Acute myeloid leukemia (AML) is characterized by recurrent chromosomal abnormalities that form the basis of the European LeukemiaNet (ELN) risk classification and serve as essential determinants of prognosis and therapeutic decision-making. Conventional metaphase karyotyping remains the diagnostic gold standard for detecting these abnormalities; however, its utility is limited by longer turnaround times, often delaying critical clinical management. Here, we present a long-read sequencing-based (LRS) low-coverage whole genome sequencing (lcWGS) approach using Oxford Nanopore Technology as a rapid and scalable alternative for cytogenetic profiling. A total of 100 diagnostic AML samples were analyzed, comprising 50 retrospectively selected cases with known adverse-risk cytogenetics and 50 prospectively enrolled patients with clinically defined de novo AML. LcWGS demonstrated robust analytical performance, identifying chromosomal aberrations with 93% sensitivity, specificity, and overall accuracy, respectively. Complex karyotypes were reliably detected, with an area under the curve (AUC) of 0.971. Reproducibility was validated through replicate sequencing at two independent laboratories (R=0.99). LcWGS-derived estimates of clone size showed moderate correlation with conventional cytogenetic assessments (R=0.54). Patients with complex karyotypes identified by lcWGS exhibited significantly shorter overall and relapse-free survival, closely mirroring outcomes defined by conventional karyotyping and underscoring the value of lcWGS for risk stratification. Median turnaround time from sample receipt to bioinformatics interpretation was approximately 34 hours, enabling delivery of actionable karyotype results within 72 hours. These findings establish lcWGS as a rapid, reproducible, and accurate platform for detecting clinically relevant chromosomal abnormalities, addressing a critical need for timely risk stratification and treatment initiation in AML.

Acute myeloid leukemia (AML) with complex karyotype is characterized by high genomic complexity, including frequent TP53 mutations and chromothripsis. Genomic rearrangements can reposition active enhancers near proto-oncogenes, leading to their aberrant expression; however, a comprehensive understanding of these events in AML is still incomplete. To facilitate the discovery of such "enhancer hijacking" events, we developed Pyjacker, a computational tool, and applied it to 39 AML samples with complex karyotype. Pyjacker identified several enhancer hijacking events in AML patient samples, including aberrant expression of MNX1, which can result from del(7)(q22q36) and is associated with hijacking of a CDK6 enhancer. MNX1 activation occurred in 1.4% of patients with AML and showed significant co-occurrence with BCOR mutations. Through a xenograft mouse model, we demonstrated that MNX1 is required for leukemia cell fitness. Pyjacker is an easy-to-use, accurate, and broadly applicable tool for identifying consequences of genomic events driving tumorigenesis, especially when germline genomic data are missing. SIGNIFICANCE:This study examines the consequences of structural alterations in AML and demonstrates that proto-oncogene activation by enhancer hijacking is an understudied pathomechanism. MNX1 overexpression demonstrates that deletions on chromosome 7q can not only lead to haploinsufficiency but also to activation of oncogenes by enhancer hijacking.

Abstract Background Deletions and partial losses of chromosome 7 (chr7) are frequent in acute myeloid leukemia (AML) and are linked to dismal outcome. However, the genomic landscape and prognostic impact of concomitant genetic aberrations remain incompletely understood. Methods To discover genetic lesions in adult AML patients with aberrations of chromosome 7 [abn(7)], 60 paired diagnostic/remission samples were investigated by whole-exome sequencing in the exploration cohort. Subsequently, a gene panel including 66 genes and a SNP backbone for copy-number variation detection was designed and applied to the remaining samples of the validation cohort. In total, 519 patients were investigated, of which 415 received intensive induction treatment, typically containing a combination of cytarabine and anthracyclines. Results In the exploration cohort, the most frequently mutated gene was TP53 (33%), followed by epigenetic regulators (DNMT3A, KMT2C, IDH2) and signaling genes (NRAS, PTPN11). Thirty percent of 519 patients harbored ≥ 1 mutation in genes located in commonly deleted regions of chr7—most frequently affecting KMT2C (16%) and EZH2 (10%). KMT2C mutations were often subclonal and enriched in patients with del(7q), de novo or core-binding factor AML (45%). Cancer cell fraction analysis and reconstruction of mutation acquisition identified TP53 mutations as mainly disease-initiating events, while del(7q) or −7 appeared as subclonal events in one-third of cases. Multivariable analysis identified five genetic lesions with significant prognostic impact in intensively treated AML patients with abn(7). Mutations in TP53 and PTPN11 (11%) showed the strongest association with worse overall survival (OS, TP53: hazard ratio [HR], 2.53 [95% CI 1.66–3.86]; P < 0.001; PTPN11: HR, 2.24 [95% CI 1.56–3.22]; P < 0.001) and relapse-free survival (RFS, TP53: HR, 2.3 [95% CI 1.25–4.26]; P = 0.008; PTPN11: HR, 2.32 [95% CI 1.33–4.04]; P = 0.003). By contrast, IDH2-mutated patients (9%) displayed prolonged OS (HR, 0.51 [95% CI 0.30–0.88]; P = 0.0015) and durable responses (RFS: HR, 0.5 [95% CI 0.26–0.96]; P = 0.036). Conclusion This work unraveled formerly underestimated genetic lesions and provides a comprehensive overview of the spectrum of recurrent gene mutations and their clinical relevance in AML with abn(7). KMT2C mutations are among the most frequent gene mutations in this heterogeneous AML subgroup and warrant further functional investigation.

Acute myeloid leukemia (AML) is a hematological malignancy characterized by a block in differentiation and accelerated proliferation of myeloid progenitor cells. Genes encoding for epigenetic regulators are among the most frequent targets for mutations and structural variations in AML, giving rise to profound epigenetic heterogeneity between and within tumors. Deletions of chromosome 5q [del(5q)] are among the most common copy number alterations in AML and are associated with extremely poor clinical outcome and therapy resistance, however the mechanisms linking del(5q) to leukemic progression are not understood. Analyzing DNA methylation profiles from 477 elderly AML patients using DNA methylome deconvolution, we discovered that del(5q) AML is an epigenetically distinct subgroup characterized by a signature of DNA hypermethylation, which we propose may be linked to dysregulation of H3K9me1/2 and overexpression of the leukemic stem cell marker, DNMT3B. Interrogation of the minimally deleted 5q region highlighted the H3K9me1/2 demethylase KDM3B as a likely target for haploinsufficiency in this subgroup. Our data suggest that del(5q) AML should be reconsidered as an epigenetically dysregulated subgroup, driven by heterozygous loss of KDM3B, and that the resulting imbalance of H3K9me1/2 may contribute to the progression of these aggressive leukemias. ### Competing Interest Statement The authors have declared no competing interest.

Acute myeloid leukemia with complex karyotype (ckAML) is characterized by high genomic complexity, including frequent TP53 mutations and chromothripsis. We hypothesized that the numerous genomic rearrangements could reposition active enhancers near proto-oncogenes, leading to their aberrant expression. We developed pyjacker, a computational tool for the detection of enhancer hijacking events, and applied it to a cohort of 39 ckAML samples. Pyjacker identified motor neuron and pancreas homeobox 1 ( MNX1 ), a gene aberrantly expressed in 1.4% of AML patients, often as a result of del([7][1])(q22q36) associated with hijacking of a CDK6 enhancer. MNX1 -activated cases show significant co-occurrence with BCOR mutations and a gene signature shared with t(7;12)(q36;p13) pediatric AML. We demonstrated that MNX1 is a dependency gene, as its knockdown in a xenograft model reduces leukemia cell fitness. In conclusion, enhancer hijacking is a frequent mechanism for oncogene activation in AML. Statement of significance This study examines the consequences of structural alterations and demonstrates that proto-oncogene activation by enhancer hijacking is an overlooked pathomechanism in AML. MNX1 overexpression demonstrates that deletions on chromosome 7q can not only lead to haploinsufficiency, but also to activation of oncogenes by enhancer hijacking, providing a novel leukemogenic mechanism. ### Competing Interest Statement UHT is currently employed at Oxford Nanopore Technologies. EJ is currently employed at AstraZeneca. LB has received honoraria from AbbVie, Amgen, Astellas, BristolMyers Squibb, Celgene, Daiichi Sankyo, Gilead, Hexal, Janssen, Jazz Pharmaceuticals, Menarini, Novartis, Pfizer, Roche, and Sanofi, as well as research support from Bayer and Jazz Pharmaceuticals. DBL received honoraria from Infectopharm GmbH. All other authors declared no conflict of interest. [1]: #ref-7

Introduction: Aberrations of chromosome 7 [abn(7)] are found in ≈ 10% of newly diagnosed acute myeloid leukemia (AML) and associate with a dismal prognosis. A large-scale comprehensive exploration of the underlying genetic heterogeneity in AML with abn(7) has yet to be performed and could add essential insights into the outcome of this poorly understood patient group. Methods: We collected diagnostic samples from 523 adult AML patients (median age 59 years) with abn(7). Whole-exome sequencing (WES) was performed to discover potentially underestimated genetic lesions in 61 paired diagnostic / remission samples. Subsequently, a gene panel including 66 genes and a SNP backbone for copy-number aberration (CNA) detection was designed and applied to the remaining 471 samples. The majority of patients (78%) were diagnosed with de novo (dn) AML, whereas 22% had secondary (s) or therapy-related (t) AML. Intensive induction treatment was administered to 80% of the patients, while 36 % underwent allogenic stem cell transplantation. Apart from 43% of patients with concomitant complex karyotype (abn(7)/CK+), 24% had -7 as a sole abnormality (-7 sole) and 13% del(7q) sole. Results: A mean of 15.3 single-nucleotide variants (SNVs) and 7.5 CNAs per patient were found by WES. Here, the most frequent recurrent SNVs were identified in TP53 (29.5%), followed by mutations (mut) in genes involved in epigenetic regulation ( DNMT3A, TET2, ASXL1, IDH2), transcription factors ( RUNX1), and genes affecting RAS-signaling ( NF1, KRAS), Figure 1. Targeted sequencing revealed 1829 SNVs with a VAF≥ 2% in 64 genes (mean: 3.8 SNVs / patient). 30% of patients harbored at least one mutation in genes located within the commonly deleted region of 7q, most frequently KMT2C, EZH2 CUX1, SAMD9L, SAMD9, LUC7L2 and BRAF. The number of driver gene mutations was higher in CK- than in CK+ patients (4.5 vs. 3.3 SNVs). We found KRAS (OR 3.76, CI 1.17-16.87, P= .044) and RUNX1 (OR 3.61, CI 1.62-8.78, P= .003) mutations to be enriched in -7 sole patients, and FLT3 mutations to be associated with del(7q) sole status (OR 0.33, CI 0.13-0.82, P= .019). With respect to previously unknown lesions, a high amount of KMT2C mutations (16.6%) and recurrent alterations in FAT1 and TACC2 were discovered (6.4% each; Fig. 1). For KMT2C - located on chr7q36.1 - a total of 98 SNVs with mainly missense (73.5%) and truncating (20.4%) mutations at known cancer hotspots were noted. KMT2Cmut was associated with dnAML and AML with maturation morphology. In the entire cohort, the most common co-occurring chromosomal alterations discovered by high-resolution CNA analysis were deletions in chromosomes 5, 17, and 12. Our approach enabled the identification of small fragment deletions (≤10Mb) affecting the TP53, NF1, and ETV6 loci in 5-9% of all cases, which were missed by conventional G-banding. Cancer Cell Fraction and Bradley-Terry models were used to simulate the sequential order of genomic aberrations. While mutations in TP53 and epigenetic-related genes were early events, -7 and del(7q) were often subclonal and SNVs in NRAS, KMT2C very late events in leukemogenesis. Survival analyses in intensively-treated patients (n= 414) revealed that 61% reached complete remission, 67% relapsed, and median overall survival (OS) was 11.9 months. Abn TP53 and high WBC count were independently associated with shorter relapse-free survival (RFS). For OS, besides older age and high WBC count, we identified abn TP53, PTPN11mut, TET2mut,-5, and -18 as poor prognostic factors in multivariate analysis (Table 1). In contrast, IDH2mutconferred an independent favorable prognostic effect for RFS and OS. Notably, abn TP53 outcompeted the prognostic impact of CK+ (Table 1). Compared to TP53wt, patients with abn TP53/CK+ and abn TP53/CK- had a similar poor outcome with median RFS of 6 and 4 months (CK+/CK-, P<.001) and OS of 6.8 and 8.6 months (CK+/CK-, P<.001). In contrast to other genomic studies in myelodysplastic syndrome, we found abn TP53 to be associated with poor outcome irrespective of the single- or multihit mutation status following definitions of the latest ICC classification (Blood, 2022). Conclusion: Our results offer novel insights into the genomic landscape and clonal trajectory of AML with abn(7). This work unravels formerly underestimated genetic lesions ( KMT2Cmut) and alterations with high prognostic impact (abn TP53 and IDH2mut) for better future risk stratification.

Background: The genomic landscape of acute myeloid leukemia (AML) has been previously mostly studied in younger patients who received intensive chemotherapy. Data in older patients receiving less intensive therapies are scarce. In addition, no genetic risk classification for older AML patients has so far been established. Aims: To characterize the genomic landscape and leukemogenic pathways of AML in older patients, and to study the clinical implications of these biological features. Methods: Targeted sequencing of 263 genes was performed in 604 patients enrolled in the randomized, multi-center phase 3 ‘ASTRAL-1’ trial (NCT02348489) evaluating the second-generation hypomethylating agent guadecitabine (SGI-110) in treatment-naïve AML pts not eligible for intensive chemotherapy in comparison to a treatment choice of decitabine, azacitidine, or low-dose cytarabine. Results: Recurrent mutations were found in ASXL1 (28%), TET2 (27%), SRSF2 (22%), DNMT3A (21%), RUNX1 (20%), TP53 (19%), NPM1 (15%) and FLT3 (13%; [internal tandem duplication (ITD) 8%, tyrosine kinase domain 6%]). DDX41mut were found in 5.5% of the patients, 61% of whom with suspected germline variant. Adverse cytogenetic features such as complex karyotype (29%), -7/del(7q) (22%), 5q/del(5q) (20%), -17/del(17p)/abn(17p) (12%) were common. Based on the 2022 ELN classification, patients were stratified into the adverse (73%), intermediate (14%) or favorable (13%) risk group. Based on the International Consensus Classification (ICC), most patients were classified as AML with myelodysplasia-related gene mutations (45%), AML with mutated TP53 (17%), AML with mutated NPM1 (16%), AML not otherwise specified (10%) and AML with myelodysplasia-related cytogenetic abnormalities (5%). A modelling algorithm yielded a stable oncogenetic tree (Figure 1A) identifying distinct leukemogenic trajectories with ASXL1, DDX41, DNMT3A, TET2 and TP53 mutations as leukemia-initiating events. ASXL1, DNMT3A, and TET2 gave rise to further clones, whereas DDX41 and TP53 terminated at the node, suggesting independence from further events that drive leukemogenesis. Supporting the ICC classification, the ASXL1 subtree contained 8 of 9 genes that define the new category of AML with myelodysplasia-related gene mutations. When assessing clinical impact, both the 2017 and 2022 ELN risk classifications failed to identify clinically meaningful prognostic groups. In Cox regression models, older age (HR 1.02 [1.01-1.04], p=.009), male sex (HR 1.32 [1.09-1.6], p=.004), ECOG score ≥2 (HR 1.55 [1.28-1.88], p<.001), higher white blood cell counts (HR 1.63 [1.34-1.97], p<.001), FLT3-ITD (HR 1.7 [1.2-2.4], p=.003), SRSF2 (HR 1.36 [1.06-1.76], p=.017), and TP53 mutations (HR 1.59 [1.24-2.05], p<.001) had an adverse impact on overall survival, whereas DDX41 mutations (HR 0.41 [0.24-0.69], p<.001) were exceptionally beneficial. To simplify the model, a subsequent backward elimination based on the Akaike information criterion led to delineation of 3 genetically defined risk groups (favorable: DDX41mut, adverse: TP53mut or FLT3-ITDpos, intermediate: all other) with predicted survival curves (Figure 1B). Summary/Conclusion: Using different modelling algorithms, our comprehensive analysis of the so far largest study in older, treatment naïve AML patients identified distinct trajectories of leukemia development, provided support for AML with mutated DDX41 as a new clinico-pathologic entity and a basis for the development of a risk stratification that may be applicable for the numerous older patients receiving less intensive therapies.Keywords: Tumorigenesis, Age, AML, Prognostic groups

To characterize the genomic landscape and leukemogenic pathways of older, newly diagnosed, non-intensively treated patients with AML and to study the clinical implications, comprehensive genetics analyses were performed including targeted DNA sequencing of 263 genes in 604 patients treated in a prospective Phase III clinical trial. Leukemic trajectories were delineated using oncogenetic tree modeling and hierarchical clustering, and prognostic groups were derived from multivariable Cox regression models. Clonal hematopoiesis-related genes (ASXL1, TET2, SRSF2, DNMT3A) were most frequently mutated. The oncogenetic modeling algorithm produced a tree with five branches with ASXL1, DDX41, DNMT3A, TET2, and TP53 emanating from the root suggesting leukemia-initiating events which gave rise to further subbranches with distinct subclones. Unsupervised clustering mirrored the genetic groups identified by the tree model. Multivariable analysis identified FLT3 internal tandem duplications (ITD), SRSF2, and TP53 mutations as poor prognostic factors, while DDX41 mutations exerted an exceptionally favorable effect. Subsequent backwards elimination based on the Akaike information criterion delineated three genetic risk groups: DDX41 mutations (favorable-risk), DDX41wildtype/FLT3-ITDneg/TP53wildtype (intermediate-risk), and FLT3-ITD or TP53 mutations (high-risk). Our data identified distinct trajectories of leukemia development in older AML patients and provide a basis for a clinically meaningful genetic outcome stratification for patients receiving less intensive therapies.

Background: Internal tandem duplications of the FLT3 gene (FLT3-ITD) occurring in 10-25% of adult acute myeloid leukemia (AML) patients (pts) represent an attractive target for monitoring of measurable residual disease (MRD), particularly in pts treated with a FLT3 inhibitor. So far, MRD monitoring in FLT3-ITD mutated (FLT3-ITD+) AML was limited by the heterogeneity of ITD length and insertion site and the number of ITD clones. We recently developed a next-generation sequencing (NGS)-based MRD assay to monitor FLT3-ITD at very high sensitivity (Blätte et al, Leukemia 2019). Aims: To evaluate the prognostic impact of the MRD status in pts 18-70 years with FLT3-ITD+ AML enrolled on the AMLSG16-10 trial (NCT01477606) combining intensive chemotherapy with midostaurin (mido) followed by mido maintenance. Methods: Using FLT3-ITD paired-end NGS (Illumina MiSeq) with a variant allele frequency (VAF) sensitivity of 10-4-10-5, 497 bone marrow (BM) and 66 blood samples from 157 FLT3-ITD+ AML were analyzed at diagnosis (Dx, n=157), after two cycles of chemotherapy (Cy2, n=142), at end of treatment (EOT, n=116), and during 3-12 months follow-up (FU, n=148). All pts achieved complete remission (CR) at Cy2. Allogeneic hematopoietic-cell transplantation in first CR (HCT_CR1) was performed in 121 (77%) pts. NPM1 and DNMT3A mutation status were available for all pts (NPM1mut, n=111; DNMT3Amut, n=74). Results: 465 ITDs were identified at Dx. Median length was 51 nucleotides (range, 9-284), median VAF 0.312% (0.006-92.26), median total VAF per pt 31.54% (0.46-92.26), and 69% of pts exhibited >1 ITD (median 2; range, 1-16). At Cy2 and EOT pts’ total ITD VAF significantly decreased (median log10 reduction: 4.6 and 4.7; P<.001, each), and MRD negativity (MRD-) was achieved in 78% and 94%, respectively. Concurrent NPM1mut favorably impacted on log10 VAF reduction (median, 4.7 vs 3.7; P<.001) and MRD- (88% vs 53%; P<.001) at Cy2. Median follow-up was 3.9 years. Achievement of MRD- at Cy2 predicted for superior 4-year rates of cumulative incidence of relapse (CIR) (4y-CIR, 29% vs 51%; P<.001) and overall survival (OS) (4y-OS, 70% vs 42%; P=.004) (Figure 1A and B), also for pts undergoing HCT_CR1 (4y-CIR, 16% vs 40%; P<.001; 4y-OS, 74% vs 48%; P=.014). Multivariate models for CIR and OS, including age, white blood cell count, BM blasts, NPM1mut, DNMT3Amut, FLT3-ITD allelic ratio, FLT3-ITD MRD+ at Cy2 and HCT_CR1 as time-dependent variable, revealed FLT3-ITD MRD- as the only consistent favorable variable for CIR (HR, 0.22; P<.001) and OS (HR, 0.37; P=.005); NPM1mut (HR, 0.17; P<.001) and HCT_CR1 (HR, 0.08; P<.001) were favorable for CIR. No significant benefit was observed at EOT mainly due to the high rate of MRD-. During FU, 16 pts converted from MRD- to MRD+ (MRDconv) and 13/16 pts relapsed within a median time of 7 days (range, 0-197 days), translating into a significantly increased relapse risk (2y-CIR, 81% vs 14%; P<.001) and inferior OS (2y-OS, 31% vs 83%; P<.001) (Figure 1C and D). In Cox regression MRDconv as time-dependent variable was of adverse impact for CIR (HR, 10.99; P<.001) and OS (HR, 4.27; P<.001). Summary/Conclusion: NGS-based FLT3-ITD MRD monitoring allows for the identification of pts at high risk of relapse and death. Combining mido with intensive chemotherapy MRD- at Cy2 and EOT was achieved in a high proportion of pts; at Cy2 MRD- was the strongest independent favorable prognostic factor for relapse risk and OS. Concurrent NPM1mut correlated with deeper molecular responses and higher rates of MRD-..During FU, MRDcon was the strongest unfavorable factor for both CIR and OS.Keywords: Flt3-ITD, Acute myeloid leukemia, MRD

Background: Epigenetic enzymes, including histone modifiers and regulators of DNA methylation, are among the most frequent targets for mutations and structural variations in acute myeloid leukemia (AML). However, studies of complex karyotype AML, an aggressive subgroup defined by ≥3 unrelated chromosome abnormalities in the absence of other class-defining genetic abnormalities, have focused largely on its genetic rather than epigenetic characteristics. Within this subgroup, the most common abnormality is a deletion in the long arm of chromosome 5; an early event in leukemogenesis, which is invariably associated with poor clinical outcome and therapy resistance. While several candidate 5q tumor suppressors have been previously investigated, the pathogenic mechanisms underlying this deletion have not yet been elucidated. Aims: Here we aimed to assess the utility of DNA methylation profiles to improve the molecular classification of AML, with a focus on patients with complex karyotype. Methods: A reference-free methylome deconvolution method (MeDeCom) was applied to a cohort of 480 older AML patients which had been profiled by Infinium MethylationEPIC array. This approach allows decomposition of bulk methylomes to reveal a set of latent methylation components (LMCs) that might otherwise be obscured within epigenetically heterogeneous tumors. To decipher their likely origin, each methylation component was then interpreted by comparison to known cell-type-specific methylomes, and association with mutational and cytogenetic features. Results: Deconvolution of the AML methylome identified a distinct hypermethylation signature enriched in del5q AML. This encouraged us to further investigate candidate 5q tumor suppressor genes from an epigenetic perspective. Based on the overlap of deleted 5q segments in own and public datasets, we defined a minimally deleted region within 5q31.2, which peaks at a locus encoding the H3K9me1/2 demethylase, KDM3B. Differential gene expression analysis comparing del5q to 5q-retaining AML identified KDM3B as the most significantly downregulated of all genes within the minimally deleted interval. We thus hypothesized that KDM3B depletion could result in an increase in H3K9me1/2 and consequently trigger de novo DNA methylation at its targets, giving rise to the observed hypermethylation signature. Supporting this model, the del5q methylation signature correlated not only with reduced KDM3B gene expression but also with increased expression of its opposing H3K9me1/2 methyltransferases, EHMT2 (G9a) and PRDM16, and their downstream effector of de novo DNA methylation, DNMT3B. Furthermore, we could support our hypothesis by comparison to AML subgroups whose epigenetic alterations have already been described: Firstly, we note that the del5q methylome shows similarity to IDH mutant AML; a subgroup in which KDM3B activity is inhibited due to its metabolic dependency on alpha-ketoglutarate. Secondly, we found a similar methylation signature in patients overexpressing MECOM (EVI-1), an oncogene which is known to drive DNA hypermethylation through cooperation with DNMT3B. Summary/Conclusion: Our findings suggest that haploinsufficiency of KDM3B might represent an underlying epigenetic event in deletion 5q AML. We hypothesize that the resulting imbalance in H3K9me1/2 could confer epigenetic plasticity that might favor leukemic progression.Keywords: Complex aberrant karyotype, DNA methylation, AML

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Deletions in the long arm of chromosome 7 (del7q) are recurrent events in acute myeloid leukemia (AML) which are associated with an unfavorable outcome. Since the search for recessive tumor suppressor genes located within the deleted region was unsuccessful, the prevalent idea currently is that haploinsufficiency of one or more tumor suppressor genes drive this leukemia. Other studies showed that structural rearrangements such as translocations and inversions can lead to the activation of oncogenes through relocation of cis-regulatory elements. A similar mechanism would also be plausible for chromosomal deletions, specifically del(7q) in AML. Aims: We investigated the hypothesis that del(7q) could activate a proto-oncogene located outside the deleted region due to restructuring of topologically associating domains and relocation of cis-regulatory elements. Methods: Whole genome sequencing (WGS), RNA sequencing (RNAseq) and Infinium MethylationEPIC array were used to study 13 patients with isolated del(7q) or monosomy 7. Epigenomic profiling using antibody-guided chromatin tagmentation with sequencing (ACT-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq) and circular chromosome conformation capture (4C) was done on primary AML samples with MNX1 activation. CRISPR/Cas9 genome editing was used to generate model systems for the putative enhancer hijacking event. Results: We identified a subgroup of del(7q)-AML that aberrantly expresses Motor Neuron and Pancreas Homeobox (MNX1), which is a key developmental homeobox gene located on chromosome 7q36.3 coding for the transcription factor MNX1. MNX1 expression is usually tightly regulated and restricted to the brain, gastrointestinal tract and pancreas. The majority of MNX1-expressing cases had breakpoints within CDK6 and upstream of MNX1, thus, the deletions juxtapose MNX1 with a region containing a strong hematopoietic enhancer. To verify the presence of an enhancer near the breakpoint, we used epigenomic techniques to map accessible chromatin and histone modifications in primary AML. We identified a putative enhancer in CDK6 and demonstrated interaction between the CDK6 region and the MNX1 promoter in these samples. To strengthen the evidence that the enhancer drives MNX1 activation, we inserted the putative enhancer upstream of MNX1 in a human induced pluripotent cell line (hiPSC). Upon differentiation into CD34+ hematopoietic stem and progenitor cells (HSPC), MNX1 was activated in the hiPSC line containing the inserted enhancer. This activation was not observed in the control, i.e. the parental hiPSC line without the inserted enhancer. Summary/Conclusion: We showed that chromosomal deletions in AML can lead to enhancer hijacking events through reshuffling of cis-regulatory elements. Specifically, we showed that MNX1 is activated in a subgroup of del(7q)-AML by CDK6 enhancer. Further validation and insights into the effects of MNX1 activation will be gained from single cells RNA sequencing (scRNA-seq) and single cell assay for transposase-accessible chromatin with sequencing (scATAC-seq) in the hiPSC line with the inserted enhancer fragment. Keywords: Gene regulation, Cytogenetic abnormalities, Acute myeloid leukemia, Epigenetic

Background: The total number of structural rearrangements in acute myeloid leukemkia (AML) is generally much lower than in other cancer types. However, AML with a complex karyotype (ckAML) is a rare AML subtype (10-15% of all cases) characterized by the presence of at least three cytogenetic alterations and has a dismal prognosis. It is still poorly understood. Many commonly deleted regions have been identified, for example in 5q, 7q, 12p and 17p, but it is still unclear how they may drive leukemia. In parallel, it has been discovered in the last decade that structural rearrangements can lead to aberrant expression of genes, for example of EVI1 in inv(3) or t(3;3), but no systematic search for such events has been undertaken. Since ckAML cases have many structural rearrangements, it is likely that some of them could lead to enhancer hijacking. Aims: We systematically searched for genes, which can be activated by enhancer hijacking in ckAML. Methods: We performed whole genome sequencing (WGS) and RNAseq of 42 ckAML cases. For each gene, we identified samples which had breakpoints located in the same topologically-associating domain, using the WGS. Then, we tested if the samples with breakpoints in the vicinity of the gene had an outlier high expression for this gene. Enhancer hijacking should lead to the expression of only the rearranged allele, so we filtered for genes with monoallelic expression, based on SNPs detected in WGS and RNAseq. Results: We detected many putative enhancer hijacking events in our cohort of 42 ckAML cases. Among the top 20 candidates were three genes, which have already been reported in the literature as being activated by enhancer hijacking in AML: EVI1, BCL11B and MNX1. This supports the reliability of our method. In addition, we detected several novel interesting candidates and we could for some of them find recurrence of these events in other cohorts. Several enhancers were hijacked, including strong hematopoietic enhancers like MYC, GATA2 or CDK6. Summary/Conclusion: We performed for the first time an exhaustive search for enhancer hijacking events in ckAML, and found both known and novel genes. This provides a better understanding of this deadly and poorly understood disease. Since these events are rare, sequencing even larger cohorts could lead to the identification of even more genes and could help estimate the frequency of these events.Keywords: AML, EVI1, TP53, Complex aberrant karyotype

Introduction: We evaluated low coverage whole genome sequencing (WGS) of acute myeloid leukemia (AML) patients using long read Oxford Nanopore Technology (ONTseq) for karyotyping and compared the results and the frequency of patients with myelodysplasia-related cytogenetic abnormalities according to previous and current AML classifications with conventional cytogenetics (CG). Methods: Diagnostic samples from peripheral blood or bone marrow of 100 AML patients were sequenced in 3 medical centers on a GridION sequencer using SQK-LSK109 kits according to the manufacturer's instructions, and analyzed by CG. Ten samples were sequenced independently in two different laboratories. Fifty samples were sequenced prospectively from newly diagnosed de novo AML patients. Sequencing reads were analyzed by an in-house bioinformatics pipeline based on publicly available tools such as nglmr aligner, samtools, igv_tools, R package ACE, the CyDAS webserver, and custom scripts for transforming ACE copy number models to karyotype formulas. CNVs from ONTseq data were mapped to 290 chromosome bands to allow comparison with CG. We compared the following cytogenetic categories: Complex karyotype (cKT) ≥ 3 chromosomal abnormalities; monosomal karyotype (mKT) as defined by Breems et al.; karyotype with myelodysplasia-related cytogenetic abnormalities that defines MRC-AML according WHO 2016 classification (MRC-KT), and ICC 2022 classification (MRCA-KT). Results: ONTseq of 100 AML patients with a 4.4-fold genome coverage (range: 2.1-6.2) allowed us to resolve CNVs at a high resolution of ≥0.1 mega-basepairs (Mbp). Analysis of the ten duplicate samples established a high reproducibility of ONTseq. 110 CNVs were detected by both laboratories at identical chromosome positions and similar length (R2=0.999). Only 8 CNVs (7%) differed considerably in length between the replicates (median length difference 2 Mbp, range 0.4-16 Mbp). Of 100 patients with a median age of 65 years (range 18-97), ≥1 chromosome band was gained or lost in 69 patients according to ONTseq analysis, while CNVs or balanced translocations were found by cytogenetics in 72 patients. Five patients had a normal karyotype by ONTseq, but not by CG, while two patients had a normal karyotype by CG but not by ONTseq. Two of these 7 discrepant patients had translocations as their sole cytogenetic abnormality [t(8;21) and t(9;11)], which cannot be detected by low coverage genome ONTseq. Patients with chromosomal abnormalities had a median of 29 (range 1-140) and 30 (range 3-136) chromosomal bands with CNVs according to ONTseq and CG, respectively. Considering all patients, gains and losses at the level of chromosomal bands correlated very well (gains: R2=0.922; losses: R2=0.923) (Figure 1). All patients were then classified according the definitions for cKT, mKT, MRC-KT, and MRCA-KT (Table 1). Overall, patients were similarly classified by ONTseq and CG results (P<0.001). MRC and MRCA-KTs were consistently identified by ONTseq and CG in 57 patients. In 2 and 2 patients, MRC and MRCA-KTs, respectively, were identified by CG only. In 1 and 1 patient, MRC and MRCA-KT, respectively, was identified by ONTseq only. The largest discrepancies were found for mKTs due to the higher resolution of ONTseq, which more often identified subchromosomal instead of whole chromosome CNVs compared to CG, which were not considered for mKT. Fifty of the 100 patients were sequenced prospectively at time of diagnosis. The median age of these patients was 70 years (range 61-97) and all patients had de novo AML. At least one CNV was detected by ONTseq in 24 (48%) and by CG in 26 (52%) patients. cKT, mKT, MRC-KT, and MRCA-KT were detected by ONTseq in 12, 7, 17, and 17 patients and by CG in 14, 11, 17, and 17 patients, respectively. The median time for library preparation for prospectively analyzed patients was 3:45 hours, for ONTseq 24 hours and for bioinformatic analysis 6:30 hours. In this setting, clinical reports can be delivered on the fourth day of sample receipt. Conclusion: Low coverage WGS with ONT provides a highly reproducible method for rapid karyotyping with high resolution. The identification of cKT and myelodysplasia-related cytogenetic abnormalities correlated very well with conventional cytogenetics, even though balanced translocations cannot be detected by our ONTseq protocol. Low coverage WGS is a promising approach for diagnostic karyotyping in AML patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The aim of this study was to characterize the mutational landscape of patients with FLT3 -mutated acute myeloid leukemia (AML) treated within the randomized CALGB 10603/RATIFY trial evaluating intensive chemotherapy plus the multi-kinase inhibitor midostaurin versus placebo. We performed sequencing of 262 genes in 475 patients: mutations occurring concurrently with the FLT3 -mutation were most frequent in NPM1 (61%), DNMT3A (39%), WT1 (21%), TET2 (12%), NRAS (11%), RUNX1 (11%), PTPN11 (10%), and ASXL1 (8%) genes. To assess effects of clinical and genetic features and their possible interactions, we fitted random survival forests and interpreted the resulting variable importance. Highest prognostic impact was found for WT1 and NPM1 mutations, followed by white blood cell count, FLT3 mutation type (internal tandem duplications vs. tyrosine kinase domain mutations), treatment (midostaurin vs. placebo), ASXL1 mutation, and ECOG performance status. When evaluating two-fold variable combinations the most striking effects were found for WT1 : NPM1 (with NPM1 mutation abrogating the negative effect of WT1 mutation), and for WT1 :treatment (with midostaurin exerting a beneficial effect in WT1 -mutated AML). This targeted gene sequencing study provides important, novel insights into the genomic background of FLT3 -mutated AML including the prognostic impact of co-mutations, specific gene–gene interactions, and possible treatment effects of midostaurin.