NPM1-mutated Acute Myeloid Leukemia Research Articles

Molecular, clinical, and therapeutic determinants of outcome in NPM1 mutated AML

AbstractAlthough NPM1-mutated acute myeloid leukemia (AML) carries a generally favorable prognosis, many patients still relapse and die. Previous studies identified several molecular and clinical features associated with poor outcomes; however, only FLT3-internal tandem duplication (ITD) mutation and adverse karyotype are currently used for risk stratification because of inconsistent results and uncertainty about how other factors should influence treatment, particularly given the strong prognostic effect of postinduction measurable residual disease (MRD). Here, we analyzed a large group of patients with NPM1 mutations (NPM1mut) AML enrolled in prospective trials (NCRI AML17 and AML19, n = 1357) to delineate the impact of baseline molecular and clinical features, postinduction MRD status, and treatment intensity on the outcome. FLT3-ITD (hazard ratio [HR], 1.28; 95% confidence interval [CI], 1.01-1.63), DNMT3A (HR, 1.65; 95% CI, 1.32-2.05), WT1 (HR, 1.74; 95% CI, 1272-2.38), and non-ABD NPM1mut (HR, 1.64; 95% CI, 1.22-2.21) were independently associated with poorer overall survival (OS). These factors were also strongly associated with MRD positivity. For patients who achieved MRD negativity, these mutations (except FLT3-ITD) were associated with an increased cumulative incidence of relapse (CIR) and poorer OS. However, apart from the few patients with adverse cytogenetics, we could not identify any group of MRD-negative patients with a CIR >40% or with benefit from allograft in first remission. Intensified chemotherapy with the FLAG-Ida regimen was associated with improved outcomes in all subgroups, with greater benefits observed in the high-risk molecular subgroups.

A 2024 Update on Menin Inhibitors. A New Class of Target Agents against KMT2A-Rearranged and NPM1-Mutated Acute Myeloid Leukemia.

Menin inhibitors are new and promising agents currently in clinical development that target the HOX/MEIS1 transcriptional program which is critical for leukemogenesis in histone-lysine N-methyltransferase 2A-rearranged (KMT2Ar) and in NPM1-mutated (NPM1mut) acute leukemias. The mechanism of action of this new class of agents is based on the disruption of the menin-KMT2A complex (consisting of chromatin remodeling proteins), leading to the differentiation and apoptosis of AML cells expressing KMT2A or with mutated NPM1. To date, this new class of drugs has been tested in phase I and II clinical trials, both alone and in combination with synergistic drugs showing promising results in terms of response rates and safety in heavily pre-treated acute leukemia patients. In this brief review, we summarize the key findings on menin inhibitors, focusing on the mechanism of action and preliminary clinical data on the treatment of acute myeloid leukemia with this promising new class of agents, particularly revumenib and ziftomenib.

FLT3 and IRAK4 Inhibitor Emavusertib in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia.

Targeting the FLT3 receptor and the IL-1R associated kinase 4 as well as the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The FLT3 and IRAK4 inhibitor emavusertib (CA4948), the MCL1 inhibitor S63845, the BCL2 inhibitor venetoclax, and the HSP90 inhibitor PU-H71 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells in vitro. AML cells represented all major morphologic and molecular subtypes, including FLT3-ITD and NPM1 mutant AML cell lines and a variety of patient-derived AML cells. Emavusertib in combination with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in MOLM-13 cells. In primary AML cells, the response to emavusertib was associated with the presence of the FLT3 gene mutation with an allelic ratio >0.5 and the presence of NPM1 gene mutations. S63845 was effective in all tested AML cell lines and primary AML samples. Blast cell percentage was positively associated with the response to CA4948, S63845, and venetoclax, with elevated susceptibility of primary AML with blast cell fraction >80%. Biomarkers of the response to venetoclax included the blast cell percentage and bone marrow infiltration rate, as well as the expression levels of CD11b, CD64, and CD117. Elevated susceptibility to CA4948 combination treatments with S63845 or PU-H71 was associated with FLT3-mutated AML and CD34 < 30%. The combination of CA4948 and BH3-mimetics may be effective in the treatment in FLT3-mutated AML with differential target specificity for MCL1 and BCL2 inhibitors. Moreover, the combination of CA4948 and PU-H71 may be a candidate combination treatment in FLT3-mutated AML.

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.

Abstract 3218: Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant AML

Abstract Background: Acute myeloid leukemia (AML) with KMT2A-rearrangements have a poor prognosis and so there is an urgent need for novel therapies. Menin inhibitors have shown promising anti-leukemic effects in KMT2A-rearranged and NPM1-mutated AML. However, the efficacy of menin inhibitors as single agents may be limited by the development of resistance mutations. Combining menin inhibitors with other epigenetic drugs may present a strategy to enhance their therapeutic potential. In this study, we performed a combination drug screen using an epigenetic drug library in KMT2A-rearranged (MV4;11) and NPM1-mutant (OCI-AML3) AML cell lines to identify synergistic therapies that can be used in conjunction with menin inhibitors. Methods: During assay optimization, MV4;11 and OCI-AML3 cells were treated with the menin inhibitor DSP-5336, and their viability was assessed. Quality control metrics were applied with Z’ values consistently greater than 0.5 for all assay plates and then the cells were subjected to a small molecule compound library screen containing a set of 932 epigenetic drugs. The screening process included a systematic evaluation of each drug's effect on cell viability in combination with the DSP-5336. Results: The combination drug screen demonstrated robust performance in both MV4;11 and OCI-AML3 cell lines, with reliable quality control metrics. Among the epigenetic drugs tested, five compounds were identified as potential hits that synergistically enhanced the efficacy of DSP-5336 across both cell lines.The five identified compounds exhibited enhanced anti-leukemic effects when combined with the menin inhibitor in either one of the cell lines. The targets of these drugs varied, including histone deacetylases, histone acetyltransferases and BRD4 inhibition, suggesting many alternate mechanisms of action involving epigenetic modulation. Remarkably, one of the 5 hits C301-3161, a KAT2A inhibitor, showed limited activity in either cell line alone but increased cell killing with DSP-5336 in both cell lines, highlighting its potential as a specific therapeutic for combining with DSP-5336 in AML patients. Conclusions: Our findings suggest combining menin inhibitors with other epigenetic drugs may improve AML treatment outcomes. The combination drug screen successfully identified four novel compounds that synergistically enhance menin inhibitor efficacy in both KMT2A-rearranged and NPM1-mutant AML cell lines. These compounds hold significant promise as potential therapeutic candidates for further investigation. Future studies should focus on elucidating the underlying mechanisms of action for each hit and evaluating their effects in preclinical animal models. Ultimately, this research may pave the way for the development of innovative combination therapeutic strategies for AML patients with KMT2A-rearragned and NPM1-mutant AML. Citation Format: Tulasigeri M. Totiger, Sana Chaudhry, Skye Montoya, Maurizio Affer, Anya K. Sondhi, Alexandra Chirino, Claude-Henry Volmar, Shaun Brothers, Justin Watts, Justin Taylor. Combination drug screen identifies novel epigenetic therapies to enhance menin inhibitor efficacy in KMT2A rearranged and NPM1 mutant AML [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3218.

Automated manufacture of ΔNPM1 TCR-engineered T cells for AML therapy

Acute myeloid leukemia (AML) is a heterogeneous malignancy that requires further therapeutic improvement, especially for the elderly and for subgroups with poor prognosis. A recently discovered Tcell receptor (TCR) targeting mutant nucleophosmin 1 (ΔNPM1) presents an attractive option for the development of a cancer antigen-targeted cellular therapy. Manufacturing of TCR-modified Tcells, however, is still limited by a complex, time-consuming, and laborious procedure. Therefore, this study specifically addressed the requirements for a scaled manufacture of ΔNPM1-specific Tcells in an automated, closed, and good manufacturing practice-compliant process. Starting from cryopreserved leukapheresis, 2E8 CD8-positive Tcells were enriched, activated, lentivirally transduced, expanded, and finally formulated. By adjusting and optimizing culture conditions, we additionally reduced the manufacturing time from 12 to 8days while still achieving a clinically relevant yield of up to 5.5E9 ΔNPM1 TCR-engineered Tcells. The cellular product mainly consisted of highly viable CD8-positive Tcells with an early memory phenotype. ΔNPM1 TCR CD8 Tcells manufactured with the optimized process showed specific killing of AML invitro and invivo. The process has been implemented in an upcoming phase 1/2 clinical trial for the treatment of NPM1-mutated AML.

Postinduction molecular MRD identifies patients with NPM1 AML who benefit from allogeneic transplant in first remission

AbstractSelection of patients with NPM1-mutated acute myeloid leukemia (AML) for allogeneic transplant in first complete remission (CR1-allo) remains controversial because of a lack of robust data. Consequently, some centers consider baseline FLT3–internal tandem duplication (ITD) an indication for transplant, and others rely on measurable residual disease (MRD) status. Using prospective data from the United Kingdom National Cancer Research Institute AML17 and AML19 studies, we examined the impact of CR1-allo according to peripheral blood NPM1 MRD status measured by quantitative reverse transcription polymerase chain reaction after 2 courses of induction chemotherapy. Of 737 patients achieving remission, MRD was positive in 19%. CR1-allo was performed in 46% of MRD+ and 17% of MRD− patients. We observed significant heterogeneity of overall survival (OS) benefit from CR1-allo according to MRD status, with substantial OS advantage for MRD+ patients (3-year OS with CR1-allo vs without: 61% vs 24%; hazard ratio [HR], 0.39; 95% confidence interval [CI], 0.24-0.64; P < .001) but no benefit for MRD− patients (3-year OS with CR1-allo vs without: 79% vs 82%; HR, 0.82; 95% CI, 0.50-1.33; P = .4). Restricting analysis to patients with coexisting FLT3-ITD, again CR1-allo only improved OS for MRD+ patients (3-year OS, 45% vs 18%; compared with 83% vs 76% if MRD-); no interaction with FLT3 allelic ratio was observed. Postinduction molecular MRD reliably identifies those patients who benefit from allogeneic transplant in first remission. The AML17 and AML19 trials were registered at www.isrctn.com as #ISRCTN55675535 and #ISRCTN78449203, respectively.

Complex karyotype but not other cytogenetic abnormalities is associated with worse posttransplant survival of patients with nucleophosmin 1-mutated acute myeloid leukemia: A study from the European Society for Blood and Marrow Transplantation Acute Leukemia Working Party.

In the 2022 European LeukemiaNet classification, patients with nucleophosmin 1 (NPM1)-mutated acute myeloid leukemia (AML) were classified in the adverse-risk category in the presence of high-risk cytogenetics (CG). Nonetheless, the impact of various CG aberrations on posttransplant outcomes remains to be unraveled. This registry study analyzed adult patients with NPM1-mutated de novo AML who underwent their first allogeneic hematopoietic cell transplantation in the first complete remission from 2005 to 2021. A total of 3275 patients were identified, 2782 had normal karyotype, 493 had chromosomal aberrations including 160 with adverse-risk CG, 72 patients had complex karyotype (CK), and 66 monosomal karyotype (MK). Overall, 2377 (73%) patients had FLT3-ITD. On univariate analysis, only FLT3-ITD, minimal/measurable residual disease (MRD) positivity and CK, but not abnormal CG, affected posttransplant outcomes. On multivariable analysis, CK was associated with lower overall survival (OS) (hazard ratio [HR] 1.72, p = .009). In the subgroup of 493 patients with aberrant CG, the 2-year leukemia-free survival (LFS) and OS were around 61% and 68%, respectively. On multivariable analysis for this subgroup, CK and MRD positivity were associated with increased risk of relapse (HR 1.7, p = .025; and 1.99, p = .003 respectively) and worse LFS (HR 1.62, p = .018; and 1.64, p = .011 respectively) while FLT3-ITD, MK, or other CG abnormalities had no significant effect. Importantly, CK negatively affected OS (HR 1.91, p = .002). In the first complete remission transplant setting, CK was found as the only cytogenetic risk factor for worse outcomes in NPM1-mutated AML. Nevertheless, even for this subgroup, a significant proportion of patients can achieve long-term posttransplant survival.

Venetoclax–based low intensity therapy in molecular failure of NPM1-mutated AML

AbstractMolecular failure in NPM1-mutated acute myeloid leukemia (AML) inevitably progresses to frank relapse if untreated. Recently published small case series show that venetoclax combined with low-dose cytarabine or azacitidine can reduce or eliminate measurable residual disease (MRD). Here, we report on an international multicenter cohort of 79 patients treated for molecular failure with venetoclax combinations and report an overall molecular response (≥1-log reduction in MRD) in 66 patients (84%) and MRD negativity in 56 (71%). Eighteen of 79 patients (23%) required hospitalization, and no deaths were reported during treatment. Forty-one patients were bridged to allogeneic transplant with no further therapy, and 25 of 41 were MRD negative assessed by reverse transcription quantitative polymerase chain reaction before transplant. Overall survival (OS) for the whole cohort at 2 years was 67%, event-free survival (EFS) was 45%, and in responding patients, there was no difference in survival in those who received a transplant using time-dependent analysis. Presence of FLT3-ITD mutation was associated with a lower response rate (64 vs 91%; P < .01), worse OS (hazard ratio [HR], 2.50; 95% confidence interval [CI], 1.06-5.86; P = .036), and EFS (HR, 1.87; 95% CI, 1.06-3.28; P = .03). Eighteen of 35 patients who did not undergo transplant became MRD negative and stopped treatment after a median of 10 months, with 2-year molecular relapse free survival of 62% from the end of treatment. Venetoclax–based low intensive chemotherapy is a potentially effective treatment for molecular relapse in NPM1-mutated AML, either as a bridge to transplant or as definitive therapy.

Influence of Bone Marrow Blast Enumeration and Co-Occurring Myelodysplasia Related Gene Mutations in NPM1-Mutated Myeloid Malignancies

Introduction Both World Health Organization (WHO 5th) and International Consensus Classification (ICC) guidelines recognize mutations in nucleophosmin 1 ( NPM1mut) as a class defining molecular event in acute myeloid leukemia (AML). WHO 5th omitted the blast percentage requirement and ICC lowered the blast threshold to 10%. NPM1mut AML without FLT3 ITD or adverse risk cytogenetics is considered favorable risk in European LeukemiaNet (ELN) 2022, however co-occurring adverse-risk myelodysplasia related mutations (MR mut) do not influence ELN 2022 risk stratification. This analysis addresses the prognostic impact of blast enumeration in NPM1mut myeloid neoplasms (MN) with &amp;lt;10% blasts and influence of co-occurring mutations in NPM1mut AML. Methods We interrogated publicly available data from adult (≥18 years) patients (pts) with chronic myeloid neoplasms included in the International Working Group for Prognosis in MDS (N= 3,323; Bernard et al. NEJMEvid 2022) and two independent AML cohorts (AMLSG [N=1540] Papaemmanuil et al. NEJM 2016; UKNCRI [N=2,113] Tazi et al. Nat. Commun. 2022). Between group differences were assessed using the Wilcoxon rank-sum test or Fisher's exact test as appropriate. Time to event endpoints were analyzed using the log-rank method. Multivariate analysis (MVA) used cox proportional hazards regression. Results NPM1 mut was identified in 17% (N=1148) of pts (AML: 30% [1109/3653], MN with &amp;lt;20% blasts: 1.2% [39/3,323]). Compared to pts with NPM1mut AML, pts with NPM1mut MN (MDS, CMML, aCML) with blast &amp;lt; 20% were older (median age 65 vs. 55 years, p &amp;lt; 0.001), had a lower median total WBC count (5 vs. 32 x10 9 /L, p &amp;lt; 0.001) and bone marrow (BM) blast percentage (8% vs. 76%, p &amp;lt; 0.001). No significant difference was observed between NPM1mut AML and MN with &amp;lt;20% blasts in median hemoglobin (9.1 vs. 9 x10 9, p: 0.44), platelets (62 vs. 66 x10 9 /L, p: 0.55), or NPM1mut variant allele frequency (VAF; 35% vs. 37%, p: 0.64). After a median follow up of 4.7 years (range: 0-12), no difference in overall survival (OS) was observed between pts with low blast (LB; &amp;lt;10%) NPM1mut MN (N=19) vs. ≥10% blasts (N=18) (median OS: 0.64 years [95% CI: 0.31-3.0] vs. 1.1 years [95% CI: 0.95-NR], p=0.32). Median leukemia free survival (LFS) was 0.81 years (95% CI: 0.67-1.5) and not significantly different between NPM1mut MN pts with &amp;lt;10 vs. ≥10% blasts (median LFS: 0.55 vs. 0.91 years, p=0.27). In pts with NPM1mut AML (N=1109), 52% (N=581) were ELN 2022 favorable-risk; 15% (N=92) had MR mut including SRSF2 (51%, N=47), STAG2 (21%, N=19), ASXL1 (9%, N=8), BCOR (9%, N=8), RUNX1 ( 8%, N=7), EZH2 (5%, N=5), SF3B1 (5%, N=5), U2AF1 (1%, N=1), and ZRSR2 (1%, N=1). Pts with MR mut were older (median age 62 vs. 53 years, p &amp;lt; 0.001), had higher BM blasts (80% vs. 72% p=0.019), and lower platelets (45 vs. 77 x10 9/L p &amp;lt; 0.001) compared to pts with NPM1mut AML without MR mut. No significant difference in NPM1mut VAF was observed (34% in both groups). After a median follow up of 3.5 years (range: 0-15), median OS was 6.2 years in pts with NPM1mut AML (95% CI: 4.2-9.7). Inferior OS was observed in ELN favorable risk NPM1mut pts with vs. without MR mut, respectively (2 vs. 8.4 years, p=0.0001). When stratified by age &amp;lt; 60 vs. ≥ 60 years, younger pts with MR mut had an increased risk of death (HR 1.7 95% CI: 1.1-2.8, p: 0.02). No OS difference was observed in older pts with or without MR mut (HR: 1.2, 95% CI: 0.80-1.68, p=0.44), however OS was significantly shorter in older pts with NPM1mut compared to younger pts (median 1.7 years vs. NR, p &amp;lt; 0.001). Patients with NPM1mut /MR mut AML had survival comparable to patients with ELN 2022 intermediate risk AML (2.0 vs. 2.1 years, p=0.35). In MVA of NPM1mutAML adjusted for MR mut, BM blast percentage, age, performance status, baseline WBC, hemoglobin, platelet count, NPM1mut VAF, and treatment setting (AMLSG or UKNCRI), the presence of an MR mut (HR 1.39 [95% CI: 1.0-1.9], p=0.04), age &amp;lt; 60 (HR 0.43 [95% CI: 0.32-0.56], p &amp;lt; 0.001), and performance status (HR 1.35 [95% CI: 1.14-1.60], p=0.0004) retained statistical significance. Conclusion Pts with low blast NPM1mut MN have outcomes similar to pts with NPM1mut AML, supporting NPM1mut as an AML defining event irrespective of BM blast enumeration. In patients with ELN favorable risk NPM1mut AML, co-occurrence of MR mut is independently associated with inferior survival, comparable to ELN intermediate risk AML. These results may inform future refinements of current consensus guidelines.

The Genomic Landscape and Its Clinical Implication in NPM1-Mutated AML Patients: A Study within the AMLSG 09-09 Clinical Trial

Background: Mutations in the Nucleophosmin 1 gene ( NPM1 mut ) represent one of the most common genetic lesions in acute myeloid leukemia (AML).Based on its characteristic clinico-pathologic features, NPM1 mutAML has been recognized as a distinct entity among the category “AML with recurrent genetic abnormalities”. According to the ELN 2022 genetic risk-stratification, NPM1 mut AML, in the absence of FLT3-ITD mutation, is associated with a favorable prognosis. However, a significant proportion of these patients (pts) relapse after intensive therapy suggesting that other co-mutations may have an impact on outcome. Aims: To comprehensively characterize the genomic landscape and leukemogenic trajectoriesin a large cohort of NPM1 mut AML pts and to investigate its prognostic and predictive impact on outcome. Methods: Targeted DNA sequencing (mean read depth: 1817) on the entire coding region of 263 genes was performed in 568 NPM1 mut AML pts (median age: 58.7 years; 18-60 years, n=317; &amp;gt;60 years, n=251). All pts were enrolled in the randomized open-label Phase 3 AMLSG 09-09 trial [NCT00893399; Döhner H et al. Lancet Haematol 2023]. In this trial, pts were assigned to intensive chemotherapy plus all- trans retinoic acid with or without gemtuzumab ozogamicin; none of the pts received midostaurin. Results: In total n=3,058 variants (variant allele frequency of ≥1%) were identified in 195/263 genes. The median number of co-mutations was 3 (range 0-11). The most common co-mutated genes were DNMT3A (49.5%), FLT3-TKD (42.8%) PTPN11 (24.8%), NRAS (22.7%) TET2 (21.7%), IDH2 (21.3%), IDH1 (18%), and FLT3-ITD (17.3%). DNMT3AR882 hotspot mutations occurred more frequently in younger pts (36.6% vs 17.1%), while there was no difference for DNMT3AnonR882 mutations between the two age groups (21.8% and 21.9%). An age-dependent difference was also identified for mutations in myelodysplasia-related genes ( ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2) as defined by the ICC, occurring with a higher incidence in older pts (30.3% vs 12.3% in younger pts). Analyzing the mutational pattern of co-mutations, we found statistically significant tertiary gene-gene interactions: e.g., NPM1- NFE2- STAG2 (p&amp;lt;.001), NPM1- IDH2- SRSF2 (p&amp;lt;.001), NPM1- CEBPA- TET2 (p&amp;lt;.001), NPM1- DNMT3AR882- NRAS (p=.002), and NPM1- ASXL1- SRSF2 (p=.004); mutual exclusivities were identified for NPM1- DNMT3AR882- DNMT3AnonR882 (p&amp;lt;.001), NPM1- IDH2- TET2 (p&amp;lt;.001), NPM1- DNMT3AR882- SRSF2 (p&amp;lt;.001), NPM1- IDH1- TET2 (p&amp;lt;.001), and NPM1- FLT3-ITD- KRAS (p&amp;lt;.002). Correlating co-mutation data with outcome, we found that DNMT3AR882 hotspot mutations confer inferior event-free (EFS) and overall survival (OS) only in younger pts (EFS, p&amp;lt;.001 vs p=.11, Figure 1a; OS, p=.003 vs p=.8), whereas DNMT3AnonR882 mutations did not impact prognosis within the two age groups. We also found a negative prognostic impact of IDH1 mutations which was restricted to younger pts (EFS, p=.05), whereas IDH2 mutations were associated with superior EFS in older pts (p=.04) and superior OS in both groups (p=.05 and p=.03). Of note, co-mutations occurring in one or more of the myelodysplasia-related genes did not impact EFS or OS (Figure 1b). In multivariable analysis (all pts) including age, WBC, LDH, allogeneic transplantation in CR1, and mutations with an incidence of at least 3% as covariables, age (HR,1.03; p&amp;lt;.001), DNMT3AR882 (HR, 1.86; p&amp;lt;.001), FLT3-ITD (HR, 1.54; p=.012), IDH1 (HR, 1.48; p=.009), MYC (HR, 1.83; p=.032), and WT1 (HR, 1.73; p=.012), were associated with an inferior EFS, while SMC3 mutation showed favorable EFS (HR, 0.44; p=.019). To further study the leukemogenic trajectories, we used an oncogenetic tree modeling algorithm, which yielded a tree with several main branches including DNMT3AR882, DNMT3AnonR882, FLT3-TKD , IDH1, IDH2, PTPN11, and TET2. These mutations might represent initiating events which predispose to additional events with further distinct branches. Conclusions: Our study provides comprehensive data on the genomic landscape and its clinical impact in pts with NPM1mut AML fit for intensive chemotherapy. The co-mutational pattern clearly differs between younger and older NPM1 mutAML pts. Using this large dataset allowed the identification of secondary and tertiary gene-gene interactions with significant impact on outcome. Further data analysis is ongoing and will be presented at the meeting.

Intensive Induction Chemotherapy Vs Hypomethylating Agents + Venetoclax (HMA/VEN) in NPM1-Mutant Newly Diagnosed Acute Myeloid Leukemia (AML) - a Multicenter Cohort Study

Introduction: Intensive induction chemotherapy (IC) with cytarabine + anthracycline remains the standard of care for younger and fit patients (pts) with acute myeloid leukemia (AML). Post-hoc analyses from clinical trials evaluating venetoclax (VEN)-based therapies in AML have identified mutations in NPM1 as being associated with high rates of durable responses. However, these trials were conducted in pts age ≥75 years or unfit for IC. Thus, it is unknown whether IC or hypomethylating agent (HMA)+VEN is the optimal frontline treatment for pts with NPM1-mutant AML. We performed a large multicenter retrospective cohort study dedicated to pts with newly diagnosed AML with NPM1 mutations, comparing treatment modalities and assessing the impact of clinical and molecular characteristics on response and survival. Methods: We included pts with newly diagnosed, NPM1-mutant AML who were treated at 4 large academic centers with IC (either conventional 7+3 or liposomal cytarabine/daunorubicin [CPX-351]) or HMA+VEN. Detection of NPM1 mutations was performed at the participating sites either by polymerase chain reaction (PCR) or next-generation sequencing (NGS) per local standards. Composite complete response (cCR) was defined as complete response (CR) + CRi (CR with incomplete count recovery) per European LeukemiaNet 2017 criteria. Overall survival (OS) was compared between groups by log-rank test. Cox regression multivariable model for OS was fitted using a stepwise backward selection to evaluate the effect of treatment, allogeneic stem cell transplantation (allo-SCT) as a time-varying covariate and additional covariates with a p&amp;lt;0.1 in the univariate model. Results: We reviewed 1132 pts with AML and included 225 pts with NPM1-mutant AML in the final analysis ( Table 1). Among these pts, 71% (n=160) and 29% (n=65) received IC and HMA+VEN, respectively. Some pts (22%) in the IC group also received midostaurin compared to 0% in the HMA+VEN group. Pts who received IC for NPM1-mutant AML were younger (median age 61 years [Range: 22 -76] vs 74 years [33 -89]; p&amp;lt;0.001) when compared to HMA+VEN and less likely to have prior myelodysplastic syndrome (MDS) or myeloproliferative neoplasms (MPN; 10% vs 20%, p=0.042), and SF3B1 mutations (3.1% vs 15%; p=0.002). There was no difference in the rate of FLT3-ITD mutations between groups (36% vs 29%; p=0.36). Rates of cCR and CR were higher for IC than HMA+VEN-treated pts (cCR: 87% vs 74%; p=0.018; CR: 83% vs 52%; p&amp;lt;0.001), as well as rates of allo-SCT (56% vs 23%; p&amp;lt;0.001). Median OS was 31 months (95% CI: 24 months [mo] - not reached [NR]) for the total cohort of pts with NPM1-mutant AML and was better in pts treated with IC vs HMA+VEN (IC: 24 mo OS 65% [95% CI: 57 - 74%] vs HMA+VEN: 24 mo OS 38% [95% CI: 26 - 55%]; p&amp;lt;0.001). While the median OS remained favorable for IC among pts age &amp;gt; 60 years with NPM1-mutant AML (34 mo [95% CI: 24 mo - NR] vs 15 mo [13 mo - 28 mo]; p=0.015), there was no difference in OS by treatment type among the pts undergoing allo-SCT (IC: median OS not reached; 24 mo OS: 72% [95% CI: 63-83%] vs HMA+VEN: median OS 29 mo; 24 mo OS 69% [95% CI: 48-100%]; p=0.55). In the univariate analysis, treatment (HMA+VEN vs IC), age, prior chemotherapy exposure, abnormal cytogenetics, SF3B1, TET2 and RNA splicing mutations were associated with worse outcomes ( Table 2). Of note, neither the presence of concurrent FLT3 or IDH1/2 mutations nor allo-SCT as a time-varying covariate had a statistically significant impact on OS. In the multivariable analysis, treatment with HMA+VEN vs. IC retained its predictive value (hazard ratio: 2.00; 95% CI: 0.92 - 3.03; p=0.006), as well as prior chemotherapy and abnormal cytogenetics ( Table 2). Conclusion: In pts with newly diagnosed NPM1-mutant AML, treatment with IC was associated with higher cCR rates and favorable OS when compared with HMA+VEN in multivariable regression analysis. However, as residual confounding cannot be excluded given the significant differences in baseline patient and disease characteristics between the two groups, a randomized clinical trial is needed to definitively verify the potential superiority of IC vs HMA+VEN in pts with newly diagnosed NPM1-mutant AML.

NPM1-Mutated Myeloid Neoplasms: Updated Outcomes with High-Intensive Chemotherapy Regardless of the Blast Percentage

Background: The most recent World Health Organization (WHO) classification has stablished NPM1-mutated myeloid neoplasms (MNs) to be acute myeloid leukemia (AML) regardless of bone marrow (BM) blast percentage. In contrast, the new International Consensus Classification (ICC) considers NPM1-mutant MNs as AML only in the presence of ≥10% BM blasts. Previous studies from our group and others have reported favorable outcomes of NPM1-mutant MNs with &amp;lt;20% blasts when treated with intensive chemotherapy. Here we seek to further evaluate clinical outcomes of this group of pts. Methods: We retrospectively re-evaluated the patient's characteristics, disease features and outcomes in patients with NPM1-mutated oligoblastic AML (&amp;lt;20% blasts) (oligo-AML). In addition, we also evaluated the clinicopathological characteristics in a cohort of patients with secondary NPM1-mutated AML (sAML), the majority of which had received treatment before progressing to AML. The Kaplan-Meier method was used to estimate the overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS). Results: We identified 54 patients with NPM1-mutated oligo-AML. Median age was 62 years (range, 19-87) and most of them were female (59%). Median bone marrow (BM) blasts was 10%. Most patients could have been classified as CMML-2 (7/54, 13%), MDS-EB1 (7/54, 13%) and MDS-EB2 (24/54, 44%), based on the 2016 WHO classification. Median NPM1 variant allele frequency (VAF) was 0.38 (range, 0.05-0.52) and 38 patients (70%) had normal karyotype. These patients harbored a median of 1 co-mutation (range, 0-5), predominantly DNA methylation pathway, such as DNMT3A (31%) or TET2 (23.5%), or RAS pathway, mainly NRAS (25.5%), but rarely exhibited mutations in other functional pathways (Figure 1). Twenty-three patients (43%) progressed to AML with &amp;gt;20% BM blasts within a median of 10.7 months. Of 54 patients, 46 patients (82%) received chemotherapy-based regimens [61% low-intensity chemotherapy (LI-Chemo) based on hypomethylating agents or low-dose cytarabine and 21% high-intensity chemotherapy (HI-Chemo)] and 21 patients (39%) received a stem cell transplant (SCT). With a median follow up of 67.6 months (LI-Chemo of 48.3 months; HI-Chemo of 71.4 months), the median OS, EFS and RFS was 30.4 months, 30.4 months and 14.7 months for the LI-Chemo group, respectively, but median OS, EFS and RFS were not reached (NR) for HI-Chemo group. However, when compared by age, patients with &amp;lt;60 years had trends to have better OS with LI-Chemo or HI-Chemo than patients aged ≥60 years (Figure 2). In addition, 5 patients received chemotherapy-based regimens with venetoclax, however improved OS was only seen when combined with HI-Chemo and SCT (n=2, with a median OS NR without deaths, likely related to younger people). In parallel, we identified 31 patients with NPM1-mutated sAML progressed from oligo-AML. Median age was 67 years (range, 31-83) and female were 58%. Median BM blasts was 40%. Median NPM1 VAF was 0.38 (range, 0.02-0.56). Most patients did not exhibit clonal cytogenetic evolution and remained with a diploid karyotype (65%). Seventy-four percent of patients (17 out of 23 tested) had known prior NPM1-mutated oligo-AML, and in 6 patients NPM1 mutation was acquired at progression. Median number of co-mutations were 3 (range, 0-7). DNTM3A (65%) or TET2 (33.3%) mainly co-occurred with RAS pathway mutations, and in particular FLT3-ITD (26.7%) or FLT3-TKD (13.3%) (Figure 1). Eleven of 31 patients (36%) received LI-Chemo regimens, whereas 15 of 31 patients (48%) had HI-Chemo regimens. Nine patients (29%) underwent SCT. With a median follow up of 39.6 months (LI-Chemo of 55.5 months; HI-Chemo of 39.4 months), the median OS, EFS and RFS was 11.4 months, 12.4 months and NR for LI-Chemo, respectively; and 13.2 months, 6.5 months and NR for HI-Chemo, respectively. No significant differences were seen depending on the number of therapies received for previous oligo-AML. Previously untreated patients who received a venetoclax-based regimen with SCT had prolonged survival (n=4, median OS NR, with median follow up of 41 months). Conclusions: NPM1-mutated acute myeloid leukemia can be treated as such irrespective of blasts burden. For these cases, especially young patients, should be treated with intensive chemotherapy and subsequent allogeneic stem cell transplant as a clear curative approach.

Genomic Analyses Unveil the Pathogenesis and Inform on Therapeutic Targeting in KMT2A-PTD AML

A partial tandem duplication (PTD) in the KMT2A gene is detected in approximately 5-10% of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Previous studies revealed that HOX-genes are highly expressed in KMT2A-PTD AML similar to KMT2A-rearranged or NPM1-mutated AML. While the overexpression of HOX-genes is a convergent oncogenic pathway shared by these molecular subtypes of AML, the mechanism by which KMT2A-PTD induces high expression of the HOX-genes remains unclear and may be distinct from that mediated by balanced KMT2A-translocations, and NPM1 mutations. We stipulated that analysis of common molecular features of KMT2A-PTD will help identifying mechanistic intersections pointing towards possible targeted therapies. In a total of 5420 AML cases 1 including 793 cases from publicly available sources (Beat AML 2 and TCGA 3), we identified 254 cases of KMT2A-PTD. Somatic mutational screening and karyotype analysis showed co-occurrence with DNMT3A (31%), FLT3-ITD (32%), IDH1/2 (26%), RUNX1 (28%), TET2 (18%), SRSF2, STAG2, U2AF1 mutations and deletion 5q. Strong leukemic initiators such as KMT2A-rearrangement, CBFB-MYH11, PML-RARA, RUNX1-RUNX1T1, trisomy 8 and NPM1 mutations were mutually exclusive with KMT2A-PTD and did not rely on acquisition of additional mutations linked to secondary AML ontogenesis ( e.g., ASXL1, RUNX1, SRSF2, and STAG2) different from KMT2A-PTD. Next, we analyzed bulk mRNA-expression between mutually exclusive KMT2A-PTD, KMT2A-rearranged or NPM1-mutant high HOX-genes expressors. While several differentially expressed genes (DEG) between KMT2A-PTD and KMT2A-rearranged cases were detected, there were few DEG between KMT2A-PTD and NPM1-mutated cases. Compared to KMT2A-rearrenged cases, KMT2A-PTD had higher expression of CD34 and KIT in addition to some HOX-genes such as HOXB2 or NKX2-3. In contrast, expression of CD1D, CD14, and immune checkpoint genes including LILRB4, CD276, CD86, and SIGLEC7 was much higher in KMT2A-rearranged than KMT2A-PTD cases. These results prompted us to analyze the hematopoietic differentiation status. For this purpose, we defined three gene signatures along the HOX-targeted genes axis, termed as HOX-primitive, HOX-transient, and HOX-committed profiles. Differentiation analysis revealed almost all of KMT2A-PTD cases could be subclassified into HOX-transient stage while most of KMT2A-rearranged cases were HOX-committed. However, NPM1-mutated cases were interspersed in all the differentiation stages along the HOX-axis. We therefore assumed that there were a few DEG shared between KMT2A-PTD and NPM1-mutated cases as some NPM1-mutated AML clustered in the HOX-transient expression profile group. Intriguingly, DEG analysis was concordant with the hierarchical clustering based on the HOX-gens axis. Indeed, CD34 and KIT were highly expressed in the HOX-transient cases, and CD1D and CD14 in the HOX-committed cases, consistent with the enrichment of KMT2A-PTD and KMT2A-rearranged AML, respectively. We can conclude that the observed signatures were unique to the HOX-stages rather than the molecular abnormalities such as KMT2A-PTD and KMT2A-rearrangements. For example, CD34 upregulation was a feature shared transversally by all HOX-transient cases, including both KMT2A-rearranged and NPM1-mutated cases. As shown in Figure, KMT2A-rearranged, KMT2A-PTD, and NPM1-mutated cases were more clearly classified in HOX-profiles than molecular abnormalities. Additionally, among the cases which molecular subtypes were not clear, those with high HOX-genes expression revealed higher MEN1 expression. In conclusion, despite molecularly different, KMT2A-PTD, KMT2A-rearranged, and NPM1-mutated AML have similar expression patterns defined by each HOX differentiation stage. Like core binding factor AML such as RUNX1-RUNX1T1 and CBFB-MYH11, AML with higher expression of HOX-genes should be classified in the same group, HOX-AML. Given the results obtained with menin inhibitors in KMT2A-rearranged and NPM1-mutated AML, our findings open an opportunity for exploiting a therapeutic vulnerability in all HOX-AML including KMT2A-PTD AML or AML with high MEN1 expression. Since HOX-AML highly express genes according to the HOX differentiation profile, stage-specific surface proteins coded by these genes would be promising targets.

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 &amp;gt;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&amp;lt;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&amp;lt;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.

Combined Single Cell Flow Cytometry and Imaging Analyses Reveal Immunomodulatory Effects Exerted By Targeted Phospho-SYK Inhibitors with Elevated Sensitivity in NPM1 Mutated AML

Acute myeloid leukemia (AML) is driven by genetic lesions that can result in addiction to signaling pathways that lead to aberrant proliferation and survival. Several new targeted drugs are being assessed for activity in AML with some recently approved for the treatment of subgroups of patients with specific genetic features, such as mutations in FLT3, IDH1 or IDH2. Entospletinib (ENTO) and lanraplenib (LANRA) are inhibitors of spleen tyrosine kinase (SYK); the latter is a next-generation SYK inhibitor and is currently being evaluated in combination with gilteritinib (GILT) in patients with relapsed or refractory FLT3-mutated AML (NCT05028751). Although SYK is rarely mutated, it has been found to cooperate with FLT3, and is highly active especially in AML patients with mutated FLT3. In addition, SYK is important for immune cell signaling through antibody constant region Fc and B cell receptors. To examine the multi-modulatory effect of pSYK inhibitors in AML, we used combined high parameter flow cytometry (HP-FC) and single cell miniaturized imaging (sc-MI) approaches to profile the immunologic, mutation, and functional protein pathway targets of ENTO and LANRA. Two different AML patient cohorts were assessed by ex vivo treatment with ENTO, LANRA and combinations of LANRA (trametinib, gilteritinib (GILT), aPD-1) over a range of time points (2h, 4h, 3d and 9d) and readouts for cellular phenotype (e.g., differentiation state, immune cell populations) and functional readouts (pSYK, viability) for each cohort were analyzed by HP-FC and sc-MI and associated with available mutation information (bulk gene and single cell RNA sequencing). Differential analyses of data from flow cytometry (AML-HP-FC Cohort) and single cell imaging (AML-sc-MI Cohort) demonstrated: 1) sensitivity to ENTO and LANRA (³ 1uM) was higher in NPM1 and FLT3 mutated AML samples, as measured via cell viability and pSYK target inhibition; sensitivity was not drastically altered in the presence of other mutations (e.g. IDH1/2, MLL); 2) lower doses of ENTO and LANRA (&amp;lt;1uM) induced proliferation in specific cell types, including progenitor myeloid cells, and T and B lymphocytes, but induced more T cell proliferation in AML samples that have low baseline proliferation; analysis with next generation bulk gene and single cell RNA sequencing suggested downregulation of STAT1expression, downregulation of MYC- and mTOR-dependent pathways, and upregulation of IL-1 pathway genes; and 3) LANRA was more broadly effective at targeting pSYK inhibition than ENTO; however, both were effective in combinations (ENTO with trametinib and GILT, and LANRA with aPD-1). In sum, analysis using collective flow and single cell imaging provides corroboratory and complementary information revealing new roles for ENTO and LANRA as immune modulating agents supporting B and T cell proliferation at lower dosages, and with demonstrated greater effect in individuals with NPM1 and FLT3 mutations. These support studies investigating the use of pSYK inhibitors in combination with other therapies to reduce tumor burden, and as an improved line of treatment for AML. Acknowledgements: We are appreciative of the patients and families for their time and participation.

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.

Only FLT3-ITD Mutation Did Not Have a Deleterious Effect on Acute Myeloid Leukemia Patients with NPM1 Mutation, but Concomitant with DNMT3A Mutation or a＜3log Reduction of MRD2 Predicted Poor Survival

Background Co-occurring mutations were observed in NPM1-mutated acute myeloid leukemia (AML) frequently, and the measurable residual disease (MRD) for NPM1 is effective for the prognosis of NPM1-mutated AML. However, the combined effects of co-occurring gene mutation at the baseline and MRD on survival are unclear. We investigated the impact of gene mutation status and NPM1 MRD on predictive value of AML patients with NPM1 mutation. Methord A single-center, retrospective cohort study was carried out in patients diagnosed as AML with NPM1 mutation in Peking University Peoples'hospital. Patients were treated by an anthracycline and cytarabine-based or homoharringtonine and cytarabine-based induction chemotherapy regimen and followed by high dose cytarabine as consolidation therapy. Patients who had the indications of allogeneic hematopoietic stem cell transplantation (allo-HSCT) underwent allo-HSCT. A small part of patients with FLT3-ITD mutation received FLT3 inhibitor. Targeted next-generation sequencing (NGS) were analyzed at diagnosis, and quantitative NPM1 mutation (A, B, and D type) were detected at diagnosis and each end of chemotherapy cycle. As MRD for NPM1 rare type (NPM1 RT) was not available, MRD, relapse free survival (RFS) or overall survival (OS) analysis were conducted in patients received chemotherapy only (NGS chemo), excluding harboring NPM1 RT. Results A total of 234 patients, with NGS results, diagnosed as NPM1-mutated AML were included. 68.4% (160/234) patients including 17 patients with NPM1 RT received chemotherapy only, and NGS chemo patients were 143. After chemotherapy, 31.6% (74/234) patients underwent allo-HSCT. 1.7% (4/234) patients died of infection or hemorrhage during induction therapy. 15.8% (37/234) patients received FLT3 inhibitor，and 92.2% (212/230) patients achieved complete remission rate (CR) or incomplete hematologic recovery (CRi). During median follow-up time of 24.9 [interquartile range (IQR) 11.6-47.8] months, 40.6% (95/234) patients died. Median transcription of NPM1 at diagnosis was 31.13 (IQR 17.23-56.91)%, and would take a median time of 4.1 (IQR 2.6-5.6) months to converted to negativity.Reduction of MRD at the end of second cycle of consolidation (MRD2) ≥3log associated with higher RFS (3-year RFS rate 66.3% vs. 11.1%, p＜0.001) and OS (3-year OS rate 78.4% vs. 32.4%, p＜0.001) than reduction＜3log, and consistent negativity of MRD also predicted higher RFS (3-year RFS rate, 98.1% vs. 42.3%, p＜0.001) and OS (3-year OS rate, 100% vs. 67.7%, p＜0.001) than those whose MRD turned from negativity to positivity. 9 genes were mutated in＞10% in 234 patients, and DNMT3A and FLT3-ITD were the most prevalence (Figure 1). Univariate analysis showed FLT3-ITD, DNMT3A and reduction of MRD2＜3log were associated with poorer RFS and OS in 143 NGS chemo patients. But In multivariate analysis, only DNMT3A mutation [RFS, hazard ratio (HR)=1.9, p =0.023; OS, HR=2.1, p =0.032] and reduction of MRD2 ≥3log（RFS, HR=0.1; OS, HR =1.9, both p＜0.001）were independent prognostic factors for survival. Four subgroups were divided by different status of DNMT3A and FLT3-ITD, that were both FLT3-ITD and DNMT3A negative, FLT3-ITD positive and DNMT3A negative, FLT3-ITD negative and DNMT3A positive, and both FLT3-ITD and DNMT3A positive. The concurrence of FLT3-ITD and DNMT3A was associated with lowest RFS and OS (3-year RFS rate 29.2%, p＜0.001; 3-year OS rate 53.5%, p＜0.001). However, patients with only FLT3-ITD mutation did not contribute to poor survival compared with patients without FLT3-ITD mutation (3-year RFS rate 71.4% vs. 74.4%, p＜0.843; 3-year OS rate 74.5% vs.75.8%, p = 0.759). Three risk group were re-stratified by the gene mutation and gene reduction of MRD2 (Figure2), and the RFS and OS of the poor risk group, defined by patients with ＜3log reduction of MRD2, or with both FLT3-ITD and DNMT3A mutation, could be improved by allo-HSCT(3-year RFS rate, 56.4% vs. 29.2%, p = 0.015; 3-year OS rate, 72.0% vs. 31.6%, p = 0.001). Conclusion In AML patients with NPM1 mutation, the deleterious effect of FLT3-ITD could be pronounced in patients concomitant with DNMT3A mutation. A＜3log reduction of MRD2 was also another independent poor prognostic factor for survival, which could be improved by allo-HSCT. Meanwhile, the conversion of MRD for NPM1 from negative to positive might be a poor prognostic factor, but it requires to be validated by multivariate analysis.

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&amp;lt;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.

APL-like Subset within NPM1-Mutated Acute Myeloid Leukemia: A Distinct Phenotypic Signature Correlating with Early-Onset Vascular Complications

Background: NPM1 mutations represent the most common genetic alteration in adult acute myeloid leukemia (AML), often in co-occurrence with FLT3-ITD and mutations in genes influencing DNA methylation ( DNMT3A, IDH1, IDH2 and TET2). Whether these genetic alterations correlate with distinct immunophenotypic and clinical features is still a matter of debate. There is growing evidence of distinct immunophenotypic subsets driven by co-occurring genetic mutations in NPM1-mutated AML, potentially accounting for the heterogeneity of clinical presentations and outcome. Mason et alhave described a subtype of NPM1-mutated AML, displaying an immunophenotypic profile resembling that of acute promyelocytic leukemia, specifically, the negativity for both CD34 and HLA-DR, and as such defined APL-like. Aims: We studied the characteristics of a series of patients with APL-like NPM1-mutated AML, focusing on the incidence of vascular events at disease onset, and investigated the impact of some markers (blood counts, coagulation parameters and LDH) reported to correlate with coagulopathy in APL. Methods: The study cohort included patients diagnosed with NPM1-mutated AML at our Centre according to conventional morphological, immunophenotypic, cytogenetic and molecular criteria. Vascular events were defined according to the revised World Health Organization (WHO) bleeding scale and to the CTCAE grading of thromboembolic events. Results: From April 2007 to May 2023, 139 patients with a diagnosis of NPM1-mutated AML were enrolled, of whom 31 (22.3%) featured by APL-like phenotype. Their characteristics are detailed in Table 1. APL-like patients were older (64 y) compared to non-APL-like (57 y, P=0.002) NPM1-mutated patients; no further difference emerged for baseline blood count parameters. Vascular complications (n=22 bleeding events and n=2 thrombotic events) were significantly more frequent in the APL-like (n=10, 34.5%) than non-APL-like (n=16, 13.6%, P=0.015) group. There was a trend for more severe (G3-G4) vascular events in APL-like (3/31, 9.7%) vs non-APL-like (3/108, 2.8%, P=0.12). Also, abnormal coagulopathy-related parameters, including INR ≥1.5 and/or fibrinogen below normal level) were more frequent in APL-like patients (27.6% versus 15.7%,). D-dimer levels resulted significantly higher in APL-like patients (median 5998 ng/ml versus 2287 ng/ml, P=.005). The D-dimer/fibrinogen ratio (DD/FBG) showed significantly higher level in APL-like (median 16.84) vs non-APL-like (4.4, P=.017) patients. Of note, in multivariate analysis, APL-like phenotype maintained a value (OR=2.67, P=0.007) on DD levels from WBC count (OR=2.37, P=0.017), thus suggesting an effect independent from leukocytosis. Also, APL-like subset maintained an age-independent impact (OR=2.77, P=0.041) on the rate of vascular events. Among the 85 patients with full molecular information, there was a significant enrichment IDH1 (36.8%, P=0.002) and IDH2 (47.6%, P=.001) mutations in APL-like vs non-APL-like (6.1% and 8.6%, respectively) patients. No difference in the incidence of TET2 mutations was observed (18.8% and 25% in APL-like and non-APL-like, respectively, P=0.744). As regards outcome, complete remission rate was 85.7% and 78.9% in APL- and non-APL-like (P=0.43). We did not observe any significant difference between APL- and non-APL-like patients in disease-free (13.2 vs 22.0 months; P=0.83) or overall (15.9 vs 15.1 months, respectively; P=0.38) survival. Conclusions: Our findings suggest APL-like signature to be a potential predictor of susceptibility to vascular events within NPM1-mutated AML. Our results deserve validation in a larger patient set and might indicate the utility of an intensive monitoring and supportive care to prevent early vascular, especially hemorrhagic, events in this patient category.