Search Results (263)

Next-generation sequencing (NGS) clonality assessment has the potential to overcome the conventional limitations of PCR-based methods. This study aimed at comparing the performance and concordance of the EuroClonality-NGS approach and the LymphoTrack^® Dx assay for immunoglobulin (IG)/T-cell receptor (TR)-marker identification at diagnosis. Across four quality-control rounds, six Italian laboratories analyzed 23 acute lymphoblastic leukemia (ALL) and five chronic lymphocytic leukemia (CLL) cases. Overall, 171 rearrangements were identified; 80.7% were detected by both methods. Among shared targets, the concordance was 89.1%, with a higher agreement for IG (IGH 100%–IGK 92.1%) than for TR loci (TRG 78.9%–TRB 81.8%). Bland–Altman analysis indicated no statistically significant systematic bias between methods [mean bias 1.99% (95% CI: −5.29 to +1.31%)]. The Spearman correlation was ρ = 0.680 (p < 0.001). Discordances (10.9%) rarely yielded suitable sensitive PCR assays for disease monitoring (20%) and were never the sole marker in individual patients. In larger independent cohorts, similar rates of no-marker cases were observed (~4–5% in adults), with higher frequencies in T-ALL and no association with blast percentage (Spearman’s ρ = 0.447, p = 0.450 in adults and ρ = 0.700, p = 0.188 in childhood). These findings support the reliability of both methods for diagnostic screening, while highlighting locus-specific variability and the importance of multi-target identification. Full article

Background: Acute myeloid leukemia (AML) is characterized by reduced antileukemic effector cells and increased immunosuppressive cell populations. Leukemia-derived dendritic cells (DC_leu), generated from 18 leukemic whole blood (WB) ex vivo using ‘Kit-M’ (clinically approved: GM-CSF + PGE1), lead to improved cytotoxicity against autologous blasts after mixed lymphocyte culture (MLC) with patients’ T-cells. Methods: We studied Kit-M-mediated effects on frequencies of tolerogenic, immunosuppressive DC (DC_tol) and correlated findings with ex vivo-achieved antileukemic effects (increased intracellular IFNγ production/degranulation, blast lysis) and patients’ clinical characteristics. Results: We show significantly decreased frequencies of DC_tol (and increased frequencies of mature DC_leu) without induced blast proliferation in Kit-M treated vs. untreated WB samples. After T-cell-enriched MLC with Kit-M pretreated vs. not pretreated, WB frequencies of regulatory (CD152+ T-cells) were significantly decreased, while ‘activated’ (IFNγ+, degranulating) non-naive, proliferating, memory, CD154+) T-cells, as well as NK and CIK-cells were (significantly) increased. We found a (significant) positive correlation of achieved improved blast lysis, frequencies of DC_leu and ‘activated’ (IFNγ+/degranulating) T- or NK/CIK cells, and a (significant) negative correlation with frequencies of DC_tol and regulatory (CD152+ T-cells). Kit-M treatment of leukemic WB increases DC_leu and decreases DC_tol, correlating with improved immune reactions/improved cytotoxicity against autologous blasts, and downregulated suppressive T-cells in samples before or after MLC. Conclusions: These findings demonstrate the potential of Kit-M (using clinically approved drug compositions) to treat AML patients to potentially overcome the immunosuppressive tumor microenvironment, leading to improved antileukemic responses—thereby stabilizing remission of the disease in AML patients. Full article

Background: Germline variants of the immune checkpoint receptor CTLA4 may modulate T-cell activation, potentially influencing post-transplant immune reconstitution and clinical outcomes. Methods: In this retrospective single-center study, 140 AML patients who underwent ASCT were stratified into three groups according to the CTLA4 rs231775 geno-types A17hom, T17Ahet, and T17hom. Clinical outcomes, including overall survival (OS) and progression-free survival (PFS), were evaluated. Multivariate analysis was performed to adjust for known prognostic covariates. Results: Baseline clinical characteristics varied according to CTLA4 genotype with a higher proportion of favorable cytogenetic risk in A17hom carriers. Comparative analysis revealed differences in survival rates, with superior outcomes in A17hom carriers. In multivariate analysis directional trends persisted across all endpoints. Conclusion: The prognostic impact of CTLA4 rs231775 on post-ASCT outcomes suggests that T-cell inhibitory signaling may contribute to anti-leukemic immune surveillance in the autologous setting. These findings provide a rationale for investigating CTLA4 inhibition as consolidation therapy following ASCT in future prospective studies, particularly in T17hom carriers, who may harbor a less favorable immune profile. CTLA4 genotyping at diagnosis may represent a practical tool to support risk stratification in AML. Full article

Background: Lymphocyte-activation gene 3 (LAG3) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) are immune checkpoint receptors and inhibitory regulators of T-cells. Methods: Here, we analyzed the prevalence and potential impact of the LAG3 gene variant rs870849 and the CTLA4 gene variant rs231775 in AML patients eligible for autologous stem cell transplantation. Results: While CTLA4 rs231775 was prevalent at reduced minor allele frequencies (MAF 0.33), LAG3 rs870849 was prevalent at elevated minor allele frequencies (MAF 0.58) in AML patients, compared to the allele frequencies in the European population (MAF 0.37 and MAF 0.39). The gene risk analysis indicated a dose-dependent risk of AML disease associated with LAG3 rs870849, but no risk associated with CTLA4 rs231775. Baseline blood count profiles differed across LAG3 genotypes, suggesting a link between LAG3 rs870849 and disease-associated levels of anemia, thrombocytopenia and peripheral blast percentage. Conslusions: The germline LAG3 variant rs870849 may be associated with AML disease risk and specific hematological disease features. Full article

Acute lymphoblastic leukemia (ALL) is a malignant neoplasm of the blood and bone marrow characterized by the uncontrolled proliferation of precursor cells of B- or T-lymphocyte lineage. Usually, the disease arises because of spontaneous mutations in bone marrow cells. Risk factors include genetic predisposition, exposure to ionizing radiation, prior chemotherapy or radiotherapy, and certain environmental factors. Clinical manifestations may include recurrent infections, anemia, and an increased tendency toward bleeding and stroke. A 12-year-old boy presented to the emergency department with a sudden decrease in visual acuity in the right eye. Best-corrected visual acuity (BCVA) in the right eye was 0.02, and intraocular pressure (IOP) was 16 mmHg. Ophthalmologic examination revealed a macular hemorrhage in the right eye. Blood samples were obtained for laboratory analysis. Complete blood count demonstrated leukocytosis with a white blood cell (WBC) count of 362.58 × 10³/µL, thrombocytopenia with a platelet (PLT) count of 87 × 10³/µL, hemoglobin (Hgb) level of 8.7 g/dL, and a red blood cell (RBC) count of 3.46 × 10⁶/µL. The patient was subsequently referred to the Department of Pediatric Hematology, where the diagnosis of acute lymphoblastic leukemia of B-cell precursor origin was confirmed. Appropriate systemic therapy targeting the underlying disease was initiated. Full article

The immunological composition of the microenvironment has shown relevance for diagnosis, prognosis, and therapy in solid tumors but remains underexplored in acute leukemias. We investigated the significance of the acute myeloid leukemia (AML) bone marrow microenvironment in predicting chemosensitivity and long-term remission in pediatric patients. We analyzed 32 non-promyelocytic pediatric AML patients at diagnosis using a NanoString PanCancer IO 360 assay, RNA sequencing, and deep-phenotype flow cytometry analyses. The findings were validated using the pediatric TARGET AML dataset. A short signature of three interferon (IFN)-related genes (GBP1, PARP12, and TRAT1) distinguished patients with chemosensitive disease and reduced minimal residual disease after induction chemotherapy. The signature stratified patients overall, and within the clinically defined “standard-risk” group, patients with high gene expression at diagnosis had significantly longer overall survival. The leukemia microenvironment associated with this signature showed enrichment of non-exhausted CD4⁺ and CD8⁺ T cytotoxic lymphocytes and expansion of CD8⁺ T effector memory cells re-expressing CD45RA (TEMRA) in patients with a favorable prognosis. Our results show the importance of the bone marrow microenvironment in pediatric AML and provide tools for a refined stratification of “standard-risk” patients, lacking adequate risk-oriented therapies. They also offer a promising guide for tackling immune pathways and exploiting immune-targeted therapies. Full article

Hematologic malignancies arise and progress within a systemic ecosystem in which the gut microbiota is an increasingly recognized, partially modifiable component. Across acute leukemias, chronic lymphocytic leukemia, plasma cell disorders, lymphomas, and clonal myeloid neoplasms, human studies consistently report reduced microbial diversity, depletion of barrier-supportive, short-chain fatty acid-producing commensals, and enrichment of Gram-negative, pro-inflammatory, or hospital-adapted taxa. These alterations are associated with pre-leukemic clonal expansion, adverse genetic and immunological features, progression from precursor conditions, and inferior outcomes after chemotherapy, immunochemotherapy, chimeric antigen receptor T-cell therapy, and allogeneic hematopoietic stem cell transplantation. Mechanistic work in animal models and ex vivo systems demonstrates that microbiota-derived signals and metabolites—including Th17/IL-17-skewing consortia and the lipopolysaccharide intermediate ADP heptose sensed by the cytosolic receptor ALPK1—can actively modulate hematopoietic stem and progenitor cell fitness, inflammatory circuits, and malignant cell survival, supporting a causal role in disease biology. At the same time, major knowledge gaps remain because most human cohorts are small, single-center, and cross-sectional, frequently rely on 16S rRNA profiling, and are vulnerable to dietary, geographic, and treatment-related confounding. Within this context, three translational domains appear particularly promising: pharmaco-microbiomics, microbiome-informed risk stratification, and rational microbiota-targeted interventions, particularly diet-based strategies and antimicrobial stewardship. Here, we provide an integrated, disease-spanning synthesis of these data, emphasizing clonal hematopoiesis and myeloid neoplasms as emerging examples of microbiota–marrow crosstalk and outlining practical priorities for embedding microbiome science into future hematologic trials. Routine microbiome profiling or empiric microbiota-directed therapies cannot yet be recommended in everyday hematology practice, but integrating microbiome science into prospective therapeutic and transplant trials offers a realistic path to improved disease modeling, biomarker development, and rational adjunctive strategies to enhance outcomes for patients with hematologic malignancies. Full article

Acute Lymphoblastic Leukemia (ALL) is a highly prevalent hematological malignancy, especially in children, for whom precise and prompt subtype identification is essential to establish suitable treatment protocols. Current deep learning-based computer-aided diagnosis (CAD) methods for identifying ALL are hindered by numerous drawbacks, such as a dependence on solely spatial feature depictions, elevated feature dimensions, computationally extensive deep learning architectures, inadequate multi-layer feature utilization, and poor interpretability. This paper introduces LUMINA-Net, a custom, lightweight, and interpretable deep learning CAD for the automated identification and subtype diagnosis of ALL using microscopic blood smear pictures. LUMINA-Net makes four principal contributions: first, it integrates a self-attention module within a lightweight custom Convolution Neural Network (CNN) to effectively capture long-range spatial relationships across clinically pertinent cytological patterns while preserving a compact design. Second, it employs a Discrete Wavelet Transform (DWT)-based wavelet pooling layer that decreases feature dimensions by up to 96.875% while enhancing the obtained depictions with spatial-spectral information. Third, it utilizes a multi-layer feature fusion strategy that combines wavelet-pooled features from two deep layers with a third fully connected layer to create a discriminating multi-scale feature vector. Fourth, it incorporates Gradient-weighted Class Activation Mapping as a dedicated explainability process to furnish clinicians with apparent visual explanations for each classification decision. Withoit the need for image enhancement or segmentation preprocessing, LUMINA-Net outperforms the competing state-of-the-art methods on the same dataset, achieving a peak accuracy of 99.51%, specificity of 99.84%, and sensitivity of 99.51% on the publicly available Kaggle ALL dataset. This demonstrates that LUMINA-Net has the potential to be a dependable, effective, and clinically interpretable CAD tool for ALL diagnosis. Full article

Background: The aminosteroid RM-581 exhibits strong antiproliferative activity against cell lines from more than 10 solid tumor cancers, including some with poor prognoses. However, RM-581’s impact has never been assessed on leukemia. Methods: Cellular responses to RM-581 were evaluated using complementary approaches. Cytotoxicity was quantified using MTS-based viability assays and drug interactions were analyzed according to the Chou-Talalay method. Flow cytometry was employed to assess apoptosis, cell cycle distribution and effects on lymphocytes subpopulations. The transcriptomic profile was investigated by mRNA sequencing to identify differentially expressed genes and associated pathways. Results: Its evaluation on six leukemia cell lines (HL-60, THP-1, JURKAT, K-562, HG-3 and JVM-2) showed that RM-581 efficiently blocked the proliferation of leukemia cells. In healthy peripheral blood lymphocytes, flow cytometry revealed a significant impact on T lymphocytes (CD3+), particularly cytotoxic T cells (CD8+), at 50 µM. In THP-1 cells, an acute monocytic leukemia cell line, RM-581 triggered apoptosis and induced G0/G1 cell cycle arrest, which was confirmed with a transcriptomic analysis of enriched pathways. The role of RM-581 as an endoplasmic reticulum (ER) stress aggravator was confirmed by observing an increase in ER stress markers, such as BIP (GRP-78), CHOP and HERP, and in unfolded protein response (UPR) effectors (PERK, IRE1α and ATF6). Conclusions: This study demonstrates that RM-581 could be a promising candidate to treat leukemia, notably through the induction of ER-stress mediated apoptosis. Full article

Background/Objectives: Acute myeloid leukemia (AML) is characterized by impaired anti-leukemic immune responses, and the ex vivo or in vivo generation of dendritic cells (DCs), including leukemic dendritic cells (DC_leu), represents a promising strategy to stimulate immune cells and improve anti-leukemic activity. Methods: This study examined the generation, phenotype and functional relevance of DCs and DC_leu produced ex vivo from blast-containing PBMNCs and whole blood (WB) in AML. Using both standard DC/DC_leu-generating protocols and available Kits. Results: We show that DC/DC_leu can be reliably generated with both methods. Generated DC/DC_leu effectively activated T cells during mixed lymphocyte cultures (MLCs), resulting in enhanced anti-leukemic cytotoxicity. Improved blast lysis correlated with specific immunological features, including higher frequencies of generated DC_leu and mature DC subsets, as well as a certain cytokine pattern after DC/DC_leu cultures or MLC. In addition, the frequencies of proliferating T cells after MLC strongly correlated with the degree of achieved blast lysis, underscoring the importance of efficient DC/DC_leu-mediated T cell stimulation. Both the frequencies of generated DC/DC_leu and the resulting blast lytic activity were linked to overall survival (OS) in AML patients. Individuals who failed to demonstrate improved blast lysis exhibited significantly reduced OS, suggesting inadequate immune responsiveness of patients in vivo. Conclusions: These findings identify phenotypic and functional immune parameters as predictors of clinical outcome and highlight the prognostic relevance of ex vivo immune profiling. This approach may help to optimize and personalize future immunotherapeutic strategies in AML. Full article

Background/Objectives: Chondrosarcoma, glioblastoma, acute myeloid leukemia, chronic lymphocytic leukemia, and cholangiocarcinoma cancers all contain mutations in the gene isocitrate dehydrogenase 2 (IDH2). The mutant IDH2 enzyme metabolizes alpha-ketoglutarate (αKG) into the potent oncometabolite D-2-hydroxyglutarate (D2HG) in the mitochondria of these cancers, leading to altered cellular metabolism. Emerging evidence suggests that mitochondrial transfer between cancer and recipient cells represents an important form of intercellular communication that may influence cellular metabolism. The presence of intercellular TNTs between IDH2-mutant chondrosarcoma cells motivated an investigation into mitochondria-associated physiological changes occurring during an intercellular exchange with immune cells. A mitochondrial transfer is a two-way process, and we hypothesized that mitochondria-associated material derived from IDH2-mutant chondrosarcoma cells is exchanged with normal cells through TNTs. We further hypothesized that disruption of the actin cytoskeleton will inhibit this transfer. Accordingly, our objectives were to (1) quantify the extent and directionality of the mitochondrial exchange between IDH2-mutant cells and wild-type cells and to modulate this process via cytoskeletal inhibitors, and (2) measure the metabolic changes associated with the coculture and mitochondrial exchange. Methods: IDH2-mutant chondrosarcoma cells were cocultured with immune cells in vitro to quantify the extent and directionality of the mitochondrial exchange, and cytochalasin B was used as a cytoskeletal inhibitor to disrupt actin-dependent transfer. Metabolic changes associated with coculture and mitochondrial exchange were assessed using Seahorse extracellular flux analysis. Results: The experimental data presented here demonstrate a bidirectional exchange of mitochondria-associated material between IDH2-mutant chondrosarcoma cells and immune cells in vitro, accompanied by metabolic alterations in both cell types. Conclusions: These findings advance our understanding of intercellular communication in the tumor microenvironment and provide a foundation for future studies examining the functional and therapeutic relevance of a mitochondrial exchange in IDH2-mutant cancers. Full article

Background: Donor lymphocyte infusion (DLI) is commonly used to prevent or treat leukemic relapse following allogeneic hematopoietic cell transplantation; however, efficacy is limited by immune exhaustion, checkpoint-mediated inhibition, and the risk of graft-versus-host disease (GvHD). Gamma delta (γδ) T-cells represent a promising “off-the-shelf” adoptive cell therapy (ACT) with favorable safety and MHC-independent cytotoxicity, yet their function is similarly constrained by the leukemic tumor microenvironment (TME). Acute exercise mobilizes cytotoxic lymphocyte subsets, and is an emerging strategy to enhance cellular immunotherapies, including DLI and expanded γδ T-cells. This study examined how exercise-mobilized lymphocytes and exercise-expanded γδ T-cells interact with TIGIT blockade to improve anti-leukemic activity. Methods: Healthy participants completed an acute cycling bout, after which peripheral blood mononuclear cells (PBMCs) and ex vivo expanded γδ T-cells were phenotyped and cytotoxicity was determined against leukemia cells with TIGIT checkpoint inhibition. The therapeutic relevance of combining TIGIT blockade with rest- or exercise-expanded γδ T-cells was further evaluated in NSG-IL15 mice challenged with K562-luc leukemia. Results: Acute exercise increased circulating CD8⁺ and γδ T-cells with higher TIGIT and PD-1 expression. Exercise-expanded γδ T-cells maintained increased PD-1 and TIGIT expression and exhibited increased co-expression of DNAM-1 and TIGIT. Exercise mobilized PBMCs and exercise-expanded γδ T-cells demonstrated enhanced cytotoxicity, further amplified by TIGIT blockade. In vivo, TIGIT-treated exercise-expanded γδ T-cells modestly improved tumor suppression and prolonged tumor-free survival compared to untreated controls. Conclusions: Exercise primes DLI and γδ T-cell products for enhanced responsiveness to TIGIT checkpoint inhibition. Targeting TIGIT likely augments DNAM-1 dependent cytotoxicity and improves anti-leukemic activity, supporting the integration of exercise-enhanced DLI and γδ T-cell therapies with immune checkpoint blockade as a safe strategy to improve relapse control in leukemia. Full article

Objective: This study aimed to investigate erythrocyte morphological alterations in hematological malignancies, with particular emphasis on structural differences among leukemia subtypes and anemia. Materials and Methods: Peripheral blood samples were obtained from 60 patients, including individuals with anemia (n = 10), acute lymphoblastic leukemia (ALL, n = 15), acute myeloid leukemia (AML, n = 15), chronic lymphocytic leukemia (CLL, n = 15), and chronic myeloid leukemia (CML, n = 5), as well as 10 healthy controls. Erythrocyte morphology was evaluated using light microscopy and scanning electron microscopy. Morphological abnormalities, including loss of biconcavity, poikilocytosis, echinocyte transformation, burr cells, and stomatocytes, were assessed in accordance with International Council for Standardization in Haematology (ICSH)-based morphological definitions. Results: Distinct erythrocyte morphological alterations were observed across disease groups. AML cases demonstrated pronounced central depression-like or perforation-like structures and hypochromasia. Lymphoid malignancies, particularly ALL and CLL, exhibited increased echinocyte formation, whereas chronic leukemias showed a higher prevalence of stomatocytes and cup-shaped cells. Quantitative scoring indicated that loss of biconcavity was most prominent in anemia, followed by AML, CML, ALL, and CLL. Poikilocytosis was most frequent in anemia, followed by ALL, CLL, AML, and CML. Conclusions: The findings indicate that erythrocyte shape alterations are more heterogeneous and prominent in lymphoid leukemias, whereas myeloid leukemias exhibit distinct ultrastructural membrane abnormalities. Although studies focusing on erythrocyte morphology in leukemia remain limited, the present results provide a foundational morphological reference dataset that may support the development and validation of artificial intelligence-based diagnostic approaches. Further studies involving larger cohorts and expanded imaging analyses are warranted to improve diagnostic accuracy and translational applicability. Full article

Introduction: Chronic lymphocytic leukemia (CLL) is a common adult leukemia often treated with fludarabine, cyclophosphamide, and rituximab (FCR). While effective, FCR can lead to therapy-related myeloid neoplasms (t-MN), including aggressive therapy-related acute myeloid leukemia (t-AML). Stem cell transplantation offers the best chance for long-term remission in these cases. Here, we report a rare case of t-AML with plasmacytoid dendritic cells (pDC-AML) developing after FCR treatment for CLL that was successfully treated with haplotransplantation. Case Presentation: A 57-year-old woman with CLL-B was treated with six cycles of FCR, achieving a complete response. Six years later, at age 63, she developed t-AML with a rare morphophenotypic subtype: acute myelomonocytic leukemia with plasmacytoid dendritic cells (pDC-AML) and monosomy 8. Diagnostic challenges included distinguishing this subtype from blastic plasmacytoid dendritic cell neoplasm (BPDCN). She was treated with high-dose cytarabine followed by haploidentical stem cell transplantation from her son. Haploidentical transplantation was prioritized due to the urgent clinical need in a patient with high-risk acute leukemia (therapy-related leukemia secondary to prior chemoimmunotherapy and failure to achieve complete remission following the standard 3 + 7 induction protocol). In this critical setting, the patient’s son was immediately available as an HLA-haploidentical donor. Prior to the performance of the haploidentical stem cell transplant from her son, no HLA-matched unrelated donor (MUD) could be identified. Another viable alternative would have been the utilization of umbilical cord blood-derived stem cells harvested from the patient’s twin granddaughters. She was closely monitored post-transplant for potential complications, including graft-versus-host disease (GVHD), post-transplant lymphoproliferative disorder, and thyroid dysfunction, all of which were ruled out during follow-up. The patient remains in complete remission 15 years after her initial CLL diagnosis and 8 years after the t-AML diagnosis and haplotransplantation. Notably, no residual CLL clone was detected at the time of t-AML development, and a benign polyclonal lymphocytosis observed between 2018 and 2020 spontaneously resolved without intervention. Conclusions: This case illustrates the potential for long-term survival in high-risk patients with therapy-related AML developed after cytotoxic treatment for lymphoid malignancies. Haplotransplantation from a semi-identical Human Leukocyte Antigen (HLA) donor proved to be a viable and effective treatment option despite the patient’s age and dual hematologic malignancies. Full article

Background/Objectives: Conventional workflows, peripheral blood smears, and bone marrow assessment supplemented by LDI-PCR, molecular cytogenetics, and array-CGH, are expert-driven in the face of biological and imaging variability. Methods: We propose an AI pipeline that integrates convolutional neural networks (CNNs) and transfer learning-based models with two explainable AI (XAI) approaches, LIME and Grad-Cam, to deliver both high diagnostic accuracy and transparent rationale. Seven public sources were curated into a unified benchmark (66,550 images) covering ALL, AML, CLL, CML, and healthy controls; images were standardized, ROI-cropped, and split with stratification (80/10/10). We fine-tuned multiple backbones (DenseNet-121, MobileNetV2, VGG16, InceptionV3, ResNet50, Xception, and a custom CNN) and evaluated the accuracy and F1-score, benchmarking against the recent literature. Results: On the five-class task (ALL/AML/CLL/CML/Healthy), MobileNetV2 achieved 97.9% accuracy/F1, with DenseNet-121 reaching 97.66% F1. On ALL subtypes (Benign, Early, Pre, Pro) and across tasks, DenseNet121 and MobileNetV2 were the most reliable, achieving state-of-the-art accuracy with the strongest, nucleus-centric explanations. Conclusions: XAI analyses (LIME, Grad-CAM) consistently localized leukemic nuclei and other cell-intrinsic morphology, aligning saliency with clinical cues and model performance. Compared with baselines, our approach matched or exceeded accuracy while providing stronger, corroborated interpretability on a substantially larger and more diverse dataset. Full article