NPM1-mutated Patients Research Articles

Early Prediction and Streamline of Nucleophosmin Mutation Status in Acute Myeloid Leukemia Using Cup-Like Nuclear Morphology.

Background and Objectives: With the advent of novel therapies for nucleophosmin gene (NPM1)-mutated acute myeloid leukemia (AML), there is a growing need for the reliable prediction of NPM1 mutations. This study explored the role of cytomorphological features in the early prediction of NPM1-mutated AML. Materials and Methods: Altogether, 212 de novo AML cases with normal karyotypes, diagnosed and treated at a single institution within 5 years (2018-2023), were retrospectively evaluated. A final diagnosis of NPM1-mutated AML, based on the World Health Organization (WHO) integrated criteria, including real-time based identification of NPM1 mutation and normal karyotype, was established in 83/212 (39.15%) cases. Results: Cup-like blasts (CLBs), a cytomorphological feature suggestive of NPM1-mutated AML, were detected in 56/83 (67%) patients. Most cases (44/56, 78.6%) had CLB ≥ 10%. In total, 27 of 83 AML NPM1-mutated patients had no CLB morphology (missed call). Additionally, two of 212 had CLB morphology without confirmed NPM1 mutation (wrong call). The positive/negative predictive values of cytomorphological evaluation for CLB ≥ 10% were 95.7%/75.6%, with sensitivity/specificity of 53%/98.5%, while the accuracy was 80.7%. We noted an increased percentage of CLBs (≥15%) in 77.8% and 50% of patients with AML without and with granulocytic maturation, respectively (the specificity for NPM1 mutation prediction was 100%). CLB was associated with fms-like tyrosine kinase 3 (FLT3) mutation (p = 0.03), but, without statistical significance for CLB ≥ 10% and CLB ≥ 15%. Conclusions: Our investigation confirmed that the morphological identification of CLB at diagnosis represents a reliable and easily reproducible tool for the early prediction of NPM1 mutations, enabling a streamlined genetic work-up for its confirmation. This may facilitate considering the early administration of individualized therapies by clinicians for specific patients.

Evolution from an antecedent chronic myeloid malignancy does not impact survival outcomes in NPM1-mutated AML.

Nucleophosmin-1 (NPM1)-mutated AML is a molecularly defined subtype typically associated with favorable treatment response and prognosis; however, its prognostic significance in AML evolving from an antecedent chronic myeloid malignancy is unknown. This study's primary objective was to determine the impact of mutated NPM1 on the prognosis of AML evolving from an antecedent chronic myeloid malignancy. We conducted a retrospective chart review including patients with NPM1-mutated de novo and sAML. sAML was defined as those with a preceding chronic-phase myeloid malignancy before diagnosis of AML. Of 575 NPM1-mutated patients eligible for inclusion in our study, 51 (8.9%) patients were considered to have sAML. The median time from diagnosis of NPM1-mutated chronic myeloid malignancy to sAML evolution was 3.6 months (0.5-79.3 months). No significant differences in leukemia-free (2-year LKFS 52.0% vs. 51.2%, p = .9922) or overall survival (2-year OS 56.3% vs. 49.4%, p = .4246) were observed between patients with NPM1-mutated de novo versus sAML. Our study suggests that evolution from a preceding myeloid malignancy is not a significant predictor of poor prognosis in the setting of an NPM1 mutation. Our study demonstrated a short time to progression to sAML in most patients, which further supports the consideration of NPM1 as an AML-defining mutation.

Influence of genetic co-mutation on chemotherapeutic outcome in NPM1-mutated and FLT3-ITD wild-type AML patients.

Nucleophosmin 1 (NPM1) gene-mutated acute myeloid leukemia (NPM1mut AML) is classified as a subtype with a favorable prognosis. However, some patients fail to achieve a complete remission or relapse after intensified chemotherapy. Genetic abnormalities in concomitant mutations contribute to heterogeneous prognosis of NPM1mut AML patients. In this study, 91 NPM1-mutated and FLT3-ITD wild-type (NPM1mut/FLT3-ITDwt) AML patients with intermediate-risk karyotype were enrolled to analyze the impact of common genetic co-mutations on chemotherapeutic outcome. Our data revealed that TET1/2 (52/91, 57.1%) was the most prevalent co-mutation in NPM1mut AML patients, followed by IDH1/2 (36/91, 39.6%), DNMT3A (35/91, 38.5%), myelodysplastic syndrome related genes (MDS-related genes) (ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1 and ZRSR2 genes) (35/91, 38.5%), FLT3-TKD (27/91, 29.7%) and GATA2 (13/91, 14.3%) mutations. Patients with TET1/2mut exhibited significantly worse relapse-free survival (RFS) (median, 28.7 vs. not reached (NR) months; p = 0.0382) compared to patients with TET1/2wt, while no significant difference was observed in overall survival (OS) (median, NR vs. NR; p = 0.3035). GATA2mut subtype was associated with inferior OS (median, 28 vs. NR months; p < 0.0010) and RFS (median, 24 vs. NR months; p = 0.0224) compared to GATA2wt. By multivariate analysis, GATA2mut and MDS-related genesmut were independently associated with worse survival. Mutations in TET1/2, GATA2 and MDS-related genes were found to significantly influence the chemotherapeutic outcome of patients with NPM1mut AML. The findings of our study have significant clinical implications for identifying patients who have an adverse response to frontline chemotherapy and provide a novel reference for further prognostic stratification of NPM1mut/FLT3-ITDwt AML patients.

Do NPM1 and FLT3-ITD mutations modify prognosis in patients treated with non-intensive regimens?

FLT3-ITD and NPM1 mutations are key to defining the genetic risk profile of acute myeloid leukemia (AML). We aimed to assess the prognostic features of the FLT3-ITD and NPM1 mutations in old and/or unfit individuals with AML treated with non-intensive therapies in the era before azacitidine-venetoclax approbation. The results of various non-intensive regimens were also compared. We conducted a retrospective analysis that included patients treated with different non-intensive regimens, between 2007 and 2020 from PETHEMA AML registry. We compiled 707 patients with a median age of 74years and median follow-up time of 37.7months. FLT3-ITD patients (N = 98) showed a non-significant difference in overall survival (OS) compared to FLT3-ITD negative-patients (N = 608) (P = 0.17, median OS was 5 vs 7.3months respectively). NPM1-mutated patients (N = 144) also showed a non-significant difference with NPM1 wild type (N = 519) patients (P = 0.25, median OS 7.2 vs 6.8 respectively). In the Cox regression analysis neither NPM1 nor FLT3-ITD nor age were significant prognostic variables for OS prediction. Abnormal karyotype and a high leukocyte count showed a statistically significant deleterious effect. Azacitidine also showed better survival compared to FLUGA (low dose cytarabine plus fludarabine). NPM1 and FLT3-ITD seem to lack prognostic value in older/unfit AML patients treated with non-intensive regimens other than azacitidine-venetoclax combination.

Abstract P25: Identification of biological components and novel clinical subsets of NPM1-mutated AML using an integrated, multi-omics approach

Abstract P25: Identification of biological components and novel clinical subsets of <i>NPM1</i>-mutated AML using an integrated, multi-omics approach

Distinct karyotypic and mutational landscape in trisomy AML.

Trisomy karyotype occurs in 5%-10% of AML. Its mutational landscape and prognostic significance are not well defined. A cohort of 156 trisomy AML patients was analysed, with reference to 615 cytogenetically normal (CN) AML patients. Trisomy AML showed distinct mutational landscape with more prevalent SMC1A, N/KRAS, ASXL1 and BCOR but fewer CEBPAbZIP and NPM1 mutations in patients ≤60, and fewer NPM1 mutations in those >60. NRAS mutations were associated with poor outcome in trisomy AML, whereas DNMT3A and FLT3-ITD mutations had neutral effect. Trisomy AML appeared biologically distinct from CN-AML.

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

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

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

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

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

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

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.

Survival improvement of patients with FLT3 mutated acute myeloid leukemia: results from a prospective 9 years cohort

Midostaurin added to intensive chemotherapy is the standard of care for acute myeloid leukemia (AML) with FLT3 mutations (FLT3mut). We analyzed the impact of midostaurin in 227 FLT3mut-AML patients included in the AML-12 prospective trial for fit patients ≤70 years (#NCT04687098). Patients were divided into an early (2012–2015) and late (2016–2020) cohorts. They were uniformly treated except for the addition of midostaurin in 71% of late group patients. No differences were observed in response rates or the number of allotransplants between groups. Outcome was improved in the late period: 2-year relapse incidence decreased from 42% vs 29% in early vs late group (p = 0.024) and 2-year overall survival (OS) improved from 47% vs 61% (p = 0.042), respectively. The effect of midostaurin was evident in NPM1mut patients (n = 151), with 2-yr OS of 72% (exposed) vs 50% (naive) patients (p = 0.011) and mitigated FLT3-ITD allelic ratio prognostic value: 2-yr OS with midostaurin was 85% and 58% in low and high ratio patients (p = 0.049) vs 67% and 39% in naive patients (p = 0.005). In the wild-type NPM1 subset (n = 75), we did not observe significant differences between both study periods. In conclusion, this study highlights the improved outcome of FLT3mut AML fit patients with the incorporation of midostaurin.

Abstract A07: Single-cell proteomic assessment of FLT3-ITD AML landscape identifies distinct resistance patterns

Abstract Acute myeloid leukemia (AML) represents a heterogeneous hematopoietic disorder characterized by accumulation of immature hematopoietic precursors with differentiation block. FLT3 internal tandem duplications (FLT3-ITD) are commonly occurring genetic alterations in AML and are associated with poor prognosis. Preclinical studies showed combining FLT3 inhibitor with MDM2 inhibitor, which is a negative regulator of p53, was synergistic in FLT3-ITD/TP53 wild type (WT) AML. We performed single-cell proteomic evaluation of leukemia landscape in FLT3-ITD patients treated with FLT3i and MDM2i to assess proteomic profiles associated with response and resistance. We performed CyTOF analysis of leukemia cells in serially collected samples from six FLT3-ITD and TP53 WT AML patients following treatment with FLT3i+MDM2i treatment. Using 51 features assessed in CyTOF, we first performed UMAP dimension reduction and clustering to identify distinct cells in leukemia compartment. Notably, the frequencies of blasts identified through CyTOF data analysis were compatible with clinical lab reports. In line with previous reports, we also detected that NPM mutant AML cells did not express CD34. Of note, CD34+ and CD34- leukemia cells were clustered together in NPM1 mut patients, indicating that they have overlapping proteomic profiles. Interestingly, the CD34+ leukemia cells were eliminated at the early time points in CR patients while the CD34- leukemia cells were still detectable after two months. On the other hand, CD34+ leukemia cells in nonresponders(NR) persisted despite therapy. These findings indicate CD34+ leukemia cells were more sensitive to the treatment compared to NPM1 mutant CD34- leukemia cells. Next, we interrogated leukemia proteomic landscape in serial samples, evaluated the therapy-induced alterations in proteomic profiles and sought to identify potential adaptive mechanisms. To this end, we performed differential expression analysis and observed that signaling pathways (p-4EBP1, p-GSK3, p-MEK1/2, p-S6) and differentiation markers(CD33, CD68,CXCR4 HLADR) were more enriched on day 8 post treatment in NR patients, revealing that compensatory signaling activity, phenotypic profiles and differentiation status could be associated with therapy response. Moreover, we found that leukemia cells in NR patients had distinct phenotypic profiles(CD11b,CD68,CXCR4), higher levels of anti-apoptotic molecules (BCL2,MCL1) and enriched survival pathways (p-GSK3, YTHDF2) compared to baseline. In contrast, we did not observe rebound increases in CR patients post-therapy. These findings demonstrate high levels of BCL2 and MCL1 and preferential survival of more differentiated cells may be associated with therapy resistance and treatment failure in NR patient treated with MDM2i and FLT3i. In conclusion, multiplexed single-cell proteomic analysis permitted longitudinal monitoring of leukemia landscape and identified proteomic alterations associated with therapy resistance. Citation Format: Li Li, Muharrem Muftuoglu, Mahesh Basyal, Naval Daver, Michael Andreeff. Single-cell proteomic assessment of FLT3-ITD AML landscape identifies distinct resistance patterns [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A07.

Outcome prediction by the 2022 European LeukemiaNet genetic-risk classification for adults with acute myeloid leukemia: an Alliance study

Recently, the European LeukemiaNet (ELN) revised its genetic-risk classification of acute myeloid leukemia (AML). We categorized 1637 adults with AML treated with cytarabine/anthracycline regimens according to the 2022 and 2017 ELN classifications. Compared with the 2017 ELN classification, 2022 favorable group decreased from 40% to 35% and adverse group increased from 37% to 41% of patients. The 2022 genetic-risk groups seemed to accurately reflect treatment outcomes in all patients and patients aged <60 years, but in patients aged ≥60 years, relapse rates, disease-free (DFS) and overall (OS) survival were not significantly different between intermediate and adverse groups. In younger African-American patients, DFS and OS did not differ between intermediate-risk and adverse-risk patients nor did DFS between favorable and intermediate groups. In Hispanic patients, DFS and OS did not differ between favorable and intermediate groups. Outcome prediction abilities of 2022 and 2017 ELN classifications were similar. Among favorable-risk patients, myelodysplasia-related mutations did not affect patients with CEBPAbZIP mutations or core-binding factor AML, but changed risk assignment of NPM1-mutated/FLT3-ITD-negative patients to intermediate. NPM1-mutated patients with adverse-risk cytogenetic abnormalities were closer prognostically to the intermediate than adverse group. Our analyses both confirm and challenge prognostic significance of some of the newly added markers.

Prognostication refinement in NPM1-mutated acute myeloid leukemia stratified by FLT3-ITD status with different induction doses of cytarabine.

We aimed to retrospectively discern the heterogeneity of outcomes from clinicopathological characteristics and next-generation sequencing (NGS) data in adult patients with NPM1-mutated (NPM1mut ) acute myeloid leukemia (AML) induced with standard-dose (SD, 100-200 mg/m2 ) and intermediate-dose (ID, 1000-2000 mg/m2 ) cytarabine arabinose (Ara-C). In the entire cohort and FLT3-ITD subgroups, multivariate Logistic and Cox regression analyses were used to analyze the comprehensive complete remission (cCR) rate after one or two induction cycles, event-free survival (EFS), and overall survival (OS). Among a total of 203 NPM1mut patients evaluable for clinical outcome, 144 (70.9%) received a first SD-Ara-C induction and 59 (29.1%) received ID-Ara-C induction. Early death was recorded in seven (3.4%) after one or two cycles of induction. Focusing analysis on the NPM1mut /FLT3-ITD(-) subgroup, independent factors showing inferior outcome were presence of TET2 mutation [cCR rate, OR=12.82 (95%CI 1.93-85.28), p=0.008; EFS, HR=2.92 (95%CI 1.46-5.86), p=0.003], increasing age [EFS, HR=1.49 (95%CI 1.10-2.02), p=0.012 by every 10-years elevation], white blood cell count ≥60 × 109 /L [EFS, HR=3.30 (95%CI 1.63-6.70), p=0.001], and ≥4 mutated genes at initial diagnosis [OS, HR=5.54 (95%CI 1.77-17.33), p=0.003]. In contrast, when focusing on the NPM1mut /FLT3-ITD(+) subgroup, factors showing superior outcome were ID-Ara-C induction [cCR rate, OR=0.20 (95%CI 0.05-0.81), p=0.025; EFS, HR=0.27 (95%CI 0.13-0.60), p=0.001] and allo-transplantation [OS, HR=0.45 (95%CI 0.21-0.94), p=0.033]. Factors showing inferior outcome included CD34(+) [cCR rate, OR=6.22 (95%CI 1.86-20.77), p=0.003; EFS, HR=2.01 (95%CI 1.12-3.61), p=0.020] and ≥5 mutated genes [OS, HR=2.85 (95%CI 1.33-6.10), p=0.007]. We conclude that TET2(+) , age, and white blood cell count convey an outcome risk modulation for AML with NPM1mut /FLT3-ITD(-) , as does CD34 and ID-Ara-C induction for NPM1mut /FLT3-ITD(+) . The findings permit re-stratification of NPM1mut AML into distinct prognostic subsets to guide risk-adapted individualized treatment.

LSC17 Score Is Complementary with Minimal Residual Disease to Stratify NPM1-Mutated Acute Myeloid Leukemia: An ALFA Study

Context. A 17-gene Leukemic Stem Cell gene expression signature (LSC17) has been shown to be prognostic in AML (Ng et al., 2016), independently of genetic risk stratification (Bill et al., 2020; Duployez et al., 2019). Whether assessment of LSC17 adds prognostic value on top of measurable residual disease (MRD) has not been explored. Objective. We analyzed the prognostic value of LSC17 in adult AML patients prospectively treated in the intensive chemotherapy ALFA-0702 trial (NCT00932412) in the context of ELN 2017 risk stratification (ELN17) and NPM1 MRD. Methods. The LSC17 score was determined by Nanostring technology (Duployez et al., 2019). NPM1 MRD negativity was defined as a 4-log reduction in peripheral blood NPM1c expression (Balsat et al., 2017; Lesieur et al., 2021). Multiple testing was adjusted for using the Benjamini-hochberg procedure. Results. The study population included 504 AML patients (237 female, 267 male) with a median age of 48 years (range 18-60). One hundred eighty-seven (37%) patients had an NPM1 mutation. ELN17 risk was favorable in 176 (35%), intermediate in 151 (30%) and adverse in 170 (34%) patients. A higher LSC17 score was positively associated with RUNX1 (median difference [md]=0.13, corrected p-value [q]=0.0005) and negatively correlated with NPM1 mutation (md=-0.14, q<0.0001) and double-mutated CEBPA (md=-0.30, q<0.0001). Regarding recurrent cytogenetic lesions, presence of monosomy 7 or del7q (md=0.29, q<0.0001), monosomy 5 or del5q (md=0.25, q=0.0004), monosomy 17, del17p or abnormal 17p (md=0.24, q=0.01), monosomal karyotype (md=0.17, q=0.02) and complex karyotype (md=0.13, q=0.03) were each associated with higher LSC17, whereas normal karyotype was associated with a lower LSC17 score (md=-0.11, q<0.0001) (Figure 1A). In the subgroup of NPM1-mutated AML (n=187), a higher LSC17 score was only correlated with FLT3-ITD (md=0.13, q=0.0002) but not DNMT3A (md=-0.0007, q=1) mutations. Patients were dichotomized into LSC17-low (n=252) and LSC17-high (n=252) groups using the median LSC17 score as cutoff. Overall, 400 (79%) patients achieved complete response (CR) or CR with incomplete platelet recovery (CRp) after one cycle of induction and an additional 31 (6%) after a second course. LSC17-high patients had a lower rate of CR/CRp after one course of induction (69% vs 89% in LSC17-low patients) in univariate (p<0.0001) or multivariate analysis (OR=0.41, p=0.001) considering ELN17 risk, age and white blood cell (WBC) count. LSC17-high patients had lower 3-year overall survival (OS) from CR/CRp (60.6%, 95% CI, 54.2-67.8%) than LSC17-low patients (75.4%, 95% CI, 70.0-81.2%, p=0.0003). In multivariate analysis considering ELN17, age and WBC, LSC17-high population had shorter DFS (HR=1.39, p=0.038) and a trend for shorter OS from CR/CRp (HR=1.40, p=0.069). In univariate analyses, LSC17-high status was associated with a higher cumulative incidence of relapse (p=0.002) without impact on non-relapse mortality (p=0.27). Considering hematopoietic cell transplantation (allo-HCT) as a time-dependent covariate, there was no interaction on OS from CR/CRp between allo-HCT in first CR and LSC17-high status (p=0.84). In NPM1-mutated patients (n=187), the rate of CR/CRp after one induction course was 90% in LSC17-high patients versus 96% in LSC17-low patients (p=0.20). In 123 NPM1-mutated patients in CR after the first induction course and with available MRD, LSC17-high status (n=42) predicted a poorer DFS (HR:2.47, p=0.005) in multivariate analysis independently of age (p=0.55), higher WBC (p=0.84), intermediate ELN17 risk (p=0.97) and MRD positivity (p=0.0002). LSC17-high status also independently predicted poorer OS from CR/CRp (HR:2.73, p=0.007) in a similar multivariate model. Three-year OS from CR was 93.1% (95% CI, 86.8-99.9%) in the 59 (48%) NPM1-mutated LSC17-low patients achieving negative MRD compared to 60.7% (95% CI, 49.8-74.0%) in those (n=64, 52%) with LSC17-high status and/or positive MRD (p=0.0001) (Figure 1B). Conclusion. The LSC17 score complements genetic risk stratification in AML patients treated intensively. Combining LSC17 and MRD assessment identifies a subset of NPM1-mutated AML with excellent long-term prognosis. 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

Dexaml-02 : A Phase II Study of Dexamethasone Added to Induction and Postremission Therapy in Older Patients with Newly Diagnosed AML. a French Innovative Leukemia Organization (FILO) Study

Background In a recent retrospective study, we have showed that the addition of dexamethasone to intensive chemotherapy was significantly associated with a better outcome in AML patients with hyperleukocytosis at diagnosis (Bertoli S, Haematologica 2018). Moreover, preclinical studies demonstrated that specific AML oncogenic alterations (RUNX1-RUNX1T1, RUNX1, NPM1, or SRSF2 mutations as well as NF-kB activation in leukemic stem cells) or therapeutic stress (cytarabine, FLT3 inhibitors) are associated with up-regulation of the inflammatory gene response and expression of the glucocorticoid receptor which induce sensitization to glucocorticoids (Récher C, Front Oncol 2021). Therefore, dexamethasone, used as a chemo sensitizer in combination with intensive chemotherapy, may improve treatment response and outcome regardless of the white blood cell count. Methods DEXAML-02 (LAM-SA 2018) is a non-randomized phase II multicenter trial to assess the impact on outcome of dexamethasone added to induction chemotherapy in older patients with AML (NCT03609060). Key inclusion criteria were: > 60 years of age, newly diagnosed AML (either de novo or therapy-related AML), favorable or intermediate cytogenetic risk according to MRC 2010 classification, ECOG performance status < 3; HCT-CI score < 3, adequate baseline organ function. Key exclusion criteria were: AML with adverse cytogenetic risk, AML arising from MDS, MPN or CMML, AML with BCR-ABL1, known active central nervous system leukemia. Induction chemotherapy regimen consisted in one cycle of idarubicin 8 mg/m²/day, D1 to D5, cytarabine 100 mg/m²/d, CIV D1 to D7, lomustine 200 mg/m²/d, orally at D1 and dexamethasone 10 mg/12h, IV, D1 to D3. The use of G-CSF, anti-fungal, anti-viral and anti-pneumocystis jiroveci prophylaxis were recommended. Patients who failed to reach complete remission (CR/CRi) after this single cycle of induction were off-study whereas CR/CRi patients were planned to receive up to 6 courses of mini-consolidation administrated every 30 to 45 days with idarubicin 8 mg/m², D1, cytarabine 50 mg/m²/12h, subcutaneous, D1 to D5, dexamethasone 20 mg/d, D1. The addition of midostaurin in patients with FLT3-ITD or FLT3-TKD mutations was allowed during induction, consolidation and maintenance. Allogeneic stem-cell transplantation was allowed after 2 to 4 cycles in patients with intermediate or adverse risk according to ELN 2017 classification. Patients with CBF-AML could receive 2-3 cycles of intermediate dose cytarabine (with dexamethasone) instead of 6 cycles of mini-consolidations according to investigator choice. Results From August 2018 to February 2020, 120 patients were included. 75 patients were male (62.5%). Median age was 70y (min-max, 61-80). Median WBC was 4.9 G/L (0.64-137). Most patients had an intermediate cytogenetic risk (91.7%). NPM1 and FLT3 mutations (per local analysis) were observed in 45 (37.5%) and 33 patients (27.5%), respectively. NGS of a 91 myeloid panel genes was centrally performed in 111 patients. 24 patients received midostaurin. Following the induction cycle, 100 patients (83.3%) achieved CR/CRi, 14 failed (11.7%) and 6 died early (5 %). In NPM1mut patients, 43 achieved CR/CRi (95.6%). During induction, grade III-IV adverse events were: infections (50.8%), mucositis (16.7%), gastro-intestinal (14.2%), hepatic (11.7%), hyperglycemia (10.8%), pulmonary (7.5%), cardiac (5.8%), cutaneous (5%) or bleedings (5%). Of the 100 CR/CRi patients, 86 entered the consolidation phase of whom 59 and 38 received 3 and 6 cycles, respectively. 23 patients received an allogeneic-SCT in first CR. The median follow up time was 31.4 months. Median RFS and OS were 20.2 and 30.7 months, respectively. In NPM1mut patients, median RFS was 20.1 months while median OS was not reached. Conclusion In older AML patients who are eligible for intensive treatment, adding dexamethasone to induction and consolidation chemotherapy is feasible and associated with a high response rate after a single induction cycle and encouraging overall survival. A historical comparison with the patient population of the LAM-SA 2007 trial (Pigneux A, JCO 2018) will be presented during the meeting to highlight a potential signal of activity. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Proteomic Characterization of Acute Myeloid Leukemia

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

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

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

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