Trabectedin is standard for r/r soft tissue sarcomas. tTF-NGR accumulates in tumor vasculature leading to tumor vascular occlusion and tumor infarction. Both compounds in sequence could trap trabectedin inside tumors and increase its efficacy, which then optimizes the pro-coagulatory activity of tTF-NGR. This report summarizes translational data and results of the safety run-in patient cohort of the TRABTRAP trial combining trabectedin plus tTF-NGR. A dose of trabectedin of 1.5 mg/m2 (24 h, day 1) combined with 1.0 mg/m2 of tTF-NGR (1 h, days 2 and 3, q day 22) represents the approx. Maximum tolerated dose (MTD) and with 0.5 mg/m2 tTF-NGR (days 2 and 3) the recommended starting dose for the randomized part of TRABTRAP. None of the 6 patients on 0.5 mg/m2 tTF-NGR had dose-limiting toxicity (DLT). Higher doses or additional days of application of tTF-NGR led to grade 3 DLT including early troponin T high sensitivity increase, a reversible non-ST-elevation myocardial infarction in one patient, and reversible thromboembolic events. Pharmacokinetics explain the difference of the MTD between the phase I study and in TRABTRAP. Experimental and clinical efficacy and tolerability of the combination between trabectedin and tTF-NGR supports the active randomized part of TRABTRAP.

Introduction and Aim. This overview summarizes the development of CD13-targeted tissue factor (tTF-NGR) from its original design to clinical phase 3 in oncology. Materials and Methods. A series of targeted tissue factors (TFs) were cloned and biotechnologically produced with various tumor endothelial cell and pericyte targets. They were tested in vitro and in vivo for pro-coagulatory activity, specific binding, tumor accumulation, vascular occlusion, inhibition of blood flow, therapeutic antitumor effects, toxicology in rodents and non-rodents, and in clinical studies. Results. The lead compound tTF-NGR showed strong in vitro and in vivo activity, factor X activation, inducing tumor infarction, tumor growth inhibition and regression. Its preclinical efficacy was independent of tumor histology (melanoma, lung, breast, sarcoma, glioblastoma). Combination with cytotoxics such as doxorubicin or trabectedin (T) yielded combinatorial effects. A phase 1 trial in end-stage cancer patients defined the Maximum Tolerated Dose (MTD) at 3 mg/m² IV daily for 5 days, q day 22. Dose-limiting toxicities (DLT, grade 3 CTCAE) at higher doses were observed as troponin T hs elevation (early myocardial hypoxia) and reversible thromboembolic events. No grade 4 or 5 events occurred. In a dose-finding cohort of 19 advanced soft-tissue sarcoma patients treated with T plus tTF-NGR, MTD was T 1.5 mg/m² over 24 h (day 1) plus tTF-NGR 1.0 mg/m² IV (days 2–3), q day 22. The recommended starting dose for phase 3 is tTF-NGR 0.5 mg/m² (days 2–3). At this dose, none of six patients had DLTs. Number of cycles per patient (RECIST result) were: 2 (PD, progressive disease), 3 (PD), 6 (PD), 12 (SD, stable disease), 14 (PR, partial response), 14 (SD). All SD patients had tumor shrinkage. Higher tTF-NGR doses or extended schedules led to grade 3 DLTs (troponin T hs rise, N-STEMI, thromboembolic events), all reversible upon anticoagulation. Again, no grade 4 or 5 toxicities were seen. Pharmacokinetics show more and longer tTF-NGR in the plasma after T which explains MTD differences between monotherapy and combination therapy. Among 11 L-sarcoma patients completing at least one cycle, the Disease Control Rate (PR plus SD) was 63.6%. Conclusions. CD13-targeted tTF-NGR demonstrates strong preclinical antitumor efficacy and a manageable safety profile with clinical anti-sarcoma activity when combined with T. A randomized phase 3 trial (1:1, T vs T plus tTF-NGR) is actively recruiting patients.

Abstract The efficient and safe transfection of hematopoietic cells is a major hurdle that limits efficacy of therapeutic approaches like RNA-interference. We therefore used our modular EL ectrostatic A ntibody si R NA T argeted therapy platform (ELART) to develop nanocarriers decorated with antibodies for delivery of siRNA into hematopoietic CD20-, CD22-, or CD33-positive cells. To analyze internalization efficacy into tumor cells, we applied nanocarriers loaded with Cy3- or Cy5-labeled siRNA and reached nearly all target cells within 1–4 h. Exposure to Cy5-labeled non-functional siRNAs resulted in reduction of the mitochondrial membrane potential and reduced viability, as detected in tetramethylrhodamine methyl ester (TMRM) and CellTiter-Glo (CTG) assays. We concluded that with our modular nanocarrier system, we can transport cytotoxic agents such as cyanine dyes when bound to siRNA, as ELART nanocarriers safely complex anionic siRNA electrostatically and releases it intracellularly within the target cell. This proof-of-concept study shows that ELART nanocarriers can transport anti-cancer agents utilizing siRNA as carrier substance.

Acute myeloid leukemia (AML) is a complex disease characterized by diverse molecular pathogenesis. Genetic alterations, including germline and somatic variants in the DEAD box helicase 41 gene ( DDX41) located on chromosome 5 play an increasingly recognized role. Recent reports indicate that 5% of intensively treated adult AML patients harbor DDX41 germline mutations ( DDX41MutGL), and their precise impact remains incompletely understood. These studies suggest that DDX41MutGL may define a distinct biological subgroup, associated with e.g. older age, male gender, low blast, and low white blood cell count (WBC). To further elucidate the role of DDX41 in AML, we performed a retrospective analysis of 906 unselected adult AML patients from the AML Cooperative Group (AMLCG) registry (2015-2022), by targeted sequencing. As DDX41MutGL are typically “null mutations”, leading to reduced DDX41 expression, we additionally investigated whether DDX41 gene expression correlates or resembles the observed germline phenotype. Our analysis encompassed >1000 independent gene expression profiles (GSE37642, GSE14468, and GSE106291) of intensively treated adult AML patients. We identified a sub-cohort with low DDX41 ( DDX41-low) expression and re-analyzed them with next-generation sequencing to detect DDX41MutGL. Additionally, we correlated gene expression data with 198 DNA methylation profiles (from patients who had undergone both analyses) to identify potential epigenetic mechanisms underlying DDX41-low expression. Among the 906 patients (median age 61 years; range 18-98 years), we identified 11 unrelated individuals with suspected DDX41MutGL (VAF > 40%). Notably, the overall frequency of DDX41MutGL in the German unselected AML patient population was merely 1%, considerably lower than previously reported (p<0.0001 in comparison to Duployez et al. 2022). Characteristically, DDX41MutGL patients in this cohort were mostly male (73%), with a median age of 67 years displaying low WBC (average 1,3G/l) and a normal karyotype (80%) at initial diagnosis. Remarkably, our analysis of independent large gene expression cohorts revealed a pattern of phenotypic association in patients with decreased DDX41 expression ( DDX41-low) closely resembling those with DDX41MutGL. These associations were consistent across different datasets and included e.g. older age, low WBC, and low blast count. Furthermore, DDX41-low patients had poor overall survival. The DDX41-low subgroup constituted almost 10% of all AML patients, surpassing the reported frequencies of DDX41MutGL. In an exploratory pilot study, we screened 48 patients exhibiting the lowest DDX41 gene expression by sequencing the coding regions for DDX41 mutations but found none, suggesting alternative mechanisms, such as copy number changes, non-coding alterations or aberrant DNA methylation patterns that may replicate the phenotypic effects associated with DDX41MutGL. To further elucidate this mechanism, we correlated the gene expression of DDX41 with all DNA methylation loci (CpG sites covered by the EPIC array) on chromosome 5 using Spearman correlation analysis (GSE106291). We plotted the correlations against the sorted chromosome length, identifying a peak at a specific location corresponding to the PCDH (Protocadherin) cluster (Figure). Thus, DDX41-low was associated with reduced methylation at the PCDH loci and linked to higher PCDH gene expression. Across several data sets, we confirmed the association between higher PCDH cluster gene expression and low DDX41 expression. In summary, our study reveals that the frequency of DDX41MutGL in an unselected population of German AML patients is considerably lower than previously reported. Additionally, we found that apart from DDX41MutGL, DDX41-low exhibits a comparable clinical profile and is linked to specific methylation and gene expression patterns, with a notable emphasis on the PCDH complex located on chromosome 5. Understanding the functional implications of PCDH genes and their interaction with DDX41 in AML may significantly advance our knowledge of AML pathogenesis. Gaining insights into DDX41 alterations may open avenues for personalized therapeutic approaches and further prognostic stratification for AML patients with distinct molecular characteristics.

ABSTRACT:Attempting to induce a complete remission before allogeneic hematopoietic cell transplant (alloHCT) is current practice in patients with acute myeloid leukemia (AML). However, benefit of remission induction strategy (RIST) before alloHCT has never been proven in a prospective trial. Potent conditioning regimens exist that allow for successful alloHCT in patients with active AML. Therefore, the ASAP trial was conducted to test RIST by salvage chemotherapy before alloHCT against immediate transplant after intensified conditioning. In total, 281 patients with AML with poor response after first induction or untreated first relapse were randomized 1:1 to RIST with high-dose cytarabine plus mitoxantrone vs immediate alloHCT with sequential conditioning after nonintensive disease control (DisC) measures, preferentially watchful waiting only. Overall survival at 5 years from randomization analyzed according to intention-to-treat was 46.1% for DisC vs 47.5% for RIST (P = .82). In multivariable Cox regression analysis, genetic AML risk according to European LeukemiaNet criteria (P < .0001), age (P = .001), and comorbidities (P = .046) predicted survival, but not treatment arm (hazard ratio, 1.08 for DisC vs RIST; P = .67). In conclusion, long-term follow-up of the ASAP trial showed no survival advantage for standard salvage chemotherapy before alloHCT as opposed to immediate alloHCT. The trial results question the general concept of RIST with intensive standard salvage therapy before alloHCT for all patients, because immediate alloHCT may reduce time in hospital and health care expenses. Novel bridging therapies that are well tolerated, and posttransplant maintenance with targeted drugs are urgently warranted, especially for adverse-risk AML, to improve outcomes after alloHCT. This trial was registered at www.ClinicalTrials.gov as #NCT02461537.

We designed and synthesised an anionic small-molecular photosensitizer (aPSM-Cy3.5) for incorporation into electrostatic antibody targeted (ELART) vesicles, consisting of a protamine-coupled antibody as targeting unit, free protamine and aPSM-Cy3.5. These nanocarriers specifically internalize into different solid tumour cell lines. Upon illumination, the aPSM component initiates the production of reactive oxygen species (ROS). Tumour cells from lung, colorectal and pancreatic cancer with internalized aPSM show decreased growth in colony forming assay. This supports the development of systemically applicable anionic ROS inducers capable of specifically targeting tumour cells.

Abstract Introduction The randomized-controlled SAL DaunoDouble trial (NCT02140242) showed no significant differences in complete remission (CR) rates or survival between 60 mg/m² and 90 mg/m² of daunorubicin (Dauno), or between single and double induction in patients (pts) with newly diagnosed AML eligible for intensive chemotherapy (Röllig et al., JCO, 2024). To further study the impact of Dauno dose-escalation and the number of induction cycles in more detail, and to evaluate possible implications for post-remission therapy, we aimed to retrospectively analyze NPM1 measurable residual disease (MRD) kinetics in this study population. Methods Pts treated within DaunoDouble were retrospectively screened for data on NPM1 MRD. MRD was assessed within standard of care on bone marrow (BM) samples using a quantitative reverse transcription polymerase chain reaction (RT-qPCR) assay at the central reference laboratory. The assay achieves a validated sensitivity of 0.001%; pts were analyzed based on a threshold of 0.1% NPM1/ABL1. Time-to-event variables were estimated using the Kaplan-Meier method. Statistical analyses were performed using R v4.5.0. Results Data on post-induction MRD were available for 79 pts with NPM1-mutated AML who achieved a CR/CRi. The median age of this cohort was 51 years (range, 24-64), 49 pts (62%) were female. Most pts (97%) had de-novo AML. According to the European LeukemiaNet (ELN) 2017 risk classification, 78%, 22%, and 0% of pts had favorable, intermediate, and adverse risk disease, respectively. A FLT3-ITD co-mutation was observed in 31% of pts. Eighteen pts (23%) underwent allogeneic hematopoietic cell transplantation (alloHCT) in CR1. The median post-induction NPM1 load showed no significant differences after induction with Dauno 60 mg/m2 versus 90 mg/m2 (0.6% vs. 0.3%, p=0.601). Likewise, rates of MRD-negative remission did not significantly differ between pts receiving Dauno 60 mg/m2 and those receiving 90 mg/m2 (27% vs. 30%, p=0.804). In contrast, when comparing one versus two induction cycles, we detected a significantly lower post-induction MRD load in pts receiving double induction (0.1% vs. 1.5%, p<0.001). Pts receiving two cycles were significantly more likely to attain MRD-negative remissions (40% vs. 15%, p=0.015). As per March 22nd 2025, after a median follow-up of 43.7 months, the median overall survival (OS) of the entire cohort was not reached, with a corresponding 2-year OS rate of 86%. The 2-year relapse-free survival (RFS) rate of the entire cohort was 59%. When analyzing the entire cohort irrespective of post-remission treatment, the RFS in pts achieving MRD-negative CR was numerically longer when compared to pts being MRD-positive (median RFS (mRFS), NR vs. 42.5 months, 2-year RFS rate, 77% vs. 52%, p=0.084). When focusing on pts with cytarabine-based consolidation in CR1, the RFS benefit in MRD-negative pts was more pronounced and significantly longer than in MRD-positive pts (median RFS, NR vs. 15.3 months, 2-year RFS rate, 78% vs. 39%, p=0.014). With respect to the benefit of alloHCT in CR1, RFS was relevantly longer in MRD-positive pts undergoing alloHCT compared to chemotherapy consolidation (2-year RFS rate 83% vs. 41%, p=0.059). In contrast, alloHCT in CR1 did not improve RFS in pts considered MRD-negative post-induction (2-year RFS rate 69% vs. 67%, p=0.597). In terms of OS, we did not observe any relevant differences in pts with positive versus negative NPM1 MRD, regardless of post-remission therapy. Conclusions Dauno dose escalation from 60 to 90 mg/m2 in first induction did not improve depth of response in terms of NPM1 MRD levels. However, pts with double induction were more likely to achieve MRD-negative remissions and attained a higher reduction in their MRD load. Pts achieving MRD-negative remissions showed improved RFS which was statistically significant in pts not undergoing alloHCT in CR1. Regarding RFS, MRD-positive pts seemed to benefit from alloHCT in CR1, whereas MRD negative pts did not. Post-induction MRD status was not predictive of OS, most likely due to retained chemosensitivity and high efficacy of salvage alloHCT in pts with relapsed NPM1-mutated AML. Our results confirm that 60 mg/m² of Dauno is equipotent to 90 mg/m² on a molecular level, and sufficient for induction therapy. In addition, we demonstrate the predictive value of NPM1 MRD on relapse risk, as well as the value of alloHCT in pts with an insufficient response to induction.

Trabectedin is a treatment option for relapsed/refractory soft tissue sarcomas (STS). CD13 is a neutral aminopeptidase expressed on invasive endothelial cells (EC) such as in the tumor vasculature. CD13-targeted tissue factor (tTF-NGR) is a recombinant pro-coagulatory fusion protein with a molecular weight of 30381.98 g/mol which accumulates in the tumor vasculature leading to tumor vascular occlusion and tumor infarction. Giving both compounds in sequence could trap trabectedin inside tumors and increase its efficacy. Vice versa trabectedin optimizes activity of tTF-NGR by externalizing phosphatidylserine (PS) on the surface of EC. PK of safety patients treated at our hospital within a multicenter trial of a combination of trabectedin and tTF-NGR in advanced STS patients refractory to 1st line systemic therapy (TRABTRAP, EudraCT 2020-005858-21) were compared with PK from our phase I monotherapy study with tTF-NGR (EudraCT 2016-003042-85). We compared standard PK parameters for tTF-NGR at identical dose levels (1.0 to 3.0 mg/m2) from TRABTRAP and phase I within the same laboratory with identical methods and calculations. Although some PK differences between phase I and TRABTRAP were more pronounced at lower dose levels, global values such as Cmax, AUC, t1/2alpha, t1/2term, and Kel indicated an increased AUC with a delayed elimination of tTF-NGR when given after trabectedin. As tTF-NGR was always given daily, we measured the remaining tTF-NGR concentrations before subsequent applications and found significantly increased values in the combination protocol (1 mg/m2: p<0.0001; 3 mg/m2: p<0.0002) when compared with the phase I values, suggesting a possible accumulation of tTF-NGR which we did not observe with monotherapy. A specific way of tTF-NGR elimination in the vascular system is the internalization of the tTF-NGR:CD13 complex into EC. Using flow cytometry and fluorescence labeling with tTF-NGR and EC in vitro, expression of CD13 on EC was slightly diminished by trabectedin exposure. Also, tTF-NGR binding to CD13 (p<0.01) and internalization (p=0.01) decreased when tested on control and trabectedin-exposed EC. Both lead to more tTF-NGR molecules remaining in the circulation, explaining part of the delayed elimination. Further, we compared functionality of tTF-NGR in the plasma of patients from phase I and TRABTRAP. On the basis of equal protein amounts and without influence of confounders such as storage, the ability of factor VIIa:tTF-NGR:CD13 to activate factor X to Xa was higher when trabectedin was given to the patients before. Among possible explanations are the decreased liver protease levels after trabectedin, which could influence the functionality of tTF-NGR. In summary, these results explain why the Maximum Tolerated Dose level for tTF-NGR established in the TRABTRAP safety cohort is lower than the 3 mg/m2 in the phase I study. Christian Schwöppe, Caroline Brand, Kathrin Hessling, Heike Hintelmann, Andrew F. Berdel, Georg Lenz, Torsten Kessler, Manfred Fobker, Christoph Schliemann, Wolfgang E. Berdel. Comparative pharmacokinetics (PK) of targeted tissue factor tTF-NGR when intravenously applied alone or in combination with preceding trabectedin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 824.

Given the selection of elderly patients with AML in first complete remission (CR1) the advantage of consolidation with allogeneic hematopoietic cell transplantation (HCT) over chemotherapy is still unclear. Newly diagnosed AML patients in CR1 aged 60-75 years were registered and a donor search initiated. After one consolidation cycle, patients with a matched donor were randomized to HCT with fludarabine/low-dose total body irradiation and cyclosporine/mycophenolate mofetil immunosuppression or conventional non-HCT. Primary outcome was restricted mean leukemia-free survival (RM-LFS) up to 5 years. Between 2010 and 2017, 245 patients (median age 67 years) were registered at CR1. After one consolidation, 26.9% of patients failed inclusion criteria. Of the 179 (73%) patients still on study, 75.4% had an HLA identical donor. Ten ineligible patients were excluded, and 125 randomized to HCT (N=83) or non-HCT (N=42). The primary outcome RM-LFS up to 5 years was 24.5 months (95% confidence interval [CI]: 18.9-30.1) in the HCT and 15.6 months (95% CI: 10.4-20.8) in the non-HCT arm (P=0.022) due to a decrease in cumulative relapse incidence from 91.1% (95% CI: 80.7-100.0) after non-HCT to 37.8% (95% CI: 27.2-48.4) after HCT (P<0.0001). The secondary endpoints RM-OS up to 5 years was 27.8 months (95% CI:22.3-33.2) in the HCT as compared to 28.6 months (95% CI: 22.2-35.0) in the non-HCT arm; non-relapse mortality at 5 years was 33.4% (95% CI: 23.0-43.9) with HCT and 0% without. In older patients with AML in CR1 5-year RM-LFS is better with HCT than with non-HCT consolidation treatment. The long-term RM-LFS benefit did not translate into a better RM-OS during the study period.

We performed a questionnaire-based cross-sectional study to analyze Acute Myeloid Leukemia (AML) long-term survivor (LTS) outcomes, including psychosocial well-being and somatic health status. Four-hundred-twenty-seven former AML patients participated (response rate, 63%) ≥5 years[y] and up to 18.6 y past their leukemia diagnosis (median, 11.3 y). Median age at study participation was 61 y (range 28y–93y), 23% had experienced disease relapse, and 63% had received allogeneic hematopoietic stem cell transplantation (alloHSCT). Overall, quality of life (QoL) and general life satisfaction (gLS) summary scores were higher in AML LTS (p < 0.001) compared to age-/sex-matched reference cohorts, although differences were small and likely not clinically relevant. However, we identified subgroups of survivors reporting impaired QoL (27%), gLS (13%) and health-related life satisfaction (hrLS; 17%). Using multivariable regression models, we identified predisposing and protective factors for each of these outcomes. Treatment with alloHSCT did not adversely impact QoL, gLS, or hrLS. In summary, global QoL and LS in AML LTS are comparable to the general population, irrespective of treatment modality, although some survivors report clinically significant impairment of global QoL and/or in specific domains. We identified factors associated with impaired outcomes (e.g., comorbidity and fatigue), delineating a subgroup of survivors with unmet needs ≥5 y after their AML diagnosis.

The outcome of patients with acute myeloid leukemia (AML) worsens with increasing age. Dichotomization into “younger” and “older” patients is clinically routine and often dictates treatment options. We aimed to delineate whether molecular genetic features and/or outcome measures support assorting patient populations by age, including division into “younger” and “older” groups. We analyzed 2823 adult AML patients enrolled onto frontline chemotherapy-based clinical protocols of two cooperative study groups from USA and Germany who were profiled molecularly via targeted sequencing platforms. Frequencies of gene mutations and cytogenetic findings were depicted in 5-year age increments. Clinical outcomes of 2756 AML patients were analyzed with respect to molecular features, genetic-risk groups and age. Age-associated distributions of gene mutations and cytogenetic abnormalities were similar in both cohorts. There was almost linear shortening of overall survival with increasing age among all patients (P < 0.001) and within 2022 European LeukemiaNet-defined genetic-risk groups, with survival decreasing as age increased (favorable-risk, P < 0.001; intermediate-risk, P < 0.001; adverse-risk, P < 0.001). Although mutational profiles and outcomes of the youngest patients differed from those of older patients, there was no age cut-off identifying “younger” and “older” patients. These findings support more age-associated flexibility for drug approval and trial eligibility.

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with demographic and genetic features. Although the associations of acquired genetic alterations with patients’ sex have been recently analyzed, their impact on outcome of female and male patients has not yet been comprehensively assessed. We performed mutational profiling, cytogenetic and outcome analyses in 1726 adults with AML (749 female and 977 male) treated on frontline Alliance for Clinical Trials in Oncology protocols. A validation cohort comprised 465 women and 489 men treated on frontline protocols of the German AML Cooperative Group. Compared with men, women more often had normal karyotype, FLT3 -ITD, DNMT3A , NPM1 and WT1 mutations and less often complex karyotype, ASXL1 , SRSF2 , U2AF1 , RUNX1 , or KIT mutations. More women were in the 2022 European LeukemiaNet intermediate-risk group and more men in adverse-risk group. We found sex differences in co-occurring mutation patterns and prognostic impact of select genetic alterations. The mutation-associated splicing events and gene-expression profiles also differed between sexes. In patients aged <60 years, SF3B1 mutations were male-specific adverse outcome prognosticators. We conclude that sex differences in AML-associated genetic alterations and mutation-specific differential splicing events highlight the importance of patients’ sex in analyses of AML biology and prognostication.

Assignment of patients diagnosed with acute myeloid leukemia (AML) to the 2022 European LeukemiaNet (ELN) favorable genetic risk group has important clinical implications, as allogeneic stem cell transplantation in first complete remission (CR) is not advised due to a relatively good outcome of patients receiving chemotherapy alone and transplant-associated mortality. However, not all favorable genetic risk patients experience long-term relapse-free survival (RFS), making recognition of patients who would most likely be cured of high importance. We analyzed 297 patients aged <60 years with de novo AML classified as 2022 ELN favorable genetic risk who achieved a CR and had RNA sequencing (RNA-seq) and gene mutation data from diagnostic samples available (Alliance trial A152010). To identify prognostically relevant transcripts that can distinguish patients cured from patients susceptible to lower or higher risk of relapse or death, we fit a regularized mixture cure model (MCM) where RNA-seq expression values were our candidate covariates. To validate the identified transcripts, we analyzed 75 patients with de novo AML aged <60 years included in the 2022 ELN favorable genetic risk group who achieved a CR in an independent test set from Gene Expression Omnibus (GSE37642). Our MCM identified 145 transcripts associated with cure or long-term RFS and 149 transcripts associated with latency or shorter-term time to relapse. The area under the curve and C-statistic were, respectively, 0.946 and 0.856 for our training set and 0.877 and 0.857 for our test set. Our results suggest that the favorable risk group includes distinct transcriptionally defined subgroups with different biological properties, which may be useful for refining this genetic risk category.

Our understanding of tumour biology has evolved over the past decades and cancer is now viewed as a complex ecosystem with interactions between various cellular and non-cellular components within the tumour microenvironment (TME) at multiple scales. However, morphological imaging remains the mainstay of tumour staging and assessment of response to therapy, and the characterization of the TME with non-invasive imaging has not yet entered routine clinical practice. By combining multiple MRI sequences, each providing different but complementary information about the TME, multiparametric MRI (mpMRI) enables non-invasive assessment of molecular and cellular features within the TME, including their spatial and temporal heterogeneity. With an increasing number of advanced MRI techniques bridging the gap between preclinical and clinical applications, mpMRI could ultimately guide the selection of treatment approaches, precisely tailored to each individual patient, tumour and therapeutic modality. In this Review, we describe the evolving role of mpMRI in the non-invasive characterization of the TME, outline its applications for cancer detection, staging and assessment of response to therapy, and discuss considerations and challenges for its use in future medical applications, including personalized integrated diagnostics.

Despite recent refinements in the diagnostic and prognostic assessment of CEBPA mutations in AML, several questions remain open, i.e. implications of different types of basic region leucin zipper (bZIP) mutations, the role of co-mutations and the allelic state. Using pooled primary data analysis on 1010 CEBPA-mutant adult AML patients, a comparison was performed taking into account the type of mutation (bZIP: either typical in-frame insertion/deletion (InDel) mutations (bZIPInDel), frameshift InDel or nonsense mutations inducing translational stop (bZIPSTOP) or single base-pair missense alterations (bZIPms), and transcription activation domain (TAD) mutations) and the allelic state (single (smCEBPA) vs. double mutant (dmCEBPA)). Only bZIPInDel patients had significantly higher rates of complete remission and longer relapse free and overall survival (OS) compared with all other CEBPA-mutant subgroups. Moreover, co-mutations in bZIPInDel patients (e.g. GATA2, FLT3, WT1 as well as ELN2022 adverse risk aberrations) had no independent impact on OS, whereas in non-bZIPInDel patients, grouping according to ELN2022 recommendations added significant prognostic information. In conclusion, these results demonstrate bZIPInDel mutations to be the major independent determinant of outcome in CEBPA-mutant AML, thereby refining current classifications according to WHO (including all dmCEBPA and smCEBPA bZIP) as well as ELN2022 and ICC recommendations (including CEBPA bZIPms).

Introduction: The ELN classification of AML incorporates cytogenetic and mutational risk profiles of AML to define separate favorable, intermediate and adverse risk groups. Recommendations for post remission treatment (PRT) intensity, most importantly allogeneic stem cell transplantation (alloSCT), are based on these risk groups. The ELN classification is regularly updated as the understanding of genetic risk factors for AML prognosis improves. In-depth genetically defined cohorts from registries or clinical trials, often reported as retrospective real-world validation cohorts, contain an inherent bias as pts were not treated according to treatment recommendations corresponding to the time interval of the respective ELN classification. Methods: 662 AML pts who received intensive induction therapy at the University Hospital Muenster between 2010 and 2022 were included in this retrospective analysis. Pts were generally classified according to ELN 2010 recommendations if treated between 2010-2016 (ELN10), and ELN 2017 recommendations if treated between 2017-2022 (ELN17). Survival was estimated according to Kaplan-Meier. Cumulative incidences of relapse (CIR) and non-relapse mortality (NRM) were calculated using Gray's method. To eliminate treatment bias, we retrospectively reclassified the risk of the ELN17 group according to ELN 2010 (ELN10re) and compared outcomes with the actual ELN10 cohort in identical risk groups. Results: Baseline characteristics of pts were evenly distributed between ELN10 (n=373) and ELN17 (n=289), except for higher proportions of therapy-related and intermediate risk AML after 2016. Median follow-up was 61 months, with 90 months for ELN10 and 42 months for ELN17 cohorts, respectively. Overall survival (OS), event-free survival (EFS) and relapse-free survival (RFS) significantly improved for ELN17 pts compared to ELN10 (4-year OS 60% vs. 40%, HR 0.59, 95% CI 0.5-0.7, p<.0001; 4-year EFS 37% vs. 21%, HR 0.68, 95% CI 0.6-0.8, p<.0001; 4-year RFS 54% vs. 35%, HR 0.61, 95% CI 0.5-0.8, p=.00026). ELN17 pts achieving a first complete remission (CR1) had a significantly reduced CIR with similar NRM compared to ELN10 (CIR, HR 0.57, CI 0.4-0.8, p<.001; NRM, HR 0.95, CI 0.6-1.6, p=.86). Generally, alloSCT as PRT was recommended for pts with ELN10 intermediate risk (HLA match-related donor) and adverse risk (HLA match-related or unrelated donor) AML. With the implementation of the ELN 2017 classification, alloSCT as PRT was recommended for pts with intermediate and adverse risk irrespective of donor relation. Comparing time intervals, ELN17 pts receiving an alloSCT either in CR1 (n=211), in primary refractory situation after at least one induction cycle (n=124) or following relapse (n=100), showed better OS and RFS (4-year OSallo 66% vs. 50%, HR 0.62, 95% CI 0.5-0.8, p=.0016; 4-year RFSallo 59% vs. 45%, HR 0.68, 95% CI 0.5-0.9, p=.0056) and lower NRM rates (HR 0.63, CI 0.4-0.9, p=.02), while CIR was unchanged (HR 0.78, CI 0.5-1.1, p=.2). To eliminate treatment bias, retrospectively reclassified ELN10re pts of the ELN17 group were compared to regularly classified ELN10 pts. Risk distribution according to ELN 2010 criteria was comparable between time intervals. The majority of ELN17 pts were retrospectively reclassified from favorable (23 pts /8% of cohort) or adverse (58 pts / 20%) to ELN10re intermediate risk, while no patient was reclassified to ELN10re adverse risk. ELN10re intermediate risk pts, treated between 2017-2022 benefited most in OS, EFS and RFS (OS, HR 0.5, 95% CI 0.4-0.7, p<.0001; EFS, HR 0.58, 95% CI 0.5-0.8, p<.0001; RFS, HR 0.47, 95% CI 0.3-0.7, p<.0001). ELN10re favorable and adverse group comparisons showed no significant difference in outcome over time intervals. Conclusion: Based on the analysis of more than 600 AML pts treated at our center from 2010-2022, we conclude that since 2017, besides approval of novel substances for conventional induction and consolidation therapies, the update of the ELN classification - moving more pts to intermediate and adverse risk groups with subsequent intensification of PRT through a higher proportion of pts proceeding to alloSCT - significantly improved the survival of AML pts. This improvement is particularly pronounced for intermediate risk pts according to ELN 2010 criteria. To the best of our knowledge, this is the first “as treated” real-world validation for the ELN 2017 update reported.

Secondary-type mutations (STM) are defined as alterations in ASXL1, BCOR, EZH2, SF3B1, SRSF2, STAG2, U2AF1, and ZRSR2 in the recent update of the WHO classification on myeloid neoplasms. The International Consensus Classification (ICC) extends its definition to also include alterations of RUNX1. Patients bearing STMs are considered adverse risk in the absence of other class-defining markers according to the recent update of the European LeukemiaNet (ELN) 2022 recommendations. However, it is unclear if all STMs convey an equally adverse prognostic effect or if individual STMs have a differential prognostic impact. We pooled data on molecular genetics, cytogenetics, and outcomes of 5311 newly diagnosed and intensively treated AML patients from previous randomized multicenter trials of the German SAL (n=1606), the German-Austrian AMLSG (n=1354, dataset from Gerstung et al., Nature Genetics 2017), the German AMLCG (n=1138), the French DATAML (n=1040), and the Czech CELL (n=173). Patients were retrospectively categorized into ELN2022. All analyses were carried out for WHO 2022 definitions (STMWHO) and ICC 2022 definitions (STMICC). We found 1485 (28%) of patients to harbor STMWHO and 1698 patients (32.0%) with STMICC. Patients with STMWHO and STMICC were significantly older than non-STM patients (median 60 vs 53 and 59 vs 52 years, p<0.001) and more frequently male (STMWHO: 62.5% and STMICC: 61.1% male vs 48.3% and 48.1%, p<0.001). STM-bearing patients had significantly higher rates of secondary AML (STMWHO: 19.8% and STMICC: 19.2% vs 6.9% and 6.3%) while de novo AML rates were significantly lower (STMWHO: 75.5% and STMICC: 75.8% vs 87.7% and 88.3%) compared to non-STM patients. Patients with STMs presented with significantly lower white blood cell count as well as significantly lower bone marrow and peripheral blood blast counts. After intensive induction therapy, 65.1% or 65.7% of patients with STMWHO or STMICC achieved complete remission (CR) compared to 77.2% or 77.7% of patients without STM according to either definition (OR 0.55 and p<0.001 for both). To test whether STMs constitute an individual risk group aside from ELN2022 adverse, these groups were separated. Patients with STMs and co-occurring defining favorable or intermediate risk features were assigned to ELN2022 favorable or intermediate risk, respectively. Patients with STM and no other class-defining alterations had longer event-free survival (EFS; STMWHO: 4.7 months, hazard ratio [HR] 1.66, p<0.001; STMICC: 4.9 months. HR 1.69, p<0.001) than ELN2022 adverse risk patients (3.0 months, HR 2.02, p<0.001, and 2.7 months, HR 2.12, p<0.001, respectively), longer relapse-free survival (RFS; STMWHO: 12.6 months, HR 1.53, p<0.001, STMICC: 11.9 months, HR 1.60, p<0.001 vs 8.0 months, HR 1.91, p<0.001 and 7.4 months, HR 1.93, p<0.01, respectively), and longer overall survival (OS; STMWHO: 14.6 months, HR 1.64, p<0.01 and STMICC: 14.7 months, HR 1.68, p<0.01 vs 9.5 months, HR 2.09, p<0.001 and 8.3 months, HR 2.22, p<0.001, respectively). The odds of achieving CR were significantly decreased for alterations in ASXL1, BCOR, SF3B1, SRSF2, STAG2, U2AF1, or RUNX1 compared to wildtype (univariable OR range: 0.38-0.68; BCOR p=0.003; all other p<0.001), with no significant differences for EZH2 (OR: 0.87, p=0.387) or ZRSR2 (OR: 0.96, p=0.882). Median EFS was significantly reduced for patients harboring alterations in ASXL1, BCOR, SF3B1, SRSF2, STAG2, U2AF1, or RUNX1 (HR range: 1.17-1.79; STAG2 p=0.017, all other p<0.001) while there was no difference for EZH2 (HR: 1.11, p=0.167) or ZRSR2 (HR: 1.17, p=0.232) compared to wildtype patients. Significantly reduced median RFS was found for patients with altered ASXL1, BCOR, SF3B1, SRSF2, U2AF1, and RUNX1 (HR range: 1.28-1.88, BCOR p=0.007, SF3B1 p=0.005, all other p<0.001) while EZH2, STAG2, and ZRSR2 showed no significant differences (HR range: 0.93-1.17). Finally, OS was significantly reduced for patients harboring alterations in ASXL1, BCOR, SF3B1, SRSF2, U2AF1, and RUNX1 (HR range: 1.26-1.87, BCOR p=0.002, all other p<0.001) again with no difference for alterations of EZH2, STAG2, and ZRSR2 (HR range: 1.16-1.28). Our analysis highlights that STMs represent an unfavorable risk level between the ELN2022 intermediate and adverse categories while contrasts between individual STMs exist, as not all alterations convey the same degree of adverse prognostic impact.

INTRODUCTION To attempt inducing a complete remission (CR) prior to allogeneic hematopoietic cell transplantation (alloHCT) in patients with active AML after first induction or untreated first relapse is considered as standard of care. This standard, however, has never been tested in a randomized controlled trial (RCT). With the availability of highly potent sequential conditioning regimens the value of CR induction prior to alloHCT is unclear for patients with poor responsive or relapsed AML. Against this background, the ASAP trial was conducted (NCT 02461537, Stelljes et al Lancet Hematology 2024). Here, we report the long-term follow-up (FU) and outcome according to genetic risk groups. METHODS To test salvage chemotherapy prior to alloHCT, patients aged between 18 and 75 years with AML and poor response after first induction or untreated first relapse and available HLA-compatible donor were randomized 1:1 to remission induction (RIST) with high-dose cytarabine plus mitoxantrone or immediate alloHCT with sequential conditioning after disease control (DisC). Disease control consisted preferentially of watchful waiting only. Overall survival (OS) from randomization according to the intention-to-treat (ITT) was the major secondary endpoint and is reported here with a median FU of 5 years. To improve molecular risk assessment available baseline samples were re-analyzed and classified according to the ELN 2022 risk classification. RESULTS 281 patients were enrolled between September 2015 and January 2022. Of 140 patients randomized to DisC 135 proceeded to alloHCT (96%) and of 141 patients randomized to RIST 128 patients (91%) were transplanted. Treatment success was documented for 82.9% (116/140) of patients in the DisC arm and 79.4% (112/141) of patients in the RIST arm, resulting in a difference for treatment success of +3.4% (95% CI, -5.8% to 12.6%) for DisC versus RIST. OS at 3 years from randomization analyzed according to ITT was 54.8% (95% CI, 46% to 63%) versus 54.5% (95% CI, 46% to 62%) (p=0.95) and at 5 years 46.1% (95% CI, 37% to 55%) versus 47.5% (95% CI, 39% to 56%) (p=0.8), for DisC versus RIST, respectively, indicating no benefit of remission induction prior to alloHCT. Among patients with treatment success, disease-free survival (DFS) was not statistically different by treatment arm (4-year DFS from treatment success, 48.3% (95% CI, 39% to 57%) for DisC versus 49.1% (39% to 58%) for RIST, p=0.9). In multivariable Cox regression models for OS from randomization, ELN risk and age significantly predicted survival, but not treatment arm. TP53-lesions were detected in 21 patients randomized to DisC and 9 patients randomized to RIST. Three-year OS since randomization in patients with adverse risk AML with TP53-lesions was 24% (95% CI, 9% to 34%) versus 33% (95% CI, 8% to 62%) (log-rank test, p=0.5) and with adverse risk AML without TP53-lesions 48% (95% CI, 33% to 62%) versus 45% (95% CI, 29% to 59%) (log-rank test, p=0.8) for DisC versus RIST, respectively. With intermediate risk AML 3-year OS was 60% (95% CI, 46% to 72%) compared to 63% (95% CI, 51% to 73%) for patients randomized to DisC or RIST (log-rank test, p=0.52), respectively. Patients with favorable risk AML (94% enrolled with untreated relapse) randomized to DisC had 3-year OS of 90% (95% CI, 64% to 97%) compared to 46% (95% CI, 18% to 70%) for patients randomized to RIST (log-rank test, p=0.01). CONCLUSIONS In summary, longer follow-up of the ASAP trial confirms our previous results and shows no survival advantage for remission induction prior to alloHCT as opposed to immediate alloHCT. This result questions the general concept of remission induction prior to alloHCT, since immediate alloHCT after sequential conditioning may reduce time in hospital and treatment exposure. More potent bridging concepts with targeted drugs prior to alloHCT are warranted especially for adverse risk AML and need to be tested in RCTs to demonstrate sustained survival advantage. Together with the profound impact of genetic risk on long-term survival the results may be interpreted as a sign that patients with poor responsive or relapsed AML benefit rather from relapse prevention after alloHCT than conventional CR induction prior to alloHCT.