RUNX1 Fusion Research Articles

The significance of CD49f expression in pediatric B-cell acute lymphoblastic leukemia.

CD49f is an adhesion molecule present on malignant lymphoblasts in B-cell acute lymphoblastic leukemia; it is associated with a poor prognosis. CD49f expression has been proposed as a marker for measurable residual disease (MRD) marker, but this marker has yet to be implemented in clinical practice. In this study, we used flow cytometry to detect CD49f expression by leukemic blasts in paired bone marrow and cerebrospinal fluid samples at diagnosis and bone marrow at day 15 of treatment. At diagnosis, 93% of bone marrow and 100% of cerebrospinal fluid lymphoblasts expressed CD49f. The intensity of CD49f expression statisticallysignificantly increased during treatment (P < .001). In MRD-negative end-of-treatment samples, only a small population of hematogones expressed CD49f. Interestingly, the intensity of CD49f expression varied among the different groups of recurrent genetic abnormalities. The ETV6::RUNX1 fusion and ETV6::RUNX1 combined with the high hyperdiploid group were associated with increased expression, whereas the Philadelphia-like group showed low CD49f expression. The lower CD49f expression at diagnosis predicted a lower MRD rate at day 15 of treatment. We concluded that CD49f can be used as an MRD marker and possible prognostic factor in B-cell acute lymphoblastic leukemia.

Preclinical efficacy of BRG1/BRM ATPase inhibitor in B lymphoblastic leukemia.

10030 Background: Acute lymphoblastic leukemia (ALL) is the most common blood cancer in children and young adults. While ALL has a high cure rate, approximately 20% of patients who initially respond to treatment relapse and prognosis post relapse remains poor. New therapeutic treatments are needed to reduce relapse rates in B-ALL pediatric patients and to minimize the toxic side effects seen with current therapies. We and others have previously shown that BRG1/BRM ATPase inhibitors have efficacy in targeting acute myeloid leukemia (AML) and one, FHD-286, is currently being evaluated for its use in AML in an ongoing clinical trial (NCT04891757). BRG1 and BRM are the ATP-dependent catalytic subunits of the SWI/SNF chromatin remodeling complex which has a critical role in controlling gene expression, cell growth and survival of AML cells. Our prior work revealed that BRG1/BRM ATPase inhibition leads to selective transient reduction of normal B cells. Further, data from Depmap also nominates SMARCA4, the gene encoding BRG1, as a dependency in B-ALL cell lines, leading us to hypothesize that BRG1/BRM inhibition may be an effective therapy for B-ALL. Methods: We treated B-ALL cell lines with varying genetic lesions ( KMT2A-rearranged: RS4;11, KOPN-8, SEM; ETV6::RUNX1 fusion: REH and DUX4-rearranged: Nalm6) with dose titrations of FHD-286 and measured cell growth over the course of six days. In addition, at 48- and 72-hours post-treatment, we assayed cells for viability, cell cycle kinetics, and apoptosis induction via flow cytometry to provide insight into FHD-286’s mechanism of action in B-ALL. Results: We found that FHD-286 treatment resulted in decreased cell growth in a dose-dependent manner across all B-ALL cell lines. While all cell lines were displayed reduced proliferation at nanomolar doses, sensitivity varied. Nalm6, REH and SEM cells were the most sensitive with low nanomolar IC50s (2-7nM). In contrast, two of the KMT2Ar B-ALL lines, RS4;11 and KOPN-8, required higher doses to achieve growth inhibition (40-70nM). To better understand the mechanism by which FHD-286 is affecting cell growth, we evaluate whether FHD-286 is targeting cell growth through the apoptotic pathway or through cell cycle arrest. Our preliminary data indicates cell growth inhibition is only slightly attributable to cell cycle arrest and apoptosis induction, leading us to explore terminal differentiation as a mechanism of slowed proliferation. Conclusions: Our results show FHD-286 can strongly inhibit B-ALL cell growth in vitro, indicating FHD-286 as a potential therapeutic agent to treat B-ALL. In vivo validation is ongoing. Further directions include defining the effect of FHD-286 on chromatin remodeling, gene expression, and exploring efficacy in combination therapy, which has been shown to increase FHD-286 efficacy against AML. Our data may ultimately justify inclusion of B-ALL patients in clinical studies of BRG1/BRM inhibitors.

RUNX1 regulates promoter activity in the absence of cognate DNA binding motifs.

Runt-related transcription factor 1 (RUNX1) plays an important role in normal haematopoietic cell development and function, and its function is frequently disrupted in leukaemia. RUNX1 is widely recognised as a sequence-specific DNA binding factor that recognises the motif 5'-TG(T/C)GGT-3' in promoter and enhancer regions of its target genes. Moreover, RUNX1 fusion proteins, such as RUNX1-ETO formed by the t(8;21) translocation, retain the ability to recognise and bind to this sequence to elicit atypical gene regulatory effects on bona fide RUNX1 targets. However, our analysis of publicly available RUNX1 chromatin immunoprecipitation sequencing (ChIP-Seq) data has provided evidence challenging this dogma, revealing that this motif-specific model of RUNX1 recruitment and function is incomplete. Our analyses revealed that the majority of RUNX1 genomic localisation occurs outside of promoters, that 20% of RUNX1 binding sites lack consensus RUNX motifs, and that binding in the absence of a cognate binding site is more common in promoter regions compared to distal sites. Reporter assays demonstrate that RUNX1 can drive promoter activity in the absence of a recognised DNA binding motif, in contrast to RUNX1-ETO. RUNX1-ETO supresses activity when it is recruited to promoters containing a sequence specific motif, while interestingly, it binds but does not repress promoters devoid of a RUNX1 recognition site. These data suggest that RUNX1 regulation of target genes occurs through multiple mechanisms depending on genomic location, the type of regulatory element and mode of recruitment.

Generation of a Zebrafish Knock-In Model Recapitulating Childhood ETV6::RUNX1-Positive B-Cell Precursor Acute Lymphoblastic Leukemia.

Approximately 25% of children with B-cell precursor acute lymphoblastic leukemia (pB-ALL) harbor the t(12;21)(p13;q22) translocation, leading to the ETV6::RUNX1 (E::R) fusion gene. This translocation occurs in utero, but the disease is much less common than the prevalence of the fusion in newborns, suggesting that secondary mutations are required for overt leukemia. The role of these secondary mutations remains unclear and may contribute to treatment resistance and disease recurrence. We developed a zebrafish model for E::R leukemia using CRISPR/Cas9 to introduce the human RUNX1 gene into zebrafish etv6 intron 5, resulting in E::R fusion gene expression controlled by the endogenous etv6 promoter. As seen by GFP fluorescence at a single-cell level, the model correctly expressed the fusion protein in the right places in zebrafish embryos. The E::R fusion expression induced an expansion of the progenitor cell pool and led to a low 2% frequency of leukemia. The introduction of targeted pax5 and cdkn2a/b gene mutations, mimicking secondary mutations, in the E::R line significantly increased the incidence in leukemia. Transcriptomics revealed that the E::R;pax5mut leukemias exclusively represented B-lineage disease. This novel E::R zebrafish model faithfully recapitulates human disease and offers a valuable tool for a more detailed analysis of disease biology in this subtype.

Acute myeloid leukemia with t(16;21)(q24;q22), CBFA2T3::RUNX1, in post-treatment setting, a case report

Abstract Introduction/Objective Acute myeloid leukemia (AML) with t(16;21)(q24;q22), CBFA2T3::RUNX1 is an uncommon entity that has been reported in both pediatric and adult patients as de novo AML or secondary to prior chemo/radiation therapy. CBFA2T3::RUNX1 AMLs share similar clinico-pathological features and gene expression profile with RUNX1::RUNX1T1 AML. Methods/Case Report We report a case of AML with CBFA2T3::RUNX1 in a 52 year old female who had history of small cell cervical cancer treated with chemotherapy (4 cycles of cisplatin and etoposide) and pelvic radiation therapy. About 17 months after completion of therapy, she presented with anemia and thrombocytopenia, normal WBC/neutrophil count, eosinophilia and increased blasts (up to 10%) in blood. Bone marrow biopsy shows AML with maturation (FAB-M2), erythropoiesis is present, whereas megakaryocytes are decreased. Flow cytometry analysis shows myeloblasts with aberrant CD19 and CD56 expression; in addition, increased CD117/tryptase positive cells are noted, mostly interstitial and more concentrated around bone trabeculae. By immunohistochemical stains, these cells are at least partially positive for CD25, negative CD2, CD30. Karyotype studies demonstrate t(16;21)(q24;q22) and trisomy 8 in all metaphases. FoundationOne heme NGS studies confirmed CBFA2T3::RUNX1 fusion, and no additional mutations were identified. Patient received induction chemotherapy with Idarubicin/Cytarabine plus GO (Gemtuzumab ozogamicin); day 14 bone marrow showed chemotherapeutic effect and she is now in morphologic remission 5 months after diagnosis. Results (if a Case Study enter NA) NA Conclusion Our case adds to the limited knowledge of CBFA2T3::RUNX1 AMLs. Our case shows morphological and immunophenotypic features similar to AML with RUNX1::RUNX1T1, with unusual presentation of peripheral eosinophilia, and increased CD117/tryptase positive cells in bone marrow, the latter has been reported occasionally in core binding factor AMLs. We emphasize the importance of comprehensive genetic studies in AML patients, lack of adverse genetic abnormalities in our patient indicates favorable risk category regardless of the post-treatment clinical setting.

A Longitudinal Single-Cell Atlas of Treatment Response in Pediatric AML

Introduction: Pediatric acute myeloid leukemia (pAML) is a heterogeneous disease in terms of driver alterations, treatment response and patient outcomes. Although approximately 90% of pAML patients respond to initial treatment, tumours relapse frequently. Despite large-scale genomic sequencing efforts, it remains elusive why tumors relapse. Previously, mutations have been identified that affect transcriptional regulation, indicating that epigenetic processes may contribute to treatment resistance in pAML. We therefore studied how tumor epigenetic patterns, transcriptional regulation and cell populations change over the course of treatment using single-cell ATAC sequencing (scATAC-seq) and single-cell RNA sequencing (scRNA-seq) in pediatric populations. Methods: We profiled 330,047 cells using scRNA-seq and 353,984 cells using scATAC-seq derived from 28 uniformly treated pAML patients, driven by either MLL fusions, CBFB fusions, RUNX1 fusions, FLT3-ITD or other aberrations (i.e. CEBPA mutations). All patients were enrolled in the AAML1031 trial (https://childrensoncologygroup.org/aaml1031), a randomized phase 3 clinical trial that investigated the addition of Bortezomib (BTZ) and Sorafenib to standard treatment modalities. Samples were obtained serially at diagnosis, at remission and at relapse and single-cell profiles were compared between these stages. Results: We identified malignant and non-malignant populations in the scATAC-seq and scRNA-seq data, showing that in all cases, malignant cells from patients at diagnosis were distinct from those taken at relapse. Major differences between diagnosis and relapse were associated with differentiation, and by inferring distinct cell populations we identified that relapsed cell populations appeared to shift towards a more primitive state. The extent of this shift was dependent on the genetic subtype and the effects were more pronounced in MLL-rearranged tumors. To identify the candidate mechanisms and transcription factors involved in this shift we studied the monocytic, lymphocytic and erythrocytic lineages in non-malignant cells and compared these to the tumor at diagnosis and relapse. Based on motif enrichment we found that across all different subgroups, transcription factors (TFs) that drive monocytic differentiation (i.e. CEBPA) have a lower activity while TFs driving erythrocytic and lymphocytic differentiation (i.e. TCF3) have a higher activity. Analysis of hematopoietic stem-like and progenitor-like cells (HSPC-like) confirmed this change, indicating that the “priming” towards other lineages is not only a result of shifts in cell populations but also a result of rewiring of early progenitors. To investigate this rewiring, we analysed regions of open chromatin that were enriched in malignant HSPCs compared to normal HSPCs across different subgroups. We found that those regions were highly specific to the driving alteration and were enriched for MEF2C binding sites in MLL-driven tumors and AP-1 binding sites in other tumors. Overall, upon relapse MEF2C expression and inferred TF activity appeared to increase, coinciding with an apparent increase in lymphoid priming and early lymphoid factors including LMO2, LYL1 and TCF3, among others. Two cases in the cohort appeared to switch lineage from a monocytic to lymphocytic phenotype and were enriched in CD19 and CD79A positive malignant cells upon relapse. Both cases shared the characteristic that MEF2C was upregulated upon relapse and showed upregulation of typical B-lymphoid transcription factors such as PAX5. Discussion: By generating a large dataset of pediatric AML patient samples collected at diagnosis, remission and relapse, we have identified changes in cellular hierarchies and transcriptional networks that occur upon relapse. Our study shows that shifts in cell populations readily occur upon treatment and are dependent on subgroup-defining alterations. Particularly MLL-rearranged tumors show a high level of plasticity that could explain the poor outcome of this group. Furthermore, we show that tumors appear to adapt their transcriptional programs towards a more “stem-like” state, with a likely higher propensity to differentiate towards other lineages such as the lymphoid lineage. We have identified MEF2C as a possible regulator of these processes, opening a new perspective to target the plasticity of these tumors.

RNA binding protein IGF2BP1 synergizes with ETV6-RUNX1 to drive oncogenic signaling in B-cell Acute Lymphoblastic Leukemia

BackgroundAcute lymphoblastic leukemia (ALL) is the most common pediatric hematological malignancy, with ETV6::RUNX1 being the most prevalent translocation whose exact pathogenesis remains unclear. IGF2BP1 (Insulin-like Growth Factor 2 Binding Protein 1) is an oncofetal RNA binding protein seen to be specifically overexpressed in ETV6::RUNX1 positive B-ALL. In this study, we have studied the mechanistic role of IGF2BP1 in leukemogenesis and its synergism with the ETV6::RUNX1 fusion protein.MethodsGene expression was analyzed from patient bone marrow RNA using Real Time RT-qPCR. Knockout cell lines were created using CRISPR-Cas9 based lentiviral vectors. RNA-Seq and RNA Immunoprecipitation sequencing (RIP-Seq) after IGF2BP1 pulldown were performed using the Illumina platform. Mouse experiments were done by retroviral overexpression of donor HSCs followed by lethal irradiation of recipients using a bone marrow transplant model.ResultsWe observed specific overexpression of IGF2BP1 in ETV6::RUNX1 positive patients in an Indian cohort of pediatric ALL (n=167) with a positive correlation with prednisolone resistance.IGF2BP1 expression was essential for tumor cell survival in multiple ETV6::RUNX1 positive B-ALL cell lines. Integrated analysis of transcriptome sequencing after IGF2BP1 knockout and RIP-Seq after IGF2BP1 pulldown in Reh cell line revealed that IGF2BP1 targets encompass multiple pro-oncogenic signalling pathways including TNFα/NFκB and PI3K-Akt pathways. These pathways were also dysregulated in primary ETV6::RUNX1 positive B-ALL patient samples from our center as well as in public B-ALL patient datasets. IGF2BP1 showed binding and stabilization of the ETV6::RUNX1 fusion transcript itself. This positive feedback loop led to constitutive dysregulation of several oncogenic pathways.Enforced co-expression of ETV6::RUNX1 and IGF2BP1 in mouse bone marrow resulted in marrow hypercellularity which was characterized by multi-lineage progenitor expansion and strong Ki67 positivity. This pre-leukemic phenotype confirmed their synergism in-vivo. Clonal expansion of cells overexpressing both ETV6::RUNX1 and IGF2BP1 was clearly observed. These mice also developed splenomegaly indicating extramedullary hematopoiesis.ConclusionOur data suggest a combined impact of the ETV6::RUNX1 fusion protein and RNA binding protein, IGF2BP1 in activating multiple oncogenic pathways in B-ALL which makes IGF2BP1 and these pathways as attractive therapeutic targets and biomarkers.

Abstract 24 Development of an In Vitro B-Cell Differentiation Model for the Characterization of the Origin and Causation of ETV6::RUNX1+ Preleukemic Cells

Abstract Introduction Pregnant women are exposed to various carcinogenic substances that can subsequently affect their progeny. Recurrent preleukemic chromosomal translocations characteristic for childhood acute lymphoblastic leukemia (ALL) frequently emerge before birth in utero. The most common translocation t (12;21) occurs in 25% of B-cell precursor ALL and results in the formation of the chimeric oncogenic transcription factor ETV6::RUNX1. Objectives We aim to analyze factors promoting the generation of preleukemic ETV6::RUNX1 translocations, investigate the biological origin of these preleukemic cells, and develop an in vitro model of early B-cell differentiation to analyze the mechanisms underlying the persistence of ETV6::RUNX1+ preleukemic cells and leukemia evolution in children. Methods Genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) (Schafer et. al., 2018) was applied to identify ETV6::RUNX1+ cord blood units (CBU). Screening results were validated by Sanger sequencing and quantified by real-time PCR (ipsogen ETV6-RUNX1 Kit, Qiagen). ETV6::RUNX1+ CBUs were thawed, sorted for CD34+ and CD19+ cells, and analyzed by qPCR. CD34+ were differentiated towards B-cell lineage on MSC feeder lines according to a modified protocol (Ichii et. al., 2008). Results We screened a cohort of 1405 cord bloods collected in 2004 and discovered that 103 (7.3%) carried the ETV6::RUNX1 fusion gene. The preleukemic translocation was detected in CD34+ stem and early B progenitor cells. For the establishment of an in vitro B-cell differentiation model, bone marrow (BM-MSCs) and CB (USSCs, CB-MSCs) stromal feeder cells were compared and the effect of different media (SCGM, DMEM/5 % FCS, MSCBM/10 % FCS) in the presence of stem cell factor (SCF), Flt-3, IL3, +/- IL7 was assessed. BM-MSCs demonstrated the highest ability to support B-lymphopoieses towards CD19+ cell differentiation in MSCBM media (~30%), primarily due to the release of IL-7 by the feeder layer. Currently, factors that may promote generation of preleukemic translocations in utero e.g. nitrosamines contained in food products, are being tested in this model. Discussion We identified the HSC/early B progenitor state as the origin of ETV6::RUNX1 translocations in CB. The establishment of an in vitro B-cell differentiation system will allow to test the impact of environmental carcinogens on the generation of ETV6::RUNX1+ pre-leukemic cells and their leukemic evolution.

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

78. Identification of a non-productive KMT2A rearrangement in B-ALL with apparent concurrent ETV6::RUNX1 and KMT2A fusions

78. Identification of a non-productive KMT2A rearrangement in B-ALL with apparent concurrent ETV6::RUNX1 and KMT2A fusions

Commonly Assessed Markers in Childhood BCP-ALL Diagnostic Panels and Their Association with Genetic Aberrations and Outcome Prediction.

Immunophenotypic characterization of leukemic cells with the use of flow cytometry (FC) is a fundamental tool in acute lymphoblastic leukemia (ALL) diagnostics. A variety of genetic aberrations underlie specific B-cell precursor ALL (BCP-ALL) subtypes and their identification is of great importance for risk group stratification. These aberrations include: ETV6::RUNX1 fusion gene, Philadelphia chromosome (BCR::ABL1 fusion gene), rearrangements of the KMT2A, TCF3::PBX1 fusion gene and changes in chromosome number (hyperdiploidy and hypodiploidy). Diagnostic panels for BCP-ALL usually include B-cell lineage specific antigens: CD19, CD10, CD20, maturation stage markers: CD34, CD10, CD38, TdT, IgM and other markers useful for possible genetic subtype indication. Some genetic features of leukemic cells (blasts) are associated with expression of certain antigens. This review comprehensively summarizes all known research data on genotype-immunophenotype correlations in BCP-ALL. In some cases, single molecules are predictive of particular genetic subtypes, i.e., NG2 with KMT2A gene rearrangements or CD123 with hyperdiploidy. However, much more information on possible genotype or prognosis can be obtained with wider (≥8-color) panels. In several studies, a quantitative antigen expression scale and advanced statistical analyses were used to further increase the specificity and sensitivity of genotype/immunophenotype correlation detection. Fast detection of possible genotype/immunophenotype correlations makes multicolor flow cytometry an essential tool for initial leukemia diagnostics and stratification.

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.

Abstract 759: Analytical performance of a novel next generation sequencing assay for myeloid MRD

Abstract The detection of molecular measurable residual disease (MRD) for acute myeloid leukemia (AML) has shown promise as a prognostic for AML. To support clinical and translational research into potential strategies for MRD monitoring applications in the future, a next-generation sequencing (NGS) assay, the Oncomine Myeloid MRD Assay (RUO), was developed. This assay utilizes Unique Molecular Tags (UMT) and Ion AmpliSeq HD™ technology to detect ultra-low frequency genetic alterations from blood or bone marrow samples. The workflow includes manual AmpliSeq HD library construction, templating on Ion Chef™, and sequencing on Ion GeneStudio™ sequencers. The assay contains both DNA and RNA targeted panels to detect SNVs, indels, fusion markers, and microhaplotype. The DNA panel covers 1256 hotspots in 33 genes, including key mutations in NPM1, FLT3, DNMT3A and full gene coverage for CEBPA and TP53. The panel achieved a limit of detection (LOD) of 0.1% when tested on 27 reference genomic DNA samples and over 40 clinical research samples. The RNA panel detects 1003 fusion isoforms for 27 fusion driver genes, including BCR-ABL1, RUNX1, CBFB-MYH11, and KTM2A (MLL) partial tandem duplications (PTDs) and fusions. The assay also includes a Microhaplotyping panel to monitor chimerism. The workflow is analytically verified and includes an optimized analysis pipeline for sensitive and accurate MRD detection. Overall, this comprehensive Myeloid MRD (RUO) assay reliably identifies DNA mutations and fusions with high sensitivity and provides a new tool for myeloid MRD research. RUO: For Research Use Only. Not for use in diagnostic procedures. Citation Format: Hua Zhang, Xinzhan Peng, Guobin Luo, Annie Kraltcheva, David Mandelman, Michelle Swimley, Eli Pazol, Jose Espinoza, Scott Mitchell, Bo Ding, Ratnesh Singh, Doug Fenger, Na Li, Andrew Hatch, Vadim Mozhayskiy, Steven Roman, Sophie Rozenzhak. Analytical performance of a novel next generation sequencing assay for myeloid MRD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 759.

Low Prevalence of ETV6::RUNX1 Fusion Gene in a Hispanic Population.

ETV6::RUNX1 is a genetic rearrangement of good prognosis in children with acute lymphoblastic leukemia (ALL). In Mexico, its prevalence is low in comparison with Caucasian populations. We developed a novel TaqMan one-step RT-qPCR approach to assess the prevalence of four genetic rearrangements in a cohort of Hispanic children with ALL from Mexico City. The prevalence of common fusion gene transcripts was as follows: TCF3::PBX1 7.7%; BCR::ABL1p190 3.3%; and KMT2A::AFF1 2.8%, and ETV6::RUNX1was observed with low prevalence (10.5%) in comparison to that reported for developed countries. This is consistent with previous findings on Mexican children with ALL and similar to those reported on children from Hispanic populations. The confirmation of a low prevalence of ETV6::RUNX1 in children of a Hispanic origin represents an advancement in the description of genetic factors of ALL in these populations.

1020 – TARGETED PROTEIN DEGRADATION DEFINES DIRECT TARGETS AND MECHANISMS OF CONTROL BY RUNX1/ETO

Loss-of-function germline mutations in the master transcription factor, RUNX1, cause familial platelet disorder with a predisposition to malignancy, while somatic mutations and chromosomal translocations targeting RUNX1 are associated with MDS and AML. Fundamentally, RUNX1 is a sequence-specific transcription factor that regulates the expression of select target genes critical for normal hematopoiesis. Thus, the ability to precisely define downstream targets of RUNX1 activity is critical to our understanding of how RUNX1 regulates hematopoietic cell fate decisions and how disruption of RUNX1 activity predisposes to cancer. However, defining direct transcriptional targets remains a challenge, because while gene expression networks are highly dynamic, our approaches to eliminate transcription factor function through genetic targeting are incredibly slow. These approaches allow the accumulation of secondary/compensatory changes in gene expression that make it difficult to distinguish direct from indirect transcriptional targets, creating a technical hurdle for target identification that must be overcome. Here, we have engineered leukemia cells to integrate an FKBP12-based degron tag into the endogenous RUNX1 or RUNX1-ETO (RE) fusion locus. This allowed the degradation of the RUNX1 or RE fusion proteins within two hours of treatment with the small molecule, dTAG. By combining rapid protein degradation with nascent transcript profiling, we defined direct RUNX1/RE target genes. Further characterization of changes in RUNX1 or RE bound enhancers following dTAG treatment allowed us to identify enhancers regulated by RUNX1 to elicit these rapid changes in gene expression. Integration of a 3xFLAG tag into the endogenous RUNX1 or RE locus allowed the identification of endogenous co-regulatory complexes recruited to RUNX1 and RE-regulated enhancers. Thus, the combination of rapid transcription factor degradation, nascent transcript profiling and mass spectrometry, is allowing us to define detailed mechanisms of transcriptional regulation at RUNX1-regulated enhancer elements. Loss-of-function germline mutations in the master transcription factor, RUNX1, cause familial platelet disorder with a predisposition to malignancy, while somatic mutations and chromosomal translocations targeting RUNX1 are associated with MDS and AML. Fundamentally, RUNX1 is a sequence-specific transcription factor that regulates the expression of select target genes critical for normal hematopoiesis. Thus, the ability to precisely define downstream targets of RUNX1 activity is critical to our understanding of how RUNX1 regulates hematopoietic cell fate decisions and how disruption of RUNX1 activity predisposes to cancer. However, defining direct transcriptional targets remains a challenge, because while gene expression networks are highly dynamic, our approaches to eliminate transcription factor function through genetic targeting are incredibly slow. These approaches allow the accumulation of secondary/compensatory changes in gene expression that make it difficult to distinguish direct from indirect transcriptional targets, creating a technical hurdle for target identification that must be overcome. Here, we have engineered leukemia cells to integrate an FKBP12-based degron tag into the endogenous RUNX1 or RUNX1-ETO (RE) fusion locus. This allowed the degradation of the RUNX1 or RE fusion proteins within two hours of treatment with the small molecule, dTAG. By combining rapid protein degradation with nascent transcript profiling, we defined direct RUNX1/RE target genes. Further characterization of changes in RUNX1 or RE bound enhancers following dTAG treatment allowed us to identify enhancers regulated by RUNX1 to elicit these rapid changes in gene expression. Integration of a 3xFLAG tag into the endogenous RUNX1 or RE locus allowed the identification of endogenous co-regulatory complexes recruited to RUNX1 and RE-regulated enhancers. Thus, the combination of rapid transcription factor degradation, nascent transcript profiling and mass spectrometry, is allowing us to define detailed mechanisms of transcriptional regulation at RUNX1-regulated enhancer elements.

Application of CD27 AND CD44 expression for prediction of ETV6::RUNX1 fusion gene presence in pediatric B-CELL precursor acute lymphoblastic leukemia

<h3>ЦЕЛЬ ИССЛЕДОВАНИЯ</h3> Оценить возможность прогнозирования наличия транслокации t(12;21)(p13;q22)/<i>ETV6::RUNX1</i> методом проточной цитометрии с использованием антигенов<i></i> CD27 и CD44 у детей с острым лимфобластным лейкозом из B-линейных предшественников (ВП-ОЛЛ). <h3>МЕТОДЫ ИССЛЕДОВАНИЯ</h3> В исследование вошло 437 случаев ВП-ОЛЛ у детей в возрасте от 1 до 18 лет. Процентное количество CD27 и CD44 позитивных опухолевых клеток, а также средняя интенсивность флуоресценции (MFI) были сопоставлены с результатами молекулярно-генетических методов исследования. Оптимизация порогового уровня для определения позитивной экспрессии антигенов CD27 и CD44 была проведена с помощью ROC-анализа. <h3>РЕЗУЛЬТАТЫ</h3> Было доказано, что транслокация t(12;21)(p13;q22)/<i>ETV6::RUNX1</i> ассоциирована с сочетанием позитивной экспрессии CD27 и сниженной, либо отсутствующей экспрессией CD44. Тем не менее, оптимальными пороговыми уровнями для определения позитивной экспрессии CD27 и CD44 в процентах оказались 80,7 и 86,1% соответственно. Использование отношения MFI CD27 к MFI CD44 показало наиболее высокий параметр диагностического отношения шансов — 953,1. <h3>ЗАКЛЮЧЕНИЕ</h3> Использование сочетания антигенов CD27 и CD44 позволило с высокой диагностической эффективностью предсказывать наличие t(12;21)(p13;q22)/<i>ETV6::RUNX1</i> при ВП-ОЛЛ у детей методом проточной цитометрии.

Detection of RUNX1-RUNX1T1 Fusion Gene in AML Patients by FISH Technique in Iraq

Background: AML with t(8;21)(q22;q22.1) is a balanced translocation that results in the fusion of RUNX1 and RUNX1T1. The translocation product is located on derivative chromosome 8. It has characteristic morphologic and immunophenotypic features and is linked with good prognosis. t(8;21)(q22;q22.1) AML is diagnostic of AML regardless of blast count. Objectives: To detect the RUNX1-RUNX1T1 fusion gene) in patients with AML by FISH technique and to investigate the relation between this chromosomal abnormality and the immunophenotypic markers CD117, cMPO, CD34, CD13, CD64 and CD33, clinical features and haematological parameters (Hb, WBC, blast percentage and platelets). Materials ad Methods: Fifty patients with de novo AML were selected sequentially from Baghdad teaching hospital in medical city from June 2020 till April 2021. History was taken and data were collected for each patient using a questionnaire form that included: name, age, sex, symptoms and physical signs. The data of haematological parameters and CD markers expression were collected from the patients’ diagnostic reports. Results:The RUNX1/RUNX1T1 fusion gene expression was positive in 4 patients representing 8% whereas 46 patients representing 92% of the samples had negative gene expression. Among the 4 positive AML patients for RUNX1/RUNX1T1 fusion gene expression, M2FAB subtype was revealed in all the cases. All the positive cases expressed CD117 and CD34 markers, while it was noted that 3 out of 4 positive cases were having cMPO and CD13 markers and one positive case expressed CD33 and CD64. Conclusion: It’s shown that RUNX1-RUNX1T1 fusion gene frequency in AML Iraqi patients is similar to international reports.

Distinct gene expression pattern of RUNX1 mutations coordinated by target repression and promoter hypermethylation in acute myeloid leukemia.

Runt-related transcription factor 1 (RUNX1) is an essential regulator of normal hematopoiesis. Its dysfunction, caused by either fusions or mutations, is frequently reported in acute myeloid leukemia (AML). However, RUNX1 mutations have been largely under-explored compared with RUNX1 fusions mainly due to their elusive genetic characteristics. Here, based on 1741 patients with AML, we report a unique expression pattern associated with RUNX1 mutations in AML. This expression pattern was coordinated by target repression and promoter hypermethylation. We first reanalyzed a joint AML cohort that consisted of three public cohorts and found that RUNX1 mutations were mainly distributed in the Runt domain and almost mutually exclusive with NPM1 mutations. Then, based on RNA-seq data from The Cancer Genome Atlas AML cohort, we developed a 300-gene signature that significantly distinguished the patients with RUNX1 mutations from those with other AML subtypes. Furthermore, we explored the mechanisms underlying this signature from the transcriptional and epigenetic levels. Using chromatin immunoprecipitation sequencing data, we found that RUNX1 target genes tended to be repressed in patients with RUNX1 mutations. Through the integration of DNA methylation array data, we illustrated that hypermethylation on the promoter regions of RUNX1-regulated genes also contributed to dysregulation in RUNX1-mutated AML. This study revealed the distinct gene expression pattern of RUNX1 mutations and the underlying mechanisms in AML development.

Integrated Genetic/Epigenetic Analysis Revealed High Heterogeneity of Acute Lymphoblastic Leukemia in Down Syndrome

BackgroundChildren with Down syndrome (DS), which caused by an extra copy of chromosome 21, have a 20-fold increased risk for acute lymphoblastic leukemia (ALL) compared with non-DS children. Recent genome-wide studies have revealed that DS-ALL have uncommon genetic alterations such as mutations in JAK2 , NRAS , and KRAS , mutations or overexpression of CRLF2 , suggesting the unique biological features of DS-ALL distinct from non-DS ALL. However, most of previous studies regarding molecular analysis of DS-ALL have been limited to genomic and transcriptomic characterizations, and no study showed comprehensive genetic/epigenetic overview in DS-ALL.Materials and MethodsOur cohort included 179 pediatric B-cell precursor ALL samples (61 DS-ALL and 118 non-DS-ALL). Biological subtypes are detailed in Table 1. We performed targeted deep sequencing for seven ALL-related genes in 61 DS-ALL, SNP array analysis in 43 DS-ALL, and whole transcriptome sequencing (WTS) in 25 DS-ALL and 118 non-DS-ALL samples. WTS data from 153 ALL in TARGET database were also used in this study as a validation cohort. Additionally, methylation array analysis was performed in 36 DS-ALL, 17 non-DS-ALL, and 20 DS patients without ALL. To validate gene mutations detected by WTS, Sanger sequencing was also conducted. To cluster gene expression profiling, we applied the hierarchical clustering method for expression profiling and consensus clustering for methylation profiling.ResultsTargeted deep sequencing revealed frequent gene mutations of JAK2 (23%) and RAS (31%) pathway. SNP array analysis elucidated several recurrent chromosomal deletions affecting actionable gene loci; IKZF1 (33%), PAX5 (30%), EBF1 (15%), CDKN2A (34%), and RB1 (7%). Gains of whole chromosome X (18%) and 8 (10%) were also recurrently identified. Expression profiling stratified 143 samples into 6 clusters (E1-E6 clusters), which were well correlated with biological subtypes of ALL (Figure 1). Intriguingly, DS-ALL cases were clustered into four clusters, suggesting the heterogeneity of DS-ALL. Cluster E3 included one DS-ALL, which was clustered to the branch of PAX5 -rearranged cases. SNP array analysis revealed this case had focal amplification of PAX5 . All DS-ALL cases with ETV6 - RUNX1 fusion were classified into cluster E4, which also included all ETV6 - RUNX1 fusion cases of non-DS-ALL. This cluster included one DS-ALL case without ETV6 - RUNX1 fusion. This particular case had a homozygous deletion of ETV6, implicating ETV6 - RUNX1 -like signature. DS-ALL with high hyperdiploidy (HeH) were classified into cluster E5, which also included most of HeH samples of non-DS-ALL. Among other 5 DS-ALL cases in this cluster, 2 cases showed IGH - CEBPA fusion and IGH - CEBPD fusion, respectively. Cluster E6 was characterized by BCR - ABL1 fusion and Ph-like expression profiles. In our cohort, we identified 7 DS-ALL cases as Ph-like signature, which included all JAK2 mutated samples. In this cluster, PAX5 mutation and IKZF1 mutation were detected in 2 DS-ALL cases, respectively. Notably, by using 250 genes in our hierarchical clustering, TARGET database samples were divided to similar 6 clusters (Figure 2). On the other hand, Methylation array analysis revealed unique hypermethylation of promoter regions of RUNX1 in DS-ALL. Methylation of this regions were confirmed in B-cell of DS without ALL, but not in non-DS-ALL. Furthermore, methylation array analysis categorized DS-ALL into 3 clusters (Figure 3). Cluster M1 included IGH - CEBPA fusion and IGH - CEBPD fusion cases. This cluster had worse prognosis than other 2 clusters.ConclusionOur integrated analysis enlightened the genetic and epigenetic heterogeneity of DS-ALL. Importantly, DS-ALL had genetic features corresponding to each biological subtypes same as non-DS-ALL. Our analyses also elucidated that DS-ALL had unique subtypes such as IGH - CEBPA fusion, IGH - CEBPD fusion, or PAX5 alterations. It may be a reason for worse prognoses of DS-ALL. Our result indicated risk classification or therapy of DS-ALL should be tailored by each biological subtype. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Whole Genome Sequencing in Combination with Transcriptome Sequencing Unravels the Pathogenetic Impact of Rare Balanced Rearrangements in Myeloid Malignancies and May Lead to Novel Personalized Therapeutic Strategies

Background: Gene fusions function as therapeutic targets, impressively shown for e.g. BCR - ABL1 and PML - RARA . Hence, the need for a comprehensive genetic analysis as a basis for personalized medicine is increasing allowing to select treatment based on genotype and to provide markers for disease monitoring. At ASH 2016 we presented targeted RNA sequencing data from 38 patients with hematological malignancies harboring rearrangements one partner gene of which had been identified by standard diagnostic work up. Using targeted RNA sequencing the second partner gene could be identified in 22/38 cases. All rearrangements involving KMT2A (n=8), JAK2 (n=1), and RARA (n=1) were resolved. However, fusion transcripts were identified in only 4/9 ETV6 rearrangements and 8/19 RUNX1 rearrangements. In the present study a subset of these cases was further evaluated.Aim: To evaluate the potential of whole genome sequencing (WGS) in combination with transcriptome (TS) sequencing to unravel the significance of rare translocations.Patient cohort and methods: Five cases with unresolved ETV6 rearrangement and 4 cases with unresolved RUNX1 rearrangement were further analyzed by WGS (~90x coverage) and TS (~61m reads). WGS was performed using the Truseq DNA PCR-Free HT sample preparation kit and RNA was prepared using TruSeq Stranded Total RNA HT (with Ribo-Zero HMR) kit for TS. Library was prepared according to manufacture's protocol using fresh/frozen samples and sequencing performed on NovaSeq instruments in multiplexed runs (Illumina, San Diego, CA). Tumor-only sample analysis was conducted with BaseSpace (BS) WGS App with default parameters for alignment and subsequent (structural) variant calling. For further downstream analysis we loaded the data into BS Variant Interpreter software to filter down and prioritize variants of interest. TS analysis was performed using BS RNAseq App with default parameters to assess gene fusions and expression (Illumina, San Diego, CA).Results: In all 9 cases WGS detected the translocation predicted by chromosome banding analysis and the genomic breakpoints (Bp) were determined. Details are depicted in the table 1. In cases 1 to 4 the genomic breakpoints were located in introns 1 or 2 of ETV6 or upstream of ETV6 . The corresponding breakpoints were upstream of CCND1, CCND2, SOX9 and PDGFRA . By TS no fusion transcripts involving ETV6 were identified. However, in line with the genomic data an overexpression of CCND1, CCND2, SOX9 and PDGFRA was observed in the respective cases by TS (figure 1). Data suggest that an enhancer in the region of ETV6 is driving the overexpression of the respective genes. In case 5 the genomic breakpoint was located in intron 2 and no ETV6 fusion transcript identified by TS. Most likely the translocation leads to a stop codon and thus to haploinsufficiency of ETV6 .In cases 6 and 7 a RUNX1 - GRXCR1 (RUNX1 exon 6 fused to GRXCR1 exon 2) and a RUNX1 - JARID2 (RUNX1 exon 2 fused to JARID2 exon 7) were detected by TS, respectively. The respective genomic breakpoints are depicted in the table 1. In cases 8 and 9 the breakpoints in RUNX1 were both located in intron 2. No RUNX1 fusion transcripts were detected by TS. Based on WGS and TS data the results of the RUNX1 translocations are most likely out of frame fusions and thus lead to loss of function of RUNX1 with an effect comparable to RUNX1 frameshift mutations.In summary the combination of TS and WGS revealed 3 different mechanisms of ETV6 - and RUNX1 -rearrangements: 1. Overexpression of the partner gene, 2. Novel fusion transcripts, and 3. Loss of function of ETV6 or RUNX1 .From the clinical point of view in case of PDGFRA overexpression therapy with a tyrosine kinase can be considered. The overexpression of CCND1 and CCND2 suggests that the mTOR inhibitor rapamycin might be a therapeutic option in these cases.Conclusions and Perspectives: Whole genome sequencing in combination with transcriptome sequencing is a promising approach to unravel the pathogenetic role of rare rearrangements. This provides new options for therapeutic interventions and monitoring of minimal residual disease on a personalized level. Studies to evaluate the potential role of WGS and TS in a future diagnostic setting have been started to improve patients outcome based on a comprehensive genetic work-up allowing optimal treatment choice and monitoring of minimal residual disease. [Display omitted] DisclosuresHaferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.