NPM1-mutated Acute Myeloid Leukemia Research Articles

Targeting and Monitoring Acute Myeloid Leukaemia with Nucleophosmin-1 (NPM1) Mutation.

Mutations in NPM1, also known as nucleophosmin-1, B23, NO38, or numatrin, are seen in approximately one-third of patients with acute myeloid leukaemia (AML). A plethora of treatment strategies have been studied to determine the best possible approach to curing NPM1-mutated AML. Here, we introduce the structure and function of NPM1 and describe the application of minimal residual disease (MRD) monitoring using molecular methods by means of quantitative polymerase chain reaction (qPCR), droplet digital PCR (ddPCR), next-generation sequencing (NGS), and cytometry by time of flight (CyTOF) to target NPM1-mutated AML. Current drugs, now regarded as the standard of care for AML, as well as potential drugs still under development, will also be explored. This review will focus on the role of targeting aberrant NPM1 pathways such as BCL-2 and SYK; as well as epigenetic regulators (RNA polymerase), DNA intercalators (topoisomerase II), menin inhibitors, and hypomethylating agents. Aside from medication, the effects of stress on AML presentation have been reported, and some possible mechanisms outlined. Moreover, targeted strategies will be briefly discussed, not only for the prevention of abnormal trafficking and localisation of cytoplasmic NPM1 but also for the elimination of mutant NPM1 proteins. Lastly, the advancement of immunotherapy such as targeting CD33, CD123, and PD-1 will be mentioned.

Heliangin acts as a covalent ligand of RPS2 that disrupts pre-rRNA metabolic processes in NPM1-mutated acute myeloid leukemia.

Although NPM1 mutations are frequently found in acute myeloid leukemia patients, therapeutic strategies are scarce and unsuitable for those who cannot tolerate intensive chemotherapy. Here we demonstrated that heliangin, a natural sesquiterpene lactone, exerts favorable therapeutic responses in NPM1 mutant acute myeloid leukemia cells, with no apparent toxicity to normal hematogenous cells, by inhibiting their proliferation, inducing apoptosis, causing cell cycle arrest, and promoting differentiation. In-depth studies on its mode of action using quantitative thiol reactivity platform screening and subsequent molecular biology validation showed that the ribosomal protein S2 (RPS2) is the main target of heliangin in treating NPM1 mutant AML. Upon covalent binding to the C222 site of RPS2, the electrophilic moieties of heliangin disrupt pre-rRNA metabolic processes, leading to nucleolar stress, which in turn regulates the ribosomal proteins-MDM2-p53 pathway and stabilizes p53. Clinical data shows that the pre-rRNA metabolic pathway is dysregulated in acute myeloid leukemia patients with the NPM1 mutation, leading to a poor prognosis. We found that RPS2 plays a critical role in regulating this pathway and may be a novel treatment target. Our findings suggest a novel treatment strategy and lead compound for acute myeloid leukemia patients, especially those with NPM1 mutations.

155 - Outcomes with Allogeneic Hematopoietic Stemcell Transplantation in NPM1-Mutated Acute Myeloid Leukemia: A Systematic Review And Meta-Analysis

155 - Outcomes with Allogeneic Hematopoietic Stemcell Transplantation in NPM1-Mutated Acute Myeloid Leukemia: A Systematic Review And Meta-Analysis

Mutational analysis of DNMT3A improves the prognostic stratification of patients with acute myeloid leukemia.

Nucleophosmin1 (NPM1) mutations are the most frequently detected gene mutations in acute myeloid leukemia (AML) and are considered a favorable prognostic factor. We retrospectively analyzed the prognosis of 605 Japanese patients with de novo AML, including 174 patients with NPM1-mutated AML. Although patients with NPM1-mutated AML showed a high remission rate, this was not a favorable prognostic factor for overall survival (OS); this is contrary to generally accepted guidelines. Comprehensive gene mutation analysis showed that mutations in codon R882 of DNA methyltransferase 3A (DNMT3AR882 mutations) were a strong predicative factor indicating poor prognosis in all AML (p < 0.0001) and NPM1-mutated AML cases (p=0.0020). Furthermore, multivariate analysis of all AML cases showed that DNMT3AR882 mutations and the co-occurrence of internal tandem duplication in FMS-like tyrosine kinase 3 (FLT3-ITD), NPM1 mutations, and DNMT3AR882 mutations (triple mutations) were independent factors predicting a poor prognosis related to OS, with NPM1 mutations being an independent factor for a favorable prognosis (hazard ratios: DNMT3AR882 mutations, 1.946; triple mutations, 1.992, NPM1 mutations, 0.548). Considering the effects of DNMT3AR882 mutations and triple mutations on prognosis and according to the classification of NPM1-mutated AML into three risk groups based on DNMT3AR882 /FLT3-ITD genotypes, we achieved the improved stratification of prognosis (p < 0.0001). We showed that DNMT3AR882 mutations are an independent factor for poor prognosis; moreover, when confounding factors that include DNMT3AR882 mutations were excluded, NPM1 mutations were a favorable prognostic factor. This revealed that ethnological prognostic discrepancies in NPM1 mutations might be corrected through prognostic stratification based on the DNMT3A status.

Cytoplasmic Expression of TP53INP2 Modulated by Demethylase FTO and Mutant NPM1 Promotes Autophagy in Leukemia Cells.

Acute myeloid leukemia (AML) with a nucleophosmin 1 (NPM1) mutation is a unique subtype of adult leukemia. Recent studies show that NPM1-mutated AML has high autophagy activity. However, the mechanism for upholding the high autophagic level is still not fully elucidated. In this study, we first identified that tumor protein p53 inducible nuclear protein 2 (TP53INP2) was highly expressed and cytoplasmically localized in NPM1-mutated AML cells. Subsequent data showed that the expression of TP53INP2 was upregulated by fat mass and obesity-associated protein (FTO)-mediated m6A modification. Meanwhile, TP53INP2 was delocalized to the cytoplasm by interacting with NPM1 mutants. Functionally, cytoplasmic TP53INP2 enhanced autophagy activity by promoting the interaction of microtubule-associated protein 1 light chain 3 (LC3) - autophagy-related 7 (ATG7) and further facilitated the survival of leukemia cells. Taken together, our study indicates that TP53INP2 plays an oncogenic role in maintaining the high autophagy activity of NPM1-mutated AML and provides further insight into autophagy-targeted therapy of this leukemia subtype.

Successful Management of NPM1-Mutated Acute Myeloid Leukemia in a Patient with Robinow Syndrome

Successful Management of NPM1-Mutated Acute Myeloid Leukemia in a Patient with Robinow Syndrome

The Impact of Mutation of Myelodysplasia-Related Genes in De Novo Acute Myeloid Leukemia Carrying NPM1 Mutation.

Background: The impact of gene mutations typically associated with myelodysplastic syndrome (MDS) in acute myeloid leukemia (AML) with NPM1 mutation is unclear. Methods: Using a cohort of 107 patients with NPM1-mutated AML treated with risk-adapted therapy, we compared survival outcomes of patients without MDS-related gene mutations (group A) with those carrying concurrent FLT3-ITD (group B) or with MDS-related gene mutations (group C). Minimal measurable disease (MMD) status assessed by multiparameter flow cytometry (MFC), polymerase chain reaction (PCR), and/or next-generation sequencing (NGS) were reviewed. Results: Among the 69 patients treated intensively, group C showed significantly inferior progression-free survival (PFS, p < 0.0001) but not overall survival (OS, p = 0.055) compared to group A. Though groups A and C had a similar MMD rate, group C patients had a higher relapse rate (p = 0.016). Relapse correlated with MMD status at the end of cycle 2 induction (p = 0.023). Survival of group C patients was similar to that of group B. Conclusion: MDS-related gene mutations are associated with an inferior survival in NPM1-mutated AML.

Overlapping features of therapy-related and de novo NPM1-mutated AML

AbstractNPM1-mutated acute myeloid leukemia (AML) shows unique features. However, the characteristics of “therapy-related” NPM1-mutated AML (t-NPM1 AML) are poorly understood. We compared the genetics, transcriptional profile, and clinical outcomes of t-NPM1 AML, de novo NPM1-mutated AML (dn-NPM1 AML), and therapy-related AML (t-AML) with wild-type NPM1 (t-AML). Normal karyotype was more frequent in t-NPM1 AML (n = 78/96, 88%) and dn-NPM1 (n = 1986/2394, 88%) than in t-AML (n = 103/390, 28%; P < .001). DNMT3A and TET2 were mutated in 43% and 40% of t-NPM1 AML (n = 107), similar to dn-NPM1 (n = 88, 48% and 30%; P > 0.1), but more frequently than t-AML (n = 162; 14% and 10%; P < 0.001). Often mutated in t-AML, TP53 and PPM1D were wild-type in 97% and 96% of t-NPM1 AML, respectively. t-NPM1 and dn-NPM1 AML were transcriptionally similar, (including HOX genes upregulation). At 62 months of median follow-up, the 3-year overall survival (OS) for t-NPM1 AML (n = 96), dn-NPM1 AML (n = 2394), and t-AML (n = 390) were 54%, 60%, and 31%, respectively. In multivariable analysis, OS was similar for the NPM1-mutated groups (hazard ratio [HR] 0.9; 95% confidence interval [CI], 0.65-1.25; P = .45), but better in t-NPM1 AML than in t-AML (HR, 1.86; 95% CI, 1.30-2.68; P < .001). Relapse-free survival was similar between t-NPM1 and dn-NPM1 AML (HR, 1.02; 95% CI, 0.72-1.467; P = .90), but significantly higher in t-NPM1 AML versus t-AML (HR, 1.77; 95% CI, 1.19-2.64; P = .0045). t-NPM1 and dn-NPM1 AML have overlapping features, suggesting that they should be classified as a single disease entity.

Supplementary Information -TG-interacting factor 1 improves risk stratification in patients with NPM1-mutated acute myeloid leukemia

Supplementary Information -TG-interacting factor 1 improves risk stratification in patients with NPM1-mutated acute myeloid leukemia

Machine Learning Allows the Identification of New Co-Mutational Patterns with Prognostic Implications in NPM1 Mutated AML - Results of the European Harmony Alliance

Background: Mutations of NPM1 are generally considered as a favorable prognostic marker in acute myeloid leukemia (AML), although patients present with distinct co-mutations that might influence outcomes. The recently updated European LeukemiaNet classification (ELN2022) does now group NPM1mut AML patients with FLT3-ITD (regardless of the allelic ratio) as intermediate prognosis, while NPM1mut without FLT3-ITD is part of the favorable group. However, the mutational landscape of NPM1mut AML is more complex and analysis of a large cohort of patients might allow further dissection to identify less frequent co-mutational patterns of prognostic and predictive importance. Aims: To identify clinically significant co-mutational patterns in NPM1mut AML to further refine risk stratification models for this subset of patients. Methods: From the HARMONY Alliance database, a total of 1093 intensively treated NPM1mut patients were selected. Patients treated with targeted therapies (e.g. FLT3 or IDH1/2 inhibitors) were not included and NPM1 MRD information was not available at this stage of the analysis. A machine learning algorithm was developed in order to identify combinations of up to 4 co-mutated genes with potential impact on overall survival (OS). A heuristic search algorithm was implemented and bootstrap sampling was applied to estimate the impact of all possible gene combinations on OS. In addition, a global dashboard was developed where it was possible to compare mutational combinations with Kaplan Meier and Cox regression models. Finally, clinically significant co-mutational patterns were summarized in a novel risk stratification model for NPM1mut AML, which was validated using a publicly available external patient cohort (Awada et al, Blood, 2021). Results: The study population of 1093 NPM1mut AML patients included 57% females and median age was 53 years. Regarding ELN2022 classification, 57% of patients were classified into the favorable, 42% into intermediate and only 1% into adverse risk groups. The most frequent co-mutations were DNMT3A (54%) and FLT3-ITD (42%), followed by NRAS, FLT3-TKD and TET2 (20% each). Mutations on IDH1 (13%) and IDH2 (15%) showed a similar behavior for all the analyses performed, so for simplification purposes we will refer to IDHmut when any of them was mutated and to IDHwt when both were wildtype. The triple combination of NPM1mut + FLT3-ITD + DNMT3Amut identified a subgroup of patients with adverse prognosis (2-year OS of 33%), similar to patients with TP53mut. Of note, not all FLT3-ITD patients carried an intermediate or adverse prognosis, as we were able to identify a subgroup (FLT3-ITD + IDHmut + DNMT3Awt) with excellent prognosis (2-year OS of 80%), which represented 4% of NPM1mut AML. However, in the absence of FLT3-ITD and the presence of DNMT3Amut, the addition of IDHmut decreased OS towards the intermediate risk (2-year OS 59%). Notably, not all DNMT3Amut patients carried an intermediate or adverse prognosis. In the absence of FLT3-ITD and with IDHwt, mutations on either NRAS, KRAS, PTPN11 or RAD21 revealed a subgroup with favorable prognosis even when DNMT3A was mutated (2-year OS 80%) and this group represented 11% of NPM1mut AML. This information is summarized in a 4-category risk stratification model (figure 1). The revised NPM1mut favorable group presented with a 3-year OS of 78%, which was 63%, 48% and 29% for intermediate-1, intermediate-2 and adverse risk groups respectively (p<0.001). Regarding 3-year relapse free survival (RFS), it was 71%, 59%, 39% and 26% accordingly (p<0.001). In the validation cohort, 3-year OS of was 73% for NPM1mut favorable group, being 59%, 40% and 22% for intermediate-1, intermediate-2 and adverse groups respectively (p<0.001). Multivariate OS analysis in NPM1mut AML identified the following independent prognostic factors: revised NPM1mut model (taking favorable group as reference, HR 1.77 for intermediate-1, HR 2.92 for intermediate-2 and HR 5.13 for adverse group, p<0.001); secondary or therapy-related AML (HR 1.93, p<0.001), age >60 years (HR 1.84, p<0.001), WBC at diagnosis >100x103/uL (HR 1.59, p<0.001), and male gender (HR 1.21, p=0.04). Conclusion/summary: Analysis of large NPM1mut AML cohorts allows the identification of clinically significant co-mutational patterns. We propose a new genetic stratification model for NPM1mut AML that identifies 4 groups with different OS and RFS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SYK Inhibitors, Entospletinib and Lanraplenib, Show Potent Anti-Leukemic Activity in Combination with Targeted Agents

Introduction: Spleen tyrosine kinase (SYK) acts as a key integrator of cellular signaling from surface contact and receptor tyrosine kinase receptors containing an immunoreceptor tyrosine-based activation motif (ITAM). In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network (TRN) linked to NPM1, HOXA9, and MEIS1 helping to suppress myeloid progenitor maturation and promote proliferation. The selective, oral SYK inhibitor, entospletinib (ENTO), has demonstrated clinical activity in HOXA9/MEIS1-driven AML with acceptable tolerability when combined with intensive induction chemotherapy. A global phase 3 trial (AGILITY, NCT05020665) is currently underway, evaluating the combination of ENTO with intensive induction/consolidation chemotherapy in patients with treatment-naive NPM1-mutated (NPM1m) AML with measurable residual disease as the primary endpoint. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency, enhanced selectivity, and more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in patients with relapsed or refractory (R/R) FLT3-mutated AML (NCT05028751). Results: ENTO exhibits anti-proliferative activity in NPM1m AML with associated HOXA9/MEIS1 dysregulation in ex vivo drug sensitivity studies. We hypothesized that SYK inhibition could synergize with other targeted therapies to enhance anti-leukemic activity at different nodes in this pathway. To address this hypothesis, the FLT3 internal tandem duplication/MLL-rearranged cell lines, MOLM13 and MV411, were treated with increasing doses of either LANRA or ENTO in combination with a small molecule MLL-Menin inhibitor (SNDX5613). Synergistic anti-proliferative effects were observed across a broad range of concentrations. No combinatorial or single-agent SNDX5613 activity was seen in the KASUMI-1 AML cell line, which lacks an MLL rearrangement. Phenotypic studies using flow cytometry were performed to assess the mechanistic response for SYK inhibition with LANRA in combination with SNDX5613. LANRA was found to increase the percentage of CD11b-positive cells and mean fluorescence intensity, while SNDX5613 led to a marked increase in CD14. In contrast to either single agent, the combination triggered early apoptosis and cell death, suggesting a more complete blockade of the HOXA9/MEIS1 transcriptional program through synergistic inhibition by orthogonal mechanisms. Furthermore, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. We also explored the potential for synergistic activity with gilteritinib (FLT3 inhibitor) and venetoclax (BCL2 inhibitor) using patient-derived AML isolates ex vivo based on the potential for SYK signaling cross talk with FLT3 itself and survival pathways. We found strong synergistic anti-proliferative activity for the combination of LANRA with either gilteritinib or venetoclax. Patient-derived xenograft (PDX) studies also demonstrated deeper reductions in leukemic burden in the peripheral blood, liver, and bone marrow after 28 days of treatment. Follow-up PDX studies assessing overall survival and pharmacodynamic activity are ongoing using an optimized dosing regimen and schedule. Summary/Conclusion: SYK is a promising therapeutic target for genetically defined subsets of AML. Two highly selective SYK inhibitors are currently undergoing clinical testing in different but complementary clinical settings. Given its central role as a modulator of leukemogenic signaling, SYK inhibition has the potential to synergize with other targeted therapies in AML. In preclinical studies, LANRA-mediated SYK inhibition shows compelling anti-proliferative and pro-apoptotic activity in combination with gilteritinib, venetoclax, and SNDX5613 in genetically defined subsets of AML.

The Prognostic Impact of FLT3 in NPM1-Mutated AML: Co-Occurrence of FLT3-ITD and FLT3-TKD Confers Poor Outcomes

Background:NPM1 and FLT3 are commonly mutated in patients with acute myeloid leukemia (AML). While FLT3 internal tandem duplication (ITD) is known to confer worse prognosis even in the setting of NPM1 according to the recent European LeukemiaNet (ELN) 2022 criteria, the prognostic impact of FLT3 tyrosine kinase domain (TKD) in this population remains unclear. One recent study suggests FLT3-TKD and NPM1 co-mutation defines a highly favorable prognostic group (Boddu et al.). Furthermore, NPM1-mutated AML with both FLT3-ITD and FLT-TKD mutations are rare and not well described. Objective: In this study, we explored the prognostic impact of FLT3-ITD and/or FLT3-TKD in patients with NPM1-mutated AML. Methods: This was a multicenter, retrospective study (Moffitt Cancer Center, Weill Cornell, and Memorial Healthcare System) of NPM1-mutated AML patients who were diagnosed and treated from 2013 to June 2022. Inclusion was restricted to NPM1-mutated AML patients who also harbored FLT3-ITD, FLT-TKD, or both (n=107). All patients have next generation sequencing (NGS) performed at diagnosis. Kaplan-Meier, univariate (log-rank), and multivariate (Cox regression) analyses were performed. Results: Among 107 patients (47M/60F, median age 62 at diagnosis, range 23-79), 85% (91/107) had de novo AML. By ELN 2017 criteria, 37.4% (40/107) had favorable risk, 50.5% (54/107) had intermediate risk, and 9.3% (10/107) had adverse risk disease. Over 90% had intermediate risk cytogenetics at the time of diagnosis. Common co-mutations included DNMT3A (53.3%), TET2 (24.3%), WT1 (12.1%), IDH2 (8.4%), and IDH1 (6.5%) (Table 1). There were 66.4% (71/107) of patients with NPM1+FLT3-ITD, 23.4% (25/107) with NPM1+FLT3-TKD, and 10.3% (11/107) with NPM1 and both FLT3-ITD and FLT-TKD (NPM1+FLT3). Patients with NPM1+FLT3-ITD achieved CR/CRi in 84.5% (60/71) similar to patients with NPM1+FLT3-TKD, 88% (22/25), p=1.0. Most patients in both groups received intensive frontline therapy (80.3% vs. 72%, p=0.408). Forty-two (59.2%, 42/71) NPM1+FLT3-ITD patients received allogeneic hematopoietic cell transplant (HCT) compared to 36% (9/25) of NPM1+FLT3-TKD patients, p=0.193. The median overall survival (mOS) was not significantly different between NPM1+FLT3-ITD (42.4 months) and NPM1+FLT3-TKD (not reach), p=0.51. The NPM1+FLT3 cohort had dismal mOS of 8.4 months, which is significantly worse compared to those with NPM1+FLT3-ITD (p<0.005) and NPM1+FLT3-TKD (p=0.01) (Figure 1). Univariate analysis showed co-occurrence of FLT3-ITD and FLT-TKD significantly impacted OS (HR 2.94, 95% CI: 1.42-6.08, p=0.004). Multivariate analysis using covariates including age over 60, AML type, mutation burden (4 or more), and co-occurrence of FLT3-ITD and FLT-TKD confirmed its prognostic significance on survival (HR 2.96, 95% CI: 1.41-6.20, p=0.004). Conclusions: Our findings suggest very poor outcomes in patients with NPM1+FLT3 and no significant difference observed in survival between NPM1+FLT3-ITD and NPM1+FLT3-TKD. The prognosis of NPM1-mutated AML is increasingly being refined as more knowledge is gained in the molecular level. More studies are needed to understand the different gene-gene interactions that underlie this unique subset. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Proteomic Characterization of Acute Myeloid Leukemia

Introduction: Acute myeloid leukemia (AML) is a disease characterized by the expansion of myeloid blasts in the bone marrow. Recently, we published the results of the first proteogenomic characterization of bone marrow blasts from 177 adult patients with AML (10.1016/j.ccell.2022.02.006) that were uniformly treated with intensive induction chemotherapy. Using mass spectrometry (MS) and next-generation sequencing we curated a multi-omics data set with global protein and gene expression, DNA mutations and chromosomal aberrations. We identified a proteomic subtype characterized by enhanced expression of mitochondrial proteins, significantly poorer overall survival and high sensitivity to inhibitors targeting mitochondrial respiration (Mito-AML). We have now expanded the proteomic data set with bone marrow biopsies from 90 additional adult patients with AML in order to facilitate the proteomic characterization of individual genetic subtypes of AML including nucleophosmin 1 (NPM1)-mutated AML. Methods: We used a label-free (LF), data-independent (DIA) MS-based proteomic workflow to profile protein expression in bone marrow-derived leukemic blasts from 90 additional patients with AML. The patient cohort was similar to our initial cohort with regard to demographic-, disease- and clinical characteristics. We applied stringent filtering, missing value imputation and batch correction to merge the two cohorts. We were able to validate the proteomic Mito-AML subtype in this merged cohort by our previously published machine-learning model. Next, we investigated proteomic differences between NPM1mut (n=89) and NPM1wt (n= 172) patients using linear models; we identified differentially regulated biological processes from the gene ontology (GO) and REACTOME gene sets as well as gene expression signatures of healthy and malignant cellular subtypes (van Galen et al.) using a gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA). Clustering of the NPM1mut proteome was done using a consensus approach on the Euclidean distances and kmeans with k = 2 was chosen based on a set of metrices, including silhouette and concordance score. Results: The global protein expression structure, represented by inter-sample Euclidean distance, was largely independent of blast maturity, genetic risk groups and mutations. In our combined cohort of 267 patients, we identified 33 Mito-AML patients, confirming a frequency of 12 % of this proteomic AML subtype. As published by us previously, Mito-AML showed poor overall survival and independent, significant risk of death in the combined cohort as well as in the 90 patient sub-cohort. Mutations in NPM1 are frequently found in de novo AML and are characterized by a more chemo-sensitive disease. In addition to proteins previously described as being differentially expressed we identified a number of previously unreported proteins including surface proteins (e.g. DmX-like protein 2 (DMXL2)), to be significantly higher expressed in NPM1mut AML. Together with our proteomic data for CD34+ progenitor cells from healthy donors our resource data set might reveal potential target structures for cellular therapies. Among the gene sets differentially enriched between NPM1mut and NPM1wt were terms associated to mitochondrial respiration and metabolism. Interestingly, Mito-AML was enriched among NPM1mut AML (48.5% in NPM1mut vs. 32% in NPM1wt), indicating that Mito-AML is a high-risk subset within NPM1mut AML. Furthermore, we identified regulation of chromosomal and nucleosome architecture amongst the top down-regulated processes in NPM1mut which is in accordance with the role of wild-type NPM1 to chaperone these structural processes. Interestingly, we observed among NPM1mut AML two sub-clusters that were either enriched for blasts with myelo-monocytic differentiation or for progenitor- and stem cell-like blasts indicating a cell-of-origin phenotype. Conclusion: We report the expansion of our previously published proteomic characterization of AML with bone marrow-derived AML blasts from 90 additional patients. We confirm the proteomic Mito-AML subtype and its poor outcome under intensive chemotherapy. We furthermore elucidate the proteomic landscape of NPM1mut AML and identifie two subgroups of NPM1mut AML that are characterized by either myelo-monocytic differentiation or a progenitor and stem cell-like blast state. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Multiparameter Flow Cytometry-Based Residual Disease Analysis Identifies Intermediate-Risk AML Patients Who May Benefit from Allogeneic Hematopoietic Stem Cell Transplantation

Background Allogeneic hematopoietic stem cell transplantation (alloSCT) is a curative therapeutic modality in acute myeloid leukemia (AML). While numerous studies have confirmed the benefit of alloSCT - improved overall survival (OS) and reduced cumulative incidence of relapse (CIR) - restricted mainly to the intermediate and high-risk groups, indications for alloSCT are becoming more restricted over time. Residual disease (RD) monitoring has been proven to be highly predictive of long-term AML outcomes and is now used commonly to guide ongoing therapy decisions. RD-based guidelines in AML propose the use of multiparameter flow cytometry-based RD (FRD) analyses in patients whose disease cannot be followed by molecular means (e.g. CBF AML, APL, NPM1-mutated AML). Methods We performed a retrospective review to evaluate the potential therapeutic benefit of alloSCT in AML patients who achieved first complete remission (CR1) and to further define this benefit based on the FRD status at remission. We performed a time-dependent analysis using the covariate of time-to-alloSCT as the primary endpoint of the study. The Mantel-Byar method was applied to compare time to event between the alloSCT and non-transplant groups, addressing time bias concerns that occur with alloSCT studies, according to the FRD status at the time of remission. Results The FRD assay has been part of our standard of care since 2015 with a sensitivity of 0.1%. The cohort analyzed included 435 patients treated between 2015 and 2018, of which 336 patients had FRD assessment at CR1. The patients were risk classified using the European Leukemia Network (ELN) 2017 classification which integrates cytogenetic and mutational analyses. The proportion of patients with positive FRD (RDpos) at the post-induction time-point was 35.1% (n=118). Negative residual disease (RDneg) status after induction occurred more frequently in de novo, favorable risk, and NPM1-mutated AMLs, than in the adverse risk or secondary AML groups. Overall, across all ELN 2017 groups, the impact of alloSCT was neutral for both OS and relapse free survival (RFS). A potential impact of alloSCT could be discerned, however, when patients were grouped by FRD status at CR assessment. Overall, in the RDpos group, alloSCT improved OS and RFS, while not benefitting the RDneg group. The impact of alloSCT was defined further by considering ELN 2017 risk groups, with an OS/RFS benefit observed in adverse risk patients (n=186; HR for OS 0.55 [0.31-0.99]; p=0.05); HR for RFS 0.57 [0.31-1.05], p=0.07), while an increased risk of death or of an RFS event was seen in favorable risk patients (n=151; HR for OS 4.3 [1.65-11.2], p=0.003; HR for RFS 2.29 [1.07-4.93], p=0.03). In the ELN 2017 intermediate risk patients (n=98), a survival benefit of alloSCT was not seen (HR for OS 1.05 [0.45-2.46], p=0.92; HR for RFS 0.86 [0.42-1.78], p=0.69), although alloSCT was associated with a lower CIR (HR 0.43 [0.20-0.90], p=0.03). However, when only RDpos patients were considered (n=27), trends towards benefit from alloSCT in OS (HR 0.52 [0.11-2.52], p=0.42), RFS (HR 0.38 [0.09-1.62], p=0.19), and CIR (HR 0.44 [0.07-2.72], p=0.38) were observed. In RDneg patients (n=53), alloSCT was associated only with a lower CIR (HR 0.18 [0.05-0.66], p=0.01). We then re-analyzed the data using the older UK-MRC risk classification of AML based exclusively on cytogenetics at diagnosis. This approach increased the patients in the intermediate group from 98 (ELN 2017) to 298 (UK-MRC). When using UK-MRC, an RFS benefit from alloSCT was observed for intermediate risk RDpos patients (n=211; HR 0.44 [0.20-0.96], p=0.04). Trends for benefit in OS (HR 0.45 [0.19-1.03], p=0.06) and CIR (HR 0.45 [0.20-1.04], p=0.06) were observed as well. In RDneg patients, however, an RFS/CIR benefit of alloSCT was not observed, while inferior OS was seen in the alloSCT group from increased non-relapse mortality. Conclusion Our data underscore the established benefit of alloSCT in adverse risk AML and suggest a potential benefit of this treatment modality in RDpos intermediate risk patients, thus confirming the utility of ongoing RD assessment in AML. Our results also indicate that ongoing sophistication in AML risk assignment, based on mutational profile at diagnosis, influences the potential benefit of alloSCT. Further studies are needed to better define the subgroup of RDpos intermediate risk patients who may benefit from alloSCT. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mutational Analysis of DNMT3A Improves the Prognostic Stratification Using NPM1/FLT3-ITD Genotypes in Patients with Acute Myeloid Leukemia

Background: Nucleophosmin 1 (NPM1) mutations are considered a favorable factor of acute myeloid leukemia (AML). However, their clinical significance in non-European regions remains unclear. DNA methyltransferase 3A (DNMT3A) mutations have been reported as a factor for poor prognosis in many cohort analyses. However, they were excluded among major prognostic factors even in the European Leukemia Net 2017 (ELN2017) classification. In this study, we analyzed the effects of NPM1 and concurrent mutations, particularly DNMT3A mutations, on prognosis. Methods: All patients in this analysis were enrolled and selected by the gene sequencing of Japanese AML (GS-JAML) conducted by Nippon Medical School. We targeted only patients aged 70 years or younger who were diagnosed after 2010 and who received standard induction therapy consisting of 7 days of standard-dose cytarabine (100-200 mg/m2 continuous infusion) and 3 days of an anthracycline antibiotic infusion (idarubicin 12 mg/m2 or daunorubicin 60-90 mg/m2). Mutation screening were performed with target-captured sequencing for the AML gene panel. We also conducted agarose gel electrophoresis for the FMS-like tyrosine kinase 3 (FLT3-ITD) mutations and used the Sanger method to screen for CEBPA and NPM1 mutations. This study was reviewed and approved by the Human Subjects Institutional Review Board (project approval number 29-07-783) of the Nippon Medical School (Tokyo, Japan). Informed consent was obtained in accordance with the Declaration of Helsinki from all participants. Results: We retrospectively analyzed the prognosis in 605 Japanese patients with de novo AML, including 174 patients with NPM1-mutated, 125 patients with DNMT3A-mutated and 127 patients with FLT3-ITD mutated AML. NPM1 mutation was not a prognostic factor either overall or in subgroups based on NPM1/tandem duplication in FLT3-ITD genotypes. Comprehensive gene mutation analysis showed that mutations in codon R882 of DNMT3A(DNMT3AR882) were a strong factor of poor prognosis for AML overall and NPM1-mutated AML. Furthermore, multivariate analysis of all AML showed that DNMT3AR882 mutations and the cooccurrence of FLT3-ITD, NPM1 mutations, and DNMT3AR882 mutations (triple mutation) were independent factors for poor prognosis related to overall survival, with NPM1 mutations being an independent factor for favorable prognosis (hazard ratio: DNMT3AR882 mutations, 1.946; triple mutation, 1.992, NPM1 mutations, 0.548). Furthermore, we evaluated the prognostic impacts of DNMT3AR882 and triple mutation and demonstrated that NPM1-mutated AML patients could be more clearly stratified into three risk groups based on DNMT3AR882/FLT3-ITD genotypes than the ELN 2017 classification. Conclusion: Our study showed that the addition of DNMT3AR882 mutations to existing prognostic models allowed for further stratification of NPM1-mutated AML. This new prognostic model might provide important clinical guidance. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Chemo-Free Bridge-to-Transplant Strategy with Venetoclax and Azacitidine for NPM1-Mutated Acute Myeloid Leukemia in Molecular Failure

Background: In patients (pts) with NPM1-mutated acute myeloid leukemia (AML) fit for intensive chemotherapy, the persistence, reoccurrence, or progression of NPM1mut transcript identifies poor chemotherapy responders that require allocation to allogeneic stem cell transplantation (alloSCT). Currently, there is no standard of care for pts in molecular failure and the proper bridging strategy to alloSCT still needs to be defined. The combination of Venetoclax (VEN) and Azacitidine (AZA) offers an alternative chemo-free strategy with favorable toxicity profile in NPM1mut AML, a molecular subset exquisitely sensitive to VEN. Aims: To characterize a series of NPM1mut AML pts in molecular failure who received VEN-AZA as bridge-to-transplant treatment strategy. Methods: We explored the off-label combination of Venetoclax (VEN) and Azacitidine (AZA) in 9 NPM1mut fit AML pts in molecular failure after intensive chemotherapy eligible for alloSCT, treated at 4 Italian Hematology Institutions. Disease was monitored on bone marrow (BM) with NPM1mut-specific qRT-PCR assay. The quantitative detection limit of the assays was 0.01%. Complete response with minimal residual disease negativity (CR-MRDneg) was defined as ratio NPM1mut/ABL x 100 transcript < 0.01 copies Results: Of the 9 pts analyzed 4 (44%) were female, median age at diagnosis was 56 years (range 41-66) and 58 years (range 43-68) at VEN-AZA therapy. Eight of the 9 pts had ELN2017 favorable-risk AML (2 pts with concomitant FLT3-ITDlow and 1 FLT3-TKD) and 1 pt had intermediate-risk AML (FLT3-ITDhigh). Pts received in median 4 (range 2-5) cycles of front line chemotherapy. Seven pts were treated for molecular relapse and 2 for molecular persistence/progression. All pts received full VEN dosage (400 mg/die or 100 mg/die if concomitant strong CYP3A4 inhibitor) and AZA 75 mg/m2/die s.c. in 5- or 7-day schedule. All pts but 1 performed VEN ramp-up. Pts received a median of 3 cycles (range 1-4). No AZA dose reduction occurred. Median VEN duration during cycle (C)1 was 25 days (range 14-28), 21 days (range 7-28) in C2, 24.5 days (range 7-28) in C3 and 25.5 days (range 19-28) in C4. VEN dosage reductions were due to hematological toxicity ≥ G3 in 60% of cases and in 40% for logistic reasons. The observed ≥ G3 toxicities were neutropenia in 4/9 (44%) pts, 1 thrombocytopenia and 1 febrile neutropenia, managed out-patient. No tumor lysis syndrome was observed. Best response to VEN-AZA was CR MRDneg (NPM1mut undetectable) in 7/9 (78%) pts, reached after a median of 2 cycles (range 1-4). Importantly, no pts relapsed ongoing therapy; 1 pt, notably the FLT3-ITDhigh pt, in CR MRDpos presented molecular progression after C3. Median time from VEN-AZA start to alloSCT was 4.3 months (range 1.7-8.1). At pre-transplant evaluation 6/9 (67%) pts were in CR MRDneg. Eight of 9 pts received myeloablative conditioning and 1 pt received reduced-intensity conditioning. With a median follow-up from alloSCT of 9.3 months (range 2.4-24.0) all patients are alive and 8/9 (89%) in CR MRDneg. Four pts presented NPM1mut reoccurrence after alloSCT: in 2 pts CsA tapering achieved molecular negativity, 1 pt underwent prophylactic therapy with sorafenib and 1pt is currently in CR MRDpos. In no pts FLT3 mutations were detected and, most importantly, no patients experienced morphological relapse. Conclusions: VEN-AZA demonstrated to be a safe and effective bridging regimen for NPM1mut AML pts in molecular failure, allowing to achieve deep responses in the majority of pts at the expense of low toxicity. These preliminary encouraging results will be explored in the GIMEMA AML2521 trial in the same patient subset (clinicaltrials.gov NCT04867928).

Bone Marrow Stromal Cell Senescence Induced By Dnmt3a-Mutant Hematopoietic Stem and Progenitor Cells Accelerates Clonal Hematopoiesis and Progression to Leukemia

Clonal hematopoiesis (CH) is commonly driven by mutation in the DNA methyltransferase DNMT3A. While DNMT3A catalyzes DNA methylation, the mechanism(s) by which DNMT3A mutations confer a clonal selective advantage to hematopoietic stem cells (HSCs), and how this can lead to development of acute myeloid leukemia (AML), is not completely understood. Non-hematopoietic cell types in the bone marrow (BM) microenvironment have recently emerged as key contributors in AML by creating a favorable environment for cell growth, including pro-inflammatory changes, accumulation of senescent cells, and the senescence-associated secretory phenotype (SASP). As these alterations have also been found in the context of normal aging, we hypothesized that they contribute to CH-mutant HSC expansion and initiation of AML. To investigate alterations in the BM microenvironment induced by Dnmt3a-mutant hematopoiesis, we transplanted Dnmt3a-mutant or control BM cells into wild-type recipient mice and performed single cell RNA-seq on hematopoietic and non-hematopoietic BM cell fractions. We identified a cellular senescence signature as being enriched in the BM stromal cell (BMSC) subsets Osteo-CAR and Adipo-CAR. We hypothesized that Dnmt3a-mutant hematopoietic stem and progenitor cells (HSPC) remodel the BM microenvironment by induction of senescence to favor their selective advantage. To test this, we developed an ex vivo co-culture system using primary murine BMSC and Dnmt3a-mutant HSPC. Primary BMSCs cultured with Dnmt3a-mutant HSPCs were found to have increased expression of senescence markers (P16, P21, b-galactosidase) and SASP (IL-6, IL-1a) compared to BMSC cultured with control wild-type (WT) HSPCs. This phenotype was similarly observed in BMSCs in vivo after Dnmt3a-mutant cells were transplanted into WT recipient mice, in addition to increased expression of the anti-apoptotic proteins BCL-2 and BCL-xL in BMSCs. We then pursued mechanistic experiments to reveal how Dnmt3a-mutant HSPCs induce senescence of BMSCs. Transwell assays supported that senescence induction was cell contact independent. Cytokine profiling of conditioned media from Dnmt3a-mutant HSPCs, and well as BM fluid of transplanted recipient mice, revealed elevated levels of the inflammatory cytokines IL-6, S100A8/9 and TNFα. We directly tested the effect of IL-6 on primary BMSC by culture with recombinant IL-6 alone and found that this was sufficient to increase b-galactosidase staining in BMSCs. To determine the extent to which deletion of senescent cells can abrogate Dnmt3a-mutant selective advantage and progression to hematologic malignancy, we used our inducible mouse model of Dnmt3a;Npm1-mutant AML (Loberg et al., Leukemia 2019). We transplanted cells carrying the Dnmt3a mutation into WT recipient mice, administered the senolytic Navitoclax or vehicle control, and then induced Npm1 mutation in vivo. We found that Navitoclax-treated mice had reduced peripheral blood myeloid cell expansion, a biomarker of Dnmt3a;Npm1-mutant transformation. Furthermore, Navitoclax-treated mice had reduced WBC and monocyte counts, reduced bone marrow engraftment, reduced myeloid colony-forming units (CFU), and maintained lower BMSC senescence. Together, we report that Dnmt3a-mutant HSPCs induce senescence of BMSCs through production of the pro-inflammatory cytokine IL-6. Depletion of these senescent cells using Navitoclax reduces myeloproliferation and transformation of Dnmt3a-mutant HSPCs to AML. We suggest that SASP may be used as a biomarker of transformation risk in individuals with CH, and that targeting the induced senescent BM microenvironment may be used prophylactically to prevent transformation to AML.

Measurable residual disease detected by flow cytometry independently predicts prognoses of NPM1-mutated acute myeloid leukemia.

Acute myeloid leukemia (AML) with NPM1 mutation is a distinct genetic entity with favorable outcomes. Nevertheless, emerging evidence suggests that NPM1-mutated AML is still a highly heterogeneous disorder. In this study, 266 patients with AML with NPM1 mutations were retrospectively analyzed to evaluate the associations between variant allele frequency (VAF) of NPM1 mutations, co-mutated genes, measurable residual disease (MRD), and patient outcomes. Multiparameter flow cytometry (MFC) and real-time quantitative polymerase chain reaction (RT-PCR) were used for monitoring MRD. Ultimately, 106 patients were included in the long-term follow-up period. Patients with high NPM1 VAF (≥ 42.43%) had poorer 2-year relapse-free survival (RFS) (55.7% vs. 70.2%, P = 0.017) and overall survival (OS) (63.7% vs. 82.0%, P = 0.027) than those with low VAF. DNMT3A mutations negatively influenced the outcomes of patients with NPM1 mutations. Patients with high DNMT3A VAF or NPM1/DNMT3A/FLT3-ITD triple mutations had shorter RFS and significantly lower OS than that in controls. After two cycles of chemotherapy, patients with positive MFC MRD results had lower RFS (MRD+ vs. MRD-:44.9% vs. 67.6%, P = 0.007) and OS (61.5% vs. 76.6%, P = 0.011) than those without positive MFC MRD results. In multivariate analysis, high NPM1 VAF (hazard ratio [HR] = 2.045; P = 0.034) and positive MRD after two cycles of chemotherapy (HR = 3.289; P = 0.003) were independent risk factors for RFS; MRD positivity after two cycles of chemotherapy (HR = 3.293; P = 0.008) independently predicted the OS of the patients. These results indicate that VAF of both NPM1 gene itself or certain co-occurring gene pre-treatment and MRD post-treatment are potential markers for restratifying the prognoses of patients AML having NPM1 mutations.

Outcomes with Allogeneic Hematopoietic Stem Cell Transplantation in NPM1-Mutated Acute Myeloid Leukemia: A Systematic Review and Meta-Analysis

Introduction Acute myeloid leukemia (AML) has poor outcomes despite long term advancement in its diagnosis and treatment. The introduction of molecular genetic markers in AML has produced a major effect on therapeutic approach used in these patients. The genetic aberration in the Nucleophosmin (NPM1) gene is the most common mutation found in AML and is associated with "favorable" prognosis. Allogenic hematopoietic stem cell transplant (Allo-HSCT) is potentially a curative treatment option. Herein, we present a systematic review and meta-analysis with the aim of exploring outcomes with allo-HSCT in NPM1-mutated AML. Methods A detailed literature search was performed for the systematic review and meta-analysis in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines. A population, intervention, comparison, and outcome table were developed, and three electronic databases (PubMed, Cochrane Register of Controlled Trials, and Clinical trials.gov) were searched using MeSH terms and keywords for "Transplantation Hematopoietic Stem Cell" AND "Nucleophosmine Protein, Human" AND "Acute Myeloid Leukemia". A total of 137 articles were screened and original studies reporting patients with AML having NPM1 mutation who underwent Allo-HSCT were included while reviews, duplicate, and non-relevant articles were excluded. A total of twelve studies were included. Quality evaluation was done using the NIH quality assessment tool. The inter-study variance was calculated using Der Simonian-Laird Estimator. The pooled analysis was conducted using the 'meta' package by Schwarzer et al. in the R programming language (version 4.16-2). A random-effects model was used to estimate the proportions with 95% confidence intervals (CI). Results A total of 769 patients from twelve studies were included for this systematic review and meta-analysis. The median age of patients was 53 (21-79) years and 46.5% (303/651) were males as reported by ten studies. The donor type was matched related donor (MRD) 38%, matched unrelated donor (MUD) 61%, haploidentical 0.6%, and cord blood donor (CBD) 0.2%. The median follow-up was 46 (2-96) months. At a median follow-up of 2 (2-5) years, the pooled overall survival (OS) was 71% (95 % CI 0.66-0.73, p= 0.15, I2=31%, n= 710) while the pooled event free survival (EFS) and pooled relapse rate (RR) was 64% (95 % CI 0.59-0.68, p= 0.06, I2=45%, n= 741) and 25% (95 % CI 0.19-0.31, p <0.01, I2=70%, n= 695), respectively. The pooled incidence of acute graft versus host disease (GvHD) was 39% (95 % CI 0.16-0.65, p<0.01, I2=96%, n= 380) and the pooled incidence of chronic GvHD was 46% (95 % CI 0.35-0.58, p= 0.02, I2=71%, n= 301). The pooled incidence of non-relapse mortality (NRM) was 15% (95 % CI 0.08-0.23, p= 0.02, I2=63%, n= 298) at a median follow-up of 2 (2-5) years. Conclusion NPMI-mutated AML has favorable outcomes with allogeneic hematopoietic stem cell transplant and has good overall and event free survival with lower incidence of relapse and non-relapse mortality. Further randomized clinical trials are needed to consolidate these findings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Gemtuzumab Ozogamicin Plus Intensive Chemotherapy for Patients with NPM1-Mutated Acute Myeloid Leukemia

Gemtuzumab Ozogamicin Plus Intensive Chemotherapy for Patients with NPM1-Mutated Acute Myeloid Leukemia