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This study investigated the importance of comprehensive genetic diagnosis in pediatric B-cell acute lymphoblastic leukemia (B-ALL). We analyzed 175 B-ALL employing karyotyping, FISH, MLPA, targeted next-generation sequencing (t-NGS), and Optical Genome Mapping (OGM). This approach achieved an 83% classification rate, identifying 17 distinct genetic subtypes. Specifically, within B-other subtype, seven different subgroups were identified (ZNF384, IGH, DUX4, NUTM1 rearrangements, PAX5 alterations, PAX5 P80R, and IKZF1 N159Y). Secondary genetic alterations were observed, with copy number alterations (CNA) present in 60% of cases and mutations detected in 70.6%. While these alterations exhibited specific associations with certain genetic subtypes, CNAs did not appear to significantly impact the prognosis within these genetic groups. HeH, ETV6::RUNX1, ZNF384-r, and PAX5 P80R exhibited excellent outcomes, contrasting with the poor prognoses observed in KMT2A-r, hypodiploidy, and CRLF2-r (5-year overall OS were 50%, 50%, and 52%, respectively). These findings underscore the value of integrated genetic diagnostics for accurate subtyping, risk stratification, and guiding personalized treatment in pediatric B-ALL. Therefore, optimizing diagnostic workflows for routine clinical practice is crucial. Our study confirms the utility of conventional techniques (karyotyping and FISH), combined with t-NGS and OGM, for comprehensive genetic diagnosis., Competing Interests: Declarations. Ethical approval: All patients provided informed consent in accordance with protocols approved by Institutional Ethics Committee of Vall d’Hebron University Hospital and in compliance with the Declaration of Helsinki. Competing interest: The authors declare no competing interests., (© 2025. The Author(s).)

In chronic lymphocytic leukemia, the reliability of next-generation sequencing (NGS) to detect TP53 variants ≤10% allelic frequency (low-VAF) is debated. We tested the ability to detect 23 such variants in 41 different laboratories using their NGS method of choice. The sensitivity was 85.6%, 94.5%, and 94.8% at 1%, 2%, and 3% VAF cut-off, respectively. While only one false positive (FP) result was reported at >2% VAF, it was more challenging to distinguish true variants <2% VAF from background noise (37 FPs reported by 9 laboratories). The impact of low-VAF variants on time-to-second-treatment (TTST) and overall survival (OS) was investigated in a series of 1092 patients. Among patients not treated with targeted agents, patients with low-VAF TP53 variants had shorter TTST and OS versus wt- TP53 patients, and the relative risk of second-line treatment or death increased continuously with increasing VAF. Targeted therapy in ≥2 line diminished the difference in OS between patients with low-VAF TP53 variants and wt- TP53 patients, while patients with high-VAF TP53 variants had inferior OS compared to wild type- TP53 cases. Altogether, NGS-based approaches are technically capable of detecting low-VAF variants. No strict threshold can be suggested from a technical standpoint, laboratories reporting TP53 mutations should participate in a standardized validation set-up. Finally, whereas low-VAF variants affected outcomes in patients receiving chemoimmunotherapy, their impact on those treated with novel therapies remains undetermined. Our results pave the way for the harmonized and accurate TP53 assessment, which is indispensable for elucidating the role of TP53 mutations in targeted treatment., Competing Interests: Bárbara Tazón‐Vega: Honoraria: Bristol Meyer Squibb. Beatriz Bellosillo: Advisory board honoraria, research support, travel support, speaker fees: Astra‐Zeneca, BMS, Janssen, Merck‐Serono, Novartis, Pfizer, Hoffman‐La Roche, ThermoFisher. Christian Brieghel: Travel grant: Octapharma. Carsten U. Niemann: Research funding and/or consultancy fees: Abbvie, AstraZeneca, Beigene, Janssen, Genmab, Lilly, MSD, CSL Behring, Takeda, Octapharma. Davide Rossi: Honoraria: AbbVie, AstraZeneca, BeiGene, BMS, Janssen, Lilly. Research grants: AbbVie, AstraZeneca, Janssen. Travel grants: AstraZeneca, Janssen. Eugen Tausch: Honoraria and research support: Abbvie, AstraZeneca, BeiGene, Janssen, Hoffmann‐La Roche; Research support from Abbvie, Roche, Gilead. Frédéric Davi: Honoraria: Janssen, AstraZeneca. Kostas Stamatopoulos: Research funding, honoraria and/or consultancy fees: Abbvie, AstraZeneca, Janssen, Lilly, Roche. Lydia Scarfo: Consultancy: AbbVie, AstraZeneca, BeiGene, Janssen, Lilly; Speaker Bureau: Octapharma. Miguel Alcoceba: Honoraria and travel grants: Janssen, AstraZeneca. Martin Andres: Consultancy, Honoraria, and travel support: AstraZeneca, Novartis, Roche, Janssen‐Cilag. Maria Gomes da Silva: Consultancy and Research Funding: Janssen Cilag, AstaZeneca, Abbvie, Roche, Takeda. Pau Abrisqueta: Consultancy and Honoraria: Janssen, Abbvie, Roche, BMS, AstraZeneca, Genmab. Panagiotis Baliakas: Honoraria: Abbvie, Gilead, Janssen. Research funding: Gilead. Paolo Ghia: Honoraria: AbbVie, Astrazeneca, BeiGene, BMS, Galapagos, Janssen, Lilly/Loxo Oncology, MSD, Roche. Research funding: AbbVie, AstraZeneca, BMS, Janssen. Richard Rosenquist: Honoraria: AbbVie, AstraZeneca, Janssen, Illumina, Roche. Renata Walewska: Travel support: AbbVie, AstraZeneca, Janssen, Beigene. Sylwia Czekalska: Honoraria: AstraZeneca. Funding: Janssen, AstraZeneca. Stephan Stilgenbauer: Advisory board honoraria, research support, travel support, speaker fees: AbbVie, Acerta, Amgen, AstraZeneca, BeiGene, BMS, Celgene, Gilead, GSK, Hoffmann‐La Roche, Infinity, Janssen, Lilly, Novartis, Sunesis, Verastem. Tiina Kahre: Honoraria: AstraZeneca. Tomasz Stokłosa: Honoraria and Research Funding: Janssen, AstraZeneca. The remaining authors have no competing interests to declare., (© 2025 The Author(s). HemaSphere published by John Wiley & Sons Ltd on behalf of European Hematology Association.)

Purpose: The study conducts a comparative analysis between two prominent methods for fabricating composites for bone scaffolds-the (solid) solvent method and the solvent-free (melting) method. While previous research has explored these methods individually, this study provides a direct comparison of their outcomes in terms of physicochemical properties, cytocompatibility, and mechanical strength. We also analyse their workflow and scalability potentials., Design/methodology/approach: Polycaprolactone (PCL) and hydroxyapatite (HA) composites were prepared using solvent (chloroform) and melting (180°C) methods, then 3D-printed using an extrusion-based 3D printer to fabricate scaffolds (8 × 8 × 4 mm). Rheology, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), accelerated degradation, mechanical/compression test, wettability/contact angle, live/dead assay, and DNA quantification (Picogreen) assays were evaluated., Findings: The study finds that scaffolds made via the solid solvent method have higher mechanical strength and degradation rate as compared to those from the melting method, while both methods ensure adequate cytocompatibility and homogenous hydroxyapatite distribution, supporting their use in bone tissue engineering., Originality: This research investigates the utility of chloroform as a solvent for PCL composite in a direct comparison with the melting method. It also highlights the differences in workflows between the two methods and their scalability implications, emphasizing the importance of considering workflow efficiency and the potential for automation in scaffold fabrication processes for bone tissue engineering applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 De Vega, Dutta, Mumtaz, Schroeder, Gerrand, Boyd and Kalaskar.)

In 2015, Catalonia introduced sickle cell disease (SCD) screening in its newborn screening (NBS) program along with standard-of-care treatments like penicillin, hydroxyurea, and anti-pneumococcal vaccination. Few studies have assessed the clinical impact of introducing NBS programs on SCD patients. We analyzed the incidence of SCD and related hemoglobinopathies in Catalonia and the change in clinical events occurring after introducing NBS. Screening 506,996 newborns from 2015 to 2022, we conducted a retrospective multicenter study including 100 screened (SG) and 95 unscreened (UG) SCD patients and analyzed SCD-related clinical events over the first six years of life. We diagnosed 160 cases of SCD, with an incidence of 1 in 3169 newborns. The SG had a significantly lower median age at diagnosis (0.1 y vs. 1.68 y, p < 0.0001), and initiated penicillin prophylaxis (0.12 y vs. 1.86 y, p < 0.0001) and hydroxyurea treatment earlier (1.42 y vs. 4.5 y, p < 0.0001). The SG experienced fewer median SCD-related clinical events (vaso-occlusive crisis, acute chest syndrome, infections of probable bacterial origin, acute anemia requiring transfusion, acute splenic sequestration, and pathological transcranial Doppler echography) per year of follow-up (0.19 vs. 0.77, p < 0.0001), a reduced number of annual emergency department visits (0.37 vs. 0.76, p < 0.0001), and fewer hospitalizations (0.33 vs. 0.72, p < 0.0001). SCD screening in Catalonia's NBS program has effectively reduced morbidity and improved affected children's quality of life.

Approximately 80% of breast cancer (BC) cases are estrogen receptor positive (ER+) and sensitive to hormone treatment; Tamoxifen is a prodrug, and its main plasmatic active metabolites are 4-hydroxytamoxifen (4-OH Tam) and endoxifen. Despite the effectiveness of tamoxifen therapy, resistance can be developed. An increment in eukaryotic initiation factor-4A complex (eIF4A) activity can result in tamoxifen-resistant tumor cells. For this work, we developed a cell variant resistant to 4-OH Tam and endoxifen, denominated MCF-7 Var E ; then, the aim of this research was to reverse the acquired resistance of this variant to tamoxifen metabolites by incorporating the natural compound auraptene. Combination treatments of tamoxifen derivatives and auraptene successfully sensitized the chemoresistant MCF-7 Var E . Our data suggest a dual regulation of eIF4A and ER by auraptene. Joint treatments of 4-OH Tam and endoxifen with auraptene identified a novel focus for chemoresistance disruption. Synergy was observed using the auraptene molecule and tamoxifen-derived metabolites, which induced a sensitization in MCF-7 Var E cells and ERα parental cells that was not observed in triple-negative breast cancer cells (TNBC). Our results suggest a synergistic effect between auraptene and tamoxifen metabolites in a resistant ER+ breast cancer model, which could represent the first step to achieving a pharmacologic strategy.

Objective: Estrogen receptor-positive (ER+) breast cancer represents about 80% of cases, tamoxifen is the election neoadjuvant chemotherapy. However, a large percentage of patients develop chemoresistance, compromising recovery. Clinical evidence suggests that high plasmatic levels of low-density lipoproteins (LDL) could promote cancer progression. The present study analyzed the effect of LDL on the primary plasmatic active Tamoxifen's metabolites resistance acquisition, 4-hydroxytamoxifen (4OH-Tam) and 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), in breast cancer ERα + cells (MCF-7)., Methods: Two resistant cellular variants, MCF-7Var-H and MCF-7Var-I, were generated by a novel strategy and their phenotype features were evaluated. Phenotypic assessment was performed by MTT assays, cytometry, immunofluorescence microscopy, zymography and protein expression analysis., Results: MCF-7Var-H, generated only with tamoxifen metabolites, showed a critical down-regulation in hormone receptors, augmented migration capacity, metalloprotease 9 extracellular medium excretion, and a mesenchymal morphology in contrast with native MCF-7, suggesting the transition towards Triple-negative breast cancer (TNBC) phenotype. In contrast, MCF-7Var-I which was generated in a high LDL media, showed only a slight upregulation in ER and other less noticeable metabolic adaptations. Results suggest a potential role of transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) in phenotypic differences observed among variants., Conclusion: LDL high or low concentrations during Tamoxifen´s metabolites chemoresistance acquisition leads to different cellular mechanisms related to chemoresistance. A novel adaptative cellular response associated with Nrf2 activity could be implicated., (© 2024 The Author(s).)

Sr50, an intracellular nucleotide-binding leucine-rich repeat receptor (NLR), confers resistance of wheat against stem rust caused by the fungal pathogen Puccinia graminis f. sp. tritici . The receptor recognizes the pathogen effector AvrSr50 through its C-terminal leucine-rich repeat domain, initiating a localized cell death immune response. However, this immunity is compromised by mutations in the effector, as in the escape mutant AvrSr50 QCMJC , which evades Sr50 detection. In this study, we employed iterative computational structural analyses and site-directed mutagenesis for rational engineering of Sr50 to gain recognition of AvrSr50 QCMJC . Following an initial structural hypothesis driven by molecular docking, we identified the Sr50 K711D single mutant, which induces an intermediate immune response against AvrSr50 QCMJC without losing recognition against AvrSr50. Increasing gene expression with a stronger promoter enabled the mutant to elicit a robust response, indicating weak effector recognition can be complemented by enhanced receptor expression. Further structural refinements led to the creation of five double mutants and two triple mutants with dual recognition of AvrSr50 and AvrSr50 QCMJC with greater immune response intensities than Sr50 K711D against the escape mutant. All effective mutations against AvrSr50 QCMJC required the K711D substitution, indicating that multiple solutions exist for gain of recognition, but the path to reach these mutations may be confined. Furthermore, this single substitution alters the prediction of AlphaFold 2, allowing it to model the complex structure of Sr50 K711D and AvrSr50 that match our final structural hypothesis. Collectively, our study outlines a framework for rational engineering of NLR systems to overcome pathogen escape mutations and provides datasets for future computational models for NLR resurrection., Competing Interests: Competing interests The authors declare no competing interests.

Patients aged 50 or above diagnosed with myeloid neoplasms (MNs) are typically not candidates for germline testing. However, approximately 8% carry pathogenic germline variants. Allogeneic haematopoietic stem cell transplantation (alloHSCT) remains an option for those aged over 50; neglecting germline testing could mask the risk for relative donor cell-derived MN. We propose a germline-augmented somatic panel (GASP), combining MN predisposition genes with a myeloid somatic panel for timely germline variant identification when initial testing is not indicated. Out of our 133 whole-exome-sequenced MN cases aged over 50 years, 9% had pathogenic/likely variants. GASP detected 92%, compared to 50% with somatic-only panel. Our study highlights the relevance of germline screening in MN, particularly for alloHSCT candidates without established germline-testing recommendations., (© 2024 British Society for Haematology and John Wiley & Sons Ltd.)

Comprehending the immune defense mechanisms of new aquaculture species, such as the Chilean meagre ( Cilus gilberti ), is essential for sustaining large-scale production. Two bioassays were conducted to assess the impact of acute and intermittent hypoxia on the antibacterial activity of juvenile Chilean meagre epidermal mucus against the potential pathogens Vibrio anguillarum and Vibrio ordalii . Lysozyme and peroxidase activities were also measured. In general, fish exposed to hypoxia showed a 9-30% reduction in mucus antibacterial activity at the end of hypoxic periods and after stimulation with lipopolysaccharide. However, following water reoxygenation, the activity of non-stimulated fish was comparable to that of fish in normoxic conditions, inhibiting bacterial growth by 35-52%. In the case of fish exposed to chronic hypoxia, the response against V. anguillarum increased by an additional 19.8% after 6 days of control inoculation. Lysozyme exhibited a similar pattern, while no modulation of peroxidase activity was detected post-hypoxia. These results highlight the resilience of C. gilberti to dissolved oxygen fluctuations and contribute to understanding the potential of mucus in maintaining the health of cultured fish and the development of future control strategies.

Background:  Polycythemia vera (PV) patients are classified as high or low thrombotic risk based on age and prior history of thrombosis. Despite adherence to treatment recommendations, vascular events remain frequent, leading us to question whether thrombotic risk stratification could be improved. We previously reported an association between thrombotic events and mutations in DTA genes ( DNMT3A, TET2, and ASXL1 ). The objective of this study was to confirm this observation in a larger series of PV patients., Methods:  PV patients with a minimum follow-up of 3 years were recruited from 8 European centers. Medical history was searched for thrombotic event recorded at any time and next-generation sequencing carried out with a myeloid panel. Multivariable logistic regression evaluated the impact of variables on thrombotic risk. Kaplan-Meier thrombosis-free survival curves were compared by the log rank test. Associations in the total cohort were confirmed in a case-control study to exclude selection bias., Results:  Of the 136 patients recruited, 74 (56.1%) had a thrombotic event, with an incidence density of 2.83/100 person-years. In multivariable analysis, DTA mutation was a risk factor for thrombotic event, being predictive for shorter thrombosis-free survival in the whole cohort ( p  = 0.007), as well as in low-risk patients ( p  = 0.039) and older patients ( p  = 0.009), but not for patients with a prediagnostic event. A gender- and age-matched case-control study confirmed the increased risk of thrombotic event for PV patients with a DTA mutation., Conclusion:  Our results support the use of molecular testing at diagnosis to help predict which PV patients are at higher risk of developing thrombosis., Competing Interests: None declared., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commercial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

Objective: To examine the association between objectively-measured preoperative physical activity with postoperative outcomes and recovery milestones in patients undergoing gynecologic oncology surgeries., Methods: Prospective cohort study of patients undergoing surgery with gynecologic oncologists who wore wearable actigraphy rings before and after surgery from 03/2021-11/2023. Exposures encompassed preoperative activity intensity (moderate- and vigorous-intensity metabolic equivalent of task-minutes [MAVI MET-mins] over seven days) and level (average daily steps over seven days). Intensity was categorized as <500, 500-1000, and >1000 MAVI MET-mins; level categorized as <8000 and ≥8000 steps/day. Primary outcome was 30-day complications. Secondary outcomes included reaching postoperative goal (≥70% of recommended preoperative intensity and level thresholds) and return to baseline (≥70% of individual preoperative intensity and level)., Results: Among 96 enrolled, 87 met inclusion criteria, which constituted 39% (n = 34) with <500 MET-mins and 56.3% (n = 49) with <8000 steps preoperatively. Those with <500 MET-mins and <8000 steps had higher ECOG scores (p = 0.042 & 0.037) and BMI (p = 0.049 & 0.002) vs those with higher activity; all other perioperative characteristics were similar between groups. Overall, 29.9% experienced a 30-day complication, 29.9% reached postoperative goal, and 64.4% returned to baseline. On multivariable models, higher activity was associated with lower odds of complications: 500-1000 MET-mins (OR = 0.26,95%CI = 0.07-0.92) and >1000 MET-mins (OR = 0.25,95%CI = 0.07-0.94) vs <500 MET-mins; ≥8000 steps (OR = 0.25,95%CI = 0.08-0.73) vs <8000 steps. Higher preoperative activity was associated fewer days to reach postoperative goal., Conclusion: Patients with high preoperative activity are associated with fewer postoperative complications and faster attainment of recovery milestones. Physical activity may be considered a modifiable risk factor for adverse postoperative outcomes., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)