NPM1-mutated (NPM1-mut) acute myeloid leukemia (AML) is generally associated with a more favorable outcome, although the presence of additional gene mutations can influence patient prognosis. We analyzed intensively-treated adult NPM1-mut AML patients included in the HARMONY Alliance database. A newly developed risk classification, which included combinations of co-mutations in FLT3-ITD, DNMT3A, IDH1/IDH2, and TET2 genes, was applied to a training cohort of NPM1-mut AML patients included in clinical trials (n = 1001), an internal validation cohort more representative of real-world settings (n = 762), and an external validation cohort enrolled in UK-NCRI trials (n = 585). The HARMONY classification considered 51.8% of the NPM1-mut AML training cohort patients as favorable, 24.8% as intermediate, and 23.4% as adverse risk, with median overall survival (OS) of 14.4, 2.2, and 0.9 years, respectively; p < 0.001), thereby reclassifying 42.7% of NPM1-mut patients into a different European LeukemiaNet (ELN) 2022 risk category. These results were confirmed both in an internal and external validation cohort. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in first complete remission (CR1) showed the highest benefit in the NPM1-mut adverse-risk subgroup. The HARMONY classification provides the basis for a refined genetic risk stratification for adult NPM1-mut AML with potential clinical impact on allo-HSCT decision-making.

Longitudinal changes of somatic nuclear and mitochondrial DNA mutations in genetically stable CLL.

Abstract IDH1 and IDH2 are frequently mutated in various cancers, including acute leukemias. However, the distinct mechanisms by which mutant IDH1 or IDH2 drive hematopoietic neoplasms remain poorly understood. Here, we analyzed DNA methylation in IDH1- and IDH2-mutant acute myeloid leukemia and found neutrophil lineage-specific epigenetic alterations in IDH1-mutant patients that went along with severely impaired neutrophil differentiation. Transcriptional analysis of normal hematopoiesis in humans and mice revealed a strong physiological upregulation of IDH1/Idh1 in myeloid progenitors. To study the functional effects of Idh1 mutations on hematopoiesis in a preleukemic setting, we used a genetically engineered inducible mouse model expressing a heterozygous Idh1 mutation under control of the endogenous promotor. Our study revealed a cell-intrinsic block in neutrophil differentiation caused by repression of myeloid transcription programs in neutrophil progenitors. This included impaired expression of Cebpe, which encodes a key transcription factor regulating neutrophil differentiation. Reactivation of Cebpe expression, by overexpression of its upstream regulator Cebpa or following treatment with hypomethylating agents, restored differentiation, indicating that the differentiation block is reversible. In summary, we found a reversible, preleukemic impairment of neutrophil differentiation in IDH1-mutant hematopoiesis that correlates with elevated IDH1 expression in myeloid progenitors and likely explains the strong association of IDH1 mutations with myeloid neoplasms.

ABSTRACT:The mechanisms that lead to extramedullary tropism of acute myeloid leukemia (eAML) remain obscure and no specific therapeutic approaches for this entity exist. Because the long-term survival of eAML is poor, a deeper understanding of the immune microenvironment and leukemia phenotypes underlying this entity is warranted. Here, we performed bulk and single-cell transcriptome profiling of 23 eAML biopsies from 10 patients with isolated extramedullary disease in skin and subcutaneous tissue. Unlike normal healthy skin, we found leukemia cutis to be heavily immune infiltrated; in extramedullary relapse after allogeneic stem cell transplantation, >90% of T/natural killer cells were donor derived. eAML-associated T cells expressed a clear signature of T-cell exhaustion, dissimilar to leukemia-associated immune populations in bone marrow relapse (n = 7) but related to acute and chronic skin inflammation. Furthermore, HLA class II was downregulated in 4 of 7 leukemia cutis specimens, consistent with an immune escape phenotype in eAML. Extramedullary and bone marrow-resident leukemia cells differed with regard to the expression of 8 homing receptor molecules (ICAM1 [encoding CD54], PECAM1 [CD31], ITGA4, ITGA6, ITGAL, ITGB4, ITGA5, and ITGAV). Serial samples obtained from 1 leukemia cutis throughout consecutive immune checkpoint blockade with ipilimumab followed by nivolumab showed a consistently high degree of overlap between local and circulating T-cell receptor sequences, suggesting that only a minority of eAML-associated T cells are leukemia specific. Our analysis reveals eAML to associate with complex changes in leukemia and T-cell gene expression profiles that suggest multiple potential avenues for therapeutic targeting.

Comparison of SF3B1-mutated MDS with del(5q) to subclonal SF3B1-mutated MDS without del(5q). (A-F) No significant differences in baseline clinical demographics or SF3B1 VAF were observed between cases with vs. without del(5q). (G,H) Whilst survival was superior in patients with clonal SF3B1 mutations, no significant difference in leukemia-free or overall survival was identified between cases of SF3B1-mutated MDS with del(5q) vs. subclonal SF3B1-mutated MDS without del(5q), suggesting survival outcomes in such cases are influenced by alternative molecular driver events. Reported p-values utilized the cox model Wald test.

Identification of cell types and tracking of mitochondrial DNA mutations in immunosuppression tapering (IST) cohort using ASAP-seq.

Abstract Immune thrombocytopenia (ITP) is an autoimmune disease mediated by platelet-reactive antibodies, leading to accelerated platelet clearance and an increased risk of bleeding. Despite multiple available therapeutic options, durable treatment-free remissions remain uncommon in patients with refractory disease. Here, we report 3 patients with multidrug refractory ITP treated with a bispecific B-cell maturation antigen (BCMA)–targeting T-cell engager, teclistamab, approved for the treatment of multiple myeloma. Fixed-duration teclistamab therapy–induced platelet response within 4, 9, and 23 days, which was sustained after treatment discontinuation. The entirety of ITP-directed therapies was tapered and discontinued, and the 3 patients remain in treatment-free remission for 8, 6, and 3 months, respectively. Responses were associated with rapid depletion of B cells and plasma cells. Toxicity was manageable and largely limited to low-grade cytokine release syndrome, transient neutropenia, and infections that were readily controlled. These observations highlight BCMA-directed bispecific antibodies as a potential therapeutic strategy in autoimmune hematologic diseases and provide a rationale for prospective clinical trials.

Chimeric antigen receptor (CAR) T cell-mediated B cell depletion has demonstrated efficacy in several autoimmune diseases. Here we report clinical and molecular data obtained during the nonrandomized phase 1 part of the phase 1/2 COMPARE trial, evaluating safety and efficacy of mivocabtagene autoleucel (miv-cel), an autologous fully human CD19 CAR T cell therapy, in rheumatoid arthritis (RA). Six patients (three men, three women) with severe, treatment-refractory, anti-citrullinated protein antibody (ACPA)-positive RA received a single infusion of miv-cel after stopping all disease-modifying antirheumatic drug treatments and after standard lymphodepletion therapy. Patients were followed for 36-52 weeks for safety and efficacy. Primary endpoints were the incidence and severity of cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS) and adverse events (AEs) within the first 4 weeks after treatment. Secondary and explorative endpoints assessed clinical efficacy and cellular and humoral immune responses. CRS occurred in all patients and was limited to grade 1 and 2 events. No ICANSs or serious AEs occurred; one dose-limiting toxicity was recorded (grade 3 transaminase elevation, resolved without sequelae). The primary endpoint was met with acceptable safety findings, allowing advancement to phase 2. CAR T cell therapy resulted in depletion of CD19+ B cells across blood and tissue, coinciding with a continuous decline of autoantibodies with seroconversion in four of the six patients for ACPAs against mutated citrullinated vimentin and five of six for rheumatoid factor immunoglobulin M. Despite cessation of immunosuppressive treatments, disease activity improved in all patients (median 34% DAS28-CRP reduction at the latest follow-up with DAS28-CRP remission and American College of Rheumatology 70% response in 3 of 6 patients). CD19 CAR T cells showed acceptable short-term tolerability in patients with treatment-refractory RA, justifying further evaluation. ClinicalTrials.gov identifier: NCT06475495 .

Deconvolution and identification of AML phenotypes in bone marrow using ASAP-seq.

WHO 2016 classification of included patients with mutated SF3B1

Molecular characteristics of SF3B1-mutated MDS. (A) Mixed violin/boxplot depicting the VAF of SF3B1 in patients with clonal vs. subclonal SF3B1 mutations. Box and whisker plot demonstrates median (solid bar), interquartile range, minimum, and maximum VAF values. (B) The number of cases based on the count of co-occurring mutations between SF3B1low vs. SF3B1high MDS. (C) Distribution of identified amino acid variants within the SF3B1 protein. Variant frequency is represented as Log2 values. Location of variants displayed within HEAT domains, conserved regions (1–20) of tandem repeats within the SF3B1 protein. Canonical MDS mutations are commonly associated with domains 4 to 7 (yellow highlight). (D) Barplot depicting clonality of SF3B1low vs. SF3B1high MDS cases. (E) Barplot depicting differences in canonical and non-canonical variants between SF3B1low vs. SF3B1high MDS. (F) Alternative SF3B1 variants identified in SF3B1low vs. SF3B1high MDS. VAF: variant allele frequency. Asterisk indicates Fisher’s exact p-value <0.05.

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

Comparison of SF3B1-mutated MDS groups with SF3B1a and SF3B1b subgroups. (A) Patients with SF3B1low and subclonal SF3B1 mutations were more frequently assigned to the SF3B1b subgroup (defined as having a co-occurring mutation in any gene including BCOR, BCORL1, NRAS, RUNX1, SRSF2, or STAG2) compared to patients with SF3B1high or clonal SF3B1 mutations, who were more frequently assigned to the SF3B1a group (defined as having any other co-mutation with SF3B1). (B,C) Leukemia-free and overall survival was shorter in patients with SF3B1b vs. SF3B1a mutations. (D) When comparing groups stratified based on SF3B1 VAF, significant survival differences were observed (E). Multivariable analysis demonstrating differences in outcomes based on SF3B1a and SF3B1b designation, clinical parameters, and SF3B1 VAF. Reported p-values utilized the cox model Wald test.

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

Quizartinib is a potent type II inhibitor of FMS-like tyrosine kinase 3 (FLT3) that demonstrated significant survival benefit among patients with newly diagnosed (ND) acute myeloid leukemia (AML) without FLT3-internal tandem duplication (FLT3-ITD) in the randomized, Phase II QUIWI trial. Here, we present the rationale and design of QuANTUM-Wild, a double-blind, randomized, placebo-controlled, Phase III study to confirm the efficacy and safety of quizartinib plus standard induction and consolidation chemotherapy and as maintenance monotherapy in adult patients with ND FLT3-ITD-negative AML. Eligible patients are randomized 2:2:1 to one of three treatment arms. Patients in Arm A (n ≈ 280) receive quizartinib plus chemotherapy during induction and consolidation (with option for transplant without study drug), followed by up to 36 cycles of single-agent maintenance therapy. Patients in Arm B (n ≈ 280) receive placebo in all treatment phases plus chemotherapy, then as maintenance monotherapy. Patients in Arm C (n ≈ 140) receive quizartinib plus chemotherapy in the induction and consolidation phases, then placebo during the maintenance phase, to specifically assess the utility of quizartinib maintenance. The primary endpoint is overall survival. Secondary endpoints include event-free survival, remission rate, and safety.Clinical trial registration: http://www.clinicaltrials.gov identifier is NCT06578247.

Cancer is characterized by complex interactions across genetic, cellular, and microenvironmental scales. However, a quantitative understanding of how these interactions shape clinical trajectories remains limited. Here, we present a multi-scale single-cell dataset from 184 treatment-naive acute myeloid leukemia (AML) patients spanning all major genetic subtypes, together with an analytical framework to dissect interactions across biological scales. We show that distinct clinical outcomes are encoded by specific cross-scale, cross-compartment interactions present at diagnosis: response to induction therapy is governed by interactions between genetic alterations and leukemic differentiation state; relapse following chemotherapy is associated with non-genetic programs linked to metabolism; and relapse after allogeneic stem cell transplantation is driven by interactions between the immune microenvironment and residual healthy hematopoiesis. Together, our study provides a framework to resolve intra- and inter-patient heterogeneity in cancer and supports a model in which clinical trajectories in AML emerge from defined interactions across biological scales.

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.

Immunosuppressive treatments are broadly used to prevent or treat graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (alloSCT). The resulting severe treatment-related immunodeficiency is the main contributor to high alloSCT-associated mortality, highlighting the medical need for non-immunosuppressive strategies to prevent GVHD. Angiogenesis represents an attractive target because its therapeutic inhibition leads to reduced inflammation without major negative effects on immunity. The major hurdle for translation is safety: current anti-angiogenic drugs lack specificity and disrupt physiological angiogenesis, which is required for repair and regeneration. In search for more specific targets, we identified carnitine palmitoyltransferase 2 (Cpt2), a key enzyme in mitochondrial long-chain fatty acid oxidation, which is selectively upregulated during acute GVHD (aGVHD)-associated pathological angiogenesis. Genetic and therapeutic inhibition of Cpt2 prevented inflammation-associated pathologic endothelial changes and preserved normal endothelial cell functions in vitro . In preclinical alloSCT models, endothelial-specific CPT2 knockout as well as therapeutic CPT2 inhibition with perhexiline reduced aGVHD-associated angiogenesis, mitigated aGVHD severity and promoted functional restoration of the endothelial phenotype. Importantly, CPT2 inhibition did not inhibit immune reconstitution, preserved the graft-versus-leukemia effect and reduced tumor growth in preclinical models. Our data indicate that Cpt2 contributes to endothelial processes driving pathological angiogenesis in aGVHD. The selective targeting of pathologic angiogenesis is a new non-immunosuppressive strategy to mitigate aGVHD.