Abstract Background Single-cell multi-omics profiling of hematopoietic malignancies frequently involves pooling of patient samples before library preparation to reduce costs. Demultiplexing and quality control of the resulting sequencing data depend on experimental design, sequencing depth, and computational methods. Existing approaches benchmark individual tools, auto-select a single best method, or apply majority voting. However, none systematically exploit disagreement patterns among orthogonal strategies as a diagnostic signal for cell quality. Results We introduce Split-flow, a modular Nextflow pipeline that runs hashing-based and SNP-based demultiplexing, and transcriptome-based doublet detection in parallel. It classifies cells into quality strata through a concordance-based decision framework. Validation on multiplexed CITE-seq data from 14 multiple myeloma patients across eight Chromium channels demonstrates high reproducibility and shows that discordant cells cluster within specific cell types and quality strata. TCR clonotype cross-referencing against VDJdb confirms that concordance-based classification enriches for biologically genuine immune receptor sequences, with a 5.3-fold enrichment of confirmed public TCR sequences in the high-confidence stratum. Downsampling analysis reveals that SNP-based methods are more depth-sensitive than hash-based approaches, supporting the recommendation to combine both strategies. The framework transfers to AML samples across three assay types (snMultiome-seq, scRNA-seq, scATAC-seq), where ATAC-based demultiplexing resolves donor assignment discordance under low hashing efficiency. Conclusions Split-flow demonstrates that combining of orthogonal preprocessing methods yields structured information about cell quality and offers a concordance-based framework that transforms this disagreement into a diagnostic signal. It introduces a preprocessing approach that can be exploited beyond hematopoietic malignancies in multiplexed single-cell applications. Graphical abstract Highlights and main findings Introduces Split-flow, a modular Nextflow DSL2 pipeline for preprocessing of multiplexed single-cell multi-omics sequencing data from hematopoietic malignancy samples via a post hoc concordance-based decision framework. Provides practical guidance for the experimental design of multiplexed single-cell multi-omics experiments, including the recommendation to combine antibody-based hashing with a SNP genotype reference for orthogonal demultiplexing. Reveals that SNP-based demultiplexing is more sensitive to sequencing depth than hash-based approaches, and that the combined strategy mitigates depth-dependent biases in cell-type recovery. Demonstrates that disagreement between demultiplexing methods contains structured diagnostic information about cell quality, with concordance categories reflecting genuine quality gradients in multiple myeloma CITE-seq samples. Validates the concordance framework using T cell receptor sequences as an orthogonal biological readout, with a 5.3-fold enrichment of confirmed public TCR sequences in the high-confidence stratum. Applies the preprocessing framework to AML patient samples across three assay types (snMultiome-seq, scRNA-seq, and scATAC-seq) and demonstrates that ATAC-based demultiplexing can resolve donor-assignment discordance.

This study aims to identify parameters predicting COVID-19 inflammation score (CIS) response and survival probability in critically ill patients with hyperinflammation treated with the Janus kinase (JAK) 1/2 inhibitor ruxolitinib. This is a single arm, non-randomized, open-label, phase-II study for frontline treatment in adults in the intensive care unit (ICU). Ninety-two critically ill COVID-19 patients with CIS ≥ 10 were treated in the RuxCoFlam trial (NCT04338958) with ruxolitinib between April 2020 and June 2021. Median ICU treatment duration was 15 days (range, 2–73). Out of 81 evaluable patients, 62 (77%) showed CIS reduction ≥ 25% on day 7 (CIS response). In multiple logistic regression analyses, higher CIS on day 0 (odds ratio (OR), 1.56; 95% confidence interval (CI), 1.01–2.41; p = 0.046) and male gender (OR, 4.76; 95% CI, 1.22–16.67; p = 0.024) were significantly associated with CIS response. Sixty-day survival probability was higher in CIS-responders compared to non-responders (74% vs. 32%; p < 0.001). Multiple Cox regression analysis revealed younger age (10-year difference) (hazard ratio (HR), 0.65; 95% CI, 0.46–0.91; p = 0.012) and CIS response (HR, 0.19; 95% CI, 0.08–0.45; p < 0.001) as significant parameters for survival probability. In conclusion, reduced risk of death in CIS-responders underlines the usefulness of CIS for the assessment of hyperinflammatory disorders, such as COVID-19, under JAK1/2 inhibitor therapy.

Introduction: Acute myeloid leukemia (AML) is a clonal malignancy characterized by specific genetic alterations that impact cell differentiation and survival programs. Only few studies have so far explored proteomic and proteogenomic disease characteristics that furthermore contribute to AML pathogenesis. Methods: We conducted a large-scale proteomic analysis on bone marrow blasts from 284 patients with newly diagnosed NPM1-mutated AML enrolled in the phase III AMLSG 09-09 trial [NCT00893399], comparing intensive chemotherapy plus all-trans retinoic acid with or without gemtuzumab ozogamicin. We quantified the expression of 7,223 proteins. Additionally, targeted DNA sequencing of 263 genes frequently mutated in myeloid malignancies was performed as published recently (Cocciardi et al., Hemasphere 2025). Results: Applying previously defined gene signatures that distinguish immature leukemic stem cell (LSC)-like states from more differentiated granulocyte-macrophage progenitor (GMP)-like promonocyte-like, and monocyte-like leukemic blasts, we identified a robust differentiation gradient as a principal source of proteomic variance. This gradient was significantly associated with genetic alterations, particularly an enrichment of FLT3-ITD (adjusted p-value <0.001) in immature disease, and the absence of DNMT3A (adjusted p-value <0.001), NRAS (adjusted p-value 0.026) and PTPN11 (adjusted p-value <0.001) mutations. Immature AML strongly correlated with BCL2 protein expression (Spearman's rho = 0.7), consistent with previous evidence of sensitivity to venetoclax, a clinically approved BCL2 inhibitor. Building on our prior observation linking venetoclax sensitivity to a mitochondrial protein-rich AML subtype, we applied the previously described mitochondrial gene signature and observed a positive correlation with immature NPM1-mutated AML (Spearman's rho = 0.48) and a negative correlation with more committed differentiation states (Spearman's rho=-0.78). Interestingly, although the Mito-score generally declined with increased differentiation, a modest increase was observed in highly differentiated, monoblast-like AML. Functional enrichment analysis using mitochondrial gene sets from the Broad Institute's MitoCarta database revealed distinct mitochondrial functional states between AML with either immature or differentiated blast characteristics. Immature AML was enriched for mitochondrial DNA translation and mitochondrial central dogma terms, whereas AML characterized by more differentiated blasts was enriched for terms related to mitochondrial metabolism, suggesting both quantitative and qualitative differences in mitochondrial biology within NPM1-mutated AML. Clinically, a high Mito-score (top quartile) was significantly associated with worse event-free survival (EFS; log-rank p-value = 0.026, HR = 4.46) and overall survival (OS; log-rank p-value = 0.03, HR = 4.74) in the entire cohort, as well as in genetically favorable-risk patients characterized by NPM1 mutations in the absence of FLT3-ITD (EFS log-rank p-value = 0.021, HR = 5.4; OS log-rank p-value = 0.065, HR = 5.24). Importantly, the prognostic value of a high Mito-score remained significant in a multivariate Cox regression model controlling for treatment arm, gender, LDH levels, and white blood cell count (multivariate p-value = 0.017), with borderline significance when accounting for age (multivariate p-value = 0.05). Conclusion: In summary, our comprehensive proteogenomic analysis of NPM1-mutated AML within a prospective clinical trial identifies differentiation trajectories as a key driver of disease heterogeneity. Immature NPM1-mutated AML was characterized by distinct genetic profiles including FLT3-ITD, high BCL2 expression, and a specific mitochondrial protein expression signature (high Mito-score). Clinically, a high Mito-score predicted poorer outcomes in not only the entire cohort but also the favorable NPM1-mutated 2022 ELN subgroup, establishing a link between differentiation states and mitochondrial biology as a relevant feature impacting patient outcome upon intensive chemotherapy treatment.

ELN recommendations for AML diagnosis, genetic risk stratification and treatment have been updated in 2022. Accordingly, FLT3-ITD+ AML is now included in the intermediate-risk category, regardless of the allelic ratio (AR), and capillary electrophoresis (CE) is the recommended detection method. In two large trials combining intensive chemotherapy with the FLT3 inhibitors midostaurin (RATIFY) and quizartinib (QuANTUM-First), the AR threshold to define FLT3-ITD+ was conventionally set at 0.05 and 0.03, respectively. CE analysis requires high level of expertise specifically to detect FLT3-ITD microclones (FLT3-ITDm, AR: >0.01,<0.05), whose clinical role is not yet well established. Here, we leveraged the EHA AML-SWG framework to explore diagnostic evaluation methods and prognostic impact of FLT3-ITDmin AML. In a first methodological part, results of CE from 63 FLT3-ITD+patient samples were blindly analyzed by 7 European labs, and reproducibility of results was assessed. The intraclass correlation coefficient (ICC) was computed as an index of interrater reliability of AR data. Overall, we observed a good ICC for ITD length determination (95%CI=0.52–0.92), but a less satisfactory ICC for AR estimation (95%CI=0.06–0.63). Next, we compared NGS and CE for FLT3-ITD status determination, by running in parallel an additional set of 48 FLT3-ITD+ AML cases. Results showed a significant correlation of AR/VAF (r=0.923,p<0.001), and a 94% concordance overlap, with only 3/48 cases with AR<0.02 detected by CE, but not by NGS. Next, we enrolled AML patients diagnosed between 2017 and 2022 with FLT3-ITD AR<0.05 by CE, and a comparator cohort of cases with AR>0.05 treated with standard chemotherapy without FLT3-inhibitors, or with less-intensive strategies. A total of658 patients with FLT3-ITD+ AML were included in our study. Of these, 212 (32%) had FLT3-ITDm (AR range:0.01-0.04), whereas 446 (68%) had an AR>0.05 (0.05-15.26). Median age at AML onset was 59 years (range 18-94). Patients with microclones were older (62 vs 58 years, p<0.001), and had a less proliferative phenotype. AML with FLT3-ITDm were also more frequently classified as “secondary” with regards to disease ontogeny (14% vs 8%, p=0.012), and had a higher frequency of myelodysplasia-related (MR) genes mutations (60% vs 30%, p<0.001), whereas no difference was noticed for NPM1. A total of 161 patients (24%, median age 76 years) were deemed unfit and were managed with less-intensive approaches, while 497 patients (76%) received intensive chemotherapy. Among the latter, complete response (CR) was achieved in 88% of cases with AR>0.05 vs. 74% of cases with FLT3-ITDm (p<0.001). CR achievement was associated with younger age (p<0.001), presence of NPM1 (p=0.018), absence of DNMT3A (p=0.035) and MR-genes (p=0.011) mutations, FLT3-ITD AR >0.05 (p<0.001), and ELN 2017/22 favorable/intermediate vs adverse categories (p=0.015/0.045). With a median follow-up of 30 months (18-41), 3-year overall survival (OS) was 58% in intensively treated patients, censoring for transplant. The multivariable analysis (MVA) for OS, including clinical and genetic variables, and ELN 2022 categories, showed that increasing age and WBC, together with NPM1 mutation had an independent prognostic role (HR=1.03, 1.03 and 0.63 respectively, p<0.001, 0.021 and 0.012, respectively). Overall, 136 intensively treated patients (27%) relapsed at a median of 8 months (range 1-56) from AML diagnosis, leading to a 3-year disease-free survival (DFS) of 47%, when censoring for transplant, with no impact of FLT3-ITD AR (HR=0.9, p=0.6). The MVA identified increased WBC and presence of NPM1mutation as independent predictors of DFS (HR=1 and 0.61, p=0.007 and 0.017, respectively). Of cases with available FLT3-ITD status at relapse (n=33), 45% remained positive, of which 64% were originally FLT3-ITDm. Dissecting their FLT3-ITD clonal make-up, the majority (91%) recapitulated the same clone harbored at onset (18% also acquiring additional clones, and 27% losing prior clones), whereas a new clone was detected in 9% of cases. Finally, the clonal burden expanded in all cases (median 0.027 vs 0.686, p<0.001), with 89% of FLT3-ITDm patients relapsing with AR >0.05. Our data show that FLT3-ITDmare associated with older age, secondary-type AML and higher frequency of MR-gene mutations vs AML with FLT3-ITD >0.05. The potential benefit of FLT3 inhibitors in patients with FLT3-ITDm needs to be evaluated in randomized clinical trials.

Acute myeloid leukemia (AML) with complex karyotype is characterized by high genomic complexity, including frequent TP53 mutations and chromothripsis. Genomic rearrangements can reposition active enhancers near proto-oncogenes, leading to their aberrant expression; however, a comprehensive understanding of these events in AML is still incomplete. To facilitate the discovery of such "enhancer hijacking" events, we developed Pyjacker, a computational tool, and applied it to 39 AML samples with complex karyotype. Pyjacker identified several enhancer hijacking events in AML patient samples, including aberrant expression of MNX1, which can result from del(7)(q22q36) and is associated with hijacking of a CDK6 enhancer. MNX1 activation occurred in 1.4% of patients with AML and showed significant co-occurrence with BCOR mutations. Through a xenograft mouse model, we demonstrated that MNX1 is required for leukemia cell fitness. Pyjacker is an easy-to-use, accurate, and broadly applicable tool for identifying consequences of genomic events driving tumorigenesis, especially when germline genomic data are missing. SIGNIFICANCE:This study examines the consequences of structural alterations in AML and demonstrates that proto-oncogene activation by enhancer hijacking is an understudied pathomechanism. MNX1 overexpression demonstrates that deletions on chromosome 7q can not only lead to haploinsufficiency but also to activation of oncogenes by enhancer hijacking.

In the European LeukemiaNet (ELN) 2022 recommendations, myelodysplasia-related (MR) gene mutations were classified as a novel adverse prognostic category for intensively treated acute myeloid leukemia (AML). To assess the prognostic impact of individual MR genes within the ELN, clinical, cytogenetic, and molecular data from 4,978 intensively treated AML patients were analyzed. Remission rates and survival outcomes were evaluated. For analyses in context of ELN2022 classification, patients carrying an MR mutation were excluded from the adverse group and analyzed separately; those with co-occurring favorable or intermediate features remained in their respective groups. Overall, 1698 patients (34.1%) harbored at least one MR mutation. Lower complete remission rates were observed in MR-mutated cases (65.7% vs 77.7%; p < 0.001) along with shorter event-free (HR 1.45; p < 0.001), relapse-free (HR 1.33; p < 0.001), and overall survival (HR 1.45; p < 0.001) were recorded. Gene-specific prognostic patterns emerged: ASXL1, RUNX1, SF3B1, and U2AF1 mutations associated with adverse risk-like outcomes; SRSF2 and STAG2 aligned with intermediate-risk; BCOR, EZH2, and ZRSR2 did not differ significantly from intermediate or adverse risk. These findings from a large cooperative cohort highlight prognostic heterogeneity among MR mutations and suggest that SRSF2 and STAG2 mutations are associated with less adverse risk patterns, comparable to intermediate-risk.

The nuclear export protein XPO1 interacts with nucleoporin 214 (NUP214) and has been implicated in the pathogenesis of SET::NUP214 acute myeloid leukemia (AML). We evaluated DEK::NUP214 (DN), characterizing a distinct AML entity, for its dependency on XPO1 in human AML models. Deletion of XPO1 in DN-positive FKH-1 cells revealed a strong dependency on XPO1. Pharmacologic inhibition of XPO1 by the second-generation selective inhibitor of nuclear export, eltanexor, in primary human and FKH-1 cells reduced XPO1 expression, disrupted co-localization of XPO1 and DN, and induced apoptosis and cell cycle arrest. Functionally, XPO1 and DN co-localized at chromatin, and this co-localization was strongly reduced by XPO1 inhibition. Loss of chromatin binding resulted in downregulation of DN target genes and pathways related to cell cycle and self-renewal. Eltanexor treatment of a patient-derived DN-AML xenograft model disrupted leukemia development, showing molecular clearance in bone marrow after a median of 377 days in eltanexor-treated mice, while control mice succumbed after a median of 244 days. In summary, XPO1 stabilizes DN at chromatin to allow the activation of its oncogenic gene signature, while targeting XPO1 treats leukemia successfully in vivo. These findings establish XPO1 as a molecular target in DEK::NUP214 AML.

Chronic kidney disease (CKD) before treatment and renal function decline during treatment are common in elderly patients receiving hypomethylating agents (HMAs) and venetoclax (VEN). This retrospective multicenter study of 130 newly diagnosed older acute myeloid leukemia (AML) patients evaluated the prognostic impact of renal function before and during the first treatment cycle. A total of 56 patients (43%) had CKD, and 49 (38%) developed acute kidney injury (AKI) during treatment. AKI occurrence was associated with laboratory tumor lysis syndrome (LTLS). CKD before treatment showed a trend towards a lower overall response rate (ORR, OR 0.5, p = 0.07) in multivariable analysis (MVA) and inferior relapse-free survival (RFS, HR 2.16, p = 0.06) in univariate analysis (UVA), but not MVA (RFS, HR 1.93, p = 0.15). CKD did not affect overall survival (OS) or event-free survival (EFS). AKI during HMA/VEN therapy was associated with significantly higher 30-day and 60-day mortality rates and emerged as an independent prognostic factor for inferior OS (HR 1.86, p = 0.01) and EFS (HR 1.81, p = 0.007). RFS did not differ significantly by AKI status. Sepsis was a more frequent cause of death in patients with vs. without AKI (33% vs. 5%). In conclusion, kidney function is a key prognostic factor in HMA/VEN-treated patients, warranting further study on treatment adjustments and supportive care.

The World Health Organization fifth edition and International Consensus Classification for myeloid neoplasms both incorporate empirical numerical thresholds to morphologic and molecular features defining certain disease entities. However, the clinical implications of these thresholds remain unclear. We analyzed a large cohort (N = 6,976) of patients with myeloid neoplasms to evaluate the impact of proposed yet different numerical thresholds for variant allele frequency of genetic mutations or hematologic parameters set forth by the World Health Organization fifth edition and International Consensus Classification for classification of SF3B1-mutated myelodysplastic neoplasms, NPM1-mutated acute myeloid leukemia (AML), and oligomonocytic chronic myelomonocytic leukemia. Our analysis demonstrated that the clonal burden of SF3B1 mutation in myelodysplastic neoplasms informs classification and prognosis. Our findings support the notion that NPM1 mutation should be AML-defining regardless of blast percentage and highlight the adverse prognostic impact of the cumulative number of myelodysplasia-related mutations in NPM1-mutated AML. Finally, we provide evidence that integrating specific molecular signatures could improve the accuracy of oligomonocytic chronic myelomonocytic leukemia classification. SIGNIFICANCE:Using comprehensive clinical and molecular profiling, this study provides a data-driven approach for evaluating numerical thresholds of variant allele frequency or hematologic parameters (i.e., blast percentage and absolute monocyte count) included in current classification schemas across a spectrum of myeloid malignancies, enabling refinement of disease classification and prognostication.

Introduction CNL is a rare myeloproliferative neoplasm (MPN) with a 5-year-survival-rate of 28 percent, and no standard of care for treatment. The majority of CNL patients harbors the membrane proximal T618I mutation in the colony-stimulating factor 3 receptor (CSF3R), which leads to constitutive activation of the JAK1/STAT3-pathway. However, JAK1/2-inhibitor Ruxolitinib achieves only a limited response in CNL patients, suggesting the involvement of additional signaling pathways. Our previous data established that CSF3RT618I is retained in the endoplasmic reticulum (ER), where it interacts with Calnexin, induces unfolded protein response (UPR) and ER-Phagy. In this study we analyzed the role of ER-stress sensor ATF6 for CSF3Rmutations mediated cell proliferation and signaling, and explored potential signaling pathways crucial for Ruxolitinib-persistent CSF3RT618I expressing cells. Methods PDI inhibitor 16F16 treatments in Ba/F3, 32D and HEK293 cells were conducted to target ATF6 and analyze effects on membrane proximal-, truncation-, and compound-mutated CSF3R, and mutant Calreticulin (CALR)del52 mediated oncogenic signaling pathways, using MTT-assays, Western Blots (WB) and co-Immunoprecipitation (IP). RNA-seq was performed with Ba/F3 cells and peripheral blood samples from CNL-patients from the German Study Group for Myeloproliferative Neoplasms (GSG-MPN) (n=28). NIH-3T3 and 32D cells expressing CSF3RT618I, CSF3RWT-KDEL and CSF3R compound mutations were used to assess the significance of altered protein structure on signaling and UPR, using confocal microscopy, MTT-assays and WB. A PamGene assay was utilized to analyze differential kinase activity in Ruxolitinib-persistent CSF3RT618I expressing Ba/F3 cells. Results RNA-seq revealed upregulation of ER-stress-sensor ATF6 and ATF6 target genes in CSF3RT618I, CSF3RW791* and CSF3RT618I+Y748* expressing Ba/F3 cells, and in CNL-patients harboring CSF3RT618I or CSF3R-compound-mutations. Compound-mutated CSF3R proteins displayed a specific pattern of UPR induction compared to CSF3RT618I, suggesting an important role of the cytoplasmic tail of CSF3R in ER-stress-sensor activation. Mechanistically, PDI inhibitor 16F16 led to decreased ATF6 activation in 32D and Ba/F3 cells, and impaired dimerization of CSF3RT618I and CSF3RW783*, but not CSF3RWT expressing, flag-/HA-tagged CSF3R constructs containing, HEK293 cells. Targeting ATF6 activation and PDI activity with 16F16 led to a decrease in cell proliferation and reduced STAT3, Src kinase and AKT activation in membrane proximal-, truncation-, and compound-mutated CSF3R, as well as Ruxolitinib-persistent CSF3RT618I, but not CSF3RWTexpressing 32D and Ba/F3 cells. PamGene assay analysis revealed enhanced activity of PKC-family-kinases in Ruxolitinib-persistent CSF3RT618I expressing Ba/F3 cells compared to non-persisters. Co-IP showed interaction of CSF3RT618I and PKC-family-kinases and WB-results corroborated a JAK-independent activation mechanism. Interestingly, 32D cells expressing thrombopoietin receptor (MPL) and CALRdel52 also showed ATF6 protein activation and UPR induction. Treatment with 16F16 led to significantly decreased cell proliferation compared to CALRWT expressing 32D cells. ConclusionsCSF3RT618I strongly induces UPR through the PDI-ATF6-axis. Activation of this pathway requires specific regions of the cytoplasmic domain of CSF3R, acting in concert with protein folding alterations induced by T618I. Our results suggest a shift to enhanced PKC-family-kinase mediated signaling as a possible mechanism for CSF3RT618I expressing cells to evade Ruxolitinib mediated JAK1/2 inhibition, potentially explaining limited treatment efficacy in CNL patients. However, 16F16 treatment significantly decreases cell proliferation mediated by various CSF3R mutants, including Ruxolitinib-persistent CSF3RT618I expressing cells, potentially through a multilayered mechanism, comprising impaired dimerization and enhanced degradation of CSF3R mutants due to blockage of the PDI-ATF6-axis. CALRdel52 mediated proliferation, as seen in MPNs such as essential thrombocythemia, can also be diminished by 16F16. These results implicate the PDI-ATF6-axis as a novel and effective treatment target for CSF3R mutations mediated CNL and other MPNs.

Myeloproliferative neoplasms (MPN) harboring concurrent mutations in more than one driver gene represent an increasingly recognized yet frequently overlooked subgroup, posing significant diagnostic and clinical challenges. Current guidelines recommend a stepwise diagnostic approach: testing of CALR and MPL only if JAK2 is negative. This sequential strategy inherently risks overlooking cases, as simultaneous mutations in JAK2, CALR, and MPL, although rare, do exist. In published, rather limited datasets, patients typically exhibited older age and elevated platelet counts compared to those with single mutations, with suboptimal responses to conventional therapies necessitating tailored strategies. The true incidence of “double-driver” mutations is likely underestimated. To characterize the molecular landscape and clinical management of “double driver” patients 3 international patient cohorts were investigated: first we analyzed 84,921 samples with suspected diagnosis of MPN. 29,372 individuals (34.59%) were positive for at least one MPN driver mutation (JAK2, MPL or CALR). 439/29,372 individuals had at least 2 (1.5%) and 3/29,372 individuals all 3 driver mutations (0.01%) detectable. In 424 evaluable individuals, M:F ratio was 1.09 with a median age of 73.2 years (range 16.9-96). Leukocyte range was 1.5-117.0 Gpt/l and hemoglobin 6.1-22.0 g/dl, indicating a high variability depending on the clinical phenotype. Patients harboring 2 mutations frequently had elevated platelet counts (median 773Gpt/l; range 22-1,956 Gpt/l). Clone size of the respective driver mutations showed a high variability (CALR: mean 22.2%; range 0.85-62%; JAK2 16.9%, range 0.83-96%; MPL 13.5%, range 0.96-88.8%), while >80% of individuals revealed clone sizes <50%. 57% of cases that had received a complete NGS myeloid panel showed additional co-mutations, most frequently in TET2, SRSF2 and ASXL1, while 43% of the analyzed cases had no further mutation detectable. These results were confirmed in a cohort of the German MPN Study Group Registry, an observational study of MPN patients with >70 participating centers (university & community hospitals and office-based hematologists): out of 4,148 MPN patients with detailed clinical annotation, 54/4,148 (1.28%) were identified with 2 and 6/4,148 (0.05%) with all 3 driver mutations. Analysis of individually selected patient cells (with 2 confirmed drivers) in colony formation assays revealed their bi-clonal nature. The type of driver mutation combination had impact on the clinical phenotype with JAK2/MPL being predominantly diagnosed as ET (45%) and MF (40%), JAK2/CALR-co-mutated cases as ET (53%) and MF (22%), CALR/MPL-mutated cases as ET (60%) and MF (40%) and triple mutated cases as PV (50%) or ET (50%). Overall, patients with 2 drivers showed a higher rate of progression (to myelofibrosis or MPN-blast phase) compared to those with 1 driver mutation (16.7% vs. 9.9%). Of note, JAK2/CALR co-mutated PMF patients showed better overall survival (median OS: JAK2/CALR not reached; JAK2: 5.4 years, CALR: not reached), while JAK2/MPL co-mutation resulted in dismal survival (median OS: JAK2/MPL: 3.8 years; JAK2 5.4 years; MPL 3.2 years). Consistently, clonal competition assays conducted with murine hematopoietic cells over 8 days revealed clonal dominance of JAK2 over CALR (day 8; p<0.0046**) while MPL-mutated cells were dominant over JAK2-clones (day 8; p=0.0115*). Transcriptome profiling of competing cell populations identified distinct inflammatory signatures depending on the driver combination that can be traced to subclonal compartments in murine competition assays in vivo and single cell sequencing. To assess for therapy responses and clinical characteristics in more detail, we investigated 56 “double driver” patients with detailed clinical annotation from 12 international MPN centers and found differential clinical and molecular responses in patients treated with Jak-inhibitor or interferon-alpha therapies, e.g. partial and complete molecular responses of MPL-clones upon IFN-containing regimens. Our data provides insight into a rare, so far overlooked patient population and indicates a need to modify the sequential diagnostic approach. Appearance of MPL and CALR mutations parallel to JAK2 impact on prognosis irrespective of clone size. Differential patterns of therapy response (e.g. to IFN) appear to be relevant for molecular responses and survival.

Background FLT3-ITD mutations are found in approximately one quarter of patients with acute myeloid leukemia (AML). One major change in the 2022 ELN risk classification is the re-classification of all FLT3-ITD mutated patients as intermediate risk independently of allelic burden or NPM1 co-mutation. Recommendations state that FLT3-ITD mutations without concurrent NPM1 mutations but with adverse-risk genetic aberrations should be classified as adverse risk. However, the prognostic impact of concurrent mutations in the newly-defined subgroup of myelodysplasia-related genes (MRG) in FLT3-ITD mutated patients remains unclear. Methods We retrospectively analysed 4078 intensively-treated AML patients within the AML HARMONY Consortium database with molecular genetic data from diagnosis including the mutational status of all nine MRG mutations (ASXL1, BCOR, EZH2, RUNX1, SF3B1, SRSF2, STAG2, U2AF1, ZRSR2), NPM1 and FLT3. All cases were annotated and organised using the OMOP common data model. Outcome was assessed by multivariate analysis (MVA) using a Cox model with backward elimination considering mutations in at least one MRG, hemoglobin levels, white blood cell counts (WBC), platelets, bone marrow blast count, age ≥60 years, TP53 mutation, NPM1 mutation, FLT3-ITD to wild type (wt) allelic ratio and complex karyotype. For multivariate analysis, data was imputed using logistic regression for binary variables and predictive mean matching for continuous variables with 5 imputations by chained equations. Results The prognostic impact of MRG mutations was analysed in 862 patients (21%) with a detectable FLT3-ITD mutation at diagnosis, and in subgroups defined by FLT3-ITD to wt allelic ratio and NPM1 mutational status. Median age was 51 years. Median follow-up was 6.2 years. 171 of these patients (20%) had at least one additional MRG mutation. Only 13 patients were treated with FLT3-inhibitors during induction therapy. The most common MRG mutations were RUNX1 (n=77), SRSF2 (n=37) and STAG2(n=32). Patients with MRG mutations were older (median age 57 vs. 49.9 years), more likely to be male (61% vs. 42%) and had lower WBC at diagnosis (median WBC 26.6 vs. 47.8*109/L). In MVA MRG mutations were neither associated with relapse free survival (RFS) (HR 1.10, 95%CI 0.85-1.41, p=0.5) nor overall survival (OS) (HR 1.21, 95%CI 0.97-1.50, p=0.085). Grouping the patients according to low (n=231) or high (n=525) FLT3-ITD allelic ratio, MRG mutations were neither predictive for RFS (low: HR 1.38, 95%CI 0.86-2.19, p=0.2; high: HR 0.80, 95%CI 0.55-1.15, p=0.2) nor OS (low: HR 1.13, 95%CI 0.73-1.75, p=0.6, high: HR 1.12, 95%CI 0.83-1.51, p=0.5). Among patients with NPM1 co-mutation (n=491), only 46 (9%) had an additional MRG mutation. In the small subgroup of FLT3/NPM1co-mutated patients MRG mutations had a similar prognostic impact on RFS (HR 0.68, 95%CI 0.41-1.12, p=0.13) and OS (HR 0.74, 95%CI 0.43-1.26, p=0.3) compared to the remaining patients by MVA. Interestingly, in FLT3-ITD mutated patients without NPM1 mutation (n=371) multivariate analysis revealed MRG mutations as an independent prognostic marker for RFS (HR 1.44, 95%CI 1.06-1.95, p=0.021) and OS (HR 1.43, 95%CI 1.08-1.89, p=0.013) besides a high FLT3-ITD to wt ratio, age and high WBC count. Both ASXL1 and RUNX1 mutations have been associated with an adverse outcome before the introduction of MRG mutations in the ELN 2022 classification. The presence of either RUNX1 or ASXL1 mutations (n=93) but not MRG mutations other than ASXL1 and RUNX1 was an independent predictor of worse RFS (HR 1.60, 95%CI 1.20-2.14, p=0.001) and OS (HR 1.32, 95%CI 1.00-1.73, p=0.046) by MVA in the full FLT3-ITD mutated cohort besides age ≥60 years, high WBC count, NPM1 wildtype status and a high FLT3-ITD to wild type ratio. Similarly, in FLT3-ITD mutated patients without NPM1 mutation, RUNX1 and/or ASXL1 mutations were associated with a worse RFS (HR 1.59, 95%CI 1.14-2.20, p=0.006) and OS (HR 1.40, 95%CI 1.04-1.89, p=0.026), while MRG mutations other than RUNX1 and ASXL1were an independent predictor of worse OS (HR 1.49, 95%CI 1.00-2.22, p=0.0496), but not RFS (HR 1.23, 95%CI 0.72-2.10, p=0.4). Conclusion In the ELN 2022 subgroup of FLT3-ITD mutated patients MRG mutations were not associated with RFS and OS. However, in the subgroup of FLT3-ITD/NPM1 wildtype patients, MRG mutations were an independent risk factor for shorter RFS and OS.

Background: C10603/RATFIY was the first AML trial to show the benefit of adding a targeted agent to intensive chemotherapy for a specific genetically determined subset (Stone R et al, NEJM 2017). The addition of the multi-kinase inhibitor midostaurin (M) to chemotherapy in previously untreated adults with FLT3-mutant AML resulted in superior event-free (EFS) and overall survival (OS) compared to placebo (P) and led to the approval of this agent in combination therapy. Subsequently, additional targeted drugs including gilteritinib for relapsed/refractory FLT3-mutant AML and quizartinib plus chemotherapy in untreated adults with AML with FLT3-ITD disease have gained approval. Long-term follow-up after reporting of the primary endpoint, especially if met, is important in oncology. Objective: We report the EFS and OS data from C10603 using 10 years of follow up to determine the persistence of a midostaurin benefit and to assess the nature of late relapses or toxicity. Methods: C10603 enrolled 717 pts (360 on the M and 357 on the P arm; median age 47.8 years (range 18-61), 398 women (55.5%) of whom 51.7% were randomized to M and 59.4% to P (p=0.04), and 89% white) from 2011-2015 with previously untreated AML who had either a FLT3-TKD or ITD mutation with allelic ratio of >0.05 to receive daunorubicin/cytarabine (3+7) induction (one reinduction permitted) followed by up to 4 consolidation cycles with cytarabine 3 g/m2 every 12h on days 1, 3 and 5. M (50 mg bid) or P was given orally on days 8-22 of each chemotherapy cycle as well as daily during one year of single-agent maintenance. Allogeneic transplantation (alloHCT) was employed at investigator's discretion; study drug was not continued post-HCT. Pts stayed on their randomized arm and were not re-randomized prior to maintenance. Pts were followed for relapse and survival after the end of protocol therapy, regardless of subsequent therapy, for 10 years. Log-rank tests, Kaplan-Meier methods, and Cox proportional hazard models were used to evaluate the treatment effect in EFS and OS, as well as the alloHCT effect. All p values are two-sided. Results: We have completed the 10-year FU on this trial and data collection has concluded. The median EFS was 8.2 months (95% CI, 5.5-11.4) on the M arm compared with 3.0 months for P (95% CI, 1.9-6.0) with a HR of 0.79 (95% CI 0.67-0.94, p=0.0067). Among the 420 CR pts who achieved complete remission (CR) by the protocol specified time of 60 days, 253 have had an event (death in 69 and relapse in 184); these events were relatively equal on each arm. OS, the co-primary endpoint, remained marginally superior for those randomized to M compared to P; the 10-year OS estimate was 43.7% (95% CI, 38.7-49.3%) vs 38.6% (95% CI, 33.6-44.4%) (p= 0.0485). The hazard ratios for OS favored randomization to M in each key biological subgroup (FLT3-ITD low or high allelic ratio or FLT3-TKD), but smaller subsets precluded statistical significance. There was a 10-year OS benefit in favor of M in men (p=0.005), but not women (p=0.9). A total of 414 transplants were performed: only 5 in the latter half of the follow-up period. Transplantation in CR1 was highly beneficial overall (10-year OS, 56% vs 35.8% without transplantation, p=0.001). Among those transplanted in CR1, randomization to M was marginally better than P (61.5% vs 49%, p=.064). After censoring for alloHCT in CR1, there was an OS trend in favor of M (51.8% v 48.0%, p=0.102). In CR1 patients who were randomized to maintenance therapy, M reduced the cumulative incidence of relapse in ELN 2017 favorable and intermediate risk pts, but not in those with adverse risk disease (HR 0.71, 0.47, and 1.01, respectively); maintenance M had no effect on OS. Only 3 pts relapsed after 5 years (all randomized to M). 25 pts randomized to M vs 13 P pts died after 5 years of follow-up. 5 pts on each arm died of a new primary cancer whereas 17 M and 5 P pts were coded as having died from treatment-related causes. None of the 3 pts who relapsed after 4 years had spliceosome mutations at diagnosis; whereas 18% (21/117) patients pts who had relapsed earlier had spliceosome mutations at diagnosis (p=0.419). Conclusions: The EFS benefit of randomization to midostaurin vs placebo when added to chemotherapy was maintained over time, although the benefit for OS was diminished, likely due in part to aging. Patient and disease factors differed between early vs late relapses, which could suggest the use of alternative therapies during distinct therapeutic stages.

Supplementary Tables 1-2, Figures Legends 1-4 from Stromal Niche Cells Protect Early Leukemic FLT3-ITD+ Progenitor Cells against First-Generation FLT3 Tyrosine Kinase Inhibitors

Dysregulated hyperinflammatory response is key in the pathogenesis in patients with severe COVID-19 leading to acute respiratory distress syndrome and multiorgan failure. Whilst immunosuppression has been proven to be effective, potential biological targets and optimal timing of treatment are still conflicting. We sought to evaluate efficacy and safety of the Janus Kinase 1/2 inhibitor ruxolitinib, employing the previously developed COVID-19 Inflammation Score (CIS) in a prospective multicenter open label phase II trial (NCT04338958). Primary objective was reversal of hyperinflammation (CIS reduction of ≥25% at day 7 in ≥20% of patients). In 184 patients with a CIS of ≥10 (median 12) ruxolitinib was commenced at an initial dose of 10 mg twice daily and applied over a median of 14 days (range, 2–31). On day 7, median CIS declined to 6 (range, 1–13); 71% of patients (CI 64–77%) achieved a ≥25% CIS reduction accompanied by a reduction of markers of inflammation. Median cumulative dose was 272.5 mg/d. Treatment was well tolerated without any grade 3–5 adverse events related to ruxolitinib. Forty-four patients (23.9%) died, all without reported association to study drug. In conclusion, ruxolitinib proved to be safe and effective in a cohort of COVID-19 patients with defined hyperinflammation.

Supplementary Figures 1-4 from Stromal Niche Cells Protect Early Leukemic FLT3-ITD+ Progenitor Cells against First-Generation FLT3 Tyrosine Kinase Inhibitors

Book Citations: Authors, Title, HemaSphere, 2023;7(S3):pages. The individual abstract DOIs can be found at https://journals.lww.com/hemasphere/pages/default.aspx. Disclaimer: Articles published in the journal HemaSphere exclusively reflect the opinions of the authors. The authors are responsible for all content in their abstracts including accuracy of the facts, statements, citing resources, etc. 2996 (especially IL-6) was demonstrated. Toxicity comprised elevated liver enzymes (8.5 %) and bleeding complications (3.3%), but no severe adverse events in relationship to Rux were reported. Forty-four pts (23.9 %) died, in most cases due to progression of Covid19.