Adult Acute Myeloid Leukemia Research Articles

Dysregulated immune and metabolic pathways are associated with poor survival in adult acute myeloid leukemia with CEBPA bZIP in-frame mutations

Acute myeloid leukemia (AML) with CEBPA bZIP in-frame mutations (CEBPAbZIP-inf) is classified within the favorable-risk group by the 2022 European LeukemiaNet (ELN-2022). However, heterogeneous clinical outcomes are still observed in these patients. In this study, we aimed to investigate the mutation profiles and transcriptomic patterns associated with poor outcomes in patients with CEBPAbZIP-inf. One hundred and thirteen CEBPAbZIP-inf patients were identified in a cohort of 887 AML patients homogeneously treated with intensive chemotherapy. Concurrent WT1 or DNMT3A mutations significantly predicted worse survival in AML patients with CEBPAbZIP-inf. RNA-sequencing analysis revealed an enrichment of interferon (IFN) signaling and metabolic pathways in those with a shorter event-free survival (EFS). CEBPAbZIP-inf patients with a shorter EFS had higher expression of IFN-stimulated genes (IRF2, IRF5, OAS2, and IFI35). Genes in mitochondrial complexes I (NDUFA12 and NDUFB6) and V (ATP5PB and ATP5IF1) were overexpressed and were associated with poorer survival, and the results were independently validated in the TARGET AML cohort. In conclusion, concurrent WT1 or DNMT3A mutations and a dysregulated immune and metabolic state were correlated with poor survival in patients with CEBPAbZIP-inf, and upfront allogeneic transplantation may be indicated for better long-term disease control.

MethScore as a new comprehensive DNA methylation-based value refining the prognosis in acute myeloid leukemia.

Changes in DNA methylation are common events in the pathogenesis of acute myeloid leukemia (AML) and have been repeatedly reported as associated with prognosis. However, studies integrating these numerous and potentially prognostically relevant DNA methylation changes are lacking. Therefore, we aimed for an overall evaluation of these epigenetic aberrations to provide a comprehensive NGS-based approach of DNA methylation assessment for AML prognostication. We designed a sequencing panel targeting 239 regions (approx. 573kb of total size) described in the literature as having a prognostic impact or being associated with AML pathogenesis. Diagnostic whole-blood DNA samples of adult AML patients divided into a training (n = 128) and a testing cohort (n = 50) were examined. The libraries were prepared using SeqCap Epi Enrichments System (Roche) and sequenced on MiSeq instrument (Illumina). Altogether, 1935 CpGs affecting the survival (p < 0.05) were revealed in the training cohort. A summarizing value MethScore was then calculated from these significant CpGs. Patients with lower MethScore had markedly longer overall survival (OS) and event-free survival (EFS) than those with higher MethScore (p < 0.001). The predictive ability of MethScore was verified on the independent testing cohort for OS (p = 0.01). Moreover, the proof-of-principle validation was performed using the TCGA dataset. We showed that comprehensive NGS-based approach of DNA methylation assessment revealed a robust epigenetic signature relevant to AML outcome. We called this signature MethScore and showed it might serve as a strong prognostic marker able to refine survival probability of AML patients.

Real-Life Multicenter Experience of Venetoclax in Combination with Hypomethylating Agents in Previously Untreated Adult Patients with Acute Myeloid Leukemia in Greece.

The landscape of first-line treatment for acute myeloid leukemia (AML) patients ineligible for intensive chemotherapy has changed remarkably after venetoclax approval. Accumulating real-world data further apprises us with more knowledgeable use. To assess the efficacy and safety challenges in the real-life setting of the combination of hypomethylated agent (HMA) and venetoclax, we conducted a multi-center retrospective study. Forty adult AML patients treated with the combination of HMA and venetoclax as a first-line treatment after full approval (2020) were included. To confirm VIALE-A results, this group was compared to a historical cohort of 17 chemotherapy-ineligible AML patients treated with HMA monotherapy before 2020. The combination of HMA-venetoclax achieved a composite complete response rate of 86.8% (p < 0.001), median overall survival, and event-free survival of 33.8 and 19.7 months, respectively, in a median follow-up of 17.8 months (pos < 0.001, HR = 0.276, CI: 0.132-0.575, pEFS = 0.004, HR = 0.367, CI: 0.174-0.773). High rates of neutropenia (90%) and consequent infection rates (57.5%) were noted. Only 55% of our patients received antifungal prophylaxis, as its use remains controversial, and invasive fungal infections were presented in 7.5%. Evidently, venetoclax-HMA yields high response rates and profound survival benefits in real life and has changed our approach to alternative chemotherapy options.

Inhibition of NRF2 enhances the acute myeloid leukemia cell death induced by venetoclax via the ferroptosis pathway

Venetoclax, an inhibitor that selectively targets B cell lymphoma-2 (BCL-2) that has been approved for treating adult acute myeloid leukemia (AML) in combination with hypomethylating agents. However, its short duration of response and emergence of resistance are significant issues. In this study, we found that the sensitivity of AML cells to venetoclax was considerably enhanced by ML385, an inhibitor of the ferroptosis factor nuclear transcription factor erythroid 2-related factor 2 (NRF2). Using AML samples, we verified that NRF2 and its target gene ferritin heavy chain 1 (FTH1) were highly expressed in patients with AML and correlated with poor prognosis. Downregulation of NRF2 could inhibit FTH1 expression and significantly enhance the venetoclax-induced labile iron pool and lipid peroxidation. By contrast, NRF2 overexpression or administration of the reactive oxygen species inhibitor N-acetylcysteine and vitamin E could effectively suppress the anti-AML effects of ML385+venetoclax. Furthermore, the ferroptosis inducer erastin increased the anti-AML effects of venetoclax. Our study demonstrated that NRF2 inhibition could enhance the AML cell death induced by venetoclax via the ferroptosis pathway. Thus, the combination of ML385 with venetoclax may offer a favorable strategy for AML treatment.

Pathogenesis and treatment of acute myeloid leukemia with FLT3 mutations

FLT3 mutations are the most frequently identified genetic abnormalities in adult acute myeloid leukemia (AML) patients, accounting for approximately 30%. FLT3-ITD mutation specifically is considered as a poor prognostic factor in AML, and allogeneic hematopoietic cell transplantation in first remission is recommended for younger patients. The recent clinical introduction of FLT3 inhibitors has been reported to improve the prognosis of patients with FLT3 mutation-positive AML. In Japan, alongside monotherapy with gilteritinib or quizartinib for relapsed/refractory patients, combination of quizartinib with intensive chemotherapy was approved in 2023 for untreated FLT3-ITD mutation-positive AML. Studies to date have demonstrated the utility of measurable/minimal residual disease evaluation targeting FLT3 mutations and the efficacy of maintenance therapy after allogeneic transplantation. However, emergence of additional genetic mutations associated with treatment resistance has been observed. Thus, FLT3 mutations are utilized not only as a prognostic factor in AML but also as a target for treatment and for response assessment. Furthermore, the development of new treatment strategies involving FLT3 inhibitors is highly anticipated to improve clinical outcomes for patients with FLT3 mutation-positive AML.

The profile and prognostic significance of bone marrow T-cell differentiation subsets in adult AML at diagnosis.

T lymphocytes in tumor microenvironment play a pivotal role in the anti-tumor immunity, and the memory of T cells contributes to the long-term protection against tumor antigens. Compared to solid tumors, studies focusing on the T-cell differentiation in the acute myeloid leukemia (AML) bone marrow (BM) microenvironment remain limited. Fresh BM specimens collected from 103 adult AML patients at diagnosis and 12 healthy donors (HDs) were tested T-cell differentiation subsets by multi-parameter flow cytometry. CD4 and CD8 T-cell compartments had different constituted profiles of T-cell differentiated subsets, which was similar between AML patients and HDs. Compared to HDs, AML patients as a whole had a significantly higher proportion of CD8 effector T cells (Teff, P = 0.048). Moreover, the T-cell compartment of AML patients with no DNMT3A mutations skewed toward terminal differentiation at the expense of memory T cells (CD4 Teff: P = 0.034; CD8 Teff: P = 0.030; CD8 memory T: P = 0.017), whereas those with mutated DNMT3A had a decrease in CD8 naïve T (Tn) and CD4 effector memory T cells (Tem) as well as an increase in CD4 central memory T cells (Tcm) (P = 0.037, 0.053 and 0.053). Adverse ELN genetic risk correlated with a lower proportion of CD8 Tn. In addition, the low proportions of CD4 Tem and CD8 Tn independently predicted poorer relapse-free survival (RFS, HR [95%CI]: 5.7 (1.4-22.2), P = 0.017 and 4.8 [1.3-17.4], P = 0.013) and event-free survival (EFS, HR [95% CI]: 3.3 (1.1-9.5), P = 0.029; 4.0 (1.4-11.5), P = 0.010), respectively. AML patients had abnormal profiles of BM T-cell differentiation subsets at diagnosis, which was related to DNMT3A mutations. The low proportions of CD4 Tem and CD8 Tn predicted poor outcomes.

Multi-omic analysis of longitudinal acute myeloid leukemia patient samples reveals potential prognostic markers linked to disease progression.

Relapse remains a determinant of treatment failure and contributes significantly to mortality in acute myeloid leukemia (AML) patients. Despite efforts to understand AML progression and relapse mechanisms, findings on acquired gene mutations in relapse vary, suggesting inherent genetic heterogeneity and emphasizing the role of epigenetic modifications. We conducted a multi-omic analysis using Omni-C, ATAC-seq, and RNA-seq on longitudinal samples from two adult AML patients at diagnosis and relapse. Herein, we characterized genetic and epigenetic changes in AML progression to elucidate the underlying mechanisms of relapse. Differential interaction analysis showed significant 3D chromatin landscape reorganization between relapse and diagnosis samples. Comparing global open chromatin profiles revealed that relapse samples had significantly fewer accessible chromatin regions than diagnosis samples. In addition, we discovered that relapse-related upregulation was achieved either by forming new active enhancer contacts or by losing interactions with poised enhancers/potential silencers. Altogether, our study highlights the impact of genetic and epigenetic changes on AML progression, underlining the importance of multi-omic approaches in understanding disease relapse mechanisms and guiding potential therapeutic interventions.

Serum podoplanin expression and frequency of thrombosis in adult de novo acute myeloid leukemia patients: An Egyptian single center study

Background Cancer-associated thrombosis although represents a significant disease worse prognosis, understanding its underlying pathophysiology remains obscure. Podoplanin and its platelet ligand C-type lectin receptor type-2 have been investigated in the past few years for their potential role as procoagulant substances as it has been shown in many studies that binding of podoplanin with platelet C-type lectin receptor type-2 results in enhancement of platelet aggregation and thrombotic insults. Aim In this study, serum podoplanin was measured in newly diagnosed acute myeloid leukemia (AML) patients and its relation to the incidence of thrombosis in these patients in addition to its relation to other clinical, laboratory, and prognostic outcomes. Patients and methods This study included 80 participants (60 newly diagnosed adult AML patients and 20 age-matched and sex-matched healthy controls). Blood counts and flow cytometry using an acute leukemia panel were performed in addition to serum podoplanin, and cytogenetic studies including conventional karyotyping and fluorescence in-situ hybridization were performed to determine the risk group. The clinical status of the patients, particularly the incidence of thrombotic events in the following 6 months, as well as the D28 outcome and 6-month survival, were determined and reported. Results Serum podoplanin was highly expressed in AML patients compared with control samples, with a highly significant difference (P=0.001). There was a significant inverse relation between serum podoplanin level, hemoglobin level, and white blood cell count (P=0.006 and 0.036, respectively). Those with thrombotic events during the course of the disease did not show statistically significant results when compared with the control group with regard to serum podoplanin levels (P=0.546). Conclusion Serum podoplanin was much higher in AML patients than in the control group (P=0.000), and it was inversely related to platelet level (P=0.036) and total leukocyte count (P=0.006). However, there was no statistically significant link between podoplanin level and the incidence of thrombotic events (P=0.546).

Clinico-Hematological Profile of Acute Myeloid Leukemia: Experience From a Tertiary Care Cancer Center in North India.

Complete diagnosis of acute myeloid leukemia (AML) requires knowledge of clinical information combined with morphologic evaluation, immunophenotyping, karyotyping, and molecular genetic testing. The study intends to evaluate the demographic profile, clinical workup, and investigation, including flow cytometric immunophenotyping, in adult and pediatric age groups of AML. This is a retrospective study of AML patients treated between January 2017 and December 2021. Clinical and demographic characteristicsand investigation findings were recorded from case files and the hematology database. A total of 896 cases of AML were registered during the given period, of which 819 cases were de-novo AML. Among those 819 cases, more than two-thirds of cases, i.e., 78.9% (N = 646), received induction chemotherapy. A significantly higher male-to-female ratio was observed (1.5:1). The median age was 22 years. The median time for diagnosis was three days and the median time for treatment intervention was four days. There were significant differences in the Eastern Cooperative Oncology Group (ECOG) performance status scores between pediatric and adult AML patients. Pediatric AML patients presented with better ECOG performance scores (ECOG performance scores 0 and 1) than adult patients (74.76% vs. 43.14%, p < 0.001). Further comparing adult vs. pediatric AML patients, normal karyotype (60.56% vs. 31.93%, p < 0.001) and NPM1 (22.25% vs. 6.72%, p < 0.001)and FLT3-ITD mutations (20.28% vs. 7.98%, p<0.001) were more common in the adult group, whereas AML-ETO (40.76% vs. 16.34%, p < 0.001) was more common in the pediatric group. The study highlights the presenting age is lower than global figures. The median time for initial diagnosis and the start of treatment is within the acceptable norms. Normal karyotype andNPM1and FLT3 mutations were common in adult AML patients, whereas AML-ETO was more common in the pediatric cohort. These findings will help plan prospective studies and see the correlation with treatment outcomes. The laboratory workup practice currently complies with the standard guidelines at our center.

DELE1 haploinsufficiency causes resistance to mitochondrial stress-induced apoptosis in monosomy 5/del(5q) AML

Monosomy 5 and deletions of the chromosome 5q (−5/del(5q)) are recurrent events in de novo adult acute myeloid leukemia (AML), reaching up to 40% of cases in secondary AML. These chromosome anomalies are associated with TP53 mutations and with very poor prognosis. Using the large Leucegene genomic and transcriptomic dataset composed of 48 −5/del(5q) patient specimens and 367 control AML, we identified DELE1 – located in the common deleted region – as the most consistently downregulated gene in these leukemias. DELE1 encodes a mitochondrial protein recently characterized as the relay of mitochondrial stress to the cytosol through a newly defined OMA1-DELE1-HRI pathway which ultimately leads to the activation of ATF4, the master transcription factor of the integrated stress response. Here, we showed that the partial loss of DELE1 expression observed in −5/del(5q) patients was sufficient to significantly reduce the sensitivity to mitochondrial stress in AML cells. Overall, our results suggest that DELE1 haploinsufficiency could represent a new driver mechanism in −5/del(5q) AML.

Prognostic significance of the frequencies of bone marrow lymphocyte subsets in adult acute myeloid leukemia at diagnosis.

Immune microenvironment plays an important role in the occurrence and development of acute myeloid leukemia (AML). Studies assessing the prognostic significance of bone marrow (BM) lymphocyte subsets' frequencies at diagnosis in patients with AML were limited. Fresh BM samples collected from 97 adult AML patients at diagnosis were tested for lymphocyte, T, CD4+ T, CD8+ T, γδT, NK, and B cell frequencies using multi-parameter flow cytometry. Low frequencies of lymphocytes, T, CD4+ T, and CD8+ T cells were associated with significantly lower rates of one-course complete remission (CR) (all p < 0.05). Moreover, the frequency of CD4+ T cells independently predicted one-course CR achievement (p = 0.021). Low frequencies of T and CD8+ T cells were significantly associated with lower relapse-free survival (RFS) rates (p = 0.032; 0.034), respectively, and a low frequency of CD8+ T cells was associated with a significantly lower overall survival (OS) rate (p = 0.028). Combination of frequency of CD8+ T cells and ELN risk stratification showed that patients with ELN-intermediate/adverse risk + high CD8+ T cell frequency had a similar RFS rate to those with ELN-favorable risk + high CD8+ T cell frequency and those with ELN-favorable risk + low CD8+ T cell frequency (p = 0.88; 0.76), respectively. The RFS rate of patients with ELN intermediate/adverse risk + low CD8+ T cell frequency was significantly lower than that of all aforementioned patients (p = 0.021; 0.0007; 0.028), respectively. The frequencies of BM lymphocyte subsets at diagnosis predicted clinical outcomes and could help improve risk stratification in AML.

Emerging and Future Targeted Therapies for Pediatric Acute Myeloid Leukemia: Targeting the Leukemia Stem Cells.

Acute myeloid leukemia (AML) is a rare subtype of acute leukemia in the pediatric and adolescent population but causes disproportionate morbidity and mortality in this age group. Standard chemotherapeutic regimens for AML have changed very little in the past 3-4 decades, but the addition of targeted agents in recent years has led to improved survival in select subsets of patients as well as a better biological understanding of the disease. Currently, one key paradigm of bench-to-bedside practice in the context of adult AML is the focus on leukemia stem cell (LSC)-targeted therapies. Here, we review current and emerging immunotherapies and other targeted agents that are in clinical use for pediatric AML through the lens of what is known (and not known) about their LSC-targeting capability. Based on a growing understanding of pediatric LSC biology, we also briefly discuss potential future agents on the horizon.

Clinical characteristics and prognostic factors analysis of core binding factor acute myeloid leukemia in real world.

Chromosomal translocations involving core binding factor (CBF) genes account for 15% of adult acute myeloid leukemia (AML) cases in China. Despite being classified as favorable-risk by European Leukemia Net (ELN), CBF-AML patients have a 40% relapse rate. This study aims to analyze clinical characteristics and prognosis of CBF-AML, compare its subtypes (inv(16) and t(8;21)), and validate prognostic factors. Retrospective analysis of 149 AML patients (75 CBF-AML, 74 non-CBF) at Peking University First Hospital (March 2012-March 2022). CBF-AML patients have significantly lower disease-free survival (DFS) (p = 0.005) and higher non-relapse mortality (NRM) (p = 0.028) compared to non-CBF AML. inv (16) and t(8;21) show distinct co-occurring gene mutation patterns, with inv(16) being prone to central nervous system (CNS) leukemia. Multivariate analysis identifies age as a risk factor for overall survival (OS) and disease free survival (DFS), kinase mutation as a risk factor for DFS and Recurrence, while WT1 mutation as a risk factor for OS and non relapse mortality (NRM) risk in t(8;21) AML. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) improves prognosis in low-risk t(8;21). Prognosis of CBF-AML is poorer than ELN guidelines suggest. inv(16) and (8;21) are separate entities with relatively poor prognoses, requiring rational risk stratification strategies. Allo-HSCT may benefit low-risk t(8;21), but further research is needed for conclusive evidence.

Linking GATA2 to myeloid dysplasia and complex cytogenetics in adult myelodysplastic neoplasm and acute myeloid leukemia

AbstractGATA binding protein 2 (GATA2) is a conserved zinc finger transcription factor that regulates the emergence and maintenance of complex genetic programs driving development and function of hematopoietic stem and progenitor cells (HSPCs). Patients born with monoallelic GATA2 mutations develop myelodysplastic neoplasm (MDS) and acute myeloid leukemia (AML), whereas acquired GATA2 mutations are reported in 3% to 5% of sporadic AML cases. The mechanisms by which aberrant GATA2 activity promotes MDS and AML are incompletely understood. Efforts to understand GATA2 in basic biology and disease will be facilitated by the development of broadly efficacious antibodies recognizing physiologic levels of GATA2 in diverse tissue types and assays. Here, we purified a polyclonal anti-GATA2 antibody and generated multiple highly specific anti-GATA2 monoclonal antibodies, optimized them for immunohistochemistry on patient bone marrow bioosy samples, and analyzed GATA2 expression in adults with healthy bone marrow, MDS, and acute leukemia. In healthy bone marrow, GATA2 was detected in mast cells, subsets of CD34+ HSPCs, E-cadherin–positive erythroid progenitors, and megakaryocytes. In MDS, GATA2 expression correlates with bone marrow blast percentage, positively correlates with myeloid dysplasia and complex cytogenetics, and is a nonindependent negative predictor of overall survival. In acute leukemia, the percent of GATA2+ blasts closely associates with myeloid lineage, whereas a subset of lymphoblastic and undifferentiated leukemias with myeloid features also express GATA2. However, the percent of GATA2+ blasts in AML is highly variable. Elevated GATA2 expression in AML blasts correlates with peripheral neutropenia and complex AML cytogenetics but, unlike in MDS, does not predict survival.

A nomogram based on clinical features and molecular abnormalities for predicting the prognosis of patients with acute myeloid leukemia.

The high clinical and molecular heterogeneity of acute myeloid leukemia (AML) has led to an unsatisfactory clinical prognosis, thus we sought to incorporate both clinical features and molecular abnormalities to construct a new prognostic model. A database search of the Gene Expression Omnibus (GEO) revealed 238 cases of adult AML. The independent risk factors were assessed using both univariate and multivariate Cox regression, as well as least absolute shrinkage and selection operator (LASSO) regression. The predictive accuracy, discriminatory power and clinical applicability of the nomogram were determined by the consistency index (C-index), calibration curves and decision curve analysis (DCA). In addition, a single-centre cohort of 135 cases was used for external validation. Multivariate Cox regression analysis showed that the independent influences on overall survival (OS) were age, type of disease, DNMT3A, IDH2 and TP53 mutations. The area under the curve (AUC) values for the training set were 0.755, 0.745 and 0.757 at 1, 2 and 3 years respectively; the AUC for the validation set were 0.648, 0.648 and 0.654 at 1, 2 and 3 years; and the AUC for the northwest China set were 0.692, 0.724 and 0.689 at 1, 2 and 3 years. The calibration and DCA indicated good consistency and clinical utility of the nomogram. Finally, younger (age <60 years) and elderly (age ≥60 years) patients were each divided into two risk groups with significantly different survival rates. A nomogram consisting of five risk factors was developed for forecasting the prognosis of AML with guaranteed reliability.

Clinical Features and Monitoring of Germline CEBPA-Mutated Carriers

Background: Despite being classified as an independent entity in World Health Organization (WHO) classification in 2016, familial cases of adult acute myeloid leukemia (AML) with germline-mutated CCAAT/enhancer-binding protein-α (CEBPA) gene have been rarely reported due to insufficient knowledge of the clinical features of this entity. Germline CEBPA-mutated carriers are at extremely high risk of developing AML but have long been ignored until the onset of AML. We aimed to fully identify the clinical features of the familial cases and healthy carriers for this entity to call for a guideline for its management. Methods: Whole exome sequencing (WES) data were analyzed in four members from one hereditary AML Chinese family, including two AML patients, one obligated carrier, and one non-consanguineous member. The whole coding region of CEBPA was sequenced in 21 members in this family and 6 members in another AML family. Deep sequencing targeting 248 genes related to myeloid malignancies was performed on 2 germline CEBPA-mutated carriers. To fully estimate the incidences of AML for each germline CEBPA mutation, we performed a thorough literature search for multiple members carrying germline CEBPA mutations. Results: Here, we report two Chinese families with multiple AML cases carrying germline CEBPA mutations (c.247C&amp;gt;T; p.Gln83*). One family had 11 cases in four consecutive generations and 5 healthy carriers, and the other had 2 cases in two generations. Together with these two families, we collected clinical data from 57 AML patients in 22 families with germline CEBPA-mutation in total. 48 out of 57 AML were confirmed carrying germline CEBPA mutations. 58.3% (28/48) of AML harbored double CEBPA mutations. The first hit frequently occurred at the N-terminal of CEBP/α (22/28, 78.6%), resulting in an exclusive expression of p30 at the second translation starting site of CEBPA (hereafter, CEBPA p30). The second hit normally located at C-terminal of CEBP/α (hereafter, CEBPA others). Germline CEBPA p30-carriershave higher incidences of AML compared to those with germline CEBPA others (80.36% vs 42.86%, P=0.0003). The age at onset is much younger in patients carrying germline CEBPA p30 than those carrying CEBPA others (18 vs 38.5, P=0.0093). Among cases with detailed treatment information, 10/36 did not receive standard chemotherapies according to the NCCN guidelines for AML, and 16/36 received transplantation, nearly half (7/16) of which went through autologous or synergetic (monozygotic twin) transplantation and maintained endurable complete remission. 41.7% (15/36) of AML had at least one episode of relapse, and recurrent relapses occurred in 1/3 (5/15) of the AML patients. Intriguing, despite the high rates of relapse, only 3/15 of AML patients died of relapses. Most familial AML cases with germline CEBPA mutations had a favorable overall survival (OS). We found the OS in AML patients with germline CEBPA p30 is better than those with CEBPA others (&amp;gt;25 years vs. 11 years, P=0.0125). There are 27 healthy germline CEBPA mutated carriers aged 14 to 88, 20 of which were selected for further analysis due to complete age information, 80% (16/20) are still within the range of age at onset of familial AML with germline CEBPA mutations. We detected a pre-leukemia clone harboring a pathogenic IDH2 variant (Arg140Gln) in 1 germline CEBPA-mutated carrier, who is currently healthy. Conclusions: The high penetrance accompanied by early onset in AML with CEBPA p30 mutation implies the mutation as a strong genetic risk factor for AML . The intensity of chemotherapy should be tailored for familial AML with germline CEBPA mutations. Our study is the most extensive clinical data for familial AML cases with germline CEBPA mutations and is the first to reveal precise incidences of AML with certain germline CEBPA mutations. These data should aid in genetic counsel and the management of AML patients and healthy carriers with germline CEBPA mutations.

DNMT3A Mutants Are Enriched in NPMc+ AML and Associated with Adverse Outcome in Childhood AML

Background: Somatic mutations in the DNA methyltransferase 3 alphagene (DNMT3A) are common in adult acute myeloid leukemia (AML), but are rare events in childhood AML (Ho, Ped Blood Cancer, 2011). Missense mutations occur most frequently in the catalytic domain at R882, resulting in loss of enzyme methylation activity and the ability of the enzyme to bind DNA. DNMT3Amutations are associated with adverse outcome in adults, while their impact is understudied in childhood AML. Our objective was to identify the prevalence of DNMT3A -mutated AML across the childhood age spectrum utilizing a large cohort of patients enrolled on pediatric trials and compare findings with adult cohorts. Methods: Pediatric patients treated on two consecutive COG trials with publicly available data were included (n=1607). Mutational profiling was performed by whole genome and transcriptome sequencing methods. Prevalence of DNMT3Amutations, co-occurring mutations, and outcomes (5-year event-free [EFS] and 5-year overall survival [OS]) were analyzed. Results: The prevalence of DNMT3Amutations amongst the entire childhood cohort was 1.2% (n=20/1607). DNMT3A mutations were absent in children &amp;lt;10 years with increased incidence up to 2.6% in patients &amp;gt;10yrs of age. All variants were missense, with the exception of one nonsense mutation and alterations at the 882 residue were the most common (70%). Most importantly, all but 4 DNMT3A mutations occurred in patients with NPM1 mutations (NPMc+; 80%). Conversely, DNMT3A mutations were seen in 9% of all NPM positive patients, demonstrating unique enrichment of this mutation within the NPM1 mutant cohort. Further, R882 mutations were seen in all but a single patient with the dual NPM/DNMT3A variant. It is notable that 11 patients harbored additional FLT3-ITD mutations (triple positive), which we have previously reported to be associated with adverse outcome in adults. In addition to mutations in NPM1 and FLT3, co-occurring variants with DNMT3A were also found in PTPN11 (35%), and WT1, IDH1/2 and MYC each at 10% frequency (Fig.A). Given the recent report of association of NPM1 mutation genotype and association of Type D mutation with adverse outcome (Pigazzi, ASH 2023), we evaluated NPM1 mutation genotype in those with cooperating DNMT3A mutation which demonstrated lack of association of DNMT3A mutations and Type D NPM1 mutations. Current risk classification in COG pediatric AML trials treat NPMc+ patients as favorable risk. Given the prevalence of DNMT3A mutations in NPMc+ patients we inquired whether DNMT3A mutations might modify outcome in this population. The 5 yr EFS estimates for NPMc+; patients with and without DNMT3A was 26.8% vs. 74.8% (p&amp;lt;0.0001; Fig.B) with a corresponding OS estimate at 5yrs of 60.3% vs. 82.2% (p=0.0169). We further concluded that presence or absence of FLT3-ITD does not impact the adverse outcome with similar 5 yr EFS for those with dual NPM1/DNMT3A or triple NPM1/DNMT3A/FLT3-ITD positive AML(20.0% vs. 30.3%respectively). These data are consistent with the adult DNMT3A data previously presented last year (Torabi; ASH 2022; abstract #306) regarding clinical implications of DNMT3A mutation in the setting of cooperating NPM1 mutation. Conclusion: Mutational profiling of children with AML identified DNMT3A mutations in adolescents and young adults and is enriched in those with NPMc+ mutations. As NPMc+ patients are considered to be favorable risk, knowledge of DNMT3A mutations would provide additional information for more precise risk stratification in childhood AML. Children with de novo AML and dual DNMT3A/NPM1 mutations remain at high risk of relapse and adverse outcome regardless of FLT3-ITD status.

Proteomic Profiling Identifies Unique IDH Mutant Signatures and Novel Therapeutic Opportunities in Adult Acute Myeloid Leukemia Patients

CONCLUSION Patients with IDH mutations are common in adult Acute Myeloid Leukemia (AML), with selective IDH inhibitors (IDHi) being recommended, but they do not always respond. We evaluated the proteome of IDH mutant AML patients to identify prognostic signatures and therapeutic targets. The expression of 429 proteins (339 total, 90 post-translational modified) were measured by Reverse Phase Protein Array in fresh samples from 805 newly diagnosed AML patients, and normalized to normal CD34+ cells. We analyzed 151 patients with IDH mutations (IDH1 = 56, IDH2 = 76, both = 19), treated with AraC-based combination therapy (N=71, including 5 with IDHi, 11 with Venetoclax (VTX)) or Hypomethylating agent based (HMA, N=65, + IDHi=5, +VTX=11, or both=2) and 3 received IDHi monotherapy, Proteins prognostic for Overall Survival (OS) were identified using pairwise LogRank tests (p&amp;lt;0.05) adjusted for False Discovery Rate (FDR), followed by unbiased hierarchical clustering. Continuous and categorial variables were compared with Wilcoxon, Kruskal-Wallis (KS) or Fisher's Exact test with simulated p-values (10000 replicates). Cox proportional hazards models (CoxPH) were developed for Uni-(UV) and Multi-variate (MV) analysis. Differentially expression (DE) analysis was performed using the KS test and pairwise Dunn's tests with p-values adjusted with FDR (p&amp;lt;0.01). Deep neural network analysis using random forest models identified proteins most predictive of cluster assignment. 97 proteins were individually prognostic for OS, and formed 3 patient clusters (Fig. 1: C1=red, C2=blue, C3=green). Most clinical and molecular features were not biased between clusters, however C2 had more cases with primary AML, diploid karyotype (kar.), intermed. cytogenetic (cyto.) risk, and IDH+FLT3_ITD or NPM1 mutants, while C3 has more cases with unfavorable cyto. risk, complex kar., and mutations in IDH+EZH2 or TP53, and ASLX1+IDH combination were more frequent in C1. Of note, C1 has the largest proportion of VTX-treated patients, while C3 has the largest proportion of IDHi-treated ones. The OS (Fig. 2) of C1 patients was better (Median: C1=42.2mo, C2=18.3mo, C3=10.7mo; p=0.0021), but no difference was noted for Complete Remission Duration (CRD) (Median: C1=72.3mo, C2=21.6mo, C3=33.4mo; p=0.7). The OS was similar when patients were filtered by specific mutations or combinations, e.g. IDH1, IDH2, and IDH combined with ASLX1, RUNX1 or TET2, or by other features, e.g. 56-70 age group, males, primary AML, intermed. cyto. risk, diploid kar., VTX or HMA without IDHi and/or VTX (HMA-TX) therapies. CoxPH models showed only C1 and C3 predictive for OS in the UV analysis (HR=1, 1.5, 2.3; p=ref., 0.22, &amp;lt;0.001), along with age, secondary AML, complex, diploid and del12 kar., HMA-TX, and IDH1, IDH2, NPM1, TP53, DNMT3a and JAK2 mutations. In the MV analysis, C1, C2 and C3 (HR=1, 2.6, 3.4; p=ref., 0.019, &amp;lt;0.001), age, secondary AML, complex and diploid kar., HMA-TX and IDH1 mutation predicted OS. Regarding CRD, no cluster was predictive in the UV model (HR=1, 1.2, 1.4; p=ref., 0.65, 0.41), but age, secondary AML, complex kar., HMA-TX, and ASLX1, EZH2 and IDH2 mutants were. In contrast, C1 and C3 (HR=1, 2.3, 2.6; p=ref., 0.13, 0.046) predicted CRD in the MV model, as well as secondary AML, HMA-TX and IDH2 mutation. epigenetics, DNA damage response, cell cycle and metabolism, with 23, 12, 12, and 11 proteins. DE analysis identified 133 proteins among 429, including: ADM, LCK and BTK up-, and TP53BP1 and PRMT1 down-regulated in C1; ANXA1 up-, and PTK2 and SRC down-regulated in C2. In C3, TGM2 is higher than normal, although lower than in C1. Inhibitors for upregulated proteins are in clinical development, potentially working as synergistic agents. Furthermore, a set of 5 proteins (SMARCB1, SMARCA2, WTAP, PIK3CB and RAD50) predicted cluster membership with 97% of accuracy, useful for quickly identifying patients and personalizing therapy. Proteomics identified unique protein profiles that were independently prognostic for outcome in IDH mutant AML patients. We identified upregulated proteins for which inhibitors are in development, suggesting potential therapeutic synergies to improve outcomes. Finally, we defined 5 proteins that accurately stratifies IDH patients for novel treatment recommendations.

Myeloid Master Regulators PU.1 and CEBPA Suppress Leukemogenesis through Downregulating Cell Cycle Related Genes in NPM1 Mutated Leukemia

Nucleophosmin1 mutation (NPM1c) is the most common mutation in adult acute myeloid leukemia (AML) and known as a driver mutation causing leukemogenesis through upregulating homeotic gene transcription. Mutated NPM1 is reported to mis-localize in the cytoplasm. However, how cytoplasmic NPM1c regulates leukemic transcription remains poorly understood. We previously reported that NPM1c reprograms homeotic and leukemogenic transcription through reshaping CTCF-defined three-dimensional topology associated domain (TAD). Also, re-localization of cytoplasmic NPM1c to the nucleus switches MIZ1/MYC repressive transcription regulatory axis to MIZ1/NPM1 active transcription regulatory axis at CDKN locus and CEBPA locus leading to cell cycle arrest and myeloid differentiation in NPM1 mutated AML. In this study, we are focused on regulation of myeloid differentiation and hypothesized upregulation of myeloid master regulators, PU.1 and CEBPA may suppress the leukemogenic transcription and induce myeloid differentiation in NPM1 mutated AML cells. To confirm NPM1c regulates TAD formation at CEBPA locus, we performed HiC-seq in OCI-AML3 with nuclear re-localized NPM1c using XPO1 inhibitor treatment and NPM1-wildtype overexpression. Nuclear re-localized NPM1 upregulates CTCF-defined TAD interaction at CEBPA locus and upregulates CEBPA and CEBPD transcription which indicates NPM1c regulates CEBPA transcription via altering CEBPA TAD formation. We then test whether activation of CEBPA or SPI1 in NPM1 C+ AML cells induced myeloid differentiation and blocked NPM1 C+ driven leukemogenesis. We transcriptionally activated SPI1 or CEBPA gene in OCI-AML3 using CRISPR-dCas9-VP160 system to determine how these myeloid regulators changed the leukemic transcription and leukemic cell phenotype. PU.1- or CEBPA- activated OCI-AML3 reduces cell proliferation, especially affecting the G2/M phase in cell cycle. They have monocyte-like nuclei with large cytoplasm and induce myeloid differentiation with upregulating both of CD11b and CD14 myeloid cell surface markers. Moreover, compared to parental OCI-AML3 cells, Pu.1- or CEBPA-activated cells showed lower colony formation ability and in xenograft mouse models produced diminished leukemic symptoms and prolonged survival. We further conducted RNA-seq to explore how PU.1 or CEBPA changed the gene transcription leading to the blockage of leukemogenesis. Between PU.1- and CEBPA-activated cells, 58% of differentially expressed genes overlap and GO or GSEA analysis revealed that both of them downregulates G2M checkpoint/mitotic-spindle cell cycle related gene sets, indicating that PU.1 or CEBPA activation controls cell cycle control rather than myeloid cell differentiation in NPM1 C+ AML. In summary, our data revealed that re-localization of NPM1 C+ into nucleus activates master myeloid transcription factors, PU.1 and CEBPA, which, in turn, suppress leukemogenesis via inducing cell cycle arrest and myeloid cell differentiation in NPM1 C+ AML.

Ubtf- Tandem Duplications Are Recurrent Somatic Genetic Events in Adult AML and Associate with Additional Structural Variants and a Distinct Gene Expression Profile (Alliance)

Background The gene upstream binding transcription factor ( UBTF ) encodesa nucleolar protein that is a key regulator of the epigenetic status of ribosomal DNA and RNA transcription. Tandem duplications (TDs) of the UBTF gene have been initially identified in pediatric patients (pts) with acute myeloid leukemia (AML) and were recently also reported as relatively rare, but recurrent genetic events in adult AML based on targeted re-sequencing efforts (1.2%-3%). Their presence was associated with dysplastic bone marrow features, and co-occurrence of select genetic features, including FLT3-internal tandem duplications (ITDs) and WT1 mutations, and inferior outcome. However, associated biologic features and affected pathways have not yet been thoroughly investigated, and may provide further insights into leukemogenesis and identify potentially targetable vulnerabilities. Thus, we set out to assess the frequency of UBTF-TDs in a large cohort of adult AML pts (n=807) treated on frontline protocols of the Alliance for Clinical Trials in Oncology with complete RNA sequencing data, comprehensive targeted mutation profiling, centrally reviewed cytogenetic data and clinical information available, and to gain further insights into UBTF-TDs via comprehensive integrated genomic profiling. Methods UBTF-TD detection was performed via the RNAseq-based RNAmut and EnFusion algorithm. To gain insights into associated molecular features, all UBTF-TD + pts underwent paired tumor-normal whole-exome sequencing. To understand associated pathways and downstream effects, differential gene expression (DGE) and gene set enrichment analyses (GSEA) were performed. Results UBTF-TDs were detected in 6/807 pts (0.7%). In line with previous studies, these 6 UBTF-TD + pts were young, with a median age of 35y (range: 19-59y). Three pts had extramedullary disease, 2 with skin and 1 with gingival involvement. All but 1 of the 6 pts were cytogenetically normal. Clinically, pts did poorly after treatment with intensive chemotherapy induction, with a CR rate of only 33%. No pt was alive 2.5 y after diagnosis. In paired tumor/normal exome sequencing, UBTF-TD + pts had a median mutation burden of 21 non-synonymous variants in coding genes (range: 17-27), with a notable presence of larger insertions/deletions (indels), loss of heterozygosity (LOH) or TDs as co-occurring somatic genetic lesions (median: 4, range: 2-5). There was a 100% co-occurrence of UBTF-TDs with FLT3-ITDs. In addition, pts harbored larger indels in WT1 (n=2), BPTF, CEBPA, Corf53, KRTAP5-7, NFE2, PTPRN2, RNF144B, SOWAHC and ZCCHC14. Also, analysis of copy-number variations and LOH revealed LOH in 5 pts, in chromosomes 13 (n=3), 15 (n=1) and 18 (n=1), suggestive of a propensity to the acquisition of structural variants. In unsupervised clustering analysis of the RNAseq data, UBTF-TD + pts clustered with FLT3-ITD + pts but formed a relatively distinct subcluster in principal component and t-distributed stochastic neighbor embedding analyses. In supervised clustering comparing 6 UBTF-TD +/ FLT3-ITD + and 196 UBTF-TD -/ FLT3-ITD + pts, 2 UBTF-TD - pts clustered together with the UBTF-TD +/ FLT3-ITD + group, suggesting either presence of an undetected TD or genetic phenocopy. UBTF expression was higher in UBTF-TD pts. DGE analysis of UBTF-TD +/ FLT3-ITD + and UBTF-TD -/ FLT3-ITD + pts (Fig 1) showed distinct upregulation of the locus of the stem cell and chemoresistance marker ALDH1A3, including the associated long non-coding RNAs AC015712.7/AC015712.2 in UBTF-TD + pts, which was confirmed by a comparison of those pts to AML pts who had neither TD (n=605). GSEA indicated a downregulation of inflammation pathways and several metabolic pathways of UBTF-TD +/ FLT3-ITD + compared to FLT3-ITD + pts (Fig 2). Conclusions UBTF-TDs are rare, but recurrent genetic events in adult AML, associated with high chemotherapy resistance. Our results highlight a susceptibility of those pts to the acquisition of structural variants and suggest a distinct associated biology, with upregulation of ALDH1A3 as possible contributing resistance mechanism. Support: U10CA180821, U10CA180882, U24CA196171; Clinicaltrials.gov Identifiers: NCT00048958 (CALGB 8461), NCT00899223 (CALGB 9665), NCT00900224 (CALGB 20202); https://acknowledgments.alliancefound.org