NPM1-mutated Acute Myeloid Leukemia Patients Research Articles

Comprehensive Molecular Profiling of NPM1-Mutated Acute Myeloid Leukemia Using RNAseq Approach.

Acute myeloid leukemia (AML) is a complex hematologic malignancy with high morbidity and mortality. Nucleophosmin 1 (NPM1) mutations occur in approximately 30% of AML cases, and NPM1-mutated AML is classified as a distinct entity. NPM1-mutated AML patients without additional genetic abnormalities have a favorable prognosis. Despite this, 30-50% of them experience relapse. This study aimed to investigate the potential of total RNAseq in improving the characterization of NPM1-mutated AML patients. We explored genetic variations independently of myeloid stratification, revealing a complex molecular scenario. We showed that total RNAseq enables the uncovering of different genetic alterations and clonal subtypes, allowing for a comprehensive evaluation of the real expression of exome transcripts in leukemic clones and the identification of aberrant fusion transcripts. This characterization may enhance understanding and guide improved treatment strategies for NPM1mut AML patients, contributing to better outcomes. Our findings underscore the complexity of NPM1-mutated AML, supporting the incorporation of advanced technologies for precise risk stratification and personalized therapeutic strategies. The study provides a foundation for future investigations into the clinical implications of identified genetic variations and highlights the importance of evolving diagnostic approaches in leukemia management.

Biological and Therapeutic Implications of NPM1-Mutated AML Receiving Allogeneic Hematopoietic Cell Transplantation

Background: The nucleophosmin 1( NPM1) gene mutations are the most common gene alteration in acute myeloid leukemia (AML) patients, occurring in approximately 30%-35% of adult AML cases. However, molecular and response to therapy heterogeneity are existed in this distinct group. Response to therapy, differing patterns of co-mutation and the prognosis of NPM1-mutated AML patients receving allogeneic hematopoietic stem cell transplantation (allo-HSCT) treatment were unknown. Aims: To evaluate prognostic factors in the NPM1-mutated AML who underwent allo-HSCT treatment. Methods: A total of 115 consecutive NPM1-mutated AML patients who underwent allo-HSCT from 2016 to 2022 were enrolled in this study. The primary endpoint was the cumulative incidence of relapse (CIR). Results: The median patient age was 46 (IQR: 36-50) years. The most frequent co-mutations were FLT3-ITD(58.3%) and DNMT3A (40.0%), followed by IDH2(18.3%), NRAS (13.9%) and IDH1 (11.3%). The overall 3-year cumulative incidence of relapse (CIR) and leukemia-free survival (LFS) was 17.3% and 77.2%. The 3-year CIR of patients with FLT3-ITD was significantly higher than that without FLT3-ITD (24.3% vs. 7.0%; P=0.023). The 3-year CIR of patients with DTA mutations ( DNMT3A, TET2, and ASXL1) was significantly higher than that without DTA mutations (24.7% vs. 11.2%; P=0.030). Patients who received matched sibling donor (MSD) allo-HSCT had the highest CIR compared with unrelated donor (URD) allo-HSCT and haploidentical (HRD) allo-HSCT (46.2%, 10.0% and 11.2%, P=0.002). The 3-year CIR in patient with pre-HSCT measurable residual disease negative (MRD-), MRD+ and non-remission was 10.9%, 30.0% and 72.2%(P<0.001). After multivariable adjustment, DTA mutations, pre-MRD+ or non-remission, MSD allo-HSCT remained statistically significantly associated with higher CIR as well as lower LFS and OS. In univariate and multivariate analyses, the cell of origin of AML did not impact the outcomes of NPM1-mutated AML patients accept allo-HSCT treatment . Conclusion: The DTA mutations ( DNMT3A, TET2, and ASXL1) were identified a distinct group of patients with adverse prognosis in NPM1-mutated AML patients who received allo-HSCT. Our results indicate that NPM1-mutated patients accept HRD/URD donors can achieve lower CIR than MSD donors in transplantation. HRD allo-HSCT may suitable for high risk patients within this group.

Molecular Relapse after First-Line Intensive Therapy in Patients with Core-Binding Factor and NPM1-Mutated Acute Myeloid Leukemia - a Filo Study

Background: Patients with Core-Binding Factor (CBF) and NPM1-mutated acute myeloid leukemia (AML) are considered as favorable or intermediate-risk according to the European LeukemiaNet (ELN) 2022 classification and allogeneic hematopoietic cell transplantation (HCT) is usually not considered in those achieving first complete remission (CR) with good molecular response. Close molecular monitoring by quantitative PCR has been widely implemented over time and approximately 30% of patients will experience molecular relapse. However, there is no recommendation on how to manage these patients due to the lack of data from large patient series or clinical trials. In this study, we retrospectively analyzed the outcome of CBF or NPM1-mutated AML patients who had MRD monitoring after having achieved first CR. Methods: All CBF and NPM1-mutated AML patients, aged 18-60 years, who had MRD molecular monitoring (RT-qPCR of RUNX1-RUNXT1 or CBFB-MYH1 transcripts or classical NPM1 mutations) after first-line therapy including induction therapy with daunorubicin/idarubicin and cytarabine followed by intermediate or high-dose cytarabine consolidation, without allogeneic HCT in first CR, were included. MRD measurements were generally performed every three months during follow-up for 24 months in either bone marrow (BM) or peripheral blood (PB). Molecular relapse was defined according to ELN recommendations (Heuser M, Blood 2021) as a conversion from MRD negativity to positivity or an increase in MRD copy numbers >1 log between two positive samples. Results: Between 2010 and 2019, 303 patients from 9 centers with newly diagnosed CBF or NPM1-mutated AML ( RUNX1-RUNX1T1, 19%; CBFB-MYH11, 27%; NPM1, 53%) and available MRD monitoring after first-line therapy were included. Among these patients, 153 (51%) never relapsed, 95 (31%) had molecular relapse, with a median of 326 days (interquartile range [IQR]: 276-438) from initial diagnosis, and 55 (18%) had upfront morphologic relapse (i.e., without prior molecular relapse detected), with a median of 388 days (IQR: 280-559). Patients with molecular and upfront morphologic relapse had higher white blood cell (WBC) count at diagnosis (12 vs. 22 vs. 30 G/l in those without relapse, molecular relapse, and morphological relapse, respectively, P=0.003) and were more likely to have a FLT3-ITD mutation (7% vs. 25% vs. 19%, respectively, P=0.001). In addition, patients with upfront morphologic relapse had a lower log-reduction of MRD during first-line treatment (4.90 vs. 4.93 vs. 3.77, respectively, P=0.009) and were more likely to have CR with positive MRD at the end of treatment in peripheral blood (2% vs. 16% vs. 32%, respectively, P<0.001). Overall survival (OS) was significantly different according to whether patients never relapsed, had molecular relapse, or had upfront morphological relapse (three-year OS of 100% vs. 75% vs. 60%, P<0.001; Figure A). Among the 95 patients with molecular relapse, 53 (56%) received preemptive therapy (median of 48 days [IQR:24-68] from molecular relapse to treatment initiation) and 42 (44%) progressed to morphologic relapse before treatment was initiated (median of 58 days [IQR:36-134] from molecular to morphologic relapse). Patients who received preemptive therapy had a better OS than those who received salvage therapy after having progressed from molecular to morphologic relapse, and those who had upfront morphologic relapse (78% vs. 51% vs. 51% at three years, respectively, P=0.01; Figure B). Preemptive therapy included upfront allogeneic HCT (n=19), intensive chemotherapy (n=21), and non-intensive therapy (n=13) with different OS rates that did not reach statistical significance (92% vs. 79% vs. 58% at three years for upfront allogeneic HCT, intensive chemotherapy, and less intensive therapy, respectively, P=0.09). Conclusion: Up to 30% of patients with CBF or NPM1-mutated AML experienced molecular relapse in our cohort and more than 50% of them received preemptive therapy. Patients treated preemptively at molecular relapse had a better OS than those treated with active disease. Although these results need to be confirmed in larger studies, it supports molecular monitoring during follow-up to start preemptive therapy before overt morphologic relapse. Other techniques for MRD monitoring are urgently needed for AML patients who cannot be monitored by qPCR.

Secondary-Type Mutations Do Not Impact the Favorable Outcome of NPM1-Mutated Acute Myeloid Leukemia Patients - Results from a Large Cohort of Intensively Treated Patients

Introduction: In 2022, the ELN risk classification for AML was updated for the second time. One of the major novelties of the ELN2022 is that all secondary-type mutations (STMs, i.e., mutations in the genes SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, and STAG2) were now added to the adverse risk characteristics. However, a pertinent question also raised by the ELN expert panel is whether STMs abrogate the positive prognostic value of co-occurring, favorable NPM1 mutations. Aim: The aim of this study was to analyze the prognostic value of STMs in AML patients (pts) who also harbor an NPM1 mutation. Methods: We investigated a pooled cohort of 936 NPM1-mutated AML pts who were treated in previously reported multicenter trials of the Study Alliance Leukemia or the AML Cooperative Group. Eligibility was determined based on diagnosis of non-APL, age ≥ 18 years, NPM1 mutation detected in targeted sequencing, curative treatment intent, and available biomaterial at diagnosis. Standard techniques for chromosome banding and fluorescence-in-situ-hybridization (FISH) were used for karyotyping. Next-generation panel sequencing was performed to detect genetic alterations that are recurrently found in myeloid neoplasms. Results: In our multicenter cohort of 936 NPM1-mutated AML pts, median follow-up for the entire cohort was 8.0 years. We found 125 patients (13.4%) harboring at least one STM ( SRSF2 [n=48; 5.1%], STAG2 [n=32; 3.2%], EZH2 [n=22, 2.4%], BCOR [n=16; 1.7%], SF3B1 [n=13; 1.4%], ASXL1 [n=12; 1.3%], ZRSR2 [n=5; 0.5%], and U2AF1 [n=4; 0,4%]). A comparison of pretreatment clinical and genetic features revealed that pts with a STM were significantly older ( p=.003, median 59 vs. 55 years), had lower white blood cell counts ( p<.001, 22.2*10 9/L vs. 39.7*10 9/L,) and platelet counts ( p<.001, 46.5*10 9/L vs. 65.0 10 9/L). The strongest pair-wise associations between gene mutations were observed between U2AF1 and RUNX1 ( p<.001) as well as SRSF2 and IDH2 ( p<.001). With respect to outcome, complete remission (CR) rate did not differ significantly between NPM1-mutated patients with or without additional STMs ( p=.41, 74.4% vs. 77.7%, OR 0.83 [95%-CI 0.54-1.29]). Median RFS for NPM1-mutated pts with STMs was 32.9 months (95%-CI: 13.0-46.0) while patients without STMs had a median RFS of 24.3 months (95%-CI: 18.7-33.3) corresponding to a HR of 1.04 ( p=0.80, 95%-CI 0.79-1.37; Figure A). Median OS for NPM1-mutated pts with or without STMs was 27.2 months (95%-CI: 14.2-49.0) and 29.1 months (95%-CI: 23.5-41.4), respectively, corresponding to a HR of 1.11 ( p=.37, 95%-CI 0.88-1.41; Figure B). To focus solely on the impact of STMs, we subsequently excluded patients with co-occurring mutations in TP53 or myelodysplasia-related cytogenetics, which all define an ELN adverse risk. Again, we observed no differences in CR rate ( p=.54, 78% vs. 75.4%), RFS ( p=.59, median 33.2 months vs. 26.6 months), or OS ( p=.33, median 27.4 month vs. 32.6 month) between NPM1-mutated patients with or without STMs. Next, we restricted our analysis to pts who are classified favorable risk according to ELN2022. Again, we found no significant outcome differences based on the STM status (unmutated vs. mutated: CR rate, 80% vs. 70.7% [ p=.072]; RFS, median 49.7 months vs. 46.0 months [ p=.702]; OS, median 45.3 months vs. 59.8 months [ p=.092]). Conclusion: NPM1 mutations rank as the second most frequent mutations in AMLand the most common in patients with a normal karyotype and serve as an established favorable prognostic marker. Our data from a large cohort demonstrate that additional STMs have no adverse effect on the clinical outcome of NPM1-mutated patients. As a result, these patients should still be considered ELN favorable risk.

Establishment of an NPM1 Mutation Copy Number Estimator for Xpert ® NPM1 Mutation Test

Objectives: The nucleophosmin ( NPM1)is the most mutated gene (~30%) in Acute Myeloid Leukemia (AML) 1. Three NPM1 mutations (type A, B, and D) represent ~84% in NPM1-mutated AML cases while other uncommon subtypes occupy ~16% 2. Xpert® NPM1 mutation, an automated cartridge-based test for measuring NPM1 mutation transcript levels (type A, B and D), is standardized to quantify the amount of mutated NPM1 relative to ABL1 control gene based on delta Ct in peripheral blood 3. Since mutated NPM1 level is crucial for risk assessment, medication selection, and ongoing therapeutic monitoring in AML 4,5, it is important to obtain the NPM1 mutation copy number (CN). The aim of this work is to develop NPM1 mutation CN estimator and to compare %NPM1 mutation/ABL1 reporting between delta Ct-based and CN-based methods. Methods: Five levels of NPM1 mutations (A, B, D) and ABL1 IVT-RNA panels as well as two lots of Xpert® NPM1 mutation tests were used to generate standard curves for CN and %CN reporting. The cell lysates from cell lines carrying either NPM1 mutation A, B, or D and AML clinical samples containing NPM1 mutations were examined to evaluate the CN and %CN between two lots of the Xpert® NPM1 mutation tests and to compare the delta Ct-based and CN-based methods for reporting %NPM1 mutation/ABL. Results: Linearity was demonstrated in Ct vs CN input for NPM1 mutation and ABL1 with R 2 above 0.96 for Lot1 and Lot2. Less than 3-fold difference was exhibited for CN and %CN across two lots of Xpert® NPM1 mutation test. Less than 3-fold difference was observed in %NPM1 mutation/ABL1 reporting between delta Ct-based and CN-based approaches. Conclusion: An NPM1 mutation copy number estimator for Xpert® NPM1 mutation test was established, which will provide diagnostic and prognostic values for NPM1-mutated AML patients.

Effect of clonal hematopoiesis in remission on hematopoiesis recovery in patients with NPM1 mutated acute myeloid leukemia after chemotherapy

Objective: To investigate the effect of clonal hematopoiesis (CH) in remission on hematopoiesis recovery in patients with NPM1 mutated acute myeloid leukemia (AML) after chemotherapy. Methods: Retrospective analysis was performed on 86 patients with NPM1(mut) AML newly diagnosed and treated in the First Affiliated Hospital of Soochow University between July 2016 and June 2019. Their clinical data and NGS test results at diagnosis were analyzed. Moreover, bone marrow samples in remission were tested using Sanger sequencing. The log-rank test was used to analyze the difference in hematopoietic recovery, and Cox proportional hazard models were used to analyze the prognostic factors affecting hematopoietic recovery. Results: The median age of the 86 NPM1(mut) AML patients was 50 years (15-69 years). There were 39 males and 47 females. Forty-one patients were induced with intensity chemotherapy ("7 + 3"), whereas 45 patients were treated with low-dose cytarabine-based induction chemotherapy. At diagnosis, The most common mutations in the patients were FLT3, DNMT3A, TET2, and IDH1/IDH2 mutations. CH-associated mutations persisted in 21 patients during remission, and the mutations were DNMT3A, TET2, ASXL1, and IDH1/IDH2. The recovery time of neutrophils in patients with CH-associated mutations in remission was consistent with that in patients without CH in remission (P=0.282) but the recovery time of platelets in patients with CH in remission was significantly longer[26 (95% CI 21-32) days vs 25 (95% CI 23-26) days, P=0.032]. Furthermore, univariate analysis indicated that age, induced chemotherapy program, and CH in remission were risk factors for platelet recovery, whereas multivariate analysis indicated that induced chemotherapy program and CH in remission were independent risk factors for platelet recovery (HR=0.454, P=0.001 and HR=0.520, P=0.027, respectively) . Conclusion: CH in remission delays the hematopoietic recovery of patients with NPM1(mut) AML after chemotherapy.

Genetic, Phenotypic, and Clinical Heterogeneity of NPM1-Mutant Acute Myeloid Leukemias.

The current classification of acute myeloid leukemia (AML) relies largely on genomic alterations. AML with mutated nucleophosmin 1 (NPM1-mut) is the largest of the genetically defined groups, involving about 30% of adult AMLs and is currently recognized as a distinct entity in the actual AML classifications. NPM1-mut AML usually occurs in de novo AML and is associated predominantly with a normal karyotype and relatively favorable prognosis. However, NPM1-mut AMLs are genetically, transcriptionally, and phenotypically heterogeneous. Furthermore, NPM1-mut is a clinically heterogenous group. Recent studies have in part clarified the consistent heterogeneities of these AMLs and have strongly supported the need for an additional stratification aiming to improve the therapeutic response of the different subgroups of NPM1-mut AML patients.

Preclinical Validation of an Advanced Therapy Medicinal Product Based on Cytotoxic T Lymphocytes Specific for Mutated Nucleophosmin (NPM1mut) for the Treatment of NPM1mut-Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) with nucleophosmin (NPM1) genetic mutations is the most common subtype in adult patients. Refractory or relapsed disease in unfit patients or after allogeneic hematopoietic stem cell transplantation (allo-HSCT) has a poor prognosis. NPM1-mutated protein, stably expressed on tumor cells but not on normal tissues, may serve as an ideal target for NPM1-mutated AML immunotherapy. The study aim was to investigate the feasibility of producing mutated-NPM1-specific cytotoxic T cells (CTLs) suitable for somatic cell therapy to prevent or treat hematologic relapse in patients with NPM1-mutated AML. T cells were expanded or primed from patient or donor peripheral blood mononuclear cells by NPM1-mutated protein-derived peptides, and tested for leukemia antigen-targeted cytotoxic activity, cytokine production and hematopoietic precursor inhibitory effect. We found that mutated-NPM1-specific CTLs, displaying specific cytokine production and high-level cytotoxicity against patients' leukemia blasts, and limited inhibitory activity in clonogenic assays, could be obtained from both patients and donors. The polyfunctional mutated-NPM1-specific CTLs included both CD8+ and CD4+ T cells endowed with strong lytic capacity. Our results suggest that mutated-NPM1-targeted CTLs may be a useful therapeutic option to control low-tumor burden relapse following conventional chemotherapy in older NPM1-mutated AML patients or eradicate persistent MRD after HSCT.

Mutant NPM1: Nuclear export and the mechanism of leukemogenesis.

Mutant NPM1: Nuclear export and the mechanism of leukemogenesis.

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

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

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

Quantitative Impact of Mutated NPM1 MRD in Patients with AML Undergoing Allogeneic Stem Cell Transplantation

Introduction: In NPM1-mutated (NPM1mut) acute myeloid leukemia (AML) the detection of measurable residual disease (MRD) using quantitative PCR methods for NPM1mut allows for risk stratification and therapy adjustments during disease course. Even though it is well understood that a persistent NPM1mut burden at allogeneic stem cell transplantation (HSCT) is associated with relapse and shorter survival, the quantitative impact of NPM1mut at HSCT has not been investigated in detail. Methods: We analyzed 81 NPM1mut AML patients (pts) who received their first allogeneic peripheral blood HSCT at a median age of 61 (range 33-76) years in morphologic complete remission (CR) or CR with incomplete peripheral recovery (CRi). Conditioning regimens were non-myeloablative (70%), reduced-intensity (11%), or myeloablative (19%) according to EBMT guidelines. At diagnosis, cytogenetics and co-mutational status of FLT3, IDH1, IDH2, DNMT3A, and CEBPA were assessed. NPM1 MRD up to 28 days prior to HSCT in CR/CRi was measured by digital droplet PCR using a competitive probe approach, and results were normalized to ABL1 (limit of detection 0.001%NPM1mut/ABL1). For patients with a positive NPM1 MRD test result, receiver operator characteristics (ROC) curves were used to analyze distinct NPM1/ABL1 MRD threshold. Presence of chronic graft-versus-host disease (GvHD) was assessed according to Glucksberg criteria. Median follow up after HSCT was 4.4 years. Results: ROC curves identified 0.01% NPM1mut/ABL1 as the most informative cut-off to define MRDpos and MRDnegpts in both, bone marrow and blood, and irrespective of conditioning intensity. Using this cut-off 31 (38%) pts were NPM1mut MRDpos at HSCT and had a significantly higher cumulative incidence of relapse (CIR, P<.001, after 2 years 4% vs 66%) and shorter overall survival (OS, P<.001, after 2 years 77% vs 38%). Patients transplanted NPM1mut MRDpos had a 20.8-times higher risk of relapse than those transplanted MRDneg. While there were no differences regarding the gene mutation profile at diagnosis in pts with or without NPM1mut MRD at HSCT (Figure 1A), pts with NPM1mut MRD at HSCT had a significantly higher diagnostic WBC (49.6 vs 17.9; P=.03), LDH (12.9 vs 6.7; P=.02), and more CD34+ cells in bone marrow at diagnosis (1.4% vs 0.5%; P=.02). We observed a negative correlation of the NPM1mut /ABL1 MRD levels before HSCT and the time to relapse; the higher the NPM1mut MRD burden at HSCT the earlier the relapse occurred (R= -0.5, P=.03). This correlation was strongest in pts with a FLT3-ITD present at diagnosis (R= -0.82, P=.002). When utilizing two cut-offs for low (≥ 0.01% but < 1%) and high (≥ 1%) NPM1mut /ABL1 MRD levels there was a significantly shorter time to relapse in relapsing AML pts with high NPM1mut MRD levels (median time to relapse: 73 days) before HSCT compared to pts with low MRD levels (median time to relapse: 149 days). In addition, compared to patients with low NPM1mut MRD levels, pts with high NPM1mut MRD levels showed a trend for shorter OS (P=.10; OS after 2 years: 77% for NPM1mut MRDnegpatients vs 48% for low NPM1mut MRDpos patients vs 21% for high NPM1mut MRDpos patients; Figure 1B), indicating that pts transplanted with higher NPM1mut MRD may be harder to salvage. Presence of a FLT3-ITD and age at HSCT did not alter the negative impact on outcomes of low or high NPM1mut MRD at HSCT. We also evaluated the prognostic impact of the presence of chronic GvHD as a surrogate marker for graft-versus-leukemia effects with respect to the NPM1mut MRD status at HSCT. In a landmark analysis for pts surviving longer than 100 days, NPM1 MRDneg (< 0.01% NPM1mut/ABL1) pts had the lowest CIR (4% after 2 years), followed by NPM1mut MRDpos pts developing a chronic GvHD (25% after 2 years) and by NPM1 MRDpos pts who did not develop a chronic GvHD and had a very high CIR (92% after 2 years, P<.001). Conclusion: AML patients with > 0.01% NPM1mut/ABL1 MRD before allogeneic HSCT had a significant higher risk of relapse and shorter OS. High WBC, LDH, and CD34+ cells in bone marrow at diagnosis associated with persisting NPM1mut MRD at HSCT. The higher the NPM1mut MRD burden at HSCT, the earlier the relapse, and the lower the chances of successful salvage therapy after relapse. Development of a chronic GvHD after HSCT was associated with a reduced risk of relapse in NPM1mut MRDpos pts, indicating that bolstering immunologic effects post-HSCT for NPM1mut MRDpos AML pts may be a promising option to improve outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

S130: NPM1 MUTATED AML: IMPACT OF CO-MUTATIONAL PATTERNS - RESULTS OF THE EUROPEAN HARMONY ALLIANCE

Background: Acute myeloid leukemia (AML) is a heterogeneous disease in terms of clinical features, outcomes and genetics. While mutations of NPM1 are usually considered as a favorable prognostic marker, the vast majority of the patients carry several co-mutations that might influence the prognosis. Therefore, a better understanding of the NPM1mut AML mutational landscape is warranted. The large cohort of AML patients collected within the European HARMONY Alliance provides an excellent basis for this purpose. Aims: To identify clinically significant co-mutational patterns in NPM1mut AML in order to establish a revised risk stratification model. Methods: From the HARMONY Alliance AML database, a total of 1001 NPM1mut intensively treated patients were selected. Clinically significant co-mutations were evaluated using graphical patterns created with the Gephi tool and confirmed by detailed survival analysis using Kaplan-Meier and Cox regression models. Finally, a novel multi-state risk stratification model for NPM1mut AML was established. Results: The study population of 1001 NPM1mut AML patients included 57% females and median age was 53 years. Regarding ELN2017 classification, 68% of patients were classified into the favorable, 29% intermediate and 3% adverse risk groups. The most frequent co-mutations were DNMT3A (54%), followed by FLT3-ITD (38%). In total, 24% of patients presented with a high allelic mutant-to-wildtype ratio ≥0.5 (FLT3-ITDhigh) while 14% had low allelic ratio <0.5 (FLT3-ITDlow). Other frequent co-mutations were NRAS (21%), TET2 (20%) and PTPN11 (15%). The triple mutation pattern of NPM1mut + FLT3-ITDhigh + DNMT3Amut identified a subgroup with adverse prognosis (2-year OS of 25%), similar to NPM1mut + TP53mut. The combination of FLT3-ITDlow + DNMT3Amut or FLT3-ITDhigh + DNMT3Awt was associated with intermediate prognosis (2-year OS of 45% and 53% respectively). Notably, mutations of NRAS, KRAS, PTPN11 or RAD21 were identified to be associated with better OS. However, in the context of NPM1mut + DNMT3Amut these mutations did not affect the prognosis when a FLT3-ITD was present. This information is summarized in a 3-category risk classification model (Figure 1). The revised NPM1mut favorable group presented with a 2-year OS of 73%, while for intermediate and adverse groups the OS was 54% and 27% respectively (p<0.001). Regarding relapse free survival (RFS), the median was not reached in the favorable group, while it was 23 months for intermediate and 6 months for adverse group (p<0.001). It should be noted that 171 patients in the NPM1mut intermediate group would be considered as favorable according to the ELN2017 criteria, as well as 162 patients in the NPM1mut adverse group were previously classified as intermediate risk. Therefore, our model was able to reclassify 33% of NPM1mut AML patients in comparison to ELN2017 criteria. Multivariate analysis of OS in NPM1mut AML identified the following independent prognostic factors: NPM1mut model (taking favorable group as reference, HR 1.6 for intermediate and HR 2.7 for adverse group, p<0.001); secondary or therapy-related AML (HR 1.8, p<0.001), WBC at diagnosis >100x103/μL (HR 1.5, p<0.001) and age >60 years (HR 1.4, p<0.001). Image:Summary/Conclusion: Analysis of large NPM1mut AML cohorts allows the discovery of co-mutation patterns associated with prognostic outcome. In accordance, we propose a new genetic stratification model for NPM1mut AML that identifies 3 groups with different OS and RFS. This model improves ELN2017 criteria as it is able to correctly reclassify 33% of NPM1mut AML patients.

Clinical Significance of Low Expression of LncRNA CASC15 in Acute Myeloid Leukemia with NPM1 Mutations

Eighty-two de novo AML patients and 18 healthy donors were included in the study. Meanwhile, seven public datasets from Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) were included to confirm the expression and methylation data of CASC15. Receiver operating characteristic (ROC) curve analysis was applied to determine the discriminative capacity of CASC15 expression to identify AML. The patients were divided into CASC15high group and CASC15low group by X-tile method, and the prognostic value of CASC15 was identified by Kaplan-Meier method and univariate and multivariate Cox regression analysis. The expression level of CASC15 was significantly decreased in BM cells of AML patients compared with healthy donors (P<0.001). ROC curve analysis suggested that CASC15 expression might be a potential biomarker to discriminate AML from controls. The expression of CASC15 was high at the early stage of hematopoiesis, and reached a peak at the stage of multipotent progenitors differentiation, then decreased rapidly, and was at a range of low level fluctuations in the subsequent process. Among FAB subtypes, CASC15 expression in M0 was significantly higher than that in M1-M7. Clinically, CASC15low patients were more likely to have NPM1 mutations than CASC15high patients (P=0.048), while CASC15high patients had a significantly higher frequency of IDH1 and RUNX1 mutations (P=0.021 and 0.014, respectively). Moreover, CASC15low group had a shorter overall survival (OS) in patients with NPM1 mutations. Furthermore, multivariate analysis confirmed that CASC15 expression was a significant independent risk factor for OS in NPM1 mutated AML patients. In addition, CASC15 methylation level in BM samples of AML patients was significantly decreased compared with healthy donors. Patients with CASC15 high methylation had poor OS and disease-free survival. The expression of CASC15 is decreased in AML, and low CASC15 expression may predict adverse prognosis in AML patients with NPM1 mutations. Moreover, CASC15 methylation level in AML is significantly decreased, and high CASC15 methylation may predict poor prognosis in AML.

Cytogenetic abnormalities in NPM1-mutated acute myeloid leukemia

NPM1 mut acute myeloid leukemia (AML) has been identified as a distinct entity of myeloid neoplasms according to the 2017 European LeukemiaNet (ELN) guidelines. It confers a favorable prognosis regardless of cytogenetic abnormalities. We evaluated 418 newly diagnosed AML patients to test the validity of this hypothesis. Seventy-four patients with NPM1 mut AML showed a good response to induction and a relatively favorable prognosis. Abnormal karyotypes were observed in 15 patients. Chromosomal abnormalities were significantly associated with a worse prognosis in NPM1 mut AML patients (5-year overall survival (OS): 38.9 ± 12.9%, p = .037; event-free survival (EFS): 33.3 ± 12.2%, p = .043, respectively). Four patients with abnormal karyotypes who underwent allogeneic hematopoietic stem cell transplantation (alloHSCT) during CR1 had longer survival than those who received chemotherapy only. Multivariable analysis revealed abnormal karyotypes independently predicted OS and EFS among NPM1 mut AML patients. In summary, cytogenetic abnormalities are strong prognostic indicators in NPM1 mut AML. Therefore, they should be classified accordingly, and alloHSCT should be performed on selected patients during CR1.

Abstract P195: Omacetaxine mepessucinate plus venetoclax show strong synergistic anti-leukemic activity in nucleophosmin (NPM1)-mutated AML patient-derived xenograft (PDX) models to support a phase 1 clinical trial

Abstract Despite different novel targeted drugs against specific genetic lesions have been developed for acute myeloid leukemia (AML) treatment, therapy in many AML subtypes should still rely on drugs possibly targetting alternative pathways essential for leukemia survival. Different biological and clinical evidences point to nucleophosmin (NPM1) gene mutations (the most frequent genetic lesion in AML) among strong predictors of sensitivity to venetoclax, a small molecule drug inhibitor of the B-cell leukemia/lymphoma-2 (Bcl-2) anti-apoptotic protein, highly expressed in most AML. Venetoclax appeared to be extremely efficacious in combination with hypomethylating agents (HMA) as first-line therapy, particularly in NPM1-mutated AML. Omacetaxine mepessucinate is a synthetic form of the plant cephalotaxine alkaloid homoharringtonine (HHT). HHT binds to the A-site cleft of ribosomes preventing the initial elongation step of protein synthesis and leading to a transient but profound inhibition of protein synthesis especially of those with a short half-life, including the antiapoptotic protein myeloid cell leukemia-1 Mcl-1, acting as mechanism of resistance following Bcl-2 inhibition. Besides its action as ‘Mcl-1 inhibitor’ (targeting its protein levels), we focused on HHT because i) it is a FDA-approved drug, in the setting of chronic myeloid leukemia (CML); ii) it showed some activity in clinical trials in AML; iii) it was shown to be a safe treatment, when evaluated also in association with chemotherapy, either standard intensive or low-dose regimen, in AML; iv) it is administered subcutaneously in its FDA-approved formulation; v) it has never been used in combination with venetoclax. Here, we sought to explore the therapeutic potential of omacetaxine mepessucinate and venetoclax in pre-clinical in vivo models, and used 2 different NPM1-mutated AML PDX (one, NPM1/FLT3-ITD double mutant; the other, DNMT3A/NPM1/FLT3-ITD triple mutant) edited to express luciferase allowing to track the disease by in vivo bioluminescence imaging (BLI) during treatment and evaluate drug response. HHT was given at 1 mg/Kg by subcutaneous administration for 2 consecutive weeks (5 days/week) and venetoclax at 100 mg/kg by gavage for 4 weeks (5 days/week), in cycles of 28 days. The anti-leukemic activity of the treatment was measured by evaluating AML growth/tissue infiltration on sacrificed animals by immunohistochemistry or flow cytometry and in vivo by BLI. Strikingly, on overt disease combinatorial HHT and venetoclax treatment resulted in a strong and synergic anti-leukemic effect with no leukemia detection in a cohort of mice sacrificed and analyzed after the first cycle, and in a significant advantage in a cohort analyzed for survival compared with single-drug or vehicle-treated animals. These data gave the support to design a pilot phase 1 clinical trial aimed at assessing safety and preliminary efficacy of omacetaxine mepessucinate plus venetoclax in relapsed/refractory NPM1-mutated AML patients. The trial is now open to patients accrual at our Hematology department. Citation Format: Federica Mezzasoma, Valeria Cardinali, Ilaria Gionfriddo, Francesca Milano, Sofia Sciabolacci, Alessio Ferrari, Marcella Sabino, Serenella Silvestri, Serena Donini, Valentina Tini, Giulio Spinozzi, Brunangelo Falini, Maria Paola Martelli. Omacetaxine mepessucinate plus venetoclax show strong synergistic anti-leukemic activity in nucleophosmin (NPM1)-mutated AML patient-derived xenograft (PDX) models to support a phase 1 clinical trial [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P195.

Mutations Highly Specific for Secondary AML Are Associated with Poor Outcomes in Patients with NPM1-Mutated ELN Favorable Risk AML

Background: NPM1 is commonly mutated in acute myeloid leukemia (AML) and represents a distinct entity under the WHO 2016 classification. It is one of the few mutations that can potentially support favorable risk by European LeukemiaNet (ELN) 2017 criteria. Mutations that are highly specific for secondary AML including SRSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, and STAG2 (sMut) (Lindsley et al.) have been shown to confer poor prognosis. The impact of these mutations on NPM1-mutated AML warrants further investigation.Objective: In this study, we explore the outcomes in patients with NPM1-mutated AML.Methods: This was a retrospective study of NPM1-mutated AML patients who were diagnosed and treated at the Moffitt Cancer Center from 2013 to March 2021. Inclusion was restricted to NPM1-mutated patients with mutation analysis (NGS) performed at diagnosis (n=159). Kaplan-Meier, univariate, and multivariate analyses were performed.Results: Among 159 patients (78M/81F, median age 63 years at diagnosis), 80.5% had de novo AML. By ELN 2017 criteria, 63.5% (101/159) had favorable risk, 27.7% (44/159) had intermediate risk, and 8.2% (13/159) had adverse risk disease. Almost 90% had intermediate risk cytogenetics at the time of diagnosis. Common co-mutations included DNMT3A (47.2%), FLT3-ITD (35.8%), TET2 (26.4%), IDH1 (17.6%), FLT3-TKD (15.1%), and IDH2 (13.8%). sMut comprised 19.5% (31/159) of patients and 20.8% (21/101) of those with ELN favorable risk. In patients with treatment response data, those with sMut never achieved CR/CRi in 35.7% (10/28) compared to 17.2% (22/128) of patients without sMut (p=0.038). The overall survival (OS) was 43.7 months with a median follow up of 35.5 months. Patients with sMut had worse OS compared to those without sMut (14.7 months vs 57.6 months, p=0.011). Among patients with favorable risk disease, OS was 11.6 months compared to not reached for those with sMut and without sMut, respectively (p<0.0001). Univariate analysis showed sMut and allogeneic hematopoietic cell transplant (HCT) significantly impacted OS (sMut: HR 3.48, 95% CI: 1.80-6.72, p<0.001; HCT: HR 0.17, 95% CI: 0.07-0.44, p<0.001). Multivariate regression using covariates including age, AML type, sMut, and HCT confirmed their prognostic significance on survival (sMut: HR 2.40, 95% CI: 1.17-4.93, p=0.017; HCT: HR 0.26, 95% CI: 0.08-0.56, p=0.002).Conclusions: Our findings suggest NPM1-mutated AML patients with sMut have significantly worse prognosis despite being classified primarily as favorable risk by ELN 2017 at diagnosis. This may have treatment implications altering the need for and/or timing of HCT. These findings should be assessed prospectively and validated in independent datasets. [Display omitted] DisclosuresHussaini: Adaptive: Consultancy, Honoraria, Speakers Bureau; Stemline: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy; Celegene: Consultancy; Decibio: Consultancy; Guidepoint: Consultancy; Bluprint Medicine: Consultancy. Talati: AbbVie: Honoraria; Pfizer: Honoraria; Astellas: Speakers Bureau; BMS: Honoraria; Jazz: Speakers Bureau. Kuykendall: Incyte: Consultancy; Novartis: Honoraria, Speakers Bureau; Protagonist: Consultancy, Research Funding; Celgene/BMS: Honoraria; Abbvie: Honoraria; Blueprint: Honoraria; Pharmaessentia: Honoraria. Padron: Blueprint: Honoraria; Incyte: Research Funding; Kura: Research Funding; Stemline: Honoraria; Taiho: Honoraria; BMS: Research Funding. Sallman: Shattuck Labs: Membership on an entity's Board of Directors or advisory committees; Syndax: Membership on an entity's Board of Directors or advisory committees; Magenta: Consultancy; Takeda: Consultancy; Kite: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Incyte: Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Intellia: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Agios: Membership on an entity's Board of Directors or advisory committees; Aprea: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sweet: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astellas: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Bristol Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees. Komrokji: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Geron: Consultancy; BMSCelgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Acceleron: Consultancy; AbbVie: Consultancy; Jazz: Consultancy, Speakers Bureau; Taiho Oncology: Membership on an entity's Board of Directors or advisory committees; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees. Lancet: AbbVie: Consultancy; Celgene/BMS: Consultancy; Daiichi Sankyo: Consultancy; ElevateBio Management: Consultancy; Millenium Pharma/Takeda: Consultancy; BerGenBio: Consultancy; Jazz: Consultancy; Agios: Consultancy; Astellas: Consultancy.

Neoantigen-Specific T-Cell Immune Responses: The Paradigm of NPM1-Mutated Acute Myeloid Leukemia

The C-terminal aminoacidic sequence from NPM1-mutated protein, absent in normal human tissues, may serve as a leukemia-specific antigen and can be considered an ideal target for NPM1-mutated acute myeloid leukemia (AML) immunotherapy. Different in silico instruments and in vitro/ex vivo immunological platforms have identified the most immunogenic epitopes from NPM1-mutated protein. Spontaneous development of endogenous NPM1-mutated-specific cytotoxic T cells has been observed in patients, potentially contributing to remission maintenance and prolonged survival. Genetically engineered T cells, namely CAR-T or TCR-transduced T cells, directed against NPM1-mutated peptides bound to HLA could prospectively represent a promising therapeutic approach. Although either adoptive or vaccine-based immunotherapies are unlikely to be highly effective in patients with full-blown leukemia, these strategies, potentially in combination with immune-checkpoint inhibitors, could be promising in maintaining remission or preemptively eradicating persistent measurable residual disease, mainly in patients ineligible for allogeneic hematopoietic stem cell transplant (HSCT). Alternatively, neoantigen-specific donor lymphocyte infusion derived from healthy donors and targeting NPM1-mutated protein to selectively elicit graft-versus-leukemia effect may represent an attractive option in subjects experiencing post-HSCT relapse. Future studies are warranted to further investigate dynamics of NPM1-mutated-specific immunity and explore whether novel individualized immunotherapies may have potential clinical utility in NPM1-mutated AML patients.

Prognostic Impact of Distinct Combinations of Molecular Mutations in AML Patients. Results of Two-Center International Cooperation Study

To analyze the prognostic impact of distinct combinations of molecular aberrations, a retrospective analysis of 620 acute myeloid leukemia (AML) was performed. The mean patient's age was 57 (16-85) years. 47% of patients were characterized by abnormal karyotype: favorable (11%), intermediate (67%), and adverse (22%) risk. We analyzed NPM1, FLT3, DNMT3A, IDH1/2, and c-KIT mutations in diagnostic samples. Molecular genetics and cytogenetics considered adverse risk for 28% of patients, favorable for 26%, and intermediate in 46% of patients. More common mutations in whole cohort of patients and in patients with normal karyotype (NK) were NPM1 (22% and 36%), FLT3- ITD (23% and 31%). In addition, DNMT3A and IDH1 mutations were found in 18% and 17% of NK-AML patients, respectively. IDH2 mutations were associated with older patients age (p=.0001). DNMT3A and NPM1 mutations were more frequently detected in patients with monocyte/myelomonocytic differentiation of leukemic blasts (p=.0001). C-kit mutations were more common in Core Binding Factor AML.The presence of NPM1 mutations were associated with low relapse rate (p=.02) and significant better disease free (DFS) and overall survival (OS), particularly in cases of single mutation (p=.05). NPM1mut AML patients with additional FLT3 -ITD mutations were characterized by the inferior DFS as compared with others combination of mutations (p=.01). Patients with DNMT3Amut / FLT3mut / NPM1wt have demonstrated more inferior DFS than DNMT3mut / FLT3wt / NPM1mut (p=.01). Moreover, in patients after allogeneic stem cell transplantations, combinations of DNMT3Amut and FLT3 -ITDmut was a significant adverse prognostic factor (p=.01). Among FLTwt patients, the favorable DFS was seen in patients with NPM1 mutation and poor outcome was estimated in those with DNMT3A mutation (p=.03). Among FLT3mut / NPM1mut patients, more favorable outcome was found in cases with additional IDH1 and IDH2 mutations. The data suggest that favorable prognosis of NPM1 mutation dominates in cases of DNMT3A but not in FLT3 mutations, but poor prognosis of FLT3 mutation prevails all others. We conclude that mutations status is important factor for risk stratifications of AML patients. DisclosuresNo relevant conflicts of interest to declare.