapy, the monocytic switch was not associated with a higher incidence of relapse and poorer prognosis in patients undergoing standard ALL treatment.

Frequency and prognostic impact of PAX5 p.P80R in pediatric acute lymphoblastic leukemia patients treated on an AIEOP-BFM acute lymphoblastic leukemia protocol.

PAX5 is a member of the paired box (PAX) family of transcription factors involved in B‐cell development. PAX5 P80R has recently been described as a distinct genetic B‐cell precursor (BCP) acute lymphoblastic leukemia (ALL) subtype with a favorable prognosis in adults. In contrast, an unfavorable outcome has been observed in children. Our aim was to determine the frequency of PAX5 P80R in childhood BCP‐ALL treated according to the Associazione Italiana Ematologia ed Oncologia Pediatrica‐Berlin‐Frankfurt‐Muenster (AIEOP‐BFM) ALL 2000 protocol and to evaluate its clinical significance within this study cohort. The analyses included 1237 patients with ALL treated in the AIEOP‐BFM ALL 2000 trial with complete information for copy number variations (CNVs) of IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A, CDKN2B, and ERG. A customized TaqMan genotyping assay was used to screen for PAX5 P80R. Sanger sequencing was used to confirm PAX5 P80R‐positive results as well as to screen for second variants in PAX5. Agilent CGH + SNP arrays (e‐Array design 85 320; Agilent Technologies) were performed in PAX5 P80R‐positive patients to verify additional CNVs. Almost 2% (20/1028) of our BCP‐ALL cohort were PAX5 P80R‐positive. White blood cell counts higher than 50 000/μl as well as male sex were significantly (P < .05) associated with PAX5 P80R. Most of the PAX5 P80R‐positive cases were 10 years of age or older. PAX5 P80R‐positive samples were enriched for deletions affecting PAX5, IKZF1, CDKN2A, and CDKN2B. Compared to PAX5 P80R ‐wildtype BCP‐ALL, PAX5 P80R‐positive patients showed a significantly reduced 5‐year overall survival (P = .042). Further studies should evaluate the interaction of PAX5 P80R with other genetic aberrations to further stratify intermediate risk pediatric BCP‐ALL.

Immunophenotypic characterization of acute lymphoblastic leukemia in a flowcytometry reference centre in Sri Lanka.

Acute lymphoblastic leukemia (ALL), is a biologically heterogeneous disease where diagnosis, treatment and prognosis is heavily dependent on the correct characterization of the immunophenotype of each case. To describe the immunophenotypic and laboratory features of a cohort of Sri Lankan children and adults with ALL and to compare them with those reported in other series. Records of 229 patients who were suspected of having acute leukaemia and referred for flow cytometry to the Asiri Hospital Sri Lanka, between August 2009 and October 2013 were reviewed. Referrals were from across the country including the National Cancer Institute. Sixty seven percent were children below 12 years of age. 80% had B-lymphoblastic leukaemia (B-ALL), 20% T- lymphoblastic leukaemia (T-ALL). In children, the peak age of diagnosis was between 2-6 years in B-ALL (61%) and 3-6 years (43.7%) in T-ALL. Incidence was commoner in males in all age groups and subtypes except in the adult B ALL group where the incidence was equal. Ninety three percent of paediatric B-ALL and 67% adult B-ALL were CD10 positive common ALL. In T-ALL cytoplasmic expression of CD3 was 100%. nTdT was the most commonly expressed immature marker, in B-ALL - 91% and in T-ALL- 69%. At least one myeloid antigen was present in 34% of B-ALL cases and 60% of T-ALL cases. This study represents the first immunophenotype based description of ALL in Sri Lanka. Age, sex distribution, ALL subtypes and the immunophenotypic profile of each subtype mirrored those of previous studies.

MicroRNA Signatures in Blood or Bone Marrow Distinguish Subtypes of Pediatric Acute Lymphoblastic Leukemia

OncomiRs are microRNAs that are associated with early onset of specific cancers. To identify microRNAs involved in pediatric acute lymphoblastic leukemia (ALL) subtypes T-ALL and B-ALL, peripheral blood and bone marrow samples were independently subjected to microarray analysis using two different high-fidelity array platforms. The unique and common gene signatures from both arrays were validated by TaqMan individual assays in 100 pediatric ALL samples. Survival studies were carried out in the test set and validation set with 50 randomly selected samples in each set. MicroRNA expression profile revealed characteristic signatures for distinguishing T and B lineages and identified 51 novel microRNAs in pediatric ALL. Interestingly, the present study also revealed endogenous similarities and differences between blood and bone marrow within each ALL subtype. When Cox regression analysis was carried out with these identified microRNAs, 11 of them exhibited expression levels significantly correlated with survival. Validation of some of the common and relevant microRNAs from both arrays showed that their targets are involved in key oncogenic signaling pathways. Thus, this study suggests that microRNAs have the potential to become important diagnostic tools for identification and monitoring clinical outcomes in ALL patients.

Low LEF1 expression is a biomarker of early T-cell precursor, an aggressive subtype of T-cell lymphoblastic leukemia.

Early T-cell precursor (ETP) is the only subtype of acute T-cell lymphoblastic leukemia (T-ALL) listed in the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia. Patients with ETP tend to have worse disease outcomes. ETP is defined by a series of immune markers. The diagnosis of ETP status can be vague due to the limitation of the current measurement. In this study, we performed unsupervised clustering and supervised prediction to investigate whether a molecular biomarker can be used to identify the ETP status in order to stratify risk groups. We found that the ETP status can be predicted by the expression level of Lymphoid enhancer binding factor 1 (LEF1) with high accuracy (AUC of ROC = 0.957 and 0.933 in two T-ALL cohorts). The patients with ETP subtype have a lower level of LEF1 comparing to the those without ETP. We suggest that incorporating the biomarker LEF1 with traditional immune-phenotyping will improve the diagnosis of ETP.