Hartmut Döhner

The 2022 ELN recommendations reclassified fms‐like tyrosine kinase‐3 ( FLT3 )‐ITD mutated acute myeloid leukemia (AML) as intermediate‐risk, irrespective of allelic ratio (AR), and designated capillary electrophoresis (CE) as the preferred detection method. However, CE requires substantial expertise, particularly to identify FLT3 ‐ITD microclones ( FLT3 ‐ITD ^m ; AR < 0.05), whose clinical significance remains uncertain. Herein, we investigated diagnostic aspects and the prognostic impact of FLT3 ‐ITD ^m . An initial methodological study performed on 48 FLT3 ‐ITD+ samples, with AR 0.001–6.00, showed overall concordance between CE and next‐generation sequencing (NGS) results ( p < 0.001), with only three microclones (AR: 0.002, 0.001, and 0.008) identified by CE and not by NGS. We next analyzed 658 AML patients diagnosed with a FLT3 ‐ITD+ AML ( n = 446 with AR ≥ 0.05, and n = 212 with a microclone) between 2017 and 2022. Patients with FLT3 ‐ITD ^m , as compared to patients with AR ≥ 0.05, were older, more often had secondary‐type AML, and displayed a higher prevalence of myelodysplasia‐related gene mutations, while NPM1 ‐mutation frequency was similar in the two groups. Complete remission rates were lower in FLT3 ‐ITD ^m (74%) than in patients with AR ≥ 0.05 (88%) treated with intensive chemotherapy without FLT3‐inhibitors (FLT3i). In the intensively treated subgroup, multivariate analyses demonstrated that age, NPM1 mutation, and allogeneic stem cell transplantation, rather than FLT3 ‐ITD AR, were independent predictors of disease‐free survival. At relapse, 15 of 33 (45%) evaluable patients remained FLT3 ‐ITD+, with 10 expanding the original microclone. In conclusion, FLT3 ‐ITD ^m are associated with older age, secondary AML, and with expansion of the FLT3 ‐ITD clone upon relapse, thereby providing a rationale for the evaluation of FLT3i in randomized clinical trials.

To describe the impact of adding a tyrosine kinase inhibitor (TKI) to intensive chemotherapy (IC) on the outcome of de novo BCR::ABL1+ acute myeloid leukemia (AML), we retrospectively analyzed the data of 212 adult AML with ≥20% bone marrow blasts, BCR::ABL1+ or t(9;22)(q34.1;q11.2), no history of previous chronic myeloid leukemia and no prior exposure to BCR-ABL1 TKI. Eighty-nine patients were treated with IC alone and 123 with IC+TKI between 1999 and 2024. Complete remission (CR) or CR with incomplete hematologic recovery (CRi) was achieved in 52/77 patients (68%) with IC and 110/123 patients (89%) with IC+TKI (P<0.0001). With a median follow-up of 66.7 months, the median overall survival (OS) was 20.5 months with IC and not reached with IC+TKI. The 3-year and 5-year OS rates were 42.1% and 38.5% with IC and 70.9% and 62.9% with IC+TKI (P<0.0001) respectively. In multivariate analyses, the addition of TKI to IC was significantly and independently associated with an improved CR/CRi rate (odds ratio: 4.74 [95% confidence interval: 2.17-10.35]; P<0.001) and OS (hazard ratio: 0.40 [0.27-0.62]; P<0.001). Also, adding TKI to IC and alloHSCT in CR1 were independent prognostic factors for relapse-free survival (HR: 0.42; 95% CI: 0.23-0.75; P=0.004 and HR: 0.31; 95% CI: 0.17-0.55; P<0.0001). The outcome of de novo BCR::ABL1+ AML is strongly improved by the addition of a TKI to IC and should become the standard of care. The classification as adverse-risk should be reconsidered in the future ELN classification.

Background: Acute myeloid leukemia (AML) is predominantly a disease of older patients with a poor long-term survival. Approval of the combination of venetoclax (VEN) with azacitidine (AZA) or decitabine (DAC) in the European Union in 2021 for the treatment of patients with newly diagnosed AML who are ineligible for induction chemotherapy has become the treatment backbone for older, medically non-fit AML patients. Based on in vitro priming of AML cells to all- trans retinoic acid (ATRA) by DAC, we had previously conducted a randomized phase II trial (DECIDER, AMLSG 14 − 09) in 200 elderly, non-fit AML patients on the effect of ATRA as add-on to DAC. Median overall survival (OS) was 8.2 months with ATRA versus 5.1 months without ATRA (hazard ratio, 0.65; 95% CI, 0.48 to 0.89; P = 0.006). Notably, ATRA did not add toxicity, the combination was active also in patients with adverse genetics, and prolonged time to treatment resistance. Investigating a triple combination of DAC, VEN and ATRA was a logical next step. This randomized double-blind phase III trial compares the efficacy of ATRA versus placebo as add-on to the backbone treatment (DAC and VEN) with respect to OS, objective best response, quality of life and safety. The accompanying translational research will contribute to identify molecular markers for drug efficacy and better tailoring epigenetic therapy. Methods: DECIDER-2 (AMLSG 32 − 21) is a prospective, randomized, double-blind, placebo-controlled, parallel group, multicenter trial. The primary study objective is to compare the efficacy of ATRA versus placebo as add-on to the backbone treatment (DAC and VEN) in terms of OS. The target population is adult AML patients unfit for standard induction chemotherapy. Patients are randomized (1:1) to receive backbone treatment in combination with either ATRA or placebo. Four interim safety analyses were planned with the involvement of the Data Monitoring Committee. Discussion: This trial investigates whether the in vitro cooperativity of DAC, VEN, and ATRA prolongs OS in unfit AML patients compared to placebo, offering a novel, low-toxicity triplet combination with a promising risk-benefit ratio. Clinical trial number: EU CT number: 2020-005495-36/2023-507461-26-00 (registered 18.11.2020); German clinical trials registry number: DRKS00023646 (registered 23.08.2022).

Acute myeloid leukemia (AML) is characterized by recurrent chromosomal abnormalities that form the basis of the European LeukemiaNet (ELN) risk classification and serve as essential determinants of prognosis and therapeutic decision-making. Conventional metaphase karyotyping remains the diagnostic gold standard for detecting these abnormalities; however, its utility is limited by longer turnaround times, often delaying critical clinical management. Here, we present a long-read sequencing-based (LRS) low-coverage whole genome sequencing (lcWGS) approach using Oxford Nanopore Technology as a rapid and scalable alternative for cytogenetic profiling. A total of 100 diagnostic AML samples were analyzed, comprising 50 retrospectively selected cases with known adverse-risk cytogenetics and 50 prospectively enrolled patients with clinically defined de novo AML. LcWGS demonstrated robust analytical performance, identifying chromosomal aberrations with 93% sensitivity, specificity, and overall accuracy, respectively. Complex karyotypes were reliably detected, with an area under the curve (AUC) of 0.971. Reproducibility was validated through replicate sequencing at two independent laboratories (R=0.99). LcWGS-derived estimates of clone size showed moderate correlation with conventional cytogenetic assessments (R=0.54). Patients with complex karyotypes identified by lcWGS exhibited significantly shorter overall and relapse-free survival, closely mirroring outcomes defined by conventional karyotyping and underscoring the value of lcWGS for risk stratification. Median turnaround time from sample receipt to bioinformatics interpretation was approximately 34 hours, enabling delivery of actionable karyotype results within 72 hours. These findings establish lcWGS as a rapid, reproducible, and accurate platform for detecting clinically relevant chromosomal abnormalities, addressing a critical need for timely risk stratification and treatment initiation in AML.

The World Health Organization fifth edition and International Consensus Classification for myeloid neoplasms both incorporate empirical numerical thresholds to morphologic and molecular features defining certain disease entities. However, the clinical implications of these thresholds remain unclear. We analyzed a large cohort (N = 6,976) of patients with myeloid neoplasms to evaluate the impact of proposed yet different numerical thresholds for variant allele frequency of genetic mutations or hematologic parameters set forth by the World Health Organization fifth edition and International Consensus Classification for classification of SF3B1-mutated myelodysplastic neoplasms, NPM1-mutated acute myeloid leukemia (AML), and oligomonocytic chronic myelomonocytic leukemia. Our analysis demonstrated that the clonal burden of SF3B1 mutation in myelodysplastic neoplasms informs classification and prognosis. Our findings support the notion that NPM1 mutation should be AML-defining regardless of blast percentage and highlight the adverse prognostic impact of the cumulative number of myelodysplasia-related mutations in NPM1-mutated AML. Finally, we provide evidence that integrating specific molecular signatures could improve the accuracy of oligomonocytic chronic myelomonocytic leukemia classification.Significance:Using comprehensive clinical and molecular profiling, this study provides a data-driven approach for evaluating numerical thresholds of variant allele frequency or hematologic parameters (i.e., blast percentage and absolute monocyte count) included in current classification schemas across a spectrum of myeloid malignancies, enabling refinement of disease classification and prognostication.

Comparison of clinical and molecular demographics and survival in patients with NPM1-mutated myeloid neoplasms with <20% blasts from the UK NCRI AML17/19 trials. (A) Density plots demonstrating no significant difference in baseline age or hematologic parameters between patients with <10% vs. 10% to 19% blasts. (B) Similarly, no significant difference was observed with respect to frequently identified mutations between patients with mutated NPM1 and <10% vs. 10% to 19% blasts. (C and D) Event-free and overall-survival was similar between patients with mutated NPM1 and <10% vs. 10% to 19% blasts. Reported p-values utilized the cox model Wald test.

Biplot of quantitative variables included in factor analysis of mixed data (FAMD) of myelodysplastic CMML cases (MD-CMML) including platelets, bone marrow blast (BM blasts) percentage, absolute neutrophil count (ANC), absolute monocyte count (AMC), relative monocytes, hemoglobin, and patient age at diagnosis. Arrow length and direction with respect to the circle indicates relative contribution to each quadrant. Closer proximity of an arrowhead to the circle indicates a stronger contribution of a variable to the quadrant.

WHO 2016 classification of included patients with NPM1-mutated myeloid neoplasms (MN)

Density plot depicting Bradley-Terry modeling of the estimated order of mutation acquisition in AML samples from the UKNCRI and AMLSG cohorts. Mutations in genes classified as MR mutations (SRSF2, ASXL1, RUNX1, U2AF1, ZRSR2, EZH2, STAG2, BCOR, SF3B1) tended to occur as earlier events relative to mutations in NPM1. VAF: Variant allele frequency