Risk factors for relapse and non-relapse mortality (NRM) in the first years after allogeneic hematopoietic stem cells transplant (allo-HSCT) for acute myeloid leukemia (AML) are well known, but their correlation with long-term survivorship is unclear. This study aims to evaluate factors that impact on long-term survival after allo-HSCT in AML. We retrospectively analyzed data of 456 consecutive patients who received a first allo-HSCT in our center, regardless of conditioning intensity, GvHD prophylaxis, donor matching and HSC source. At transplant, two hundred and four patients (44%) were in CR1, 62 patients (14%) in CR>1, 27 (6%) in CR MRD+ and 163 patients (36%) in active disease (AD). Two-hundred and thirty-nine patients (52%) were alive 2 years after allo-HSCT. Median follow-up was 6.1 years. The 5 years OS was 63% for CR1, 38% for CR>1 and 25% for AD. However, in patients alive and disease free 2 years after allo-HSCT the probability of being alive for 3 more years was 86% for CR1, 72% for CR>1 and 79% for AD, with no statistically significant differences between CR>1 and AD. Other variables associated with better outcomes included younger age, more recent transplantation, and having a female donor for a male recipient. Relapse remained the main cause of death, even among long-term survivors. In conclusion, the negative impact of advanced disease at the time of allo-HSCT appears to progressively reduce after transplantation, highlighting the excellent long-term outcomes of patients who remain disease-free at 2 years after allo-HSCT, regardless of pre-transplantation disease status.

Abstract Stress conditions influence hematopoietic stem cell (HSC) fate and lineage output, underscoring hematopoietic flexibility to external stimuli. In β-thalassemia (Bthal), chronic bone marrow (BM) stress since anemia and ineffective erythropoiesis remodels the BM microenvironment, affecting HSC biology (Aprile et al Blood 2020). We recently showed that BM HSCs/MPPs (multipotent progenitors) in pediatric Bthal patients are activated and primed towards erythroid differentiation, with reduced stemness pathways, altered inflammatory signaling (e.g. TGFβ, TNFα-NFKB), and decreased dormancy signatures (Lidonnici MR et al., Nat Commun 2025). In the Tiget-Bthal clinical trial, HSC lentiviral gene therapy (GT) led to initial transfusion independence for most patients, correlating with engraftment of gene-corrected HSCs (Marktel S. et al. Nat Med 2019). However, a subset of pediatric patients showed poor engraftment with a drop in genetically marked cells post-GT despite optimal transduction efficiency and vector copy number (VCN) in the administered drug product and adequate conditioning, requiring transfusional support. We hypothesize that HSC heterogeneity and the status of BM microenvironment influence repopulating capacity and the consequent hematopoietic reconstitution in GT-treated patients. To investigate HSC diversity in nine Bthal patients treated with gene therapy, we performed single-cell RNA sequencing (scRNAseq) on CD34+ cells and HSCs/MPPs from BM pre-GT, mobilized peripheral blood (mPB, GT source), and BM post-GT. On the basis of clinical outcome and molecular follow-up, the patients here are referred to as with high (HE) or low engraftment (LE) of genetically modified cells. No difference in HSPC (hematopoietic stem and progenitor cell) composition in the CD34+ compartment was observed across timepoints or between HE and LE patients, consistently with immunophenotypic analyses. Gene set enrichment analysis using stemness signatures and MSigDB gene sets identified differences in HSCs/MPPs across samples. Specifically, mPB HSCs/MPPs from LE patients showed lower dormancy scores and reduced TNFα via NFkB signaling enrichment compared to HE patients. Integration with HSC meta-programs (Zeng et al., bioRxiv 2023) revealed that LE HSCs/MPPs had reduced enrichment for the inflammatory memory HSC program (HSC-iM), consistent with downregulation of TNFα via NFkB signaling. Analysis of differentially expressed genes by DESeq2 identified upregulation of IFN-responsive genes in LE mPB HSCs/MPPs. These data suggest diverse responses to inflammatory stimuli in a heterogeneous HSC population. Post-GT analysis showed that BM HSCs/MPPs in LE patients were more quiescent, exhibited reduced cycling, and showed an elevated hypoxic signature. These cells were enriched for the expression of dormancy-associated genes, as well as HSC-iM genes, including those related to the IFN-γ and IL-1 signaling pathways. Measurement of BM plasma cytokines (IFN-γ and IL-1b) correlated with HSC/MPP transcriptomic profiles, linking high basal homeostatic inflammatory microenvironment to stem cell status. It is noteworthy that TNFα, IFN-γ, and IL-1b levels were already altered in the BM plasma of Bthal patients compared to healthy controls. These data suggest that inflammatory stress, likely induced in Bthal BM by chronic stimulus to erythroid proliferation and hypoxia, may drive the affected HSC into dormancy as a protective adaptation to protect the integrity of the stem cell pool. Finally, re-analysis of vector integration sites (Calabria et al. Nature 2024), stratified by patients' groups, confirmed fewer active HSPCs in LE compared to HE patients, indicating reduced contribution of genetically modified HSPCs to hematopoiesis. Overall, our data indicate that the degree of HSC dormancy in the stem cell source and prevalence of inflammatory transcriptional signatures are critical to preserving HSC function and ensuring effective engraftment and hematopoietic reconstitution. Ongoing analyses using specific inflammatory-related signatures will delineate HSC subset composition and underscore heterogeneity concerning activation, lineage commitment, stemness, and inflammation. Understanding how distinct HSC subsets respond to inflammatory cues in vivo will guide strategies to predict patient responses and refine conditioning regimens or ex vivo manipulation protocols, ultimately improving gene therapy efficacy for β-thalassemia.

The hematopoietic stem cell and multipotent progenitor (HSC/MPP) pool dynamically responds to stress to adapt blood output to specific physiological demands. In β-thalassemia (Bthal), severe anemia and ineffective erythropoiesis generate expansion of erythroid precursors and a chronic stress status in the bone marrow (BM) microenvironment. However, the response to the BM altered status at the level of the HSC/MPP compartment in terms of lineage commitment has not been investigated. Bulk and single-cell RNA-sequencing reveal that Bthal HSCs/MPPs are expanded and activated with enhanced priming along the whole Ery differentiation trajectory. Consistently, HSC/MPP showed an altered TGFβ expression and autophagy transcriptional signatures along with a declined dormancy state. We discovered that the altered TGFβ signaling fosters the Ery potential of HSCs by reducing their autophagic levels, and in vivo stimulation of autophagy is sufficient to rescue the imbalance of the HSC compartment. Our findings identify the interplay between TGFβ and HSC autophagy as a key driver in the context of non-malignant hematopoiesis.

Introduction: Secondary involvement of the CNS is a direful complication of large B cell lymphomas (LBCL), which can occur at diagnosis or relapse. Treatment of SCNSL differs from systemic lymphomas and is based on high dose chemotherapy (CHT) with drugs that penetrate the blood-brain barrier, HDMTX in particular, followed by autologous stem cell transplantation (ASCT). However, 60% of pts is refractory to or relapses after this treatment, 2/3 of relapsed pts do not receive further salvage therapy, and responses are anecdotal. CAR-T is an effective salvage treatment for LBCL whereas experience in SCNSL is limited to a few registry studies, where response to prior lines and the number of SCNSL pts who result unsuitable candidates for CAR-T is roughly reported. Herein we report selection and management of consecutive pts with SCNSL referred to 3 Italian CAR-T-cell centres, providing more detailed information for pts who actually received CAR-T. Methods: SCNSL pts were evaluated for CAR-T eligibility between 07/22 and 03/25. Selected pts were offered a holding therapy according to extension of disease and prior treatments. Pts in CNS remission after holding were evaluated for CAR-T eligibility by a multidisciplinary team of experts. Eligible candidates underwent lymphocytoapheresis (Flu-Cy) and bridging therapy until commercial CAR-T infusion. Results: 23 pts were referred to holding: 6 partial-brain irradiation, 4 intrathecal CHT, 2 polatuzumab vedotin, 5 ibrutinib, 1 HD-ifosfamide-based CHT, 2 MATRix regimen, 1 thiotepa-based ASCT, combinations of these strategies in 2. 7 pts experienced progressive disease after holding, were deemed not eligible for CAR-T and died within one month. The other 16 achieved a remission of CNS disease and were eligible for CAR-T. All pts had ECOG 0-1, and all but one were refractory to prior HDMTX. Only one pt experienced G3-4 CRS, all CRS cases were successfully treated with tocilizumab and steroids, anakinra was used in 2 cases. ICANS were treated with steroids, plus anakinra in 2 cases. G≥3 ICANS occurs in two pts: one required pre-emptive ICU admission but resolved after 3 days, while the second pt had G1 CRS, developed refractory epilepticus status, and died 18 days after CAR-T infusion. Although recommended washout period was respected, the latter event could be explained by multiple intrathecal CHT instillations performed before CAR-T. Four pts had ≥1 cytopenia at 3 months. At a median follow up of 8 months (IQR: 3-12), 12/16 infused pts remain relapse-free; 3 had a systemic relapse, while none experienced CNS recurrence. Conclusion: Varied presentation and high aggressivity of SCNSL impede the use of a uniform treatment for these pts. SCNSL refractory to HDMTX-based polyCHT can benefit significantly from a tailored multimodal holding based on the pt's history and extension of disease. Only pts with good PS and responsive to holding therapy should be offered CAR-T to avoid superfluous toxicity.

Haematopoietic stem cell (HSC) gene therapy (GT) may provide lifelong reconstitution of the haematopoietic system with gene-corrected cells1. However, the effects of underlying genetic diseases, replication stress and ageing on haematopoietic reconstitution and lineage specification remain unclear. In this study, we analysed haematopoietic reconstitution in 53 patients treated with lentiviral-HSC-GT for diverse conditions such as metachromatic leukodystrophy2,3 (MLD), Wiskott-Aldrich syndrome4,5 (WAS) and β-thalassaemia6 (β-Thal) over a follow-up period of up to 8 years, using vector integration sites as markers of clonal identity. We found that long-term haematopoietic reconstitution was supported by 770 to 35,000 active HSCs. Whereas 50% of transplanted clones demonstrated multi-lineage potential across all conditions, the remaining clones showed a disease-specific preferential lineage output and long-term commitment: myeloid for MLD, lymphoid for WAS and erythroid for β-Thal, particularly in adult patients. Our results indicate that HSC clonogenic activity, lineage output, long-term lineage commitment and rates of somatic mutations are influenced by the underlying disease, patient age at the time of therapy, the extent of genetic defect correction and the haematopoietic stress imposed by the inherited disease. This suggests that HSCs adapt to the pathological condition during haematopoietic reconstitution.

Background: Invasive fungal infections (IFIs) represent a major cause of morbidity among allogeneic hematopoietic stem cell transplantation (allo-HSCT). Isavuconazole (ISA) is a broad-spectrum triazole with favorable safety profile. Objectives and design: Herein, we evaluate the real life coadministration of ISA and sirolimus in allo-HSCT recipients in a single-center retrospective analysis, describing clinical efficacy, safety, and therapeutic drug monitoring (TDM) of both drugs. Methods: All consecutive allo-HSCT recipients who received the coadministration of ISA and sirolimus for at least 2 weeks between July 2017 and December 2022 were included in this retrospective analysis. TDM was longitudinally performed during treatment. IFIs were classified according to the revised European Organization for Research and Treatment of Cancer/Mycoses Study Group consensus criteria. Results: A total of 51 recipients were included in the analysis. A total of 17 patients received ISA as continuous antifungal treatment for IFI diagnosed before transplant: one patient experienced a probable invasive pulmonary aspergillosis, and one patient switched from ISA to liposomal amphotericin B for a possible IFI. A total of 34 patients started ISA as antifungal therapy for IFI diagnosed after transplant. Sixteen of 34 were treated for a proven/probable breakthrough IFI during mold-active prophylaxis: 6/16 patients died for IFI after a median of 51 days of ISA. Eighteen of 34 started ISA as empirical therapy for a possible IFI: 15/18 patients were alive with resolution of infection after 6 weeks, 1 died for disease progression, and 2 had empirically changed antifungal therapy due to pneumonia progression. Clinical and radiological response rate was 68% after 90 days from IFI diagnosis. No toxicities related to drug–drug interaction have been registered in patients reaching concomitant therapeutic levels of ISA and sirolimus. Conclusion: The coadministration of ISA and sirolimus was safe and feasible in this cohort, confirming favorable clinical efficacy in patients with multiple-drug coadministration. Keywords allogeneic transplantation , IFI , isavuconazole , sirolimus , TDM