NPM1-mutated acute myeloid leukemia is genetically heterogeneous, and risk assessment remains focused exclusively on FLT3-ITD despite the potential role of other co-mutations. We retrospectively analyzed 1,360 adults from a multicenter cohort to map age-resolved co-mutation architecture and prognostic impact. Co-mutations were present in 97% of patients (median = 3 mutations) with DNMT3A (45%), FLT3-ITD (42%), TET2 (27%), and IDH2 (20%) predominating. RTK/RAS partners were enriched in younger adults, whereas myelodysplasia-related genes (MR-genes), epigenetic lesions and higher mutation burden accumulated in the elderly. Pairwise co-mutations mapped to distinct clinical phenotypes with NPM1+FLT3-ITD showing a hyperproliferative profile, and NPM1+SRSF2/TET2 associated to older patients with cytopenic disease. In the 688 patients receiving upfront intensive therapy, NPM1 type Non-ABD exhibited a tendency toward poorer OS compared with type A. In competing-risk models, SRSF2 and DNMT3A were associated with a higher cumulative incidence of relapse (CIR). Consistently, multivariable analyses showed that increasing age and SRSF2 independently conferred adverse risk across OS, RFS, and EFS; KRAS adversely impacted OS, whereas FLT3-OTHER was associated with improved OS; and DNMT3A with inferior RFS and EFS but not OS. These findings show that co-mutation signatures in NPM1-mutated AML are age-structured and clinically meaningful, refining risk beyond FLT3-ITD.

Acute myeloid leukemia (AML) is a heterogeneous malignancy with a poor prognosis. Genetic and molecular profiling help guide treatment decisions, including the use of allogeneic hematopoietic stem cell transplantation (allo-HSCT), to reduce relapse risk. This study evaluated the impact of individual and co-mutational genetic profiles in AML patients in first complete remission after receiving allo-HSCT using data from the PETHEMA registry. A retrospective analysis assessed overall survival and relapse-free survival (RFS). Cox regression identified significant variables used to develop a risk score based on hazard ratios, incorporating age, AML type, transplant timing, and genetic/molecular alterations. A total of 717 patients (median age 56.5 years) were included, most classified as adverse risk by ELN2022 criteria. Both ELN2017 and ELN2022 risk classifications were validated. Multivariate analysis showed that DNMT3A, SF3B1, TP53, and WT1 mutations were linked to shorter RFS, whereas FLT3-ITD mutations correlated with prolonged RFS. These findings were integrated into a proposed prognostic score, which was validated. Therefore, this study highlights the prognostic importance of genetic mutations in AML patients undergoing allo-HSCT. These insights could inform pre-transplant strategies, including donor selection and conditioning regimens, as well as post-transplant maintenance therapy.

NPM1-mutated acute myeloid leukemia (AML) is genetically well-defined, but clinical outcomes remain heterogeneous, suggesting that quantitative clonal features may refine current risk stratification. We analyzed 688 intensively treated NPM1-mutated AML integrating variant allele frequency (VAF), mutation order, and clonal architecture inferred by PyClone and ClonEvol. Co-mutations were present in 97% of patients (median = 3 per case), dominated by DNMT3A (49%), FLT3-ITD (46%), TET2 (22%), and IDH2 (20%). Prognostic modelling of NPM1 VAF identified an optimal cut-off of 31.44%, defining NPM1high and NPM1low groups. NPM1low correlated with splicing-related alterations and independently predicted inferior overall (HR = 1.46; p = 0.037) and relapse-free survival (HR = 1.40; p = 0.036). Gene-specific VAF analyses revealed divergent effects across partners, high DNMT3A, FLT3-OTHER, KRAS, and PTPN11 burdens were adverse, whereas high IDH2 VAF was protective. Combined models showed that patients with NPM1high and favorable co-mutation VAFs had the best outcomes, while dual unfavorable burdens conferred the poorest survival. Mutation ordering inferred from VAFs positioned NPM1 after epigenetic and splicing lesions but before signaling and transcription-factor mutations. Non-canonical orders, such as early FLT3-OTHER/TKD or WT1 prior to NPM1, significantly stratified outcomes. Clonal reconstruction revealed predominantly linear evolutionary trajectories (84.3%), with increased mutational burden and clonal diversity associating with inferior survival. Notably, intra-clonal co-localization of NPM1 with IDH1 or TET2 was associated with improved outcomes, whereas co-localization with WT1 predicted dismal prognosis. These results demonstrate that quantitative and structural dimensions of clonality refine the biological and prognostic landscape of NPM1-mutated AML beyond mutational status alone.

NPM1 mutations are among the most frequent genetic alterations in acute myeloid leukemia (AML) and have been associated with several immunophenotypic features. This study aims to characterize the immunophenotypic profile of this entity at diagnosis using multiparametric flow cytometry and to evaluate the impact of phenotype classifications on prognosis. Immunophenotypic analysis enabled the identification and characterization of various populations previously described in this entity, including immature (CD117+ HLA-DR+), neutrophil-committed (CD117+/heterogeneous HLA-DR-), and monocytic (with variable CD117 expression, HLA-DR+, and CD64+) leukemic cells, which were identified as leukemic expansions, as well as populations presenting immunophenotypic alterations. Cases were distributed in seven immunophenotypic patterns based on R-classification that considers the presence of the three previously described leukemic cell populations but independently of their proportion and relative distribution. Most of the patients included in this study (30, 33%) exhibited an immature and monocytic leukemic cells population (pattern 4), followed by those patients (16, 18%) with a predominant expansion of immature leukemic cells (pattern 1), and those (16, 18%) characterized by a neutrophil-committed leukemic population (pattern 2). Less frequent patterns included (11, 12%) neutrophil-committed and monocytic cells population (pattern 6), (7, 8%) coexistence of the three populations (pattern 7), (6, 7%) immature and neutrophil-committed leukemic cells population (pattern 5), and (5, 5%) predominant expansion of monocytic leukemic cells population (pattern 3). Our analysis indicated that cases with monocytic leukemic cells population predominance exhibit a trend towards a lower cumulative incidence of relapse compared to other leukemic populations, either as the predominant population or across different immunophenotypic patterns. However, differences did not reach statistical significance. These findings help us to contribute with additional information about the biological and immunophenotypic signature of NPM1-AML.

The aim of our study was to analyze the incidence, co-mutation pattern, and prognostic impact of CEBPA gene mutations in a large multicenter consecutive series of 1367 adult patients AML patients treated with non-IT modalities. A total of 83 patients (6.1%) had mutations in CEBPA gene. Among these, 34 (2.5%) harbored mutations located in bZIP domain (bZIP in-frame N = 6) and 49 (3.6%) in other regions of the gene (other CEBPAmut). Genes most frequently co-mutated in these CEBPAmut patients were TET2 (45.8%, N = 38), SRSF2 (42.2%, N = 35), and ASXL1 (40.9%, N = 34). Using the Bradley-Terry model we identified that mutations in MDS-related genes, in TP53, and in epigenetic regulators appear to occur earlier. In contrast, genes involved in activating cell signaling appeared to occur later than CEBPAmut. Overall Survival (OS) of non-IT AML patients was analyzed according to the type of CEBPAmut. Median OS was 11.6 months in CEBPA-bZIP patients compared to 9.0 and 6.9 for patients with other CEBPAmut or CEBPAwt, respectively. When selecting 1129 AML patients treated with HMA or HMA-based combinations, CEBPA-bZIP patients had a median survival time of 11.6 months (range 9.6-NR) and 2.5 years survival probability of 20.1% which were outcomes comparable to remaining ELN2024 favorable patients. We concluded that our series of non-IT AML patients within the PETHEMA registry confirm a low percentage of CEBPA mutated cases which are frequently co-mutated with MDS related genes. Those CEBPA-bZIP cases harbor a similar median OS than those belonging to favorable ELN2024 risk category.

Phenocopies are leukemias that mirror the transcriptional programs, signaling dependencies and often the clinical behavior of established genetic entities, yet lack their defining lesions. In acute lymphoblastic leukemia (ALL), RNA sequencing (RNAseq) classifiers identify phenocopy subtypes including BCR::ABL1-like, ETV6::RUNX1-like, ZNF384-rearranged-like and KMT2A-rearranged-like, which reproduce canonical expression patterns and may share drug vulnerabilities. Their emergence is reshaping taxonomy, but World Health Organization and International Consensus Classifications differ on which phenocopies merit entity status. Phenocopies can refine risk stratification within "B-other" ALL by integrating expression with genotype, copy-number alterations and measurable residual disease assessment. It may also broaden access to pathway-directed therapies (ABL, JAK-STAT, menin) for patients without sentinel fusions but with convergent circuitry. Adoption remains constrained by RNAseq availability, expertise and lesion-centric regulation. Prospective studies are needed to establish the clinical utility of newly described phenocopies, extending the advances made in subclassification and targeted treatment of BCR::ABL1-like ALL.

BACKGROUND:Acute myeloid leukemia (AML) harboring BCR::ABL1 is considered a separate diagnostic entity and is classified as adverse in the risk score of the European LeukemiaNet. However, its prognosis could change with the addition of tyrosine kinase inhibitors (TKI) to chemotherapy. METHODS:In this study, the authors interrogated Programa Español de Tratamientos en Hematología (PETHEMA) AML epidemiologic registry to shed light on the outcome of patients with AML harboring BCR::ABL1. RESULTS:Fifty-seven patients with newly diagnosed AML harboring BCR::ABL1 were included, 40 of them treated intensively. Fifteen patients received TKI and 19 did not receive TKI, median overall survival (mOS) was 15.7 months (95% CI, 3.9-27.6 months) and 12.3 months (95% CI, 0.4-24.1 months), respectively (p = .28). Median relapse-free survival was not reached versus 7.6 months (95% CI, 3-12.2 months) in patients who did or did not receive TKI (p = .029). Age, TKI treatment, allogeneic bone marrow transplantation and date of diagnosis were included in the Cox regression analysis, and no independent prognostic factors for OS were found. Comparison of BCR::ABL1 AML patients with the intensively treated global PETHEMA AML cohort (N = 2107) showed that patients who received a TKI had a median overall survival similar to that of the intermediate‑risk group. CONCLUSIONS:This study shows improved outcomes with the addition of TKI to chemotherapy and supports re-classification of AML patients harboring BCR::ABL1 in the intermediate-risk group. Nonetheless, given the small sample size, larger studies are needed to confirm these findings.

QUIWI (NCT04107727) was a phase II, randomized, double-blind, placebo-controlled trial evaluating quizartinib or placebo added to induction and consolidation chemotherapy and/or allogeneic hematopoietic cell transplantation (allo-HCT), followed by maintenance, in newly diagnosed FLT3-ITD– negative acute myeloid leukemia (AML). This post hoc analysis assessed the impact of allo-HCT, modeled as a time-dependent variable, performed in first composite complete remission (CRc1) on overall survival (OS) and disease-free survival (DFS) according to treatment arm. Among 273 randomized patients, 32.2% in the quizartinib arm and 30.1% in the placebo arm underwent allo-HCT in CRc1. Quizartinib improved OS and DFS compared with placebo regardless of allo-HCT status. In Cox models with allo-HCT as a timedependent covariate, quizartinib remained associated with improved OS (HR 0.59; p=0.008) and DFS (HR 0.67; p=0.03), whereas allo-HCT was not significantly associated with OS (HR 0.91; p=0.62) and showed a numerical DFS benefit (HR 0.73; p=0.08). Multivariable analyses confirmed quizartinib as an independent favorable factor for OS (HR 0.56; p=0.046) and DFS (HR 0.60; p=0.04). No additional safety signals were observed. In patients with newly diagnosed FLT3-ITD–negative AML achieving CRc1, quizartinib improved OS and DFS in the overall population. Notably, the clinical benefit of quizartinib was observed regardless of allo-HCT, and appeared more evident in patients who did not proceed to transplant.

In a large multicenter real-world cohort, we aimed to evaluate outcomes of FLAG-Ida salvage therapy for relapsed/refractory (R/R) acute myeloid leukemia (AML) and validated the SALFLAGE prognostic score. We analyzed 1079 adults with R/R AML treated across 112 PETHEMA institutions over 26 years (1998-2024), including patients with primary refractory disease (36.9%) and first relapse episode (63.1%), with a median age of 52 years. Complete remission composite (CRc) was achieved 56.8%, including complete remission (CR) in 51.0%, CR with incomplete recovery in 4.0%, and morphological-free-state in 1.8%, enabling 35.2% of patients and 62% of responders to proceed to allogeneic transplantation without morphological disease. With median follow-up of 50.9 months, median overall survival (OS) was 10.2 months, with 5-year OS rate of 21.6%. Prior allogeneic transplantation (HR 0.54; p < 0.001) and relapse-free interval ≥ 1 year (HR 0.75; p = 0.024) independently predicted improved OS, whereas modified high-risk cytogenetics including t(8; 21) (HR 3.58; p < 0.001), FLT3-ITD mutation at primary diagnosis (HR 1.61; p < 0.001), and age ≥ 60 (HR 1.43; p < 0.001) conferred inferior OS. Validation of the SALFLAGE score demonstrated moderate discrimination (C-index 0.67), with 5-year survival of 38.4%, 27.2%, and 12.7% across risk categories (p < 0.001). Outcomes improved over periods (1998-2005 vs. 2006-2016 vs. 2017-2024): 30-day mortality was 6.9% vs. 9.3% vs. 5.0%, respectively (p = 0.030), and median OS was 7.8 versus 9.4 versus 11.1 months, respectively (p = 0.16). We confirm FLAG-Ida as a reference salvage regimen in fit R/R AML and validate the SALFLAGE score in this setting.

The 2024 European LeukemiaNet (ELN 2024) genetic risk classification was proposed to assess prognosis in patients with acute myeloid leukemia (AML) receiving less-intensive hypomethylating agent (HMA)-based regimens. However, its applicability to real-world patients remains unclear. We retrospectively analyzed 669 adult patients with AML from the PETHEMA registry treated in routine practice with HMA monotherapy (n = 414) or HMA-venetoclax (VEN) (n = 255). Molecular profiling was performed centrally among the Programa Español de Tratamientos en Hematología (PETHEMA) laboratory network. Median age was 75 years, and 48% had secondary AML (s-AML). Across the full cohort, ELN 2024 stratified patients into prognostic groups across both regimens, but overall survival (OS) was consistently shorter with HMA monotherapy than with HMA-VEN (median OS, 6.5 vs 11.2 months). Favorable risk patients treated with HMA-VEN achieved a median OS exceeding 18.4 months, whereas adverse risk patients had an OS of 7.7 months. ELN 2024 provides meaningful stratification in nonintensive AML, especially with HMA-VEN. However, prognostic accuracy is limited in HMA monotherapy and s-AML, particularly those arising from a myeloid neoplasm (sM-AML). Incorporating cytogenetic risk and sM-AML as adverse risk improved discrimination (area under the curve at 24 months, 0.71 vs 0.76). Refinement incorporating clinical history, cytogenetics, emerging mutations, and treatment response is needed to improve risk prediction in real-world populations.

Cytogenetic and molecular studies are the standard of care in the diagnosis of myelodysplastic neoplasms (MDS) and acute myeloid leukemia (AML), which are characterized by a highly heterogeneous genetic landscape. This complexity challenges accurate characterization, risk stratification, and treatment decision-making. Optical Genome Mapping (OGM) is emerging as a high-resolution technique capable of detecting cryptic structural variants (SVs) and copy number changes (CNVs). In this study, we analyzed 150 MDS and AML patients using standard diagnostic methods and complemented by OGM to assess its utility in resolving complex karyotypes and uncovering hidden genomic aberrations. The results revealed novel alterations and refined previous cytogenetic results (e.g., breakpoints, translocation partners) in 80% of cases, involving clinically relevant genes such as MECOM, KMT2A, and NUP98. Importantly, in complex karyotypes, undefined marker chromosomes and chromoanagenesis events were resolved by OGM, uncovering genomic aberrations with potential clinical relevance. Overall, our findings demonstrate that OGM enables a more comprehensive genomic characterization of patients, uncovering cryptic genomic alterations and resolving complex karyotypes in AML and MDS. The identified abnormalities have shown significant and potential clinical implications. Additionally, the discovery of novel alterations could offer insights into previously unknown pathogenic mechanisms, enhancing our understanding of AML and MDS pathogenesis.

Background: Most adult patients with acute myeloid leukemia (AML) will relapse after achieving a first complete remission (CR) or show primary resistance. The prognosis of relapsed/refractory (R/R) AML remains poor, with no universally accepted salvage regimen. FLAG-Ida (fludarabine, high-dose cytarabine, idarubicin, and G-CSF) is used due to its potential to induce remissions suitable for allogeneic stem cell transplantation (allo-HCT), the main potentially curative option in this setting. Methods: We retrospectively analyzed 1,093 adult patients with non-M3 AML from the PETHEMA registry (NCT02006004) treated with FLAG-Ida between 1997 and 2024 for their first R/R episode. Eligible patients had either primary refractory disease (defined as >5% bone marrow blasts or persistent extramedullary disease after one or two identical induction cycles) or relapsed after first CR/CRi. Responses were assessed using ELN 2022 criteria. Median follow-up was 52.0 months. Results: Median age at first salvage was 52 years (range 16–77); 168 (16%) had secondary AML, and the median leukocyte count at initial AML diagnosis was 11.5 ×10⁹/L. Primary refractory disease was present in 404 (37%), early relapse (relapse-free interval [RFI] <1 year) in 431 (39%), and late relapse (RFI ≥1 year) in 258 (24%). Prior transplantation was reported in 336 (32%), including 220 (21%) allogeneic and 116 (11%) autologous. According to MRC 2010 classification, 105 (10%) were low-, 563 (53%) intermediate-, and 299 (27%) high-risk. Among evaluable cases, NPM1, FLT3-ITD, and IDH1/2 mutations were present in 194 (18%), 174 (16%), and 77 (7%), respectively; and 77 (7%) had inv(16). A total of 558 patients (51%) achieved CR, 43 (4%) CRi, and 19 (2%) MLFS, resulting in a composite CR (CRc) rate of 620 (57%). Partial response was seen in 57 (5%), 323 (30%) had no response, and 93 (8%) induction death. Allo-HCT in CRc was performed in 403 (37%). Median overall survival (OS) for the entire cohort was 10.4 months (95% CI, 9.5–11.7), with 2- and 5-year OS rates of 30% and 21%, respectively. Patients with late relapse (n=258) had superior median OS (14.9 months, 95% CI 14.1–19.5) compared to those with early relapse (n=431, 8.2 months) or primary refractory disease (n=404, 10.2 months; p<0.001). Prior allo-HCT (n=220) was associated with better OS (21.6 months, 95% CI 14.4–29.0) than no prior transplant (8.4 months) or autologous transplant (14.2 months; p<0.001). Regarding molecular subgroups, FLT3-ITD-mutated (n=174) had worse OS (8.0 months, 95% CI 6.8–10.4) than FLT3-ITD wild-type (12.2 months; p<0.001). In contrast, IDH1/2-mutated (n=77) showed improved OS (18.0 months, 95% CI 12.0–NR) versus IDH1/2 wild-type (10.5 months; p=0.004). Patients with inv(16) showed 2- and 5-year OS rates of 65% and 47%, respectively, and higher median OS of 58.4 months (95% CI, 25.3–NR) versus 9.7 months in the rest of the cohort (p<0.001). Regarding treatment periods, 102 (9%) received FLAG-Ida before 2006, 482 (44%) between 2006–2016, and 509 (47%) between 2017-2024. Median OS improved from 8.0 months pre-2006 to 9.9 in 2006–2016 and 11.1 in 2017-2024 (p=0.20). The 30 days survival improved in the last period as compared to the previous one: 91%, 86%, and 92% (p=0.008). The rate of allo-HCT increased from 23%, 34% and 42% across eras (p<0.001). Prognostic stratification in three groups using the previously published SALFLAGE score (based on age, relapse type, prior SCT, FLT3-ITD status, and MRC cytogenetics) was validated in 764 patients: low- (n=206, 27%), intermediate- (n=227, 30%), and high-risk (n=331, 43%). Median OS was 18.9 months in the low-, 12.3 in the intermediate-, and 7.4 in the high-risk group (p<0.001), with 2-year OS rates of 45%, 35%, and 25%, and 5-year OS rates of 36%, 25%, and 15%, respectively. Conclusion: This study represents the largest series of R/R AML patients treated with FLAG-Ida to date. Despite the inherent limitations of retrospective analyses, the inclusion of all patients captured in the PETHEMA registry provides a comprehensive real-world overview. FLAG-Ida achieved 57% CRc rate with relatively low induction death rate (8%). Outcomes improved over time, with median OS reaching 11.1 months and allo-HCT rates increasing to 42% after 2016. The SALFLAGE score was validated and reliably stratified patients into prognostic groups with significantly different survival. These findings reinforce FLAG-Ida as a key salvage option for fit R/R AML patients.

Recent studies have demonstrated the association between constitutional ring chromosome 21 (r(21)c) and the development of B-cell acute lymphoblastic leukemia (B-ALL) with intrachromosomal amplification of chromosome 21 (iAMP21). iAMP21 acts as a driver which is often accompanied by secondary alterations that influence disease progression. Here, we report an atypical case of iAMP21 B-ALL with a unique molecular profile in the context of r(21)c. The onset of B-ALL occurred significantly earlier than previously reported in iAMP21-ALL, likely due to the presence of r(21)c. Only scarce cases of iAMP21 with concomitant PAX5 fusions have been reported. Through an extensive genomic characterization, the novel WWOX::PAX5 as well as 13q12.2 deletion involving FLT3 overexpression was found. These findings suggest that r(21)c may induce chromosomal instability on chromosome 21, triggering chromothripsis and leading to iAMP21-ALL. This case provides valuable insights to unravel the complex interplay between germline and somatic genetic alterations in leukemia. Moreover, it underscores the need for thorough genetic evaluation and multidisciplinary management in patients with syndromic presentation, particularly when rare genetic events may contribute to hematologic malignancies.

While allogeneic stem cell transplantation (allo-SCT) is the preferred consolidation for high and most intermediate-risk acute myeloid leukemia (AML) patients in first remission, the role of autologous SCT (auto-SCT) vs. chemotherapy (CT) when allo-SCT is not feasible or indicated, remains controversial. We conducted a real-world, retrospective cohort study using the PETHEMA AML registry to compare auto-SCT and CT. Multivariate Cox regression and propensity score matching (PS-matching) were used to adjust for confounding factors. A total of 1272 patients in first remission and who received 2 consolidation courses were included (615 receiving additional CT cycles and 657 undergoing auto-SCT). Overall, 78.08

Quizartinib pharmacokinetics in FLT3-ITD negative acute myeloid leukemia (AML) remain largely unexplored. This study aims to validate a population pharmacokinetics model (popPK) for quizartinib in plasma samples of FLT3-ITD negative AML patients. To do so, an ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS) method has been developed and validated for the quantification of quizartinib. Plasma samples were collected from FLT3-ITD negative newly diagnosed AML patients undergoing quizartinib therapy at induction in the QUIWI phase II clinical trial [NCT04107727, PETHEMA group] between March 2020 and February 2022. The UPLC-MS/MS method was developed and validated. A previously described popPK model was validated using external validation techniques and implemented using the software NONMEM v7.5. The developed UPLC-MS/MS method demonstrated high accuracy and precision with a linear range of 6 to 200 ng/mL, with relative standard deviation between 3–11 and accuracy of 88–97

This study has two main objectives. First, to evaluate a feature selection methodology based on SEQENS, an algorithm for identifying relevant variables. Second, to validate machine learning models that predict the risk of complications in patients with acute myeloid leukemia (AML) using data available at diagnosis. Predictions are made at three time points: 90 days, six months, and one year post-diagnosis. These objectives represent fundamental steps toward the development of a tool to assist clinicians in therapeutic decision-making and provide insights into the risk factors associated with AML complications. A dataset of 568 patients, including demographic, clinical, genetic (VAF), and cytogenetic information, was created by combining data from Hospital 12 de Octubre (Madrid, Spain) and Instituto de Investigación Sanitaria La Fe (Valencia, Spain). Feature selection based on an enhanced version of SEQENS was conducted for each time point, followed by the comparison of four classifiers (XGBoost, Multi-Layer Perceptron, Logistic Regression and Decision Tree) to assess the impact of feature selection on model performance. SEQENS identified different relevant features for each prediction horizon, with Age, TP53, − 7/7Q, and EZH2 consistently relevant across all time points. The models were evaluated using 5-fold cross-validation, XGBoost achieve the highest average ROC-AUC scores of 0.81, 0.84, and 0.82 for 90-day, 6-month, and 1-year predictions, respectively. Generally, performance remained stable or improved after applying SEQENS-based feature selection. Evaluation on an external test set of 54 patients yielded ROC-AUC scores of 0.72 (90-day), 0.75 (6-month), and 0.68 (1-year). The models achieved performance levels that suggest they could serve as therapeutic decision support tools at different times after diagnosis. The selected variables align with the European LeukemiaNet (ELN) 2022 risk classification, and the SEQENS-based feature selection effectively reduced the feature set while maintaining prediction accuracy.

PURPOSE:Quizartinib, an oral, selective, second-generation, type-II FMS-like tyrosine kinase 3 (FLT3) inhibitor with high binding affinity to internal tandem duplication (ITD) and wild-type (WT) FLT3, has shown early clinical activity as monotherapy in patients with relapsed/refractory FLT3-ITD-negative AML. The phase III QuANTUM-First trial showed that quizartinib significantly prolonged survival versus placebo when added to standard chemotherapy, followed by single-agent maintenance, in patients with newly diagnosed (ND) FLT3-ITD-positive AML. We investigated the safety and efficacy of quizartinib in patients with ND FLT3-ITD-negative AML. METHODS:The phase II, randomized, double-blind, placebo-controlled QUIWI trial enrolled patients age 18-70 years with ND FLT3-ITD-negative (mutant-to-WT allelic ratio <0.03) AML. Patients were randomly assigned 2:1 to receive standard induction and consolidation chemotherapy combined with either quizartinib 60 mg once daily or placebo, followed by single-agent maintenance with quizartinib or placebo. The primary end point was event-free survival (EFS). Secondary end points included overall survival (OS) and safety. RESULTS:Overall, 273 patients were randomly assigned to quizartinib (n = 180) or placebo (n = 93). At data cutoff, median EFS was 20.4 months and 9.9 months in the quizartinib and placebo arms, respectively (P = .046). Median OS was not reached and 29.3 months in the quizartinib and placebo arms, respectively (P = .012); 3-year OS rates were 60.8% and 45.7%. The most frequently reported adverse events (any grade) were fever, rash, diarrhea, and mucositis. CONCLUSION:The addition of quizartinib to standard chemotherapy was associated with significantly longer EFS and OS than placebo in patients with ND FLT3-ITD-negative AML.

Background/Objectives: This PETHEMA PCR-LMA study aimed to evaluate whether mutations detected by NGS (VAF cut-off of ≥5%) correlate with NPM1, FLT3-ITD, FLT3-TKD, IDH1, and IDH2 mutations detected using conventional PCR (analytical sensitivity 3%) in a nationwide network of seven reference laboratories. Methods: Between 2019 and 2021, 1685 adult AML patients with at least one centralized sample (NGS or PCR) at primary diagnosis or relapse/refractory episode were included. Results: During this period, 1288 paired NGS/PCR samples (1094 at diagnosis, 103 at relapse and 88 at refractoriness) were analyzed. Considering PCR the gold-standard, for NPM1 NGS sensitivity was 98.5% and specificity 98.9%, for FLT3-ITD 73.8% and 99.6%, for FLT3-TKD 84.5% and 99.3%, for IDH1 98.7% and 98.7%, and for IDH2 99.1% and 97.7%, respectively. Overall concordance rate of positive results between NGS (and PCR was 95% (262/276) for NPM1, 72% (149/206) for FLT3-ITD, 74% (49/66) for FLT3-TKD, 87% (77/89) for IDH1 and 84% (107/127) for IDH2. Overall, median days from sample reception until report were 7 for PCR and 28 for NGS. Conclusions: This study shows high concordance between NPM1 and IDH results using PCR and NGS. However, sensible important discrepancies are observed for FLT3 mutations. In our context, rapid screening for these druggable mutations should be performed by conventional PCR.

Introduction FMS-like tyrosine kinase 3 (FLT3) plays a critical role in hematopoiesis. Circulating levels of its ligand, the soluble FLT3 ligand (FL), have been shown to correlate with the degree of bone marrow aplasia following cytotoxic treatments. Notably, FL can also be secreted by leukemic blasts, potentially promoting leukemic cell proliferation via autocrine signaling mechanisms, thereby potentially contributing to resistance. In this context, we aimed to investigate the impact of FL kinetic profiles during induction chemotherapy on clinical outcomes in newly diagnosed, FLT3-ITD negative acute myeloid leukemia (AML) patients enrolled in the QUIWI trial [NCT04107727]. In this trial median event-free survival (EFS) was 20.4 months and 9.9 months in the Quizartinib (Quiz) and placebo (PBO) arms, respectively (P=0.045). Median overall survival (OS) was not reached and 29.3 months in the Quiz and PBO arms, respectively (P=0.01). Methods This was a preplanned subanalysis conducted within the PETHEMA QUIWI trial, a multicenter, prospective, randomized, double-blind, placebo-controlled phase II study. The trial enrolled patients with newly diagnosed AML who received standard 3+7 induction chemotherapy (idarubicin and cytarabine), followed by quizartinib or placebo from day +8 to day +21, between September 2019 and October 2022. Plasma levels of FL (expressed in pg/mL) were measured by ELISA before starting and on day +15 of induction therapy. Based on the prior study by Peterlin et al. (2019), we defined two subgroups according to FL levels on +15: High Level (High, ≥1000 pg/mL) and Low Level (Low, <1000 pg/mL). Clinical outcomes evaluated included complete remission (CR) or CR with incomplete hematologic recovery (CRi), and measurable residual disease (MRD) after induction, EFS, OS, and duration of CR/CRi (DoR). Results A total of 111 out of 273 FLT3 -ITD negative AML patients enrolled in the QUIWI were included in this study, with a median age of 56 years (range 19–70). In total, 219 plasma samples were analyzed during induction (108 on day +1 before starting chemotherapy and 111 on day +15). Median baseline FL levels were 0.7 pg/mL (0–1116), and 1012 pg/mL (range 0–1176) on day +15. Overall Low FL on +15 was observed in 53 patients (48.2%) and High FL in 57 (51.8%). Median age was 59 years (range 25-70) in the Low FL group vs. 57 years (range 19–69) in the High FL group (p=0.19). Median bone marrow blast percentage was 42% (14–93) in Low FL vs. 44.5% (3–97) in High FL (p=0.71), and median white blood cell count was 4.23 ×10⁹/L (0.56–181.34) in Low FL and 3.1 ×10⁹/L (0.5–181.86) in High FL (p=0.37). ELN 2022 risk classification was unfavorable in 70.6% of patients with Low FL vs. 58.2% in High FL (p=0.37), MRC 2010 cytogenetic was adverse in 31.4% of patients with Low FL vs. 32.7% in High FL (p=0.99), and the diagnosis was secondary AML in 21.6% of patients with Low FL vs. 16.4% in High FL (p=0.66). The proportion of patients achieving CR/CRi after induction was higher in the High FL group (85.5% vs. 64.7%; p=0.0241), and among those patients, the MRD negativity was superior in the High FL vs. the Low FL group (65.8% vs. 33.3%; p=0.0127). The median OS was 19.8 months in the Low FL group and not reached in the High FL group (p=0.17). Patients with Low FL had a median EFS of 8.0 months and it was not reached in patients with High FL (p<0.01). Median DoR was 14.6 months in Low FL vs. not reached in High FL group (p=0.03). By treatment arm, 34/62 (54.8%) of Quiz and 21/44 (47.7%) of PBO patients showed FL ≥1000 pg/mL at day +15 (p=0.47). Among High FL patients, the 4-year OS, EFS, and DoR in Quiz vs. PBO arms were: 58.2% vs. 47.6%; p=0.29; 58.1% vs. 42.9%; p=0.60; and 68.2 vs. 74.9%; p=0.83, respectively. Among Low FL patients, the 4-year OS, EFS, and DoR in Quiz vs. PBO arms were: 48.1% vs. 39.1%; p=0.80; 33.3% vs. 21.7%; p=0.69; and 56.2 vs. 35.3%; p=0.24, respectively. Conclusion In this QUIWI trial subanalysis, plasma FL level ≥1000 pg/mL at day +15 of intensive induction chemotherapy was strongly associated with improved CR/CRi, OS, EFS and DoR. The Quiz arm showed a numerically higher proportion of patients achieving ≥1000 pg/mL FL level, and we observed a trend for prolonged DoR among Low FL patients randomized to Quiz. Larger studies are needed to elucidate the biology and prognostic impact of FL levels after chemotherapy in AML.