Secondary autoimmune and inflammatory diseases (SAIDs) are underrecognized and poorly described after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The standardization of management is hindered by the scarcity of data on epidemiology, pathogenesis, and clinical outcomes. To improve the evidence base, we performed a retrospective multicenter EBMT database study of patients who underwent allo-HSCT between 2005 and 2019 for either hematological malignancy or severe aplastic anemia with available information on SAIDs. We included 129 cases of SAIDs and 14,617 controls. The 5-year incidence of SAIDs was 0.9% (95% confidence interval (CI): 0.7-1.1), and the 10-year incidence was 1.1% (95% CI: 0.9-1.3). The median time from allo-HSCT to the diagnosis of SAIDs was 442 days [interquartile range (IQR): 242-1082]. Overall survival after the onset of SAIDs was 83.4% (95% CI 74.6-89.3) at 2 years and 73.4% (95% CI: 62-82) at 5 years. In multivariate analysis, risk factors significantly associated with SAIDs were bone marrow failure (Hazard Ratio (HR): 3.41 [95% CI, 1.55-7.52], p = 0.002), female donor to male patient (HR 1.77 [95% CI, 1.15-2.73], p = 0.009), and the presence of chronic GvHD (HR 1.61 [95% CI, 1.04-2.51], p = 0.034). SAIDs after allo-HSCT are rare but clinically significant entities requiring further investigations, exploring treatment and prevention strategies, as well as improving diagnostic approaches.

Allogeneic hematopoietic cell transplantation (allo-HCT) remains the only curative option for many hematologic malignancies, yet transplant-related toxicity and relapse continue to constrain long-term benefit. Prognostic tools based on clinical variables alone show limited transportability across centers. We hypothesized that integrating immunogenetic architecture, captured by HLA Evolutionary Divergence (HED), into machine-learning survival models would improve prediction of graft-versus-host disease–free/relapse-free survival (GRFS). We developed SMART, a time-dependent framework that estimates dynamic GRFS probabilities after allo-HCT. We analyzed 16,028 adults transplanted between 2010–2022. Model development used the French SFGM-TC registry (N = 13,979), split into training (N = 9,840) and held-out test (N = 4,139) sets. Random survival forests, XGBoost-Cox, and elastic-net Cox models were trained using 9 clinical predictors, with or without 10 recipient/donor locus-specific HED features (19 predictors total) and externally evaluated in 616 patients from five U.S. centers with complete data. Across algorithms, discrimination for this composite endpoint was modest (c-index < 0.60), but the addition of HED consistently improved performance and enabled reproducible stratification into low-, intermediate-, and high-risk groups based on the cumulative hazard score. Model-based simulations uncovered a non-linear (U-shaped) association between total HED and GRFS, with optimal outcomes at intermediate HED levels (~ 75th percentile). In haploidentical transplantation (N = 2,056), outcomes were maximized when donor and recipient HED were concordant (“match like with like”). In 9/10 mismatched unrelated donors (N = 1,326), HLA-B mismatches showed the greatest HED sensitivity. Integrating immunogenetics with clinical data improves GRFS risk modeling and supports HED as an actionable feature for donor selection and pre-transplant risk stratification. SMART is available for research use.

IntroductionAn increasing number of studies focus on anti-tumor immune responses. In acute myeloid leukemia (AML), blasts and immune cells, such as T cells, are frequently studied together in the same leukocyte sample. While hemolysis is the clinical standard for leukocyte isolation because it preserves the native leukocyte composition, Ficoll-based density gradient centrifugation is widely used in research and biobanking.MethodsUsing hemolysis as a reference, we assessed whether Ficoll processing introduces biases into AML and immune cell profiling through analytical methods frequently applied in AML research.ResultsFlow cytometry analyses showed that Ficoll systematically alters the composition of AML samples by enriching lymphocytes and AML blasts while depleting granulocytes. In RNA sequencing analysis, Ficoll isolation altered the expression of 1,136 genes, notably leading to an overestimation of the expression of leukemic stem cell gene sets. Immunogenomic deconvolution highlighted that Ficoll leads to an overestimation of CD8+ T-cell and monocyte abundances. Mutation calling from RNA-seq data revealed substantial discrepancies between methods, including failure to detect a clinically relevant DNMT3A R882 mutation in a Ficoll-processed sample. While Ficoll isolation had minimal impact on ex vivo AML blast expansion or chemotherapy response, it appeared to affect AML engraftment in NSG mice, possibly through the enrichment of T cells mediating graft-versus-host disease.DiscussionTogether, these findings show that Ficoll isolation introduces bias in the cellular and molecular characterization of AML samples while having only a moderate impact on functional assays. We provide recommendations on which method to use depending on the investigator's objectives.

The European LeukemiaNet (ELN) 2022 classification categorized both t(9;11)(p21.3;q23.3) and isolated del(7q) in acute myeloid leukemia (AML) as intermediate-risk when treated with intensive chemotherapy. However, their prognostic relevance in the context of allogeneic hematopoietic cell transplantation (allo-HCT) needs further validation. This retrospective, registry-based analysis from the EBMT assessed outcomes in adults with AML who underwent allo-HCT in first complete remission between 2010 and 2022. In the first cohort, data from 141 patients with t(9;11) were analyzed, of whom 23% had additional adverse cytogenetic abnormalities (ACA), primarily complex karyotype. Most had de novo AML (72%), had received myeloablative conditioning (57%), and peripheral blood stem cells (88%). After a median follow-up of 3 years, there were no significant differences in 2-year relapse incidence (22% vs. 18.2%, p=0.85), leukemia-free survival (66% vs. 76%, p=0.42), or overall survival (72% vs. 75%, p=0.68) between patients with non-adverse t(9;11) and those with additional ACA. The second cohort included 250 patients: 84 with del(7q), 95 with monosomy 7, and 71 with del(5q), and all without additional ACA. Most had de novo AML (59%) and had received reduced-intensity conditioning (65%). After similar follow-up, survival outcomes did not differ significantly across the groups (2-year leukemia-free survival: 61% vs. 59% vs. 52% for del(7q), monosomy 7 and del(5q), respectively). In conclusion, these findings suggest that the prognostic value of t(9;11) as intermediate-risk remains consistent in the setting of allo-HCT regardless of additional ACA, whereas del(7q), even without additional ACA, confers a risk comparable to monosomy 7 and del(5q).

Acute myeloid leukemia (AML) is a heterogeneous malignancy whose characterization relies on immunophenotyping and molecular profiling. While hemolysis is recommended for leukocyte isolation in clinical diagnostics, Ficoll-based density gradient centrifugation is widely used in research and biobanking. Here, we evaluated the impact of Ficoll isolation on commonly performed analyses of AML samples. Ficoll altered flow cytometry-based characterization by systematically enriching lymphocytes and AML blasts while depleting granulocytes. The increased T-cell content impaired AML engraftment in NSG mice, as T cells mediated terminal graft-versus-host disease. Although Ficoll had minimal impact on ex vivo AML blast expansion or chemotherapy response, RNA sequencing identified 1,136 differentially expressed genes compared with hemolysis, with Ficoll-processed samples notably leading to an overestimation of leukemic stem cell gene set expression. Immunogenomic deconvolution highlighted that Ficoll leads to an overestimation of CD8+ T-cell and monocyte abundances in sequenced samples. Mutation calling from RNA-seq data revealed substantial discrepancies between methods, including failure to detect a clinically relevant DNMT3A R882 mutation in a Ficoll-processed sample. Together, these findings support the systematic use of hemolysis to preserve cellular diversity and avoid unpredictable biases introduced by Ficoll-based isolation. ### Competing Interest Statement The authors have declared no competing interest.

ABSTRACT:Chronic graft-versus-host disease (cGVHD) remains a major hurdle to the success of hematopoietic stem cell transplantation, directly affecting patient morbidity and mortality. Impaired regulatory T-cell (Treg) recovery in patients with cGVHD has led to clinical studies aiming to increase peripheral Treg numbers. We conducted phase 1 dose-escalation clinical trials testing the feasibility and safety of using freshly isolated donor-derived Treg infusions in steroid-refractory/dependent cGVHD. The phase 1 was extended to a preliminary phase 2 trial, resulting in a total of 33 treated patients. We report that Treg purification from donor leukapheresis using CliniMACS were feasible and that Treg infusions were safe. Importantly, Treg infusions resulted in improved symptoms, particularly at higher Treg doses. Global responses were observed in 71% of patients, and 52% of patients had at least a 2-point improvement in the cGVHD severity scale. Furthermore, improvement in cGVHD symptoms resulted in reductions in corticosteroids, ruxolitinib, and mycophenolate in 58%, 83%, and 33% of patients, respectively, whereas calcineurin inhibitors were discontinued in 75% of patients. Exploratory analyses revealed the detection of infused Treg clonotypes up to 12 months after infusion and suggest increased Treg numbers in circulation. We observed increases in serum levels of interleukin-7 and interferon gamma and decreases in soluble CD13 and suppression of tumorigenicity 2 over time, which were not statistically significant after adjustment for multiple comparisons. Although these studies were not powered to assess efficacy, they suggest potential therapeutic benefits of donor-derived Treg in cGVHD treatment and highlight the need for larger phase 2 clinical trials. The trials were registered at www.clinicaltrials.gov as #NCT02385019 and #NCT03683498.

Ex vivo T-cell activation is critical in both basic immunology and clinical applications, such as CAR-T cell therapy. CD3/CD28 antibody-coated beads and soluble antibody complexes are widely used, yet direct comparisons remain limited and often contradictory. We longitudinally profiled human T cells stimulated with two widely used activation reagents, one bead-based and the other pre‑formed soluble antibody complexes, to clarify these differences. Both approaches supported robust expansion and stable CD4/CD8 ratios, indicating comparable proliferative capacity. Beads induced earlier, stronger activation and rapid effector memory differentiation, whereas the soluble antibody complex reagent promoted slower activation and preserved central memory subsets. Upon restimulation, however, both conditions efficiently reactivated and converged toward effector memory differentiation with sustained TIM‑3 expression, consistent with chronic stimulation. This convergence suggests that restimulation is a major determinant of long‑term phenotype, potentially overriding differences introduced by the initial activation reagent. Together, our findings reconcile prior inconsistencies and demonstrate that while expansion is comparable, activation and differentiation diverge across these commonly used reagents, providing a framework to tailor activation strategies to specific immunotherapy outcomes.

BACKGROUND:Despite improvements in survival outcomes for acute myeloid leukemia (AML), limited evidence is available on health-related quality of life (HRQoL) and health problems experienced by long-term survivors. PATIENTS AND METHODS:This international, cross-sectional study evaluated HRQoL, comorbidities, and lifestyle behaviors among long-term AML survivors enrolled from 24 centers across 6 countries. Health-related quality of life was assessed using the SF-36 and the EORTC QLQ-C30 questionnaires, while comorbidities were measured with an adapted version of the validated self-administered comorbidity questionnaire. Lifestyle factors, including physical activity, diet, smoking, alcohol consumption, and body mass index, were also assessed. RESULTS:Overall, 225 AML survivors were enrolled, with a median time since diagnosis of 8.8 years (IQR 6.4-11.9) and a median age of 58.9 years (IQR 49.0-67.0). Compared with the general population, AML survivors exhibited clinically relevant impairments in SF-36 physical functioning (Δ = -8.09, P < .001) and role physical scales (Δ = -11.09, P < .001), as well as clinically relevant lower physical component summary scores (Δ = -3.94, P < .001). Survivors treated with alloSCT reported worse HRQoL profiles compared with those treated with autoSCT or chemotherapy only. Comorbidities were highly prevalent (88.5%), with impaired vision, back pain, and arthrosis/arthritis being the most frequent. Analysis of lifestyle behaviors showed that 66.2% of AML survivors were physically inactive, 80.2% did not meet dietary recommendations, and 55.3% were overweight/obese. Multivariate analysis identified physical inactivity as the only independent factor associated with worse HRQoL (β = -6.3, P < .001). CONCLUSION:Our study shows that AML survivors experience physical limitations and a high comorbidity burden even many years after diagnosis, and it provides insights to better inform survivorship care programs. Further research examining the relationship between physical activity and HRQoL in long-term AML survivors is warranted.

Acute myeloid leukemia (AML) includes genetically defined subsets. In allogeneic hematopoietic cell transplantation (allo-HCT), the frequency and prognosis of gene-gene interactions may differ from those of patients treated with chemotherapy alone. In this study, adult patients (N = 952) with AML allografted between 2015 and 2023, with available next generation sequencing (NGS) at diagnosis were included. Most frequent mutations were DNMT3A (24%), FLT3-ITD (21%), NPM1 (21%), RUNX1 (16%), NRAS (16%), TET2 (14%), and IDH2 (12%). Multiple correspondence analysis identified distinct groups of co-occurring mutations. Outcome analysis was performed on 646 AML patients allografted in first complete remission (CR1). Six non-overlapping groups were constructed: 1) TP53 mutation (N = 47); 2) NPM1 mutation (N = 129); 3) FLT3-ITD and/or DNMT3A mutation (N = 128); 4) SRSF2 and/or ASXL1 and/or RUNX1 mutation (SAR group) (N = 132); 5) IDH1 and/or IDH2 and/or TET2 mutation (N = 43); and 6) all ten genes unmutated (N = 167). In multivariable analysis, TP53 mutation, adverse karyotype, and age negatively affected leukemia-free survival (LFS) and overall survival (OS). OS was additionally negatively affected when the ten genes were unmutated. Notably, outcomes were excellent for SAR mutations (2-year LFS 76%, OS 84%), indicating allo-HCT in CR1 can overcome their adverse risk at diagnosis.

Background Socioeconomic status (SES) is a known factor frequently associated with health-related quality of life (HRQoL) in oncology, yet its impact in the setting of myelodysplastic syndromes/neoplasms (MDS) remains underexplored. Patients with higher-risk MDS may be particularly vulnerable in this respect, considering the high burden of symptoms and functional impairments imposed by the disease already at the time of diagnosis. Objectives The primary objective of this analysis was to examine the prevalence of clinically important problems and symptoms in newly diagnosed patients with higher-risk MDS by their SES. The secondary objective was to assess the risk of impaired functioning and symptoms by different SES groups. Methods We conducted a cross-sectional analysis of adult patients with newly diagnosed higher-risk MDS (according to the IPSS-R) enrolled in a large international prospective observational study by the GIMEMA. The SES was determined using three variables: level of education, employment status, and living arrangements. Each variable was scored as 0 (for low level of education, living alone, or no income) or 1 (for intermediate/high level of education, living with others, or receiving a salary/pension). The final SES index was computed by summing the scores for each variable, and each patient was classified into three categories: low (score 0-1), middle (score 2), and high SES (score 3). To assess HRQoL, all participants completed the EORTC QLQ-C30 at study entry. Prevalence of clinically important problems and symptoms were assessed using established thresholds for the QLQ-C30 (Giesinger JM et al. J Clin Epidemiol 118:1-8, 2020). This prevalence reflects the number of patients in each SES group reporting clinically important problems or symptoms that limit their daily lives, cause worry to them, or require help or care. Logistic regression models were used to assess the risk of impaired functioning or symptoms in the low and middle SES groups vs the high SES group. These models were adjusted for the following potential confounders: age, sex, time since diagnosis, transfusion dependency, ECOG performance status, and HCT comorbidity index. Results Overall, 521 patients with higher-risk MDS were analyzed. The SES was available for 504 patients, who had a median age of 73 years (IQR 65.9-78.8) and a median time since diagnosis of 0 weeks (IQR 0-4.3). Almost half of them were classified as high SES (45.4%), 41.5% as middle SES, and 13.1% as low SES. Prevalence of clinically important problems was higher in the low SES group with respect to middle and high SES groups, across all the QLQ-C30 scales. For example, more than half of patients (59.1%) in the low SES group reported a clinically important level of fatigue, while this prevalence was 47.4% and 39.3% in the middle and high SES groups, respectively (p=0.013). Prevalence of clinically important pain was 53%, 39.2% and 27.1% in low, middle and high SES, respectively (p<0.001). Three out of four (74.2%) patients in the low SES group had a clinically important dyspnea, while this was 58.9% and 57.6% in the middle and high SES, respectively (p=0.045). In the functioning scales, the prevalence of clinically important problems in physical functioning was similar in the low (77.3%) and middle (74.2%) SES groups, but lower in the high SES group (60.3%) (p=0.002). For the cognitive functioning scale, the prevalence of clinically important problems was 43.9%, 32.5% and 25.8% in the low, middle and high SES groups, respectively (p=0.015). Logistic regression analyses revealed a similar trend, with low SES more likely to report impaired functioning or symptoms than those with high SES, independently of potential confounders. For example, patients in the low SES group had a statistically significant higher risk of reporting impaired pain (OR=2.84, 95% CI 1.55-5.24; p<0.001), dyspnea (OR=1.98, 95% CI 1.06-3.83; p=0.037) and cognitive functioning (OR=1.86, 95% CI 1.00–3.42; p=0.047). Conclusions We observed that newly diagnosed patients with higher-risk MDS who have a low SES tend to report worse HRQoL outcomes compared to those with middle or high SES, suggesting that these patients are most in need of special attention. Future research should investigate whether such socioeconomic inequalities may also impact long-term treatment outcomes.

In this multicenter retrospective study, we analyzed the EEG characteristics of 17 patients with possible central nervous system (CNS) graft-versus-host disease (pCNS-GvHD). All EEGs were abnormal. Most (11 of 17 patients) showed background activity slowing. Sporadic epileptiform discharges were rare (2 patients) and observed only in chronic pCNS-GvHD. Sporadic nonepileptiform discharges, often generalized, frontally predominant, and triphasic, were common (15 of 17 patients). Two patients presented generalized rhythmic delta activity, one showed lateralized rhythmic delta activity, and one exhibited lateralized periodic discharges. Background activity slowing was statistically associated with higher 1-yr overall mortality (P = 0.026). These findings suggest that EEG may serve as a prognostic tool in CNS-GvHD.NEW & NOTEWORTHY This retrospective study is the first to describe EEG features of central nervous system involvement in graft-versus-host disease (CNS-GvHD). It shows that CNS-GvHD consistently associates with EEG abnormalities at peak disease severity. Epileptiform discharges are rare and mostly occur in chronic CNS-GvHD. Finally, it identifies a statistically significant association between background rhythm frequency and 1-yr survival, suggesting EEG as a potential prognostic tool for CNS-GvHD.

Sinusoidal obstruction syndrome (SOS) formerly known as Veno-occlusive disease (VOD) is a potentially fatal complication that occurs mainly after haematopoietic cell transplantation, especially allogeneic transplantation. The liver is the principal organ affected, though other organs, such as the lungs, may also be involved to a lesser extent. The condition is characterised by obstruction of the hepatic venules, leading to sinusoidal congestion, hepatic ischaemia and, in severe cases, fulminant liver failure. Recent refined diagnostic criteria, published by the European Society for Blood and Marrow Transplantation in 2023, provide a more accurate method of detecting SOS/VOD, allowing earlier intervention and better stratification of patients according to the severity of their disease. This article focuses on liver SOS/VOD and discussing key risk factors, new diagnostic methods and therapeutic strategies, with an emphasis on the early use of defibrotide, which remains the reference treatment for severe SOS/VOD.

Multiple myeloma (MM) is an incurable hematologic malignancy arising from clonal plasma cells, with poor long-term outcomes due to inevitable relapse after conventional therapies. Chimeric antigen receptor (CAR) T-cell immunotherapy targeting B-cell maturation antigen (BCMA) has shown remarkable efficacy in relapsed patients. Conventional CARs employ single-chain variable fragments (scFvs), whereas single-domain antibodies (sdAb or VHHs) offer advantages such as small size, high stability, and potentially reduced immunogenicity. We designed and evaluated a novel anti-BCMA nanoCAR-T based on the VHH Nb17, compared with the conventional scFv-based CAR-T CT103a. Nb17 demonstrated strong BCMA binding and was incorporated into a CAR construct. Both nanoCAR-T and CT103a were generated via lentiviral transduction of primary T cells. Their cytotoxicity, cytokine secretion, degranulation, memory phenotype, and gene expression were assessed in vitro, along with antitumor activity in vivo. Nb17-nanoCAR-T demonstrated specific cytotoxicity, cytokine release (IL-2, TFNa, IFNg), and CD107a degranulation comparable to CT103a. Transcriptomic analysis revealed overlapping pathways between both CARs. Upon rechallenge, both CARs showed enhanced proliferation compared with untransduced T cells. In vivo, Nb17-nanoCAR-T and CT103a eradicated tumors in NSG mice. These findings demonstrate Nb17-nanoCAR-T exhibits potent anti-myeloma efficacy comparable to scFv-based CAR-T, supporting its potential as a promising therapeutic alternative.