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Mutations 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 FLT3and involve the PAN3locus. 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 cisinteractions between the FLT3promoter and another enhancer located distally to the deletion breakpoints, with subsequent allele-specific upregulation of FLT3expression, 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 FLT3involvement in leukemogenesis by upregulation via chromatin remodeling and enhancer hijacking. These data further emphasize the role of FLT3as a driver gene in BCP ALL.

Mutations 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.

Whilst the molecular pathogenesis of childhood B-cell precursor (BCP) acute lymphoblastic leukemia (ALL) has been studied extensively, its 3D chromatin landscape remains poorly explored. Genome-wide chromosome conformation capture methods have provided the tools to investigate the different units of chromatin organization, such as transcriptionally active (A) and inactive (B) compartments, topologically associating domains (TADs), and fine-scale chromatin loops and enhancer-promoter interactions. The aim of this study is to elucidate the chromatin architecture and topological gene regulation in childhood BCP ALL. To date, 29 primary patient samples were included, comprising the high hyperdiploid (HeH) (n=11), ETV6:: RUNX1-positive (n=8), BCR:: ABL1-positive (n=2), TCF3:: PBX1-positive (n=2), DUX4-rearranged (n=2), intrachromosomal amplification of chromosome 21 (iAMP21) (n=1), KMT2A-rearranged (n=1), near-haploid (n=1) and near-triploid (n=1) genetic subtypes. Leukemic blast cells obtained at diagnosis were analyzed using Micro-C, a high-resolution variation of Hi-C (average number of total reads = 1.4 billion, highest resolution = 5 kb) combined with pair-end sequencing. Chromatin contact heatmaps were generated for each case using Juicer and Cooler. A/B compartments were identified using FanC at 500 kb resolution and visualized in the software IGV, while TAD calling was performed by Juicer, Domaincaller and Insulation Score. Differential chromatin interaction loop calling was made using Pareidolia and Mustache, and structural variant (SV) calling was carried out by EagleC. Preliminary principal component analysis of the 29 cases, based on the first two eigenvectors of the contact matrix (500 kb resolution), showed that HeH, ETV6:: RUNX1-positive, TCF3:: PBX1-positive and DUX4-rearranged cases each clustered based on their chromatin 3D organization. Furthermore, the A/B compartments of 11 HeH and 8 ETV6:: RUNX1-positive cases were analyzed at 500 kb resolution and compartment shifts among the two subtypes were annotated. A total of 390 shifts were detected, where activating shifts (from B to A compartment) happened more often in HeH (263 shifts) than in ETV6:: RUNX1-positive cases (127 shifts). Analysis of TAD boundary strength at 25 kb resolution revealed that HeH cases displayed significantly weaker boundaries compared to ETV6:: RUNX1-positive cases. TAD boundary strength showed no bias towards the frequently gained or non-gained chromosomes in HeH ALL. By merging individual heatmaps of all HeH and ETV6:: RUNX1-positive cases using Cooler, we created subtype-specific profiles and compared the intensity of chromatin interactions between the two genetic subtypes. Chromatin interaction intensity analysis was then combined with previously published RNA-sequencing data to identify transcriptional dysregulation events that could be associated with chromatin interaction changes. Preliminary results show that there was a chromatin loop missing close to the well-known leukemia-related gene IKZF1in HeH compared to ETV6:: RUNX1-positive cases; this gene also showed lower expression in the RNA-sequencing data. FLT3was associated with weakened chromatin interactions and down-regulated in ETV6:: RUNX1-positive cases compared to HeH, in agreement with its known high expression in HeH. Finally, we performed screening of SVs using EagleC and Micro-C heatmaps in HeH and ETV6:: RUNX1-positive samples. Out of the 19 included cases, previous whole-genome sequencing (WGS) data were available for 16. We detected 75 SVs, of which 50 were intrachromosomal rearrangements and 25 were translocations. Micro-C heatmaps allowed visual detection of SVs smaller than 1 Mb and permitted identification of the type of SVs. WGS detected 61% of the SVs found in the HeH samples with Micro-C and 51% of those in the ETV6:: RUNX1-positive cases. In summary, we present the first high-resolution genome-wide map of chromatin 3D organization in pediatric ALL. Our results indicate that different subtypes of childhood BCP ALL have distinct 3D chromatin landscapes and that abnormal chromatin architectures affect the regulation of leukemia-related genes.

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High hyperdiploid (HeH) B-cell precursor acute lymphoblastic leukemia (BCP ALL) is characterized by a very specific nonrandom gain of chromosomes; a feature distinct from other types of aneuploid tumor types that usually display very heterogeneous gains and losses of chromosomes. Gains of chromosomes X, 4, 6, 10, 14, 17, and 18 are seen in more than 75% of cases of HeH childhood ALL, and of chromosome 21 in 100% of cases. In contrast to many aneuploid malignancies, there has been little evidence of chromosomal instability (CIN) in HeH ALL and the mechanisms leading to these chromosomal gains remain unknown. The aim of this project was to determine the level of genomic heterogeneity in HeH ALL. In order to do this, we performed low-pass whole genome sequencing (WGS) of single cells isolated from diagnostic bone marrow samples from HeH ALL patients to investigate cell-to-cell heterogeneity. Single nuclei in G 1phase from nine diagnostic childhood HeH ALL samples were isolated using fluorescence-activated cell sorting and DNA libraries were constructed for low-pass WGS. Copy number analysis for each individual cell was performed in-house using the software programs AneuFinder and Ginkgo. Homolog inheritance of chromosomes gained or lost was determined by screening for heterozygous variants and calculation of the variant allele frequencies (VAF). Sequencing 2,572 single cells showed that the nine HeH ALL patients were all relatively homogenous at the trunk of their evolution tree and that the bulk of chromosomal gains were stable and unchanging. Structural aberrations, visible as partial chromosome copy number changes, were detected at diagnosis in most cases, with a greater number of structural aberrations detected in patients with greater numbers of sub-clones (defined as three or more cells presenting with the same numerical and structural aberrations). Unique numerical chromosomal aberrations detected in two or fewer cells were relatively random and therefore indicative of nondisjunction events in recent cell divisions rather than being part of the clonal evolution of the leukemia. These results indicate very low heterogeneity in HeH ALL and suggests that the genome of these leukemias is relatively stable.

Introduction.Pediatric B-cell precursor acute lymphoblastic leukemia (BCP ALL) is the most common pediatric hematological malignancy and it remains an important cause of morbidity and mortality in children. In this study, we performed an allele-specific expression (ASE) analysis of pediatric BCP ALL with the aim to investigate the relationship between cis-regulatory mutations and gene expression patterns.

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.

No relevant conflicts of interest to declare.

Whilst a great deal is known about acquired somatic aberrations associated with the diagnosis and prognosis of acute leukaemias, relatively little is known about the effects of germline variation. A recent study reported a link between germline variations in the zinc finger (ZnF) binding domain array of the PR domain-containing 9 (PRDM9) gene, regulator of meiotic recombination, with the development of acute lymphoblastic leukaemia (ALL) in children. An excess of rare PRDM9alleles that affect meiotic recombination events were seen in parents of children affected with B-cell precursor ALL (pre-B-ALL), and in an independent cohort of children with pre-B-ALL; in particular in aneuploid and infant ALL. In the present study, we carried out Sanger sequencing to investigate variation of PRDM9alleles in a cohort of parents (n=59) with children diagnosed with high hyperdiploid ALL (HeH; 51-67 chromosomes) (n=31) and a control cohort of individuals (n=66) from the south of Sweden. Whereas similar numbers of rare PRDM9alleles were observed in both groups, a larger number of rare alleles that affect recombination events were observed in the parents from the family cohort with children diagnosed with HeH ALL compared to individuals of the population control cohort (15.3% vs. 4.5%, P=0.0414). Two-thirds of the parents transmitted the rare allele to their affected children. A previously unreported ZnF repeat was also detected in two individuals of the control cohort but it was not detected in the family cohort. A statistically significant difference in frequency of rare alleles affecting recombination events between the two groups indicates a true association between PRDM9allelic forms and HeH ALL in an independent cohort. Thus, our results confirm the previous findings that PRDM9may play a role in the development of childhood ALL.

AML is currently classified using a combination of morphology and major cytogenetic abnormalities. There is considerable heterogeneity with respect to secondary cytogenetics, molecular mutations, response to therapy, remission rate, and overall survival. New molecular therapies are being developed constantly and are tested in vitro using patient samples in order to balance the drug-patient relationship. Use of the same drug produces patient specific responses, whereas different molecular drugs give drug specific responses. In order to rationalise and enhance the drug-patient relationship we have, using MTS assays, tested a range of molecular inhibitors against FLT3, PKC, HSP90, aminopeptidases and NFκB as well as modified nucleoside analogues on a panel of myeloid cell lines. These cell lines have also been profiled for gene expression. The combination of drug response and gene expression levels, were analysed by statistical algorithms including SAM, ANOVA and T-test to identify genes that differentiated between molecular responses or the overall therapeutic index (TI). Forty one genes distinguished between the molecular responses of FLT3, PKC, HSP90, and NFkB inhibitors. Around 100 genes were needed for the Ara-C response. The gene expression profiles of 187 diagnostic AML samples were also available with full clinical and molecular analysis. Some of these samples have been tested for in vitro responses to the molecular therapies, such as those targeted to FLT3 or HSP90. The TI gene list was used to analyse AML sub-groups including elderly patients (>60 yrs old); younger patients (<60 yrs old); cytogenetic risk groups, and those patients with FLT3 mutations (either ITD or TKD mutations) in an attempt to identify patients who may respond to specific molecularly targeted therapy. The Ara-C genes were used to predict whether a CR response was achieved. In patients less than 60 yrs old this correlation was not significant due to the great majority of patients achieving a complete remission. However, this was significant (p<0.031) in patients >60 yrs. Around 12% of patients were predicted to be responsive to HSP90 inhibitors whilst 18% might respond to FLT3 inhibitors. Several patients were in both groups, so 17% of patients were sensitive to a least one of the drugs. Whilst the potential added value of identifying patients sensitive to one of these drugs was only an additional 1–3%, this study shows the potential of using expression data to identify patients for specific molecular therapies and increasing the possibility of achieving a remission.

AML is currently classified using a combination of morphology and major cytogenetic abnormalities. There is considerable heterogeneity with respect to secondary cytogenetics, molecular mutations, response to therapy, remission rate, and overall survival. New molecular therapies are being developed constantly and are tested in vitrousing patient samples in order to balance the drug-patient relationship. Use of the same drug produces patient specific responses, whereas different molecular drugs give drug specific responses.

In AML, the majority of patients <60 years of age will enter remission but at least 50% will subsequently relapse, therefore the monitoring of minimal residual disease (MRD) during treatment has become an important issue. Current molecular markers for MRD are mainly limited to the RT-PCR detection of the fusion genes resulting from recurrent translocations which paradoxically are mostly limited to favourable risk groups who are least likely to relapse leaving the majority of the patients, including those with a normal karyotype, without a molecular marker suitable for monitoring. Two hundred and twenty patients have been assessed by gene expression profiling using Affymetrix U133A chips and the data analysed with the aim of identifying novel MRD markers for patients who do not currently have a suitable marker. As an initial “proof of principle”, we have identified possible MRD markers for patients with either t(15;17), t(8;21) or inv(16) and correlated with changes in expression of these markers with clinical changes as measured by established molecular MRD markers (PML-RARα or WT1). Of the expression profile from 22,283 probe sets in 29 cases of t(15;17), 20 genes were identified which had at least a two fold over expression which was unique to the t(15;17) subgroup. Of these several of the probe sets were related to the same gene, but from the reduced gene list 2 (HGF and ILGF binding protein) were selected for quantitation by quantitative PCR. Similarly the expression profile identified 20 genes which were unique to the 15 cases of t(8;21), and 20 genes which were unique to the 19 cases of inv(16). These included ETO and MYH11 representing the respective 3′ end of the respective fusion transcript. Three other genes (PRAME, POU4F1, and IL5RA) were selected for the t(8;21) cases and ST18, CLIP-170 and MNI for the inv(16) cases. When the relative quantitative expression of each of these “unique” genes was correlated with the expression of the established markers of minimal residual disease (PML-RARα or WTI) there was good correlation. These data suggest that gene expression profiling can identify ‘unique’ genes which can be used to develop specific markers for minimal residual disease monitoring for a larger proportion of cases of AML than is currently available.