ITD Mutations Research Articles

Measurable residual disease monitoring in AML with FLT3-ITD treated with intensive chemotherapy plus midostaurin

AbstractMeasurable residual disease (MRD) monitoring in acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITDpos]) has been hampered by the broad heterogeneity of ITD mutations. Using our recently developed FLT3-ITD paired-end next-generation sequencing (NGS)–based MRD assay with a limit of detection of 10−4 to 10−5, we evaluated the prognostic impact of MRD at different time points in 157 patients with FLT3-ITDpos AML who were enrolled in the AMLSG16-10 trial and who were treated with a combination of intensive chemotherapy and midostaurin, followed by midostaurin maintenance. Achievement of MRD negativity (MRDneg) after 2 cycles of chemotherapy (Cy2), which was observed in 111 of 142 (78%) patients, was predictive of superior 4-year rates of cumulative incidence of relapse (CIR) (4y-CIR; 26% vs 46%; P = .001) and overall survival (OS) (4y-OS; 70% vs 44%; P = .012). This survival advantage was also seen among patients who underwent allogeneic hematopoietic-cell transplantation during first complete remission (4y-CIR, 14% vs 39%; P = .001; 4y-OS, 71% vs 49%; P = .029). Multivariate models for CIR and OS after Cy2 revealed FLT3-ITD MRDneg as the only consistent favorable variable for CIR (hazard ratio [HR], 0.29; P = .006) and OS (HR, 0.39; P = .018). An NPM1 co-mutation correlated with a deeper molecular response as reflected by stronger MRD reduction and a higher rate of FLT3-ITD MRDneg after Cy2. During follow-up, conversion from MRDneg to MRDpos was a strong, independent factor for inferior CIR (HR, 16.64; P < .001) and OS (HR, 4.05; P < .001). NGS-based FLT3-ITD MRD monitoring allows for the identification of patients at high risk for relapse and death following treatment with intensive chemotherapy and midostaurin. Using NGS-based technology, FLT3-ITD emerges as a novel, highly clinically relevant target for MRD monitoring. This trial was registered at www.ClinicalTrials.gov as #NCT01477606.

Prognostic impact of 'multi-hit' versus 'single hit' TP53 alteration in patients with acute myeloid leukemia: results from the Consortium on Myeloid Malignancies and Neoplastic Diseases.

While there is clear evidence to suggest poorer outcome associated with multi-hit (MH) TP53 mutation compared to single-hit (SH) in lower-risk myelodysplastic syndrome (MDS), data are conflicting in both higher-risk MDS and acute myeloid leukemia (AML). We conducted an indepth analysis utilizing data from 10 US academic institutions to study differences in molecular characteristics and outcomes of SH (n= 139) versus MH (n= 243) TP53MTAML. Complex cytogenetics (CG) were more common in MH compared to SH TP53MT AML (p <0.001); whereas ASXL1 (p= <0.001), RAS (p<0.001), splicing factor (p= 0.003), IDH1/2 (p= 0.001), FLT3 ITD (p= <0.001) and NPM1 (p= 0.005) mutations significantly clustered with SH TP53MT AML. Survival after excluding patients who received best supportive care alone was dismal but not significantly different between SH and MH (event free survival [EFS]: 3.0 vs 2.20 months, p= 0.22/ overall survival [OS]: 8.50 vs 7.53 months, respectively, p= 0.13). In multivariable analysis, IDH1 mutation and allogeneic hematopoietic stem cell transplantation (allo-HCT) as a time-dependent covariate were associated with superior EFS (HR; 0.44, 95% CI: 0.19-1.01, p= 0.05/ HR; 0.34, 95% CI: 0.18-0.62, p<0.001) and OS (HR; 0.24, 95% CI: 0.08-0.71, p= 0.01/ HR; 0.28, 95% CI: 0.16-0.47, p<0.001). While complex CG (HR; 1.56, 95% CI: 1.01-2.40, p= 0.04) retained unfavorable significance for OS. Our analysis suggests that unlike in MDS, multihit TP53MT is less relevant in independently predicting outcomes in patients with AML.

FLT3-ITD regulation of the endoplasmic reticulum functions in acute myeloid leukemia.

The FLT3-ITD mutation represents the most frequent genetic alteration in newly diagnosed acute myeloid leukemia (AML) patient and is associated with poor prognosis. Mutation result in the retention of a constitutively active form of this receptor in the endoplasmic reticulum (ER) and the subsequent modification of its downstream effectors. Here, we assessed the impact of such retention on ER homeostasis and found that mutant cells present lower levels of ER stress due to the overexpression of ERO1α, one of the main proteins ofthe protein folding machinery at the ER. Overexpression of ERO1α resulted essential for ITD mutant cells survival and chemoresistance and also played a crucial role in shaping the type of glucose metabolism in AML cells, being the mitochondrial pathway the predominant one in those with a higher ER stress (non-mutated cells) and the glycolytic pathway the predominant one in those with lower ER stress (mutated cells). Our data indicate that FLT3 mutational status dictates the route for glucose metabolism in an ERO1α depending on manner and this provides a survival advantage to tumors carrying these ITD mutations.

Abstract 3626: Day 21 early bone marrow response does not offer high concordance with response bone marrow in the induction chemotherapy setting of FLT3 mutant acute myeloid leukemia

Abstract Purpose: Day 14 early bone marrow biopsies are routinely initiated to determine need for reinduction chemotherapy, but poorly predict true response and risk overtreatment. Day 21 (D21) bone marrow biopsies have been performed in the context of trials utilizing FLT3-targeted therapies in combination with induction chemotherapy (Stone et al. NEJM 2017). However, the utility of D21 bone marrow responses is not well defined. Methods: We retrospectively identified 26 newly diagnosed FLT3 mutant AML patients who underwent induction chemotherapy with midostaurin and Next Generation Sequencing (NGS) of bone marrow aspirate within a week of diagnosis. Results: 19/26 (73%) of patients had both D21 and response bone marrow biopsy. Of the remaining 7 patients, 4 patients did not have D21 bone marrow, 2 died of unrelated complications prior to response bone marrow, and 1 did not have recovery marrow available for assessment. Our cohort included 13 (50%) men with a median age of 57 years (range, 21 - 73 years). All patients had ECOG of 0 - 1. 96% of cases were de novo AML and 81% had normal cytogenetics. FLT3 ITD mutations were detected in 17 (65%) patients with a median FLT3 ITD ratio of 0.305 (range, 0.03 - 1) and median FLT3 VAF of 23.8% (range, 3.2 - 80.6%). 7 patients (26.9%) underwent additional allogeneic stem cell transplant. In the 19 patients evaluable for analysis, 13/13 (100%) patients with blast % ≤ 5 at D21 achieved a complete response (CR) based on recovery marrow. 4/6 (67%) patients with blast % &amp;gt; 5 at D21 which were considered to have residual disease based on pathology review (median blast % 11 (range, 6 - 17%) ultimately achieved a CR. The 2 patients that did not achieve a CR has a blast % of 29% and 9% on D21 marrow. Thus, the positive predictive value of D21 early bone marrow response corresponding with the response bone marrow was only 33% in our patient cohort. Of the 4 patients that did not have D21 marrow but had recovery marrows available for review, the CR rate was 100%. No other significant differences in initial disease characteristics were seen. There were no differences proportion of co-mutations in NPM1, DNMT3A, TP53, IDH2, and IDH1, and higher FLT3 ITD ratio nor FLT3 VAF corresponded with ability to clear D21 bone marrow. Additionally, there were no differences in minimal residual disease (MRD) and overall survival (OS) between the groups. Conclusions: Our study suggests early bone marrow response in the context of FLT3-mutant disease is not dependent on inherent disease characteristics and does not correspond to overall response to targeted induction chemotherapy. The utility of these marrows in is this population needs to be studied further in a larger cohort to better understand the clinical necessity to avoid unnecessary cost/procedures to patients and even more importantly unnecessary reinductions. Citation Format: Vincent Lok, Su Bin Hahn, Nikesh N. Shah, Daniel A. Kerr, Ivan M. Borrello, Eduardo M. Sotomayor, David M. Swoboda. Day 21 early bone marrow response does not offer high concordance with response bone marrow in the induction chemotherapy setting of FLT3 mutant acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3626.

Unpredicted transformation of acute myeloid leukemia with translocation (16;16) (p13; q22): a case report and review of the literature

IntroductionThe transformation of acute myeloid leukemia with translocation (16;16) (p13; q22) from AML M2 to acute monocytic leukemia (AML M5) during therapy is a rare clinical occurrence, and this is the first time it has been reported.Clinical complainA 19-year-old male patient was admitted for severe fatigue with anemic manifestation and weight loss, for more than 1 month, with exacerbation of the condition in the last 2 days.DiagnosisA primary diagnosis was made for AML M2 with t (16;16) (p13; q22) established on bone marrow (BM) morphology. A consequential detection of FLT-3 ITD mutation was done. At day 28 follow-up after induction and maintenance therapy, the diagnosis of AML M2 was maintained with a high bone marrow (BM) blast count, prompting the initiation of a more aggressive treatment protocol. After 1 month of implementing the recent protocol, the patient remains morphologically resistant with a notable transformation of bone marrow infiltration by an abnormal monocytic population (monoblasts and promonocytes). The final diagnosis of transforming FLT3-mutated AML with t (16;16) (p13; q22) was established.InterventionAfter the initial diagnosis of AML M2 with t (16;16) (p13; q22), the patient received the 3 + 7 induction protocol. The 2nd induction protocol initiated after the second evaluation and morphological resistance was the FLAG Adrian protocol. The 3rd protocol after transformation to AML M5 was 1 cycle of the MEC protocol. Anti-FLT3 treatment was considered.OutcomesThe patient was maintained on the 3rd protocol of chemotherapy. Unfortunately, he was admitted to the ICU unit complaining of neutropenic fever and severe sepsis where he died before final re-evaluation and the anti-FLT3 treatment initiation.ConclusionAML with t (16;16) (p13; q22) characterized by favorable outcome. However, identifying additional chromosome abnormality or genetic aberration, especially FLT3 gene mutation, is recognized as an important factor influencing final disease outcome. Therefore, early detection of FLT3 mutations will allow comprehensive disease course prediction and targeted therapy that might achieve longer and more durable remissions.

Quantum-First Trial: FMS-like Tyrosine Kinase 3-Internal Tandem Duplication ( FLT3-ITD)-Specific Measurable Residual Disease (MRD) Clearance Assessed through Induction (IND) and Consolidation (CONS) Is Associated with Improved Overall Survival (OS) in Newly Diagnosed (nd) FLT3-ITD+ AML Patients (pts)

Background Detection of MRD by flow cytometry or gene fusion transcript quantitation increasingly is used to guide treatment decisions in AML. Molecular monitoring of recurrent gene mutations for MRD detection is an alternative strategy but, other than for nucleophosmin 1 ( NPM1) gene quantitation, remains controversial. FLT3-ITD is among the most common recurrent mutations in AML and confers a worse prognosis vs FLT3-wild-type and FLT3-TKD+ AML. Routine use of FLT3-ITD detection in remission as a predictor of relapse risk or OS has been limited by the low sensitivity of both conventional PCR-based detection methods and broad NGS platforms in clinical practice settings. More recently, FLT3-ITD-specific PCR-NGS assays such as getITD (PMID: 31089248) show greater sensitivity and ease of interpretation that point to eventual routine clinical application. However, the clinical value of these measurements has not been evaluated prospectively in large scale randomized controlled trials of FLT3 inhibitors. The phase 3 QuANTUM-First study (NCT02668653) evaluated the novel, potent, and highly selective type II FLT3 inhibitor quizartinib (Quiz) in nd FLT3-ITD+ AML pts and demonstrated that Quiz added to intensive IND and CONS, ± transplant, followed by single-agent continuation (CONT) therapy (Tx) resulted in a significant improvement in OS (PMID: 37116523). We analyzed if FLT3-ITD-specific MRD in QuANTUM-First pts impacted the clinical outcome or the benefits provided by Quiz in nd FLT3-ITD+ AML pts. Methods Genomic DNA, isolated from bone marrow aspirates or peripheral blood from pts after achievement of remission after 1 or 2 courses of IND and at end of CONS (prior to transplant or CONT cycle 1 day 1 [C1D1] for transplant pts and prior to CONT C1D1 for non-transplant pts), was analyzed with a FLT3-ITD PCR-NGS assay specifically developed for this trial (PMID: 31722002). ITD mutations detected after IND were cross-validated against the ITD detected at enrollment for each patient. Using a custom bioinformatics program, ITD mutations were identified, and variant allele frequencies (VAFs) were calculated with a sensitivity of 10 −5. MRD was classified as undetectable (using the 0 cutoff) or detectable above or below a 10 −4 predefined cutoff (based on lower limit of quantification for the assay). Comparisons of complete response (CR), composite complete response (CRc=CR+CRi), and the rate of pts achieving CRc during IND with no MRD between arms were made using a stratified Cochran-Mantel-Haenszel test. Comparison of FLT3-ITD VAF during IND and during CONS between arms was made using Wilcoxon rank sum test. All P-values were not adjusted for multiplicity. Results In QuANTUM-First, 539 nd FLT3-ITD+ AML pts were randomized to Quiz (n=268) or placebo (PBO; n=271). Of the 539 randomized pts, 368 (68.3%) achieved CRc after 1 or 2 courses of IND, and MRD analysis was performed on 321 (87.2%) of these pts (162 pts in Quiz and 159 pts in PBO) using samples collected at the time of response assessment during IND, before further Tx. MRD was also assessed in 337 pts (172 pts in Quiz and 165 pts in PBO) by end of up to 4 cycles of CONS prior to CONT/maintenance Tx. Of these pts, 166 additionally received transplant (87 in Quiz, 79 in PBO). The % of pts in CRc at end of IND with FLT3-ITD MRD of &amp;lt;10 −4 was similar between study arms (25.4% Quiz vs 21.8% PBO, nominal P = 0.3430), however, a greater % of pts in CRc had undetectable MRD with Quiz than PBO (12.3% vs 7.0%, nominal P = 0.0403). Among pts with CRc at end of IND, the median best FLT3-ITD VAF by end of CONS was lower (0% versus 0.0017%; nominal P = 0.0006) with Quiz vs PBO (Fig 1) and, using the 0 cutoff at end of IND, a longer OS was observed with Quiz vs PBO, regardless of MRD status, with a greater effect with Quiz (HR, 0.789 in MRD− pts; HR, 0.749 in MRD+ pts; Fig 2). In MRD+ pts, the median OS was not reached with Quiz and 35.4 months with PBO (Fig 2). Performing this analysis with an MRD cutoff of 10 ‒4 yielded similar findings (HR, 0.696 in MRD− pts; HR, 0.800 in MRD+ pts). Additional data about MRD impact on pts ± transplant in this trial will be presented. Conclusions These findings demonstrate the potential prognostic utility of FLT3-ITD-specific MRD measurements in the clinical management of pts with FLT3-ITD+ AML. Our data suggest that long-term OS benefits conferred by Quiz in part derive from a deep and sustained reduction of the FLT3-ITD+ leukemia burden.

Evaluation of Neddylation and Apoptosis-Related Gene Expression in Patients with Acute Myeloid Leukemia

Introduction Regulated cell death (RCD) is a process commonly dysregulated in patients (pts) with acute myeloid leukemia (AML), resulting in the survival of genetically unstable cells. Apoptosis, as the best-known form of RCD, interacts with neddylation, a process that contributes to tumorigenesis and apoptosis resistance. Aims This study was conducted to determine the expression of the selected genes involved in the neddylation pathway and to assess their clinical importance along with selected interacting proapoptotic genes. In addition, we correlated the expression levels of these genes with known prognostic factors in AML and determined their impact on the survival of AML pts. Methods The expression of selected proapoptotic genes BIK, BAX, BAK, BCL2L11, BBC3 , PMAIP1 CASP3, CASP7, and neddylation-related genes: CUL1, CUL2 , CUL4A, CUL5, CUL7, CUL9, NEDD8 was determined in duplicates in bone marrow samples of newly diagnosed AML pts. Real-Time PCR was carried out using the TaqMan chemistry and the QuantStudio7 thermal cycler (Applied Biosystems-Thermo Fisher Scientific). Gene expression was normalized using reference genes selected by the Normirazor tool. The normalization was performed by ∆Ct = Ct (reference) - Ct (mRNA of interest). This approach results in higher values for higher mRNA expression. Differential expression analysis was performed using a Welch t-test. Kaplan-Meier method with the log-rank test was used to assess overall survival (OS). Results In a prospective study, we included 40 newly diagnosed AML pts (mean age 62; range 26-87) with a median (m) follow-up of 6.7 months (95% CI: 4.5-9.1). High-risk AML was present in 52.5%, intermediate-risk in 32.5%, and low-risk in 15% of pts, according to European LeukemiaNet (ELN) 2022 risk stratification. NPM1 mutation was present in 23%, whereas FLT3 ITD or TKD mutation in 25% of pts. Intensive treatment was introduced in 43% of pts, while 25% were treated with azacitidine+venetoclax. Non-intensive treatment with azacitidine in monotherapy was administered to the remaining pts. BAK expression was detected in 70%, BIK in 98%, while expression of the other genes was found in all pts. In the high-risk group, we detected lower expression of both proapoptotic BAX (fold change - FC=0.7, p=0.01) and BIK gene (FC=0.4, p=0.01), as well as lower expression of CUL9 (FC=0.7, p=0.03). Accordingly, in pts with NPM1 mutation we revealed upregulation of BIK (FC=6.1, p=0.001), and CUL9 (FC=1.6, p=0.01). Whereas pts with FLT3 mutation had lower expression of proapoptotic BBC3 (FC=0.5, p=0.01), and neddylation-related CUL1 (FC=0.6, p=0.01). In cases of initial bone marrow blasts infiltration above median value (&amp;gt;49%) BCL2L11 CUL5 , NEDD8 and PMAIP1were downregulated ( p=0.001-0.02), while CUL7 ( p=0.00) and CASP3 ( p=0.04) were upregulated. Patients with initial white blood cell count &amp;gt;20 G/l had lower expression of BCL2L11 ( p=0.005), and CUL4A ( p=0.02), while higher expression of CASP7 ( p=0.04). The mOS in the study cohort was not reached. OS was longer in pts with higher (&amp;gt; -2.061, cutoff was determined with CutOff Finder) BAX expression (log-rank: p=0.03) [Figure 1]. The mOS was not reached in this group, whereas the mOS in pts with lower BAX expression was 1.3 months (95% CI: 0.6-1.3). In univariate Cox regression analysis, higher BAX expression (HR 0.31, 95%CI: 0.11-0.84, p=0.022), higher albumin level (HR 0.13, 95%CI: 0.04-0.41, p=0.0004), and intensive treatment (HR 0.18, 95%CI: 0.04-0.69, p=0.012) were factors influencing the outcome. In the multivariate model for OS, higher expression of BAX (HR 0.03, 95%CI: 0.003-0.33, p=0.004) retained its significant protective effect in the context of established prognostic factors [Table 1]. Conclusions To our knowledge, we were the first to evaluate such a complex spectrum of neddylation and apoptosis-related genes in AML. We revealed dysregulation of these genes in AML. Our data indicate that upregulation of BAX at the diagnosis is associated with longer OS. Moreover, we reported significantly lower BAX expression in ELN high-risk group. Thus, indicating BAX may be a potential prognostic factor in AML. Our preliminary results did not prove significant differences in the effect of neddylation gene expression levels on the prognosis of AML patients. As studies are emerging on the potential of neddylation-targeted therapies, we are conducting further research to verify the effect of neddylation on AML.

Optimizing FLT3 Inhibitor Use in Adult Acute Myeloid Leukemia with FLT3 Mutations Using Proteomics

CONCLUSION FLT3 mutations are the most frequent genetic alteration in adult Acute Myeloid Leukemia (AML). The use of FLT3 inhibitors (FLT3I) has improved outcome among FLT3 mutant patients, but not all benefit. We developed a proteomic-based approach to stratify patients according to their response to FLT3I therapy. The expression of 429 proteins (339 total, 90 post-translational modified) was measured by Reverse Phase Protein Arrays (RPPA) in fresh samples from 805 newly diagnosed AML patients, with levels normalized to normal CD34+ cells. Of 137 with FLT3 ITD or D835 mutations, 54 who were treated with FLT3I (Sorafenib=22, Quizartinib=11, Midostaurine=6, Gilteritinib=4 and Crenolanib=1) combined with other agents (Hypomethylating agents (HMA)=23, HMA+Venetoclax (VTX)=5, Idarrubicin+Cytarabine (AraC)+Purine Analogs (IA+Pur)=19, IA+Pur+VTX=2, AraC=3, Fludarabine+AraC (FA)=1, none=1) were selected for analyses. Proteins prognostic for Overall Survival (OS) and Complete Remission Duration (CRD) were identified using pairwise LogRank tests (p &amp;lt; 0.05) adjusted for False Discovery Rate, followed by unbiased hierarchical clustering. Continuous and categorial variables were compared with Wilcoxon, Kruskal-Wallis or Fisher's Exact test with simulated p-values (10000 replicates). Cox proportional hazards models (CoxPH) were developed for Uni-(UV) and Multi-variate (MV) analysis. Deep neural network analysis using random forest models identified proteins most predictive of cluster assignment. 28 proteins were individually prognostic for both OS and CRD and divided patients into 3 clusters (Fig. 1: C1=red, C2=blue, C3=green). The distribution of nearly all other clinical and molecular features did not differ between clusters, including: age, gender, race, cytogenetic risk and AML group. Co-occurrence with other mutations was not biased, except for FLT3+TET2 and FLT3+TET2+NPM1 mutants, more frequent in C1. OS was significantly longer (Fig. 2), in C1 (Median: C1&amp;gt;84mo, C2=14.8mo, C3=11.4mo; p&amp;lt;0.001) as was CRD (Median: C1&amp;gt;84mo, C2=8.6mo, C3=5.7mo; p&amp;lt;0.05). A similar OS pattern was observed for mutation combos, including with NPM1 or DNMT3a but not if IDH, CEBPa, TET2, or ASXL1 mutation were present. It was also seen when patients were divided by gender, age groups (except &amp;gt;70yo), primary, but not secondary AML, diploid karyotype and intermediary cytogenetic risk, and with Sorafenib or IA+PR therapies. In the UV CoxPH model for OS, C1, C2 and C3 were predictive (HR=1, 4.1, 7.5; p=ref., 0.037, 0.009), along with age, secondary AML, complex karyotype, Trisomy 6 and IDH1 mutation. In the MV model for OS, only C1, C2 and C3 (HR=1, 3.3, 4.8; p=ref., 0.011, &amp;lt;0.001), and age were predictive. In the CRD UV model, C1 and C3 (HR=1, 3.3, 4.8; p=ref., 0.072, 0.022), age, secondary AML, and mutations in ASLX1, PTPN11, RAS, TP53 and WT1 were predictive. Only C1 and C3 were predictive in the MV model for CRD (HR=1, 2.9, 6.3; p=ref., 0.16, 0.04). The predictive proteins, are biased towards specific cellular processes, mainly: proliferation and metabolism (RHEB, GSKA_B, SDHA, AKT1, AKT2 and TYRO3), cell cycle and DNA damage response (CDKN2A, DDB1, SIRT6, SLFN11 and WEE1), cellular adhesion and cytoskeleton organization (MYH9.pS1943, PAK4 and PXN), and apoptosis (AIFM1, BBC3 and BCL2L11). Key differences between clusters included: higher FOS in C1, and higher CDKN2A, MYH9.pS1943, PXN, PAK4, SDHA, BBC3 in both C2 and C3, with higher levels of these in C3. Since PAK4, PXN and MYH9 are connected to cellular motility/migration and inhibitors for those proteins are available, there is a therapeutic opportunity to overcome FLT3I resistance in C2/C3 patients. Moreover, we identified a set of 6 proteins (FOS, AKT1, PXN, SMARCA2, CDKN2A and DPF2) that predicted cluster assignment with 97.5% accuracy, making rapid patient stratification possible in the clinical setting. Protein profiling identified cohorts of FLT3 mutant patients that achieved the highest benefit from FLT3I addition, regardless of all other clinical or molecular characteristics. We identified targetable proteins that are elevated in adverse signatures, and hypothesized that these may be targets for modulation to improve FLT3I response. Moreover, a small set of 6 proteins accurately classifies patients and could be used clinically to triage patients for treatment recommendation.

Should the European Leukemia Net 2022 Adverse Risk Classification of NPM1 mutated Acute Myeloid Leukemia be Revisited? Among Adverse Risk Cytogenetics, Solely Complex Karyotype Is Associated with Worse Post-Transplant Survival: A Study from the EBMT Acute Leukemia Working Party

Introduction The European LeukemiaNet (ELN) 2022 risk classification has identified acute myeloid leukemia (AML) with Nucleophosmin 1 (NPM1) mutation as a distinct entity associated with a favorable prognosis in those with normal cytogenetics (CG) and absent FLT3 ITD. Furthermore, the ELN 2022 reclassified AML patients with mutated NPM1 and high risk CG as adverse risk (AR) regardless of their FLT3 ITD status. We performed this analysis to evaluate the impact of the different AR CG aberrations on post-transplant outcomes in that setting. Methods: In this retrospective registry based analysis from the EBMT, we included adult patients (≥ 18 years) with NPM1 mutated de novo AML, intermediate or AR CG, and known FLT3 ITD mutation status, who received their first allogeneic hematopoietic cell transplantation (HCT) in first complete remission between 2005 and 2021. Multivariate analysis adjusting for differences between the groups (according to CG) was performed using a Cox proportional hazards regression model. Results: We identified a total of 3275 patients (54% females). FLT3 ITD was present in 2377 patients (73%); significantly higher in those with normal CG (75% versus 57%). Normal CG was seen in 2782 patients (85%), while CG aberrations were noted in 493 patients, the most common of which was trisomy 8 (141 patients). Adverse risk CG were seen in 160 patients (4.9%) including complex karyotype (CK) in 72 patients and monosomal karyotype (MK) in 66 patients. The median age was 54 [IQR 45-62]. No significant difference was noted between those with normal and those with abnormal CG with regards to baseline patient and transplant characteristics: HCT comorbidity index (CI) was £2 in 79% of patients in both groups, minimal measurable disease at transplant was positive in 42% and 44%, donor type was matched sibling (MSD) in 32% and 34%, unrelated (UD) in 57% and 58%, or haploidentical in 11% and 9%, stem cell source was peripheral blood in 89% and 89%, conditioning was myeloablative in 52% and 49% respectively. The median follow up for alive patients was 35 months (IQR 33-36). During this follow up a total of 961 patients died (48.3% due to disease relapse). In univariate analysis, post-transplant outcomes were not significantly affected when comparing normal to abnormal CG, or intermediate versus adverse risk CG. Patients with a normal karyotype had a 2-year relapse rate of 23%, NRM of 13%, LFS 63%, OS of 71%, and GRFS of 51%. Conversely, patients with aberrant CG had a 2-year relapse rate of 27%, NRM 12%, LFS 61%, OS of 68%, and GRFS of 48%. On multivariate analysis, CK as well as FLT3 ITD was associated with a worse OS with HR of 1.7 (95% CI 1.13-2.56) and 1.18 (95% CI 1.01-1.39), respectively. When analyzed by subgroup by univariate analysis according to FLT3 ITD status, the impact of CK was maintained for those with FLT3 wild type: 2-year LFS 39.6% vs. 70.5%; p=0.006, and 2-year OS 49% vs. 75.3%, p=0.012. Similarly for patients with FLT3 ITD, CK was associated with a higher risk of relapse (45.7% vs. 26.5%, p=0.021), and worse OS (52.7% vs. 67.4%; p=0.047) with no significant effect on other outcomes. Within patients with chromosomal aberrations, CK was the only significant predictor of worse LFS (HR 1.58 [1.06-2.34]), and OS (HR 1.85 [1.22-2.82]). FLT3 ITD mutation status, CG risk category, as well as the various CG abnormality subgroups had no significant effects (Notably, MK was not associated with a significant impact on LFS [60.9% versus 61.4%; p-value 0.7], OS [67.7% versus 68.2%; p-value 0.97] or GRFS [51.5% versus 47.8%; p-value 0.61]. Conclusion: These data suggest a need to revisit the ELN 2022 risk stratification of AML patients with mutated NPM1 and CG aberrations. In the transplant setting for patients in first remission, only CK among other AR CG appears to predict worse outcomes, although a significant proportion of patients with CK can still achieve long term post-transplant survival.

The Shifting Prognosis of FLT3 Mutations in Acute Myeloid Leukemia in the Era of Targeted Therapy: A Real-World Study Using Large-Scale Electronic Health Record Data

Background: Mutations in FLT3 occur in 20-30% of acute myeloid leukemia (AML), and their prognostic implications have varied over time with improved understanding of FLT3 (Döhner et al., Blood, 2022). However, since 2017, the US Food and Drug Administration (FDA) has approved three FLT3 inhibitors, and few studies have examined the prognosis of FLT3 mutations in the era of targeted therapy. Additionally, a real-world oncology data analysis suggested that novel therapies may have greater effects than clinical trials showed, due to the inclusion of previously ineligible patients (Liu et al., Nature, 2021). Therefore, we examined the relative prognosis of FLT3-mutated AML before and after the FDA approval date of the first FLT3-inhibitor, midostaurin. Methods: Patients were included if both FLT3-ITD (internal tandem duplication) and FLT3 D835 mutations were tested within 14 days of AML diagnosis at the Stanford Cancer Center. Data came from databases associated with Stanford's electronic health record (EHR), except that overall survival (OS) data was supplemented with external databases like the Social Security Death Index, and diagnosis dates came from the Stanford Cancer Registry. A FLT3 mutation was defined as either an ITD or D835 variant. The FDA approval of midostaurin served as a real-world, unplanned experiment illustrating the impact of FLT3 inhibitors' availability on the prognosis of FLT3-mutated AML. Thus, using a difference-in-differences approach, we built a logistic regression model predicting one-year overall survival using the variables FLT3 mutation, diagnosis before vs. after the midostaurin approval date, and an interaction between those terms. The model was adjusted for patient age at diagnosis, NPM1 mutation presence, and other interaction terms, and for that model, patients who were lost to follow-up before one year were excluded. Cox proportional hazards regression was used to make interpretation comparable to clinical trial hazard ratios, and Kaplan-Meier (KM) curves were used to explore age subgroups. Patients were excluded if they received gilteritinib, sorafenib, or midostaurin but were diagnosed before midostaurin's approval. Results: Between April 2008 and September 2022, 522 patients met inclusion criteria, and 120 (23%) had an ITD mutation only, 22 (4%) had a D835 mutation only, and 8 (2%) had both mutations at diagnosis, and the remainder were negative for FLT3 mutation. FLT3 mutation was not significantly associated with overall survival (p = 0.55), but the interaction between FLT3 and diagnosis after midostaurin's approval was associated with a lower chance of death in multiple logistic regression (Table 1, odds ratio [OR] 0.237, p = 0.0044), implying that a FLT3 mutation had a better prognosis after midostaurin's approval. This trend was present in patients both under age 60 (OR 0.153, p = 0.043; KM curves in Figure 1) and at least age 60 (OR 0.273, p = 0.04). In a Cox regression, the interaction term was similar (HR 0.54, p = 0.021), with a HR that compared favorably to 0.78, the HR seen in phase III clinical trials studying midostaurin or quizartinib at diagnosis (Stone et al., NEJM, 2017; Erba et al., Lancet, 2023). Younger patients with FLT3-mutated AML after midostaurin's approval were more likely to have a record of FLT3 inhibitor prescription (79% under age 60 vs. 56% at least age 60, p = 0.07). Conclusions: The relative prognosis of FLT3 mutations in AML has improved, including in older patients, likely due to the approval of FLT3 inhibitors, and the relative survival is more favorable than what was described in major clinical trials. Gathering real-world data to estimate prognosis of FLT3 mutations is crucial for accurate risk stratification, and this can be done more easily with EHR data.

FLT3-ITD Induces Immune Escape in AML Via up-Regulating CD47 Expression and Decreased Phagocytic Ability of Macrophages

The FLT3-ITD mutation is frequently observed in acute myeloid leukemia (AML) and confer a poor prognosis. FLT3 inhibitors such as Quizartinib could prolong survival. However, very few relapsed patients are cured. Little attention has been paid to the characteristics of the immune microenvironment in FLT3-ITD AML. Within the bone marrow, Flt3/ITD animals demonstrated significantly increased dendritic cells and T cells levels compared to Flt3/WT mice, which suggested that FLT3-ITD can modulate the immune microenvironment in AML. Here, we found that FLT3-ITD cell lines (MV-4-11 and MOLM-13) induced less LDH release compared to FLT3-WT cell lines (HEL and K562) ( Figure a), indicating less macrophage killing. CD47 gene is an integrin related protein, which acts as an “anti phagocytosis” signal, and it interacts with signal-regulatory protein alpha (SIRPalpha) on macrophages, so that tumor cells expressing CD47 can escape being destroyed by macrophages. Further Flow Cytometry results showed that the CD47 Mean fluorescent intensity (MFI) of the FLT3-ITD group was significantly higher than those of the control group ( Figure b). Therefore, we can ask a hypothesis that FLT3-ITD in tumor cells may leads to decreased phagocytic capacity of macrophages and immune tolerance. To test our hypothesis, the FLT3 wildtype (FLT3-OE, FLT3 over expression) or ITD mutant (FLT3-ITD) stable overexpression HEL/K562 cells were constructed by means of lentiviral transfections. In the FLT3-ITD group, the MFI levels of CD47 was higher than FLT3-OE group and FLT3 NC (FLT3 Normal control) group ( Figure c and d). Green-fluorescent (CFSE-stained) Hel and K562 were then added to cultures of macrophages induced by Peripheral Blood Mononuclear Cell stained with the red dye PKH26 (ratio tumor cell:macrophages, 5:1). Flow Cytometry results showed FLT3-ITD decreased the Phagocytic Capacity of Macrophages ( Figure e and f). The TRANSFAC database was used to predict the transcription factor binding sites of the CD47 promoter. We found that HOXB5 may regulate the expression of CD47 at the transcriptional level. Compared with the FLT3-NC and FLT3-OE group, the relative expression level of the CD47 gene in the FLT3-ITD group was significantly upregulated ( Figure g, p &amp;lt; 0.05). To ascertain the mechanism that HOXB5 regulated CD47, we analysed the location CD47 gene on the chromosome, as well as the sequence of promoter and first non-encoding exon of HOXB5 (−2000 ~ 100 bp) by Jaspar online tool (http://jaspar.genereg.net/). Mechanistically, Dual luciferase reporter gene assay and ChIP experiments suggest HOXB5 directly activate CD47( Figure h and i). Compared to the monotherapy group, Combination therapy with CD47 inhibitor and Quizartinib (AC220) significantly promoted the phagocytic ability of M1-like macrophages against MOLM13 cells ( Figure j and k). The mouse model of AML was constructed by injection of 5x106 FLT3-ITD cells via the tail vein. Mouse models further demonstrate that combination therapy with CD47 inhibitor and Quizartinib significantly reduces the tumor burden and improves survival rate. We first revealed that FLT3-ITD can up-regulates CD47 to promote immune escape of macrophages. These evidences indicates that combination therapy with CD47 inhibitor and FLT3-ITD inhibitor can be a promising therapeutic method for impressive treatment of FLT3-ITD AML patients.

Multiomic Single-Cell Sequencing May Distinguish BCR-ABL-Mutated Acute Myeloid Leukemia (AML) from Blast Crisis Chronic Myelogenous Leukemia (BC-CML)

Introduction The existence of BCR-ABL+ AML as an entity distinct from BC-CML is controversial. The WHO recently recognized BCR-ABL+ AML, but with the caveat that it is poorly characterized (Khoury, 2022). Clinically, BCR-ABL+ AML is associated with high risk of relapse (Neuendorff, 2018). Further, acquired BCR-ABL mutations have been identified as a cause of relapse on FLT3 inhibitors (McMahon, 2019). We hypothesize that BCR-ABL+ AML can be distinguished as a distinct biologic entity from BC-CML by the observation of BCR-ABL as a sub-clonal rather than a founder mutation in leukemogenesis. Methods To test the utility of multi-omic single cell (SC) sequencing in determining tumor phylogeny and identification of BCR-ABL+ AML vs BC-CML, we analyzed bone marrow samples from 4 patients (pts): 1 relapsed AML with clinical development of a new BCR-ABL fusion at the time of relapse, and 3 newly diagnosed BC-CML (2 myeloid, 1 lymphoid). We performed high-throughput SC DNA sequencing with simultaneous cell-surface immunophenotyping and targeted RNA sequencing for BCR-ABL fusion proteins (Mission Bio Tapestri). All SC DNA sequencing included pt-specific primers for BCR-ABL DNA breakpoints, which were previously identified using nanopore-based technology for sequencing long strands of DNA. Results We analyzed 19,197 single cells (median 1995 cells/pt). Multi-omic SC technology differentiated BCR-ABL+ AML from BC-CML by confirming the fusion arose as a branch mutation from a founding leukemic clone and was not present in all leukemic populations. In Patient 1, who was sampled at time of AML relapse, we identified pathogenic FLT3 ITD and NRAS Q61K mutations. FLT3 co-mutated with either the BCR-ABL DNA breakpoint or NRAS in the same single cell and clone, while the BCR-ABL DNA breakpoint and NRAS were not co-mutated. Phylogenetic reconstruction determined FLT3 to be a founding mutation, which later branched into FLT3/BCR-ABL and FLT3/ NRAS co-mutant subclones. Further, the FLT3-ITD/BCR-ABL and FLT3-ITD/ NRAS subclones had distinct immunophenotypes. Blasts harboring the BCR-ABL DNA breakpoint were predominantly identified in the CD117+/CD33+ population (60.2% of cells) compared to CD34+ (16.4% of cells, p &amp;lt;0.001) and blasts with monocytic differentiation (19.1% of cells, p = 0.02). Notably, BCR-ABL+ cells were not identified in T cells. NRAS mutations were predominantly identified in both CD34+ and monocytic blasts. Together, these data support BCR-ABL arising late within the evolutionary history of this pt's AML. By contrast, in Patients 2 and 3 with myeloid BC-CML, the BCR-ABL DNA breakpoint and corresponding BCR-ABL fusion were detectable in all cell populations, including blasts as well as immunophenotypically normal T cells, B cells, and macrophages. In Patient 2, we identified an IDH2 R132C mutation co-mutated with BCR-ABL. Phylogenetic reconstruction determined the IDH2 mutation arose as a branch from a founding BCR-ABL+clone. Unlike the BCR-ABL breakpoint, which was present in all cell populations, the IDH2 mutation was only identified in immunophenotypically-defined CD33+/CD34+/CD117+ myeloid blasts and was absent from bystander T cells and macrophages. Finally, Patient 4had a clinical diagnosis of lymphoid BC-CML. Like Patients 2 and 3, the BCR-ABL DNA breakpoint and corresponding RNA fusion were present in all immunophenotypically-defined blast populations. Unlike Patients 2 and 3, however, BCR-ABL was absent from immunophenotypically normal CD4+ and CD8+ T cells, macrophages, and monocytes. Additional pts are needed to determine whether this pattern is consistent with all lymphoid BC-CML or whether this pt's biology may have been closer to BCR-ABL+ acute lymphoblastic leukemia (ALL). Conclusions Multi-omic SC technology can distinguish BCR-ABL+ AML from myeloid BC-CML and provide direct confirmation that lineage-restriction of BCR-ABL potentially clarifies the diagnosis of discrete Ph+ leukemias. Future studies are needed to understand the role of RT-PCR based BCR-ABL1 testing as a marker of minimal residual disease in pts with BCR-ABL+ subclones, the impact of BCR-ABL as a potential driver of AML resistance, and the utility of BCR-ABL inhibitors in leukemias with late-arising BCR-ABL+ clones. Support: U10CA180821, U10CA180882, U24CA196171; https://acknowledgments.alliancefound.org. Clinicaltrials.gov Identifiers: NCT00048958, NCT00899223, NCT00900224

Streamlining the Detection of FLT3 Internal Tandem Duplication (FLT3-ITD) Using the Cepheid Genexpert® Automated System

Objectives: The development of targeted therapies for Acute Myeloid leukemia (AML) demands fast and reliable diagnostic tests. Specifically, FMS-like tyrosine kinase 3 (FLT3-ITD), comprising nearly 25% of all AML cases, is the most common targetable mutation in AML. Current diagnosis of FLT3-ITD by PCR involves numerous laborious steps, with bench-top steps involving time-consuming whole blood processing, DNA isolation, plate-based PCR and subsequent fragment analysis by capillary electrophoresis. Consequently this adds significant overhead, time-consuming personnel training, and numerous steps which could lead to errors, all of which increase the resource burden and time to result. The objective of this study was to create an automated FLT3-ITD diagnostic prototype that is faster, requires less hands-on time that can detect all clinically relevant ITD lengths. Methods: We have developed a simplified FLT3-ITD prototype assay using the Cepheid GeneXpert® automated platform for FLT3-ITD detection. The prototype substantially streamlines the process, using a less than 30 minutes off-board time to prepare a universal blood lysate that is compatible with all Cepheid Hematology Oncology products. Once the sample is loaded into the cartridge and inserted into the instrument, the cartridge performs automated DNA isolation, purification, and qPCR. A Taqman-based probe for ABL is utilized in the qPCR step as a PCR control to ensure that the blood processing and qPCR was successful and avoid wasted downstream steps. Quantitative fragment analysis can easily be performed on the PCR product using capillary electrophoresis (CE) for precise allelic ratio (AR) callout. Additionally, the PCR product is compatible be run on a bioanalyzer or standard Agarose Gel-Electrophoresis if available. Results: The developed automated FLT3-ITD diagnostic prototype demonstrated significant improvements over the existing methodologies with substantially reduced time to result and simplicity in use. To assess the sensitivity of this prototype, we used MV4-11 cancer cells harboring a 30 bp ITD and CRISPR-CAS engineered human cell lines with 3 bp, 21 bp, and 75 bp FLT3 ITD insertions, comprising the most commonly detected ITD lengths spiked into EDTA blood as a test material. For these ITD's, we observed sensitivity and linearity down to 0.01-0.02 by CE for the ITDs tested. Importantly, the prototype detected all lengths of ITD mutations with inserts from 3bp to 300 bp using plasmid test materials. Conclusion: We have successfully developed a novel FLT3-ITD diagnostic prototype that has several major advantages over existing methodologies. The prototype significantly reduces the time and hands-on effort required for analysis, streamlining the diagnostic process for same day results. The larger sample volume used allows improved sensitivity to ensure accurate detection, even at low allelic frequencies. Additionally, the prototype requires fewer reagents in that sample prep and qPCR are performed in a single cartridge and run hands-free. Downstream analysis of amplified products can be easily performed and validated using capillary electrophoresis, bioanalyzer, or gel electrophoresis. Finally, because this prototype uses the same universal blood lysate as other AML tests, it has the potential to improve future diagnostic therapeutic interventions in the clinic.

Retinoic acid and proteotoxic stress induce AML cell death overcoming stromal cell protection

BackgroundAcute myeloid leukemia (AML) patients bearing the ITD mutation in the tyrosine kinase receptor FLT3 (FLT3-ITD) present a poor prognosis and a high risk of relapse. FLT3-ITD is retained in the endoplasmic reticulum (ER) and generates intrinsic proteotoxic stress. We devised a strategy based on proteotoxic stress, generated by the combination of low doses of the differentiating agent retinoic acid (R), the proteasome inhibitor bortezomib (B), and the oxidative stress inducer arsenic trioxide (A).MethodsWe treated FLT3-ITD+ AML cells with low doses of the aforementioned drugs, used alone or in combinations and we investigated the induction of ER and oxidative stress. We then performed the same experiments in an in vitro co-culture system of FLT3-ITD+ AML cells and bone marrow stromal cells (BMSCs) to assess the protective role of the niche on AML blasts. Eventually, we tested the combination of drugs in an orthotopic murine model of human AML.ResultsThe combination RBA exerts strong cytotoxic activity on FLT3-ITD+ AML cell lines and primary blasts isolated from patients, due to ER homeostasis imbalance and generation of oxidative stress. AML cells become completely resistant to the combination RBA when treated in co-culture with BMSCs. Nonetheless, we could overcome such protective effects by using high doses of ascorbic acid (Vitamin C) as an adjuvant. Importantly, the combination RBA plus ascorbic acid significantly prolongs the life span of a murine model of human FLT3-ITD+ AML without toxic effects. Furthermore, we show for the first time that the cross-talk between AML and BMSCs upon treatment involves disruption of the actin cytoskeleton and the actin cap, increased thickness of the nuclei, and relocalization of the transcriptional co-regulator YAP in the cytosol of the BMSCs.ConclusionsOur findings strengthen our previous work indicating induction of proteotoxic stress as a possible strategy in FLT3-ITD+ AML therapy and open to the possibility of identifying new therapeutic targets in the crosstalk between AML and BMSCs, involving mechanotransduction and YAP signaling.

A new cyclin-dependent kinase-9 inhibitor A09-003 induces apoptosis in acute myeloid leukemia cells with reduction of myeloid cell leukemia sequence-1 protein

Acute myeloid leukemia (AML) is the most common type of hematological disease in adults, and has a very poor outcome [1]. Based on its wide range of efficacy in AML models, a small-molecule inhibitor of the anti-apoptotic protein BCL-2, venetoclax (ABT-199/GDC-0199), was developed for clinical trials. However, venetoclax showed limited monotherapy activity [2]. The overexpression of myeloid cell leukemia sequence-1 protein (Mcl-1)—due to mutations in Fms-like tyrosine kinase 3 internal tandem duplication (FLT-3 ITD)—was considered to be the main reason for low efficacy of venetoclax in clinical trials [3-5]. To achieve venetoclax sensitization in AML, targeting CDK-9 with venetoclax is a promising therapeutic strategy.In this study, we developed A09-003 as a potent inhibitor of CDK-9, with an IC50 value of 16 nM. A09-003 inhibited cell proliferation in various leukemia cell lines. In particular, the proliferation inhibitory effect of A09-003 was most potent in MV4-11 and Molm-14 cells, harboring the FLT-3 ITD mutation with a high expression profile of Mcl-1. Marker analysis revealed that A09-003 reduced CDK-9 phosphorylation and reduced RNA polymerase II activity with decreased Mcl-1 expression. Finally, combining A09-003 with venetoclax induced apoptotic cell death in a synergistic manner. In summary, this study shows the potential of A09-003 in AML therapy.

Midostaurin in addition to intensive chemotherapy in acute myeloid leukemia with t(8;21) and KIT and/or FLT3- ITD mutations: results of the SAL MIDOKIT trial.

Not available.

Identification of New Purpuroine Analogues from the Arctic Echinodermata Pteraster militaris That Inhibit FLT3-ITD+ AML Cell Lines.

Isolation of bioactive products from the marine environment is considered a very promising approach to identify new compounds that can be used for further drug development. In this work we have isolated three new compounds from the purpuroine family by mass-guided preparative HPLC; purpuroine K-M. These compounds where screened for antibacterial- and antifungal activity, antibiofilm formation and anti-cell proliferation activity. Additionally, apoptosis-, cell cycle-, kinase binding- and docking studies were performed to evaluate the mechanism-of-action. None of the compounds showed activity in antibacterial-, antibiofilm- or antifungal assays. However, one of the isolated compounds, purpuroine K, showed activity against two cell lines, MV-4-11 and MOLM-13, two AML cell lines both carrying the FTL3-ITD mutation. In MV-4-11 cells, purpuroine K was found to increase apoptosis and arrest cells cycle in G1/G0, which is a common feature of FLT3 inhibitors. Interactions between purpuroine K and the FLT3 wild type or FLT3 ITD mutant proteins could however not be elucidated in our kinase binding and docking studies. In conclusion, we have isolated three novel molecules, purpuroine K-M, one of which (purpuroine K) shows a potent activity against FLT3-ITD mutated AML cell lines, however, the molecular target(s) of purpuroine K still need to be further investigated.

TIGIT blockade repolarizes AML-associated TIGIT+ M2 macrophages to an M1 phenotype and increases CD47-mediated phagocytosis

BackgroundLeukemia-associated macrophages (LAMs) represent an important cell population within the tumor microenvironment, but little is known about the phenotype, function, and plasticity of these cells. The present study provides an...

Impact of FLT3-ITD Mutation Status and Its Ratio in a Cohort of 2901 Patients Undergoing Upfront Intensive Chemotherapy: A PETHEMA Registry Study.

FLT3−ITD results in a poor prognosis in terms of overall survival (OS) and relapse-free survival (RFS) in acute myeloid leukemia (AML). However, the prognostic usefulness of the allelic ratio (AR) to select post-remission therapy remains controversial. Our study focuses on the prognostic impact of FLT3−ITD and its ratio in a series of 2901 adult patients treated intensively in the pre-FLT3 inhibitor era and reported in the PETHEMA registry. A total of 579 of these patients (20%) harbored FLT3−ITD mutations. In multivariate analyses, patients with an FLT3−ITD allele ratio (AR) of >0.5 showed a lower complete remission (CR rate) and OS (HR 1.47, p = 0.009), while AR > 0.8 was associated with poorer RFS (HR 2.1; p < 0.001). Among NPM1/FLT3−ITD-mutated patients, median OS gradually decreased according to FLT3−ITD status and ratio (34.3 months FLT3−ITD-negative, 25.3 months up to 0.25, 14.5 months up to 0.5, and 10 months ≥ 0.5, p < 0.001). Post-remission allogeneic transplant (allo-HSCT) resulted in better OS and RFS as compared to auto-HSCT in NPM1/FLT3−ITD-mutated AML regardless of pre-established AR cutoff (≤0.5 vs. >0.5). Using the maximally selected log-rank statistics, we established an optimal cutoff of FLT3−ITD AR of 0.44 for OS, and 0.8 for RFS. We analyzed the OS and RFS according to FLT3−ITD status in all patients, and we found that the group of FLT3−ITD-positive patients with AR < 0.44 had similar 5-year OS after allo-HSCT or auto-HSCT (52% and 41%, respectively, p = 0.86), but worse RFS after auto-HSCT (p = 0.01). Among patients with FLT3−ITD AR > 0.44, allo-HSCT was superior to auto-HSCT in terms of OS and RFS. This study provides more evidence for a better characterization of patients with AML harboring FLT3−ITD mutations.

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