Institute of Haematology and Blood Transfusion

Anthracyclines are an essential component of induction therapy for acute myeloid leukemia (AML), but their optimal dosing and the associated risk for cardiotoxicity remain under debate. The DaunoDouble trial compared daunorubicin at 60 (Dauno60) versus 90 mg/m2 (Dauno90) in combination with cytarabine (100 mg/m2 for 7 days) in newly diagnosed AML patients aged 18-65 years. Cardiac function was assessed by left ventricular ejection fraction (LVEF) and cardiac biomarkers (high-sensitivity troponin T [hsTnT], NT-proBNP) before treatment and on Day 15 in 317 randomized patients. Median LVEF declined significantly from 65% [IQR 60%-68.5%] to 61% [IQR 58%-67.8%] across all patients (p < 0.01), without significant differences between treatment arms. NT-proBNP levels measured after induction therapy correlated negatively with LVEF at the same time point (ρ = -0.24, p = 0.02), but did not change significantly during induction-neither between Day 1 and 15 nor between treatment arms. HsTnT levels increased significantly from 5 [IQR 4-8] to 8 ng/L [IQR 6-14] across all patients (p < 0.01), with higher post-induction values in the Dauno90 group (9.5 ng/L [IQR 7-14]) compared to Dauno60 (7 ng/L [IQR 5-14]; p < 0.01). In exploratory subgroup analyses, post-induction hsTnT levels were also significantly higher in patients with obesity and arterial hypertension. These findings provide evidence of a dose-dependent cardiotoxic effect of daunorubicin, already apparent at standard induction doses, and underscore the importance of early cardiac monitoring. Long-term follow-up will be essential to determine the clinical significance of these early changes. Trial Registration: ClinicalTrials.gov identifier: NCT02140242.

Circular RNAs play a crucial role in cell development and serve as biomarkers in many diseases. Nevertheless, the function of many circular RNAs remains unknown. This function can be inferred from sponging and silencing interactions with micro RNAs and messenger RNAs. We recently proposed a network-based circRNA functional annotation tool, circGPA. However, validation data for RNA interactions are often sparse and predicted interactions contain many false positives. To address this issue, we propose an extended algorithm named circGPAcorr, which uses expression data to weight the interactions, resulting in more precise functional annotation. To assess the significance of the results, the p-value is calculated using reduction to circGPA, a generating-polynomial-based method. We show that the problem is #P-hard, and thus computationally difficult. The circGPAcorr algorithm is tested on publicly available myelodysplastic syndromes expression data, providing gene ontology annotations that align with the literature on myelodysplastic syndromes. At the same time, we demonstrate its performance in the circRNA-disease annotation task.

Trauma is a leading global cause of mortality, and systemic inflammatory response syndrome (SIRS) remains a significant complication, contributing to adverse outcomes. Neutrophils, as first responders to tissue injury, undergo substantial phenotypic and functional changes following trauma. This study investigates neutrophil subpopulations defined by CD16 and CD62L expression in trauma patients, focusing on their correlation with clinical biomarkers, trauma severity, and functional properties. We included 50 non-infectious trauma patients, categorized into SIRS and Non-SIRS groups, and 43 elective surgery patients as controls. Neutrophil subsets were analyzed at two time points (TP1 and TP2) using flow cytometry. Functional assays evaluated phagocytosis, oxidative burst, mitochondrial function, and degranulation. Correlations between neutrophil subpopulations and clinical markers, including lactate, creatine kinase, Injury Severity Score, and Trauma and Injury Severity Score, were examined. Patients with SIRS exhibited higher proportions of banded neutrophils and CD16lowCD62Llow neutrophils at TP1, alongside reduced levels of mature neutrophils. Elevated lactate and creatine kinase levels positively correlated with banded neutrophils and CD16lowCD62Llow neutrophils, while negatively correlating with mature neutrophils CD16highCD62Lhigh and hypersegmented neutrophils CD16highCD62Llow. Hypersegmented neutrophils were more prevalent in Non-SIRS patients at TP1 and in SIRS patients at TP2. Banded neutrophils showed a positive correlation with Injury Severity Score and an inverse correlation with Trauma and Injury Severity Score (TRISS), whereas hypersegmented neutrophils were negatively associated with ISS and positively correlated with TRISS. These correlations likely reflect the pro-inflammatory role of banded neutrophils and the inflammation-resolving function of hypersegmented neutrophils. CD16lowCD62Llow neutrophils displayed impaired phagocytosis, oxidative burst, and degranulation capacity, indicating functional deficiencies. This study highlights the dynamic changes in neutrophil subpopulations in trauma and their association with systemic inflammation and clinical severity. Increased banded neutrophils correlate with SIRS and metabolic stress, whereas hypersegmented neutrophils may contribute to resolving inflammation. CD16lowCD62Llow neutrophils exhibit functional impairments, warranting further investigation. Monitoring neutrophil subpopulations could aid in identifying trauma patients at risk for non-infectious SIRS and guide therapeutic interventions.

BackgroundTimely resolution of innate immune responses activated by surgical intervention is crucial for patient recovery. While cytokines and innate immune cells are critical in inflammation resolution, the specific role of IL-18 in these processes remains controversial and underexplored.MethodsWe investigate determinants of successful recovery using peripheral blood samples from orthopedic surgery (ORT) patients (n = 33) at T0 (before surgery), T1 (24 h after surgery) and T2 (3 days after surgery). Monocytes from ORT patients underwent immunophenotyping together with bulk transcriptomic analysis. We found that IL-18 strongly defines the recovery immune signature. These results were further validated in vitro by comparing IL-18 and TNF-α effects on monocytes, and in 3D human intestine organoids together with single cell (sc)-RNAseq analysis.ResultsTranscriptomics of ORT monocytes revealed upregulation of ITG family integrins, namely ITGB3 and ITGB5, CXCL family chemokines, notably CXCL1-3, CXCL5, and SCL/TAL1 factor controlling differentiation and migration, but not pro-inflammatory genes. Similar changes were observed in IL-18 stimulated healthy donor monocytes in vitro, including an increase in CD11b, CD64, and CD86 levels, accompanied by increased phosphorylation of Akt but not NFκB. These changes were attenuated in the presence of TNF-α, thus showing a unique role of IL-18 when acting alone without its most frequent paired cytokine TNF-α. We further confirmed that IL-18 induces monocyte-macrophage transition and migration using human intestinal organoids. Finally, TNF-α/IL-18 ratio showed a high predictive value of clinical severity in septic patients.ConclusionsWe propose a novel role of IL-18 on monocyte migration and macrophage transition characterizing successful orthopedic surgery recovery, as well as the ratio of IL-18/TNF-α as a novel marker of inflammation resolution, with potential implications for patient monitoring and therapeutic strategies.

BackgroundNatural killer (NK) cell-based therapies represent a promising approach for acute myeloid leukemia (AML) relapse, yet their efficacy is hindered by immunosuppressive factors such as transforming growth factor beta (TGF-β) in the tumor microenvironment. This study investigated the effects of TGF-β on NK cell cytotoxicity and migration using 2D and 3D co-culture models that mimic the leukemic microenvironment.MethodsTGF-β production was evaluated in AML-derived leukemic cell lines and mesenchymal stromal cells (hTERT-MSCs) using ELISA. Bulk RNA sequencing (RNA-seq) was performed to analyze global gene expression changes in TGF-β-treated primary human NK cells. NK cell cytotoxicity and migration were assessed in 2D monolayer and 3D spheroid co-cultures containing hTERT-MSCs and leukemic cells using flow cytometry and confocal microscopy.ResultsBoth leukemic cells and MSCs produced TGF-β, with increased levels observed in MSCs after co-culture with primary AML blasts. RNA sequencing revealed that TGF-β altered key gene pathways associated with NK cell cytotoxicity, adhesion, and migration, supporting its immunosuppressive role. In functional assays, TGF-β exposure significantly reduced NK cell-mediated cytotoxicity in a time-dependent manner and impaired NK cell infiltration into 3D spheroids, particularly in models incorporating MSCs. Additionally, MSCs themselves provided a protective environment for leukemic cells, further reducing NK cell effectiveness in 2D co-cultures.ConclusionTGF-β suppresses both NK cell cytotoxicity and migration, limiting their ability to eliminate leukemic cells and infiltrate the bone marrow niche (BMN). These findings provide novel insights into TGF-β–mediated immune evasion mechanisms and provide important insights for the future design of NK-based immunotherapies and clinical trials.

Introduction: CNS relapse occurs in around 5% of patients with systemic diffuse large B-cell lymphoma (DLBCL) and its prognosis is poor. The optimal strategy for CNS prophylaxis is not established. The CLSG-CNS-01 trial compared CNS prophylaxis with high doses of intravenous (i.v.) methotrexate (MTX) and intrathecal (i.t.) MTX in systemic DLBCL. Methods: This randomized, multicenter, prospective phase 3 trial was registered at ClinicalTrials.gov (NCT02777736). Patients with systemic DLBCL aged between 18 and 72 years were treated with 6 cycles of R-CHOP+2xR or DA EPOCH-R+2xR. Patients with intermediate (2–3 risk factors) and high risk (4–6 risk factors) for CNS relapse were randomly assigned (1:1) to CNS prophylaxis with either 2 doses of MTX 3g/m2 i.v. (arm A) or 6 doses of MTX 12 mg i.t. (arm B). Low risk patients (0–1 risk factor) for CNS relapse were not randomized and did not receive CNS prophylaxis (arm C). Primary objective was to compare cumulative incidence of CNS relapse between arms A and B. Major secondary objectives included: overall response rate (ORR), progression-free and overall survivals (PFS, OS) and treatment toxicity. Results: Overall 100 patients were enrolled between 2015 and 2024: 30 were randomly assigned to arm A and 31 to arm B; 39 patients did not receive prophylaxis (arm C). Median age of patients was 61 years (range 27–72) and 54% were male. CNS relapses occurred in 3 (3%) patients (arm A 1, arm B 2) during the median follow-up of 54.9 months. CNS relapse after MTX i.v. occurred later (5.2 years after the initiation of treatment). Comparison of 5year cumulative incidence of CNS relapse between randomized arms A and B did not reach statistical significance (0% vs. 8.7%, HR 1.521, p = 0.72). ORR was not significant among arms A, B, C (83.3% vs. 83.8% vs. 94.8%, p = 0.20). The 5year PFS was comparable in arms A and B (45.3% and 57.4%), HR 0.66, p = 0.20. CNS prophylaxis (i.v. and i.t.) significantly increased neutropenia grade ≥ 3 (12.61% vs. 18.48 % vs. 3.59 %, p < 0.0001) with the highest rate of infections grade ≥ 3 in arm A (4.95% vs. 0.95% vs. 0.80%, p = 0.0046). Other toxicities grade ≥ 3 occurred most frequently in arm A (p = 0.0039). Overall 29 patients died (arm A 16, arm B 10, arm C 3). Infections (arm A 5 vs. arm B 2) and unknown causes (arm A 4 vs. arm B 2) indicated the major difference between randomized arms. This observation resulted in significantly worse 5year OS in arm A versus B (47.2% vs. 72.4%, HR 0.46, p = 0.04). Conclusions: CNS prophylaxis with MTX i.v. or i.t. did not eliminate CNS relapse, but MTX i.v. delayed its occurrence. Cumulative incidence of CNS relapse did not differ significantly between i.v. and i.t. MTX prophylaxis, however, the number of randomized patients was low. MTX i.v. was significantly associated with worse OS, probably due to the toxicity. Research funding declaration: This work was supported by grant AZV NU21-03-00411 from the Ministry of Health of the Czech Republic and by the Cooperation Program, research area “Oncology and Haematology” Keywords: chemotherapy; aggressive B-cell non-Hodgkin lymphoma No potential sources of conflict of interest.