NPM1-mutated Patients Research Articles

Prognostic Relevance of NPM1 and FLT3 Mutations in Acute Myeloid Leukaemia, Longterm Follow-Up-A Single Center Experience.

Simple SummaryIn acute myeloid leukemia, molecular genetics abnormalities, particularly NPM1 and FLT3 mutations, have a recognized prognostic role in the ELN risk classification and guide treatment decisions, especially since the availability of FLT3-targeted drugs. The NILG-AML 01/00 protocol uses a modified induction approach (ICE) and delivers the most active cytostatic agents at maximal doses in consolidation (a high dose of ARAC plus idarubicin) with autologous stem cell support. It calls for allogeneic transplant only in ELN high-risk patients including NPM1-wt FLT3-ITD-mutated patients. The results obtained from 171 patients showed that the median survival was not reached, and 5y-OS was 58% +/− 4. The prognostic influence of co-mutated FLT3 was overcome, and the efficacy of this treatment reduced the need for early consolidation with an allogeneic transplant in double-mutated patients, in their first complete remission. These data could represent the benchmark against which results of therapeutic programs using second-generation FLT3-targeted drugs should be compared.The prognosis of acute myeloid leukemia depends on genetic aberrations, particularly NPM1 and FLT3-ITD mutations. The targeted drugs’ availability has renewed interest in FLT3 mutations, but the impact of these genetic alterations using these treatments is yet to be confirmed. Our objective was to evaluate the results obtained with the intensified NILG-AML 01/00 protocol (ClinicalTrials.gov Identifier: NCT 00400673) in 171 unselected patients (median age, 54.5 years, range 15–74) carrying the FLT3 (ITD or TKD) and/or NPM1 mutations. The CR rate and 5-y survival were 88.3% and 58% +/− 4, respectively, significantly higher in the NPM1-mutated (CR 93.9%, p: 0.0001; survival 71% +/− 6, p: 0.0017, respectively). In isolated ITD patients, the CR was lower (66.7%, p: 0.0009), and the 3 years-relapse-free survival worse (24%, p: <0.0002). The presence of ITD, irrespective of the allelic ratio, or TKD mutation, did not significantly affect the survival or relapse-free survival among the NPM1-co-mutated patients. Our data indicate that a high dose of ARAC plus idarubicin consolidation exerts a strong anti-leukemic effect in NPM1-mutated patients both with the FLT3 wild-type and mutated AML, while in the NPM1 wild-type and FLT3-mutated, the therapeutic effect remains unsatisfactory. New strategies incorporating target therapy with second-generation inhibitors will improve these results and their addition to this aggressive chemotherapeutic program merits testing.

Contemporary outcomes in IDH-mutated acute myeloid leukemia: The impact of co-occurring NPM1 mutations and venetoclax-based treatment.

Isocitrate dehydrogenase 1 or 2 (IDH1 or IDH2) mutations occur frequently in newly diagnosed (ND) acute myeloid leukemia (AML) often with co-occurring NPM1 mutations, which may influence treatment outcomes. Detailed analysis of IDH-mutated AML treated with venetoclax and influence of co-occurring NPM1 mutations remains unclear. This retrospective single-center cohort study evaluated clinical and molecular demographics,response and survival, and impact of co-occurring NPM1 mutations in patients with IDH1 or IDH2-mutated AML. 556 patients with IDH1, IDH2, and/or NPM1 mutated AML were included. Patients with IDH1mut AML (N= 119) were more likely to have older age, sAML, ELN-adverse risk disease, and adverse-risk cytogenetics compared to those with IDH2mut (N= 229) or IDHwt /NPM1mut AML (N= 208). In multivariate analysis, patients with IDH2mut (HR 0.61 [95%CI: 0.43-0.88], p value: .007) or IDHwt /NPM1mut (HR 0.65 [95% CI: 0.45-0.94], p value: .024) AML had a decreased risk of death versus IDH1mut AML. Venetoclax-based lower-intensity regimens partially abrogated the detrimental effect of IDH1mut with similar OS observed between IDH1mut /NPM1wt , IDH2mut /NPM1wt , and IDHwt /NPM1mut AML. With regards to the influence of IDHmut /NPM1mut cases, IC improved survival in IDH2mut /NPM1mut versus IDH2mut /NPM1wt AML (HR: 0.54 [95% CI: 0.2644-1.082], p value: .077), while venetoclax-based therapy improved survival in IDH1mut /NPM1mut versus IDH1mut /NPM1wt AML (HR: 0.094 [95% CI: 0.01-0.74], p value: .0056). Differing outcomes were observed in IDH1mut versus IDH2mut or NPM1mut AML which were influenced by co-occurring NPM1 mutations and partially abrogated with venetoclax-based therapy. Given the differing biology and survival in IDH1mut AML, investigations incorporating molecularly targeted therapies such as IDH inhibitors remain warranted in this subgroup.

Quantification of mutant-allele expression at isoform level in cancer from RNA-seq data.

Even though the role of DNA mutations in cancer is well recognized, current quantification of the RNA expression, performed either at gene or isoform level, typically ignores the mutation status. Standard methods for estimating allele-specific expression (ASE) consider gene-level expression, but the functional impact of a mutation is best assessed at isoform level. Hence our goal is to quantify the mutant–allele expression at isoform level. We have developed and implemented a method, named MAX, for quantifying mutant–allele expression given a list of mutations. For a gene of interest, a mutant reference is constructed by incorporating all possible mutant versions of the wild-type isoforms in the transcriptome annotation. The mutant reference is then used for the RNA-seq reads mapping, which in principle works similarly for any quantification tool. We apply an alternating EM algorithm to the read-count data from the mapping step. In a simulation study, MAX performs well against standard isoform-quantification methods. Also, MAX achieves higher accuracy than conventional gene-based ASE methods such as ASEP. An analysis of a real dataset of acute myeloid leukemia reveals a subgroup of NPM1-mutated patients responding well to a kinase inhibitor. Our findings indicate that quantification of mutant–allele expression at isoform level is feasible and has potential added values for assessing the functional impact of DNA mutations in cancers.

P490: SNORNA-DERIVED RNAS (SDRNAS) ARE IMPORTANT DRIVERS OF LEUKEMOGENESIS AND LEUKEMIC MAINTENANCE IN AML

Background: Small non-coding RNAs play important roles in leukemogenesis. The canonical function of small nucleolar RNAs (snoRNAs) is to facilitate 2’-O-methylation of rRNA via a target site-specific sequence and thus promote ribosomal biogenesis and function. Recently, we have shown that a subset of small non-coding RNAs, i.e. C/D box snoRNAs are overexpressed in acute myeloid leukemia (AML) and required for AML1/ETO-driven leukemia initiation and maintenance. Depletion of snoRNP protein components or even single snoRNAs can potently inhibit AML cell growth or decrease clonogenicity, and the levels of many snoRNAs positively correlate with leukemic stem cell frequency in AML patients. Over the past years, it has been shown that snoRNAs can be further processed into shorter snoRNA-derived RNA fragments (sdRNAs), but the expression patterns of sdRNAs in AML, as well as mechanistic and functional implications, remain widely elusive. Aims: In this study, we investigated sdRNA expression patterns, functions and mechanisms in AML and healthy hematopoiesis. Methods: We prospectively characterized snoRNA and sdRNA expression in 96 AML patient samples, as well as in healthy hematopoietic stem and progenitor cells and differentiated white blood cells by small RNA sequencing. mRNA sequencing was performed to investigate connections between snoRNAs, sdRNAs and the transcriptome. Further, we depleted AML cells of different snoRNA loci by CRISPR/Cas9-based knock-out and performed lentiviral rescue overexpression of the respective sdRNA or full-length snoRNA. Results: We discovered that more than 120 snoRNAs are further processed into sdRNA fragments in AML. Single snoRNAs often evolve into multiple sdRNA isoforms. Interestingly, sdRNA expression varied significantly between different AML patients. Correlation of sdRNA and full-length snoRNA expression with patient characteristics, clinical outcome and mutational status revealed numerous implications. Those include e.g. a distinct pattern of enriched sdRNAs in NPM1-mutated patients. Expression of several sdRNAs was associated with poor overall, event-free and relapse-free survival in AML. We identified sdRNAs that were overexpressed in AML as compared to HSCs and found characteristic expression patterns among differentiated white blood cells. Notably, ratios of sdRNAs and their host snoRNA were not stable across different samples, suggesting an active mechanism regulating sdRNA processing and thus their downstream pathways. Gene set enrichment analyses based on mRNA and sdRNA correlations suggested distinct cell type specific properties. Forced expression of several sdRNAs rescued the reduced clonogenic potential observed upon depletion of the respective host snoRNA locus. This effect was often identical or even more pronounced than the rescue with the respective snoRNA precursor. Of note, overexpression of distinct sdRNAs in AML cell lines enhanced clonogenic potential. Summary/Conclusion: SnoRNA-derived sdRNAs are a common feature of AML with characteristic expression patterns determined by mutation status and clinical features. Targeting certain sdRNAs or factors involved in sdRNA processing or function might constitute promising novel therapeutic leverage points in AML.

Association of Next Generation Sequencing Based Genotypic Profiling with MICM Characteristics in NPM1 Mutated Acute Myeloid Leukemia

To explain the clinicobiological heterogeneity of NPM1 mutated (NPM1mut) acute myeloid leukemia (AML) by analyzing the association between next-generation sequencing (NGS) profiles and MICM characteristics in patients with this AML subtype. Data of 238 NPM1mut patients with available NGS information on 112 genes related to blood disease was collected, and χ2 test and nonparametric test were used to analyze the distribution association between NGS-detecting mutations and conventional MICM parameters. In entire NPM1mut cohort, totaling 240 NPM1 mutation events were identified, of whom 10 (10/240, 4.2%) were missense mutations, which did not involve any W288 or W290 locus and were found exclusively in NPM1mut/FLT3-ITD- group. All but one of these missense mutations (9/10, 90%) were accompanied by AML subtype-defining recurrent cytogenetic or molecular abnormalities, of which 7 cases were in the low risk and 2 in the high risk. NPM1mut occurred solely as an insertion/deletion (indel) type in the NPM1mut/FLT3-ITD+ group. The incidence of favorable plus unfavorable karyotypes in NPM1mut/FLT3-ITD- group was higher than in NPM1mut/FLT3-ITD+ group (6.4% vs. 0, P=0.031). The positive rates of CD34 and CD7 in NPM1mut/FLT3-ITD+ group were significantly higher than in NPM1mut/FLT3-ITD- group (CD34: 47.9% vs. 20.6%, P<0.001; CD7: 61.5% vs. 29.9%, P<0.001). Logistic analysis showed that FLT3-ITD independently predicted for CD34+ and CD7+ [odds ratio (OR)=5.29, 95%CI: 2.64-10.60, P<0.001; OR=3.47, 95%CI: 1.79-6.73, P<0.001; respectively]. Ras-pathway mutations independently predicted for HLA-DR+ (OR=4.05, 95%CI: 1.70-9.63, P=0.002), and KRAS mutation for MPO- (OR=0.18, 95%CI: 0.05-0.62, P=0.007). TET2/IDH1 mutations independently predicted for CD34- and CD7- (OR=0.26, 95%CI: 0.11-0.62, P=0.002; OR=0.30, 95%CI: 0.14-0.62, P=0.001; respectively), and MPO+ (OR=3.52, 95%CI: 1.48-8.38, P=0.004). DNMT3A-R882 independently predicted for CD7+ and HLA-DR+ (OR=3.59, 95%CI: 1.80-7.16, P<0.001; OR=13.41, 95%CI: 4.56-39.45, P<0.001; respectively), and DNMT3A mutation for MPO-(OR=0.35, 95%CI: 1.48-8.38, P=0.004). Co-existing FLT3-ITD in NPM1mut AML independently predicts for CD34+ and CD7+, co-existing Ras-pathway mutation for HLA-DR+ and MPO-, co-existing TET2/IDH1 mutation for CD34-, CD7-, and MPO+, and co-existing DNMT3A mutation for HLA-DR+, CD7+, and MPO-, thereby providing a new mechanism explanation for the immunophenotypic heterogeneity of these AML patients.

ELN 2017 classification significantly impacts the risk of early death in acute myeloid leukemia patients receiving intensive induction chemotherapy.

Early mortality remains a challenging therapeutic facet of the initial induction phase of intensive chemotherapy in patients with acute myeloid leukemia (AML). The impact of standard molecular evaluation and risk category of the European LeukemiaNet (ELN) 2017 classification model on early mortality has not been rigorously evaluated thus far. We reviewed the medical records of 320 consecutive adult patients with newly diagnosed AML treated with intensive induction chemotherapy in our center from 2007 to 2021. The median age was 56years; 33 patients (10%) died during induction. Patient age, white blood cell count, hemoglobin level, platelet level, creatinine, uric acid, lactate dehydrogenase serum levels, and FLT3-ITD and CEBPA mutational status did not significantly impact early mortality. NPM1mut patients had a lower likelihood of early death compared to NPM1wt (5% versus 13%; p = 0.023) whereas patients with high-risk cytogenetic studies experienced higher rates of induction mortality compared with intermediate and favorable risk patients (20% versus 8 and 7%, respectively; p = 0.049). Adverse risk ELN 2017 was significantly more likely to die during induction compared with intermediate and favorable risk patients (20% versus 10 and 4%, respectively; p = 0.001). Patients treated in 2007-2011 experienced a significantly higher rate of induction death compared with patients in 2012-2021 (17% versus 8%; p = 0.039). Multivariate analysis confirmed adverse ELN 2017 [odds ratio (OR), 6.7; 95% confidence interval (CI), 1.74-25.3; p = 0.006) and treatment timeframe (OR, 0.35; 95% CI, 0.14-0.85; p = 0.019) as pivotal predictors of early mortality. ELN 2017 is a robust prognosticator of early mortality in intensively treated AML patients.

Molecular, clinical, and prognostic implications of PTPN11 mutations in acute myeloid leukemia

AbstractPrognostic factors associated with chemotherapy outcomes in patients with acute myeloid leukemia (AML) are extensively reported, and one gene whose mutation is recognized as conferring resistance to several newer targeted therapies is protein tyrosine phosphatase non-receptor type 11 (PTPN11). The broader clinical implications of PTPN11 mutations in AML are still not well understood. The objective of this study was to determine which cytogenetic abnormalities and gene mutations co-occur with PTPN11 mutations and how PTPN11 mutations affect outcomes of patients treated with intensive chemotherapy. We studied 1725 patients newly diagnosed with AML (excluding acute promyelocytic leukemia) enrolled onto the Cancer and Leukemia Group B/Alliance for Clinical Trials in Oncology trials. In 140 PTPN11-mutated patient samples, PTPN11 most commonly co-occurred with mutations in NPM1, DNMT3A, and TET2. PTPN11 mutations were relatively common in patients with an inv(3)(q21q26)/t(3;3)(q21;q26) and a normal karyotype but were very rare in patients with typical complex karyotype and core-binding factor AML. Mutations in the N-terminal SH2 domain of PTPN11 were associated with a higher early death rate than those in the phosphatase domain. PTPN11 mutations did not affect outcomes of NPM1-mutated patients, but these patients were less likely to have co-occurring kinase mutations (ie, FLT3-ITD), suggesting activation of overlapping signaling pathways. However, in AML patients with wild-type NPM1, PTPN11 mutations were associated with adverse patient outcomes, providing a rationale to study the biology and treatment approaches in this molecular group. This trial was registered at www.clinicaltrials.gov as #NCT00048958 (CALGB 8461), #NCT00899223 (CALGB 9665), and #NCT00900224 (CALGB 20202).

The Mutated Gene Sequenced by NGS and the Correlation of Their Coexistent Mutual Exclusiveness in NPM1 Mutated Acute Myeloid Leukemia

To analyze the clinicobiological heterogeneity of NPM1 mutated (NPM1mut) acute myeloid leukemia (AML) detected by next generation sequencing (NGS) and their coexistence and mutual exclusivity relationship in the AML subtype. The NGS data based on 112 genes related to blood disease in 238 newly diagnosed patients with NPM1mut were collected. The χ2 test and non-parametric test were used to analyze the distribution correlation between the genes in the mutational spectrum. Among all the patients, at least one co-mutation was detected out. The median number per case of the mutated genes, including NPM1mut was 4.5 (range 2－14), among them, there were 5.0 (range 2－10) for NPM1mut/FLT3-ITD+ and 4.0 (range 2－14) for NPM1mut/FLT3-ITD- cases, but it was no significant difference between the two groups (P=0.378). A total of 240 NPM1 mutational events were detected out in entire 238 NPM1mut patients, of which 10 (4.2%) were missense mutations, and were all found in NPM1mut/FLT3-ITD- patients. Most (9/10, 90%) of these NPM1 missense mutations were accompanied by AML subtype-defining cytogenetic or molecular abnormalities, of which 7 patients were in low risk or 2 in high risk. The most common NPM1mut coexisting mutations were DNMT3A (104, 43.7%), followed were FLT3-ITD (95, 39.9%) and FAT1 (57, 23.9%), FLT3-ITD and DNMT3A showed significant coexistence (P=0.005). FLT3-ITD showed significantly reciprocal exclusivity with FLT3-nonITD (P<0.001), NRAS (P<0.001), PTPN11 (P=0.017) and IDH1 (P=0.005), and showed an exclusivity inclination with KRAS (P=0.073). In addition, FLT3-nonITD along with KRAS (P=0.035), NRAS along with KRAS (P=0.008) and PTPN11 (P=0.039) coexisted significantly. Prognoses of AML involving less common NPM1 missense mutations should be stated on a case by case basis. The mutational landscape and co-occurrence and mutual exclusivity correlations of NPM1mut AML provide a mechanism explaining biological diversity and clinical heterogeneity in this AML subset.

Does RAD21 Co-Mutation Have a Role in DNMT3A Mutated AML? Results of Harmony Alliance AML Database

Does RAD21 Co-Mutation Have a Role in DNMT3A Mutated AML? Results of Harmony Alliance AML Database

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.

ELN 2017 Classification Significantly Impacts on the Risk of Early Death in Acute Myeloid Leukemia Patients Receiving Intensive Induction Chemotherapy

Background. Early mortality remains a challenging therapeutic facet of the initial induction phase of intensive chemotherapy in patients with acute myeloid leukemia (AML). The impact of standard molecular evaluation and risk category of the European LeukemiaNet (ELN) 2017 classification model on early mortality have not been rigorously evaluated thus far.Aim. Evaluate the impact of standard clinical, laboratory, cytogenetic, and molecular parameters on the likelihood of early death following induction chemotherapy in a cohort of uniformly treated AML patients receiving standard intensive induction chemotherapy.Methods. We reviewed the medical records of 320 consecutive adult patients with newly diagnosed AML treated with intensive induction chemotherapy in our center from 2007 to 2021. The Kaplan-Meier method was used to determine overall survival and the log-rank test was used to compare differences between groups. All tests were two-sided and a p-value < 0.05 was considered statistically significant.Results. From October 2007 to January 2021, 320 adult patients with AML were treated with standard anthracycline and cytarabine-based intensive induction chemotherapy . Median age was 56 years (range, 18 to 92); 33 patients (10%) died during induction. As outlined in table 1, patient age, white blood cell count, hemoglobin level, platelet level, creatinine, uric acid, lactate dehydrogenase serum levels, FLT3-ITD and CEBPA mutational status did not significantly impact on early mortality. NPM1 mut patients had a lower likelihood of early death compared to NPM1 wt (5% versus 13%; p=0.023) whereas patients with high-risk cytogenetic studies experienced higher rates of induction mortality compared with intermediate and favorable risk patients (20% versus 8% and 7%, respectively; p=0.049). Adverse risk ELN 2017 were significantly more likely to die during induction compared with intermediate and favorable risk patients (20% versus 10% and 4%, respectively; p=0.001). Patients treated in 2007-2011 experienced a significantly higher rate of induction death compared with patients in 2012-2021 (17% versus 8%; p=0.039). Multivariate analysis confirmed adverse ELN 2017 [odds ratio (OR), 6.7; 95% confidence interval (CI), 1.77-25.3; p=0.005) and treatment timeframe (OR, 0.36; 95% CI, 0.15-0.87; p=0.023) as pivotal predictors of early mortality.Conclusion. Our data underscore the tight association of the ELN 2017 risk model with early induction mortality in AML patients receiving intensive induction chemotherapy. With the introduction of novel agents as well as innovative regimens combining these agents, it is becoming clear that better prognostic tools are needed at the provider level to navigate the rapidly changing therapeutic landscape seen in current management of AML patients. Our findings further underpin the importance of integrated prognostication models and their profound implications on the immediate peri-induction setting in AML patients. [Display omitted] DisclosuresAvigdor: Gilead: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Research Funding; Janssen: Research Funding; Takeda: Consultancy, Honoraria.

The correlation of next-generation sequencing-based genotypic profiles with clinicopathologic characteristics in NPM1-mutated acute myeloid leukemia

The purpose of this study was to analyze the association between next-generation sequencing (NGS) genotypic profiles and conventional clinicopathologic characteristics in patients with acute myeloid leukemia (AML) with NPM1 mutation (NPM1mut). We selected 238 NPM1mut patients with available NGS information on 112 genes related to blood diseases using the χ2 and Mann-Whitney U tests and a multivariable logistic model to analyze the correlation between genomic alterations and clinicopathologic parameters. Compared with the NPM1mut/FLT3-ITD(−) group, the NPM1mut/FLT3-ITD(+) group presented borderline frequent M5 morphology [78/143 (54.5%) vs. 64/95 (67.4%); P = 0.048], higher CD34- and CD7-positive rates (CD34: 20.6% vs. 47.9%, P < 0.001; CD7: 29.9% vs. 61.5%, P < 0.001) and a lack of favorable−/adverse-risk karyotypes (6.4% vs. 0%; P = 0.031). In the entire NPM1mut cohort, 240 NPM1 mutants were identified, of which 10 (10/240, 4.2%) were missense types. When confining the analysis to the 205 cases with NPM1mut insertions/deletions-type and normal karyotype, multivariable logistic analysis showed that FLT3-ITD was positively correlated with CD34 and CD7 expressions (OR = 5.29 [95% CI 2.64–10.60], P < 0.001; OR = 3.47 [95% CI 1.79–6.73], P < 0.001, respectively). Ras-pathway mutations were positively correlated with HLA-DR expression (OR = 4.05 [95% CI 1.70–9.63], P = 0.002), and KRAS mutations were negatively correlated with MPO expression (OR = 0.18 [95% CI 0.05–0.62], P = 0.007). DNMT3A-R882 was positively correlated with CD7 and HLA-DR expressions (OR = 3.59 [95% CI 1.80–7.16], P < 0.001; OR = 13.41 [95% CI 4.56–39.45], P < 0.001, respectively). DNMT3A mutation was negatively correlated with MPO expression (OR = 0.35 [95% CI 1.48–8.38], P = 0.004). TET2/IDH1 mutations were negatively correlated with CD34 and CD7 expressions (OR = 0.26 [95% CI 0.11–0.62], P = 0.002; OR = 0.30 [95% CI 0.14–0.62], P = 0.001, respectively) and positively correlated with MPO expression (OR = 3.52 [95% CI 1.48–8.38], P = 0.004). In conclusion, NPM1mut coexisting mutations in signaling pathways (FLT3-ITD and Ras-signaling pathways) and methylation modifiers (DNMT3A and TET2/IDH1) are linked with the expressions of CD34, CD7, HLA-DR and MPO, thereby providing a mechanistic explanation for the immunophenotypic heterogeneity of this AML entity.

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.

Validation of FOXM1 As a Therapeutic Target in Acute Myeloid Leukemia

Objectives:Forkhead box M1 (FOXM1) is a transcription factor of the Forkhead family that induces genes critical in executing the mitotic program. The FOXM1 regulatory network is a major predictor of adverse survival outcomes across 18000 human tumor expression signatures (Gentles. Nat Med 2015). FOXM1 interacts with nucleophosmin (NPM) in cancer cells and NPM determines the cellular localization of FOXM1. In the OCI-AML3 cell line and in AML primary samples with mutant alleles of NPM1, we have shown that FOXM1 and mutant NPM are re-localized to the cytoplasm (Khan. Leukemia 2017). We postulated that nuclear export of FOXM1 may underlie the chemotherapy responsiveness in NPM1mut patients. We conducted a multi-institution study to investigate the nuclear expression level of FOXM1 as a predictor of chemotherapy resistance in AML and validate it as a therapeutic target.Methods:Adult patients diagnosed with intermediate-risk AML were identified at Northwestern Memorial and the University of Illinois Hospitals. A total of 102 patients were included in the study with equivalent representation from each institution. Clinical data were collected by chart review, and corresponding bone marrow biopsy samples were retrieved under an IRB approved protocol. Tissue sections were stained with a FOXM1 antibody and scanned images were analyzed using HALO 2.0 software. All biopsy specimens were reviewed by a hematopathologist for adequacy. The percentage of total nuclei expressing FOXM1, nuclear FOXM1 intensity (optical density), and the FOXM1 nuclear:cytoplasmic ratio were quantified. Statistical analysis was performed by the Design and Analysis Core, UIC.Results:Patient characteristics are summarized in Table 1. Out of 102 patients, 77 (75%) achieved a complete remission with or without count recovery (CR/CRi). We used lines of induction therapy needed to achieve remission as a measure of chemotherapy resistance. Patients were stratified as needing 1 or >1 line of induction therapy. Induction therapy included cytarabine and anthracycline in 82 cases and hypomethylator in 19 cases.We found that patients needing >1 line of induction therapy had substantially higher levels of nuclear FOXM1 in their diagnostic bone marrow biopsy compared to patients who responded to first-line therapy (20.4% vs. 11.4% positive nuclei; p= 0.066). In logistic regression analysis, the percentage of FOXM1 positive nuclei (OR 1.85 for 10% increase in positive nuclei, p=0.02) and average nuclear intensity (OR 2.18 for 0.1 U increase in OD, p=0.05) were able to predict resistance to first-line chemotherapy. Due to variation in post-induction therapies, the institutions were considered separately for survival analysis. Nuc:Cyto FOXM1 ratio was able to predict inferior overall survival in a single institution cohort analyzed (n=45) in a model incorporating standard clinicopathologic prognostic factors. Multivariate regression analysis is shown in Table 2.We then proceeded to validate nuclear FOXM1 as a therapeutic target in AML. Short hairpin knockdown of FOXM1 in AML cell lines results in decreased colony size and number (KG-1 shFOXM1: 0.35 normalized to control, p=0.01; MV4-11 shFOXM1: 0.61, p=0.03). We have shown that proteasome inhibitors stabilize HSP70 which is a negative regulator of FOXM1 (Halasi. JBC 2016). The novel proteasome inhibitor ixazomib was tested for in vitro effect on FOXM1 in AML cells. Ixazomib inhibits the transcriptional activity of FOXM1 using a luciferase reporter cell line with inducible FOXM1. Treatment with ixazomib significantly decreases FOXM1 mRNA and inhibits FOXM1 protein expression in AML cell lines and patient samples (n=12) resulting in increased apoptosis by caspase cleavage. This is consistent with the FOXM1 auto-regulation loop (Halasi. Cell Cycle 2009). The therapeutic efficacy of ixazomib in AML was then tested in vivo. Single agent treatment with ixazomib compared to vehicle in an murine xenograft model of AML showed significant reduction in tumor volume after 3 weeks of treatment (0.89cm3 vs 1.62cm3, n=8 per group) and downregulation of FOXM1 expression.Conclusion:Our work shows for the first time that nuclear FOXM1 confers chemotherapy resistance in a clinically homogenous group of intermediate risk AML patients. Moreover we show that ixazomib, a well-tolerated novel proteasome inhibitors has therapeutic efficacy in AML models that correlates with suppression of FOXM1. [Display omitted] DisclosuresKhan:Teva: Speakers Bureau; Takeda: Research Funding; Gilead: Consultancy.

Dactinomycin induces complete remission associated with nucleolar stress response in relapsed/refractory NPM1-mutated AML

Acute myeloid leukemia (AML) with mutated NPM1 accounts for one-third of newly diagnosed AML. Despite recent advances, treatment of relapsed/refractory NPM1-mutated AML remains challenging, with the majority of patients eventually dying due to disease progression. Moreover, the prognosis is particularly poor in elderly and unfit patients, mainly because they cannot receive intensive treatment. Therefore, alternative treatment strategies are needed. Dactinomycin is a low-cost chemotherapeutic agent, which has been anecdotally reported to induce remission in NPM1-mutated patients, although its mechanism of action remains unclear. Here, we describe the results of a single-center phase 2 pilot study investigating the safety and efficacy of single-agent dactinomycin in relapsed/refractory NPM1-mutated adult AML patients, demonstrating that this drug can induce complete responses and is relatively well tolerated. We also provide evidence that the activity of dactinomycin associates with nucleolar stress both in vitro and in vivo in patients. Finally, we show that low-dose dactinomycin generates more efficient stress response in cells expressing NPM1 mutant compared to wild-type cells, suggesting that NPM1-mutated AML may be more sensitive to nucleolar stress. In conclusion, we establish that dactinomycin is a potential therapeutic alternative in relapsed/refractory NPM1-mutated AML that deserves further investigation in larger clinical studies.

Fludarabine, High-Dose Cytarabine and Idarubicin-Based Induction May Overcome the Negative Prognostic Impact of FLT3-ITD in NPM1 Mutated AML, Irrespectively of FLT3-ITD Allelic Burden.

Simple SummaryThe prognostic relevance of molecular aberrations in acute myeloid leukemia (AML) has been prevalently tested in patients receiving conventional 3+7 induction. Recently, there has been a renewed interest in intensified inductions, but very few data are available on the impact of the most frequent genetic alterations with these alternative treatments. We analyzed a large multicentric cohort of younger AML patients harboring NPM1 and FLT3-ITD mutations receiving an intensified fludarabine-containing regimen (FLAI). Our data suggest that in NPM1 mut patients, FLAI may overcome the prognostic influence of co-mutated FLT3-ITD. The increased efficacy of this treatment seems to reduce the need for early consolidation with allogeneic transplant in double-mutated patients. Our data strongly support FLAI as an ideal backbone for combination with innovative targeted drugs, in order to further improve patients’ outcome.The mutations of NPM1 and FLT3-ITD represent the most frequent genetic aberration in acute myeloid leukemia. Indeed, the presence of an NPM1 mutation reduces the negative prognostic impact of FLT3-ITD in patients treated with conventional “3+7” induction. However, little information is available on their prognostic role with intensified regimens. Here, we investigated the efficacy of a fludarabine, high-dose cytarabine and idarubicin induction (FLAI) in 149 consecutive fit AML patients (median age 52) carrying the NPM1 and/or FLT3-ITD mutation, treated from 2008 to 2018. One-hundred-and-twenty-nine patients achieved CR (86.6%). After a median follow up of 68 months, 3-year overall survival was 58.6%. Multivariate analysis disclosed that both NPM1mut (p < 0.05) and ELN 2017 risk score (p < 0.05) were significant predictors of survival. NPM1-mutated patients had a favorable outcome, with no significant differences between patients with or without concomitant FLT3-ITD (p = 0.372), irrespective of FLT3-ITD allelic burden. Moreover, in landmark analysis, performing allogeneic transplantation (HSCT) in first CR proved to be beneficial only in ELN 2017 high-risk patients. Our data indicate that FLAI exerts a strong anti-leukemic effect in younger AML patients with NPM1mut and question the role of HSCT in 1st CR in NPM1mut patients with concomitant FLT3-ITD.

Frequency and Stimulation of NPM1-Specific Immune Responses By Anti-PD1 Antibodies in NPM1-Mutated AML

Nucelophosmin1 (NPM1) is one of the most commonly mutated genes in AML, represents a distinct entity according to the WHO and is commonly associated with a favorable prognosis. We described specific immune responses against immunogenic epitopes derived from the mutational region of NPM1 in AML and specific immune responses against leukemic progenitor and stem cells (LPC/LSC). Immune responses play an increasing role in AML treatment options. However, the role of Immuncheckpoint inhibition is still controversially discussed in AML. In this work, we investigated NPM1 specific immune responses but also responses against other leukemia associated antigens (LAA) like PRAME (P300), Wilms' Tumor 1 (WT1) and RHAMM (R3) in an extended cohort. We investigated these immune responses in LSC/LPC using colony-forming immunoassays (CFI) and ELISpots. We examine whether immuncheckpoint inhibition using the anti-programmed death 1 (anti-PD-1) antibody might increase immune responses against stem cell like cells, comparing NPM1-mutated (NPM1mut) to NPM1 wildtype (NPM1wt) patients. In AML NPM1mut patients, specific immune responses of cytotoxic T cells against the epitope NPM1 showed a mean reduction of colonies in CFI of 27%, for P300 of 38%, for WT1 of 32% and for R3 of 42%. NPM1wt patients showed a mean reduction of colonies in CFI of for P300 of 28%, for WT1 of 28% and for R3 of 41%. Results are comparable, for NPM1mut and NPM1wt patients, there were immune responses seen in all epitopes. When adding the anti-PD-1 antibody to CFI, in NPM1mut patients the mean additional inhibition using NPM1 as target was 47%, and for the other epitopes: P300 23%, WT1 27% and R3 26%. For NPM1WT patients, the additional inhibition with the addition of anti-PD-1 to CFI was for P300 25%, for WT1 34% and for R3 23%. For the NPM1 epitope, 8 of 10 NPM1mut patients showed an immune response and 3/10 showed reduction in CFI of &amp;gt; 50%. The NPM1 epitope displayed an additional reduction with anit-PD-1 in all 10 NPM1mut Patients and in 6 of 10 a reduction of &amp;gt; 50%. Thus, especially the NPM1 specific immune responses are strong in NPM1mut patients by adding anti-PD-1. Taken together, the anti-PD-1 antibody increases specific T cell responses of LAA-stimulated CTL and the cytotoxic effect of T cells against LPC/LSC. The effect was strongest in NPM1mut patients against the immunogenic epitope derived from the mutational region of NPM1 and even stronger when adding anti-PD-1. These data suggest there could be a NPM1 mutation directed immunotherapeutic approach using LAA-directed vaccination strategies in NPM1-mutated AML and combination with anti-PD-1 antibodies might open new application possibilities. Disclosures Greiner: BMS: Research Funding. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding.

The Natural History of NPM1MUT Measurable Residual Disease (MRD) Positivity after Completion of Chemotherapy in Acute Myeloid Leukemia (AML)

The Natural History of NPM1MUT Measurable Residual Disease (MRD) Positivity after Completion of Chemotherapy in Acute Myeloid Leukemia (AML)

Impact of DNMT3a Status on Post Induction NPM1 MRD Predictive Value on Survival in Elderly AML Patients Treated Intensively

Minimal residual disease is now a powerfull surrogate marker to assess response to chemotherapy in acute myeloid leukemia (AML). In younger adults, NPM1 MRD has recently demonstrated to be a favorable predictive marker for EFS and OS independently of fms-like tyrosine kinase-3 internal tandem duplications (FLT3-ITD) status. However, there is very few datas regarding predictive value of NPM1 MRD in elderly patients treated with intensive chemotherapy. Moreover, numerous studies have suggested the negative impact of DNMT3a mutation in NPM1 AML patients, especially in those with concurrent FLT3-ITD mutation. In this study, we aimed to investigate the impact of DNMT3a status on post induction NPM1 MRD1 predictive value for survival in a retrospective cohort of AML patients aged over 60 years old treated intensively. A total of 138 patients treated for NPM1 mutated AML in two French institutions (Lyon, Lille) were analyzed retrospectively. Median age of the entire cohort was 66.1 years old (range 60-78.2). An FLT3-ITD mutation was evidenced in 52 of 138 patients (37.6%) with a median FLT3-ITD AR of 0.53 (range, 0.05-3). With a median follow-up of 19.61 months (0.07-128.4), the overall CR rate was 89.9% with no influence of DNMT3a or FLT3 mutational status on the probability of CR. In this elderly cohort of NPM1mut patients, a 4log reduction of NPM1 bone marrow (BM) MRD1 was associated with better outcome (median OS: NR vs 13.4 months, HR=0.35, p&amp;lt;0.01)(Figure A). Overall, DNMT3 status did not influence the probability of having a ≥ 4log MRD1 reduction after induction. However, only 9/44 (20.4%) FLT3-ITD patients reached ≥ 4log MRD1 reduction whereas 38/80 FLT3wt (47.5%) were good molecular responders (p&amp;lt;0.001). FLT3-ITD mutated patients who achieved a 4log reduction had a superior outcome compared to those who did not (HR=0.34; 95% CI, 0.16 to 0.70; P &amp;lt;0.001). Similarly, NPM1mut FLT3wt patients with a 4log reduction in NPM1 BM-MRD1 had a longer OS (3-year OS, 68.1%; 95% CI, 48.8 to 82.9) than those without good molecular response (3-year OS, 46.5%; 95% CI, 30.2 to 61.7)(Figure B). DNMT3a negative patients who achieved a 4log reduction had a superior outcome to those who did not reached at least a 4log reduction (HR=0.23; 95% CI, 0.07 to 0.72; P &amp;lt;0.001). However, postinduction NPM1 MRD1 reduction was not predictive of OS and leukemia free survival (LFS) in DNMT3amut patients. DNMT3amut patients has a very poor LFS which was even worst in poor NPM1 MRD1 responders compared to those who reached at least 4log reduction (median LFS: 8.3 months vs 17.4 months, HR = 0.48, 95% CI, 0.25-0.91, p=0.023)(Figure C). In multivariate analysis, only DNMT3a mutational status and a 4-log reduction in NPM1 BM-MRD were significantly associated with survival. Based on these results, we identified among NPM1 positive patients 3 groups with distinct prognosis, based on FLT3-ITD, DNMT3a status and NPM1 BM-MRD post induction response (NPM1 scoring system)(Figure D). When compared to ELN 2017 intermediate risk group (AUC=0.695), NPM1 scoring system (NPM1 SS) was more accurate for OS prediction in patients within intermediate (AUC=0.833) and unfavorable (AUC=0.863) NPM1 SS risk group. However, there was no significant difference in AUC between NPM1 SS favorable and ELN 2017 favorable risk group. These results confirm that post-induction NPM1 MRD1 is a reliable tool to assess disease outcome in elderly AML patients. However, the presence of DNMT3a also identify a subgroup of patients at very high risk of relapase, despite good molecular responses. As hematopoietic stem cell transplantation (HSCT) might improve OS in elderly patients, DNMT3a positive AML elderly patients should be considered for HSCT or post induction maintenance strategies, even within the favorable ELN risk group. Figure Disclosures Sujobert: Gilead/Kyte: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sunesis: Research Funding.

AML-291: MRD Level After Induction Therapy in NPM1-Mutated Patients Identifies Those at High Risk of Relapse and Defines Indications for HSCT

Context Patients with NPM1 AML mutation are included in ELN favorable or intermediate genetic risk categories, although a proportion of these patients will relapse. Thus, it is necessary to identify negative prognostic factors for patientss with NPM1 and to evaluate if ASCT could overcome them. Objective To determine the clinical significance of post-remission MRD level NPM1 mutation and correlation with treatment outcome in AML patients. Design Twenty-seven patients with NPM1mut in CR were included in the study. All patients had WT1 overexpression. Additional genetic abnormalities were revealed in 12 patients ([hyperexpression of BAALC (n=4), IDH1/2 (n=4), FLT3ITD (n=8), DNMT3AR882C (n=1), ASXL1(n=1), RUNX1(n=1), aberrant karyotype (n=4)]. Monitoring of minimal residual disease was studied after induction («7+3) and consolidation courses («HiDAC»). Thirteen patients underwent HSCT. Median follow-up was 12 months (2.2–109 months). Results CIR (median 7.25 months vs not reached), RFS (median 7.25 months vs not reached) and OS (median 7.25 vs 41.80 months) were shorter in patients with NPM1mut reduction after an induction course less than 3.85 log (specificities 81.8%, sensitivities 100%) (p=0.001, p=0.0036, and p=0.05, respectively). Patients with NPM1+FLT3- status had similar results with this cut-off (median RFS 9.6 months vs not reached, p=0.037). Cutoff levels of NPM1mut 4.2 log (specificities 83.3%, sensitivities 83.3%) after the first consolidation course separated the cohort into two prognostic groups (14 and 13 patients) as well: median CIR 7.25 vs 41.80 months (p=0.02) and median OS 13 months vs not reached, p=0.05, respectively). MRD status after consolidation therapy in comparison to the MRD status after induction therapy has improved only in two patients but with relapse in 17.7 months and 9.7 months. HSCT improved CIR and OS in the group of patients with cut-off less than 3. 85 log (p=0.016, p=0.03). In the multivariate model with MRD cut-off, age, genetic abnormalities, FLT3 status, and WBC only post-induction cut-off level NPM1 were independent prognostic factors for RFS (HR:0.07; 95% CI: 0,006–0.955; p=0.04). Conclusions NPM1mut-positive patients have adverse prognosis if the post-induction reduction of expression of these genes is less than 3.85 log. This cut-off separated ELN favorable NPM1mut AML patients. Unfavorable prognosis of MRD cut-off-positive patients during AML treatment could be overcome with HSCT in the first CR.