Tyrosine kinase inhibitors are recommended as maintenance therapy following allogeneic stem cell transplantation (HSCT) for Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL), but optimal medication, dose, and duration remain a subject of ongoing study. We provide the final analysis of a prospective randomized trial comparing prophylactic and minimal residual disease (MRD)-triggered imatinib maintenance. The two patient cohorts did not differ significantly in terms of cumulative incidence of relapse (CIR; 14% vs. 18%), non-relapse mortality (NRM; 12% vs. 11%), leukemia-free survival (LFS) 64% vs. 69%, and overall survival (OS) at 10 years (68% vs. 71%). Endpoints were assessed every 6 weeks within the trial from 4 weeks after SCT until EOS at week 55 and after that according to local standards. In summary, this provides a framework for MRD-based treatment of patients for maintenance after HSCT with excellent long-term outcome after 10 years in both groups.

IntroductionAllogeneic hematopoietic cell transplantation (alloHCT) recipients are at increased risk of severe COVID-19. Although vaccination is strongly recommended, immune responses may vary and can be affected by clinical and treatment-related factors.MethodsIn this longitudinal, single-center study, we analyzed IgG, IgM, and IgA antibody kinetics to severe acute respiratory syndrome-Corona virus-2 (SARS-CoV-2) spike-1, receptor binding domain (RBD), and nucleocapsid proteins in 354 alloHCT patients and 647 healthy controls by ELISA. A total of 1,364 serum samples were collected before and after one, two, and three doses of mRNA, adenovirus vector, and/or protein-based vaccines. Clinical parameters, including age, conditioning regimen, immunosuppressive therapy, graft-versus-host disease (GVHD), donor type, time since transplantation, diagnosis, and laboratory parameters were evaluated for their influence on antibody responses.ResultsAnti-spike-1 IgG responses increased progressively: 27% of patients versus 28% of controls seroconverted after the first vaccination, 65% versus 59% after the second, and 93% versus 98% after the third. After three doses, no significant differences in IgG prevalence or reactivity remained between groups. IgM and IgA responses were infrequent. Multivariate analysis identified diagnosis, shorter interval between transplantation or vaccination and antibody testing, and lower lymphocyte count as negative predictors after the first vaccination. No negative predictors were detected after the third dose. Antibody levels remained stable for at least six months following the third vaccination. Breakthrough infections occurred but were mild.ConclusionsAlloHCT recipients achieve humoral immunity comparable to healthy individuals after three vaccine doses, supporting efficacy and safety of repeated SARS-CoV-2 vaccination in this vulnerable population.

ABSTRACT:Lenalidomide, a maintenance treatment in multiple myeloma first-line therapy, increases the risk of secondary malignancies, including B-cell precursor acute lymphoblastic leukemia (B-ALL). We present a comprehensive molecular characterization of 57 patients with lenalidomide-associated B-ALL (LenB-ALL), revealing 3 mutational subgroups: (1) TP53mt (30%); (2) IDH2mt (p.R140Q) (23%); and (3) other, including NRAS/KRASmt. Remarkably, IDH2 R140Q mutations were highly enriched in LenB-ALL compared with those in primary B-ALL (P< .001). Furthermore, IKZF1 intragenic deletions, often subclonal and likely RAG recombinase-mediated, were observed in 54% (7/13) of IDH2mt patients with LenB-ALL. IDH2 mutations were not restricted to the leukemic clone: they persisted during measurable residual disease-negative remission and were identified in lymphoid as well as myeloid cell populations using fluorescence-activated cell sorting and single-cell RNA sequencing. This indicates a preleukemic origin of the IDH2 mutation within the context of clonal hematopoiesis. Transcriptomic and DNA methylation analyses revealed a distinct gene expression profile and a DNA hypermethylation phenotype in IDH2mt LenB-ALL, including IDH2mt-specific as well as lenalidomide-associated features. We propose that lenalidomide promotes the expansion of IDH2-mutated clonal hematopoiesis and, via IKAROS downregulation, induces a maturation arrest at the B-cell precursor stage. Subsequent genetic or epigenetic alterations render leukemogenesis independent of ongoing lenalidomide exposure. All these data define IDH2mt B-ALL as a distinct molecular subtype that is markedly overrepresented after lenalidomide treatment and highlight clonal hematopoiesis as a key contributing factor in the development of LenB-ALL.

Three patients with refractory MS received CD19-targeting chimeric antigen receptor (CAR)-T-cells (UKT-CART19.1) after standard lymphodepletion and were followed-up for 3, 7 and respectively 12 months. All showed neuroinflammation on magnetic resonance imaging (MRI) and/or clinical deterioration prior to treatment. UKT-CART19.1 was well tolerated, stabilized clinical impairment, and reduced intrathecal antibody levels and inflammatory lesions on MRI imaging. Transient B-cell depletion was followed by reconstitution with predominately naïve B-cells. We propose that CD19-targeting CAR-T-cells are safe and effective in refractory MS warranting further exploration in clinical trials. Notably, this report provides first evidence of neuroimaging responses to CAR-T-cell therapy in MS.

Introduction Anti-CD19 Chimeric Antigen Receptor T cell therapy (CART19) is a promising treatment option for refractory autoimmune diseases (AD). These patients are often heavily pretreated with multiple lines of immunosuppression, increasing their susceptibility to infections. In this context, immunologic reconstitution, infectious complications, and the implementation of specific prophylactic measures are subjects of ongoing discussion. We investigated the immune recovery and immunologic remodeling in AD patients treated with CART19. Methods All patients were treated under a hospital exemption and the study was approved by the local ethics committee (771/2023BO2). Autologous T cells were collected and transduced with an anti-CD19, 2nd-generation CAR lentiviral vector containing a CD3-ζ signaling domain (Miltenyi Biotec, Germany), before infusion following lymphodepletion with fludarabine and cyclophosphamide. We analyzed immune reconstitution (cell counts, immunoglobulin levels, vaccination titers) and infectious complications within one year post-therapy, censoring patients at disease progression (PD) or follow-up completion (1 year). Febrile neutropenia, colonization, and resident flora were excluded as infections. Spectral flow phenotyping was performed to assess immunologic changes after CART19 treatment in ADs. Results Systemic sclerosis was the most common indication (n = 6), followed by antisynthetase syndrome, rheumatoid arthritis, and granulomatosis with polyangiitis (each n = 1). The median age was 50 years (range: 30–69), with 66.7% female patients. Most were heavily pretreated, with a median of 6 previous therapies. At CART therapy, 44.4% had stable disease and 55.6% had PD. Early infections (<30d) occurred in 44.4%, resulting in a density of 1.85 per 100 patient-days, which later declined to 0.33 infections. Until day 30, bacterial infections were predominant; only one patient developed a non-invasive fungal infection. Later a shift from bacterial (80.0%) to predominantly viral infections (87.5%) was observed. Following CART19 infusion, blood cell lineages recovered rapidly, with neutrophil counts (>500/µL) reached by a median of 11 days (with G-CSF). At day 180, AD patients had significantly higher CD4+ T- and CD19+ B-cell counts compared to our previously published DLBCL cohort (CD4+: 312 vs. 126/µl, CD19+: 135 vs. 0/µl, Mann-Whitney p=.04/<.001). Humoral recovery was also rapid (Median IgG d+180: 616/µl) with only one patient receiving intravenous immunoglobulin substitution (IVIG), planned for discontinuation. To assess whether vaccination titers remain adequate following CART19, we analyzed titers for the standard vaccines—tetanus, measles, diphtheria and hepatitis B. No patients received vaccinations against the analyzed pathogens between the time of CART infusion and titer assessment. Interestingly, there was no significant difference in titers between the time points except for tetanus (approximately d180) (Wilcoxon, p=.03). One patient exhibited even higher titers after CART 19 treatment, most likely due to ongoing, potent B cell depleting therapies prior lymphodepletion. Spectral flow phenotyping revealed no significant differences in the proportions of CD8+ and CD19+ relative to CD45+ cells between previously investigated healthy individuals and patients at day 180 post CART19 treatment (Mann-Whitney U, p=<.001-.006). However, significant alterations were observed in the composition of lymphocyte subsets. CART therapy resulted in significantly lower proportions of CD4+ T cells and γδ effector memory T cells, while regulatory T cells (Tregs) and activated T cells were significantly increased (Mann-Whitney U, p=<.001-.02). Moreover, CD19+ B cells in CART19 patients were predominantly immature, with a significantly reduced proportions of both switched and unswitched B cells (Mann-Whitney U, p=.006-.02). Conclusion Our findings implicate effective immune reconstitution with manageable infectious risk following CART19 treatment in ADs. Vaccination titers remained stable and IgG levels were largely sufficient, suggesting preserved humoral immunity. Compared to healthy individuals, an increase in Tregs, a reduction in CD4+ T cells and γδ effector memory T cells, and a B cell compartment skewed toward immature phenotypes may reflect a beneficial immune remodeling. Additional data on immune changes relative to the preCART19 baseline will be presented at the meeting.

ABSTRACT:Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy in treating relapsed and refractory (R/R) B-cell neoplasms, such as diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM). Despite its success, the long-term effects and sequelae of CAR T cells on the immune system remain underexplored. This study presents a 1-year follow-up analysis of 52 patients (42 with R/R DLBCL and 10 with R/R MM) treated with anti-CD19- and B-cell maturation antigen-targeted CAR T cells, focusing on immune reconstitution and infectious complications. Our findings reveal that CAR T-cell therapy leads to profound depletion of B and T cells. CD4+ T cells and CD19+ B cells exhibited impaired regeneration after treatment. Infections were more frequent during the first 30 days. In the short-term follow-up, density of infections within 100 days at risk was 1.8 in patients with DLBCL and 4.6 in patients with MM, with bacterial infections predominating in this early period after CAR T-cell infusion. In addition, we observed a shift to viral infections in the long-term follow-up, alongside with a decline in infection density to 0.1 in patients with DLBCL and 0.4 infections per 100 days at risk in patients with MM, respectively. Severe cytokine release syndrome was associated with a higher risk of late-onset infections. These findings highlight the importance of close monitoring and prophylactic measures in patients undergoing CAR T-cell therapy to reduce infection risks and enhance immune recovery.

Background: Dual-targeted CAR-T cell therapies may help overcome mechanisms of resistance associated with antigen escape. We evaluated an investigational, autologous CD19/CD22-targeted second-generation CAR construct incorporating a 4-1BB costimulatory domain and fully human scFv domains. Here, we report outcomes in adult patients with relapsed/refractory (R/R) B-cell malignancies. Methods: Nine adult patients with R/R B-cell malignancies received an investigational CD19/CD22-targeted CAR-T cell product since January 2020. Treatment was performed based on recommendations of the interdisciplinary Cell Therapy Board at the University Hospital Tübingen, and after extensive counseling with the patient´s informed consent based on hospital exemption for advanced therapy medicinal products (ATMP) treatment. For CD19/CD22 CAR-T cell production lymphocytes were transduced with a lentiviral vector encoding a human anti-CD19 scFv M19217-1 and human anti-CD22 scFv 16p17 (kindly provided by Miltenyi Biotec) and cells amplified in the Miltenyi Prodigy systems in our academic GMP facility at the University Hospital. Lymphodepletion was performed using fludarabine (25 mg/m²/day, days –5 to –3) and cyclophosphamide (1000 mg/m², day –3). Seven patients received autologous T-cells; two received donor-derived T-cells from prior allogeneic stem cell transplant donors in a dose of 3x10E6 CD19/CD22 CAR-T cells/kg bodyweight. Results: Diagnoses included B-cell acute lymphoblastic leukemia (B-ALL, n=3), mantle cell lymphoma (MCL, n=1), Richter transformation–DLBCL (n=2), primary mediastinal B-cell lymphoma (PMBCL, n=1), post-transplant lymphoproliferative disorder (PTLD, n=1), and diffuse large B-cell lymphoma (DLBCL, n=1). Best responses included complete remission (CR) in 6 patients (66%) and progressive disease (PD) in 3 (33%). All B-ALL patients achieved CR; among aggressive B-cell lymphomas, CR was achieved in 3 of 6 patients (50%). According to Kaplan-Meier estimates, the median follow-up was 25.7 months (range 0.8–60.4 months) with a median progression-free survival (PFS) of 13.6 months (29.8 months in B-ALL; 7.5 months in lymphoma). The median overall survival (OS) was 60.4 months, (60.4 months in B-ALL; 13.8 months in lymphoma). CAR-T cell persistence was assessed throughout the follow-up period. In two ALL cases, repeated CAR-T cell infusions induced renewed complete remission (CR) after relapse associated with loss of CAR-T cell persistence: one following MRD relapse, and one following relapse with cerebral leukemic infiltration. Safety: Grade 4 neutropenia occurred in 8 patients (89%), with a median duration of 10 days. Cytokine release syndrome (CRS) was observed in 7 patients (77%), including Grade 3–4 CRS in 2 patients (22%), with a median onset at day 1 and duration of 3 days. Immune effector cell–associated neurotoxicity syndrome (ICANS) occurred in 2 patients (22%), including one Grade 3–4 event. Reported complications included one case each of immune effector cell-associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS), Candida sepsis, Grade III intestinal GvHD, and squamous cell carcinoma. The main causes of death were infection (n=1) and progressive disease (n=2). Conclusion: The investigational CD19/CD22-targeted CAR-T cell product demonstrated encouraging efficacy with a manageable safety profile in heavily pretreated adult patients with relapsed/refractory B-cell malignancies, including post-transplant settings. Dual targeting may offer therapeutic advantages in overcoming antigen escape and achieving durable remissions. Based on promising results, a Phase I dose-finding and efficacy study of a third-generation CAR-T cell product (MB-CART19.22), is planned in adult and pediatric patients with relapsed or refractory B-cell malignancies.

Importance: CAR-T cells show high efficacy in B-cell neoplasia and non-malignant B-cell driven autoimmune disorders, but therapy-related toxicity can limit treatment success. The identification of biomarkers informing patient selection and pre-emptive measures is important for enhancing treatment safety and improving clinical outcome. Objective: To analyze the influence of pre-treatment cytopenia and clonal hematopoiesis on clinical outcome after B-cell targeting CAR-T-cell therapy in malignant and autoimmune disorders. Design, Participants: This retrospective real-life analysis includes all adult patients with leukemia/lymphoma (L/L, 49), multiple myeloma ((MM, n=38 (n=1 coexistent L)), autoimmune diseases (AID, 11) treated at the University Hospital Tübingen with CD19, CD19/CD22 or BCMA-targeted CAR-T cells between March 2022 and March 2025, with last follow-up in April 2025. We analyzed routine clinical data for blood/CAR-T-cell counts, ICANS, Immune effector Cell-Associated Neurotoxicity Syndrome, CRS, Cytokine Release Syndrome, IEC-HS, Immune Effector Cell–Associated Hemophagocytic Syndrome, overall survival, OS, non-relapse mortality, NRM, remission induction, RI, progression-free survival, PFS and relapse-free survival, RFS, and retrospectively analyzed clonal hematopoiesis (CH) by next generation sequencing (NGS). Main Outcome(s) and Measure(s): Low pre-treatment blood counts most commonly occurring as pre-treatment neutropenia (pNP+) associated with enhanced NRM and reduced OS. Furthermore, pNP+ patients showed higher CAR-T cell expansion but no improvement in disease control. Detection of clonal hematopoiesis (CH+) further impaired outcome in pNP+ patients, but not by itself. AID patients show better CAR-T-cell tolerability than L/L/MM patients, but one AID patient retrospectively identified as pNP+CH+ showed lethal outcome. Results: Twenty-nine of 98 (29.6%) patients showed pNP+ (MM: 47.4%, L/L: 20.4%, AID: 9.1%) and twenty four of 64 (37.5%) patients CH (L/L: 47%, MM: 31%, AID: 45%, with TET2, DNMT3A, PPM1D and TP53 as most mutated genes). pNP+ vs. pNP- (regular pre-treatment neutrophil counts) showed shorter OS (p=0.006) and enhanced non-relapse mortality (NRM, p=0.006), with post-therapy early (p<0.001) and late neutropenia (p=0.001) and infections (p=0.011). Interestingly, similar RI (p=0.174), PFS (p=0.698) and RFS (p=0.605) were observed despite higher CAR-T cell counts (pNP+ vs. pNP-, day +35: 9.5% vs. 0.9%, p=0.0002; day +100: 0.60% vs. 0.17%, p=0.002). CH co-detection impaired OS and NRM in pNP+ patients but not alone or in pNP- (p=0.024, p=0.018) likely due to higher rates of early neutropenia (p=0.002). pNP+CH+ versus pNP+CH- patients furthermore showed enhanced CAR-T cell expansion (day +10, p<0.001, day +35, p=0.042) and B-cell aplasia persistence (day +180, p=0.049). AID patients tolerated CAR-T-cell therapy better than L/L/MM patients showing less posttreatment neutropenia (p=0.029), thrombocytopenia (p=0.009), severe CRS (p=0.035), ICANS (L/L/MM: 14/87; AID: 0/11) and infections (p=0.031). However, one pNP+CH+ AID patient succumbed with cytopenia, severe infections and immune effector cell hemophagocytic syndrome (IEC-HS). Conclusions and Relevance: Combined pre-treatment cytopenia and clonal hematopoiesis should be evaluated as risk factors for CAR-T-cell therapy morbidity and mortality in prospective studies.

Background and Significance For many patients with AML and higher risk MDS, allogeneic hematopoietic stem cell translplantation (HSCT) is the only curative treatment. The introduction of reduced intensity conditioning (RIC) regimen has greatly increased the number of patients eligible for alloHSCT. While Fludarabine plus Busulfan (FluBu) is a very popular RIC regimen, data from a randomized controlled trial have demonstrated superior overall survival (OS) with Treosulfan (FluTreo) instead of Busulfan as alkylating agent (3 year OS 67 vs. 56%; Beelen et al., Am J Hematol, 2022). However, the favorable toxicity profile of RIC alloHSCT still comes at the price of a higher relapse risk as compared to myeloablatve conditioning (2 years relapse risk 6% higher; Alfaro Moya et al., Eur J Haematol, 2023). Since relapses remain the main cause of mortality after alloHSCT, there is an urgent need to reduce this risk. We hypothesized that the addition of the Bcl-2-Inhibitor Venetoclax to FluTreo RIC can enhance antileukemic efficacy, hence reducing the relapse risk, without adding relevant toxicity. Regarding toxicity, this expectation is based on the favorable safety profile of Venetoclax observed in the first line setting in elderly and comorbid patients, as well as on data from a phase I trial (Garcia et al., Blood Adv, 2021). The trial found a recommended phase II dose of 400 mg of Venetoclax for 7 days. Increased antileukemic activity is assumed based on preclinical data, showing synergism of Venetoclax with Fludarabine and alyklating agents in vitro (Valdez et al., Oncotarget, 2022). Taking into account the superiority of Treosulfan over Buslulfan, we also expect VenFluTreo to produce more favorable results than VenFluBu. Study Design and Methods The trial VeStAL has the EU Trial Number (2025-521372-62-00). It is conducted as mononcentric, single-arrm phase II trial. General study design of this safety trial and analysis of the primary endpoint, overall survival at day 28 after alloHSCT, follows a Simon Optimal Design. This design includes 27 patients and tests the alternative hypothesis of rate of early mortality of 10% against a null hypothesis of an early mortality of 30% (5% one-sided probability of tye I error, 80% power). Stage 1 enrolls 6 patients. If ≤2 deaths within 28 days after alloHSCT are observed, the trial will be ended. Otherwise, stage 2 enrolls further 21 patients. DSMB-Meetings are held after 6 and 14 patients have reached the primary endpoint. The study meets its primary endpoint, if ≤4 deaths are observed within 28 days after alloHSCT. Enrolled are patients planned for alloHSCT (with peripheral blood stem cells) with AML, MDS/AML or higher risk MDS (ICC 2022), who present with a clinically controlled myeloid neoplasm. This is defined as having either responded to induction ± consolidation therapy (at least morphological leukemia free state) in cases with AML or MDS/AML or being treated with upfront alloHSCT in cases with MDS or MDS/AML. For patients with MDS, response to pre-treatment is not required. Excluded are patients with relapsed disease, extramedullary myeosarcoma, history of alloHSCT or patients who are scheduled for alloHSCT with bone marrow stem cells or with a haploidentical donor. Patients who are enrolled receive 7 days of Venetoclax ( day -8 to -2, 400 mg p.o.), 5 days of Fludarabine (day -6 to -2, 30 mg/m2i.v.) and 3 days of Treosulfan (day -4 to -2, 10 mg/m2i.v.). Immunosuppression and infusion of allogeneic stem cells on day 0 is performed according to standard of care and local practice. However, a minimum time interval of 54 hours (3 half lives) between the last dose of Venetoclax and infusion of allogeneic stem cells is to be adhered to. Patients are followed up until day 100. As mentioned above, the primary endpoint is overall survival on day 28 after alloHSCT. Since this is a safety trial, the key secondary endpoint is the incidence of AEs CTCAE Grade ≥4 until day 28. Further important secondary endpoints include rate of delayed engraftment on day 28 and overall survival, non-relapse mortality and cumulative incidence of relapse, as well as cumulative incidence of Grade III and IV GvHD on day 100. A scientific companion program includes spatial assessment of the bone marrow microenvironment under therapy and monitoring of chimerism via single nucleotide polymorphism using digital PCR. The study was submitted via CTIS on 30.07.2025 and will start enrollment early in 2026.

BACKGROUND:Allogeneic hematopoietic cell transplantation (alloHCT) is an established curative treatment for hematological malignancies and other severe blood disorders. However, alloHCT is also known for its significant side effects. SUMMARY:Here we review recent advances in targeted molecular therapy, immunotherapy, infectiology, and diagnostics that have enhanced the tolerability and efficacy of alloHCT, expanding its use to less fit and elderly patients. We analyze developments in conditioning regimens, donor selection, and the management of graft versus host disease (GVHD) and infections and discuss posttransplantation strategies to prevent relapse. KEY MESSAGE:In a fresh perspective, alloHCT can serve as a platform to enhance the potential of emerging targeted and immune therapies.

Therapeutic progress has improved the overall survival of patients treated with allogeneic hematopoietic cell transplantation (alloHCT). Thus, the impact on quality of life (QoL) becomes increasingly relevant. However, QoL is not monitored regularly in clinical practice, and most trials stop QoL assessments early post-alloHCT, missing long-term dynamics. To address this knowledge gap, we conducted a cross-sectional survey of 214 adult alloHCT recipients (average age 53 y, 42.5% female, median follow-up 56 months) to evaluate QoL using patient-reported outcome measurements (PROMs), spanning a period from 30 days to over 10 years post-transplant. Participants completed the EORTC QLQ-C30 and FACT-BMT at a single follow-up timepoint to investigate QoL-related factors. Comparing long-term follow-up patients (beyond year 3, n = 125) with short-term follow-up patients (day 30 to month 12, n = 89) shows significantly better long-term QoL outcomes (P = 0.016). However, PROM symptom scales indicate moderate fatigue and insomnia rates in long-term survivors. Better QoL was associated with male gender, lower ECOG, RIC conditioning, no relapse, no ongoing immunosuppression and full-time work. Summarized, while we observe encouraging long-term outcomes, our data suggest that QoL recovery remain highly individual. We strongly recommend the use of PROMs to enhance our understanding of long-term survivorship post-alloHCT.

Distinct diagnostic entities within BCR :: ABL1- positive acute lymphoblastic leukemia (ALL) are currently de fi ned by the International Consensus Classi fi cation of myeloid neoplasms and acute leukemias (ICC): "lymphoid only", with BCR :: ABL1 observed exclusively in lymphatic precursors, vs "multilineage", where BCR :: ABL1 is also present in other hematopoietic lineages. Here, we analyzed transcriptomes of 327 BCR :: ABL1- positive patients with ALL (age, 2-84 years; median, 46 years) and identi fi ed 2 main gene expression clusters reproducible across 4 independent patient cohorts. Fluorescence in situ hybridization analysis of fl uorescence-activated cell-sorted hematopoietic compartments showed distinct BCR :: ABL1 involvement in myeloid cells for these clusters (n = 18/18 vs n = 3/16 patients; P < .001), indicating that a multilineage or lymphoid BCR :: ABL1 subtype can be inferred from gene expression. Further subclusters grouped samples according to cooperating genomic events (multilineage: HBS1L deletion or monosomy 7; lymphoid: IKZF1 (-/-) or CDKN2A / PAX5 deletions/hyperdiploidy). A novel HSB1L transcript was highly speci fi c for BCR :: ABL1 multilineage cases independent of HBS1L genomic aberrations. Treatment on current German Multicenter Study Group for Adult ALL (GMALL) protocols resulted in comparable disease-free survival (DFS) for multilineage vs lymphoid cluster patients (3-year DFS: 70% vs 61%; P = .530; n = 91). However, the IKZF1 (-/-) enriched lymphoid subcluster was associated with inferior DFS, whereas hyperdiploid cases showed a superior outcome. Thus, gene expression clusters de fi ne underlying developmental trajectories and distinct patterns of cooperating events in BCR :: ABL1- positive ALL with prognostic relevance.

BACKGROUND:Reactivation of viral infections, in particular cytomegalovirus (CMV) and adenovirus (ADV), cause morbidity and non-relapse-mortality in states of immune deficiency, especially after allogeneic hematopoietic cell transplantation (allo-HCT). Against the background of few available pharmacologic antiviral agents, limited by toxicities and resistance, adoptive transfer of virus-specific T-cells (VST) is a promising therapeutic approach. METHODS:We conducted a single-center retrospective analysis of adult patients treated with ADV- or CMV-specific T-cells in 2012-2022. Information was retrieved by review of electronic health records. Primary outcome was a response to VST by decreasing viral load or clinical improvement. Secondary outcomes included overall survival and safety of VST infusion, in particular association with graft-versus-host disease (GVHD). RESULTS:Ten patients were included, of whom four were treated for ADV, five for CMV, and one for ADV-CMV-coinfection. Cells were derived from stem cell donors (6/10) or third-party donors (4/10). Response criteria were met by six of 10 patients (4/4 ADV, 2/5 CMV, and 0/1 ADV-CMV). Overall survival was 40%. No infusion related adverse events were documented. Aggravation of GVHD after adoptive immunotherapy was observed in two cases, however in temporal association with a conventional donor lymphocyte infusion and a stem cell boost, respectively. CONCLUSION:In this cohort, CMV- and ADV-specific T-cell therapy appear to be safe and effective. We describe the first reported case of virus-specific T-cell therapy for CMV reactivation not associated with transplantation but with advanced HIV infection. This encourages further evaluation of adoptive immunotherapy beyond the context of allo-HCT.

Abstract Distinct diagnostic entities within BCR::ABL1-positive acute lymphoblastic leukemia (ALL) are currently defined by the International Consensus Classification of myeloid neoplasms and acute leukemias (ICC): “lymphoid only”, with BCR::ABL1 observed exclusively in lymphatic precursors, vs “multilineage”, where BCR::ABL1 is also present in other hematopoietic lineages. Here, we analyzed transcriptomes of 327 BCR::ABL1-positive patients with ALL (age, 2-84 years; median, 46 years) and identified 2 main gene expression clusters reproducible across 4 independent patient cohorts. Fluorescence in situ hybridization analysis of fluorescence-activated cell-sorted hematopoietic compartments showed distinct BCR::ABL1 involvement in myeloid cells for these clusters (n = 18/18 vs n = 3/16 patients; P < .001), indicating that a multilineage or lymphoid BCR::ABL1 subtype can be inferred from gene expression. Further subclusters grouped samples according to cooperating genomic events (multilineage: HBS1L deletion or monosomy 7; lymphoid: IKZF1-/- or CDKN2A/PAX5 deletions/hyperdiploidy). A novel HSB1L transcript was highly specific for BCR::ABL1 multilineage cases independent of HBS1L genomic aberrations. Treatment on current German Multicenter Study Group for Adult ALL (GMALL) protocols resulted in comparable disease-free survival (DFS) for multilineage vs lymphoid cluster patients (3-year DFS: 70% vs 61%; P = .530; n = 91). However, the IKZF1-/- enriched lymphoid subcluster was associated with inferior DFS, whereas hyperdiploid cases showed a superior outcome. Thus, gene expression clusters define underlying developmental trajectories and distinct patterns of cooperating events in BCR::ABL1-positive ALL with prognostic relevance.

Introduction Patients with Richter's transformation (RT) of chronic lymphocytic leukemia (CLL) to diffuse large B cell lymphoma (DLBCL) show poor prognosis despite complex chemotherapy and hematopoietic cell transplantation (HCT). Targeted agents, bispecific antibodies and CAR-T cells have successfully entered the therapeutic landscape of B-cell malignancies but yet have no established role in the treatment of RT. Here, we report successful longterm complete remission after treatment with academically manufactured dual CD19/CD22 CAR T cells in two patients showing RT relapse after allogeneic HCT. We hypothesize that patients with aggressive RT may be more efficiently treated with academically produced CD19/CD22 CAR T cells that target more than one B cell antigen and thus can reduce antigen escape and the risk of therapy resistance. Methods Two patients were treated at the University Hospital Tübingen between February and December 2023. The treatment was approved by the local Cellular Therapy Board and conducted after informed consent. CD19/CD22 targeting CAR-T cells were manufactured in a 12 day production process as previously reported in our academic Good Manufacturing Practice (GMP) laboratory using autologous mononuclear cells, transduced with a human anti-CD19/anti-CD22 lentiviral construct (Miltenyi Biotec) and amplified using the CliniMACS Prodigy platform (Phely et al. JAMA Onc 2024). CAR T cells were applied following lymphodepletion with fludarabine and cyclophosphamide and in vivo expansion was monitored by flow cytometry. Results Patient 1 (64 years, male) and Patient 2 (42 years, male) both experienced DLBCL-RT two years post-CLL diagnosis. Both received chemotherapies, rituximab, ibrutinib and HCT and suffered from multiple relapses. Patient 1 relapsed for a second time after HCT in 2023 with severe meningeal involvement, treated with steroids, local therapy and pirtobrutinib before receiving CAR T cells in December 2023. Patient 2, diagnosed with CLL 13 years ago, relapsed with CLL one year after HCT and a few months later he developed DLBCL-RT. Treatment with R-CHOP followed by lenalidomide and rituximab induced complete remission. One year later, a 1st RT relapse was treated with ibrutinib which induced sustained remission over 7 years. In March 2021 a 2nd RT-relapse occurred and was treated with rituximab, polatuzumab and bendamustine. After several months of apparent complete remission, the patient relapsed for the 3rd time. He was bridged with the prior regimen and radiotherapy, before receiving treatment with CAR-T cells in March 2023. Both patients tolerated the treatment well and remain in complete remission at 17 and 7 months post-infusion. No severe infections occurred. Patient 1 experienced grade I cytokine release syndrome (CRS), treated with tocilizumab, and showed rapid CAR T cell expansion. Patient 2 had grade I CRS and plexus neuritis, also treated with tocilizumab and dexamethasone, with moderate CAR T cell expansion. Patient 2 was later diagnosed with anal carcinoma, treated surgically. Discussion Both patients had aggressive RT, characterized by high LDH, low platelet counts, large tumors, and refractoriness to multiple treatments. RT occurs in about 2% of CLL patients, with poor responses to chemotherapy and a median survival of ten months. BTK inhibitors provide some benefit but are often short-lived, highlighting the need for new therapies. Anti-CD19 CAR-T therapy shows promise, though relapses often involve CD19 loss, leading to a short progression free survival, necessitating strategies like dual-targeting CAR-T cells. Dual-targeting CAR-T therapy, which targets both CD19 and CD22, can prevent antigen escape, enhancing treatment efficacy. This strategy has shown promise in other B-cell malignancies (Nguyen et al. Cancer Med 2023). On-site GMP production enables rapid and fresh CAR-T cell availability without cryopreservation or shipping delays. This report is the first to discuss dual-targeting CAR-T therapy in RT, with promising outcomes despite a small sample size and short follow-up. These initial findings support further clinical trials to validate the efficacy and safety of this approach in RT patients. Acknowledgement WB and CL are equally shared last authors.