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In chronic lymphocytic leukemia, the reliability of next-generation sequencing (NGS) to detect TP53 variants ≤10% allelic frequency (low-VAF) is debated. We tested the ability to detect 23 such variants in 41 different laboratories using their NGS method of choice. The sensitivity was 85.6%, 94.5%, and 94.8% at 1%, 2%, and 3% VAF cut-off, respectively. While only one false positive (FP) result was reported at >2% VAF, it was more challenging to distinguish true variants <2% VAF from background noise (37 FPs reported by 9 laboratories). The impact of low-VAF variants on time-to-second-treatment (TTST) and overall survival (OS) was investigated in a series of 1092 patients. Among patients not treated with targeted agents, patients with low-VAF TP53 variants had shorter TTST and OS versus wt- TP53 patients, and the relative risk of second-line treatment or death increased continuously with increasing VAF. Targeted therapy in ≥2 line diminished the difference in OS between patients with low-VAF TP53 variants and wt- TP53 patients, while patients with high-VAF TP53 variants had inferior OS compared to wild type- TP53 cases. Altogether, NGS-based approaches are technically capable of detecting low-VAF variants. No strict threshold can be suggested from a technical standpoint, laboratories reporting TP53 mutations should participate in a standardized validation set-up. Finally, whereas low-VAF variants affected outcomes in patients receiving chemoimmunotherapy, their impact on those treated with novel therapies remains undetermined. Our results pave the way for the harmonized and accurate TP53 assessment, which is indispensable for elucidating the role of TP53 mutations in targeted treatment., Competing Interests: Bárbara Tazón‐Vega: Honoraria: Bristol Meyer Squibb. Beatriz Bellosillo: Advisory board honoraria, research support, travel support, speaker fees: Astra‐Zeneca, BMS, Janssen, Merck‐Serono, Novartis, Pfizer, Hoffman‐La Roche, ThermoFisher. Christian Brieghel: Travel grant: Octapharma. Carsten U. Niemann: Research funding and/or consultancy fees: Abbvie, AstraZeneca, Beigene, Janssen, Genmab, Lilly, MSD, CSL Behring, Takeda, Octapharma. Davide Rossi: Honoraria: AbbVie, AstraZeneca, BeiGene, BMS, Janssen, Lilly. Research grants: AbbVie, AstraZeneca, Janssen. Travel grants: AstraZeneca, Janssen. Eugen Tausch: Honoraria and research support: Abbvie, AstraZeneca, BeiGene, Janssen, Hoffmann‐La Roche; Research support from Abbvie, Roche, Gilead. Frédéric Davi: Honoraria: Janssen, AstraZeneca. Kostas Stamatopoulos: Research funding, honoraria and/or consultancy fees: Abbvie, AstraZeneca, Janssen, Lilly, Roche. Lydia Scarfo: Consultancy: AbbVie, AstraZeneca, BeiGene, Janssen, Lilly; Speaker Bureau: Octapharma. Miguel Alcoceba: Honoraria and travel grants: Janssen, AstraZeneca. Martin Andres: Consultancy, Honoraria, and travel support: AstraZeneca, Novartis, Roche, Janssen‐Cilag. Maria Gomes da Silva: Consultancy and Research Funding: Janssen Cilag, AstaZeneca, Abbvie, Roche, Takeda. Pau Abrisqueta: Consultancy and Honoraria: Janssen, Abbvie, Roche, BMS, AstraZeneca, Genmab. Panagiotis Baliakas: Honoraria: Abbvie, Gilead, Janssen. Research funding: Gilead. Paolo Ghia: Honoraria: AbbVie, Astrazeneca, BeiGene, BMS, Galapagos, Janssen, Lilly/Loxo Oncology, MSD, Roche. Research funding: AbbVie, AstraZeneca, BMS, Janssen. Richard Rosenquist: Honoraria: AbbVie, AstraZeneca, Janssen, Illumina, Roche. Renata Walewska: Travel support: AbbVie, AstraZeneca, Janssen, Beigene. Sylwia Czekalska: Honoraria: AstraZeneca. Funding: Janssen, AstraZeneca. Stephan Stilgenbauer: Advisory board honoraria, research support, travel support, speaker fees: AbbVie, Acerta, Amgen, AstraZeneca, BeiGene, BMS, Celgene, Gilead, GSK, Hoffmann‐La Roche, Infinity, Janssen, Lilly, Novartis, Sunesis, Verastem. Tiina Kahre: Honoraria: AstraZeneca. Tomasz Stokłosa: Honoraria and Research Funding: Janssen, AstraZeneca. The remaining authors have no competing interests to declare., (© 2025 The Author(s). HemaSphere published by John Wiley & Sons Ltd on behalf of European Hematology Association.)

Vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic (VEXAS) is a haemato-inflammatory syndrome genetically defined by somatic mutations in the X-linked UBA1 gene, typically Val/Thr/Leu substitutions at the Met41 hotspot. Clinical manifestations are heterogeneous and refractory to most haemato-rheumatological treatments. To date, no guidelines exist for the management of VEXAS, and scarce is the evidence on methodology and clinical significance of longitudinal UBA1 clonal burden evaluation upon therapy. Here, we validated a method to quantify UBA1 clonal burden and explored its applicability in patients with VEXAS. Given the different treatment interactions, droplet digital polymerase chain reaction (ddPCR) may allow for informed therapeutic decisions and implementation of personalized strategies., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Purpose: To determine the optimal daunorubicin dose and number of 7 + 3 induction cycles in newly diagnosed AML, this randomized controlled trial compared a once daily dose of 60 mg/m 2 with 90 mg/m 2 daunorubicin in the first 7 + 3 induction and one versus two cycles of 7 + 3 induction., Patients and Methods: Patients age 18-65 years with newly diagnosed AML were randomly assigned to 60 versus 90 mg/m 2 daunorubicin once daily plus cytarabine. Patients with marrow blasts below 5% on day 15 after first induction were randomly assigned to receive a second induction cycle or no second induction cycle., Results: Eight hundred and sixty-four patients with a median age of 52 years were randomly assigned. After a preplanned interim analysis showing no significant difference in response between 60 and 90 mg/m 2 , all consecutive patients received 60 mg/m 2 daunorubicin once daily. The proportion of good early responders was 44% versus 48% ( P = .983) with a composite complete remission (CRc) rate of 90% versus 89% after induction ( P = .691); the 3-year relapse-free survival (RFS) after 60 versus 90 mg/m 2 once daily was 54% versus 50% ( P = .561), and the 3-year overall survival (OS) was 65% versus 58% ( P = .242). Among 389 good responders, CRc rates at the end of induction were 87% after single induction and 85% after double induction. The 3-year RFS was 51% versus 60% (hazard ratio [HR], 1.3; P = .091), and the 3-year OS was 76% versus 75% after single versus double induction (HR, 1.0; P = .937)., Conclusion: The use of 90 mg/m 2 daunorubicin once daily in the context of classical 7 + 3 induction does not significantly improve early response and does not lead to higher remission rates or longer survival than 60 mg/m 2 once daily. In patients with a good early response after first induction, a second induction has only a limited impact on RFS and does not result in an OS benefit.

Aim: Tandem-duplications of the UBTF gene (UBTF-TDs) have recently been identified as a new genetic driver in young individuals with acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS). Disease in these newly defined subgroups is characterized by poor response to standard intensive chemotherapy and inferior survival of the affected patients. However, a thorough analysis of bone marrow histomorphology of UBTF-mutated neoplasia has not been undertaken thus far., Methods and Results: In this retrospective study, we investigated the characteristic histopathological features of a cohort comprising 14 paediatric MDS patients with an excess of blasts (MDS-EB) and UBTF-TD. Bone marrow biopsies from these patients revealed hypercellularity and severe dysplasia across all three haematopoietic lineages. In particular, a marked hyperplastic megakaryopoiesis characterized by the presence of frequent micromegakaryocytes and a high number of monolobulated cells forming small clusters was observed. Additionally, erythropoiesis was left-shifted, with numerous blastoid precursors. The granulopoietic precursors displayed prominent UBTF-positive nucleoli., Conclusion: The unique combination of these histomorphological features strongly suggests a possible UBTF aberration. It will allow initiating the appropriate genetic testing to confirm the presence of UBTF-TD and identify potential additional genetic alterations. Such molecular profiling will not only contribute to a better understanding of the disease mechanism, but also facilitate more rational treatment approaches for these high-risk paediatric MDS patients., (© 2024 The Author(s). Histopathology published by John Wiley & Sons Ltd.)

Since 2017, targeted therapies combined with conventional intensive chemotherapy have started to improve outcome of patients with acute myeloid leukemia (AML). However, even before these innovations outcomes with intensive chemotherapy have improved, which has not yet been extensively studied. Thus, we used a large pan-European multicenter dataset of the HARMONY Alliance to evaluate treatment-time dependent outcomes over two decades. In 5359 AML patients, we compared the impact of intensive induction therapy on outcome over four consecutive 5-year calendar periods from 1997 to 2016. During that time, the 5- year survival of AML patients improved significantly, also across different genetic risk groups. In particular, the 60-day mortality rate has dropped from 13.0% to 4.7% over time. The independent effect of calendar periods on outcome was confirmed in multivariate models. Improvements were documented both for patients.

In 2022, the European LeukemiaNet (ELN) risk stratification for patients with AML has been updated. We aimed to validate the prognostic value of the 2022 ELN classification (ELN22) evaluating 1,570 newly diagnosed AML patients (median age, 56 years) treated with cytarabine-based intensive chemotherapy regimens. As compared to the 2017 ELN classification (ELN17), allocating 595 (38%), 413 (26%) and 562 (36%) patients to the favorable, intermediate and adverse risk category, ELN22 risk was favorable, intermediate, and adverse in 575 (37%), 410 (26%), and 585 (37%) patients, respectively. Risk group allocation was revised in 340 patients (22%). Most patients were re-classified into ELN22 intermediate or ELN22 adverse risk group. The allocation of patients according to the ELN22 risk categories resulted in a significantly distinct event-free survival (EFS), relapse-free survival (RFS), and overall survival (OS). As compared to ELN17, reallocation according to the ELN22 recommendations resulted in a significantly improved prognostic discrimination for OS (3-year area under the curve (AUC) 0.71 vs. 0.67). In patients with ELN22 favorable risk AML, co-occurring MR gene mutations did not significantly impact outcome. Within the ELN22 adverse risk group, we observed marked survival differences across mutational groups (5-year OS rate of 21% and 3% in patients with myelodysplasia-related (MR) gene mutations and TP53-mutated patients, respectively). In patients harboring MR gene mutations, EZH2-, STAG2- and ZRSR2-mutated patients showed an intermediate-like OS. In patients with secondary AML and those who underwent allogeneic HCT, EFS and OS significantly differed between ELN22 risk groups, while prognostic abilities of ELN17 and ELN22 classifications were similar. In conclusion, the ELN22 risk stratification improves prognostic discrimination in a large cohort of intensively treated AML patients. Given the heterogeneous outcome in patients with MR gene alterations, ranging between those of intermediate and adverse risk patients, we suggest reevaluation of risk allocation in these patients., (Copyright © 2024 American Society of Hematology.)

Balanced rearrangements involving the KMT2A gene (KMT2Ar) are recurrent genetic abnormalities in acute myeloid leukemia (AML), but there is lack of consensus regarding the prognostic impact of different fusion partners. Moreover, prognostic implications of gene mutations co-occurring with KMT2Ar are not established. From the HARMONY AML database 205 KMT2Ar adult patients were selected, 185 of whom had mutational information by a panel-based next-generation sequencing analysis. Overall survival (OS) was similar across the different translocations, including t(9;11)(p21.3;q23.3)/KMT2A::MLLT3 (p = 0.756). However, independent prognostic factors for OS in intensively treated patients were age >60 years (HR 2.1, p = 0.001), secondary AML (HR 2.2, p = 0.043), DNMT3A-mut (HR 2.1, p = 0.047) and KRAS-mut (HR 2.0, p = 0.005). In the subset of patients with de novo AML < 60 years, KRAS and TP53 were the prognostically most relevant mutated genes, as patients with a mutation of any of those two genes had a lower complete remission rate (50% vs 86%, p < 0.001) and inferior OS (median 7 vs 30 months, p < 0.001). Allogeneic hematopoietic stem cell transplantation in first complete remission was able to improve OS (p = 0.003). Our study highlights the importance of the mutational patterns in adult KMT2Ar AML and provides new insights into more accurate prognostic stratification of these patients., (© 2024. The Author(s).)

Leukemias with ambiguous lineage comprise several loosely defined entities, often without a clear mechanistic basis. Here, we extensively profile the epigenome and transcriptome of a subgroup of such leukemias with CpG Island Methylator Phenotype. These leukemias exhibit comparable hybrid myeloid/lymphoid epigenetic landscapes, yet heterogeneous genetic alterations, suggesting they are defined by their shared epigenetic profile rather than common genetic lesions. Gene expression enrichment reveals similarity with early T-cell precursor acute lymphoblastic leukemia and a lymphoid progenitor cell of origin. In line with this, integration of differential DNA methylation and gene expression shows widespread silencing of myeloid transcription factors. Moreover, binding sites for hematopoietic transcription factors, including CEBPA, SPI1 and LEF1, are uniquely inaccessible in these leukemias. Hypermethylation also results in loss of CTCF binding, accompanied by changes in chromatin interactions involving key transcription factors. In conclusion, epigenetic dysregulation, and not genetic lesions, explains the mixed phenotype of this group of leukemias with ambiguous lineage. The data collected here constitute a useful and comprehensive epigenomic reference for subsequent studies of acute myeloid leukemias, T-cell acute lymphoblastic leukemias and mixed-phenotype leukemias., (© 2024. The Author(s).)

Clinical research relies on high-quality patient data, however, obtaining big data sets is costly and access to existing data is often hindered by privacy and regulatory concerns. Synthetic data generation holds the promise of effectively bypassing these boundaries allowing for simplified data accessibility and the prospect of synthetic control cohorts. We employed two different methodologies of generative artificial intelligence - CTAB-GAN+ and normalizing flows (NFlow) - to synthesize patient data derived from 1606 patients with acute myeloid leukemia, a heterogeneous hematological malignancy, that were treated within four multicenter clinical trials. Both generative models accurately captured distributions of demographic, laboratory, molecular and cytogenetic variables, as well as patient outcomes yielding high performance scores regarding fidelity and usability of both synthetic cohorts (n = 1606 each). Survival analysis demonstrated close resemblance of survival curves between original and synthetic cohorts. Inter-variable relationships were preserved in univariable outcome analysis enabling explorative analysis in our synthetic data. Additionally, training sample privacy is safeguarded mitigating possible patient re-identification, which we quantified using Hamming distances. We provide not only a proof-of-concept for synthetic data generation in multimodal clinical data for rare diseases, but also full public access to synthetic data sets to foster further research., (© 2024. The Author(s).)

The VEXAS syndrome, a genetically defined autoimmune disease, associated with various hematological neoplasms has been attracting growing attention since its initial description in 2020. While various therapeutic strategies have been explored in case studies, the optimal treatment strategy is still under investigation and allogeneic cell transplantation is considered the only curative treatment. Here, we describe 2 patients who achieved complete molecular remission of the underlying UBA1 mutant clone outside the context of allogeneic HCT. Both patients received treatment with the hypomethylating agent azacitidine, and deep molecular remission triggered treatment de-escalation and even cessation with sustained molecular remission in one of them. Prospective studies are necessary to clarify which VEXAS patients will benefit most from hypomethylating therapy and to understand the variability in the response to different treatment strategies., (© 2024. The Author(s).)

Despite recent refinements in the diagnostic and prognostic assessment of CEBPA mutations in AML, several questions remain open, i.e. implications of different types of basic region leucin zipper (bZIP) mutations, the role of co-mutations and the allelic state. Using pooled primary data analysis on 1010 CEBPA-mutant adult AML patients, a comparison was performed taking into account the type of mutation (bZIP: either typical in-frame insertion/deletion (InDel) mutations (bZIP InDel ), frameshift InDel or nonsense mutations inducing translational stop (bZIP STOP ) or single base-pair missense alterations (bZIP ms ), and transcription activation domain (TAD) mutations) and the allelic state (single (smCEBPA) vs. double mutant (dmCEBPA)). Only bZIP InDel patients had significantly higher rates of complete remission and longer relapse free and overall survival (OS) compared with all other CEBPA-mutant subgroups. Moreover, co-mutations in bZIP InDel patients (e.g. GATA2, FLT3, WT1 as well as ELN2022 adverse risk aberrations) had no independent impact on OS, whereas in non-bZIP InDel patients, grouping according to ELN2022 recommendations added significant prognostic information. In conclusion, these results demonstrate bZIP InDel mutations to be the major independent determinant of outcome in CEBPA-mutant AML, thereby refining current classifications according to WHO (including all dmCEBPA and smCEBPA bZIP) as well as ELN2022 and ICC recommendations (including CEBPA bZIP ms )., (© 2024. The Author(s).)

With the availability of effective targeted agents, significant changes have occurred in the management of patients with acute myeloid leukemia (AML) over the past several years, particularly for those considered unfit for intensive chemotherapy. While testing for measurable residual disease (MRD) is now routinely performed in patients treated with intensive chemotherapy to refine prognosis and, possibly, inform treatment decision-making, its value in the context of lower-intensity regimens is unclear. As such regimens have gained in popularity and can be associated with higher response rates, the need to better define the role of MRD assessment and the appropriate time points and assays used for this purpose has increased. This report outlines a roadmap for MRD testing in patients with AML treated with lower-intensity regimens. Experts from the European LeukemiaNet (ELN)-DAVID AML MRD working group reviewed all available data to propose a framework for MRD testing in future trials and clinical practice. A Delphi poll served to optimize consensus. Establishment of uniform standards for MRD assessments in lower-intensity regimens used in treating patients with AML is clinically relevant and important for optimizing testing and, ultimately, improving treatment outcomes of these patients., (© 2023 Wiley Periodicals LLC.)

Genetic lesions of IKZF1 are frequent events and well-established markers of adverse risk in acute lymphoblastic leukemia. However, their function in the pathophysiology and impact on patient outcome in acute myeloid leukemia (AML) remains elusive. In a multicenter cohort of 1606 newly diagnosed and intensively treated adult AML patients, we found IKZF1 alterations in 45 cases with a mutational hotspot at N159S. AML with mutated IKZF1 was associated with alterations in RUNX1, GATA2, KRAS, KIT, SF3B1, and ETV6, while alterations of NPM1, TET2, FLT3-ITD, and normal karyotypes were less frequent. The clinical phenotype of IKZF1-mutated AML was dominated by anemia and thrombocytopenia. In both univariable and multivariable analyses adjusting for age, de novo and secondary AML, and ELN2022 risk categories, we found mutated IKZF1 to be an independent marker of adverse risk regarding complete remission rate, event-free, relapse-free, and overall survival. The deleterious effects of mutated IKZF1 also prevailed in patients who underwent allogeneic hematopoietic stem cell transplantation (n = 519) in both univariable and multivariable models. These dismal outcomes are only partially explained by the hotspot mutation N159S. Our findings suggest a role for IKZF1 mutation status in AML risk modeling., (© 2023. The Author(s).)

Aberrant innate immune signaling has been identified as a potential key driver of the complex pathophysiology of myelodysplastic neoplasms (MDS). This study of a large, clinically and genetically well-characterized cohort of treatment-naïve MDS patients confirms intrinsic activation of inflammatory pathways in general mediated by caspase-1, interleukin (IL)-1β and IL-18 in low-risk (LR)-MDS bone marrow and reveals a previously unrecognized heterogeneity of inflammation between genetically defined LR-MDS subgroups. Principal component analysis resolved two LR-MDS phenotypes with low (cluster 1) and high (cluster 2) levels of IL1B gene expression, respectively. Cluster 1 contained 14/17 SF3B1-mutated cases, while cluster 2 contained 8/8 del(5q) cases. Targeted gene expression analysis of sorted cell populations showed that the majority of the inflammasome-related genes, including IL1B, were primarily expressed in the monocyte compartment, consistent with a dominant role in determining the inflammatory bone marrow environment. However, the highest levels of IL18 expression were found in hematopoietic stem and progenitor cells (HSPCs). The colony forming activity of healthy donor HSPCs exposed to monocytes from LR-MDS was increased by the IL-1β-neutralizing antibody canakinumab. This work reveals distinct inflammatory profiles in LR-MDS that are of likely relevance to the personalization of emerging anti-inflammatory therapies., (© 2023. The Author(s).)

We retrospectively studied 97 acute myeloid leukemia patients with trisomy 19 (median age at diagnosis 57 years; range, 17- 83 years) treated between 2001 and 2019 within two multicenter study groups. Trisomy 19 occurred alone in ten (10.5%) patients, with additional abnormalities being present in non-complex karyotypes in eight (8%) patients and in complex karyotypes in 79 (82%) patients. Altogether, karyotypes characterized by trisomies only were present in 27 (28%) patients. Data on response and outcome of intensively treated patients were available for 92 cases. The median follow-up was 6.4 years (95% confidence interval [95% CI]: 2.9-9.0 years). The complete remission (CR) rate after induction therapy was 52% (48 patients); the early death rate was 10% (n=9). Notably, patients with trisomy 19 as the sole abnormality had a CR rate of 89%. Allogeneic hematopoietic stem cell transplantation (allo-HCT) was performed in 34 (35%) patients (CR, n=19; active disease, n=15). Five-year relapse-free and overall survival rates were 26% (95% CI: 16-43%) and 20% (95% CI: 13-31%), respectively. Overall survival rates were significantly higher in patients with trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only (P=0.05). An Andersen-Gill model including allo-HCT as a time-dependent covariable on overall survival revealed that trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only was a favorable factor (hazard ratio [HR]=0.47; P=0.021); higher age at diagnosis had an adverse impact (10 years difference; HR=1.29; P=0.002), whereas allo-HCT did not have a beneficial impact (odds ratio=1.45; P=0.21). In our cohort, patients with trisomy 19 as the sole abnormality or within karyotypes characterized by trisomies only had a high CR rate and better clinical outcome.

The European LeukaemiaNet (ELN) measurable residual disease (MRD) working group has published consensus guidelines to standardize molecular genetic MRD testing of the t(8;21)(q22;q22.1) RUNX1::RUNX1T1, inv(16)(p13.1q22) CBFB::MYH11, t(15;17)(q24.1;q21.2) PML::RARA, and NPM1 type A markers. A study featuring 29 international laboratories was performed to assess interlaboratory variation in testing and the subsequent interpretation of results, both crucial to patient safety. Most participants in this study were able to detect, accurately quantify, and correctly interpret MRD testing results, with a level of proficiency expected from a clinical trial or standard-of-care setting. However, a few testing and interpretive errors were identified that, in a patient setting, would have led to misclassification of patient outcomes and inappropriate treatment pathways being followed. Of note, a high proportion of participants reported false-positive results in the NPM1 marker-negative sample. False-positive results may have clinical consequences, committing patients to unneeded additional chemotherapy and/or transplant with the attendant risk of morbidity and mortality, which therefore highlights the need for ongoing external quality assessment/proficiency testing in this area. Most errors identified in the study were related to the interpretation of results. It was noted that the ELN guidance lacks clarity for certain clinical scenarios and highlights the requirement for urgent revision of the guidelines to elucidate these issues and related educational efforts around the revisions to ensure effective dissemination., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Immunogenetic association studies may give rise to new hypotheses on the immune surveillance of cancer. We hypothesized that certain combinations of killer immunoglobulin-like receptor (KIR) and HLA genotypes may enhance natural killer (NK) cell immunity against nascent acute myeloid leukemia (AML) and, thereby, lead to a skewed genotype distribution among patients. For this purpose, we analyzed KIR and HLA genotypes of 1767 German patients with AML and compared the results with that of the data of 51 890 German volunteers who had registered with German bone marrow donor file (DKMS). Patient samples were retrieved from the Collaborative Biobank and the biorepository of the Study Alliance Leukemia. All samples were genotyped with high-resolution amplicon-based next-generation sequencing. Because of the large number of controls, this study was very sensitive to detect the impact of KIR genotype. Knowledge on KIRs and their cognate HLA ligands allowed for testing of several hypotheses of NK cell-mediated endogenous leukemia surveillance. We did not find significant differences between the 2 cohorts in regard to the presence or absence of single KIR genes. When grouped based on telomeric or centromeric gene content, the major haplotypes A/A, A/B, and B/B were equally distributed among patients and control subjects. Using information on KIRs and their HLA ligands, we further tested receptor-ligand models and summation models without revealing markedly significant differences between patients and controls, albeit we observed a trend pointing at a minor protective effect of a low number of inhibitory KIR/KIR-ligand pairs. The results suggest that the KIR/KIR-ligand genotype has no effect on the susceptibility for the development of de novo AML., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Tandem-duplication mutations of the UBTF gene (UBTF-TDs) coding for the upstream binding transcription factor have recently been described in pediatric patients with acute myeloid leukemia (AML) and were found to be associated with particular genetics (trisomy 8 (+8), FLT3-internal tandem duplications (FLT3-ITD), WT1-mutations) and inferior outcome. Due to limited knowledge on UBTF-TDs in adult AML, we screened 4247 newly diagnosed adult AML and higher-risk myelodysplastic syndrome (MDS) patients using high-resolution fragment analysis. UBTF-TDs were overall rare (n = 52/4247; 1.2%), but significantly enriched in younger patients (median age 41 years) and associated with MDS-related morphology as well as significantly lower hemoglobin and platelet levels. Patients with UBTF-TDs had significantly higher rates of +8 (34% vs. 9%), WT1 (52% vs. 7%) and FLT3-ITD (50% vs. 20.8%) co-mutations, whereas UBTF-TDs were mutually exclusive with several class-defining lesions such as mutant NPM1, in-frame CEBPA bZIP mutations as well as t(8;21). Based on the high-variant allele frequency found and the fact that all relapsed patients analyzed (n = 5) retained the UBTF-TD mutation, UBTF-TDs represent early clonal events and are stable over the disease course. In univariate analysis, UBTF-TDs did not represent a significant factor for overall or relapse-free survival in the entire cohort. However, in patients under 50 years of age, who represent the majority of UBTF-mutant patients, UBTF-TDs were an independent prognostic factor for inferior event-free (EFS), relapse-free (RFS) and overall survival (OS), which was confirmed by multivariable analyses including established risk factors such as age and ELN2022 genetic risk groups (EFS [HR: 2.20; 95% CI 1.52-3.17, p < 0.001], RFS [HR: 1.59; 95% CI 1.02-2.46, p = 0.039] and OS [HR: 1.64; 95% CI 1.08-2.49, p = 0.020]). In summary, UBTF-TDs appear to represent a novel class-defining lesion not only in pediatric AML but also younger adults and are associated with myelodysplasia and inferior outcome in these patients., (© 2023. The Author(s).)

Background: Increasingly large and complex biomedical data sets challenge conventional hypothesis-driven analytical approaches, however, data-driven unsupervised learning can detect inherent patterns in such data sets., Methods: While unsupervised analysis in the medical literature commonly only utilizes a single clustering algorithm for a given data set, we developed a large-scale model with 605 different combinations of target dimensionalities as well as transformation and clustering algorithms and subsequent meta-clustering of individual results. With this model, we investigated a large cohort of 1383 patients from 59 centers in Germany with newly diagnosed acute myeloid leukemia for whom 212 clinical, laboratory, cytogenetic and molecular genetic parameters were available., Results: Unsupervised learning identifies four distinct patient clusters, and statistical analysis shows significant differences in rate of complete remissions, event-free, relapse-free and overall survival between the four clusters. In comparison to the standard-of-care hypothesis-driven European Leukemia Net (ELN2017) risk stratification model, we find all three ELN2017 risk categories being represented in all four clusters in varying proportions indicating unappreciated complexity of AML biology in current established risk stratification models. Further, by using assigned clusters as labels we subsequently train a supervised model to validate cluster assignments on a large external multicenter cohort of 664 intensively treated AML patients., Conclusions: Dynamic data-driven models are likely more suitable for risk stratification in the context of increasingly complex medical data than rigid hypothesis-driven models to allow for a more personalized treatment allocation and gain novel insights into disease biology., (© 2023. The Author(s).)

NPM 1-mutated acute myeloid leukemia (AML) shows unique features. However, the characteristics of "therapy-related" NPM1-mutated AML (t-NPM1 AML) are poorly understood. We compared the genetics, transcriptional profile, and clinical outcomes of t-NPM1 AML, de novo NPM1-mutated AML (dn-NPM1 AML), and therapy-related AML (t-AML) with wild-type NPM1 (t-AML). Normal karyotype was more frequent in t-NPM1 AML (n = 78/96, 88%) and dn-NPM1 (n = 1986/2394, 88%) than in t-AML (n = 103/390, 28%; P < .001). DNMT3A and TET2 were mutated in 43% and 40% of t-NPM1 AML (n = 107), similar to dn-NPM1 (n = 88, 48% and 30%; P > 0.1), but more frequently than t-AML (n = 162; 14% and 10%; P < 0.001). Often mutated in t-AML, TP53 and PPM1D were wild-type in 97% and 96% of t-NPM1 AML, respectively. t-NPM1 and dn-NPM1 AML were transcriptionally similar, (including HOX genes upregulation). At 62 months of median follow-up, the 3-year overall survival (OS) for t-NPM1 AML (n = 96), dn-NPM1 AML (n = 2394), and t-AML (n = 390) were 54%, 60%, and 31%, respectively. In multivariable analysis, OS was similar for the NPM1-mutated groups (hazard ratio [HR] 0.9; 95% confidence interval [CI], 0.65-1.25; P = .45), but better in t-NPM1 AML than in t-AML (HR, 1.86; 95% CI, 1.30-2.68; P < .001). Relapse-free survival was similar between t-NPM1 and dn-NPM1 AML (HR, 1.02; 95% CI, 0.72-1.467; P = .90), but significantly higher in t-NPM1 AML versus t-AML (HR, 1.77; 95% CI, 1.19-2.64; P = .0045). t-NPM1 and dn-NPM1 AML have overlapping features, suggesting that they should be classified as a single disease entity., (© 2023 by The American Society of Hematology.)

Background: Infectious complications reflect a major challenge in the treatment of patients with acute myeloid leukemia (AML). Both induction chemotherapy and epigenetic treatment with hypomethylating agents (HMA) are associated with severe infections, while neutropenia represents a common risk factor. Here, 220 consecutive and newly diagnosed AML patients were analyzed with respect to infectious complications dependent on treatment intensity and antifungal prophylaxis applied to these patients., Patients and Methods: We retrospectively analyzed 220 patients with newly diagnosed AML at a tertiary care hospital between August 2016 and December 2020. The median age of AML patients undergoing induction chemotherapy (n = 102) was 61 years (25-76 years). Patients receiving palliative AML treatment (n = 118) had a median age of 75 years (53-91 years). We assessed the occurrence of infectious complication including the classification of pulmonary invasive fungal disease (IFD) according to the EORTC/MSG criteria at diagnosis and until day 100 after initiation of AML treatment. Furthermore, admission to intensive care unit (ICU) and subsequent outcome was analyzed for both groups of AML patients, respectively., Results: AML patients subsequently allocated to palliative AML treatment have a significantly higher risk of pneumonia at diagnosis compared to patients undergoing induction chemotherapy (37.3% vs. 13.7%, P < 0.001) including a higher probability of atypical pneumonia (22.0% vs. 10.8%, P = 0.026). Furthermore, urinary tract infections are more frequent in the palliative subgroup at the time of AML diagnosis (5.1% vs. 0%, P = 0.021). Surprisingly, the incidence of pulmonary IFD is significantly lower after initiation of palliative AML treatment compared to the occurrence after induction chemotherapy (8.4% vs. 33.3%, P < 0.001) despite only few patients of the palliative treatment group received Aspergillus spp.-directed antifungal prophylaxis. The overall risk for infectious complications at AML diagnosis is significantly higher for palliative AML patients at diagnosis while patients undergoing induction chemotherapy have a significantly higher risk of infections after initiation of AML treatment. In addition, there is a strong correlation between the occurrence of pneumonia including atypical pneumonia and pulmonary IFD and the ECOG performance status at diagnosis in the palliative AML patient group. Analysis of intensive care unit (ICU) treatment (e.g. in case of sepsis or pneumonia) for both subgroups reveals a positive outcome in 10 of 15 patients (66.7%) with palliative AML treatment and in 15 of 18 patients (83.3%) receiving induction chemotherapy. Importantly, the presence of infections and the ECOG performance status at diagnosis significantly correlate with the overall survival (OS) of palliative AML patients (315 days w/o infection vs. 69 days with infection, P 0.0049 and 353 days for ECOG < 1 vs. 50 days for ECOG > 2, P < 0.001, respectively) in this intent-to-treat analysis., Conclusion: The risk and the pattern of infectious complications at diagnosis and after initiation of AML therapy depends on age, ECOG performance status and subsequent treatment intensity. A comprehensive diagnostic work-up for identification of pulmonary IFD is indispensable for effective treatment of pneumonia in AML patients. The presence of infectious complications at diagnosis contributes to an inferior outcome in elderly AML patients., (© 2022. The Author(s).)

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

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by high rate of relapse and mortality. Current chemotherapies whilst successful in eradicating blasts, are less effective in eliminating relapse-causing leukemic stem cells (LSCs). Although LSCs are usually identified as CD34+CD38- cells, there is significant heterogeneity in surface marker expression, and CD34- LSCs exist particularly in NPM1mut AMLs. By analyzing diagnostic primary DNMT3AmutNPM1mut AML samples, we suggest a novel flow cytometry sorting strategy particularly useful for CD34neg AML subtypes. To enrich for LSCs independently of CD34 status, positive selection for GPR56 and negative selection for NKG2D ligands are used. We show that the functional reconstitution capacity of CD34- and CD34+ LSCs as well as their transcriptomes are very similar which support phenotypic plasticity. Furthermore, we show that although CD34+ subpopulations can contain next to LSCs also normal and/or preleukemic hematopoietic stem cells (HSCs), this is not the case in CD34-GPR56+NKG2DL- enriched LSCs which thus can be isolated with high purity. Finally, we show that patients with AML, who retain at the time of diagnosis a reserve of normal and/or preleukemic HSCs in their bone marrow able to reconstitute immunocompromised mice, have significantly longer relapse-free and overall survival than patients with AML in whom functional HSCs are no longer detectable., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Achievement of complete remission signifies a crucial milestone in the therapy of acute myeloid leukemia (AML) while refractory disease is associated with dismal outcomes. Hence, accurately identifying patients at risk is essential to tailor treatment concepts individually to disease biology. We used nine machine learning (ML) models to predict complete remission and 2-year overall survival in a large multicenter cohort of 1,383 AML patients who received intensive induction therapy. Clinical, laboratory, cytogenetic and molecular genetic data were incorporated and our results were validated on an external multicenter cohort. Our ML models autonomously selected predictive features including established markers of favorable or adverse risk as well as identifying markers of so-far controversial relevance. De novo AML, extramedullary AML, double-mutated CEBPA, mutations of CEBPA-bZIP, NPM1, FLT3-ITD, ASXL1, RUNX1, SF3B1, IKZF1, TP53, and U2AF1, t(8;21), inv(16)/t(16;16), del(5)/del(5q), del(17)/del(17p), normal or complex karyotypes, age and hemoglobin concentration at initial diagnosis were statistically significant markers predictive of complete remission, while t(8;21), del(5)/del(5q), inv(16)/t(16;16), del(17)/del(17p), double-mutated CEBPA, CEBPA-bZIP, NPM1, FLT3-ITD, DNMT3A, SF3B1, U2AF1, and TP53 mutations, age, white blood cell count, peripheral blast count, serum lactate dehydrogenase level and hemoglobin concentration at initial diagnosis as well as extramedullary manifestations were predictive for 2-year overall survival. For prediction of complete remission and 2-year overall survival areas under the receiver operating characteristic curves ranged between 0.77-0.86 and between 0.63-0.74, respectively in our test set, and between 0.71-0.80 and 0.65-0.75 in the external validation cohort. We demonstrated the feasibility of ML for risk stratification in AML as a model disease for hematologic neoplasms, using a scalable and reusable ML framework. Our study illustrates the clinical applicability of ML as a decision support system in hematology.

The development and regulation of malignant self-renewal remain unresolved issues. Here, we provide biochemical, genetic, and functional evidence that dynamics in ribosomal RNA (rRNA) 2'-O-methylation regulate leukemia stem cell (LSC) activity in vivo. A comprehensive analysis of the rRNA 2'-O-methylation landscape of 94 patients with acute myeloid leukemia (AML) revealed dynamic 2'-O-methylation specifically at exterior sites of ribosomes. The rRNA 2'-O-methylation pattern is closely associated with AML development stage and LSC gene expression signature. Forced expression of the 2'-O-methyltransferase fibrillarin (FBL) induced an AML stem cell phenotype and enabled engraftment of non-LSC leukemia cells in NSG mice. Enhanced 2'-O-methylation redirected the ribosome translation program toward amino acid transporter mRNAs enriched in optimal codons and subsequently increased intracellular amino acid levels. Methylation at the single site 18S-guanosine 1447 was instrumental for LSC activity. Collectively, our work demonstrates that dynamic 2'-O-methylation at specific sites on rRNAs shifts translational preferences and controls AML LSC self-renewal., Significance: We establish the complete rRNA 2'-O-methylation landscape in human AML. Plasticity of rRNA 2'-O-methylation shifts protein translation toward an LSC phenotype. This dynamic process constitutes a novel concept of how cancers reprogram cell fate and function. This article is highlighted in the In This Issue feature, p. 247., (©2022 The Authors; Published by the American Association for Cancer Research.)

Functional perturbations of the cohesin complex with subsequent changes in chromatin structure and replication are reported in a multitude of cancers including acute myeloid leukemia (AML). Mutations of its STAG2 subunit may predict unfavorable risk as recognized by the 2022 European Leukemia Net recommendations, but the underlying evidence is limited by small sample sizes and conflicting observations regarding clinical outcomes, as well as scarce information on other cohesion complex subunits. We retrospectively analyzed data from a multi-center cohort of 1615 intensively treated AML patients and identified distinct co-mutational patters for mutations of STAG2, which were associated with normal karyotypes (NK) and concomitant mutations in IDH2, RUNX1, BCOR, ASXL1, and SRSF2. Mutated RAD21 was associated with NK, mutated EZH2, KRAS, CBL, and NPM1. Patients harboring mutated STAG2 were older and presented with decreased white blood cell, bone marrow and peripheral blood blast counts. Overall, neither mutated STAG2, RAD21, SMC1A nor SMC3 displayed any significant, independent effect on clinical outcomes defined as complete remission, event-free, relapse-free or overall survival. However, we found almost complete mutual exclusivity of genetic alterations of individual cohesin subunits. This mutual exclusivity may be the basis for therapeutic strategies via synthetic lethality in cohesin mutated AML., (© 2023. The Author(s).)

We retrospectively studied 125 patients with acute myeloid leukemia and trisomy 4 (median age at diagnosis, 58 years; range, 16-77 years) treated between 2000 and 2019 within a multicenter study. Trisomy 4 was the sole abnormality in 28 (22%) patients and additional abnormalities were present in 97 (78%) patients. Twenty-two (22%) and 15 (15%) of 101 tested patients harbored NPM1 and FLT3-ITD mutations. Two (3%) of 72 tested patients had double CEBPA mutations. Data on response to intensive anthracycline-based induction therapy were available for 119 patients. Complete remission was achieved in 67% (n=80) and the early death rate was 5% (n=6). Notably, patients with trisomy 4 as sole abnormality had a complete remission rate of 89%. Allogeneic hematopoietic cell transplantation was performed in 40 (34%) patients, of whom 19 were transplanted in first complete remission. The median follow-up of the intensively treated cohort was 5.76 years (95% confidence interval [95% CI]: 2.99-7.61 years). The 5-year overall survival and relapse-free survival rates were 30% (95% CI: 22-41%) and 27% (95% CI: 18-41%), respectively. An Andersen-Gill regression model on overall survival revealed that favorable-risk according to the European LeukemiaNet classification (hazard ratio [HR]=0.34; P=0.006) and trisomy 4 as sole abnormality (HR=0.41; P=0.01) were favorable factors, whereas age with a difference of 10 years (HR=1.15; P=0.11), female gender (HR=0.74; P=0.20) and allogeneic hematopoietic cell transplantation (HR=0.64; P=0.14) did not have an significant impact. In our cohort, patients with trisomy 4 as their sole abnormality had a high complete remission rate and favorable clinical outcome. Allogeneic hematopoietic cell transplantation did not seem to improve overall survival.

The detection of clonal hematopoiesis (CH) in patients with hematologic neoplasms who are undergoing a cellular therapy is common. The most frequently used cellular therapy procedures include autologous and allogeneic hematopoietic stem cell transplantation (HSCT) and, more recently, chimeric antigen receptor (CAR) T‑cell therapy. All three procedures differ fundamentally in terms of harvesting and manufacturing aspects as well as usage of the respective cell product. Therefore, the importance of CH in relation to the respective treatment method must be evaluated and assessed differently. In autologous HSCT, the extent of previous cytotoxic therapy significantly contributes to the high prevalence of CH. The clinically most important aspect is the development of secondary neoplasms from a pre-existing CH clone and the potential risk for enhanced cardiovascular side effects. In allogeneic HSCT, the donor selection with respect to the age largely determines the probability for the presence of CH. In this setting, the development of secondary malignancies only plays a minor role compared to the autologous HSCT. In fact, the induction of a graft versus host (GvH) or a graft versus leukemia (GvL) effect and its influence on progression-free and overall survival seem to be of possible clinical relevance. The CAR T‑cell therapy is closely linked to inflammatory reactions regarding its mode of action and the associated side effects. In this context CH might be closely linked to the effectiveness and side effects of the CAR T‑cell therapy. Initial data reported a high prevalence of CH in patients before CAR T‑cell therapy and indicated an increased rate of inflammatory side effects, although no negative effect on survival has yet been demonstrated., (© 2022. The Author(s).)

Background: Diagnosis of acute myeloid leukemia (AML) is associated with poor outcome in elderly and unfit patients. Recently, approval of the BCL-2 inhibitor venetoclax (VEN) in combination with hypo-methylating agents (HMA) led to a significant improvement of response rates and survival. Further, application in the relapsed or refractory (r/r) AML setting or in context of allogeneic stem cell transplantation (alloHSCT) seems feasible., Methods and Patients: Fifty-six consecutive adult AML patients on VEN from January 2019 to June 2021 were analyzed retrospectively. Patients received VEN either as first-line treatment, as subsequent therapy (r/r AML excluding prior alloHSCT), or at relapse after alloHSCT. VEN was administered orally in 28-day cycles either combined with HMA or low-dose cytarabine (LDAC)., Results: After a median follow-up of 11.5 (range 6.1-22.3) months, median overall survival (OS) from start of VEN treatment was 13.3 (2.2-20.5) months, 5.0 (0.8-24.3) months and 4.0 (1.5-22.1) months for first-line, subsequent line treatment and at relapse post-alloHSCT, respectively. Median OS was 11.5 (10-22.3) months from start of VEN when subsequent alloHSCT was carried out. Relapse-free survival (RFS) for the total cohort was 10.2 (2.2 - 24.3) months. Overall response rate (composite complete remission + partial remission) was 51.8% for the total cohort (61.1% for VEN first-line treatment, 52.2% for subsequent line and 42.8% at relapse post-alloHSCT). Subgroup analysis revealed a significantly reduced median OS in FLT3-ITD mutated AML with 3.4 (1.9-4.9) months versus 10.4 (0.8-24.3) months for non-mutated cases, (HR 4.45, 95% CI 0.89-22.13, p = 0.0002). Patients harboring NPM1 or IDH1/2 mutations lacking co-occurrence of FLT3-ITD showed a survival advantage over patients without those mutations (11.2 (5-24.3) months versus 5.0 (0.8-22.1) months, respectively, (HR 0.53, 95% CI 0.23 - 1.21, p = 0.131). Multivariate analysis revealed mutated NPM1 as a significant prognostic variable for achieving complete remission (CR) (HR 19.14, 95% CI 2.30 - 436.2, p < 0.05). The most common adverse events were hematological, with grade 3 and 4 neutropenia and thrombocytopenia reported in 44.6% and 14.5% of patients, respectively., Conclusion: Detailed analyses on efficacy for common clinical scenarios, such as first-line treatment, subsequent therapy (r/r AML), and application prior to and post-alloHSCT, are presented. The findings suggest VEN treatment combinations efficacious not only in first-line setting but also in r/r AML. Furthermore, VEN might play a role in a subgroup of patients with failure to conventional chemotherapy as a salvage regimen aiming for potential curative alloHSCT., (© 2022. The Author(s).)

The EUROPE phase 2 trial investigated the predictive value of biomarkers on the clinical efficacy of single agent romiplostim (ROM) treatment in patients with lower-risk myelodysplastic neoplasms (LR-MDS) and thrombocytopenia within the 'European Myelodysplastic Neoplasms Cooperative Group' (EMSCO) network. A total of 77 patients with LR-MDS and a median platelet count of 25/nl were included, all patients received ROM at a starting dose of 750 μg by SC injection weekly. Thirty-two patients (42%) achieved a hematologic improvement of platelets (HI-P) with a median duration of 340 days. Neutrophil (HI-N) and erythroid (HI-E) responses were observed in three (4%) and seven (9%) patients, respectively. We could not confirm previous reports that HI-P correlated with baseline endogenous thrombopoietin levels and platelet transfusion history, but SRSF2 mutation status and hemoglobin levels at baseline were significantly linked to HI-P. Sequential analysis of variant allelic frequency of mutations like SRSF2 did not reveal an impact of ROM on clonal evolution in both responders and non-responders. In summary, our study confirms the safety and efficacy of ROM in LR-MDS patients and may allow to better define subgroups of patients with a high likelihood of response., (© 2022. The Author(s).)

Background: The role of allogeneic hematopoietic cell transplantation (alloHCT) in acute myeloid leukemia (AML) with mutated IDH1/2 has not been defined. Therefore, we analyzed a large cohort of 3234 AML patients in first complete remission (CR1) undergoing alloHCT or conventional chemo-consolidation and investigated outcome in respect to IDH1/2 mutational subgroups (IDH1 R132C, R132H and IDH2 R140Q, R172K)., Methods: Genomic DNA was extracted from bone marrow or peripheral blood samples at diagnosis and analyzed for IDH mutations with denaturing high-performance liquid chromatography, Sanger sequencing and targeted myeloid panel next-generation sequencing, respectively. Statistical as-treated analyses were performed using R and standard statistical methods (Kruskal-Wallis test for continuous variables, Chi-square test for categorical variables, Cox regression for univariate and multivariable models), incorporating alloHCT as a time-dependent covariate., Results: Among 3234 patients achieving CR1, 7.8% harbored IDH1 mutations (36% R132C and 47% R132H) and 10.9% carried IDH2 mutations (77% R140Q and 19% R172K). 852 patients underwent alloHCT in CR1. Within the alloHCT group, 6.2% had an IDH1 mutation (43.4% R132C and 41.4% R132H) and 10% were characterized by an IDH2 mutation (71.8% R140Q and 24.7% R172K). Variants IDH1 R132C and IDH2 R172K showed a significant benefit from alloHCT for OS (p = .017 and p = .049) and RFS (HR = 0.42, p = .048 and p = .009) compared with chemotherapy only. AlloHCT in IDH2 R140Q mutated AML resulted in longer RFS (HR = 0.4, p = .002)., Conclusion: In this large as-treated analysis, we showed that alloHCT is able to overcome the negative prognostic impact of certain IDH mutational subclasses in first-line consolidation treatment and could pending prognostic validation, provide prognostic value for AML risk stratification and therapeutic decision making., (© 2022. The Author(s).)

The aim of this study was to characterize the mutational landscape of patients with FLT3-mutated acute myeloid leukemia (AML) treated within the randomized CALGB 10603/RATIFY trial evaluating intensive chemotherapy plus the multi-kinase inhibitor midostaurin versus placebo. We performed sequencing of 262 genes in 475 patients: mutations occurring concurrently with the FLT3-mutation were most frequent in NPM1 (61%), DNMT3A (39%), WT1 (21%), TET2 (12%), NRAS (11%), RUNX1 (11%), PTPN11 (10%), and ASXL1 (8%) genes. To assess effects of clinical and genetic features and their possible interactions, we fitted random survival forests and interpreted the resulting variable importance. Highest prognostic impact was found for WT1 and NPM1 mutations, followed by white blood cell count, FLT3 mutation type (internal tandem duplications vs. tyrosine kinase domain mutations), treatment (midostaurin vs. placebo), ASXL1 mutation, and ECOG performance status. When evaluating two-fold variable combinations the most striking effects were found for WT1:NPM1 (with NPM1 mutation abrogating the negative effect of WT1 mutation), and for WT1:treatment (with midostaurin exerting a beneficial effect in WT1-mutated AML). This targeted gene sequencing study provides important, novel insights into the genomic background of FLT3-mutated AML including the prognostic impact of co-mutations, specific gene-gene interactions, and possible treatment effects of midostaurin., (© 2022. The Author(s).)

Measurable residual disease (MRD) detected by multiparametric flow cytometry (MFC) is associated with unfavorable outcome in patients with AML. A simple, broadly applicable eight-color panel was implemented and analyzed utilizing a hierarchical gating strategy with fixed gates to develop a clear-cut LAIP-based DfN approach. In total, 32 subpopulations with aberrant phenotypes with/without expression of markers of immaturity were monitored in 246 AML patients after completion of induction chemotherapy. Reference values were established utilizing 90 leukemia-free controls. Overall, 73% of patients achieved a response by cytomorphology. In responders, the overall survival was shorter for MRD pos patients (HR 3.8, p = 0.006). Overall survival of MRD neg non-responders was comparable to MRD neg responders. The inter-rater-reliability for MRD detection was high with a Krippendorffs α of 0.860. The mean time requirement for MRD analyses at follow-up was very short with 04:31 minutes. The proposed one-tube MFC approach for detection of MRD allows a high level of standardization leading to a promising inter-observer-reliability with a fast turnover. MRD defined by this strategy provides relevant prognostic information and establishes aberrancies outside of cell populations with markers of immaturity as an independent risk feature. Our results imply that this strategy may provide the base for multicentric immunophenotypic MRD assessment., (© 2022. The Author(s).)

Although extramedullary manifestations (EMs) are frequent in patients with acute myeloid leukemia (AML), they are often not detected during clinical workup and neither imaging- nor molecularly based diagnostic strategies are established to reveal their existence. Still, the detection of EM is essential for therapeutic decision-making, as EM present with aggressive and resistant disease and since mutational profiling might render patients within a different risk category, requiring personalized therapeutic strategies. Here, we report the case of an AML patient presenting with AML bone marrow (BM) infiltration and molecularly distinct EM at time of diagnosis followed by multiple EM relapses while undergoing several intensive chemotherapies including allogeneic hematopoietic cell transplantations (alloHCTs). 18 Fluorodesoxy-glucose positron emission tomography ( 18 FDG-PET)-imaging revealed EM sites in the mediastinum, duodenum, skin, and in retroperitoneal tissue, whereas recurrent BM biopsies showed continuous cytomorphologic and cytogenetic remission after alloHCT. To investigate the molecular background of the aggressive character of extramedullary disease and its differential treatment response, we performed amplicon-based next generation sequencing. An exon 4 (c.497&#95;498insGA) frameshift RUNX1 mutation was exclusively found in all of the patient's EM sites, but not in the BM or in peripheral blood samples at time of EM reoccurrence. In addition, we detected an exon 13 (c.3306G>T) ASXL1 point mutation only in the retroperitoneal tumor tissue at the time of the fourth relapse. In contrast to the patient's intermediate-risk BM AML at diagnosis according to ELN2017, EM sites showed molecular adverse-risk features implicating intensified strategies like cellular therapies. Notably, disease relapse could only be detected by imaging throughout the course of disease. This case demonstrates both the necessity of continuous molecular profiling of EM to reveal differential molecular composition of EM and BM-derived AML, supposedly leading to divergent susceptibility to established therapies, as well as recurrent 18 FDG-PET-imaging for detecting residual disease and assessment of treatment response in case of EM AML., Competing Interests: Competing interests: The authors declare that there is no conflict of interest. C.T. is the CEO and co-owner of AgenDix GmbH., (© The Author(s), 2022.)

Monitoring of measurable residual disease (MRD) in patients with acute myeloid leukemia (AML) is predictive of disease recurrence and may identify patients who benefit from treatment intensification. Current MRD techniques rely on multicolor flow cytometry or molecular methods, but are limited in applicability or sensitivity. We evaluated the feasibility of a novel approach for MRD detection in peripheral blood (PB), which combines immunomagnetic preenrichment and fluorescence-activated cell sorting (FACS) for isolation of CD34+ cells with error-reduced targeted next-generation sequencing (NGS). For clinical validation, we retrospectively analyzed 429 PB and 55 bone marrow (BM) samples of 40 patients with AML or high-risk MDS, with/without molecular relapse based on CD34+ donor chimerism (DC), in complete remission after allogeneic stem cell transplantation. Enrichment of CD34+ cells for NGS increased the detection of mutant alleles in PB ∼1000-fold (median variant allele frequency, 1.27% vs 0.0046% in unsorted PB; P < .0001). Although a strong correlation was observed for the parallel analysis of CD34+ PB cells with NGS and DC (r = 0.8601), the combination of FACS and NGS improved sensitivity for MRD detection in dilution experiments ∼10-fold to levels of 10-6. In both assays, MRD detection was superior using PB vs BM for CD34+ enrichment. Importantly, NGS on CD34+ PB cells enabled prediction of molecular relapse with high sensitivity (100%) and specificity (91%), and significantly earlier (median, 48 days; range, 0-281; P = .0011) than by CD34+ DC or NGS of unsorted PB, providing additional time for therapeutic intervention. Moreover, panel sequencing in CD34+ cells allowed for the early assessment of clonal trajectories in hematological complete remission., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Background: Guidance regarding adjuvant treatment decisions in stage II colorectal cancer (CRC) remains uncertain due to lack of predictive clinical or molecular markers. Recently, postoperative circulating tumour (ct)DNA has been demonstrated to be a strong prognostic marker in early colon cancer., Patients and Methods: CIRCULATE enrols patients with stage II microsatellite stable CRC in Germany (AIO) and Austria (ABCSG). Within the AIO, screening is supported by ColoPredict Plus 2.0, a molecular registry, and screening platform for interventional trials. Patient-specific mutations are centrally analysed by next generation sequencing in the resected primary tumour. A postoperative plasma sample is subsequently screened for the specific mutation(s). ctDNA positive (ctDNApos) patients are randomised (2:1) chemotherapy (capecitabine, oxaliplatin added an investigator's choice) or to follow-up (control group). ctDNA negative (ctDNAneg) patients are randomised (1:4) to be followed-up within CIRCULATE (control group) or outside the trial. Patients in the control group remain blinded to the ctDNA results. The primary objective is to compare disease free survival (DFS) of ctDNApos patients with chemotherapy or control. To demonstrate a treatment effect with a hazard ratio of 0.617 (3-year DFS rates 42.5% vs. 25%), 231 ctDNApos and estimated 2079 ctDNAneg patients are randomised. Secondary aims include to compare overall survival and DFS in the ctDNApos and ctDNAneg patient cohorts and ctDNA kinetics., Conclusion: The CIRCULATE trial may establish ctDNA for adjuvant treatment decision in stage II colon cancer - and with the secondary objectives - support a ctDNA guided follow up in colon cancer stage II and beyond., Competing Interests: Declaration of Competing Interest GF received honoraries for lectures from Amgen, Bayer, BMS, Falk Foundation, Merck, MSD, Roche, Servier, Shire, honoraries for advisory boards from Amgen, Bayer, BMS, Merck, MSD, Pierre Fabre, Roche, Sanofi-Aventis, Servier, Shire and research funding from Merck. ARS received honoraries for lectures fromAmgen, AstraZeneca, Aurikamed, BMS, Celgene, iomedico, Lilly, Merck Serono, MCI Global, med publico, MSD, Pfizer, promedicis, Roche, Sanofi-Aventis, Servier , honoraries for advisory boards from Amgen, AstraZeneca, Baxalta, BMS, Celgene, Merck Serono, MSD, Onkowissen.de, Pierre Fabre, Pfizer, Roche, Servier, Onkowissen.de , travel expenses from AstraZeneca, Celgene, Ipsen, MCI Global, Merck Serono, MSD, onkowissen.de, Pierre Fabre, Roche, Servier and research funding for clinical studies from Roche and Ipsen, and compensation for clinical trials / research from Amgen, Alexion, Astra Zeneca, Celgene, Ipsen, Lilly, Roche, Servier, AIO Studien gGmbH, Agricola, PPD Global Limited UK, Mologen Berlin, Universität München, Universität Erlangen, Universität Köln, Pharma Consulting Group AB Schweden, Syneed medidata GmbH, Rafael-Pharmaceutics; BioNTech. LW has stocks from Guardant Health, received honoraries for lectures from Roche;Bristol-Myers Squibb; Lilly; Novocure; Merck Serono; Amgen; Pierre Fabre; Servier; MSD, honoraries for advisory boards from Bristol-Myers Squibb; Roche; Lilly; Novocure; Merck Serono; Takeda; MSD; Amgen; PharmaMar; Bayer, received research funding from Roche and Novocure and travel expenses from Ipsen; Pfizer; Merck Serono; Astra Zeneca. NvB received honoraries for lectures from Forum für Medizinische Fortbildung (FomF)CT has partly ownership at AgenDix GmbH and received honoraries for advisory boards from JAZZ, Novartis, honoraries for lectures from Novartis, Astellas, JAZZ, Janssen; Thermo and research funding from Novartis, Bayer and JAZZ. AT received honoraris for lecture from Amgen, AstraZeneca, BMS, Celgene, iomedico, Lilly, Merck Serono, MCI Global, med publico, MSD, Pfizer, promedicis, Roche, Sanofi-Aventis, Servier and support for clinical studies / research from Amgen, Alexion, Astra Zeneca, Celgene, Ipsen, Lilly, Roche, Servier, AIO Studien gGmbH, BioNTech. JW, CL, ALK,SW,DEA, and MK declared no conflicts of interest., (Copyright © 2021. Published by Elsevier Inc.)