To identify genetic variants that influence myeloproliferative neoplasm (MPN) phenotype, we undertook a two-stage case-only genome-wide association study using cohorts from the UK (including UK Biobank), Spain, Germany and Italy. MPN subtype [essential thrombocythemia (ET); polycythemia vera (PV)] were compared to each other, to healthy controls and stratified analyses was performed based on chromosome 9p aberrations, JAK2 V617F mutation burden and sex. The ET versus PV analysis identified known associations: (i) at HBS1L-MYB that increased ET risk (PMETA=7.93x10-6, OR=1.28) and reduced PV risk (PMETA=9.43x10-5, OR=0.81) and (ii) at GFI1B-GTF3C5 that predisposed to PV only (PMETA=1.43x10-9, OR=1.38). Two further linked intronic SNPs, rs2425786 and rs2425788, at CDH22/CD40 were significant in females only (PMETA=2.67x10-8) with predisposition to PV (PMETA=0.0006, OR=1.3) and reduction of ET risk (PMETA=7.82x10-5, OR=0.75). Associations with JAK2, TERT, ATM, TET2, PINT, GFI1B and SH2B3 were confirmed (PMETA<5x10-8) and nine further loci were replicated (PMETA<0.05). A polygenic risk score consisting of 48 SNPs from 31 loci demonstrated moderate discriminative performance for ET and PV (AUC=0.718) and was improved by optimization for disease subtype (AUCET=0.724 and AUCPV=0.755). Overall, our results reveal that multiple germline variants influence MPN phenotype with HBS1L-MYB and a novel sex-specific association with CDH22/CD40 being the strongest determinants.

The mechanisms that lead to extramedullary tropism of acute myeloid leukemia (eAML) remain obscure and no specific therapeutic approaches for this entity exist. As the long-term survival of eAML is poor, a deeper understanding of the immune microenvironment and leukemia phenotypes underlying this entity is warranted. Here, we performed bulk and single-cell transcriptome profiling of 23 eAML biopsies from 10 patients with isolated extramedullary disease in skin and subcutaneous tissue. Unlike normal healthy skin, we found leukemia cutis to be heavily immune-infiltrated; in cases of extramedullary relapse following allogeneic stem cell transplantation, >90% of T/NK cells were donor-derived. eAML-associated T cells expressed a clear signature of T cell exhaustion, dissimilar to leukemia-associated immune populations in bone marrow relapse (n=7), but related to acute and chronic skin inflammation. Further, HLA class II was down-regulated in 4 of 7 leukemia cutis specimens, consistent with an immune escape phenotype in cases of eAML. Extramedullary and bone marrow-resident leukemia cells differed with regard to the expression of 8 homing receptor molecules (ICAM1 (encoding CD54), PECAM1 (CD31), ITGA4, ITGA6, ITGAL, ITGB4, ITGA5, and ITGAV). Serial samples obtained from one leukemia cutis case throughout consecutive immune checkpoint blockade with ipilimumab followed by nivolumab showed a consistently high degree of overlap between local and circulating T cell receptor (TCR) sequences, suggesting that only a minority of eAML-associated T cells are leukemia-specific. Our analysis reveals eAML to associate with complex changes in leukemia and T cell gene expression profiles that suggest multiple potential avenues for therapeutic targeting.

Marginal zone lymphomas (MZLs) are a heterogeneous group of low-grade B-cell neoplasms classified into different entities by the current lymphoma classifications. They share some features, but differ significantly in clinical presentation, associated inflammatory conditions, anatomical sites of involvement, and molecular alterations. Etiopathogenesis is strongly linked to chronic antigenic stimulation and specific infections or autoimmune disorders for extranodal disease. Genetic hallmarks include constitutive NF-κB activation, and common trisomies 3 and 18, alongside subtype-specific lesions such as translocations in extranodal MZL and recurrent KLF2/NOTCH2 mutations in both nodal and splenic MZL, and deletions involving chromosome 7q, predominantly observed in splenic MZL. Diagnosis can be challenging due to overlapping features with other lymphomas like follicular lymphoma and lymphoplasmacytic lymphoma; integrating morphology, immunophenotype, and molecular data is essential. Transformation to aggressive DLBCL occurs in 3-15% of cases and is associated with the accumulation of genetic lesions, particularly in cell cycle, NF-κB, and epigenetic regulators, with subtype-specific drivers like TNFAIP3, TP53, and CDKN2A/B alterations. The tumor microenvironment plays a critical but understudied role, influenced by chronic antigen stimulation and involving complex interactions with immune cells that can promote immune suppression and influence therapeutic response. Understanding the heterogeneity of MZLs across their classification, genetic landscapes, and interaction with the microenvironment is crucial for accurate diagnosis, prognosis, and the development of effective targeted therapies.

IgM-Monoclonal gammopathy of undetermined significance (IgM-MGUS) and asymptomatic Waldenström (aWM) are precursor conditions of symptomatic Waldenström macroglobulinemia (sWM) with an annual 1.5-12% risk of progression. Although clinical prognostic models exist for risk stratification, it remains challenging to distinguish asymptomatic patients who will eventually progress from those who will not. Hence, the characterization of genomic features that shape disease progressors could potentially improve risk stratification. We performed whole-exome sequencing on 232 samples from 139 patients, including 9 patients with sequential samples. We observed an increasing mutation burden through the stages of disease evolution. Genes such as CD79B, ARID1A and CREBBP were more often mutated in the aWM progressed (aWMpr) compared to the non-progressor aWM group (aWMst) while MYD88L265 variant allele frequency (VAF) was significantly higher in aWMpr compared to aWMst patients. In addition, IgM-MGUS patients with MYD88WT genotype showed a distinct genomic profile compared to the MYD88MUT patients. Furthermore, the presence of more aneuploidies showed a significant association with a higher risk of progression to the symptomatic disease. Overall, our study shows that genomic profiling of patients' tumor at the time of aWM diagnosis might represent an improved strategy for identifying patients at high risk to progression who could benefit from earlier intervention.

Neutrophils interact with the external milieu in both tissue and blood microenvironments and are emerging as important regulators of blood coagulation 1-3. In this study, we explored whether complement induces Neutrophil Extracellular Trap (NET) formation and related blood coagulation using human donor-derived neutrophils. Complement C1q induces NETosis in Lipopolysaccharide (LPS) O127 primed neutrophils, while LPS alone does not induce NETosis. Bulk RNA sequencing revealed a unique LPS-driven altered neutrophil state and complement sensitivity for NETosis was found to be transcriptionally- dependent. Using an arrayed CRISPR Knockout screen in the neutrophil-like differentiated HL60 cells (dHL60), we identified that SCARF1 and Complement Receptor 3 are required for C1q-NETosis. Since NETs contain pro-coagulatory components such as DNA and histones, we investigated whether C1q-related NETs influenced blood coagulation. LPS+ C1q NETs were associated with reduced coagulation activity compared to LPS treatment alone. We further found that LPS upregulated Tissue Factor expression and coagulation-related activity in neutrophils. Furthermore, neutrophils secrete anticoagulant proteins, including Protein C and Tissue Factor Pathway Inhibitors, during C1q-mediated NET formation that functionally regulates NET-related coagulation. C1q- NETs also activate the coagulation factors FXII and FXI, facilitating both intrinsic coagulation and kallikrein-dependent bradykinin production. This study elucidates how NETs regulate both pro-coagulatory and anti-coagulatory components that may influence pathophysiology of disease.

Aging is a critical risk factor for platelet hyperreactivity and thrombosis, yet the mechanisms involved remain poorly understood. This study investigates the role of ubiquitination in platelet function during aging. We identified heightened platelet reactivity in aged mice and human donors. Proteomic analysis of ubiquitin (Ub)-modified proteins and western blot revealed a reduction in overall ubiquitination in aged platelets, correlated with increased expression of deubiquitinating enzymes (DUBs). Notably, USP25 was significantly upregulated in platelets from aged individuals. Functional assays indicated that USP25 deficiency impairs platelet function and delays arterial thrombus formation. Mechanistic investigations integrating ubiquitin-modified proteomics and mass spectrometry demonstrated that USP25 enhances platelet hyperreactivity by stabilizing talin-1 through deubiquitination, maintaining its levels across various tissues, including the liver and spleen. Additionally, AZ1, a USP25/28 inhibitor, effectively suppressed platelet functions in both aged human and mouse models, and decrease age-dependent platelet hyperreactivity and thrombus formation. Collectively, the findings delineate a remodeling of platelet ubiquitination during aging and establish USP25-mediated talin-1 stabilization as a key modulator of platelet hyperactivity in the elderly.

Diffuse large B-cell lymphoma (DLBCL) poses a challenge in hematology given its varied symptoms, and the complex interplay between disease and treatment effects on health-related quality of life (HRQoL). The phase 3 POLARIX study (NCT03274492) demonstrated superior progression-free survival (PFS) and a similar safety profile with polatuzumab vedotin plus rituximab, cyclophosphamide, doxorubicin and prednisone (Pola-R-CHP) vs rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) in patients with previously untreated DLBCL. Here, we evaluate HRQoL through patient-reported outcome (PRO) instruments to fully characterize the patient experience in the POLARIX study. Changes from baseline in HRQoL, lymphoma symptoms, and gastrointestinal (GI) symptoms were assessed, as well as incidence and severity of common symptoms by PROs vs clinician-reported adverse events (AEs). Baseline characteristics of PRO-evaluable patients (N = 874) were consistent. Comparison between PROs and clinician-reported AEs revealed a notable discordance; patients generally reported a higher incidence of symptoms than clinicians, emphasizing the need for patient-centric tools to accurately capture the patient experience. Both treatments exhibited rapid and sustained improvements in HRQoL and lymphoma symptoms, with the most substantial improvements seen in global health status/QoL, lymphoma symptoms, fatigue, role, emotional, and social functioning. GI symptoms (diarrhea, constipation, nausea, and vomiting) were generally similar between treatment arms and returned to baseline levels after treatment completion. These HRQoL data underscore the complementarity of PROs, as an adjunct to clinician-reported AEs, in evaluating the efficacy and tolerability of new treatments, including Pola-R-CHP, which may represent a new benchmark for patient-reported HRQoL in previously untreated DLBCL.

Chromosomal rearrangements that generate novel fusion genes are a hallmark of acute myeloid leukemia (AML). Depletion experiments in cell line models have suggested that their continued expression is required for maintaining their leukemic phenotype and that fusion genes therefore represent ideal cancer-specific therapeutic targets. However, to which extent this result holds true for the different stages of hematopoietic development in primary cells and whether therapeutic agents can be efficiently delivered to those cells is still unclear. In this study, we demonstrate that primary AML cells harboring the chromosomal translocation t(8;21) are critically dependent on the corresponding fusion gene, RUNX1::RUNX1T1, to suppress differentiation and maintain stemness. Silencing RUNX1::RUNX1T1 expression using siRNA-loaded lipid nanoparticles induces substantial changes in chromatin accessibility, thereby redirecting the leukemia-associated transcriptional network towards a myeloid differentiation program. Single-cell analyses reveal that this transcriptional reprogramming is associated with the depletion of immature stem and progenitor-like cell populations, accompanied by an expansion of granulocytic and eosinophilic/mast cell-like populations with impaired self-renewal capacity. These findings underscore the essential role of RUNX1::RUNX1T1 in sustaining AML and highlight the therapeutic potential of targeting fusion gene expression in primary AML cells.

The CASSIOPEIA trial (NCT02541383) demonstrated superior progression-free survival (PFS) with the addition of daratumumab to bortezomib, thalidomide, and dexamethasone (D-VTd) induction/consolidation and with daratumumab maintenance versus observation in transplant-eligible newly diagnosed multiple myeloma (NDMM) patients. The companion study, CASSIOPET, assessed the prognostic value of pre-maintenance (PM) PET/CT response based on the standardized Deauville score on PFS and overall survival (OS) in addition to bone marrow (BM) minimal residual disease (MRD) detection by multiparameter flow cytometry (MFC) at 10-5 level. PM PET/CT was available for 225 patients: 112 patients treated with daratumumab after D-VTd (59) or VTd (53) and 113 patients followed by observation after D-VTd (56) or VTd (57). At PM, 92% of the 175 baseline PET-positive patients achieved PET-negativity, with a longer PFS in univariate analysis (HR, 0.48; P = 0.019) and a major trend of prolonged OS (HR, 0.37; P = 0.056). In univariate analysis, patients who achieved both PET and MFC negativity were found to have a better PFS (HR, 0.39; P<0.0001) than those who had at least one positive result. In daratumumab-treated patients, PM PET-negativity was associated with prolonged PFS and OS in univariate analysis (HR, 0.35; P = 0.0023 and HR, 0.32, P = 0.033, respectively) and double MFC and PET-negativity was independently associated with PFS by multivariate analysis (HR, 0.39, P = 0.0006). This study confirms the prognostic relevance of a PM PET response in NDMM patients treated with daratumumab in addition to MRD detection by MFC at the BM level.

The prognostic heterogeneity of multiple myeloma is mainly driven by genomic features of myeloma cells. The International Myeloma Society (IMS) / International Myeloma Working Group (IMWG) recently proposed a high-risk (HR) genomic model in order to have a consensus definition of genomic risk. We performed NGS panel in 6528 new diagnosed myeloma patients (NDMM) and 1583 patients at first relapse, between 2019 and 2024. We observed that 22.4% of patients at diagnosis and 36.7% at first relapse were HR according to the Consensus Genomic Staging (CGS). Clinical data were available for 2695 patients at diagnosis. After a median follow-up of 35 months, the median PFS was 30 months for HR NDMM patients, vs 51 months for standard-risk (SR) (p<0.0001). HR cytogenetic criteria from the Revised-ISS score were not able to discriminate patients in HR nor SR IMS/IMWG genomic subgroups. Looking at each criteria independently, we found that the presence of del(17p), TP53 mutation, biallelic del(1p32), or the combination of intermediate risk cytogenetics (gain 1q, del(1p32), t(4;14), t(14;16), t(14;20)) significantly reduces the PFS compared with standard-risk patients. Moreover, patients cumulating several criteria had an even worse prognosis. Among SR patients according to the genomic definition with normal creatinine, median PFS of those with high beta2-microglobulin was not significantly different from patients with normal beta2-microglobulin level. This study validates the IMS/IMWG genomic definition of high-risk myeloma in a large cohort of patients diagnosed from 2019.

Acute myeloid leukemias (AMLs) have an overall poor prognosis with many high-risk cases co-opting stem-cell gene regulatory programs, yet the mechanisms through which this occurs remain poorly understood. Increased expression of the stem-cell transcription factor, MECOM, underlies one key driver mechanism in largely incurable AMLs. How MECOM results in such aggressive AML phenotypes remains unknown. To address existing experimental limitations, we engineered and applied targeted protein degradation with functional genomic readouts to demonstrate that MECOM promotes malignant stem-cell-like states by directly repressing pro-differentiation gene regulatory programs. Remarkably and unexpectedly, a single node in this network, a MECOM-bound cis-regulatory element located 42 kb downstream of the myeloid differentiation regulator CEBPA is both necessary and sufficient for maintaining MECOM-driven leukemias. Importantly, targeted activation of this regulatory element promotes differentiation of these aggressive AMLs and reduces leukemia burden in vivo. These findings suggest a broadly applicable approach for functionally dissecting oncogenic gene regulatory networks to inform improved therapeutic strategies.

The recent approval of adeno-associated virus (AAV)-based gene therapies for haemophilia A (HA) represents a major advancement in the management of this X-linked bleeding disorder, offering multi-year bleed protection and improved quality of life over factor VIII (FVIII) replacement. However, challenges remain-including concerns over long-term durability of expression and the difficulty of packaging the oversized FVIII transgene into AAV vectors. To address these limitations, we developed AAV8-Bi8, a liver-directed gene therapy encoding Bi8, a novel 54.5 kDa FVIII mimetic antibody. Bi8 is expressed as a compact, single-chain tandem scFv and is delivered via a 4.4 kb expression cassette packaged within AAV8 capsids-well within the vector packaging capacity. In vitro, Bi8 demonstrated FVIII mimetic activity and effectively corrected FVIII-deficient human plasma to levels comparable with emicizumab, the current market standard. In vivo, a single administration of AAV8-Bi8 in FVIII-deficient mice resulted in dose-dependent, durable expression of Bi8, complete phenotypic correction of bleeding, and therapeutic equivalence to both emicizumab-treated and wild-type animals. Importantly, no toxicity or anti-drug antibody responses were observed. This approach, based on delivering FVIII mimetic antibodies through AAV rather than truncated FVIII transgenes, could provide a more flexible and efficient platform for gene therapy in haemophilia A. AAV8-Bi8 has the potential to offer sustained, life-long haemostatic control, including in patients who have developed inhibitors to FVIII.

Talquetamab, a GPRC5D-targeting bispecific antibody for relapsed/refractory multiple myeloma (RRMM), plus daratumumab may lead to deeper and more durable responses than either therapy alone. In the phase 1b TRIMM-2 study, patients with RRMM (at least 3 prior lines of therapy or double refractory to a proteasome inhibitor and an immunomodulatory drug) received subcutaneous talquetamab 0.4 mg/kg weekly (QW; "QW cohort") or 0.8 mg/kg every other week (Q2W; "Q2W cohort") plus daratumumab 1800 mg per the approved schedule. The primary end point was safety. Secondary end points included overall response and duration of response. Progression-free survival was an exploratory end point. Sixty-five patients (median 5 prior lines of therapy; 61.5% triple-class refractory; 24.6% bispecific antibody-exposed) received talquetamab plus daratumumab (QW, n = 14; Q2W, n = 51; median follow-up: 18.6 months). Most common adverse events were oral events, skin events, cytokine release syndrome, and infections. Grade 3/4 events occurred in 81.5%. Two patients had dose-limiting toxicities, both in the Q2W cohort (grade 3 stomatitis/oral mucositis and grade 3 maculopapular rash). Responses occurred in 71.4% (QW cohort) and 82.4% (Q2W cohort) of patients. Median progression-free survival was 23.3 and 21.2 months, respectively, in each cohort. Pharmacodynamic results suggest the immunomodulatory action of daratumumab contributes to a conducive environment for talquetamab by reducing immunosuppressive cells. Talquetamab plus daratumumab demonstrated promising efficacy outcomes in heavily pretreated patients, with a safety profile consistent with each agent as monotherapy. ClinicalTrials.gov ID: NCT04108195.

In this response-adapted clinic trial with daratumumab monotherapy for elderly newly diagnosed multiple myeloma (MM), we identified target antigen expression, a plasma cell phenotype, and an activated immune microenvironment (iTME) as critical features associated with response to CD38 monoclonal antibody therapy. Here, patients achieving a partial response (PR) after 2 cycles continued daratumumab, otherwise lenalidomide or bortezomib was added. This strategy resulted in an overall response rate of 97% and low rates of adverse events, with 37% of patients able to continue daratumumab monotherapy. Importantly, we found that higher CD38 expression, plasma cell gene expression programming, and an activated iTME were associated with patients who were able to continue daratumumab therapy alone. In contrast, patients requiring the addition of lenalidomide or bortezomib had increased expression of adhesion, TNF signaling, KRAS signaling, and B cell programs as well as an immunosuppressed iTME. Tracking of clonal dynamics illustrated the selection of subclones enriched for de novo resistance gene expression programs after only two cycles of daratumumab monotherapy. Upon relapse, daratumumab refractory MM cells were characterized by the expansion of pre-existing minor subclones with mixed transcriptomic programs containing the plasma cell phenotype with decreased CD38 and maintenance of resistance programs, suggesting development of acquired resistance involves an uncoupling of transcriptional programs present in therapy naïve tumors. To our knowledge, this is the first study to demonstrate the effectiveness of response-adapted daratumumab treatment and describe critical biomarkers of single-agent daratumumab sensitivity in vulnerable, therapy naïve MM patients. NCT04151667.

Telomere length shortening has been associated with genomic instability and acquisition of molecular lesions, but these processes have not been systematically studied across large cohorts of myeloid neoplasia (MN). As proof of concept for a novel, cross-validated WGS-based method of telomere content (TC) determination combined with mutations, transcriptomics, and functional assays, we studied TC in correlation with specific molecular features of a large cohort (n=1804) of MN patients including acute myeloid leukemia (AML) and myelodysplastic syndrome. When compared to healthy subjects and patients with non-clonal diseases such as persistent polyclonal B cell lymphocytosis, both MN and non-malignant controls with clonal disease, such as paroxysmal nocturnal hemoglobinuria and aplastic anemia, exhibited decreased TC. Furthermore, we show that TC is lowered in adult MN abrogating correlation with age with considerable TC diversification among certain morphologic and molecular subtypes. For instance, AML harbored the lowest TC. Furthermore, MN originating from a more mature cell of origin (e.g., APL), and those characterized by hyperproliferative driver mutations (e.g., RAS pathway genes) had lower TC, possibly indicating a loss of telomere maintenance capacity. In contrast, MN subtypes arising in a context of profound genetic alterations, such as TP53 mutations and complex karyotype, exhibited a relatively higher/preserved TC compared to other mutations. This phenomenon did not involve alternative lengthening processes but was rather consistent with an increased TC due to preserved activity of the telomerase complex. Our results describe a common and genotype-specific telomeric make-up of a large cohort of patients with MN providing a molecular benchmark for future therapeutic targeting of the telomere machinery.