NPM1 Status Research Articles

NPM1 Minimal Residual Disease Detection at Any Time by Clinical Next Generation Sequencing Predicts Poor Survival in Acute Myeloid Leukemia

Abstract Introduction/Objective Acute Myeloid Leukemia (AML) is a deadly disease that accounts for 80% of adult leukemia cases and carries a 5-year survival rate of only 31.7%. Historically, prognosis hinged heavily on cytogenetic stratification, but more recently minimal residual disease (MRD) has emerged as a crucial prognostic marker. Flow cytometry, PCR, and NGS can all be employed for MRD detection, but each carries its own limitations. Though technically challenging, NGS has the advantage being able to simultaneously assay multiple mutations, thus providing for broader applicability. NPM1 is the most prevalently mutated gene in AML (25-35%). In this study, one of the largest of its kind, we investigate the feasibility and impact of NPM1 MRD detection by clinical NGS. Methods/Case Report IRB approval was obtained. We reviewed the medical record and clinical laboratory data sets from AML patients treated at Moffitt Cancer Center who had NPM1 status assessed by clinical NGS, Testing was performed at LabPMM (San Diego, CA). DNA was extracted, and NPM1 exon 12 was amplified. PCR products were sequenced on an Illumina MiSeq. The clinically reportable assay sensitivity was 5 x 10-5 though lower levels were detected on review of raw data. Kaplan-Meier analysis was conducted for overall survival assessment. Since patients often had multiple tests, we divide patients into those with NPM1+ at any time and NPM1 always negative. Results (if a Case Study enter NA) There was a total of 140 patients. Overall, 346 tests were conducted, with an average of 2.5 tests/patient. There were 85 patients (60%) who underwent serial testing. MRD+ patients (n=80) had a mean age of 66 years, while MRD- patients (n=60) had a mean age of 58 years. MRD+ patients exhibited a mean overall survival of 29.3 months versus 43.6 months for MRD- patients (p=0.005.) Conclusion Through our study encompassing a large patient cohort of 140 patients and nearly 350 testing data points, we confirm the feasibility and clinical utility of NPM1 MRD evaluation by NGS. The significantly poorer survival in patients who were MRD+ at any time during their disease provides a scientific rationale for serial NGS testing to enable risk stratification of NPM1+ AML patients.

Exploring the Prevalence and Prognostic Impact of Wilms Tumor 1 Exon 7 Mutation Status with Combinations of FLT3-ITD and NPM1 Mutations as Potential Molecular Biomarkers in Acute Myeloid Leukemia Patients with Normal Cytogenetics.

This study explores the prognostic impact of FLT3-ITD, NPM1, and WT1 mutations both independently and in combination in Cytogenetically Normal Acute Myeloid Leukemia (CN-AML) patients as they exhibit varying clinical outcomes. 150 CN-AML patients were selected to assess the prevalence and prognostic significance of WT1 mutations in combination with FLT3-ITD and NPM1 status using polymerase chain reaction (PCR) followed by Sanger sequencing. WT1 exon 7 mutations were present in 12.6% of patients. Elderly individuals, with a mean age of 49.4 years, were more prone to NPM1 mutations, though the association was not statistically significant (p=0.094). Significant associations were observed between lactate dehydrogenase (LDH) and hemoglobin (Hb) levels with FLT3-ITD and NPM1 mutations (p=0.003 and p=0.04, respectively). The M4 subtype exhibited the highest prevalence of WT1 mutations (p=0.0036). Patients with NPM1 mutations had a higher overall survival rate compared to NPM1 wild-type cases (p=0.057). There was no significant correlation between overall survival and WT1 and FLT3-ITD mutations. Regarding relapse-free survival, NPM1 mutation cases exhibited a higher survival probability compared to NPM1 wild-type cases. Similarly, WT1 mutated cases had a higher survival probability compared to WT1 wild-type cases, although these differences were not statistically significant. The combined mutation statuses of NPM1/FLT3-ITD with WT1 did not yield significant outcomes. The study suggests that larger cohort studies may reveal more relevant associations, given the relatively small cohort in this study. This study found a significant association between patient survival outcomes and NPM1 mutation status, as well as the combined FLT3-ITD and NPM1 status. Profiling both NPM1 and FLT3-ITD mutations at the time of diagnosis serves as a robust prognostic marker in AML treatment. WT1 mutation status did not show a significant association with patient outcomes. Larger population studies may provide more relevant insights.

Improvement in Survival of Patients with FLT3 Mutated Acute Myeloid Leukemia: Results from a Retrospective Canadian Cohort

Introduction: FLT3 internal tandem duplication (ITD) mutations have been historically associated with inferior outcomes in patients with acute myeloid leukemia (AML) and an intermediate risk status as per the European LeukemiaNet (ELN) 2022 guidelines. In 2017, the US FDA granted approval to midostaurin for frontline treatment and a year later, gilteritinib for use in relapsed/refractory (R/R) AML. These agents began to be widely used in our provincial leukemia program in early 2018 as per the label indications. In this study, we compared clinical outcomes for patients with FLT3-ITD mutated AML before and after this change in British Columbia. Methods: We identified patients in our program database diagnosed with FLT3-ITD mutated AML from January 1, 2010 to December 31, 2021. Only patients receiving intensive chemotherapy with cytarabine and anthracycline were included. Patients were divided into two eras: 2010-2017 (era 1) and 2018-2021 (era 2). The two eras were compared in terms of patient characteristics, genetics, rate of complete remission (CR) and CR with incomplete count recovery (CRi), receipt of allogeneic stem cell transplant (alloSCT) and the overall and relapse free survival (OS and RFS). OS was calculated from diagnosis to death from any cause and was not censored at the time of alloSCT. RFS was calculated from CR/CRi to relapse. Patients without an event during the study period were censored at the time of last follow-up. Categorical variables were compared using Chi-square and Fisher exact tests and continuous variable were compared using paired T tests. Kaplan-Meier survival method and log rank test were used to estimate and compare survival. Uni- and multivariate analyses were performed in a Cox proportional regression model and alloSCT was considered as a time dependent covariate. Results: 169 patients were included (era 1=101, era 2=68). The median duration of follow up of the entire cohort was 17 months (range 0.5-148.6 months) and follow-up of surviving patients (n=70) was longer in era 1 vs. era 2 (median 88 months vs. 32 months, p<0.001). The median age of the entire cohort was 60 years (range 26-79 years) and it was comparable in both eras. Most patients had normal cytogenetics (era 1: 76%; era 2: 69%). NPM1 was mutated in 69/101 (68%) and 38/68 (56%) patients in eras 1 and 2 respectively. Due to lack of genetic data for era 1, we did not risk stratify based on the recent ELN classification. The rate of CR/CRi was 58/101 (57%) vs. 40/68 (59%) after induction chemotherapy and 78/101 (77%) vs. 58/67 (87%) (p=0.09) at any time in eras 1 and 2 respectively. AlloSCT was performed in 58% and 72% of patients in the two cohorts. Midostaurin was used in 82% and gilteritinib was used in 26% of era 2 patients in the second era. OS was significantly longer in era 2 (median OS 73 months vs. 11 months, HR (death) 6.2, p=0.012) (Figure). The 2-year OS rate was higher in era 2 (57%) than in era 1 (39%). In patients achieving CR/CRi, median RFS was longer in era 2 (51 vs. 16 months, p=0.43), although this was not statistically significant. In univariate Cox regression analysis the following factors were associated with improved OS: use of midostaurin (HR 0.487, 95% CI 0.304-0.779, p=0.003), diagnosis in era 2 (HR 0.583, 95% CI 0.38-0.895, p=0.014) and receipt of alloSCT (HR 0.346,) 95% CI 0.219-0.545, p<0.001). Other parameters including age, sex, NPM1 status and cytogenetics were not significant. In multivariate analysis including era, midostaurin and alloSCT, only the receipt of alloSCT was associated with significantly improved OS (HR 0.362, 95% CI 0.229 - 0.572, p<0.001) whereas use of midostaurin (HR=0.465, CI 0.207-1.042, p=0.063) and era (HR 1.12, 95% CI 0.538-2.34, p=0.76) were not significant. Conclusions: Our results show thatOS for patients with FLT3-ITD AML has improved in patients diagnosed in a more recent treatment era. Patients diagnosed after 2018 were more frequently treated with AlloSCT and FLT3-inhibitors, which may have resulted in improvements in OS. Our findings support the assignment of FLT3-ITD AML to theintermediate risk category as per the current ELN guidelines. A limitation of this study is the differences in follow-up time between eras, which may influence the event rate between the groups.

Characteristics and prognostic impact of IDH mutations in AML: a COG, SWOG, and ECOG analysis

AbstractSomatic mutations in isocitrate dehydrogenase (IDH) genes occur frequently in adult acute myeloid leukemia (AML) and less commonly in pediatric AML. The objective of this study was to describe the prevalence, mutational profile, and prognostic significance of IDH mutations in AML across age. Our cohort included 3141 patients aged between <1 month and 88 years treated on Children’s Cancer Group/Children’s Oncology Group (n = 1872), Southwest Oncology Group (n = 359), Eastern Cooperative Oncology Group (n = 397) trials, and in Beat AML (n = 333) and The Cancer Genome Atlas (n = 180) genomic characterization cohorts. We retrospectively analyzed patients in 4 age groups (age range, n): pediatric (0-17, 1744), adolescent/young adult (18-39, 444), intermediate-age (40-59, 640), older (≥60, 309). IDH mutations (IDHmut) were identified in 9.2% of the total cohort (n = 288; IDH1 [n = 123, 42.7%]; IDH2 [n = 165, 57.3%]) and were strongly correlated with increased age: 3.4% pediatric vs 21% older, P < .001. Outcomes were similar in IDHmut and IDH-wildtype (IDHWT) AML (event-free survival [EFS]: 35.6% vs 40.0%, P = .368; overall survival [OS]: 50.3% vs 55.4%, P = .196). IDH mutations frequently occurred with NPM1 (47.2%), DNMT3A (29.3%), and FLT3-internal tandem duplication (ITD) (22.4%) mutations. Patients with IDHmut AML with NPM1 mutation (IDHmut/NPM1mut) had significantly improved survival compared with the poor outcomes experienced by patients without (IDHmut/NPM1WT) (EFS: 55.1% vs 17.0%, P < .001; OS: 66.5% vs 35.2%, P < .001). DNTM3A or FLT3-ITD mutations in otherwise favorable IDHmut/NPM1mut AML led to inferior outcomes. Age group analysis demonstrated that IDH mutations did not abrogate the favorable prognostic impact of NPM1mut in patients aged <60 years; older patients had poor outcomes regardless of NPM1 status. These trials were registered at www.clinicaltrials.gov as #NCT00070174, #NCT00372593, #NCT01371981, #NCT00049517, and #NCT00085709.

Association of FLT3 and NPM1 mutations in patients with acute myeloid leukemia with metabolomic patterns determined by mass spectrometry.

e19003 Background: Patients diagnosed with Acute Myeloid Leukemia (AML) are categorized into risk groups based on cytogenetic and molecular abnormality tests, which determine the specific treatment regimes. Since mutation tests take time and some emergency patients require immediate treatment, a method to provide fast clinical data that will be the basis for initial treatment regime is needed. Aim of the study is to discover patterns associated with FLT3 and NPM1 mutations in AML patients, which will provide a fast clinical information to clinicians. The hypothesis is based on the previous studies which indicate a link between NPM1 and FLT3 mutations with metabolism. The mutations were chosen based on their high frequency in AML patients and their critical role in risk group determination. Methods: All AML patients (n = 42) and healthy individual samples (n = 16) were obtained from Ankara University Hematology Department. Patient groups were divided into five groups based on the mutation status. Pattern determination of the mutations was achieved by LC-MS/MS measurements of amino acid and acyl carnitine panels that are highly associated with metabolism. After preprocessing of the raw data, univariate (ANOVA) and multivariate analyses (PCA and PLS-DA) were performed to define class discrimination between sample groups. The remarkable analytes that have significant power in the discrimination were determined by VIP analysis. The developed model was validated with K-Fold cross validation method and permutation test. The most significant pathways in class discrimination were identified with pathway analysis. Visualization was accomplished via Metaboanalyst 5.0 software. Results: Principal Component Analysis (PCA) showed that 79% of the total variance of the sample groups was explained by the model. In order to increase class discrimination, Partial Least Squares-Discriminant Analysis (PLS-DA) was performed. R2Y and Q2 were found as 0.845 and 0.619, respectively. PLS-DA model was validated with K-fold analysis and permutation test. In all the validation experiments, a low cross-validation error was observed. In VIP analysis, the most significant analytes that cause the class discrimination were found as C0 carnitine, glutamic acid, aspartic acid, respectively. In the pathway enrichment analysis performed with these analytes, aminoacyl t-RNA biosynthesis, arginine biosynthesis, valine-leucine-isoleucine biosynthesis were found as statistically significant pathways responsible for the class discrimination. Conclusions: A preliminary model based on the targeted metabolomics approach was developed for the prediction of mutation status of NPM1 and FLT3 proteins in AML patients. Proposed model has a high fit value, validity, and strong predictive power. The reliability and validity of the model can be further increased by future multicenter studies.

FLT3-ITD Allelic Ratio and NPM1 Mutation Do Not Impact Outcomes in Acute Myeloid Leukemia Patients with FLT3-ITD after Allogeneic Hematopoietic Stem Cell Transplantation: A Retrospective Propensity Score- Matching Study

FLT3-ITD mutation has consistently been correlated with poor outcomes in AML patients. Allo-HSCT (allogeneic hematopoietic stem cell transplantation) plays a major role in curing blood diseases. Whether allo-HSCT can eliminate the detrimental effects of FLT3-ITD mutation in AML patients remains debatable. In addition, studies showed that FLT3-ITD allelic ratio (AR) and NPM1 mutation appear to further influence the prognostic utility of FLT3-ITD in patients with FLT3-ITD-mutated AML. The influence of NPM1 mutation and AR on FLT3-ITDmut patients remains unclear in our database. To compare the survival outcomes following allo-HSCT between FLT3-ITDmut and FLT3-ITDwt patients and further analyze the influence of NPM1 and AR on outcomes. 118 FLT3-ITDmut patients and 497 FLT3-ITDwt patients with allo-HSCT were matched 1:3 on the propensity score using a nearest-neighbor matching with a caliper size of 0.2. 430 AML patients were considered, including 116 FLT3-ITDmut patients and 314 FLT3-ITDwt patients. OS (overall survival) and LFS (leukemia-free survival) of FLT3-ITDmut patients were similar to FLT3-ITDwt (2-year OS:78.5% vs 82.6%, P=0.374; 2-year LFS: 75.1% vs 80.8%, P= 0.215). A cut-off of 0.50 was applied to define subgroups with a low or high AR of FLT3-ITD, no significant CIR (cumulative incidence of relapse) and LFS differences were observed between the low AR and high AR groups (2-year CIR: P=0.617; 2-year LFS: P=0.563). CIR and LFS were comparable when patients were grouped according to the presence or absence of NPM1 and FLT3-ITD (2-year CIR: P=0.356; 2-year LFS: P=0.159). Additionally, the CIR and LFS of FLT3-ITDmut and FLT3-ITDwt patients tended to be different after MSD-HSCT (matched-sibling donor hematopoietic stem cell transplantation) (2-year CIR: P= 0.072; 2-year LFS: P= 0.084), however, the differences were not observed in patients with haplo-HSCT (2-year CIR: P= 0.59; 2-year LFS: P= 0.794). The presence of MRD before transplantation and non-CR1 were risk factors related to inferior outcomes in a multivariate analysis, regardless of FLT3-ITD or NPM1 status. Our results suggested that allo-HSCT, especially haplo-HSCT, may overcome the adverse effect of FLT3-ITD mutation, regardless of the NPM1 status or AR. Allo-HSCT could be an ideal option for AML patients with FLT3-ITD.

Abstract 5560: Prognostic effect of co-mutations on patients with NPM1+ acute myeloid leukemia (AML) enrolled in the Connect® Myeloid registry

Abstract Introduction: The 2017 World Health Organization classification of myeloid neoplasms and acute myeloid leukemia (AML) recognizes AML with mutated NPM1 as a distinct entity with favorable prognosis. However, as mutations other than NPM1 may further influence disease prognosis, here we assess the prognostic effect of NPM1 co-mutations in patients (pts) with AML enrolled in the Connect® Myeloid Registry (NCT01688011). Methods: The Registry is a large, US, multicenter, prospective observational cohort study of pts with newly diagnosed AML, myelodysplastic syndromes, idiopathic cytopenia of undetermined significance, or myelofibrosis. In this analysis, pts with AML ≥55 years of age were grouped by known NPM1 status (NPM1+ vs NPM1‒). The frequency of gene mutations was analyzed among patients with NPM1+ AML and NPM1‒ AML. Overall survival (OS) was evaluated using the Cox model adjusted for age and mutations in TP53 and RUNX1. Results: Overall, 123 pts with NPM1+ AML and 360 pts with NPM1‒ AML were included. Most pts were male (NPM1+, 61.5%; NPM1−, 60.7%) and White (NPM1+, 78.0%; NPM1−, 85.6%); median (range) age was 67 (55-87) and 71 (55-97) years for the NPM1+ and NPM1− groups, respectively. After adjusting for age, pts with NPM1+ AML had significantly longer median OS than pts with NPM1‒ AML (26 vs 15 months; HR [95% CI]: 0.58 [0.44-0.76]; P &amp;lt; 0.01; Table). Pts with NPM1+ vs NPM1‒ AML had a lower frequency of certain key mutations such as TP53 (1.6% vs 18.6%) and RUNX1 (0.8% vs 14.8%). The significant survival benefit with NPM1+ vs NPM1‒ initially observed after adjusting for age was not observed after further adjusting for presence of TP53 and RUNX1 mutations (NPM1+ vs NPM1‒; 23 vs 19 months; HR [95% CI]: 0.75 [0.48-1.17]; P = 0.21). Conclusions: TP53 plus RUNX1 mutation was identified as a pattern of co-mutational burden that were observed less frequently and potentially contributed to the improved OS of pts with NPM1+ AML and poorer OS of pts with NPM1‒ AML. NPM1+n = 123 NPM1‒n = 210 Overall survival, months Adjusted for age 26.0 15.0 Hazard ratio (95% CI) 0.58 (0.44-0.76) Adjusted for age, TP53, RUNX1 23.0 19.0 Hazard ratio (95% CI) 0.75 (0.48-1.17) Frequency of gene mutations, n (%)TP53 2 (1.6) 39 (18.6) ASXL1 3 (2.4) 37 (17.6) FLT3-ITD 44 (35.8) 36 (17.1) TET2 20 (16.3) 34 (16.2) DNMT3A 29 (23.6) 33 (15.7) IDH2 11 (8.9) 33 (15.7) RUNX1 1 (0.8) 31 (14.8) NRAS/KRAS 11 (8.9) 30 (14.3) IDH1 18 (14.6) 18 (8.6) CEBPA 4 (3.3) 15 (7.1) WT1 5 (4.1) 14 (6.7) FLT3-TKD 18 (14.6) 13 (6.2) PHF6 0 12 (5.7) PTPN11 4 (3.3) 12 (5.7) SF3B1 2 (1.6) 8 (3.8) C-KIT 1 (0.8) 7 (3.3) EZH2 1 (0.8) 4 (1.9) ETV6 0 3 (1.4) MLL-PTD 1 (0.8) 1 (0.5) Other 30 (24.4) 114 (54.3) Citation Format: Jaroslaw P. Maciejewski, Daniel A. Pollyea, Gail J. Roboz, Karen Seiter, Bart L. Scott, Irene S. DeGutis, Pavel Kiselev, Edward Yu, Ali McBride, Willem Heydendael, Harry P. Erba. Prognostic effect of co-mutations on patients with NPM1+ acute myeloid leukemia (AML) enrolled in the Connect® Myeloid registry. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5560.

Prognostic impact of FLT3-ITD, NPM1 mutation and CEBPA bZIP domain mutation in cytogenetically normal acute myeloid leukemia: a Hokkaido Leukemia Net study.

Mutation status of FLT3, NPM1, and CEBPA is used to classify the prognosis of acute myeloid leukemia, but its significance in patients with cytogenetically normal (CN) AML is unclear. We prospectively analyzed these genes in 295 patients with CN-AML and identified 76 (25.8%) FLT3-ITD, 113 (38.3%) NPM1 mutations, and 30 (10.2%) CEBPA biallelic mutations. We found that patients with FLT3-ITD had a poor prognosis at any age, while patients with CEBPA biallelic mutation were younger and had a better prognosis. FLT3-ITD and NPM1 mutations were correlated, and the favorable prognostic impact of being FLT3-ITD negative and NPM1 mutation positive was evident only in patients aged 65years or more. For CEBPA, 86.7% of the patients with biallelic mutation and 9.1% of patients with the single allele mutation had in-frame mutations in the bZIP domain, which were strongly associated with a favorable prognosis. Multivariate analysis showed that age < 65years, FLT3-ITD and CEBPA bZIP in-frame mutation were independent prognostic factors. The results suggest that analyzing these gene mutations at diagnosis can inform selection of the optimal intensity of therapy for patients with CN-AML.

Allogeneic Hematopoietic Cell Transplantation vs Standard Consolidation Chemotherapy in Patients With Intermediate-Risk Acute Myeloid Leukemia

The ideal postremission strategy in intermediate-risk acute myeloid leukemia (AML) in first complete remission (CR) has been a matter of debate. To explore the optimal therapy for patients with intermediate-risk AML after first complete remission. This investigator-initiated, open-label, 2-armed, phase 3 randomized clinical trial assessed patients at 16 hospitals in Germany from February 2, 2011, until July 1, 2018. Key eligibility criteria included cytogenetically defined intermediate-risk AML according to Medical Research Council classification, first CR or CR with incomplete blood cell count recovery after conventional induction therapy, age of 18 to 60 years, and availability of a human leukocyte antigen (HLA)-matched sibling or unrelated donor. A detailed statistical analysis plan was written and finalized on July 7, 2020. Data were exported for analysis on April 13, 2021. Patients were randomized 1:1 to receive allogeneic hematopoietic cell transplantation (HCT) or high-dose cytarabine for consolidation and salvage HCT only in case of relapse. Strata for randomization included age (18-40 vs 41-60 years), NPM1 and CEBPA variation status, and donor type (unrelated vs related). End points included overall-survival as the primary outcome and disease-free survival, cumulative incidence of relapse, treatment-related mortality, and quality of life measured according to the Medical Outcomes Study 36-Item Short-Form Health Survey as secondary outcomes. A total of 143 patients (mean [SD] age, 48.2 [9.8] years; 81 [57%] male) with AML who fulfilled the eligibility criteria were randomized. In the intention-to-treat analysis, the probability of survival at 2 years was 74% (95% CI, 62%-83%) after primary allogeneic HCT and 84% (95% CI, 73%-92%) after consolidation chemotherapy (P = .22). Disease-free survival after HCT at 2 years was 69% (95% CI, 57%-80%) compared with 40% (95% CI, 28%-53%) after consolidation chemotherapy (P = .001). Allogeneic HCT during the first CR was associated with a cumulative incidence of relapse at 2 years of 20% (95% CI, 13%-31%) compared with 58% (95% CI, 47%-71%; P < .001). Nonrelapse mortality at 2 years after primary allogeneic HCT was 9% (95% CI, 5%-19%) and 2% (95% CI, 0%-11%) after consolidation chemotherapy (P = .005). Similar outcomes were observed when analyses were confined to the 96 patients at intermediate risk according to the European Leukemia Network classification. Most importantly, all 41 patients relapsing after consolidation chemotherapy (36 hematologic, 4 molecular, and 1 extramedullary) proceeded to allogeneic HCT. No significant differences in health-related quality of life measures were observed between groups. Primary allogeneic HCT during first CR was not associated with superior overall survival compared with consolidation chemotherapy in patients 60 years or younger with intermediate-risk AML during the first CR and an available donor. ClinicalTrials.gov Identifier: NCT01246752.

Treatment Patterns and Outcomes of Patients with NPM1+ Acute Myeloid Leukemia (AML) Enrolled in the CONNECT® Myeloid Disease Registry

Background Mutations in NPM1 (NPM1+) occur in ∼30% of patients with AML. NPM1+ has been associated with favorable outcomes following intensive chemotherapy (IC) and demonstrated positive prognostic value in the absence of concurrent FLT3-internal tandem duplication (FLT3-ITD) mutations. Recently, several studies have reported differences in outcomes in patients with NPM1 mutations based on age and treatment. In this analysis we evaluated treatment patterns and outcomes in AML based on NPM1 mutational status and primary induction therapy in patients who were enrolled in the CONNECT® Myeloid Disease Registry (NCT01688011). Methods Patients aged 55 years or older with AML were grouped by known NPM1 status (NPM1+ vs NPM1‒) and induction therapy (non-IC vs IC). Overall survival (OS) and event-free survival (EFS) were evaluated using the Cox model adjusted for age. For the EFS analyses, an event was defined as death, disease progression, or refractory to therapy. Results A total of 483 patients with known NPM1 status were assessed (NPM1+, n = 123; NPM1‒, n = 360). Most patients were male (NPM1+, 61.5%; NPM1‒, 60.7%) and white (NPM1+, 78.0%; NPM1‒, 85.6%; Table). Median age, Eastern Cooperative Oncology Group performance status (ECOG PS), and frailty group were similar in the NPM1+ and NPM1‒ groups. Favorable and adverse European LeukemiaNet (ELN) risk scores at baseline were different in the NPM1+ and NPM1‒ groups. The majority of patients received non-IC regimens as induction therapy (NPM1+, 69.1%; NPM1-, 75.0%). Overall, patients with NPM1+ AML had significantly longer median OS (26 mo) compared with patients with NPM1- AML (15 mo) (HR [95% CI]: NPM1+ vs NPM1- 0.58 [0.44-0.76]; P < 0.01). When analyzed by type of induction regimen (non-IC vs IC), although OS remained numerically longer for patients in the NPM1+ groups, the differences were not statistically significant. Among patients who received non-IC regimens, median OS was 19 and 14 mo in the NPM1+ and NPM1- groups, respectively (Figure). Among patients who received IC regimens, median OS was 24 and 16 mo in the NPM1+ and NPM1- groups, respectively. There was a significant improvement in EFS for patients in the NPM1+ group vs the NPM1- group (20 mo vs 11 mo; hazard ratio [HR] [95% CI], NPM1+ vs NPM1- 0.61 [0.47-0.80]; P < 0.01). This was also true for patients treated with non-IC regimens (15 vs 11 mo in the NPM1+ and NPM1- groups, respectively; HR [95% CI], NPM1+ vs NPM1- 0.67 [0.46-1.00]; P = 0.048). In contrast with the non-IC treated regimens, in patients treated with IC regimens, NPM1+ status was not associated with significantly longer EFS compared to NPM1- status. Patients with known NPM1 status were further evaluated for additional mutations and the following differences were noted: the frequency of mutations in tumor protein p53 (TP53) (1.6% vs 18.6%), RUNX1 (0.8% vs 14.8%), ASXL1 (2.4% vs 17.6%) IDH2 (8.9% vs 15.7%), and NRAS/KRAS (8.9% vs 14.3%) was lower in patients with NPM1+ vs NPM1- AML, respectively, whereas the frequency of mutations in FLT3-ITD (35.8% vs 17.1%), FLT3- tyrosine kinase domain (TKD) (14.6% vs 6.2%), DNMT3A (23.6% vs 15.7%), and IDH1 (14.6% vs 8.6%) was higher in patients with NPM1+ vs NPM1- AML, respectively. Conclusions Overall, patients with NPM1+ AML had longer OS and EFS than patients with NPM1‒ AML. This was observed among patients with NPM1+ AML for both non-IC and IC induction regimens. Patterns of co-mutations differed between the NPM1+ and NPM1- groups and is a likely contributing factor to differences between outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The FLT3-ITD Allelic Ratio and NPM1 Mutation Do Not Impact Outcomes in AML Patients with FLT3-ITD after Allo-HSCT: A Retrospective Propensity-Score Matching Study

Introduction: FLT3-ITD occurred in about 25% of adult patients with acute myeloid leukemia (AML), which has been correlated with poor outcomes mainly in cytogenetically normal AML, thus being one of the most frequently affected genes in AML. Whether allogeneic hematopoietic stem cell transplantation (allo-HSCT) can eliminate the detrimental effects of FLT3-ITD mutation in AML patients remain debatable. Studies from our group and others showed that allo-HSCT may overcome the adverse effect of FLT3-ITD on survival outcomes, comparable outcomes were not confirmed in other studies. Additionally, high allelic ratio was correlated with inferior outcomes while NPM1 mutation was associated with favorable outcomes in several studies, but their association with prognosis was not observed in other studies. Previous study from our group showed comparable outcomes between FLT3-ITDmut and FLT3-ITDwt patients, however, the influence of NPM1 mutation and allelic ratio on FLT3-ITDmut patients remains unclear in our database. Hence, we designed a retrospective matched-pair study to compare the survival outcomes between FLT3-ITDmut and FLT3-ITDwt patients who underwent allo-HSCT and further analyzed the impact of NPM1 and allelic ratio on outcomes. Methods: 118 FLT3-ITDmut patients and 497 FLT3-ITDwt patients undergoing allo-HSCT were observed. Each FLT3-ITDmut patients was matched with three FLT3-ITDwt patients using the nearest neighbor-matching method with a caliper size of 0.2. Age and sex of patients, disease status, cytogenetics, pre-MRD, and follow-up days were included in the propensity score model. Results: 116 FLT3-ITDmut patients and 314 FLT3-ITDwt patients were included with a median (range) age of 35 years (1-64). FLT3-ITDmut patients showed higher WBC counts at diagnosis (P<0.001) and NPM1 mutation rate (P<0.001). CIR and LFS of FLT3-ITDmut patients were similar to FLT3-ITDwt patients (CIR:16.9% vs 14.2%, P= 0.266; LFS: 75.1% vs 77.7%, P= 0.399). 32 patients and 41 patients showed NPM1 mutation in FLT3-ITDmut and FLT3-ITDwt patients, respectively. We divided them into four groups based on the status of FLT3-ITD and NPM1 status, no significant difference in CIR and LFS was observed between FLT3-ITDwt NPM1wt, FLT3-ITDwt NPM1mut, FLT3-ITDmut NPM1wt and FLT3-ITDmut NPM1mut patients (FLT3-ITDwt NPM1wt vs FLT3-ITDwt NPM1mut vs FLT3-ITDmut NPM1wt vs FLT3-ITDmut NPM1mut: CIR: 14.8% vs 9.4% vs 16.4% vs 17.8%, P= 0.570; LFS: 76.9% vs 84.4% vs 79.5% vs 67.1%, P= 0.176). 49 patients with available allelic ratio were divided into low allelic ratio (n=25) and high allelic ratio groups (n=24) according to a cut-off 0.50. High allelic ratio was not correlated with inferior outcomes in this study (high allelic ratio vs low allelic ratio: CIR: 21.1% vs 18.1%, P= 0.617; LFS: 70.6% vs 73.7%, P= 0.563). To further analyze the differences between different donor type, we compared the outcomes of 318 haploidentical transplantation and 98 sibling-matched transplantation patients, respectively. FLT3-ITDmut patients had higher CIR and lower LFS than FLT3-ITDwt patients among sibling-matched transplantation (CIR:24.7% vs 12.0%, P= 0.064; LFS:68.2% vs 83.5%, P= 0.0502), but no difference was observed in haploidentical transplantation (CIR: 13.7% vs 15.0%, P= 0.996; LFS:77.8% vs 75.9%, P= 0.885). FLT3-ITD mutation didn't become a prognostic factor in survival outcomes when all patients received transplantation. Prognostic factors with P< 0.1 in univariate analysis were included in multivariate analysis. Apart from pre-MRD, and disease status influencing OS and CIR, another factor affecting OS was the gender of patients. Age played a prognostic role in NRM while pre-MRD and disease status also became the significant factors influencing LFS. Conclusion: Our results showed that allo-HSCT, especially haploidentical HSCT, may overcome the adverse effect of FLT3-ITD mutation, regardless of the NPM1 status or allelic ratio. Allo-HSCT could be an ideal option for AML patients with FLT3-ITD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic Impact of the 2022 European Leukemia Net Risk Classification in Patients with Acute Myeloid Leukemia Undergoing Allogeneic Stem Cell Transplantation

Introduction: For most acute myeloid leukemia (AML) patients (pts) an allogeneic stem cell transplantation (HSCT) offers the highest chance of cure. Recently, the European LeukemiaNet (ELN) published updated recommendations on the risk classification in AML pts. In addition to the risk at diagnosis, which is now based on cytogenetics and the mutation status of several genes, a dynamic adjustment according to the measurable residual disease (MRD) status in favorable (fav) and intermediate (int) risk pts at informative time points - such as prior to an allogeneic HSCT - was recommended. A validation of the new ELN2022 risk classification has not been reported. Methods: We retrospectively analyzed 522 AML pts who received an allogeneic HSCT between 2000 and 2021 in first complete remission (CR, 54%), second CR (11%), CR with incomplete count recovery (CRi, 13%), or relapsed/refractory (22%) at our center. Median age at HSCT was 59 (range 16-76) years (y), 53% of pts were younger than 60 y. Conditioning regimens were myeloablative (27%), reduced-intensity (23%) or non-myeloablative (50%). At diagnosis, cytogenetics, and the mutation status of CEBPA, NPM1, and FLT3-ITD were assessed. Using next-generation sequencing we analyzed a panel of 54 recurrently mutated genes in myeloid malignancies (MiSeq platform, Illumina). In pts with material available in remission prior to HSCT, the MRD status based on NPM1mutations, BAALC, MN1, and WT1 expression was evaluated as published previously (Jentzsch 2021). MRD positivity (MRDpos) was defined as positivity of any of the analyzed markers. Median follow up after HSCT was 3.0 y. Results: ELN2022 risk at diagnosis was 22% fav, 26% int, and 52% adverse (adv). Older pts (≥ 60 y at HSCT) had significantly worse ELN2022 genetic risk (P<.001). Comparing the ELN2017 and the new ELN2022, 83% (fav), 70% (int), and 90% (adv) of pts kept their allocation, while 15% and 2% of fav ELN2017 risk pts changed to int and adv ELN2022, 30% of int ELN2017 risk pts changed to adv ELN2022 & 1% and 10% of adv ELN2017 risk pts changed to fav and int ELN2022. Analyzing the c-statistic (area under the curves [AUC]) produced by the ELN2017 and ELN2022 for relapse (AUC2017 = 0.64 vs AUC2022 = 0.64, P=.21) and overall survival (OS) prediction (AUC2017 = 0.60 vsAUC2022 = 0.57, P=.06), the ELN2022 did not perform significantly better. However, the ELN2022 risk at diagnosis impacted cumulative incidence of relapse/progression (CIR, P<.001) and OS (P<.001, Figure 1), which was especially seen in pts < 60 y (CIR P<.001, OFS P<.001), but less so in pts ≥ 60 y at HSCT (CIR P=.002, OS P=.10). At diagnosis, outcomes did not differ significantly with respect to the specific genetic abnormality defining the ELN2022 fav or the ELN2022 int risk. Also a high or low FLT3-ITD allelic ratio (0.5 cut) within the ELN2022 int risk did not result in distinct outcomes (CIR P=.13, OS P=.30). In contrast, within pts harboring adv ELN2022 risk, CIR (P=.05) and OS (P<.001) significantly differed according to the presence of specific genetic abnormalities; i.e. a complex karyotype (at 3 y: CIR 59%, OS 30%), other adverse cytogenetic aberrations (at 3 y: CIR 50%, OS 54%), adverse-risk gene mutation (i.e.ASXL1, RUNX1, or TP53, at 3 y: CIR 47%, OS 58%), or secondary AML-like gene mutations (at 3 y: CIR 30%, OS 73%). When we adjusted fav and int risk pts according to the MRD status at HSCT as proposed by the ELN2022 recommendations, 49% of fav risk pts at diagnosis had persisting MRD and were reclassified as int risk at HSCT while 53% of int risk pts at diagnosis were MRD negative and reclassified as fav risk at HSCT. Outcomes of MRD-adjusted fav risk pts improved (at 3 y: CIR 12%, OS 75%), while that of MRD-adjusted int risk pts (at 3 y: CIR 53%, OS 49%) was similar to that of adv risk pts transplanted in morphologic CR/CRi (at 3 y: CIR 47%, OS 54%). Conclusion: In the context of an allogeneic HSCT, the proposed risk assessments by the ELN2022 significantly impacted outcomes at diagnosis as well as after adjustment according to MRD status prior to HSCT of AML pts. However, it did not perform significantly better than the previous ELN2017 risk stratification. The FLT3-ITD allelic ratio did not significantly impacted outcomes within the ELN2022 int risk, justifying the removal from the risk stratification. Noteworthy, in pts receiving allogeneic HSCT, secondary AML-like gene mutations did not associate with adverse outcomes when no other adverse-risk characteristics were present. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Venetoclax in Combination with Hypomethylating Agents or Low-Dose Cytarabine for Relapsed/Refractory Acute Myeloid Leukemia: A Retrospective Single-Centre Experience

Background. Relapsed/refractory (R/R) acute myeloid leukemia (AML) is a largely unmet medical condition with an overall dismal outcome owing to the lack of standardized, effective treatment approaches. The only curative option for R/R AML patients (pts) is allogeneic hematopoietic stem cell transplantation (HSCT), that can be exploited in a minority of otherwise eligible pts due to poor efficacy and high toxicity of salvage regimens used to induce leukemia remission as bridge-to-transplant. Recently, a number of targeted agents with relatively favorable toxicity profiles have been explored in clinical trials for R/R AML pts. Since its first accelerated approval by the Food and Drugs Administration in 2018, the Bcl-2 inhibitor venetoclax (VEN), in combination with hypomethylating agents (HMA) or low dose cytarabine (LDAC), has steadily become the standard therapeutic approach for newly diagnosed AML in elderly or unfit pts, while its role in R/R disease is not well defined yet. Aim. To gain insights into the potential effectiveness of VEN-based regimens in patients with R/R AML. Patients. We retrospectively analyzed the clinical outcomes of 67 R/R AML pts treated at our Institution with venetoclax-based regimens on an off-label basis, between March 2018 and April 2022. Fifty-eight pts (87%) carried a de-novo AML diagnosis, 6 pts (9%) had AML secondary to myelodysplastic syndrome and 3 pts (4%) had therapy-related AML. Median age was 58y (33-74y); 30 pts (45%) were aged >60, while 7 pts (10%) were aged >70y at VEN initiation. Thirty-eight pts (56%) were treated for first (n=32) or second relapse (n=6); last complete remission (CR) duration was <6 mo in 18 pts, 6-12 mo in 16 pts and >12 m (12-56) o in 4 patients; 11 pts (16%) had relapsed after HSCT; remaining 18 pts (27%) were treated for primary refractory disease. All pts received conventional immunophenotypic and cytogenetic characterization and at least NPM1, FLT3, CBFB/MYH11 and RUNX1/RUNX1T1 mutation/rearrangement detection. Next-generation sequencing (NGS) data were available for 56 pts (83%); specifically, 40 pts (primary refractory, n=18; relapsed, n=22) had NGS analysis carried out on bone marrow samples shortly before VEN initiation, while in 16 pts NGS was carried out on baseline diagnostic specimens. VEN partner drug was azacitidine in 45 pts (68%), LDAC in 15 (22%) and decitabine in 7 (10%). Thirty-nine pts (58%) were treated with an intention-to-transplant (ITT). Median follow-up duration was 11.8 mts (1.1 - 44). Results. Overall, we report a composite complete response (CCR) rate of 55% (n=37); 10 of complete responders (27%) had incomplete peripheral counts recovery. Minimal residual disease (MRD) negativity was achieved in 21/37 pts (56%). All CR invariably occurred during the first 8 weeks of treatment. Although it did not reach significance level (p=0.065), there was considerable variation in CCR rate among treatment settings: relapsed pts, 66%; primary refractory, 50%; post-HSCT relapse, 27%. Conventional risk stratification parameters (age, leukocyte counts, ELN 2017 risk category, NPM1/FLT3 mutational status, secondary disease, last CR duration) were poorly predictive of CR probability. A positive trend towards higher CCR rate, although not statistically significant, was found for NPM1 (CCR rate: 57%) and IDH1/2 mutation (66%) while FLT3/Ras pathway mutations were detrimental (23%). Median overall survival (OS) but not event-free survival (EFS), differed significantly among settings: relapsed pts, 11.9 mo; refractory, 8.6 mo; post-HSCT, 6.7 mo. (p=0.030). Stratifying pts based on NPM1 and FLT3-ITD status resulted in markedly different EFS: NPM1mut/FLT3-ITDneg, not reached; NPM1wt/FLT3any, 6.2 mo; NPM1mut, FLT3-ITDpos, 3.4 mo (p=0.031); as well as different OS: NPM1mut/FLT3-ITDneg, not reached; NPM1wt/FLT3any, 10.5 mo; NPM1mut, FLT3-ITDpos, 5.6 mo (p=0.003). Among the ITT population, HSCT actualization rate was 66%. Four pts are currently long-term survivors with follow-up exceeding 24 mo without receiving HSCT; notably, they all carried a NPM1mut/FLT3-ITDneg/Ras pathwayneg genotype. Conclusions: Overall, these findings suggest a role for venetoclax-based regimens in R/R AML patients and support the design of prospective studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prognostic Impact of NPM1 and FLT3-ITD Mutations in Patients Treated with Non-Intensive Regimens: A Pethema Registry Study

Introduction: Acute myeloid leukemia (AML) is the most common acute leukemia of adults, with median age at diagnosis of 68 years. Mutations in FMS-like tyrosine kinase-3 internal tandem duplication (FLT3-ITD) are associated with a higher risk of relapse and inferior overall survival (OS) in younger patients treated with intensive chemotherapy, but the prognostic relevance among older patients treated with non-intensive approaches is not well established. As well, the role of nucleophosmin (NPM1), and its interaction with FLT3-ITD status and allelic ratio (AR) remains unclear among elderly. We aimed to assess the prognostic impact of FLT3-ITD and NPM1 mutations in older/unfit newly diagnosed AML patients treated with non-intensive front-line therapies. Methods: We conducted a retrospective analysis that included 707 patients treated with different non-intensive regimens in the PETHEMA AML registry, between 2007 and 2020. FLT3-ITD and NPM1 mutations were studied by fragment length analysis. The karyotype information was available in 615 patients. Associations of demographic and clinical parameters in patients harboring FLT3-ITD and NPM1 mutations was performed using Mann-Whitney-U-test for continuous and Chi-square test for discrete variables. Overall survival (OS) and relapse curves were constructed using Kaplan-Meier and a log-rank test. All analyses were performed by SPSS. Results: The median age was 74 years (range: 43-91), and 410 patients (58%) were male. ECOG status was 0/1 in 474 patients (72%) and 2 in 124 patients (18.9%). FLT3-ITD mutation was identified in 99 patients (14%). When available, 33 patients (40.7%) showed FLT3-ITDlow (AR <0.5). NPM1 mutation was detected in 144 patients (20%). Karyotype was abnormal in 301 patients (42.7%). Regarding the frontline therapy 328 (46.3%) patients received hypomethylating agents (HMA); 353 (49.9%) fludarabine + low dose cytarabine (LDAC) (FLUGA); 11 (1.6%) fludarabine + LDAC + oral idarubicin (FLAG-IDA Lite); and 15 (2.1%) LDAC. Mortality at 2 years was 77.6% and median OS 6 months. FLT3-ITD patients (N=99) showed a non-significant difference in OS compared to FLT3 wild-type (FLT3WT) patients (N=608): median OS 5 vs 7.3 months (p=0.17). There were no differences when comparing low and high AR: FLT3-ITDlow (N=34) vs FLT-ITDhigh (N=47): median OS 5.5 vs 4.9 months (p=0.64). Survival in NPM1-mutated patients (N=144) was similar than in NPM1WT patients (N=519): median OS 7.2 vs 6.8 months (p=0.24). OS according to ELN 2017 risk stratification, considering only NPM1 and FLT3-ITD mutations, was similar in subgroups low risk (N=117), intermediate risk (N=31), high risk (N=23): median OS 7.6 vs 5.4 vs 7.2 (p=0.26) (Fig. 1). In the subgroup of normal karyotype (N=314), FLT3-ITD patients (N=64) showed a trend towards lower survival as compared to FLT3WT patients (N=249): median OS 5.4 vs 10.9 months (p=0.058), with no impact of AR: FLT3-ITDlow (N=19) vs. FLT3-ITDhigh (N=32) median OS 5.8 vs 4.8 months (p=0.73). NPM1 mutated patients (N=102) showed a non-significant difference with NPM1WT patients (N=196): median OS 7.2 vs 10.3 months (p=0.66). OS according to ELN 2017 considering only NPM1 and FLT3-ITD status was similar in subgroups: low risk (N=80), intermediate risk (N=24), high risk (N=12): median OS 2.3 vs 11 vs 2.5 months, respectively (p=0.64). AML patients treated with azacitidine (N=281) had longer median OS of 10 months (p=0.005; 95% CI 1.03 to 1.37) vs decitabine (N=47) median OS 6.1 months and FLUGA median OS 5 months (N=352) (Fig. 2). Conclusions: Data from this large cohort of AML patients treated with non-intensive regimens underlines the limitations of the ELN 2017 risk stratification in this setting. Nonetheless, there was a trend towards worse OS in patients with normal karyotype and FLT3-ITD mutation (irrespectively of the allelic burden) as compared to FLT3WT patients. Figure 1. Overall survival according to ELN 2017 risk stratification Figure 2. Overall survival regarding non-intensive regimens. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

AML-234 Prognostic Value of FLT3-ITD Residual Disease in Acute Myeloid Leukemia

FLT3-internal tandem duplications (FLT3-ITD) are among the most common genetic molecular abnormalities in acute myeloid leukemia (AML) patients. Here, the prognostic impact of the presence of minimal residual disease (MRD) of FLT3-ITD in AML patients is assessed by next-generation sequencing (NGS). The prognostic significance of FLT3-ITD in AML in relation to other concurrent gene mutations and allelic mutational burden has remained a subject of scientific controversy. Detection of FLT3-ITD MRD by RQ-PCR is restricted by patient-specific variables, such as sequence, position, and length. However, systematic studies analyzing the applicability of FLT3-ITD MRD detection with NGS techniques are currently lacking. Here, we evaluate the impact of the presence of FLT3-ITD MRD detected by NGS on treatment outcome in the context of current prognostic factors at diagnosis and MRD status measured by multiparameter flow cytometry (MFC) and mutant NPM1. In 161 de novo AML patients with an FLT3-ITD enrolled in the HOVON-SAKK clinical trials, NGS was performed at diagnosis and ultra-deep in CR after induction chemotherapy. Presence of FLT3-ITD MRD was correlated to incidence of relapse and overall survival (OS). NGS-based FLT3-ITD MRD was present in 47 of 161 (29%) AML patients. Presence of FLT3-ITD MRD was associated with increased risk of relapse (4-year CIR, 75% FLT3-ITD MRD vs. 33% no FLT3-ITD MRD; P<0.001) and inferior OS (4-year OS, 31% FLT3-ITD MRD vs. 57% no FLT3-ITD MRD; P<0.001). In multivariate analysis, detection of FLT3-ITD MRD in CR confers independent prognostic significance for relapse (hazard ratio, 3.55; P<0.001) and OS (hazard ratio 2.51; P<0.001). Presence of FLT3-ITD MRD exceeds the prognostic value of most of the generally accepted clinical and molecular prognostic factors, including the FLT3-ITD allelic ratio at diagnosis and MRD status of mutant NPM1 and MFC. Presence of FLT3-ITD MRD in CR detected by NGS after induction chemotherapy identifies AML patients with an increased risk of relapse and inferior OS that outweighs the significance of currently accepted prognostic factors. This finding offers support for the use of FLT3-ITD MRD as a clinically relevant biomarker for dynamic disease risk assessment in AML.

Overcoming Resistance: FLT3 Inhibitors Past, Present, Future and the Challenge of Cure

Simple SummaryDespite the recent approval of some FLT3 inhibitors by drug regulatory agencies, treatment guidelines for FLT3-mutated AML still require allogeneic transplantation as a necessary procedure to treat the disease in first or second CR, due to the high relapse incidence related to the use of these drugs. The study of the heterogeneity of leukemogenesis and resistance mechanisms related to the use of FLT3 inhibitors, alone or in combination, represents one of the additional challenges in attempting to achieve the eradication of the mutated FLT3 leukemic clone. The analysis and knowledge of these pathways might drive future approach in this setting.FLT3 ITD and TKD mutations occur in 20% and 10% of Acute Myeloid Leukemia (AML), respectively, and they represent the target of the first approved anti-leukemic therapies in the 2000s. Type I and type II FLT3 inhibitors (FLT3i) are active against FLT3 TKD/ITD and FLT3 ITD mutations alone respectively, but they still fail remissions in 30–40% of patients due to primary and secondary mechanisms of resistance, with variable relapse rate of 30–50%, influenced by NPM status and FLT3 allelic ratio. Mechanisms of resistance to FLT3i have recently been analyzed through NGS and single cell assays that have identified and elucidated the polyclonal nature of relapse in clinical and preclinical studies, summarized here. Knowledge of tumor escape pathways has helped in the identification of new targeted drugs to overcome resistance. Immunotherapy and combination or sequential use of BCL2 inhibitors and experimental drugs including aurora kinases, menin and JAK2 inhibitors will be the goal of present and future clinical trials, especially in patients with FLT3-mutated (FLT3mut) AML who are not eligible for allogeneic transplantation.

Depth of Response to Intensive Chemotherapy Has Significant Prognostic Value among Acute Myeloid Leukemia (AML) Patients Undergoing Allogeneic Hematopoietic Stem-Cell Transplantation with Intermediate or Adverse Risk at Diagnosis Compared to At-Risk Group According to European Leukemia Net 2017 Risk Stratification.

Simple SummaryAcute myeloid leukemia (AML) is a devastating but potentially curable disease. The updated version of the European Leukemia Net (ELN) 2017 genetic risk stratification is used as the standard for the prognosis and classification of AML. In the present study, we evaluated the prognostic value of the ELN 2017 criteria on post-hematopoietic stem-cell transplantation (HSCT) outcomes and compared it with pre-HSCT measurable residual disease (MRD) status, determined by Wilms tumor gene 1 (WT1) expression. We classified the patients as intermediate (INT) risk and adverse (ADV) risk. We found that the ELN 2017 risk classification did not effectively predict post-HSCT outcomes in patients with INT or ADV risk. The pre-HSCT WT1 level predicted post-HSCT relapse better than ELN 2017 and had a more prominent prognostic value in the ELN INT risk group than in the ADV risk group.We evaluated the prognostic efficiency of the European Leukemia Net (ELN) 2017 criteria on the post-transplant outcomes of 174 patients with intermediate (INT; n = 108, 62%) or adverse (ADV) risk (n = 66, 38%) of acute myeloid leukemia; these patients had received the first allogeneic hematopoietic stem-cell transplantation (HSCT) at remission. After a median follow-up period of 18 months, the 2 year OS, RFS, and CIR after HSCT were estimated to be 58.6% vs. 64.4% (p = 0.299), 50.5% vs. 53.7% (p = 0.533), and 26.9% vs. 36.9% (p = 0.060) in the INT and ADV risk groups, respectively. Compared to the ELN 2017 stratification, pre-HSCT WT1 levels (cutoff: 250 copies/104 ABL) more effectively segregated the post-HSCT outcomes of INT risk patients compared to ADV risk patients regarding their 2 year OS (64.2% vs. 51.5%, p = 0.099), RFS (59.4% vs. 32.4%, p = 0.003), and CIR (18.9% vs. 60.0% p < 0.001). Indeed, high WT1 levels were more prominent in INT risk patients than in ADV risk patients. Notably, FLT3-ITD had the greatest impact on post-HSCT outcomes among all the ELN 2017 criteria components; patients in the FLT3-ITD mutant subgroups exhibited the worst outcomes regardless of their allelic ratios or NPM1 status compared to the pre-HSCT WT1 level of other INT and ADV risk patients.

P392: PHARMACOLOGICAL INHIBITION OF SYK CONFERS ANTI-PROLIFERATIVE AND NOVEL ANTI-TUMOR IMMUNE RESPONSES IN AML

Background: Spleen tyrosine kinase (SYK) acts as a key integrator of signals from cell surface receptors containing an immunoreceptor tyrosine-based activation motif to boost cellular proliferation. In acute myeloid leukemia (AML), SYK serves as a relay to an oncogenic transcriptional regulatory network linked to NPM1, HOXA9 and MEIS1. The selective, orally bioavailable SYK inhibitor entospletinib (ENTO) has demonstrated clinical activity and tolerability in HOXA9/MEIS1-driven AML. ENTO is currently being investigated in a global Phase 3 trial, AGILITY (NCT05020665), in combination with intensive induction/consolidation chemotherapy in patients (pts) with treatment-naive NPM1-mutated (NPM1m) AML. Lanraplenib (LANRA) is a next-generation SYK inhibitor with similar potency and selectivity to ENTO but with more favorable pharmacologic properties that is currently being evaluated in combination with gilteritinib in pts with relapsed or refractory FLT3-mutated AML (NCT05028751). Aims: To investigate the effects of ENTO and LANRA in T-cell responses in AML. Methods: All pt-derived bone marrow (BM) and peripheral blood samples were obtained in accordance with IRB (OHSU#004422) approval. Results from gene expression profiling studies (RNA sequence [RNA-seq]) in bone marrow/peripheral blood mononuclear cells (MNCs) derived from 152 pts with AML, for which matching NPM1 status and ex vivo sensitivity to ENTO was available, previously published by Tyner et al (2018), were reanalyzed using trimmed mean of M values normalization and differential expression using edgeR. ENTO/LANRA sensitivity was assessed ex vivo after 72 hours in culture at 37°C by MTS assay. T-cell functional assays were performed by culturing MNCs with anti-CD3 and treating with ENTO or LANRA, singly and in combination with anti-PD1. CD3+ T-cell proliferation as measured by Ki67 expression and pSYK activation were assessed by quantitative single-cell immunofluorescence microscopy. Six formalin-fixed, paraffin-embedded BM biopsies from newly diagnosed AML pts were assessed for spatial expression of mRNA using a digital spatial profiling method. Results: In AML, mutations in NPM1 with co-expression of HOXA9/MEIS1 at baseline predicted anti-proliferative activity to ENTO in ex vivo drug sensitivity studies. Accordingly, treatment of leukemic cells with either ENTO or LANRA inhibited SYK auto-phosphorylation in a dose-dependent manner. Pathway analysis of archival RNA-seq data from pts enrolled in the Leukemia and Lymphoma Society BEAT AML Master Protocol (NCT03013998 [BAML-16-001-S6]) revealed gene expression signatures at baseline associated with the observed sensitivity to ENTO, including significant enrichment in the expression of genes associated with leukemogenesis, myeloid differentiation and immune regulation. This was supported by T-cell functional studies, which demonstrated that ENTO and LANRA, singly as well as in combination with anti-PD1, could restore T-cell proliferation in primary AML pt samples that exhibited suppression. Lastly, Cancer Transcriptome Atlas analysis on archival BM biopsies obtained from these AML pts showed a strong correlation between immune checkpoint response and gene expression associated with immune pathway signaling. Summary/Conclusion: Inhibition of SYK is a promising therapeutic approach in NPM1m AML. Our studies illustrate that ENTO and LANRA may restore T-cell proliferation in a subset of AML pts with dysfunctional T-cell responses, suggesting a novel mechanism of action. Additional studies are required to fully understand the mechanism of SYK-mediated antitumor immune responses in AML.

Somatic mutation-associated risk index based on lncRNA expression for predicting prognosis in acute myeloid leukemia

ABSTRACT Objectives: Genomic instability has several implications for acute myeloid leukemia (AML) prognosis. This article aims to construct a somatic mutation-associated risk index (SMRI) of genomic instability for AML to predict prognosis and explore the potential determinants of AML prognosis. Methods: We obtained differentially expressed lncRNAs from genomic instability subtypes and selected six lncRNAs to construct the SMRI through multivariate Cox regression analysis. The median SMRI classified patients into high and low SMRI groups. Kaplan–Meier survival analysis was used to clarify the prognostic differences of SMRI subtypes. Receiver operating characteristic curve analysis was performed to elucidate the value of SMRI as a prognostic indicator. Gene set variation analysis, tumor mutation burden (TMB) analysis, immune infiltration, and immune checkpoint expression analysis were performed to investigate possible causes for the differences in prognosis of SMRI subtypes. Results: The high SMRI group exhibited a poor prognosis, which was characterized by elevated levels of TMB, mutation counts (TP53, NPM1, DNMT3A, and FLT3-TKD), CD8+ T cell infiltration, and immune checkpoint (PD-1, PD-L2, CTLA4, LAG3) expression. The SMRI was still associated with prognosis, even after adjustment for age, sex, cytogenetic risk, DNMT3A status, FLT3 status, and NPM1 status. Gene set variation analysis showed that AML with FLT3-ITD mutation, CEBPA mutation, and LSCs (leukemia stem cells) were enriched in the high SMRI group. Conclusion: Our research suggests that the SMRI derived from genomic instability subtypes is a useful biomarker for predicting prognosis and may be beneficial for improving the clinical outcome of patients with AML.

A clinicopathological experience in acute myeloid leukemia: Effects of clinical data and status of FLT3, CEBPA and NPM1 on prognosis.

The purpose of the study was to analyze the expression of nucleophosmin (NPM1), CCAT/enhancer-binding protein alpha (CEBPA), and FMS-like tyrosine kinase 3 (FLT3) with immunohistochemistry and evaluate the relationship with clinicopathologic data with special emphasis on prognosis in bone marrow biopsy specimens diagnosed with acute myeloid leukemia (AML). Bone marrow biopsies of 104 patients who were diagnosed with AML were re-evaluated for diagnosis and subclassification. Immunohistochemically, anti-NPM1, anti-CEBPA, and anti-FLT3 antibodies were applied to slides prepared from formalin-fixed paraffin-embedded tissues. Sixty-three of these patients had their follow-up in our institutional hematology clinic and these patients' clinical, biochemical, and radiological data were obtained and analyzed from patient files. These data were analyzed with survival times statistically. Except for age, no significant effect of clinical data on prognosis was detected. Immunohistochemical results were also statistically compared with clinical data. No correlation was found between overall survival and disease-free survival with the expression of anti-CEBPA or anti-NPM1 antibodies. However, immunohistochemical reactivity for anti-FLT3 antibody was found to be a poor prognostic factor and statistically significant. Also, when the expression of FLT3 was analyzed with that of NPM1 or CEBPA, a correlation (dependent on the expression of FLT3) was found with disease-free survival. FLT3 is an independent prognostic factor for AML. CEBPA and NPM1 should be considered as good prognostic factors only in the absence of FLT3 abnormalities.