Acute Myeloid Leukemia Genome Research Articles

Evaluation of Acute Myeloid Leukemia Genomes using Genomic Proximity Mapping.

Cytogenetic analysis encompasses a suite of standard-of-care diagnostic testing methods that is routinely applied in cases of acute myeloid leukemia (AML) to assess chromosomal changes that are clinically relevant for risk classification and treatment decisions. In this study, we assess the use of Genomic Proximity Mapping (GPM) for cytogenomic analysis of AML diagnostic specimens for detection of cytogenetic risk variants included in the European Leukemia Network (ELN) risk stratification guidelines. Archival patient samples (N=48) from the Fred Hutchinson Cancer Center leukemia bank with historical clinical cytogenetic data were processed for GPM and analyzed with the CytoTerra® cloud-based analysis platform. GPM showed 100% concordance for all specific variants that have associated impacts on risk stratification as defined by ELN 2022 criteria, and a 72% concordance rate when considering all variants reported by the FH cytogenetic lab. GPM identified 39 additional variants, including variants of known clinical impact, not observed by cytogenetics. GPM is an effective solution for the evaluation of known AML-associated risk variants and a source for biomarker discovery.

Acute Myeloid Leukemia Genomics: Impact on Care and Remaining Challenges.

Acute Myeloid Leukemia Genomics: Impact on Care and Remaining Challenges.

The first human acute myeloid leukemia genome ever fully sequenced.

The first human acute myeloid leukemia genome ever fully sequenced.

Development of Circulating Tumor DNA (ctDNA) for Molecular Measurable Residual Disease (MRD) in Acute Myeloid Leukemia (AML)

Introduction: Despite advances in therapeutic options, the majority of AML patients experience relapsed or refractory disease. Standard-of-care (SOC) methods for MRD detection (multiparametric flow cytometry and single gene or structural variant molecular assays) are hampered by (1) inadequate lower limits of detection (LOD), (2) paucity of recurrent mutations across AML genomes, (3) insufficient numbers of identified mutations to characterize clonal evolution, and/or (4) need for invasive bone marrow biopsy. Herein we develop and apply non-invasive circulating tumor DNA (ctDNA) liquid biopsies using a customized Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq) approach to simultaneously quantify MRD and characterize clonal dynamics in AML. Methods: To increase the number of tumor-specific mutations per case for MRD detection and improve the LOD for ctDNA-MRD, we performed whole exome sequencing (WES) on purified blasts and paired T cells (latter as germline control) from 24 patients with AML (n = 20 previously untreated, n = 4 with relapsed/refractory disease) and designed personalized hybrid capture panels. We combined the personalized panels with a shared hybrid capture panel of 58 recurrently mutated (‘canonical‘) AML genes covering 225kB as a composite approach to generate patient-specific AML-CAPP-Seq assays. We applied AML-CAPP-Seq to track single nucleotide variants (SNVs) in 468 biological samples from the 24 AML patients and 21 healthy control subjects. We compared the performance of MRD detection in cellular (bone marrow mononuclear cell/BMMC, peripheral blood mononuclear cell/PBMC) and cell-free (BM-ctDNA, PB-ctDNA) compartments against SOC clinical MRD assays as well as patient specific clinical outcomes. Results: We observed a median of 30 SNVs (IQR 16.5-40.25) per patient from WES, compared to only 2 (IQR 1-3.25) if using the AML-focused canonical panel alone (p < 0.0001) ( Fig. 1A). This number of variants enabled sensitive molecular disease monitoring in 96% (23/24) of patients with a LOD of < 0.01% tumor fraction from typical quantities of cell-free DNA. In contrast, when using the canonical panel alone, only 83% (20/24) of patients had 1 or more SNV to allow MRD detection. Notably, only 12.5% (3/24) of patients in this cohort had a feature amenable to SOC molecular MRD methods. Upon comparing quantitative levels of MRD in each biological compartment, we found that tumor burden in PB-ctDNA, BM-ctDNA and BMMC samples were highly correlated (r > 0.92, p < 0.0001) with similar amounts of disease. Interestingly, PB-ctDNA contained quantitatively more tumor DNA than corresponding PBMC samples (p = 0.0002), most pronounced at low tumor burdens (median 8.2-fold enrichment when mean tumor allele fraction in PBMCs was < 1%). These results suggest that PB-ctDNA effectively captures total body BM disease and mutational burden in AML and serves as a superior analyte for MRD monitoring compared to PBMCs. When evaluating the performance of AML-CAPP-Seq against clinical SOC methods, our assay correctly detected molecular disease in 99.6% (237/238) of samples known to contain measurable disease, including 98.2% (54/55) of samples at timepoints of clinical MRD positivity during clinical remission (CR). However, restricting trackable SNVs to only ‘canonical’ genes led to significant degradation of performance. Specifically, only 69.1% (38/55) of samples with positive MRD during CR by clinical SOC assay were detected by ctDNA if canonical genes alone were employed (p = 0.0001). Intriguingly, when using the complete gene panel, AML-CAPP-Seq also identified molecular MRD in 37.5% (30/80) of samples with undetectable MRD by SOC assays. Finally, stratifying patients who achieved CR after 1st treatment line based on PB-ctDNA MRD significantly predicted relapse free survival (p = 0.046, hazard ratio (HR) = 4.5). Moreover, attaining an MRD negative CR in PB-ctDNA at any timepoint was strongly prognostic of overall survival (p = 0.0004, HR = 8.5) ( Fig. 1B). Conclusions: ctDNA is a promising biomarker for ultrasensitive detection and tracking of MRD in AML without need for invasive bone marrow assessment. Tracking mutations beyond canonical AML genes improves performance of such assays. ctDNA analysis has numerous areas of clinical utility, including response and relapse prediction, evaluation of clonal dynamics, and exploration of mechanisms of therapy resistance.

Early Identification of TP53 mutation in Myelodysplastic Neoplasms and Acute Myeloid Leukemia Via Point-of-Care p53 Immunohistochemistry

Background: Myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML) are heterogeneous diseases that often carry a poor prognosis. Once MDS or AML is diagnosed, first-line therapy choices include hypomethylating agent/venetoclax, induction chemotherapy, and/or targeted therapeutics as appropriate. Traditional “7+3” induction therapy has limited efficacy in in patients with TP53 disruption. Mutations in TP53 occur in at least 8% of de novo AML genomes, but turnaround time for next-generation sequencing (NGS) can be 10-14 days. Hence, it is possible that patients with TP53 mutation(s) will receive a regimen that will not be effective as it will not be known whether a patient has a TP53 mutation soon after diagnosis. Furthermore, health care disparities are amplified by the lack of readily available precision diagnostics in low- and middle-income countries (LMIC), leading to worse outcomes. Early identification of TP53 mutation through p53 immunohistochemistry (IHC) testing might allow for more timely prognostication and intervention. Methods: The UMass Chan Leukemia Registry and UMass Hematopathology archives identified patients with MDS or AML with TP53 disruption between 2011 and 2023. Immunohistochemical staining for p53 was performed on sections of bone marrow core biopsies by VENTANA CONFIRM anti-p53 (DO-7) primary antibody. Images were scored by manual counting by at least 2 separate hematopathologists. TP53 mutational analysis was performed as previously described (Patel SA , ..., Gerber JM. Leuk Lymphoma 2021; 62: 3348-60). For TP53 exome sequencing, the entire coding region from codons 1-393 of isoform A were covered, with coverage depth > 500X. The concordance between p53 IHC and the referent test ( TP53 mutational testing as the gold standard) was assessed. Results: Query of theUMass Leukemia Registry and UMass Hematopathology archives yielded 86 clinically annotated patients with MDS and AML with TP53 disruption. Among 42 patients with MDS (or its subentities) with TP53 disruption, 61 bone marrow biopsies had concurrently available data for p53 IHC and TP53 mutational testing. (Some cases were sequential biopsies from the same patient). Among 44 patients with AML (or its subentities) with TP53 disruption, 36 bone marrow biopsies had concurrently available data for p53 IHC and TP53 mutational testing. For patients with MDS with TP53 disruption, the sensitivity of p53 IHC testing was 68.1% and specificity was 93.3% (Panel A). For patients with AML with TP53 disruption, the sensitivity of p53 IHC testing was 75.8% and specificity was 100% (Panel B). Merged MDS and AML analysis showed sensitivity of 71.3% and specificity of 93.8%. When using a cutoff of ≥ 5% for p53 IHC testing, positive predictive value (PPV) was 100%. Conclusion: Our data show that p53 IHC testingdemonstrates high specificity and PPV but low sensitivity and negative predictive value (NPV), suggesting that IHC may be a valuable “rule-in” test. A cutoff value of ≥ 5% for IHC confers 100% PPV. This suggests that a positive IHC result may justify p53 IHC-adapted first-line choice of therapy, but a negative result does not exclude the possibility of TP53 mutation. IHC testing is a more cost-effective measure and is more readily available than NGS, which would make it an attractive alternative especially across global markets and in geographic regions marked by health care inequities, due to inability to access genomic testing. Limitations to our study include the relatively small sample size, as well as presence of TP53 mutations that prevent protein expression and thus prevent IHC positivity (i.e. stop codons and/or post-transcriptional modifications). Furthermore, results from our study may be distinct from other centers' studies given different inclusion criteria and non-uniform criteria for p53 staining analysis. However, our study retains value in that the use of a high-fidelity point-of-care p53 IHC staining method may lead to better outcomes with reduced financial toxicity and may inform first-line therapeutics if the IHC is positive. Further large-scale studies will be needed to confidently establish the p53 IHC staining method as the preferred surrogate for TP53 mutational testing. Additional work needs to be performed to assess the link between p53 staining pattern and TP53 allelic state. Earlier precision treatment may improve outcomes in this exceptionally high-risk population.

Prevalence and significance of DDX41 gene variants in the general population

AbstractGerm line variants in the DDX41 gene have been linked to myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) development. However, the risks associated with different variants remain unknown, as do the basis of their leukemogenic properties, impact on steady-state hematopoiesis, and links to other cancers. Here, we investigate the frequency and significance of DDX41 variants in 454 792 United Kingdom Biobank (UKB) participants and identify 452 unique nonsynonymous DNA variants in 3538 (1/129) individuals. Many were novel, and the prevalence of most varied markedly by ancestry. Among the 1059 individuals with germ line pathogenic variants (DDX41-GPV) 34 developed MDS/AML (odds ratio, 12.3 vs noncarriers). Of these, 7 of 218 had start-lost, 22 of 584 had truncating, and 5 of 257 had missense (odds ratios: 12.9, 15.1, and 7.5, respectively). Using multivariate logistic regression, we found significant associations of DDX41-GPV with MDS, AML, and family history of leukemia but not lymphoma, myeloproliferative neoplasms, or other cancers. We also report that DDX41-GPV carriers do not have an increased prevalence of clonal hematopoiesis (CH). In fact, CH was significantly more common before sporadic vs DDX41-mutant MDS/AML, revealing distinct evolutionary paths. Furthermore, somatic mutation rates did not differ between sporadic and DDX41-mutant AML genomes, ruling out genomic instability as a driver of the latter. Finally, we found that higher mean red cell volume (MCV) and somatic DDX41 mutations in blood DNA identify DDX41-GPV carriers at increased MDS/AML risk. Collectively, our findings give new insights into the prevalence and cognate risks associated with DDX41 variants, as well as the clonal evolution and early detection of DDX41-mutant MDS/AML.

Outcomes for Acute Myeloid Leukemia Relapse after Allogeneic Hematopoietic Cell Transplantation Remain Poor in the Modern Era

Background: Relapse of acute myeloid leukemia (AML) following allogeneic hematopoietic cell transplantation (allo-HCT) has historically been associated with dismal prognosis with most patients failing to achieve subsequent remission and estimated 2-year overall survival (OS) of <15 %. Rapid progress in the understanding of AML biology and genomics resulted in recent FDA approval of several novel small molecules and immunotherapies for the treatment of front-line and relapsed/refractory AML, as early as 2017. We hypothesized that the availability of novel therapies for AML relapsing after allo-HCT has resulted in an improved OS in recent years. Methods: Patients with AML ≥18 years old who underwent allo-HCT at Stanford University between the years 2010 and 2021 and subsequently relapsed were included in this study. Patients were divided into two eras based on year of relapse: 2010-2016 (early era) and 2017- 2022 (later era). Descriptive statistics were used to characterize patients across the two cohorts; Kaplan Meier method was used to evaluate OS. Results: A total of 258 adults with AML relapsed following allo-HCT; 142 (55%) transplanted in the early era and 116 (45%) in the later era. Relative to the early era, patients who relapsed in the later era were more likely to be Hispanic (19.8% vs. 8.5%, P=0.01), have adverse risk leukemia (64.7% vs 52.1%, P=0.039), poorer performance status as defined by KPS <90 (46.6% vs. 16.9%, p<0.001), an HCT-comorbidity index of ≥ 3 (43.1% vs. 28.2%, P=0.012), and receive alternative donor transplant (haploidentical donors 11.2% vs 3.5%, P=0.002; umbilical cord blood 9.5% vs. 4.2%, P=0.002). Post-HCT maintenance therapy was used more frequently in the later era (16.4% vs. 2.1%, P=0.002). Median age, sex, and conditioning intensity did not significantly differ between the two cohorts. The median time to AML relapse post-HCT was similar in the early vs late era (149 days vs. 172 days, P=0.003). The management of relapse differed significantly across eras (Figure 1), as expected, with patients in the later era more commonly receiving targeted therapy with FLT3 inhibitors and IDH1/2 inhibitors (25% vs. 7.7%), hypomethylating agents (HMA) (51.7% vs. 35.2%) and BCL2 inhibitors (32.8% vs 3.5%) and less likely receiving cytotoxic chemotherapy (21.6% vs 34.5%). The use of donor lymphocyte infusion increased over time (16.9% vs. 12.1%) as did second allo-HCT (10.3% vs. 7%). Despite increased use of novel therapies in the later era, there was no improvement in median OS compared to the early era (5.1 months versus 4.5 months, p=0.43) or 2-year OS following post-HCT relapse (19.1% vs. 12.4%, P=0.26), (Figure 2). Across both eras, relapsed AML was the most common cause of death in this cohort, attributed to 83.7% of all deaths. Conclusion: Despite the greater use of novel therapies to treat post-HCT relapse of AML in recent years, OS remains poor and has not improved. Relapsed AML following allo-HCT remains a significant clinical challenge despite advances in modern AML therapeutics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Role of Hottip Lncrna m6a Modification in AML Genome Organization and Leukemogenesis

Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with the dysregulation of HOX genes. MLLr+ AML is characterized by aberrant expression of HOX genes and the HOX cofactor MEIS1. HOTTIP, a HOXA-associated long non-coding RNA (lncRNA), is significantly up-regulated in MLLr+ AML. Aberrant overexpression of Hottip dramatically perturbs hematopoietic stem cell (HSC) self-renewal leading to mouse AML-like disease through maintaining a leukemic profiling. Recent studies show that N6-methyladenosine (m6A), as the most prevalent internal modification in eukaryotic coding and non-coding RNAs, plays important roles in normal and malignant hematopoiesis. There are several potential m6A modified sites in HOTTIP exon regions and 3'UTR region by re-analyzing m6A-methylated RNA immunoprecipitation sequencing (m6A-meRIP-seq) data in leukemia. However, it is still unclear whether HOTTIP m6A modification plays a critical role in AML genome organization and AML leukemogenesis. Our HOTTIP ChIRP-MASS Spectrometry (ChIRP-MS) data shows that HOTTIP directly interacts with m6A methyltransferase METTL3/METTL14 and CTCF/cohesin chromatin organization proteins in MOLM13 AML cells. To investigate whether HOTTIP lncRNA is methylated by METTL3, we perform m6A-eCLIP-seq and METTL3-eCLIP-seq in MOLM13 cells. Our data indicates that there are several m6A modified sites co-bound with METTL3 in HOTTIP exon and 3'UTR regions. Our m6A-eCLIP-qPCR data further confirms that depletion of METTL3 significantly decreases HOTTIP m6A modification levels at HOTTIP RNA m6A core motif GGACU regions (g1, g2 and g3) by comparing shScramble and shMETTL3 cells. It suggests that HOTTIP lncRNA contains specific m6A modified sites methylated by METTL3. Next, to further examine whether and which HOTTIP m6A sites is required for the interaction of HOTTIP RNA with CTCF protein, we have performed in vitro RNA methylation and RNA binding protein assay by synthesizing biotinylated HOTTIP RNA fragments in g1, g2 and g3 regions. Subsequently, these biotin-HOTTIP-g1/g2/g3 RNAs are methylated with METTL3/METTL14 recombinant proteins and then incubated with GST control or GST-CTCF fusion in vitro. Our data shows that m6A modification of HOTTIP-g2 or HOTTIP-g3 RNA fragment enhances its interaction with GST-CTCF protein, suggesting that HOTTIP m6A modification plays a critical role in coordinating with CTCF-mediated genome organization. To investigate the function of HOTTIP m6A modification in AML leukemogenesis, we have generated CRISPR-dCas13-ALKBH5 (dALK) system to specifically demethylate HOTTIP m6A sites (g1, g2 and g3) by co-transfecting gRNA vector and the catalytically inactive Cas13 (dCas13) fused with m6A demethylase AlkB homolog 5 (ALKBH5) vector. Targeting dALK to specific m6A site in HOTTIP lncRNA results in site-specific loss of m6A modification. Although loss of HOTTIP m6A modification never affects the stability and transcription of HOTTIP in dALK clones, loss of HOTTIP m6A modification significantly affects the transcription of leukemic signature genes HOXA9/13 and WNT target (β-catenin and MYC etc) in dALK-g2 or dALK-g3 cells. Additionally, our HOTTIP ChIRP-qPCR shows that loss of HOTTIP m6A modification affects HOTTIP binding at WNT target loci (β-catenin and MYC loci) in dALK-g2 or dALK-g3 cells. Critically, our data find that loss of HOTTIP m6A modification significantly impairs cell proliferation and cell cycle in dALK-g2 or dALK-g3 cells compared with ctrl cells. To further elucidate whether HOTTIP m6A modification is required for AML leukemogenesis in vivo, we xenograft ctrl and dALK clones into NOD-scid IL2Rgammanull (NSG) mice. Our data indicates that mice transplanted with ctrl or dALK-g1 cells dies around 40 days after transplantation, while those receiving dALK-g2 or dALK-g3 cells survives significantly longer up to 55 days. Critically, we also find that ctrl recipient mice have significantly higher human CD45+ cell chimerism in the BM than mice receiving dALK-g2 or dALK-g3 AML cells. Thus, loss of the specific HOTTIP m6A modification site reduces the AML leukemic burden in vivo. In summary, this research will provide a prospective insight for the role of HOTTIP-m6A-CTCT axis in AML genome organization and leukemogenesis.

HOTTIP-dependent R-loop formation regulates CTCF boundary activity and TAD integrity in leukemia

SUMMARYHOTTIP lncRNA is highly expressed in acute myeloid leukemia (AML) driven by MLL rearrangements or NPM1 mutations to mediate HOXA topologically associated domain (TAD) formation and drive aberrant transcription. However, the mechanism through which HOTTIP accesses CCCTC-binding factor (CTCF) chromatin boundaries and regulates CTCF-mediated genome topology remains unknown. Here, we show that HOTTIP directly interacts with and regulates a fraction of CTCF-binding sites (CBSs) in the AML genome by recruiting CTCF/cohesin complex and R-loop-associated regulators to form R-loops. HOTTIP-mediated R-loops reinforce the CTCF boundary and facilitate formation of TADs to drive gene transcription. Either deleting CBS or targeting RNase H to eliminate R-loops in the boundary CBS of β-catenin TAD impaired CTCF boundary activity, inhibited promoter/enhancer interactions, reduced β-catenin target expression, and mitigated leukemogenesis in xenograft mouse models with aberrant HOTTIP expression. Thus, HOTTIP-mediated R-loop formation directly reinforces CTCF chromatin boundary activity and TAD integrity to drive oncogene transcription and leukemia development.

Vitamin C Enhances PARPi Efficacy for the Treatment of AML

Poly-ADP-ribose polymerase inhibitors (PARPi) are currently in clinical trial to determine their therapeutic efficacy for the treatment of acute myeloid leukemia (AML). We have shown that vitamin C (VitC), an essential micronutrient and co-factor of Ten-Eleven translocation (TET) proteins, enhances AML sensitivity to PARPi, potentially due to an increased dependency on base-excision repair (BER) enzymes needed to remove TET-catalyzed oxidized methylcytosine bases via active DNA demethylation. TET2 is the most frequently mutated TET gene in patients with AML, and vitamin C treatment can mimic genetic restoration of TET2 function, leading to DNA demethylation, differentiation, and leukemia cell death. Whether vitamin C efficacy in combination with PARPi depends on the level of TET2 functional alleles is not yet known and may stratify whether TET2 wild-type or mutant patients should be targeted by vitamin C adjuvant therapy.We have generated primary murine AML-ETO9a+ and MLL-AF9+ leukemia models with Tet2 +/+, Tet2 +/- and Tet2 -/- alleles to determine the Tet2-dependent efficacy of PARPi treatment when combined with vitamin C. Furthermore, we have performed CRISPR gene knockout and drug library screening in human AML cell lines in combination with vitamin C treatment, and tested a panel of 10 AML cell lines with titrating concentrations of PARPi (Olaparib, Talazoparib, Veliparib and Rucaparib) alone or in combination with vitamin C (L-ascorbic acid) mimicking physiological to pharmacological in vivo doses. Primary murine AML cells and human cell lines were assayed for colony-forming capacity, differentiation, cell cycling, viability and effects on DNA methylation, levels of oxidized 5-mC and gene expression upon combination treatment in vitro and in vivo. TET2 mutant PDX and primary murine AMLs treated in vivo with L-ascorbate (4g/kg) and Olaparib (50mg/kg) by daily IP injection were also monitored for disease burden, cellular differentiation and survival.Vitamin C is known to drive the TET-catalyzed iterative oxidation of 5-methylcytosine (5-mC) leading to the formation of 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC). We show that VitC-PARPi combination treatment causes an accumulation of oxidized 5-mC intermediates in the AML genome that correlates with increased yH2AX formation in mid-S phase and cell cycle stalling. Vitamin C reduces the IC 50 of Olaparib and Talazoparib by greater than 10-fold in human AML cells lines and primary murine leukemia cells, and treatment in combination promotes myeloid differentiation and blocks colony-forming capacity greater than either alone. In both our in vitro and in vivo studies, Tet2 +/- AML cells exhibit increased sensitivity to vitamin C treatment alone or in combination with PARPi compared to either Tet2 +/+ or Tet2 -/- cells, suggesting that patients with TET2 haploinsufficiency, which represents the majority of TET2 mutant cases, could benefit the most from combined treatment.Our findings confirm that vitamin C can act synergistically with PARPi to block AML cell viability, reduce colony-forming capacity, and decrease leukemia burden in PDX and primary murine leukemia models in a TET2 allelic dose-dependent manner. The combinatorial effect works at clinically relevant concentrations of PARPi, and low-pharmacological doses of vitamin C. These studies suggest that vitamin C can be used as a non-toxic therapeutic adjuvant to PARPi therapy for the treatment of AML. DisclosuresNeel: Northern Biologics, LTD: Current equity holder in publicly-traded company, Other: Co- Founder; SAB: Other: Co-Founder; Navire Pharma: Consultancy, Current equity holder in publicly-traded company; Jengu Therapeutics: Consultancy, Current equity holder in publicly-traded company, Other: Co-Founder; Arvinas, Inc: Consultancy, Current equity holder in publicly-traded company; Recursion Pharma: Current equity holder in publicly-traded company.

Genetic and Transcriptional Contributions to Relapse in Normal Karyotype Acute Myeloid Leukemia.

To better understand clonal and transcriptional adaptations after relapse in patients with acute myeloid leukemia (AML), we collected presentation and relapse samples from six normal karyotype AML cases. We performed enhanced whole-genome sequencing to characterize clonal evolution, and deep-coverage single-cell RNA sequencing on the same samples, which yielded 142,642 high-quality cells for analysis. Identifying expressed mutations in individual cells enabled us to discriminate between normal and AML cells, to identify coordinated changes in the genome and transcriptome, and to identify subclone-specific cell states. We quantified the coevolution of genetic and transcriptional heterogeneity during AML progression, and found that transcriptional changes were significantly correlated with genetic changes. However, transcriptional adaptation sometimes occurred independently, suggesting that clonal evolution does not represent all relevant biological changes. In three cases, we identified cells at diagnosis that likely seeded the relapse. Finally, these data revealed a conserved relapse-enriched leukemic cell state bearing markers of stemness, quiescence, and adhesion. SIGNIFICANCE: These data enabled us to identify a relapse-enriched leukemic cell state with distinct transcriptional properties. Detailed case-by-case analyses elucidated the complex ways in which the AML genome, transcriptome, and immune microenvironment interact to evade chemotherapy. These analyses provide a blueprint for evaluating these factors in larger cohorts.This article is highlighted in the In This Issue feature, p. 1.

Descriptive and Functional Genomics in Acute Myeloid Leukemia (AML): Paving the Road for a Cure.

Simple SummaryAcute myeloid leukemia (AML) accounts for 7.6% of hematopoietic malignancies with a long-term survival of less than 20%. Better understanding its physiopathology and finding new treatments remain important issues. In the current review, the authors discuss how genetic engineering technologies improvement allowed a better genetic characterization of AML. Such molecular dissection of the AML genome had two direct clinical impacts: a prognostic contribution by defining a new molecular classification of AML which guides therapeutic regimen intensity, and a therapeutic impact by allowing the identification of new therapeutic targets. New genome editing tools and animal models have also paved the way for a better understanding of AML leukemogenesis. Their impact is also summarized in this review. The combination of descriptive and functional genetics may ultimately be the key to improving the prognosis of this dismal disease.Over the past decades, genetic advances have allowed a more precise molecular characterization of AML with the identification of novel oncogenes and tumor suppressors as part of a comprehensive AML molecular landscape. Recent advances in genetic sequencing tools also enabled a better understanding of AML leukemogenesis from the preleukemic state to posttherapy relapse. These advances resulted in direct clinical implications with the definition of molecular prognosis classifications, the development of treatment recommendations based on minimal residual disease (MRD) measurement and the discovery of novel targeted therapies, ultimately improving AML patients’ overall survival. The more recent development of functional genomic studies, pushed by novel molecular biology technologies (short hairpin RNA (shRNA) and CRISPR-Cas9) and bioinformatics tools design on one hand, along with the engineering of humanized physiologically relevant animal models on the other hand, have opened a new genomics era resulting in a greater knowledge of AML physiopathology. Combining descriptive and functional genomics will undoubtedly open the road for an AML cure within the next decades.

Next-generation sequencing reveals gene mutations landscape and clonal evolution in patients with acute myeloid leukemia

ABSTRACT Objectives The study aims to understand geneome diversification and complexity that developed in Acute myeloid leukemia (AML). Methods Next-generation sequencing (NGS) was used to identify the genetic profiles of 22 genes relevant to hematological malignancy in 204 patients with de novo non-M3 AML. Results At time of initial diagnosis, at least one mutation was identified in 80.9% of patients (165/204). The most commonly mutated gene was NPM1 (22.1%), followed by ASXL1 (18.1%), TET2 (18.1%), IDH2 (15.7%), CEBPA (14.7%), FLT3-ITD (13.2%) and DNMT3A (11.8%). Mutations landscape analysis indicated several patterns of co-occurring and mutual exclusive gene mutations. Some correlation was observed between gene mutations and clinicohematological features. Multivariate analysis showed that age >60 years, karyotypes, IDH2 and KIT mutations were the independent unfavorable prognostic factors for OS; NPM1-mut/ FLT3-ITD-wt was independently correlated with prolonged OS; whereas the independent poor risk factors for RFS were karyotypes, high WBC and RUNX1 mutation. According to different genotype demonstrated by multivariate analysis, 163 patients with intermediate-risk cytogenetics were classified into three subgroups: patients with NPM1-mut/ FLT3-ITD-wt or biallelic CEBPA mutation as favorable risk, patients with KIT, IDH2, TP53 or NRAS mutations as unfavorable risk, and the remaining was the intermediate risk. We also obtain information of clonal evolution during leukemia progression by observing five patients who underwent repeat NGS at relapse in our cohort. Conclusion NGS techniques is a useful tool for discovering related gene mutations and clonal evolution in AML genomes, leading to novel targeted therapeutic approaches that could improve patients outcomes.

Targeting IDH Mutations in AML: Wielding the Double-edged Sword of Differentiation.

The genomic characterization of acute myeloid leukemia (AML) by DNA sequencing has illuminated subclasses of the disease, with distinct driver mutations, that might be responsive to targeted therapies. Approximately 15-23% of AML genomes harbor mutations in one of two isoforms of isocitrate dehydrogenase (IDH1 or IDH2). These enzymes are constitutive mediators of basic cellular metabolism, but their mutated forms in cancer synthesize an abnormal metabolite, 2- hydroxyglutarate, that in turn acts as a competitive inhibitor of multiple gene regulatory enzymes. As a result, leukemic IDH mutations cause changes in genome structure and gene activity, culminating in an arrest of normal myeloid differentiation. These discoveries have motivated the development of a new class of selective small molecules with the ability to inhibit the mutant IDH enzymes while sparing normal cellular metabolism. These agents have shown promising anti-leukemic activity in animal models and early clinical trials, and are now entering Phase 3 study. This review will focus on the growing preclinical and clinical data evaluating IDH inhibitors for the treatment of IDH-mutated AML. These data suggest that inducing cellular differentiation is central to the mechanism of clinical efficacy for IDH inhibitors, while also mediating toxicity for patients who experience IDH Differentiation Syndrome. Ongoing trials are studying the efficacy of IDH inhibitors in combination with other AML therapies, both to evaluate potential synergistic combinations as well as to identify the appropriate place for IDH inhibitors within existing standard-of-care regimens.

A comprehensive review of genetic alterations and molecular targeted therapies for the implementation of personalized medicine in acute myeloid leukemia.

Acute myeloid leukemia (AML) is an extremely heterogeneous disease defined by the clonal growth of myeloblasts/promyelocytes not only in the bone marrow but also in peripheral blood and/or tissues. Gene mutations and chromosomal abnormalities are usually associated with aberrant proliferation and/or block in the normal differentiation of hematopoietic cells. So far, the combination of cytogenetic profiling and molecular and gene mutation analyses remains an essential tool for the classification, diagnosis, prognosis, and treatment for AML. This review gives an overview on how the development of novel innovative technologies has allowed us not only to detect the genetic alterations as early as possible but also to understand the molecular pathogenesis of AML to develop novel targeted therapies. We also discuss the remarkable advances made during the last decade to understand the AML genome both at primary and relapse diseases and how genetic alterations might influence the distinct biological groups as well as the clonal evolution of disease during the diagnosis and relapse. Also, the review focuses on how the persistence of epigenetic gene mutations during morphological remission is associated with relapse. It is suggested that along with the prognostic and therapeutic mutations, the novel molecular targeted therapies either approved by FDA or those under clinical trials including CART-cell therapy would be of immense importance in the effective management of AML.

The Role of Somatic Mutations in Acute Myeloid Leukemia Pathogenesis.

Acute myeloid leukemia (AML) is characterized by attenuation of lineage differentiation trajectories that results in impaired hematopoiesis and enhanced self-renewal. To date, sequencing studies have provided a rich landscape of information on the somatic mutations that contribute to AML pathogenesis. These studies show that most AML genomes harbor relatively fewer mutations, which are acquired in a stepwise manner. Our understanding of the genetic basis of leukemogenesis informs a broader understanding of what initiates and maintains the AML clone and informs the development of prognostic models and mechanism-based therapeutic strategies. Here, we explore the current knowledge of genetic and epigenetic aberrations in AML pathogenesis and how recent studies are expanding our knowledge of leukemogenesis and using this to accelerate therapeutic development for AML patients.

CBFβ-SMMHC Affects Genome-wide Polycomb Repressive Complex 1 Activity in Acute Myeloid Leukemia

Mutations and deletions of polycomb repressive complex (PRC) components are increasingly recognized to affect tumor biology in a range of cancers. However, little is known about how genetic alterations of PRC-interacting molecules such as the core binding factor (CBF) complex influence polycomb activity. We report that the acute myeloid leukemia (AML)-associated CBFβ-SMMHC fusion oncoprotein physically interacts with the PRC1 complex and that these factors co-localize across the AML genome in an apparently PRC2-independent manner. Depletion of CBFβ-SMMHC caused substantial increases in genome-wide PRC1 binding and marked changes in the association between PRC1 and the CBF DNA-binding subunit RUNX1. PRC1 was more likely to be associated with actively transcribed genes in CBFβ-SMMHC-expressing cells. CBFβ-SMMHC depletion had heterogeneous effects on gene expression, including significant reductions in transcription of ribosomal loci occupied by PRC1. Our results provide evidence that CBFβ-SMMHC markedly and diversely affects polycomb recruitment and transcriptional regulation across the AML genome.

Not Only Mutations Matter: Molecular Picture of Acute Myeloid Leukemia Emerging from Transcriptome Studies

The last two decades of genome-scale research revealed a complex molecular picture of acute myeloid leukemia (AML). On the one hand, a number of mutations were discovered and associated with AML diagnosis and prognosis; some of them were introduced into diagnostic tests. On the other hand, transcriptome studies, which preceded AML exome and genome sequencing, remained poorly translated into clinics. Nevertheless, gene expression studies significantly contributed to the elucidation of AML pathogenesis and indicated potential therapeutic directions. The power of transcriptomic approach lies in its comprehensiveness; we can observe how genome manifests its function in a particular type of cells and follow many genes in one test. Moreover, gene expression measurement can be combined with mutation detection, as high-impact mutations are often present in transcripts. This review sums up 20 years of transcriptome research devoted to AML. Gene expression profiling (GEP) revealed signatures distinctive for selected AML subtypes and uncovered the additional within-subtype heterogeneity. The results were particularly valuable in the case of AML with normal karyotype which concerns up to 50% of AML cases. With the use of GEP, new classes of the disease were identified and prognostic predictors were proposed. A plenty of genes were detected as overexpressed in AML when compared to healthy control, including KIT, BAALC, ERG, MN1, CDX2, WT1, PRAME, and HOX genes. High expression of these genes constitutes usually an unfavorable prognostic factor. Upregulation of FLT3 and NPM1 genes, independent on their mutation status, was also reported in AML and correlated with poor outcome. However, transcriptome is not limited to the protein-coding genes; other types of RNA molecules exist in a cell and regulate genome function. It was shown that microRNA (miRNA) profiles differentiated AML groups and predicted outcome not worse than protein-coding gene profiles. For example, upregulation of miR-10a, miR-10b, and miR-196b and downregulation of miR-192 were found as typical of AML with NPM1 mutation whereas overexpression of miR-155 was associated with FLT3-internal tandem duplication (FLT3-ITD). Development of high-throughput technologies and microarray replacement by next generation sequencing (RNA-seq) enabled uncovering a real variety of leukemic cell transcriptomes, reflected by gene fusions, chimeric RNAs, alternatively spliced transcripts, miRNAs, piRNAs, long noncoding RNAs (lncRNAs), and their special type, circular RNAs. Many of them can be considered as AML biomarkers and potential therapeutic targets. The relations between particular RNA puzzles and other components of leukemic cells and their microenvironment, such as exosomes, are now under investigation. Hopefully, the results of this research will shed the light on these aspects of AML pathogenesis which are still not completely understood.

Advances in Acute Myeloid Leukemia Genomics, Where Do We Stand in 2018?

The aim of this review is to summarize the data on commonly mutated genes and genomic pathways in acute myeloid leukemia (AML) with a focus on recently approved targeted therapies. AML is a heterogeneous disease with recurrent cytogenetic and genomic abnormalities that define the disease biology and pathogenesis. Classification of the disease categories and their prognostication was updated in the past 2 years to reflect the most recent advances in understanding the complex disease biology of AML. This review highlights major updates in the World Health Organization classification, including cytogenetic re-classifications, provisional entities, and updates to the European Leukemia Net (ELN) AML risk group stratification. An overview of pivotal studies that used novel sequencing techniques to define the mutational landscape of AML is also provided. In these studies, mutations are classified into subgroups based on functional pathways and are used to understand various interactions and mutual exclusivity of some mutations, suggesting important roles in disease evolution and AML pathogenesis. The complex interactions between mutations can dictate outcomes as well as possibly predict disease phenotypes after correcting for clinical variables. CONCLUSION: Genomic testing in AML using next generation sequencing has become widely available and a new standard of care for all patients. Therefore, it is vital to use novel methods to incorporate these data in clinical decision making.

From DNA Sequencing to Clinical Trials: Finding New Targeted Drugs for Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by clonal expansion of myeloid progenitor cells, resulting in disturbed hematopoiesis and bone marrow failure. For decades, AML therapy was relatively straightforward: clinicians assessed whether the patient was fit or unfit for standard chemotherapy and selected the treatment from among limited choices. With the advent of high-throughput sequencing technologies, significant progress has been made in unraveling the AML genome and understanding leukemogenesis driven by recurrent mutations in signaling and kinase pathways, DNA methylation, and spliceosome complex genes. We are now poised to see our research-based advances translate clinically into the treatment of patients with AML. As recently as within the last 2 years, the United States Food and Drug Administration (FDA) approved eight novel therapies for patients with AML. In this review, we discuss recently approved agents targeting fms-like tyrosine kinase 3 (FLT3), isocitrate dehydrogenase (IDH), B-cell lymphoma-2 (BCL-2), and other promising novel AML agents that are in late stages of clinical development.