BACKGROUND:NPM1 is a disease-defining gene in the diagnosis of acute myeloid leukemia (AML) and is important for measurable residual disease (MRD) assessment. Over 50 different NPM1 mutations have been described, but only the 3 most common are routinely monitored during follow-up. METHODS:We developed a multiplex droplet digital polymerase chain reaction (PCR) assay for measurement of both variant allele frequencies (VAF) and mRNA transcripts of 10 different NPM1 mutations, using one generic probe, one generic NPM1 reverse primer, and 10 mutation-specific NPM1 forward primers. ABL1 expression and AP3B1 VAF were used as references. The performance of the assay was tested in diagnosis and follow-up samples from patients with an NPM1-mutated AML. RESULTS:Our assay shows negligible false-positive signals and high assay precision, leading to low limits of detection of at least 0.01%. The assay can easily be expanded to cover more NPM1 mutations by adding extra mutation-specific forward primers to the primer mix. Overall, a good correlation between mutant NPM1 expression and VAF was found. However, we also observed discrepant variable ABL1 expression levels, especially in AML patients with fms-related receptor tyrosine kinase 3-internal tandem duplications co-mutations. CONCLUSION:We developed a robust and extremely flexible mRNA- and gDNA-based multiplex droplet digital PCR NPM1 assay. Because the AML tumor load is better reflected by mutant NPM1 VAF than expression level, we recommend using the gDNA-based mutant NPM1 MRD assay with a VAF detection limit of 0.01%. For MRD signals below 0.01%, our more sensitive mRNA-based method can be used, although further research has to prove its clinical impact.

Immunotherapy has significantly improved treatment outcomes for cancer patients within the past decade, with breakthrough results using immune checkpoint inhibitors (ICIs), most notably those targeting the PD-1/PD-L1 inhibitory axis. Nevertheless, many patients and tumor types do not respond to current ICIs, and next-generation drugs are urgently needed. Dual Signaling Protein 216 (DSP216) is a new ligand-based immunotherapeutic-a dual HLA-G and CD47 ICI. DSP216 was designed to exclusively bind to cells co-expressing the immune checkpoints CD47 and HLA-G, thereby mitigating ICI activity towards normal cells expressing only CD47 or HLA-G and associated side effects. Computational chemistry was used to optimize DSP216 affinity to HLA-G with the aim to achieve the desired binding mode and DSP216 binding to CD47+/HLA-G+ and CD47+/HLA-G- cancer cells, PBMCs, and RBCs was tested. Functional blocking of the CD47 and HLA-G axis was investigated in macrophage polarization and phagocytosis assays and NK cytotoxicity assays. DSP216 with an 'active' (DSP216a), but not with an 'inactive' Fc (DSP216i), triggered CD16-signaling in a reporter cell line, and the combination of checkpoint blockade and ADCC by DSP216a potentiated NK-mediated cytotoxicity. These encouraging findings support the continued preclinical evaluation of DSP216.

Loss-of-function mutations in BCOR, a subunit of the non-canonical Polycomb Repressive Complex 1.1 (PRC1.1), are frequently observed in acute myeloid leukemia (AML) and associate with adverse risk. Paradoxically, leukemic stem cell viability in BCOR wild-type AMLs strongly depends on PRC1.1 activity. Here, we use BCOR and KDM2B degron models to study PRC1.1 dependency in leukemic cells and find that BCOR is a bridging factor tethering the catalytic and chromatin-binding moieties of the PRC1.1 complex. BCOR degradation induces a quick localized loss of H2AK119ub at PRC1.1 target genes, whereas PRC2-induced H3K27me3 remains unaffected. Degron-mediated depletion of BCOR or KDM2B induces a rapid but time-dependent transcriptional induction, whereby late-upregulated genes are more heavily decorated with H3K27me3 compared to early-upregulated genes. Combined PRC1.1 inactivation and PRC2 inhibition further amplifies gene induction, suggesting collaborative yet distinct control over target genes. Strikingly, both JARID2/AEBP2 and SUZ12 knockout cells, devoid of PRC2.2 or PRC2.1/PRC2.2 respectively, retain PRC1.1 loss-induced transcriptional activation, underscoring that PRC1.1 can repress target genes independently of a downstream PRC2.2-canonical PRC1 repressive axis. Finally, combined targeting of PRC1.1 and PRC2 induces differentiation of leukemic cells, emphasizing that co-targeting PRC1.1 and PRC2 represents a promising strategy to improve treatment of AML patients.

Abstract High‐dose chemotherapy (CT), followed by autologous hematopoietic cell transplantation (auto‐HCT) or consolidation CT, are postremission strategies for European LeukemiaNet 2022 (ELN2022) favorable‐ or intermediate‐risk patients with acute myeloid leukemia (AML) in complete remission (CR) without measurable residual disease (MRD). We conducted a retrospective, nonrandomized analysis to compare the efficacy and safety of auto‐HCT versus CT in newly diagnosed AML patients aged 18–65 years treated within two recent HOVON–SAKK–Nordic trials. From a total of 1005 patients with ELN2022 favorable‐ or intermediate‐risk AML, 224 received busulfan/cyclophosphamide (Bu/Cy), followed by auto‐HCT, and 199 received mitoxantrone/etoposide‐based CT. The 5‐year relapse‐free survival (RFS) and overall survival (OS) were comparable between auto‐HCT and CT (RFS: 60% vs. 56%, P = 0.70; OS: 72% vs. 71%, P = 0.76). In multivariable analysis, auto‐HCT was not associated with a significant difference in RFS (HR 0.80, 95% CI 0.56–1.13, P = 0.20) or OS (HR 0.91, 95% CI 0.60–1.37, P = 0.64). Median time to platelet and neutrophil recovery was shorter with auto‐HCT than CT (platelet: 42 vs. 57 days, neutrophil: 13 vs. 39 days). Subgroup analysis revealed that patients achieving CR after the second induction cycle or with baseline white blood cell counts >20 × 10 9 /L had longer RFS with auto‐HCT compared to CT (HR 0.17, P = 0.003 and HR 0.48, P = 0.02), which was not observed for OS. Focusing on ELN2022 favorable‐risk patients (excluding NPM1 mut MRD‐positive) and MRD‐negative intermediate‐risk patients, RFS and OS were not different between consolidation strategies. Collectively, auto‐HCT yielded similar RFS and OS to mitoxantrone/etoposide‐based CT in favorable‐ and intermediate‐risk AML, particularly in MRD‐negative patients, with faster hematologic recovery.

BACKGROUND:T-cell prolymphocytic leukemia (T-PLL) is a rare, aggressive mature T-cell malignancy with limited therapeutic options. Alemtuzumab remains the backbone of therapy, and fit responders are consolidated with allogeneic stem cell transplantation (alloSCT). However, contemporary population-level outcomes remain poorly defined. METHODS:All T-PLL cases diagnosed between 2001 and 2023 were identified from the nationwide Netherlands Cancer Registry. Overall survival (OS) and survival after relapse (OS2) were analyzed in 220 patients. Detailed treatment-line data were available for patients diagnosed from 2014 onward (n = 120), enabling assessment of treatment sequencing, response, and progression-free survival (PFS). RESULTS:Median age at diagnosis was 71 years; 56% were male. Overall, 54% received systemic therapy, of whom 25% underwent transplantation. Median OS was 13.5 months; 2- and 5-year OS were 36% and 10%, with no improvement over time. Alemtuzumab was the predominant frontline therapy and was associated with higher response and survival than other regimens. Consolidation with alloSCT provided the greatest likelihood of durable disease control, although only one-third of treated patients were eligible. Survival after relapse remained poor (median OS2, 5-7 months), and later treatment lines rarely produced durable benefit. Nearly half of patients were initially managed with observation; long-term survival remained limited after progression. CONCLUSIONS:Alemtuzumab followed by alloSCT remains the most effective strategy for fit patients with T-PLL, yet long-term survival is uncommon and has not improved over two decades. High relapse rates and poor salvage outcomes underscore the need for novel targeted and immune-based approaches and optimized post-remission strategies.

Potency is a critical specification for CAR-T cells and is typically determined by co-culture of CAR-T cells with target cell lines. However, cell line heterogeneity hampers standardization and can lead to analytical variation impacting potency test results. Further, both the assay and cell line maintenance are labor, time, and resource intensive. Here, we developed and validated a versatile, fully standardized, and GMP-compliant CAR-T cell potency assay. This assay utilizes antigen-loaded beads instead of target cell lines and was fully validated for CD19 and qualified for CD7 and HER2 CAR-T cells. Incubation of streptavidin beads with recombinant biotinylated antigen yielded a dose-dependent bead loading. Subsequent incubation of CAR-T cells with increasing amounts of antigen-loaded beads yielded a dose-dependent secretion of IFN-γ, whereas non-loaded or MOCK antigen-loaded beads did not significantly trigger IFN-γ secretion. Notably, as assessed for CD19 CAR-T cells, cryopreserved CAR-T cells yielded lower potency than fresh CAR-T cells and potency results correlated with the total amount of CAR-T cells in the test sample. Therefore, the assay was fully standardized using a fixed amount of 50 k CAR-T cells per test, a fixed amount of antigen loaded onto the beads (1 pg/bead), and a fixed amount of 500 k antigen-loaded beads. A quantitative and statistically substantiated potency threshold for batch release was established for fresh as well as cryopreserved CAR-T cell drug products This fully standardized assay protocol and validation strategy provides a facile potency assay for CAR-T cell drug products that can be implemented for essentially any antigen of interest.

ABSTRACT:Newly diagnosed mantle cell lymphoma (MCL) is commonly treated with rituximab (R), combined with anthracycline-based chemotherapy, with or without autologous stem cell transplantation (ASCT). Although R maintenance (RM) in clinical trials has been shown to prolong overall survival (OS), its impact at the population level remains largely unknown. This study evaluated the effect of RM on the outcome of patients with MCL. Patients aged ≥18 years, who were diagnosed with MCL between 1989 and 2020 were identified using the Netherlands Cancer Registry and were categorized into periods that reflected R and RM implementation (1989-2000, 2001-2014, 2015-2020). The treatment strategies were categorized as R-CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone); R-CHOP, followed by high-dose cytarabine (intensive) and ASCT; and other. The primary end point was 5-year OS. A multivariable analysis (MVA) was performed using Cox regression. Among 4751 patients, the 5-year relative survival (RS) improved from 38% (1989-2000) to 47% (2001-2014) and 60% (2015-2020) (P< .01), irrespective of age (≤65 years: 32% vs >65 years: 20% increase over time). Patients with progression within 12 months had a 2-year OS rate of 25%. Since 2014, RM implementation reached 80% in younger patients and 50% in older patients. RM was associated with improved OS, especially for patients in partial remission (PR) after induction treatment with R-CHOP. In the MVA of patients treated with R-CHOP, RM was independently associated with reduced mortality (hazard ratio, 0.69; 95% confidence interval, 0.53-0.90). The RS in MCL improved by >20% in 30 years. Early disease progression remains associated with poor outcome. RM was associated with improved survival, especially among patients who achieved PR following R-CHOP.

Chimeric Antigen Receptor (CAR)-T cell therapy holds considerable promise for the treatment of CD7+ T cell malignancies. However, a major challenge limiting clinical development of CD7-targeted CARs has been fratricide, a process of self-cytotoxicity caused by the shared expression of CD7 on malignant and healthy cells. Current solutions, including CD7 gene editing, intracellular retention or cell-sorting strategies, add significant complexity and cost, thereby limiting the feasibility and cost-effectiveness of this therapy. In this study, we evaluated whether fratricide can instead be overcome by tailoring standard manufacturing protocols to generate effective CAR-T cell products. Specifically, we report the GMP-compliant development of UMCG-001, a non-proprietary, academically generated, third-generation ligand-based CD7-targeting autologous CAR-T product. Production of UMCG-001 was explored using standard manufacturing protocols with various cytokine mixtures and GMP-grade human plate lysate (HPL). A GMP-compliant process was optimized with both healthy donor and patient material and subsequently transferred to our GMP facility. The anti-leukemic activity of UMCG-001 was assessed in vitro and in vivo, followed by in-depth multi-omics characterization to evaluate the impact of the fratricide phase on product quality. Supplementation with human platelet lysate during the ex vivo expansion phase effectively restored expansion and improved viability of fratricide-driven CAR⁺CD7Low/Neg cells. These unedited cells exhibited robust CD7-specific antileukemic activity and achieved 90

The B-cell lymphoma 2 (BCL-2) inhibitor venetoclax (VEN) in combination with hypomethylating agents (HMAs) has improved treatment outcomes for acute myeloid leukemia (AML) patients unfit for intensive chemotherapy and is increasingly used in the relapsed/refractory setting. However, primary resistance remains a significant challenge, affecting 20%-35% of treatment-naïve and around 50% of previously treated AML patients. To investigate the mechanisms driving primary resistance to VEN-HMA therapy, we analyzed genetic, transcriptomic, BCL-2 family protein expression, and ex vivo drug sensitivity data from 101 AML patients and correlated these profiles with clinical outcomes to VEN-HMA. Our study found that blasts from refractory patients exhibit an elevated BCL-XL/BCL-2 protein expression ratio, an immature CD34+CD38- phenotype, and frequent TP53 mutations. Consistent with the high ratio of BCL-XL/BCL-2, resistant samples showed increased ex vivo sensitivity to the dual BCL-2/BCL-XL inhibitor navitoclax. In addition, SMAC mimetics were effective in refractory blasts, which correlated with high TNF gene expression in these cells. Ex vivo treatment with the combination of navitoclax and SMAC mimetics further enhanced the eradication of VEN-HMA refractory blasts, although toxicity was also observed in healthy CD34+ cells. In conclusion, our integrative analysis identifies molecular signatures associated with primary VEN-HMA resistance and highlights BCL-2/BCL-XL inhibition and SMAC mimetics as therapeutic strategies to target resistance.

Newly diagnosed (ND) acute myeloid leukemia (AML) with KMT2A rearrangement (KMT2Ar), or NPM1 mutated (NPM1m) in patients aged ≥65 years is associated with poor treatment outcomes. Intensive chemotherapy (IC), comprising ‘7+3’ with cytarabine consolidation remains the standard-of-care (SoC) therapy in those ‘fit’ enough to undergo such therapy.Bleximenib is a menin inhibitor designed to target KMT2Ar and NPM1m AML by inducing leukemia cell differentiation and cell death. No menin inhibitors are currently approved for ND KMT2Ar or NPM1m AML patients eligible for IC.Investigational use of bleximenib in combination with SoC anti-leukemic treatments is supported by preclinical and early clinical evidence. In the Phase 1 ALE1002 study (NCT05453903), an acceptable safety profile and high rates of response were observed with bleximenib in combination with ‘7+3’ in participants (pts) with ND KMT2Ar or NPM1m AML.HOVON 181 AML / AMLSG 37-25 is a Phase 3, randomized, double-blind, placebo-controlled, global multicenter study evaluating efficacy and safety of bleximenib vs placebo in combination with SoC remission induction and consolidation IC followed by maintenance therapy in adults with ND KMT2Ar or NPM1m AML (EU CT number 2025-52276715).Eligible pts are aged ≥18 years with ND KMT2Ar or NPM1m AML (≥10% blasts per 2022 International Consensus Classification criteria) and considered eligible for IC. Other inclusion criteria include Eastern Cooperative Oncology Group performance status ≤2 and adequate hepatic and renal function. Exclusion criteria include prior chemotherapy for AML, including hypomethylating agents; known active leukemic involvement of the central nervous system; prior solid organ transplant; any significant cardiac disorder ≤6 months prior to randomization; chronic respiratory disease requiring supplemental oxygen; and uncontrolled active infections including hepatitis B/C and HIV.875 pts will be randomized to 1 of 3 study arms. In Arm 1, pts will receive induction therapy with bleximenib in combination with cytarabine plus daunorubicin or idarubicin. Those achieving complete remission (CR), CR with partial or incomplete hematologic recovery, or morphologic leukemia-free state after completing induction therapy are eligible to receive either up to 3 cycles of consolidation with cytarabine plus bleximenib or allogeneic stem cell transplantation, depending on risk category, followed by up to 24 cycles of bleximenib maintenance. Pts in Arm 2 will receive the same induction and consolidation treatment as Arm 1, and placebo during maintenance. In Arm 3, pts will receive placebo instead of bleximenib throughout, with the same IC schedule as Arms 1 and 2.The primary endpoint is event-free survival. Secondary endpoints include overall survival, rate of CR without measurable residual disease, duration of CR, and safety.Enrollment is planned to begin in late 2025.

Therapy-related acute myeloid leukemia (t-AML) and AML with myelodysplasia-related changes (AML-MRC) are associated with poor outcomes. The liposomal formulation of cytarabine and daunorubicin (CPX-351) improved complete remission (CR) and CR with incomplete hematologic recovery (CRi) rates and overall survival (OS) compared with ‘standard’ induction (7+3) chemotherapy in a phase-III trial for patients aged 60-75 years. However, 7+3 dosing varies among trials and in clinical practice and it remains unknown whether CPX-351 is superior to 7+3 double-induction regimens including intermediate-dose cytarabine, as the one employed in the HOVON-SAKK-Nordic clinical trials. To address this question, we conducted a post-hoc analysis on t-AML/AML-MRC patients aged ≥60 years enrolled in three HOVON-SAKK-Nordic trials and defined a subset of patients that met the eligibility criteria of the CPX-351 trial and compared their outcomes with those of the CPX-351 arm using reconstructed survival data. CR/CRi rates were higher in the higher-intensity 7+3 cohort (67.8%) compared with CPX-351 (47.7%) with similar median OS between the two cohorts (10.1 months versus 8.9 months respectively, HR = 0.99; 95% CI 0.78-1.26, p=0.95). Thirty-day mortality (4.4% for higher-intensity 7+3 versus 5.9% for CPX-351) and adverse events, including febrile neutropenia (61% for higher-intensity 7+3 versus 68% for CPX-351), were comparable. The data suggest that obligatory double-induction may achieve outcomes similar to CPX-351 in these patients and provide a strong rationale for ongoing clinical trials comparing these regimens.

Acute myeloid leukemia (AML) exhibits extensive clonal and functional heterogeneity, limiting the ability of current genotype-based classifications to predict treatment response. We applied multi-omics factor analysis (MOFA) in a cohort of AML cases, integrating mutational status, proteomics, metabolomics, and two ex vivo drug screens comprising 115 agents targeting key metabolic and epigenetic pathways. A five-factor model captured interpretable, cross-modal axes of variation. One axis, Factor 2, defined a monocytic/inflammatory program with acylcarnitine accumulation and broad drug insensitivity. Projection of factor scores into four independent AML cohorts showed that Factor 2 reproducibly associated with reduced venetoclax sensitivity and enrichment of RAS-related signatures. This RAS-like phenotype extended beyond canonical RAS- mutant cases to include genotypically wild-type samples with high Factor 2 scores, indicating that proteomic and metabolomic integration reveals pathway activation not captured by genotype alone. Leveraging microenvironmental features of Factor 2 high disease, we further identified selective sensitivities to FABP4 inhibition and the POLG inhibitor alovudine, revealing candidate strategies for venetoclax-resistant AML.

Azacitidine/venetoclax is the standard treatment for patients with acute myeloid leukemia (AML) unfit for intensive chemotherapy. Cytochrome P450 3A4 (CYP3A4) is the major metabolizing enzyme for venetoclax, and its inhibition can boost venetoclax. In the HOVON 171 phase 2 trial, patients with AML were treated with azacitidine/venetoclax/cobicistat. This 2-stage, open-label, multicenter phase 2 trial included a crossover run-in phase followed by an ongoing extension phase. In cycle 1, patients received standard-dose azacitidine/venetoclax. In cycle 2, cobicistat was added, and venetoclax was reduced to 50 mg. The primary end point was pharmacokinetic equivalence, defined as 90% confidence interval (CI) of geometric mean ratios (GMRs) >0.8 for area under the curve over 24 hours (AUC0-24h) and maximum plasma concentration (Cmax). Polymorphisms in genes encoding for CYP enzymes were determined. In vitro assays were performed to assess cobicistat's impact on the antileukemic effect of azacitidine/venetoclax in AML cell lines. In 13 evaluable patients, cobicistat-boosted venetoclax at 50 mg achieved higher exposure than standard 400-mg dosing. GMRs were 2.0 (90% CI, 1.4-2.8) for AUC0-24h and 1.4 (90% CI, 1.0-2.0) for Cmax. In the intention-to-treat population, 65% achieved complete remission (CR) or CR with incomplete hematologic recovery. Interpatient variability in venetoclax exposure because of CYP3A4 polymorphisms was reduced by cobicistat. No unexpected toxicities were observed. In in vitro, cobicistat enhanced azacitidine/venetoclax antileukemic effects. In conclusion, cobicistat enhances and optimizes venetoclax exposure, enabling an eightfold dose reduction while maintaining efficacy. The potentiated antileukemic activity positions cobicistat as a promising complementary agent in AML therapy. This trial was registered at www.clinicaltrials.gov as NCT06014489.

Among acute myeloid leukemia (AML) patients, a subgroup remains notoriously refractory to current treatment options, with underlying mechanisms poorly understood. Here, using a multi-omics approach, we reveal that this resistant patient subgroup is characterized by high expression of the oncogenic TP73 isoform ΔNp73, exhibiting similarly poor outcomes as TP53-mutant AML. ΔNp73, which lacks a transcriptional activation domain but retains chromatin-binding properties, competes with TP53 for specific gene targets, thereby downregulating TP53 signaling. We demonstrate that the transcription factor CEBPA controls ΔNp73 expression in AML cells by binding to an intragenic enhancer region. Genetic or pharmacological inhibition of the transcriptional activity of CEBPA with guanfacine reduces ΔNp73 levels and restores drug sensitivity involving ferroptosis-mediated cell death, acting synergistically with venetoclax. Our study sheds light on a previously undercharacterized poor-risk subgroup of AML, which may support patient stratification and inform treatment considerations.

BACKGROUND:Clonal hematopoiesis (CH) is an independent risk factor for cardiovascular disease (CVD). Targeted next generation sequencing (NGS) studies have highlighted the contribution of smaller clones to CVD, particularly for DNMT3A and TET2 CH, the most frequent CH mutations. DNMT3A CH occurs more frequently in women than men. Whether sex affects the association between CH and CVD is unknown. METHODS:We included 5508 participants from the Lifelines cohort, who had previously undergone targeted NGS. Our cohort is enriched for blood count abnormalities. We investigated the sex-specific associations between CH and prior myocardial infarction (MI) or coronary artery calcium (CAC) and evaluated the association of sex or prior MI with clonal expansion. RESULTS:We identified 2103 participants carrying CH. DNMT3A CH occurred more frequently in women than men (OR 1.29; p = 5.2 × 10-4). Women with DNMT3A CH had a higher odds of prior MI (OR 1.77; p = 2.1 × 10-2), while men did not (OR 0.98; p = 8.8 × 10-1; Pinteraction = 4.4 × 10-2). Sex or prior MI did not affect clonal expansion. DNMT3A clone size positively associated with age- and sex-adjusted CAC percentile scores in women (B 28.97; p = 9.7 × 10-3), but not in men (B 2.34; p = 8.3 × 10-1; Pinteraction = 8.9 × 10-2). Women with a higher-than-average DNMT3A clonal expansion had higher CAC percentile scores compared to women with lower-than-average clonal expansion, independent of initial DNMT3A clone size (B 16.76; p = 1.1 × 10-2). CONCLUSIONS:DNMT3A CH is associated with previous MI and higher atherosclerotic burden only in women, highlighting the need to better understand the sex-specific risks that CH may confer on CVD.