Validating genetic variants in innate immunity linked to infectious events in acute myeloid leukemia post-induction chemotherapy.

Infectious events, such as sepsis and invasive fungal disease (IFD), pose significant risks in patients with acute myeloid leukemia (AML). Previous studies, including our own, have suggested a potential role of single nucleotide polymorphisms (SNPs) within the innate immune system in influencing individual infection susceptibility. However, many of these associations lack validation in independent cohorts. This study sought to validate the impact of 11 candidate SNPs across 6 genes (TLR2, TLR4, Dectin-1, DC-SIGN, PTX3, L-Ficolin) in an independent cohort of patients. Two cohorts with newly diagnosed AML patients receiving intensive induction chemotherapy were analyzed: a stratification cohort comprising 186 patients and a validation cohort consisting of 138 patients. Multiple SNPs in each cohort were found to be associated to infectious complications, notably the DC-SIGN SNP rs4804800 demonstrated a significant association with sepsis in both cohorts. SNPs within the PTX3 and Dectin-1 genes were linked to IFD development in one cohort each. This study represents the first validation study of candidate genes associated with infectious events in AML patients after intensive induction chemotherapy. Identifying genetic predispositions to infections could significantly impact the management of antimicrobial prophylaxis and treatment in AML patients.

GLA/DRST real-world outcome analysis of CAR T-cell therapies for large B-cell lymphoma in Germany.

CD19-directed chimeric antigen receptor (CAR) T cells have evolved as a new standard-of-care (SOC) treatment in patients with relapsed/refractory (r/r) large B-cell lymphoma (LBCL). Here, we report the first German real-world data on SOC CAR T-cell therapies with the aim to explore risk factors associated with outcomes. Patients who received SOC axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) for LBCL and were registered with the German Registry for Stem Cell Transplantation (DRST) were eligible. The main outcomes analyzed were toxicities, response, overall survival (OS), and progression-free survival (PFS). We report 356 patients who received axi-cel (n = 173) or tisa-cel (n = 183) between November 2018 and April 2021 at 21 German centers. Whereas the axi-cel and tisa-cel cohorts were comparable for age, sex, lactate dehydrogenase (LDH), international prognostic index (IPI), and pretreatment, the tisa-cel group comprised significantly more patients with poor performance status, ineligibility for ZUMA-1, and the need for bridging, respectively. With a median follow-up of 11 months, Kaplan-Meier estimates of OS, PFS, and nonrelapse mortality (NRM) 12 months after dosing were 52%, 30%, and 6%, respectively. While NRM was largely driven by infections subsequent to prolonged neutropenia and/or severe neurotoxicity and significantly higher with axi-cel, significant risk factors for PFS on the multivariate analysis included bridging failure, elevated LDH, age, and tisa-cel use. In conclusion, this study suggests that important outcome determinants of CD19-directed CAR T-cell treatment of LBCL in the real-world setting are bridging success, CAR-T product selection, LDH, and the absence of prolonged neutropenia and/or severe neurotoxicity. These findings may have implications for designing risk-adapted CAR T-cell therapy strategies.

Azacitidine in combination with shortened venetoclax treatment cycles in patients with acute myeloid leukemia.

The combination of venetoclax with hypomethylating agents is currently the standard of care for elderly patients with acute myeloid leukemia (AML) ineligible for intensive chemotherapy. Despite its favorable efficacy, clinical use is often associated with post-remission cytopenia, frequently necessitating treatment delays and dose modifications. This study aims to evaluate the efficacy and safety of shortened venetoclax treatment durations. A multicenter analysis was conducted involving 20 adult AML patients receiving venetoclax (7 or 14 days with 9 and 11 patients, respectively) combined with 5-azacitidine (5-7 days) between 2021 and 2024. The cohort included patients from four German academic centers all treated in first line. Outcome measures included bone marrow response, transfusion dependence, overall survival (OS) and progression-free survival (PFS). Median age was 73.5 years, with 70% of patients having secondary AML. Adverse molecular risk was observed in 75% of patients. The overall response rate (ORR) was 100%, with a composite complete remission rate of 78%. No significant differences in response rates were observed between the 7-day and 14-day venetoclax regimens. Median OS for the cohort was 15 months. Infection-related complications were observed in 55% of patients, with severe sepsis in 20% of cases. In this cohort, shortened venetoclax regimens demonstrated efficacy comparable to standard treatment protocols, with a potential reduction in hematologic toxicity. These findings support the individualization of treatment regimens to optimize clinical outcomes while potentially minimizing adverse effects.

Isoform localization of Dectin-1 regulates the signaling quality of anti-fungal immunity.

Dectin-1 is recognized as a major receptor for fungal ß-glucans and contributes to anti-fungal immunity. Human monocyte populations express Dectin-1 isoforms A and B, which differ by the presence of a stalk region and its N-linked glycosylation site. Here, we analyzed the expression of both isoforms in human monocyte-derived cells. The cellular localization on cell lines stably expressing either Dectin-1 isoform A or B was studied by flow cytometry and confocal laser scanning microscopy. Intracellular protein signaling and cytokine production were analyzed by immunoblotting and cytometric bead array, respectively. Monocyte-derived cells showed cell type-specific expression of the two isoforms. Glycosylated Dectin-1 isoform A was predominantly localized at the cell surface, non-glycosylated isoform B was retained intracellularly. Inhibition of glycosylation resulted in efficient abrogation of cell surface expression of isoform A. Signaling quality following Dectin-1 stimulation was reduced in isoform B cells. Differential isoform specific cytokine secretion was observed by cytometric bead array. We show here that n-glycosylation of Dectin-1 is crucial for its cell surface expression and consequently signal transduction. Taken together, unique cytokine secretion and varying expression levels of human Dectin-1 isoforms on monocyte-derived cells may indicate distinct isoform usage as a cell type-specific mechanism of regulating anti-fungal immunity.

Cell transformation by FLT3 ITD in acute myeloid leukemia involves oxidative inactivation of the tumor suppressor protein-tyrosine phosphatase DEP-1/ PTPRJ.

Signal transduction of FMS-like tyrosine kinase 3 (FLT3) is regulated by protein-tyrosine phosphatases (PTPs). We recently identified the PTP DEP-1/CD148/PTPRJ as a novel negative regulator of FLT3. This study addressed the role of DEP-1 for regulation of the acute myeloid leukemia (AML)-related mutant FLT3 internal tandem duplication (ITD) protein. Our experiments revealed that DEP-1 was expressed but dysfunctional in cells transformed by FLT3 ITD. This was caused by enzymatic inactivation of DEP-1 through oxidation of the DEP-1 catalytic cysteine. In intact cells, including primary AML cells, FLT3 ITD kinase inhibition reactivated DEP-1. DEP-1 reactivation was also achieved by counteracting the high levels of reactive oxygen species (ROS) production detected in FLT3 ITD-expressing cell lines by inhibition of reduced NAD phosphate (NADPH)-oxidases, or by overexpression of catalase or peroxiredoxin-1 (Prx-1). Interference with ROS production in 32D cells inhibited cell transformation by FLT3 ITD in a DEP-1-dependent manner, because RNAi-mediated depletion of DEP-1 partially abrogated the inhibitory effect of ROS quenching. Reactivation of DEP-1 by stable overexpression of Prx-1 extended survival of mice in the 32D cell/C3H/HeJ mouse model of FLT3 ITD-driven myeloproliferative disease. The study thus uncovered DEP-1 oxidation as a novel event contributing to cell transformation by FLT3 ITD.

Synergistic Effects of the RARalpha Agonist Tamibarotene and the Menin Inhibitor Revumenib in Acute Myeloid Leukemia Cells with KMT2A Rearrangement or NPM1 Mutation.

Inhibition of menin in acute myeloid leukemia (AML) harboring histone-lysine-N-methyltransferase 2A rearrangement (KMT2Ar) or the mutated Nucleophosmin gene (NPM1c) is considered a novel and effective treatment approach in these patients. However, rapid acquisition of resistance mutations can impair treatment success. In patients with elevated retinoic acid receptor alpha (RARA) expression levels, promising effects are demonstrated by the next-generation RARalpha agonist tamibarotene, which restores differentiation or induces apoptosis. In this study, the combination of revumenib and tamibarotene was investigated in various KMT2Ar or NPM1c AML cell lines and patient-derived blasts, focusing on the potential synergistic induction of differentiation or apoptosis. Both effects were analyzed by flow cytometry and validated by Western blot analysis. Synergy calculations were performed using viability assays. Regulation of the relevant key mediators for the MLL complex were quantified by RT-qPCR. In MV4:11 cells characterized by the highest relative mRNA levels of RARA, highly synergistic induction of apoptosis is demonstrated upon combination treatment. Induction of apoptosis by combined treatment of MV4:11 cells is accompanied by pronounced induction of the pro-apoptotic protein BAX and a synergistic reduction in CDK6 mRNA levels. In MOLM13 and OCI-AML3 cells, an increase in differentiation markers like PU.1 or a decreased ratio of phosphorylated to total CEBPA is demonstrated. In parts, corresponding effects were observed in patient-derived AML cells carrying either KMT2Ar or NPM1c. The impact of revumenib on KMT2Ar or NPM1c AML cells was significantly enhanced when combined with tamibarotene, demonstrating synergistic differentiation or apoptosis initiation. These findings propose promising strategies for relapsed/refractory AML patients with defined molecular characteristics.

Lymphoma-associated hemophagocytic lymphohistiocytosis (LA-HLH): a scoping review unveils clinical and diagnostic patterns of a lymphoma subgroup with poor prognosis.

Hemophagocytic lymphohistiocytosis (HLH) is a severe hyperinflammatory syndrome driven by pathologic activation of cytotoxic T-lymphocytes and macrophages. Despite advances in diagnostics and management, adult patients with lymphoma-associated HLH (LA-HLH) harbor particularly poor prognosis and optimal treatment remains challenging. As systematic data on LA-HLH are scarce, we aimed to synthesize research evidence by thorough analysis of the published literature in PubMed (MEDLINE-database) within the context of a scoping review. Of 595 search results, 132 articles providing information on 542 patients were reviewed and analyzed. Median patient age was 60 years (range, 18-98) with male predominance (62.7%). B- and T-NHL were equally represented (45.6% and 45.2%), Hodgkin's lymphoma was reported in 8.9% of the cases. The majority of patients (91.6%) presented in Ann-Arbor-Stages III and IV, and bone marrow infiltration was observed in a significant proportion of patients (61.5%). Soluble CD25 levels were markedly elevated (median 10,000 U/ml), with levels beyond 10,000 U/ml indicating unfavorable prognosis for 30-day and overall survival. 66.8% of the patients died after median 5.1 months. LA-HLH remains a clinical challenge requiring specialized management. Timely diagnosis and appropriate lymphoma-specific treatment are of utmost importance to enhance patient outcomes.

Effective bridging strategies prior to infusion with tisagenlecleucel results in high response rates and long-term remission in relapsed/refractory large B-cell lymphoma: findings from a German monocentric study.

BACKGROUND: Incorporating chimeric antigen receptor (CAR)-T cell therapy into relapsed or refractory large B-cell lymphoma (rr LBCL) treatment algorithms has yielded remarkable response rates and durable remissions, yet a substantial portion of patients experience progression or relapse. Variations in outcomes across treatment centers may be attributed to different bridging strategies and remission statuses preceding CAR-T cell therapy. PATIENTS: Twenty-nine consecutive adult patients receiving tisagenlecleucel (tisa-cel) for rr LBCL from December 2019 to February 2023 at Jena University Hospital were analyzed. RESULTS: The median age was 63, with a median of 3 prior treatments. Twenty patients (69%) were refractory to any systemic therapy before CAR-T cell treatment. Following leukapheresis, 25 patients (86%) received bridging therapy with the majority undergoing chemotherapy (52%) or combined modality therapy (32%). Radiotherapy (RT) was part of the bridging strategy in 44%, with moderately hypofractionated involved site RT (30.0 Gy/2.5 Gy) being applied most frequently (64%). Post-CAR-T infusion, the objective response rate at 30 days was 83%, with 55% achieving complete response. Twelve-month progression-free (PFS) and overall survival (OS) were 60% and 74%, respectively, with a median follow up of 11.1 months for PFS and 17.9 months for OS. Factors significantly associated with PFS were chemotherapy sensitivity pre-leukapheresis and response to bridging. CONCLUSION: The study underscores the importance of minimal tumor burden at CAR-T initiation, emphasizing the need for suitable bridging regimens. The findings advocate for clinical trials and further real-world analyses to optimize CAR-T cell therapy outcomes by identifying the most effective bridging strategies.

Anti-CD19 CAR-T cells are effective in severe idiopathic Lambert-Eaton myasthenic syndrome.

Lambert-Eaton myasthenic syndrome (LEMS) is an autoantibody-mediated disease of the neuromuscular junction characterized by muscular weakness. Autoantibodies to presynaptic P/Q-type voltage-gated calcium channels (VGCCs) induce defective neuromuscular function. In severe cases, current immunosuppressive and immunomodulatory treatment strategies are often insufficient. First reports show beneficial effects of anti-CD19 chimeric antigen receptor (CAR)-T cell therapy in patients with autoantibody-mediated myasthenia gravis. We report a patient with isolated idiopathic LEMS treated with autologous anti-CD19-CAR-T cells. In this patient, CAR-T infusion leads to expansion of predominantly CD4+ CAR-T cells with a terminally differentiated effector memory cells re-expressing CD45RA (TEMRA)-like phenotype indicating cytotoxic capabilities and subsequent B cell depletion. VGCC antibody titers decrease, resulting in a clinical improvement of LEMS symptoms, e.g., 8-fold increase in walking distance. The patient does not show relevant side effects except for cytokine release syndrome grade 2 and intermittent neutropenia suggesting that anti-CD19 CAR-T cell therapy may be a treatment option in patients with LEMS.

Impact of NOD2 polymorphisms on infectious complications following chemotherapy in patients with acute myeloid leukaemia.

We sought to investigate the relationship between polymorphisms of the NOD2 gene and infectious complications following intensive induction chemotherapy in patients with acute myeloid leukaemia (AML). We hypothesised that single nucleotide polymorphisms (SNPs) of the NOD2 gene are associated with a higher rate of infections during the phase of severe neutropenia. In 131 AML patients receiving induction therapy, the presence of the three most frequent polymorphisms of NOD2 (Arg702Trp, Gly908Arg, Leu1007fsinsC) was analysed. SNP analyses by means of genomic PCR incorporating fluorescence-labelled probes with characteristic melting curves were performed using the LightCycler platform. Our data suggest a significantly lower probability of mucositis or enteritis in AML patients lacking any of the three evaluated NOD2 polymorphisms. Furthermore, bloodstream cultures of AML patients carrying either a missense or a frameshift mutation of NOD2 were significantly more frequently tested positive concerning Streptococcus spp. In contrast, the presence of NOD2 polymorphisms had no impact on such important infectious complications as systemic inflammatory response syndrome or sepsis, the rate of central venous catheter infections or the incidence of pneumonia including fungal infections. Our data represent one of the first reports investigating the impact of polymorphisms of the innate immune system on infectious complications in patients with neutropenia following chemotherapy. A correlation between NOD2 polymorphisms and infectious events in AML patients is demonstrated.

Impact of treatment intensity on infectious complications in patients with acute myeloid leukemia.

BACKGROUND: Infectious complications reflect a major challenge in the treatment of patients with acute myeloid leukemia (AML). Both induction chemotherapy and epigenetic treatment with hypomethylating agents (HMA) are associated with severe infections, while neutropenia represents a common risk factor. Here, 220 consecutive and newly diagnosed AML patients were analyzed with respect to infectious complications dependent on treatment intensity and antifungal prophylaxis applied to these patients. PATIENTS AND METHODS: We retrospectively analyzed 220 patients with newly diagnosed AML at a tertiary care hospital between August 2016 and December 2020. The median age of AML patients undergoing induction chemotherapy (n = 102) was 61 years (25-76 years). Patients receiving palliative AML treatment (n = 118) had a median age of 75 years (53-91 years). We assessed the occurrence of infectious complication including the classification of pulmonary invasive fungal disease (IFD) according to the EORTC/MSG criteria at diagnosis and until day 100 after initiation of AML treatment. Furthermore, admission to intensive care unit (ICU) and subsequent outcome was analyzed for both groups of AML patients, respectively. RESULTS: AML patients subsequently allocated to palliative AML treatment have a significantly higher risk of pneumonia at diagnosis compared to patients undergoing induction chemotherapy (37.3% vs. 13.7%, P < 0.001) including a higher probability of atypical pneumonia (22.0% vs. 10.8%, P = 0.026). Furthermore, urinary tract infections are more frequent in the palliative subgroup at the time of AML diagnosis (5.1% vs. 0%, P = 0.021). Surprisingly, the incidence of pulmonary IFD is significantly lower after initiation of palliative AML treatment compared to the occurrence after induction chemotherapy (8.4% vs. 33.3%, P < 0.001) despite only few patients of the palliative treatment group received Aspergillus spp.-directed antifungal prophylaxis. The overall risk for infectious complications at AML diagnosis is significantly higher for palliative AML patients at diagnosis while patients undergoing induction chemotherapy have a significantly higher risk of infections after initiation of AML treatment. In addition, there is a strong correlation between the occurrence of pneumonia including atypical pneumonia and pulmonary IFD and the ECOG performance status at diagnosis in the palliative AML patient group. Analysis of intensive care unit (ICU) treatment (e.g. in case of sepsis or pneumonia) for both subgroups reveals a positive outcome in 10 of 15 patients (66.7%) with palliative AML treatment and in 15 of 18 patients (83.3%) receiving induction chemotherapy. Importantly, the presence of infections and the ECOG performance status at diagnosis significantly correlate with the overall survival (OS) of palliative AML patients (315 days w/o infection vs. 69 days with infection, P 0.0049 and 353 days for ECOG < 1 vs. 50 days for ECOG > 2, P < 0.001, respectively) in this intent-to-treat analysis. CONCLUSION: The risk and the pattern of infectious complications at diagnosis and after initiation of AML therapy depends on age, ECOG performance status and subsequent treatment intensity. A comprehensive diagnostic work-up for identification of pulmonary IFD is indispensable for effective treatment of pneumonia in AML patients. The presence of infectious complications at diagnosis contributes to an inferior outcome in elderly AML patients.

Long-Term Survivors after Failure of Chimeric Antigen Receptor T Cell Therapy for Large B Cell Lymphoma: A Role for Allogeneic Hematopoietic Cell Transplantation? A German Lymphoma Alliance and German Registry for Stem Cell Transplantation Analysis.

The outcome of patients with large B cell lymphoma (LBCL) who relapse or progress after CD19-directed chimeric antigen receptor T cell therapy (CAR-T) administered as salvage therapy beyond the second treatment line is poor. However, a minority of patients become long-term survivors despite CAR-T failure. The German Lymphoma Alliance (GLA) has proposed a hierarchical management algorithm for CAR-T failure in LBCL, aimed at allogeneic hematopoietic cell transplantation (alloHCT) as definite therapy in eligible patients. The purpose of this study was to investigate characteristics, relapse patterns, and management strategies in long-term survivors after CAR-T failure, with a particular focus on the feasibility and outcome of alloHCT. This was a retrospective analysis of all evaluable patients with a relapse/progression event (REL) observed in a previously reported GLA sample between November 2018 and May 2021. REL occurred in 214 of 356 patients (60%) who underwent CAR-T for LBCL in the previous GLA study. An evaluable dataset was available for 143 of these 214 patients (67%). Twenty-six of 143 patients (18%) survived 12 months or longer from REL, 109 (76%) died within the first year after REL, and 8 (6%) were alive but had not reached the 12-month landmark. Long-term survivors had more favorable pre-CAR-T features, had a longer interval between CAR-T and REL, and had more often received a tumor biopsy after CAR-T failure, whereas the choice of the first salvage regimen had no impact. AlloHCT was feasible in 40 of 53 patients (75%) intended and resulted in a 12-month post-transplantation overall survival of 36% in those patients who underwent transplantation with sensitive or untreated REL. AlloHCT after CAR-T failure in LBCL is feasible and may be an important contributor to long-term survival, although selection bias must be taken into account. Thus, alloHCT should be considered as a reasonable treatment option for eligible patients in this setting. However, because the overall outlook after CAR-T failure remains poor, novel effective therapeutic approaches are needed, either to allow long-term disease control per se or to improve the preconditions for successful alloHCT.

Ponatinib may overcome resistance of FLT3-ITD harbouring additional point mutations, notably the previously refractory F691I mutation.

Fms-like tyrosine kinase (FLT3) mutations are the most frequent mutations in patients with acute myeloid leukaemia (AML) that confer a poor prognosis. Constitutively active FLT3-ITD (internal tandem duplications) mutations define a promising target for therapeutic approaches using small molecule inhibitors. However, several point mutations of the FLT3 tyrosine kinase domain (FLT3-TKD) have been identified to mediate resistance towards FLT3 tyrosine kinase inhibitors (FLT3-TKI), including secondary mutations of FLT3. We investigated the cellular effects of the recently characterised FLT3-TKI ponatinib (AP24534) on murine myeloid cells transfected with FLT3-ITD with or without additional point mutations of the FLT3-TKD including the (so far) multi-resistant F691I mutation. Ponatinib effectively induced apoptosis not only in the parental FLT3-ITD cell line but also in all stably transfected subclones harbouring additional FLT3-TKD point mutations (N676D, F691I or G697R). These observations correlated with a strong inhibition of FLT3-ITD and its downstream targets STAT5, AKT and ERK1/2 upon ponatinib incubation, as determined by Western blotting. We conclude that ponatinib represents a promising FLT3-TKI that should be further investigated in clinical trials. The targeted therapy of FLT3-ITD-positive AML with ponatinib might be associated with a lower frequency of secondary resistance caused by acquired FLT3-TKD mutations.

Clinical experience with venetoclax in patients with newly diagnosed, relapsed, or refractory acute myeloid leukemia.

BACKGROUND: Diagnosis of acute myeloid leukemia (AML) is associated with poor outcome in elderly and unfit patients. Recently, approval of the BCL-2 inhibitor venetoclax (VEN) in combination with hypo-methylating agents (HMA) led to a significant improvement of response rates and survival. Further, application in the relapsed or refractory (r/r) AML setting or in context of allogeneic stem cell transplantation (alloHSCT) seems feasible. METHODS AND PATIENTS: Fifty-six consecutive adult AML patients on VEN from January 2019 to June 2021 were analyzed retrospectively. Patients received VEN either as first-line treatment, as subsequent therapy (r/r AML excluding prior alloHSCT), or at relapse after alloHSCT. VEN was administered orally in 28-day cycles either combined with HMA or low-dose cytarabine (LDAC). RESULTS: After a median follow-up of 11.5 (range 6.1-22.3) months, median overall survival (OS) from start of VEN treatment was 13.3 (2.2-20.5) months, 5.0 (0.8-24.3) months and 4.0 (1.5-22.1) months for first-line, subsequent line treatment and at relapse post-alloHSCT, respectively. Median OS was 11.5 (10-22.3) months from start of VEN when subsequent alloHSCT was carried out. Relapse-free survival (RFS) for the total cohort was 10.2 (2.2 - 24.3) months. Overall response rate (composite complete remission + partial remission) was 51.8% for the total cohort (61.1% for VEN first-line treatment, 52.2% for subsequent line and 42.8% at relapse post-alloHSCT). Subgroup analysis revealed a significantly reduced median OS in FLT3-ITD mutated AML with 3.4 (1.9-4.9) months versus 10.4 (0.8-24.3) months for non-mutated cases, (HR 4.45, 95% CI 0.89-22.13, p = 0.0002). Patients harboring NPM1 or IDH1/2 mutations lacking co-occurrence of FLT3-ITD showed a survival advantage over patients without those mutations (11.2 (5-24.3) months versus 5.0 (0.8-22.1) months, respectively, (HR 0.53, 95% CI 0.23 - 1.21, p = 0.131). Multivariate analysis revealed mutated NPM1 as a significant prognostic variable for achieving complete remission (CR) (HR 19.14, 95% CI 2.30 - 436.2, p < 0.05). The most common adverse events were hematological, with grade 3 and 4 neutropenia and thrombocytopenia reported in 44.6% and 14.5% of patients, respectively. CONCLUSION: Detailed analyses on efficacy for common clinical scenarios, such as first-line treatment, subsequent therapy (r/r AML), and application prior to and post-alloHSCT, are presented. The findings suggest VEN treatment combinations efficacious not only in first-line setting but also in r/r AML. Furthermore, VEN might play a role in a subgroup of patients with failure to conventional chemotherapy as a salvage regimen aiming for potential curative alloHSCT.