B-cell Precursor Acute Lymphoblastic Leukemia Cases Research Articles

RNA-seq–based miRNA signature as an independent predictor of relapse in pediatric B-cell acute lymphoblastic leukemia

AbstractAberrant micro-RNA (miRNA) expression profiles have been associated with disease progression and clinical outcome in pediatric cancers. However, few studies have analyzed genome-wide dysregulation of miRNAs and messenger RNAs (mRNAs) in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To identify novel prognostic factors, we comprehensively investigated miRNA and mRNA sequencing (miRNA-seq and mRNA-seq) data in pediatric BCP-ALL samples with poor outcome. We analyzed 180 patients, including 43 matched pairs at diagnosis and relapse. Consensus clustering of miRNA expression data revealed a distinct profile characterized by mainly downregulation of miRNAs (referred to as an miR-low cluster [MLC]). The MLC profile was not associated with any known genetic subgroups. Intriguingly, patients classified as MLC had significantly shorter event-free survival (median 21 vs 33 months; log-rank P = 3 ×10−5). Furthermore, this poor prognosis was retained even in hyperdiploid ALL. This poor prognostic MLC profiling was confirmed in the validation cohort. Notably, non-MLC profiling at diagnosis (n = 9 of 23; Fisher exact test, P = .039) often changed into MLC profiling at relapse for the same patient. Integrated analysis of miRNA-seq and mRNA-seq data revealed that the transcriptional profile of MLC was characterized by enrichment of MYC target and oxidative phosphorylation genes, reduced intron retention, and low expression of DICER1. Thus, our miRNA-mRNA integration approach yielded a truly unbiased molecular stratification of pediatric BCP-ALL cases based on a novel prognostic miRNA signature, which may lead to better clinical outcomes.

Constitutional and Somatic Variants in ARID5B in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

INTRODUCTION Acute lymphoblastic leukemia (ALL) is the most common malignancy in children, with the high hyperdiploid (HeH) subtype accounting for approximately 25% of B-cell precursor (BCP) ALL cases. It has been shown that germline variants in the ARID5B gene in chromosome band 10q21.2 are associated with increased risk of BCP ALL, in particular HeH ALL, and that the risk alleles result in lower expression of ARID5B in hematopoietic cells. ARID5B codes for a protein involved in regulating gene expression and chromatin remodeling. We have previously reported somatic deletions in the ARID5B locus in two cases of HeH ALL, but the overall frequency of acquired copy number changes and rearrangements involving ARID5B in BCP ALL remains unknown. Here, we have investigated constitutional and somatic ARID5B variants in pediatric BCP ALL, with a particular focus on HeH cases. METHODS Constitutional variants We studied four known risk single nucleotide polymorphisms (SNPs) in the ARID5B locus (rs7090445, rs7089424, rs7073837 and rs10740055) in HeH ALL cases heterozygous for the risk SNP and with trisomy 10. These were investigated in three different cohorts, including a total of 92 cases informative for rs7090445, 92 cases for rs7089424, 119 cases for rs7073837 and 66 cases for rs10740055. The genotype and relative allele frequencies were ascertained from SNP array or whole genome sequencing (WGS) data. One-sided binomial tests were applied to investigate whether the risk allele was more often in the duplicated chromosome than the non-risk allele. Somatic variants For somatic variants, we ascertained copy number status based on SNP array and/or WGS analysis in the ARID5B region in a total of 466 pediatric HeH ALL cases and structural rearrangements based on WGS in 77 cases. We also studied, using SNP array analysis, somatic copy number variants in a separate cohort consisting of 590 non-HeH BCP ALL cases. To compare the proportions of deletions in the HeH cohort and in the other genetic subtypes, we used Fisher's Exact two-sided test. RESULTS Constitutional variants All four risk SNPs showed a significantly higher proportion of risk allele duplication (one-sided binominal test); rs7090445 ( P=0.009), rs7089424 ( P=0.005), rs7073837 ( P=0.03) and rs10740055 ( P=0.04). This validates that there is a clonal selection for HeH blast cells with gain of the chromosome 10 homologue carrying the risk allele as opposed to gain of the homologue that carries the non-risk allele. Somatic variants Somatic deletions targeting ARID5B were found in 9/466 cases (1.9%) of HeH cases. The deletions covered different parts of ARID5B, with no minimally deleted region, suggesting that the functional outcome was downregulation of ARID5B expression. In the cohort with non-HeH BCP ALL (other genetic subtypes), 4/590 somatic deletions were found (0.68%). There was no statistically significant difference between the frequency of copy number aberrations targeting ARID5B between HeH and the other genetic subtypes of ALL (P=0.09). WGS analysis of the HeH cases revealed one translocation and one missense mutation in 2/77 cases (2.6%). The translocation involved the PAN3 and ARID5B genes and was also present in RNA sequencing data from this case. CONCLUSIONS We show that our previous finding that HeH ALL constitutionally heterozygous for ARID5B risk alleles and with an acquired trisomy 10 more commonly duplicates the chromosome 10 homologue carrying the risk allele holds true in a much larger cohort. Furthermore, somatic deletions involving ARID5B are recurrent in pediatric BCP ALL.

Reshaping MRD Detection Strategies: Next-Generation Sequencing and High DNA Input Identify Previously Missed Measurable Residual Disease in Peripheral Blood of B-Cell Precursor Acute Lmyphoblastic Leukemia

Background: In acute lymphoblastic leukemia (ALL), measurable residual disease (MRD) is the most important prognostic factor. MRD is traditionally quantified in bone marrow aspirates (BM) using real-time quantitative polymerase chain reaction (RQ-PCR) according to EuroMRD standards. Since the emergence of modern molecular methods, it has been a continuous effort to enable MRD assessment from peripheral blood (PB). The less invasive material collection would allow closer MRD monitoring and solve technical challenges of BM aspiration, such as skewed or non-representative MRD values due to hemodilution and unequal distribution of leukemic cells. While several studies demonstrated comparable MRD values of BM and PB in T-ALL, in B-cell precursor ALL (BCP-ALL) MRD determination from PB is still a challenge. Mean MRD levels in PB are considerably lower than in BM and frequently escape detection. Additionally, paired samples show a highly variable MRD level ratio in both pediatric and adult cases (Kotrova et al, Leukemia 2020). Objective: BCP-ALL cases with known MRD positivity in the BM likely also harbor MRD positive cells in the PB, even when missed by traditional methods. With more sensitive assays, either by increased DNA input or by application of amplicon-based next generation sequencing (NGS), MRD detection from PB may improve in these cases. Methods: We retrospectively selected PB - BM sample pairs of 74 BCP-ALL patients with BM MRD positivity (43 with quantifiable MRD positivity and 31 with positivity below quantitative range) and sufficient leftover DNA from a cohort of patients treated according to GMALL protocols, where conventional RQ-PCR of clonal immunoglobuline heavy chain (IGH) VJ/DJ gene rearrangements failed to detect PB MRD. MRD had been determined by standard RQ-PCR with 1.5 µg DNA in BM and PB, respectively, according to the EuroMRD guidelines (van der Velden et al, Leukemia 2007). PB samples were re-tested for MRD with high DNA input RQ-PCR using 6.6 µg DNA and amplicon-based NGS, each using 6.6 µg DNA, as well as standard input NGS. NGS analyses of corresponding PB and BM samples were performed with the EuroClonality amplicon-based NGS assay (Brüggemann et al, Leukemia 2019). Identification and quantification of leukemia-derived clonotypes was done using ARResT/Interrogate. Results: In total, 74 BCP-ALL patients with pre/c-B-ALL (n=60) and pro-B-ALL (n=14) were included. Patient age ranged from 19 - 71 years with a median of 44.5. In all included samples (56 in first-line therapy and 18 salvage treatment), BM was MRD positive and PB was MRD negative in standard RQ-PCR at a sensitivity of at least 10 -4. To assess the effect of high DNA input alone, RQ-PCR was repeated in PB with high DNA input for 59 patients and 63 IGH VJ/DJ markers. At least one positive signal was detected in 19/59 patients (32%) and 20/63 markers (32%) (FIGURE 1A). NGS was performed for 73 PB samples and 84 markers using standard and high DNA input. With standard DNA input, MRD could be detected for 19/73 patients (26%) and 21/84 markers (25%) (FIGURE 1A). In the high input approach, MRD was detected in 36/73 patients (49%) and 40/84 markers (48%) (FIGURE 1A). A quantifiable MRD value was identified in 17 patients and 19 markers. All markers with quantifiable NGS MRD positivity in PB showed higher MRD values in the corresponding BM sample and MRD values below 10 -4 (FIGURE 1B). No correlation of MRD in paired samples could be demonstrated (Pearson correlation coefficient: 0.09) (FIGURE 1B). Conclusion: In BCP-ALL, MRD detection from PB is challenging with conventional methods. Increased DNA input and amplicon-based NGS allowed detection of MRD in a subset of PB samples that were negative in standard RQ-PCR. With identical DNA input, NGS showed a higher sensitivity than RQ-PCR and best results were achieved when high DNA input and NGS were combined, identifying MRD in nearly half of the PB samples. These results demonstrate that PB can be a valuable tool for early MRD detection in BCP-ALL when adjusting traditional MRD assessment strategies. However, in half of the samples, MRD was still missed by high input NGS. Even with assays improved for sensitivity, PB therefore cannot fully replace BM for MRD assessment in BCP-ALL yet and should rather be considered to complement BM analyses or in cases where BM is not accessible.

Flow Sorting, Whole Genome Amplification and Next-Generation Sequencing as Combined Tools to Study Heterogeneous Acute Lymphoblastic Leukemia.

Next-generation sequencing (NGS) methods have been introduced for immunoglobulin (IG)/T-cell receptor (TR) gene rearrangement analysis in acute lymphoblastic leukemia (ALL) and lymphoma (LBL). These methods likely constitute faster and more sensitive approaches to analyze heterogenous cases of ALL/LBL, yet it is not known whether gene rearrangements constituting low percentages of the total sequence reads represent minor subpopulations of malignant cells or background IG/TR gene rearrangements in normal B-and T-cells. In a comparison of eight cases of B-cell precursor ALL (BCP-ALL) using both the EuroClonality NGS method and the IdentiClone multiplex-PCR/gene-scanning method, the NGS method identified between 29% and 139% more markers than the gene-scanning method, depending on whether the NGS data analysis used a threshold of 5% or 1%, respectively. As an alternative to using low thresholds, we show that IG/TR gene rearrangements in subpopulations of cancer cells can be discriminated from background IG/TR gene rearrangements in normal B-and T-cells through a combination of flow cytometry cell sorting and multiple displacement amplification (MDA)-based whole genome amplification (WGA) prior to the NGS. Using this approach to investigate the clonal evolution in a BCP-ALL patient with double relapse, clonal TR rearrangements were found in sorted leukemic cells at the time of second relapse that could be identified at the time of diagnosis, below 1% of the total sequence reads. These data emphasize that caution should be exerted when interpreting rare sequences in NGS experiments and show the advantage of employing the flow sorting of malignant cell populations in NGS clonality assessments.

Diagnostic utility of whole genome sequencing in adults with B-other acute lymphoblastic leukemia.

Genomic profiling during the diagnosis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) in adults is used to guide disease classification, risk stratification, and treatment decisions. Patients for whom diagnostic screening fails to identify disease-defining or risk-stratifying lesions are classified as having B-other ALL. We screened a cohort of 652 BCP-ALL cases enrolled in UKALL14 to identify and perform whole genome sequencing (WGS) of paired tumor-normal samples. For 52 patients with B-other, we compared the WGS findings with data from clinical and research cytogenetics. WGS identified a cancer-associated event in 51 of 52 patients, including an established subtype defining genetic alterations that were previously missed with standard-of-care (SoC) genetics in 5 of them. Of the 47 true B-other ALL, we identified a recurrent driver in 87% (41). A complex karyotype via cytogenetics emerges as a heterogeneous group, including distinct genetic alterations associated with either favorable (DUX4-r) or poor outcomes (MEF2D-r and IGK::BCL2). For a subset of 31 cases, we integrated the findings from RNA sequencing (RNA-seq) analysis to include fusion gene detection and classification based on gene expression. Compared with RNA-seq, WGS was sufficient to detect and resolve recurrent genetic subtypes; however, RNA-seq can provide orthogonal validation of findings. In conclusion, we demonstrated that WGS can identify clinically relevant genetic abnormalities missed with SoC testing as well as identify leukemia driver events in virtually all cases of B-other ALL.

Analysis of non-alcoholic fatty liver disease during induction therapy for B-cell precursor acute lymphoblastic leukemia in children and adolescents

A prospective evaluation of non-alcoholic fatty liver disease (NAFLD) during induction therapy for acute lymphoblastic leukemia (ALL) has not been performed. Herein, we prospectively investigated the frequency, risk factors, and outcomes of NAFLD during induction therapy in children and adolescents with B-cell precursor ALL (BCP-ALL). This study enrolled 74 newly diagnosed BCP-ALL cases aged 1 year and older who were admitted to our department between January 2011 and December 2020. Median age was 6.6 years (1.3-17.5 years). Plain computed tomography (CT) of the upper abdomen was performed before induction therapy, and on days 15 and 29 after initiation of induction therapy. Patients with a liver/spleen CT ratio <0.9 were defined as having NAFLD. The frequency of NAFLD was 73%. Patients with NAFLD had a higher rate of hypertriglyceridemia. There was no significant difference in 5-year overall survival and event-free survival (EFS) between patients with and without NAFLD. However, after restricting the target age to 10 years and older, 5-year EFS was significantly higher in patients with NAFLD than in those without (88.5 vs. 42.9%, respectively, P=0.037). Similarly, 5-year cumulative incidence of relapse (CIR) was significantly lower in patients with NAFLD than in those without it (5-year CIR, 6.3 vs. 57.1%, respectively, P=0.013). Patients with NAFLD exhibit better outcomes including 5-year EFS and CIR. Further studies are necessary.

Clinical characteristics and outcomes of childhood B-ALL with ZNF384 and MEF2D rearrangements

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has many subtypes with diverse clinical and biological features and outcomes. Next generation sequencing has revealed several novel subtypes, including the ZNF384 and MEF2D rearrangements. The clinical characteristics and outcomes of the largest series of BCP-ALL cases with ZNF384 and MEF2D rearrangements in an international collaborative study are described here. Patients with ZNF384 rearrangements appear to express various leukemic phenotypes, including BCP-ALL (with or without abnormal expression of myeloid markers) and B/myeloid mixed phenotype acute leukemia. We provide strong evidence that among BCP-ALL patients with a ZNF384 fusion, the partner gene is associated with demographic features and influences the outcome; particularly the EP300-ZNF384 fusion is associated with a low risk of relapse. MEF2D rearrangements have been primarily described in children and young adults with BCP-ALL. Previous research has suggested that patients with MEF2D-BCL9 fusion have a high risk of relapse. Despite having the MEF2D-HNRNPUL1 fusion gene, the prognosis was favorable. Improved diagnostic genomic testing will enable future prospective studies to clarify the clinical significance of the ZNF384 and MEF2D rearrangements in childhood and young adult BCP-ALL.

Deciphering the Somatic and Germline Structural Variation Landscape in Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia By Whole Genome Optical Mapping

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent pediatric cancer. The most common numerical and structural chromosomal alterations are high hyperdiploidy (HHD, 51-67 chromosomes) and t(12;21)(p13;q22) leading to ETV6::RUNX1 fusion. However, for a full-blown leukemia, secondary alterations are required. Furthermore, it is increasingly recognized that germline variants may enhance ALL susceptibility in ~4% of the patients (Bloom et al. 2019, Klco & Mullighan 2020). In large-scale genomic studies, the mutational landscape of somatic and germline single nucleotide variants and small indels has been well described in BCP-ALL. However, a comprehensive analysis of somatic and germline structural variants (SVs) in ETV6::RUNX1 and HHD tumors is still lacking. Recent feasibility studies showed that whole genome optical mapping (WGOM) is a novel method for reliable SV detection which could abrogate conventional cytogenetics and array based methods in the future (Neveling et al. 2021, Mantere et al. 2021). The aim of the study was to analyze the structural variation landscape in pediatric BCP-ALL patients by employing WGOM to gain significant insights on leukemia predisposition and development. Methods: We studied 60 pediatric patients diagnosed with ETV6::RUNX1 (n=30) or HHD (n=30) BCP-ALL with a median age at diagnosis of 3.9 (range 1.7-17) and a male to female ratio of 1.14:1. We collected tumor material at initial diagnosis and included fibroblast or remission material as matched non-tumor control of each patient. Ultra-high molecular weight DNA was extracted from 120 samples and labeled by a sequence specific enzyme. Fluorescently labeled DNA was analyzed using a Saphyr instrument (Bionano Genomics). We integrated comprehensive datasets of 60 matched tumor/non-tumor genomes to identify somatically acquired SVs >500bp. Corresponding data of standard methods as cyto- (karyotype, fluorescence in situ hybridization) and molecular genetics (SNP-array) of the leukemia samples were compared to WGOM data. In addition, the prevalence of rare germline SVs (<1% of 160 healthy control samples [Bionano] and minor allele frequency <0.01 in dbVar) occurring in known cancer predisposition genes was analyzed in the non-tumor samples. Results: WGOM was able to detect 94% (438/464) of previously observed events by conventional methods, including ETV6::RUNX1 and high hyperdiploid hallmark alterations in all BCP-ALL cases. Beyond that, we discovered 895 additional SVs with WGOM not detected by standard cyto- and molecular genetics. WGOM detected a median of 25 (range 0-70) somatic SVs secondary to the hallmark alterations in ETV6::RUNX1 tumors compared to 13 (range 0-28) SVs in HHD cases. We identified 60 recurrently altered regions occurring in ≥3 BCP-ALL cases including 12p13.2 (ETV6, 19/60), 9p13.2 (PAX5, 13/60), 9p21.3 (CDKN2A/B, 12/60), 11p12 (RAG2, 10/60), 20p12.2 (MKKS/SLXIP, 10/60) or 3q13.2 (CD200/BTLA, 8/60). We detected ETV6::RUNX1 subtype specific recurrent focal deletions in ATF7IP (10/30), BTG1 (10/30), NSD2 (5/30) and TBL1XR1 (4/30). Interestingly, we discovered recurrent SVs affecting putative novel genes involved in BCP-ALL leukemogenesis such as STAG2, GPN3 and UBA2 which are affected in ~10% of the BCP-ALL patients. Strikingly, WGOM revealed in total 9 novel potential in-frame fusion transcripts in both tumor subtypes, including a potential fusion of the cohesin complex members PDS5B::STAG2, arising through a t(X;13)(q25;q13.1) in an ETV6::RUNX1 patient (Figure 1). In addition, we detected 28 rare germline SVs targeting 22 known cancer predisposing genes in 35% (21/60) of the pediatric patients. 40% (9/22) of the affected genes were involved in hematopoietic or lymphoid organ development as ETV6, FANCA, IKZF1, ITCH or PTPRC. In 18% (11/60) of the children, the identified germline alteration potentially altered a coding region of a known cancer predisposing gene including a duplication of CIITA in an ETV6::RUNX1 patient. Conclusions: By applying the novel technology WGOM, we could detect a significant amount of previously hidden SVs in our BCP-ALL cohort in addition to conventional methods. We further generated a comprehensive landscape of somatic and germline SVs in childhood BCP-ALL, revealing recurrently altered regions and novel potential players in ALL leukemogenesis and predisposition. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Multi-Cohort Gene Expression Classifier (ALLCatchR) Identifies B-Precursor ALL Subtypes and Their Developmental Trajectories across Age Groups

The molecular landscape of B cell precursor acute lymphoblastic leukemia (BCP-ALL) describes >20 disease subtypes defined by initiating genomic alterations and corresponding gene expression signatures. Some subtypes represent phenocopies with shared gene expression signatures but distinct or absent genomic drivers. The majority of BCP-ALL subtypes have been acknowledged as diagnostic entities by the currently revised classifications of hematologic malignancies (WHO-HAEM5 / International Consensus Classification). Thus, unified diagnostic approaches are needed to systematically identify subtypes in individual BCP-ALL cases in clinical routine. Gene expression signatures represent the shared functional equivalent of heterogeneous genomic driver alterations. Therefore, we established a gene expression-based classifier to identify BCP-ALL subtypes and related clinical and biological phenotypes. We analyzed a total of n=3477 BCP-ALL gene expression profiles obtained from transcriptome sequencing of adult (n=1253) and pediatric patients (n=2224) including publicly available (n=2074; Gu Z et al. Nat. Genet. 2019; Schmidt B. et al. Blood Adv. 2022) and own data sets (n=1403) from adult and pediatric European study groups (GMALL, AIEOP-BFM, CELL). Raw read counts from 6 cohorts representing heterogeneous library preps (i.e., stranded/unstranded; mRNA/total RNA), sequencing platforms/depths, and gene count quantifications were included. All samples had been previously allocated to molecular BCP-ALL subtypes based on integration of independent genomic profiling, gene fusion calling and gene expression. For classification, data with defined subgroup allocation were randomly split into training (n=1831; 70%) and testing sets (n=785; 30%) and two hold-out studies (n=305). To define gene expression signatures of 21 BCP-ALL subtypes, we used training set samples and extracted specific gene sets employing different feature selection algorithms as well as ranking of genes in individual subtypes. Based on these definitions we established a BCP-ALL subtype classifier (ALLCatchR), which integrates machine learning models (support vector machines) and nearest-neighbors associations of single sample gene set enrichment analyses (Fig. A). ALLCatchR performed molecular subtype allocation for the testing set at high sensitivity (0.934±0.165) and specificity (0.997±0.005) on average across subtypes. A similar performance was achieved in the two hold-out cohorts despite completely independent data structures which have not been shown to the classifier (sensitivity: 0.946±0.097 / specificity: 0.996±0.007). Overall accuracies of 0.952 and 0.92 for testing and hold-out-data sets indicate that ALLCatchR reliably identifies molecular BCP-ALL subtypes in mixed adult / pediatric cohorts based on gene expression profiles alone (Fig. A, below). Among n=44/1090 (4%) samples of the combined hold-out/testing set with ambiguous subtype allocation (deemed 'unclassified'), ALLCatchR provides candidate subtype allocations (sensitivity: 0.671±0.393 / specificity: 0.978±0.036) for confirmation based on genomic driver alterations. In addition, we modified our classifier to predict baseline variables such as patient's sex (accuracy: 0.991), immunophenotype (accuracy: 0.935) or blast count (R=0.633). To define the corresponding B cell differentiation stage of origin, we compared BCP-ALL subtypes to differentiation stage-specific gene sets extracted from 7 FACS-sorted lymphopoietic subsets from healthy bone marrow donors (n=4). We observed defined BCP-ALL subtype-specific enrichment patterns which can be grouped along 4 major differentiation stages (Pro-B to Pre-B-II-Large, Fig. 1B) with a high degree of similarity in independent adult and pediatric cohorts. Thus, B cell developmental trajectories underlying BCP-ALL subtypes are dissected at high resolution by gene expression profiling and differentiation stages may favor the selection of specific leukemogenic drivers. ALLCatchR is a gene expression classifier for 21 established BCP-ALL molecular subtypes, which performs at independently validated cohorts with an accuracy of >90% and allows imputation of clinical baseline variables as well as underlying B cell developmental trajectories. It provides a reliable basis for systematic diagnostic approaches in pediatric and adult BCP-ALL patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Genome-Wide CRISPR-Cas9 Screen Reveals GATOR1 Complex Is a Critical Regulator of Glucocorticoid Sensitivity in B-Cell Precursor Acute Lymphoblastic Leukemia

Glucocorticoids (GCs) are essential components of B-cell precursor acute lymphoblastic leukemia (B-ALL) therapy. Upon GC binding, GC receptor (GR) translocates to the nucleus and activates GR target genes. While GCs reportedly induce cell death via apoptosis and/or cell cycle arrest, their molecular mechanisms remain largely unknown. To identify molecules/pathways governing GC sensitivity in B-ALL cells, we performed genome-wide CRISPR-Cas9 dropout screens in the presence or absence of dexamethasone (Dex) using two human B-ALL lines (NALM-6 and MUTZ-5). To do so, we employed a baboon envelope pseudotyped lentiviral vector system, which enabled highly efficient transduction of human B cells (Sasaki et al. Blood 2022). We identified a total of 90 and 46 genes, whose single-guide RNAs (sgRNAs) were significantly enriched upon Dex treatment in NALM-6 and MUTZ-5, respectively. A total of 15 genes were identified in both lines, and among them included those previously implicated in GR resistance, such as NR3C1 and TBL1XR1. Strikingly, sgRNAs targeting DEPDC5, NPRL2 and NPRL3, each encoding components of the GATOR1 complex, a negative regulator of mTORC1, were significantly enriched in the presence of Dex in both lines. To validate these findings, we depleted each gene via lentivirus-based E2-Crimson-tagged sgRNA and assessed effects on cell growth over 11 days with or without Dex. Cells expressing sgRNA targeting either DEPDC5, NPRL2 or NPRL3, but not those transduced with empty vector, demonstrated a proliferative advantage relative to non-transduced cells in the presence of Dex. Importantly, Dex treatment significantly reduced phospho-P70S6K levels, a readout of the mTOR pathway activation, in GATOR1-intact cells, but not in NPRL2 or DEPDC5 knockout cells, suggesting that Dex suppresses mTORC1-mediated growth signals via the GATOR1 complex. We next asked whether Dex/GATOR1-mediated mTORC1 suppression is mediated by transcriptional activity of GR. To do so, we established a NALM-6 line deficient in endogenous GR in which a GR mutant lacking DNA binding capacity was exogenously expressed. Dex treatment did not reduce phospho-P70S6K levels in these cells, suggesting that the Dex/GATOR1-mediated mTORC1 suppression seen in GATOR1-intact cells primarily depends on transcriptional capacity of GR. Given that loss-of-function mutations of DEPDC5 were reported in therapy-refractory B-ALL cases and that GATOR1 complex reportedly functions in amino acid metabolisms, our findings suggest a potential link between GC sensitivity and amino acid metabolism in B-ALL cells.

Association between Dysfunction of the Nucleolar Stress Response and Multidrug Resistance in Pediatric Acute Lymphoblastic Leukemia.

Simple SummaryPediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse or are refractory to chemotherapy despite the low frequency of TP53 mutations. The nucleolar stress response is one of the mechanisms that activate P53 by ribosomal protein L11 (RPL11). We hypothesized that the lack of nucleolar stress response is related to chemoresistance and relapse in some pediatric BCP-ALL cases. We revealed that clinical BCP-ALL therapeutics, such as 6-mercaptopurine, methotrexate, daunorubicin, and cytarabine, induced the nucleolar stress response, and its treatment susceptibility was dependent on the nucleolar stress response. Furthermore, we observed decreased RPL11 expression at relapse in seven children with BCP-ALL in comparison to that at onset. Our findings provide new insights into the anti-leukemia mechanism in BCP-ALL and multidrug resistance and relapse via the nucleolar stress response, suggesting that the nucleolar stress response may be a potential therapeutic strategy to predict chemosensitivity and improve chemoresistance in pediatric BCP-ALL.Approximately 20% of pediatric patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) relapse or are refractory to chemotherapy despite the low frequency of TP53 mutations. The nucleolar stress response is a P53-activating mechanism via MDM2 inhibition by ribosomal protein L11 (RPL11). We analyzed the role of the nucleolar stress response using BCP-ALL cell lines and patient samples by drug sensitivity tests, Western blotting, and reverse transcription polymerase chain reaction. We revealed that the nucleolar stress response works properly in TP53 wild-type human BCP-ALL cell lines. Next, we found that 6-mercaptopurine, methotrexate, daunorubicin, and cytarabine had anti-leukemic effects via the nucleolar stress response within BCP-ALL treatment. Comparing the samples at onset and relapse in children with BCP-ALL, RPL11 mRNA expression decreased at relapse in seven of nine cases. Furthermore, leukemia cells with relapse acquired resistance to these four drugs and suppressed P53 and RPL11 expression. Our findings suggest that the nucleolar stress response is a novel anti-leukemia mechanism in BCP-ALL. As these four drugs are key therapeutics for BCP-ALL treatment, dysfunction of the nucleolar stress response may be related to clinical relapse or refractoriness. Nucleolar stress response may be a target to predict and improve the chemotherapy effect for pediatric BCP-ALL.

P312: EMPLOYING WHOLE GENOME OPTICAL MAPPING TO CHARACTERIZE THE LANDSCAPE OF STRUCTURAL VARIANTS IN B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

Background: B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most frequent pediatric cancer. The most common subtypes are ETV6::RUNX1+ BCP-ALL, which harbor a reciprocal translocation between chromosome 12 and 21 leading to a juxtaposition of ETV6 to RUNX1, and high hyperdiploid (HHD) BCP-ALL harboring 51-67 chromosomes. In large-scale genomic studies, the mutational landscape of somatic single nucleotide variants and indels as well as copy number gains and losses based on array CGH have been well described in both subtypes. However, a comprehensive analysis of somatic structural variants (SVs) is still lacking which may reveal significant new insights on tumor development, progression, and treatment options in BCP-ALL. Aims: We aimed to primarily employ the novel technology whole genome optical mapping (WGOM) to unmask the landscape of structural variants in BCP-ALL. Firstly, we determined the concordance of chromosomal abnormalities identified by WGOM in comparison to standard techniques. Superiorly, we explored the landscape of somatic aberrations in ETV6::RUNX1+ and HHD tumors to identify recurrent alterations and novel SVs affecting genes not yet related to BCP-ALL pathogenesis. Methods: We extracted ultra-high molecular weight (UHMW) DNA of 13 ETV6::RUNX1+ and 18 HHD BCP-ALL with a tumor cell content of 25-96% at diagnosis. Additionally, we isolated DNA of fibroblasts or remission material of each pediatric patient as matching non-tumor control. Informed consent was obtained from all participants. DNA was fluorescently labeled by a sequence specific enzyme and subsequently linearized and imaged using a Saphyr instrument (Bionano Genomics). Using this approach, we generated comprehensive datasets of 31 matched tumor/non-tumor pairs, allowing the identification of somatically acquired SVs >500bp. In addition, corresponding karyotype, fluorescence in situ hybridization and SNP-array data of the leukemia samples were integrated and compared to WGOM data. Results: We validated 95% of 233 events observed by standard techniques with WGOM. All hallmark alterations including ETV6::RUNX1 fusion and high hyperdiploidy were identified by WGOM. In addition, we detected a median of 11 (1-36) somatically acquired events in leukemic cells with ETV6::RUNX1 fusion, compared to 3 (1-10) events in HHD cases. WGOM identified recurrent somatic structural aberrations occurring in at least three BCP-ALL cases, including focal deletions affecting ETV6 (9/31), PAX5 (5/31), ARPP21 (3/31), CD200/BTLA (3/31) and RAG2 (3/31). We detected subtype specific recurrent focal deletions disrupting ATF7IP (7/13) and BTG1 (5/13) in ETV6::RUNX1+ subclones whereas deletion of the CDKN2A/B locus (3/18) was only observed in HHD BCP-ALL. Interestingly, we discovered novel recurrently deleted regions on 12q24.11 (GPN3, 4/31) and Xq25 (STAG2, 3/31) in all BCP-ALL cases as well as focal loss of 19q13.11 (UBA2, 3/13) specific for ETV6::RUNX1+ subclones using WGOM. Moreover, complex three-way translocations in ETV6::RUNX1+ tumors were discovered by WGOM, including t(6;8;12)(p12.3;q24.23;q24.21) that leads to the disruption of MED13L and HMGCLL1. Summary/Conclusion: Our study showed that WGOM detects SVs as translocations and aneuploidies in BCP-ALL as good as conventional methods with a 95% concordance. However, WGOM allows a higher resolution and more precise localization of chromosomal breakpoints compared to standard techniques. Hence, we identified novel recurrent somatically acquired SVs including a deletion on Xq25 affecting STAG2, which might play an important role in pediatric BCP-ALL leukemogenesis.

Characterization of Immunophenotypic Aberrancies with Respect to Common Fusion Transcripts in B-Cell Precursor Acute Lymphoblastic Leukemia: A Report of 986 Indian Patients

Objective:Based on the immunophenotype, acute lymphoblastic leukemia (ALL) can be categorized into B-cell or T-cell lineages. B-cell precursor ALL (BCP-ALL) cases show various genetic/molecular abnormalities, and varying frequencies of chimeric fusion transcripts in BCP-ALL cases are reported from different parts of the world. We studied the immunophenotypic aberrancy profiles of a large number of BCP-ALL cases with respect to various common chimeric fusion transcripts.Materials and Methods:Flow cytometric immunophenotyping and multiplex reverse-transcription polymerase chain reaction assays were performed for 986 BCP-ALL cases.Results:Among 986 BCP-ALL cases, the incidence of various fusion transcripts was 38.36% in adult cases and 20.68% in pediatric cases. Adult BCP-ALL patients with t(9;22)(BCR-ABL1) fusion transcripts and expression of aberrant myeloid markers were significantly older at presentation (p=0.0218) with male preponderance (p=0.0246) compared to those without aberrant myeloid expression. In pediatric patients with the t(12;21)(ETV6-RUNX1) chimeric fusion transcript, aberrant expression of CD13 was observed in 39.13%, CD33 in 36.95%, and CD117 in 8.69% of patients, respectively. Pediatric BCP-ALL patients with the ETV6-RUNX1 fusion transcript and expression of aberrant myeloid markers were not significantly different compared to those without with respect to demographic and clinical/hematological characteristics (p=0.5955). Aberrant myeloid markers were rarely or never expressed in pediatric and adult BCP-ALL patients with the t(4;11)(KTM2A-AF4) and t(1;19)(TCF3-PBX1) fusion transcripts.Conclusion:Aberrant myeloid markers were frequently expressed among BCP-ALL patients with the t(9;22)(BCR-ABL1) and t(12;21)(ETV6-RUNX1) fusion transcripts. However, BCP-ALL patients with the t(4;11)(KTM2A-AF4) and t(1;19)(TCF3-PBX1) fusion transcripts rarely or never expressed aberrant myeloid markers. Aberrant myeloid CD markers can be used in predicting chimeric fusion transcripts at baseline so as to plan appropriate tyrosine kinase inhibitor therapy in cases of BCP-ALL with specific chimeric fusion transcripts. This study has delineated the relationship of chimeric fusion transcripts with the aberrant expression of myeloid markers in a large cohort of BCP-ALL cases.

Glucocorticoid receptor gene mutations confer glucocorticoid resistance in B-cell precursor acute lymphoblastic leukemia

Glucocorticoid (GC) is a key drug in the treatment of B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and the initial GC response is an important prognostic factor. GC receptors play an essential role in GC sensitivity, and somatic mutations of the GC receptor gene, NR3C1, are reportedly identified in some BCP-ALL cases, particularly at relapse. Moreover, associations of somatic mutations of the CREB-binding protein (CREBBP) and Wolf-Hirschhorn syndrome candidate 1 (WHSC1) genes with the GC-resistance of ALL have been suggested. However, the significance of these mutations in the GC sensitivity of BCP-ALL remains to be clarified in the intrinsic genes. In the present study, we sequenced NR3C1, WHSC1, and CREBBP genes in 99 BCP-ALL and 22 T-ALL cell lines (32 and 67 cell lines were known to be established at diagnosis and at relapse, respectively), and detected their mutations in 19 (2 cell lines at diagnosis and 15 cell lines at relapse), 26 (6 and 15), and 38 (11 and 15) cell lines, respectively. Of note, 14 BCP-ALL cell lines with the NR3C1 mutations were significantly more resistant to GC than those without mutations. In contrast, WHSC1 and CREBBP mutations were not associated with GC resistance. However, among the NR3C1 unmutated BCP-ALL cell lines, WHSC1 mutations tended to be associated with GC resistance and lower NR3C1 gene expression. Finally, we successfully established GC-resistant sublines of the GC-sensitive BCP-ALL cell line (697) by disrupting ligand binding and DNA binding domains of the NR3C1 gene using the CRISPR/Cas9 system. These observations demonstrated that somatic mutations of the NR3C1 gene, and possibly the WHSC1 gene, confer GC resistance in BCP-ALL.

Genetic and genomic analysis of acute lymphoblastic leukemia in older adults reveals a distinct profile of abnormalities: analysis of 210 patients from the UKALL14 and UKALL60+ clinical trials.

Despite being predominantly a childhood disease, the incidence of acute lymphoblastic leukemia (ALL) has a second peak in adults aged 60 years and over. These older adults fare extremely poorly with existing treatment strategies and very few studies have undertaken a comprehensive genetic and genomic characterization to improve prognosis in this age group. We performed cytogenetic, single nucleotide polymorphism (SNP) array and next-generation sequencing (NGS) analyses on samples from 210 patients aged ≥60 years from the UKALL14 and UKALL60+ clinical trials. BCR-ABL1-positive disease was present in 26% (55/210) of patients, followed by low hypodiploidy/near triploidy in 13% (28/210). Cytogenetically cryptic rearrangements in CRLF2, ZNF384 and MEF2D were detected in 5%, 1% and <1% of patients, respectively. Copy number abnormalities were common and deletions in ALL driver genes were seen in 77% of cases. IKZF1 deletion was present in 51% (40/78) of samples tested and the IKZF1plus profile was identified in over a third (28/77) of cases of B-cell precursor ALL. The genetic good-risk abnormalities high hyperdiploidy (n=2), ETV6-RUNX1 (no cases) and ERG deletion (no cases) were exceptionally rare in this cohort. RAS pathway mutations were seen in 17% (4/23) of screened samples. KDM6A abnormalities, including biallelic deletions, were discovered in 5% (4/78) of SNP arrays and 9% (2/23) of NGS samples, and represent novel, potentially therapeutically actionable lesions using EZH2 inhibitors. Outcome remained poor with 5-year event-free and overall survival rates of 17% and 24%, respectively, across the cohort, indicating a need for novel therapeutic strategies.

UNEXPECTED CONCOMITANCE OF ETV6-RUNX1 AND BCR-ABL1P210 IN A CHILD WITH B-CELL PRECURSOR ACUTE LYMPHOBLASTIC LEUKEMIA

ETV6-RUNX1 and BCR-ABL1 are recurrent primary events in pediatric B-cell precursor acute lymphoblastic leukemia (B-ALL), being the former the most common one. Elegant studies have shown that these are founder events, but additional genetic events are needed for leukemia development. Nonetheless, these common primary alterations are typically mutually exclusive. The presence of both ETV6-RUNX1 and BCR-ABL1 is a very rare event which has been previously reported in only two B-ALL cases, being one pediatric and one adult patient. Herein, we report a rare pediatric B-ALL case with simultaneous occurrence of ETV6-RUNX1 and BCR-ABL1 . A 5-year-old boy was admitted to Hospital Federal da Lagoa, Rio de Janeiro, Brazil. At clinical investigation, he presented hepatosplenomegaly, a white blood cell count of 0.7×10 9 /L, 94% of blasts, Hb of 6.5 g/L, and platelet count of 16×10 9 /L. The patient was treated according to the AIEOP-BFM protocol. He presented a poor prednisone response (at day 8; >1000 circulating lymphoblasts), the minimal residual disease at day 33 was negative (0.08%), and he was considered in complete remission. Four months after the diagnosis, the child is alive with negative levels of residual disease. The immunophenotyping was characterized by nTdT (low), CD10, CD13, CD19, CD21, CD22, CD24, CD33, CD34, CD38 and CD45, cCD66c, cCD79, CD81, CD123, and CD58 positive cells in 94.1% of lymphoblast cells and a diploid profile (DNA index of 1.09). ETV6-RUNX1 and BCR-ABL1 p210 were identified by reverse transcriptase PCR (RT-PCR). The presence of both rearrangements was confirmed by sanger sequencing. Moreover, additional copy number alterations (CNA) were evaluated by multiplex ligation-dependent probe amplification (MLPA) using SALSA MLPA P335-C1. Deletions affecting IKZF1 (exons 2 and 3), CDKN2A (exons 2 and 5), CDKN2B (exon 2), PAX5 (exon 1), and ETV6 (exons 2, 3, 5 and 8) were detected. In consonance with previous studies, these CNAs are the most frequently secondary events found in patients with ETV6-RUNX1 . Of note, IKZF1 deletions are enriched in patients with BCR-ABL1 . Due to the lack of material for FISH analysis, the clonality of both biomarkers could not be verified. However, we observed a low load of BCR-ABL1 p210 transcript in comparison with the endogenous gene, suggesting that this gene fusion could be present in subclones. In conclusion, to the best of our knowledge, this is the second pediatric case identified with ETV6-RUNX1 and BCR-ABL1 gene rearrangement in concomitance, but is the first case reported with the p210 transcript, which is extremely rare in B-ALL. Previous studies have shown that the additional genetic events found can impact the usually favorable prognosis of ETV6-RUNX1 – positive B-ALL. The clinical significance for these rare patients with both rearrangements remains unclear due to the small number of reported cases to date. Therefore, it is very important to describe more cases which will ultimately enable future studies with larger series and clarify this enigmatic subgroup.

The Proteogenomic Landscape of High Hyperdiploid and ETV6/RUNX1-Positive Childhood Acute Lymphoblastic Leukemia

Introduction. High hyperdiploidy (51-67 chromosomes) and ETV6/RUNX1 -fusion are the two most common primary genetic events in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL), together found in more than half of the cases. In this study, we performed a proteogenomic analysis of these two subtypes, with the aim of understanding the interplay between somatic genomic aberrations, in particular copy number variants (CNVs), and gene and protein expression in leukemogenesis.Materials and methods. Eighteen high hyperdiploid and nine ETV6/RUNX1 -postive childhood BCP-ALL cases were subjected to whole exome or whole genome sequencing, SNP-array analysis, RNA-sequencing, and proteome analyses using high-resolution isoelectric focusing liquid chromatography mass spectrometry. Furthermore, SNP array analysis and RNA-sequencing data for an additional 29 high hyperdiploid and 31 ETV6/RUNX1 -positive cases were analyzed.Results. Expression data for 12,314 genes and 8,480 proteins were obtained, with 8,224 genes/proteins overlapping between the datasets. There was a clear linear relationship between the fold changes on the transcript and protein levels for high hyperdiploid cases over ETV6/RUNX1 -positive cases (Spearman correlation coefficient=0.62). For individual gene/protein pairs, genes and proteins involved in specialized functions such as hematopoiesis and amino acid metabolism showed high correlations, indicating a general regulation on the RNA level, whereas genes and proteins involved in basic cell functions such as the spliceosome, the ribosome, and oxidative phosphorylation showed lower correlations. Unsupervised principal component analyses showed separate clustering of high hyperdiploid and ETV6/RUNX1 -positive cases on both the RNA and protein levels. Dosage effects related to the chromosomal gains in high hyperdiploid ALL were clearly apparent, affecting genes both in cis (i.e., loci in the CNV) as well as in trans (i.e., loci outside the CNV). These effects were lower on the protein level than on the RNA level, showing additional layers of control for protein expression. A total of 2,627 genes and 1,098 proteins were differentially expressed between high hyperdiploid and ETV6/RUNX1 -positive BCP-ALL. Alternative splicing events were also investigated and 787 differentially expressed isoforms were identified. Allele-specific expression (ASE) analysis was performed to investigate the cis-effect of gene expression and 18 ASE events were observed in high hyperdiploid cases. Enrichment analyses on the whole gene/protein dataset showed upregulation of translation and proteasome-associated genes and proteins in high hyperdiploid cases, likely associated with an increased protein turn-over and possibly associated with an aneuploidy stress response. Overexpression of the cytokine receptors FLT3 and CRLF2 was also seen and the cytokine receptor interaction pathway was enriched. RAG1/RAG2-associated somatic recombination was enriched in ETV6/RUNX1 -positive BCP-ALL, in line with previous studies showing frequent activation of this pathway. Furthermore, pathways related to chromatin modifications and chromosome structure were markedly downregulated in high hyperdiploid BCP-ALL, suggesting a general deregulation of transcriptional control. In particular, low levels of CTCF and the cohesin complex, which are important regulators of chromatin structure, were seen in both gene and protein expression data.Conclusions. In this study, we have characterized the proteogenomic landscape of the two most common forms of childhood ALL. Our data provides new insight into pathogenesis of high hyperdiploid and ETV6/RUNX1 -postive BCP-ALL and may be used to identify novel targets for treatment. DisclosuresNo relevant conflicts of interest to declare.

High PDGFRA Expression Does Not Serve As Effective Therapeutic Target in ERG-Deleted B-Cell Precursor Acute Lymphoblastic Leukemia

Background and aimsSeveral oncogenic aberrations of receptor tyrosine kinases occur in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here, we describe a subgroup of pediatric patients lacking common chromosomal aberrations but with high gene expression of platelet-derived growth factor receptor alpha (PDGFRA). Oncogenic fusions involving the tyrosine kinase domain of PDGFRA are recurrent in eosinophilic leukemia and are responsive to kinase inhibitors (Cools et al. N Engl J Med 2003). One case with a FIP1L1-PDGFRA fusion was reported in adult BCP-ALL (Roberts et al. J Clin Oncol 2016). Therefore, we tested for genomic aberrations underlying the overexpression of PDGFRA and evaluated the inhibition of PDGFRα as treatment option in pediatric BCP-ALL.MethodsExpression profiling was performed using Affymetrix U133 plus 2.0 arrays. BCR-ABL1 -like cases were identified using the 110 probe set expression signature (Den Boer et al. Lancet Oncol 2009). High PDGFRA was defined as log2 expression of probe set 203131_at >5.9. FISH was performed using Cytocell FIP1L1/CHIC2/PDGFRA probes. Targeted locus amplification (de Vree et al. Nat Biotechnol 2014) was performed using custom-designed primers. Cells were treated for 10 minutes with 10 ng/mL PDGF-BB with or without 10 µM imatinib or 1 µM CP673451 and analyzed by western blot. Cell viability after 4 day exposure to imatinib (0.2µM - 1.6µM - 12.5µM) or CP673451 (0.02µM - 0.16µM - 1.25µM) was assessed by flow cytometry.ResultsGene expression analysis of 654 pediatric ALL cases identified high expression of PDGFRA in 26 of 574 BCP-ALL cases (4.5%). High PDGFRA expression was exclusively found in cases belonging to the B-other (n=22) or BCR-ABL1 -like (n=4) subtypes, and not present in ETV6-RUNX1, high hyperdiploid, TCF3-PBX1, BCR-ABL1 and KMT2A -rearranged BCP-ALL subtypes or T-ALL. PDGFRA expression was confirmed by RT-qPCR, and also found in the BCP-ALL cell line Nalm6. Using RT-PCR we did not detect any of the six reported PDGFRA fusions, which involve FIP1L1, STRN, BCR, CDK5RAP2, ETV6, and KIF5B, nor did we detect novel structural aberrations affecting PDGFRA by FISH. We studied the PDGFRA locus at high resolution by targeted locus amplification. Following DNA cross-linking, digestion, and proximity-based re-ligation, targeted amplification was performed from viewpoints in intron 2 and exon 15 of PDGFRA. In the eosinophilic cell line EOL1 we confirmed an interstitial deletion on chromosome 4 which rearranges FIP1L1 intron 12 to PDGFRA exon 12. The results obtained from Nalm6 and primary BCP-ALL samples with high PDGFRA expression did not indicate genomic aberrations of PDGFRA.ERG deletions were present in 10 of 16 PDGFRA -high cases (63%), compared with 0% in PDGFRA -low BCP-ALL cases (n=71; p<0.001). Vice versa, all ERG-deleted cases expressed high levels of PDGFRA . Recently, a novel BCP-ALL subtype was described with rearrangements of the transcription factor DUX4 and deletion of ERG (Lilljebjörn et al. Nat Commun 2016; Yasuda et al. Nat Genet 2016; Zhang et al. Nat Genet 2016). Furthermore, the high PDGFRA-expressing cell line Nalm6 carries a DUX4 rearrangement (Yasuda et al. Nat Genet 2016) and PDGFRA is highly expressed in DUX4 BCP-ALL and its transcription start site directly bound by DUX4 (Zhang et al. Nat Genet 2016). Therefore, we regard it as highly likely that the PDGFRA-high cases represent this novel DUX4- rearranged subtype.Because transcription factors are difficult to target with small molecule inhibitors, PDGFRα inhibition could represent a treatment option. Exposure to the PDGFR inhibitors imatinib and CP673451 reduced phosphorylation of PDGFRα in Nalm6 and primary BCP-ALL cells. However, ex vivo drug exposure revealed no cytotoxic effect of the PDGFR inhibitors on PDGFRA-high BCP-ALL cells, either in the presence or absence of the ligand PDGF-BB. Interestingly, we found a marked ex vivo sensitivity towards prednisolone in PDGFRA-high cases, in line with the good prognosis reported for ERG -deleted/DUX4-rearranged BCP-ALL.ConclusionsWe found high PDGFRA expression, in the absence of genomic aberrations of the PDGFRA locus, in a subgroup of BCP-ALL patients with frequent ERG deletions. PDGFR inhibition caused abrogation of PDGFRα phosphorylation but was not cytotoxic in PDGFRA-high expressing BCP-ALL cells. Therefore, high PDGFRA expression does not serve as effective therapeutic target in ERG -deleted BCP-ALL. DisclosuresNo relevant conflicts of interest to declare.

Epigenetic Modification of Death Receptor Genes for TRAIL and TRAIL Resistance in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia.

Immunotherapies specific for B-cell precursor acute lymphoblastic leukemia (BCP-ALL), such as anti-CD19 chimeric antigen receptor (CAR) T-cells and blinatumomab, have dramatically improved the therapeutic outcome in refractory cases. In the anti-leukemic activity of those immunotherapies, TNF-related apoptosis-inducing ligand (TRAIL) on cytotoxic T-cells plays an essential role by inducing apoptosis of the target leukemia cells through its death receptors (DR4 and DR5). Since there are CpG islands in the promoter regions, hypermethylation of the DR4 and DR5 genes may be involved in resistance of leukemia cells to immunotherapies due to TRAIL-resistance. We analyzed the DR4 and DR5 methylation status in 32 BCP-ALL cell lines by sequencing their bisulfite PCR products with a next-generation sequencer. The DR4 and DR5 methylation status was significantly associated with the gene and cell-surface expression levels and the TRAIL-sensitivities. In the clinical samples at diagnosis (459 cases in the NOPHO study), both DR4 and DR5 genes were unmethylated in the majority of cases, whereas methylated in several cases with dic(9;20), MLL-rearrangement, and hypodiploidy, suggesting that evaluation of methylation status of the DR4 and DR5 genes might be clinically informative to predict efficacy of immunotherapy in certain cases with such unfavorable karyotypes. These observations provide an epigenetic rational for clinical efficacy of immunotherapy in the vast majority of BCP-ALL cases.

Multicolor flow cytometry immunophenotyping and characterization of aneuploidy in pediatric B-cell precursor acute lymphoblastic leukemia.

The aim of this study was to assess the incidence of DNA aneuploidy in Polish children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) and the relationship between aneuploidy and immunological phenotype, age, leukocyte count, S-phase fraction (SPF) and early response to induction chemotherapy assessed by the percentage of residual blast cells in bone marrow aspirates. The study group consisted of 267 patients. DNA content and immunophenotype were assessed in the bone marrow before treatment using multicolor flow cytometry (FC). DNA aneuploidy was detected in 50/267 (19%) patients. High hyperdiploidy was found to be associated with lower leukocyte count (p = 0.006) and common ALL immunophenotype. Flow cytometry analysis revealed that high hyperdiploid BCP-ALL patients showed significantly higher expression of CD9, CD20, CD22, CD58, CD66c, CD86 and CD123 antigens as compared to other groups of ploidy. In contrast, CD45 showed decreased expression. The percentage of leukemic blasts at diagnosis was lower in high hyperdiploid BCP-ALL cases than in diploid (79% vs. 85.7%, p = 0.001). The difference in minimal residual disease (MRD) levels on day 15 and 33 of induction therapy between analyzed groups was not significant.This study showed that high hyperdiploidy is associated with lower WBC count and specific immunological phenotype. Flow cytometric evaluation of expression of selected antigens can be used for fast identification of markers of aneuploidy in pediatric BCP-ALL, before genetic tests results are available. Understanding the biological significance of aneuploidy in leukemia can potentially be exploited therapeutically using targeted therapies against specific blast cell subclones.