Calprotectin, a calcium- and zinc-binding protein that comprises the subunits S100A8 and S100A9, has been extensively studied as a biomarker of gastrointestinal (GI) inflammation through fecal and serum analyses. However, its role in intestinal tissue remains poorly understood because of the limited availability of biopsy specimens. In this study, we analyzed S100A8 and S100A9 messenger RNA (mRNA) expression in 579 intestinal biopsy specimens from allogeneic stem cell transplantation recipients and observed a strong association with acute GI graft-versus-host disease (aGI-GVHD; P< .001). Neutrophil infiltration correlated with the severity of aGI-GVHD (P< .001), and calprotectin expression was strongly linked to Toll-like receptor 4 (TLR4; P< .001) and TLR2 (P< .001) expression. Both TLR4 and aGI-GVHD were associated with elevated calprotectin mRNA levels (P< .001). When patients received broad-spectrum antibiotics at disease onset, calprotectin expression was suppressed (S100A8, P = .001; S100A9, P = .01). GI site-specific differences in calprotectin expression were identified: during severe aGI-GVHD, levels increased up to 30-fold in the small intestine and up to fivefold in the large intestine with respect to mild or no aGI-GVHD, whereas under homeostasis, the large intestine exhibited higher baseline calprotectin (P = .001). The high clinical relevance of this finding is evident from the observation that calprotectin expression was prognostic for transplant-related mortality. Our study suggests that (1) calprotectin is a potential biopsy biomarker in aGI-GvHD and (2) calprotectin expression and neutrophil infiltration possibly indicate translocation of microbiota, which (3) may be modulated by antibiotics.

Immune thrombocytopenia (ITP) is characterized by the overproduction of antiplatelet autoantibodies. Although B-cell depletion therapies show promise in ITP, their high relapse rates suggest a potential de novo breakdown of tolerance during an early stage of B-cell development. Here, we investigated how central B-cell tolerance mechanisms affect autoantibody production in ITP. Paired single-cell RNA/B-cell receptor (BCR) sequencing and bulk BCR sequencing revealed reduced V-J genomic distances in immunoglobulin kappa-chain (IGK) genes within bone marrow and peripheral B cells from patients with ITP, along with decreased expression of recombination-activating gene in the immature B cells, suggesting insufficient receptor editing. Single-cell antibody cloning demonstrated increased autoreactive and polyreactive naïve B cells in ITP, indicating defective central B-cell tolerance. Through an in vivo study, we established a causal link between receptor editing defects and antiplatelet antibody production, validating the immature B-cell stage as the key phase of dysregulation. These findings suggest that insufficient receptor editing of immature B cells triggers central B-cell tolerance deficiency and autoantibody accumulation in ITP.

To identify genetic variants that influence myeloproliferative neoplasm (MPN) phenotypes, we undertook a 2-stage patient-only genome-wide association study. MPN subtypes (essential thrombocythemia [ET]; polycythemia vera [PV]) were compared with each other to healthy controls and stratified analyses was performed for chromosome 9p aberrations, JAK2 V617F mutation burden, and sex. The ET vs PV analysis identified known associations: (1) at HBS1L-MYB that increased ET risk (Pmeta = 7.93 × 10-6, odds ratio [OR] = 1.28) and reduced PV risk (Pmeta = 9.43 × 10-5, OR = 0.81) and (2) at GFI1B-GTF3C5 that predisposed to PV only (Pmeta = 1.43 × 10-9, OR = 1.38). Two further linked intronic variants, rs2425786 and rs2425788, at CDH22/CD40 were significant in females only (Pmeta = 2.67 × 10-8), with predisposition to PV (Pmeta = .0006, OR = 1.3) and reduction of ET risk (Pmeta = 7.82 × 10-5, OR = 0.75). A polygenic risk score consisting of 48 variants from 31 loci demonstrated moderate discriminative performance for ET and PV (area under the curve [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. Because 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 extramedullary relapse after allogeneic stem cell transplantation, >90% of T/natural killer 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. Furthermore, HLA class II was downregulated in 4 of 7 leukemia cutis specimens, consistent with an immune escape phenotype in 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 1 leukemia cutis throughout consecutive immune checkpoint blockade with ipilimumab followed by nivolumab showed a consistently high degree of overlap between local and circulating T-cell receptor 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, anatomic 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, 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 such as follicular and lymphoplasmacytic lymphomas; integrating morphology, immunophenotype, and molecular data is essential. Transformation to aggressive diffuse large B-cell lymphoma occurs in 3% to 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 including 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.

Immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (IgM-MGUS) and asymptomatic Waldenström macroglobulinemia (WM; aWM) are precursor conditions of symptomatic WM with an annual 1.5% to 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 229 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 with the stable aWM (aWMst) group, whereas MYD88L265 variant allele frequency was significantly higher in patients with aWMpr than those with aWMst. In addition, patients with IgM-MGUS with MYD88WT genotype showed a distinct genomic profile compared with 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. 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, whereas 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, we identified that SCARF1 and complement receptor 3 are required for C1q-NETosis. Given NETs contain procoagulatory 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 with 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 regulate NET-related coagulation. C1q-NETs also activate the coagulation factors XII and XI, facilitating both intrinsic coagulation and kallikrein-dependent bradykinin production. This study elucidates how NETs regulate both procoagulatory and anticoagulatory components that may influence the 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. Notably, ubiquitin specific peptidase 25 (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 Ub-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 decreased 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 older population.

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 demonstrated superior progression-free survival and a similar safety profile with polatuzumab vedotin plus rituximab, cyclophosphamide, doxorubicin, and prednisone (Pola-R-CHP) vs R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) 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. This trial was registered at www.clinicaltrials.gov as NCT03274492.

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, the extent to which 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 small interfering RNA (siRNA)-loaded lipid nanoparticles induces substantial changes in chromatin accessibility, thereby redirecting the leukemia-associated transcriptional network toward 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 demonstrated superior progression-free survival (PFS) with the addition of daratumumab to bortezomib, thalidomide, and dexamethasone (D-VTd) induction/consolidation, and with daratumumab maintenance vs observation in transplant-eligible patients with newly diagnosed multiple myeloma (NDMM). The companion study, CASSIOPET, assessed the prognostic value of premaintenance (PM) positron emission tomography (PET)/computed tomography (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 bortezomib, thalidomide, and dexamethasone (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 (P = .019) and a major trend of prolonged OS (P = .056). In univariate analysis, patients who achieved both PET and MFC negativity were found to have a better PFS (P < .0001) than those who had at least 1 positive result. In daratumumab-treated patients, PM PET negativity was associated with prolonged PFS and OS in univariate analysis (P = .0023 and P = .033, respectively), and double MFC and PET negativity was independently associated with PFS by multivariate analysis (P = .0006). This study confirms the prognostic relevance of a PM PET response in patients with NDMM treated with daratumumab in addition to MRD detection by MFC at the BM level. This trial was registered at ww.clinicaltrials.gov as #NCT02541383.

The prognostic heterogeneity of multiple myeloma is mainly driven by the genomic features of myeloma cells. The International Myeloma Society (IMS)/International Myeloma Working Group (IMWG) recently proposed a high-risk (HR) genomic model to have a consensus definition of genomic risk. We performed next-generation sequencing in the form of a panel on samples from 6528 patients with newly diagnosed multiple myeloma (NDMM) and 1583 patients at first relapse between 2019 and 2024. We observed that 22.4% of patients at diagnosis and 36.7% of patients at first relapse were classified as high risk according to the Consensus Genomic Staging. Clinical data were available for 2695 patients at diagnosis. After a median follow-up of 35 months, the median progression-free survival (PFS) was 30 months for patients with HR NDMM and 51 months for standard-risk (SR) patients (P< .0001). The HR cytogenetic criteria from the Revised- International Staging System score were not able to differentiate between HR and SR patients based on the IMS/IMWG genomic subgroups. Looking at each criterion 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 reduced the PFS when compared with SR patients. Moreover, patients with several cumulating criteria had an even worse prognosis. Among SR patients, classified according to the genomic definition with normal creatinine, the median PFS for those with high β2-microglobulin was not significantly different from that of patients with normal β2-microglobulin level. This study validated the IMS/IMWG genomic definition of HR myeloma in a large cohort of patients diagnosed from 2019 onwards.

Acute myeloid leukemias (AMLs) have an overall poor prognosis with many high-risk cases co-opting stem cell gene regulatory programs, but the mechanisms through which these programs are propogated remain poorly understood. The increased expression of the stem cell transcription factor, MECOM, underlies a key driver mechanism in largely incurable AMLs. However, 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 prodifferentiation gene regulatory programs. Remarkably and unexpectedly, a single node in this network, a MECOM-bound cis-regulatory element located 42 kilobase (kb) downstream of the myeloid differentiation regulator CEBPA is both necessary and sufficient for maintaining MECOM-driven leukemias. Importantly, the 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 hemophilia A (HA) represents a major advancement in the management of this X-linked bleeding disorder, offering multiyear 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-kilodalton FVIII-mimetic antibody. Bi8 is expressed as a compact, single-chain tandem, single-chain fragment variable, and is delivered via a 4.4-kilobase 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 antidrug 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 HA. AAV8-Bi8 has the potential to offer sustained, lifelong hemostatic control, including in patients who have developed inhibitors to FVIII.

Talquetamab, a G protein-coupled receptor class C group 5 member D-targeting bispecific antibody for relapsed/refractory multiple myeloma (R/R MM), plus daratumumab, may lead to deeper and more durable responses than either therapy alone. In the phase 1b TRIMM-2 study, patients with R/R MM (at least 3 previous 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 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 previous 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 of 18.6 months). Most common adverse events were oral events, skin events, cytokine release syndrome, and infections. Grade 3 or 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 patients with heavily pretreated disease, with a safety profile consistent with each agent as monotherapy. This trial was registered at www.clinicaltrials.gov as #NCT04108195.