B-cell maturation antigen (BCMA) is the main target for chimeric antigen receptor (CAR)-T cells in multiple myeloma (MM), demonstrating promising outcomes. However, unlike what happens with CART19 in lymphoblastic leukemia and non-Hodgkin's lymphoma, a high proportion of patients will relapse after CAR-T BCMA therapy due to insufficient antigen expression, low CAR-T cell persistence and/or T-cell exhaustion. In other B cell malignancies, second-generation anti-CD19 4-1BB CARs with CD28-transmembrane domain (TMD) have shown high efficacy and a favorable toxicity profile. We have developed a second-generation CD8α-TM BCMA-4-1BBζ CAR-T product, ARI0002h (Cesnicabtagene-autoleucel) for patients with relapsed/refractory MM. We hypothesized that replacing the TMD of ARI0002h with a CD28-TMD could increase efficacy and reduce tumor escape while maintaining a tolerable toxicity profile. We generated CAR-T cells using T-cells isolated from buffy coats and evaluated the efficacy and fitness of CAR-Ts at day 8-10 of expansion against several MM cell lines. In vitro analyses included cytotoxicity, proliferation, cytokine secretion, T-cell subset markers, activation and exhaustion profiling, metabolomic assays, and RNA-seq after multiple tumor challenges. In in vivo xenograft studies using NSG mice, with tumor cells expressing GFP-ffLuc, disease progression was monitored weekly via bioluminescence imaging. Despite showing similar in vitro performance regarding cytotoxicity, proliferation and cytokine production, ARI2h-TM28 outperforms ARI0002h in a low BCMA expression setting and achieves superior in vivo tumor control and survival in relapse models with antigen downregulation. Furthermore, ARI2h-TM28 showed an optimized metabolic profile, more oxidative and energetic compared with ARI0002h, with downregulation of proinflammatory genes in CD8 T cells, contributing altogether both to reduced exhaustion and increased persistence of the CARs, improving their efficacy in preclinical models. Incorporating a CD28-TMD into the ARI0002h CAR enhances tumor control even in relapse models with downregulation of the target antigen, offering improved long-term disease management. This modification increases potency against MM tumor cell lines with both normal and reduced BCMA expression, demonstrating superior metabolic endurance and in vivo activity.

Autophagy intensity decreases in most blood malignancies, promoting cancer cell proliferation. Acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) are two main variants of leukemia. LC3, an important autophagy gene, and its protein, MAP1LC3B, precisely predict autophagy initiation and progression. The main objective of this systematic review is to investigate the correlation between changes in the expression of the LC3 gene in patients with ALL and AML. We performed a literature search in PubMed, Scopus, and Web of Science database in November 20, 2025, using the keywords and MeSH terms. All searches were restricted to sources in the English language, andwe focused on human case-control studies where definitive diagnosis was performed using hematological parameters. Initial identification included 1282 articles. Duplicates were removed, leaving 821 papers for screening. From these, 787 articles were removed for inappropriate titles and abstracts, leaving 34 for further review. Ultimately, 10 papers met the criteria for this systematic review. Five AML investigations demonstrated considerably lower LC3 gene expression than healthy controls. Downregulation was similar in one research. Upregulation was seen in two investigations. In two papers, ALL patients had considerably lower expression than healthy controls. Upregulation was significant in one study and non-significant in another one. The LC3 gene appears to be downregulated more consistently in AML, suggesting potential diagnostic or prognostic value. However, findings in ALL are inconsistent, and no definitive conclusion can be drawn. Further high-quality studies are required to clarify the role of LC3 in acute leukemias.

The inherent fluorescent properties of anti-leukemic drugs offer unique advantages for real-time therapeutic tracking and optimization. In this study, we systematically screened the absorption and emission spectra of 82 leukemia-related compounds, identifying 28 autofluorescent drugs suitable for fluorescence-based optical concentration monitoring. Excitation and emission parameters were evaluated across various solvents (DMSO, fetal bovine serum, and culture media), revealing solvent-dependent spectral changes, intensity variations, and effect on detection limits. These 28 compounds were further assessed for cytotoxicity screening in case of drug-naive and drug-resistant K562 leukemia lymphoblast cells. By correlating their spectral properties with cytotoxic responses, our study establishes a robust framework for fluorescence-assisted drug profiling, enabling pharmacokinetic insights, resistance prediction, and informed therapeutic adjustments. These findings underscore the translational potential of fluorescence-based methodologies in supporting precision medicine for leukemia treatment.

Abstract Background Infections are common complications in pediatric allogeneic hematopoietic stem cell transplantation (HSCT), with pneumonia often leading to poor outcomes. Data on pediatric patients with pneumonia post-HSCT are limited. This study describes the clinical characteristics and outcomes of affected patients, including those who survived and those who did not. Methods In this prospective cohort study, pediatric patients with pneumonia following HSCT were enrolled between July 2022 and February 2025 across nine HSCT units in Colombia, Mexico, Argentina, and Chile. Cases were identified through active surveillance. Pneumonia was defined as the presence of fever and radiologic infiltrates. The primary outcome was pneumonia-related mortality, as determined by site investigators. Patients were followed until symptom resolution or death. Results 107 patients were included (median age 3.7 years; 57% male), with lymphoblastic leukemia as the most common HSCT indication. Pneumonia developed 83 days post-HSCT (median; IQR 22–237) (Table 1). Bacterial infections predominated, especially within the first 30 days post-transplant (Figure 1a). Pneumonia-related mortality occurred in 28 patients (26%), often between 30 and 100 days post-HSCT (Figure 1b). Mortality was associated with reduced-intensity conditioning (Table 1) and in those presenting with hypotension or hypoxemia (Table 2). Lower death risk was observed in patients presenting with cough or upper respiratory symptoms. Deceased patients were more often neutropenic and lymphopenic, frequently had adenovirus-associated pneumonia and showed certain radiographic features in X-ray, such as central and peripheral infiltrates, as well as diffuse opacities (Table 2). Fifty-three and 44% of patients required intensive care and mechanical ventilation, respectively (Table 3). Conclusion Pneumonia post-HSCT is a major complication in children with a high mortality rate. This well characterized cohort revealed clinical, temporal, etiological, laboratory, and radiologic factors associated with mortality. Continued collaboration is essential to enhance the dataset, identify and validate independent predictors of mortality, and propose effective preventive strategies. Disclosures Almudena Laris Gonzalez, MD, MSc, Astra Zeneca: Advisor/Consultant|GSK: Honoraria|Sanofi: Advisor/Consultant

In Silico-driven engineering of Halomonas elongata L-asparaginase: Towards enhanced proteolytic resistance in lymphoblastic leukemia

Background: Chronic inflammation and the development of cancer are closely linked, with components that comprise the tumour microenvironment—including proinflammatory cytokines—exerting essential tumourigenic effects. These proinflammatory cytokines include IL-1β, which has been reported to be overexpressed in several cancers and shown to activate several signalling pathways. These pathways may involve kinases such as AKT (serine/threonine kinase) and Src (Proto-oncogene tyrosine-protein kinase), and have a broad capacity to activate nuclear factors, including NF-κB (Nuclear Factor kappa-light-chain-enhancer of activated B cells), which can regulate the transcription of genes encoding proteins such as cIAP1 (Cellular Inhibitor of Apoptosis Protein 1), Bcl-2 (B-cell lymphoma 2), and cyclin D1, thereby regulating processes like apoptosis and cell cycle inhibition. Objectives: The aim of this study was to investigate the role of IL-1β (Interleukin-1 beta) in regulating cell death and proliferation in RS4:11 leukaemic lymphoblasts via the PI3K (Phosphoinositide 3-kinase)/AKT/Src/NF-κB pathway using an in vitro experimental approach. Methods: We employed flow cytometry to determine the expression levels and phosphorylation status of various proteins; proliferation was assessed using the CCK-8 kit, and apoptosis was evaluated with the Annexin V kit. Results: Our findings indicate that the IL-1β-activated signalling pathway modulates these cellular processes in leukaemic lymphoblasts. Conclusions: We therefore conclude that IL-1β exerts significant effects on cell death and proliferation in leukaemic lymphoblasts through the PI3K/AKT/NF-κB pathway, with the study’s findings indicating that an inflammatory environment may promote such lymphoblasts to acquire neoplastic characteristics. As such, the proteins involved in the effects evaluated in this work could be considered as potential therapeutic targets for the treatment of Acute Lymphoblastic Leukaemia (ALL).

Identification of TPR::FGFR1 fusion in an unusual case of concurrent acute B lymphoblastic leukemia and T-lymphoblastic lymphoma: Case report and review of literature

Title: Role of primary VTE prophylaxis in pediatric leukemia, lymphoma, and solid tumors: A systematic review and meta-analysis.

Novel bispecific epitope anti-CD5 nanobody CAR-T cells for refractory or relapsed T-cell malignancies

Oncofetal RNA-binding proteins IGF2BP suppress innate immunity signaling pathways in leukemia stem cells

ESAM is functionally involved in the activity of cancer stem-like cells in human acute myeloid leukemia

Immunophenotypic identification of ETP-like T-ALL using CD1a, CD165 and CD184.

Venous thromboembolism (VTE) is a serious complication of pediatric acute lymphoblastic leukemia (ALL), primarily occurring during induction therapy and associated with acquired hemostatic abnormalities. However, the contribution of leukemic lymphoblasts to hypercoagulability remains unexplored. This study aimed to determine whether leukemic lymphoblasts express hemostatic factors that promote a hypercoagulable state and to assess the functional impact of lysed lymphoblasts on thrombin and fibrin generation.We examined the expression of 28 hemostatic factors at both mRNA and protein levels in four pediatric leukemic cell lines (T-ALL and B-ALL) and normal lymphocytes using RT-PCR and immunoblotting. To evaluate the overall functional effect, we conducted thrombin and fibrin generation assays by adding cell lysates to platelet-poor plasma in the absence of exogenous tissue factor or phospholipids.Leukemic lymphoblasts constitutively expressed the procoagulants tissue factor, factor VIII and factor XIIIa; the coagulation inhibitors antithrombin, ADAMTS13 and TFPI; and the pro-fibrinolytic and antifibrinolytic proteins uPA, TAFI, and α2-AP. Lysed, but not intact, leukemic lymphoblasts enhanced thrombin and fibrin generation, indicating a procoagulant state. Additionally, leukemic lysates exhibited a hypo-fibrinolytic state, as evidenced by prolonged fibrin clot lysis times.These findings suggest that leukemic lymphoblasts actively contribute to a hypercoagulable state in pediatric ALL by simultaneously increasing procoagulant activity and impairing fibrinolysis. This study provides novel insights into the mechanisms underlying VTE risk in pediatric ALL, highlighting the role of leukemic lymphoblasts in disrupting the hemostatic balance. · Leukemic lymphoblasts express hemostatic system factors at gene and protein levels.. · Leukemic lymphoblasts lysis directly contributes to hypercoagulability and hypofibrinolysis in thrombin and fibrin generation assays..

Label-free classification of single-cell lymphoma is essential for advancing cancer diagnostics. This study presents a method combining scattering imaging flow cytometry (SIFC) with machine learning to classify normal CD4+ T lymphocytes and T lymphoblastic leukemia (SUP-T1) cells based on distinct scattering patterns. Scattering data were captured using a microfluidic chip integrated with the laser-based SIFC system, and different lymphoma cells were classified based on the feature values of scattering images by means of machine learning. The results of five-fold cross-validation demonstrate that the model has excellent discriminatory power and reliability, with AUC values ranging from 0.945 to 0.964 (average: 0.957), validation set accuracies ranged from 86.3% to 92.0% (average: 89.0%), sensitivity, indicating correct identification of SUP-T1 cells, ranged from 89.0% to 93.0% (average: 91.6%), and specificity, indicating correct identification of CD4+ T lymphocytes, ranged from 83.0% to 91.5% (average: 86.4%). These findings indicate that SIFC, combined with machine learning, offers a promising approach for label-free, high-accuracy, and low-cost single-cell classification, with potential applications in cancer diagnostics.

Therapeutic adaptations: Rationale, achievements and limits. Experience of GFAOP

Research on CD9 expression has been extensive in B lymphoblastic leukemia, with fewer studies focusing on acute myeloid leukemia (AML). We investigated the usefulness of CD9 in differentiating normal from abnormal myeloid progenitors, as well as expression in normal cell types and in AML. Flow cytometry was used to assess the level of CD9 expression on normal and leukemic myeloid blasts and other normal bone marrow populations. Geometric mean fluorescence intensity levels and expression patterns were compared among cell types and AML subtypes. In normal subsets (n = 69), the level of CD9 expression was lowest in mature B cells, myeloid blasts, promyelocytes, and neutrophils, with intermediate expression in monocytes and highest in hematogones (stages 1 and 2). Committed myeloid progenitors (CMPs) had lower expression than hematopoietic stem cells (HSCs). CD9 typically has higher expression in AML (n = 58) compared to normal myeloid blasts and promyelocytes, and it is differentially expressed in AML, with the highest expression in PML::RARA AML. Aberrant CD9 expression can be useful differentiating normal from abnormal myeloid progenitors, with the highest level of expression in AML with PML::RARA in our cohort. There was differential expression between HSCs and CMPs in the small numbers studied. Normal mature B cells can be used as an internal negative control in most cases.

Two types of plasmacytoid dendritic cell (pDC) proliferation disease are acknowledged so far by the 5th edition of the World Health Organization Classification of Haematolymphoid Tumors: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) and mature pDC proliferation associated with myeloid neoplasms (MPDCP) in which pDC is part of the malignant clone. We aim to investigate pDC proliferation associated with non-myeloid acute leukemia (AL). A retrospective analysis of all cases admitted in our center with a diagnosis of non-myeloid AL from September 2020 to April 2023 was performed to select cases with pDCs greater than 2% of bone marrow by flow cytometry (FCM). We conducted comprehensive analyses encompassing FCM immunophenotyping, histopathological examination, morphological assessment, cytogenetic studies, and molecular genetic profiling across all cases. Proliferation of pDCs was detected in 10 (0.88%) out of 1140 non-myeloid AL patients by FCM, including 4/944(0.42%) cases of B lymphoblastic leukemia (B-ALL), 3/95 (3.16%) cases of T lymphoblastic leukemia (T-ALL) and 3/101 (2.97%) cases of acute leukemia of ambiguous lineage (ALAL) (p = 0.009). The 4 cases of B-ALL were all Philadelphia Chromosome positive (Ph+). PDCs in 3 out of 10 patients expressed CD56 (37.5%), 8/10 expressed CD7 (80%), 9/10 expressed CD303 (90%), all expressed CD304 (100%), and 5 of 8 evaluable cases were positive for CD34 (62.5%). In cases in which pDCs expressed CD7 and/or CD56, the blast cells all expressed CD7 and/or CD56 as well; the pDCs in all B-ALL patients expressed CD19. FCM dot plot in 2 of the B-ALL-pDC showed a spectrum from blast cells to pDCs: CD303 and CD304 gradually emerged as CD34 disappeared. Among the 8 patients who underwent bone marrow biopsy, pDCs exhibited two distinct distribution patterns: pure interstitial infiltration in 6 cases (75%) and focal/scattered clusters against an interstitial background in 2 cases (25%). NRAS showed recurrent mutations at identical genomic positions. Each NRAS variant (c.35G>A and c.38G>T) was detected twice across three patients. FCM could effectively detect pDC proliferation in non-myeloid leukemia, which occurred at a significantly higher incidence in T-ALL and ALAL than in B-ALL. In B-ALL, it was associated with the Ph + subtype. PDCs and blast cells shared some lymphoid antigens that were uncommon in AML-pDC or BPDCN. In the bone marrow, pDCs were predominantly characterized by an interstitial infiltration pattern.

A retrospective analysis was conducted on the clinical data of acute B lymphoblastic leukemia (B-ALL) patients with TAF15::ZNF384 fusion gene positive at Hebei Yanda Ludaopei Hospital from December 2018 to February 2022. The patients were followed up until December 2024 to analyze their clinical characteristics and outcomes. A total of 6 patients were included, including 4 males and 2 females, aged 16 to 47 years. The follow-up period ranged from 10 days to 65 months. All 6 patients sought medical attention due to incomplete remission (CR) after treatment at an external hospital or short-term recurrence after CR. Five of the 6 patients presented with chromosomal abnormalities at initial diagnosis, including 4 with t(12;17) (p13;q12) chromosomal translocation. Immunophenotyping showed the B lymphatic system markers CD19, CD22, and cytoplasmic CD79a were positive in all cases, accompanied by positive myeloid markers such as CD33 or CD31. Four patients showd positive expression of the lymphoid maker CD10. Among the 6 patients, 4 patients achieved CR after receiving chimeric antigen receptor T-cell (CAR-T) therapy, and maintained CR after bridging allogeneic hematopoietic stem cell transplantation (allo-HSCT). B-ALL patients harboring the TAF15::ZNF384 fusion gene often present with complex chromosomal abnormalities at the initial diagnosis and the immunophenotype is often characterized by B lymphatic system with positive myeloid markers. CAR-T immunotherapy followed by allo-HSCT may offer a promising approach to improving their prognosis.

Purpose: To evaluate the uniformity of dose distribution in the target (throughout the patient’s body) for 39 sick children during total body irradiation. Material and methods: Total body irradiation (TBI) of 39 children with lymphoblastic leukemia was performed on the Clinac iX electron accelerator (Varian) with a photon radiation 6 MV. The field size at the diaphragm was 40 × 40 cm. The patient’s position from the isocenter of the accelerator is 550 cm. The single dose is 1,0 Gy, the total dose is 12,0 Gy. The number of fractions is 6. Irradiation was carried out behind plexiglas with protective cerrobend blocks on the lungs. To ensure uniformity of dose distribution, boluses of tissue-equivalent material were used. The dose rate is low and ranges from 0,05 to 0,10 Gy/min, the calculation of irradiation plans is performed on the Eclipse (Varian) planning system. Irradiation is performed with two opposite wide fields with the patient in the lying on the right side position on the therapeutic table. Vacuum mattresses are used as a patient fixation device. A quantitative analysis of patient irradiation plans was carried out using dose– volume histograms with the use of the homogeneity index HI and assessments of dose loads on critical structures (heart, lungs, kidneys, eye lenses). Results: It is shown that for 96 % of patients, doses in all organs are distributed uniformly within a tolerance of 10 %, a similar picture is observed for a tolerance of 15 %. The dose homogeneity of the HI dose distribution within the target volume was estimated with and without taking into account boluses. In the presence of boluses, the average value of the homogeneity index HI is lower than in their absence. This means that the use of tissue-equivalent boluses contributes to an increase in the homogeneity of dose distribution and is absolutely justified.

Background/AimCell lines serve as valuable in vitro models to study altered cellular signaling pathways, to identify mutations in key oncogenic genes, and to test potential antitumor drugs. The Jurkat cell line, for example, has provided important information about various signaling pathways in lymphoblastic leukemia, establishing most of what is currently known about T-cell receptor (TCR) signaling. However, many aspects of the genome modification of this cell line have not yet been analyzed. To identify genes of potential biological and clinical relevance in acute T-lymphoblastic leukemia (T-ALL), we performed an array comparative genomic hybridization (aCGH) approach on the widely used Jurkat cell line and examined the association of the detected copy number alterations (CNAs) with cancer hallmarks and T-ALL pathogenesis.Materials and MethodsCells were harvested by using trypsin/EDTA from culture flasks to extract genomic DNA. aCGH experiments were performed on an Agilent microarray platform using the SurePrint G3 Cancer CGH + SNP Microarray 4×180 K. Functional enrichment analysis of all CNAs was performed with the R package g:Profiler2. The association of these alterations with key cancer hallmarks was analyzed using the Cancer Hallmarks web-tool.ResultsOur analysis revealed several novel CNAs, including losses at 5p15.2, 6q27, 10q22.2, 14q11.2, 18q11.2-q12.1, and Xp22.33, as well as gains at 2p11.2, 7p21.2, 7q21.2 and 18p11.32. Genes within these regions were associated with important oncogenic pathways, including ‘sustained proliferative signaling’, ‘tumor suppressor evasion’, and ‘angiogenesis promotion’.ConclusionThese findings suggest that Jurkat cells may serve as a valuable model for identifying new targets for cancer research. Further studies are required to confirm the phenotypic implications of these variants, which may open new avenues for exploring the functional impact of these alterations and their potential role in the development of therapies.