We report on 8 patients with ankyrin repeat domain 26 (ANKRD26)-related thrombocytopenia 2 (ANKRD26-RT) with elevated bone marrow myeloblasts and dysmegakaryopoiesis, without somatic genetic abnormalities or progression to malignancy during long-term observation, findings which may constitute inherent ANKRD26-RT biology rather than progression to myeloid malignancy.

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic cell transplantation (HCT). Palifermin, a recombinant N-truncated keratinocyte growth factor (KGF), protects epithelial tissues, including the thymus and gut. Although high-dose KGF prevents GVHD in preclinical models, lower doses of palifermin were ineffective in humans. We conducted a phase 1/2 trial evaluating high-dose palifermin for preventing severe chronic GVHD (CGVHD) in matched unrelated donor T-cell replete peripheral blood HCT after reduced-intensity conditioning (RIC). Using a 3+3 design, we determined the recommended phase 2 dose (RP2D), followed by an expansion phase. Palifermin (180-720 μg/kg) was given on day -7 before HCT. All 31 patients received fludarabine/cyclophosphamide RIC with tacrolimus, methotrexate, and sirolimus for GVHD prophylaxis. Palifermin was well tolerated, with self-limiting rash and pancreatic enzyme elevations as notable grade 3/4 adverse events. The RP2D was 720 μg/kg. Remarkably, no patients at this dose developed grade 2 to 4 acute GVHD (AGVHD [0/19]), although severe CGVHD rates (primary end point) remained unchanged compared to historical controls. Posttransplant lymphocyte phenotyping suggests palifermin modulates regulatory and naïve CD4+ T-cell numbers. These findings indicate that high-dose palifermin with RIC is safe and may prevent AGVHD, although it did not affect CGVHD rates in this study. This trial was registered at www.ClinicalTrials.gov as #NCT02356159.

Coagulation factor Va (FVa) is the cofactor component of the prothrombinase complex required for rapid generation of thrombin from prothrombin in the penultimate step of the coagulation cascade. In addition, FVa is a target for proteolytic inactivation by activated protein C (APC). Like other protein-protein interactions in the coagulation cascade, the FVa-APC interaction has long posed a challenge to structural biology and its molecular underpinnings remain unknown. A recent cryogenic electron microscopy (cryo-EM) structure of FVa has revealed the arrangement of its A1-A2-A3-C1-C2 domains and the environment of the sites of APC cleavage at R306 and R506. Here, we report the cryo-EM structure of the FVa-APC complex at 3.15 Å resolution in which the protease domain of APC engages R506 in the A2 domain of FVa through electrostatic interactions between positively charged residues in the 30-loop and 70-loop of APC and an electronegative surface of FVa. The auxiliary γ-carboxyglutamic acid and epidermal growth factor domains of APC are highly dynamic and point to solvent, without making contacts with FVa. Binding of APC displaces a large portion of the A2 domain of FVa and projects the 654VKCIPDDDEDSYEIFEP670 segment as a "latch," or exosite ligand, over the 70-loop of the enzyme. The latch induces a large conformational change of the autolysis loop of APC, which in turn promotes docking of R506 into the primary specificity pocket. The cryo-EM structure of the FVa-APC complex validates the bulk of existing biochemical data and offers molecular context for a key regulatory interaction of the coagulation cascade.

Sickle cell hemoglobin C (HbSC) disease results from compound heterozygosity of hemoglobin S (HbS) and hemoglobin C (HbC), comprising 30% of sickle cell disease (SCD). HbC induces red blood cell (RBC) dehydration/xerocytosis, which promotes sickling. HbSC-SCD causes significant morbidity despite being milder than homozygous HbSS-SCD. Current research/treatment strategies have focused on HbSS-SCD, whereas patients with HbSC are deprived of disease-modifying/transformative therapies because of lack of preclinical models. We generated HbSC mice, which resemble human HbSC-SCD: HbSC erythrocytes showed marked xerocytosis. Anemia, hemolysis, inflammation, and organ damage were milder than HbSS mice but hypoxia/reperfusion injury was similar. Retinopathy developed at higher frequency than HbSS mice (66.7% vs 16.7%; P < .05), as in patients with HbSC-SCD. Although HbSC RBCs sickled at lower oxygen tension than HbSS RBCs, they did not completely recover deformability after hypoxia/reoxygenation. Using the HbSC mice, we studied the mechanism by which hydroxyurea causes significant clinical benefit in patients with HbSC-SCD, despite minimal/modest increases in fetal Hb (HbF). We found hydroxyurea had distinct non-HbF and HbF effects. Hydroxyurea did not increase HbF in adult HbSC/HbSS mice but reduced RBC reactive oxygen species, ferryl Hb, and Heinz-body formation, thereby reducing membrane damage; however, RBC hydration was unaffected. When given to unborn pups before γ-globin expression was switched off, and continued postnatally, we could induce HbF in both HbSC and HbSS mice (higher HbF in HbSS vs HbSC mice). Minimal increases in HbF (∼1%) improved HbSC RBC hydration. Peak HbF levels of 7% in HbSC mice abrogated sickling. Overall, this HbSC model will help bridge the knowledge gap in mechanistic/therapeutic studies in this neglected disease.

Heavy menstrual bleeding (HMB) is a widespread occurrence among women of reproductive age and inflicts a substantial impact on their well-being and on health care expenses. To better characterize the genetic architecture of HMB, we conducted a meta-analysis of the summary statistics of genome-wide association studies (GWAS) from 5 biobanks that included up to 84 633 HMB cases and 598 195 controls from several ancestries. Of the 21 signals significantly associated with HMB in a discovery GWAS meta-analysis that combined 4 biobanks, 20 had a concordant direction of effect in the remaining cohort, including 10 that were significantly replicated. By combining the discovery and replication data sets, 15 additional signals were identified in subsequent meta-analyses. These genetic analyses identified 36 signals (33 novel) that were significantly associated with HMB, and gene prioritization techniques (eg, transcriptome-wide association studies, polygenic priority score) subsequently revealed likely causal genes. Notable discoveries included the strong protective effect of the F5-Leiden variant (rs6025-T; odds ratio, 0.75; P = 6.8 × 10-33); variants at the FSHB and LHB/CGB loci, both involved in hormone production regulation; and several signals near genes involved in the Wnt/β-catenin signaling pathway. We also observed strong and significant genetic correlations with disorders of the female genital tract, including uterine fibroids, endometriosis, or ovarian cysts. Overall, we identified 33 novel genetic loci associated with HMB, thereby significantly improving our understanding of the genetic etiology of this condition, which may provide new targets for the development of therapeutic strategies.

Chronic graft-versus-host disease (cGVHD) is characterized by dysregulation of the adaptive immune system, including an aberrant B-cell homeostasis after allogeneic hematopoietic stem cell transplantation (allo-SCT). It is uncertain, however, whether this B-cell dysregulation is a result of manifest cGVHD or develops as a sign of aberrant B lymphopoiesis after allo-SCT before cGVHD becomes apparent. To gain insight into the development of B-cell dysregulation before the onset of cGVHD, we analyzed B-cell subpopulations by multiparameter flow cytometry on days 90, 180, and 356 after allo-SCT in a prospective study design. After completion of follow-up, patients were assigned retrospectively to 3 groups according to onset of GVHD: (1) no GVHD (n = 17); (2) acute GVHD (aGVHD) without subsequent cGVHD (n = 32); and (3) cGVHD (n = 59). Although CD21lowCD11c+ B cells were increased in all groups, the frequency of CD20-CD38hi plasmablasts was significantly elevated already 90 days after allo-SCT in patients who subsequently developed cGVHD, compared to patients without GVHD or with aGVHD only (median of CD19+ cells, 5.9% vs 2.2% vs 2.2%; P = .0016 and .0304, respectively). Detailed molecular analysis of expanded plasmablasts revealed a dominance of the immunoglobulin A isotype, with molecular evidence for recent generation in mucosal sites and markers for intestinal homing. A large fraction of the clonally expanded plasmablasts produced antibodies that bound to subgroups of commensals known to produce short-chain fatty acids. In summary, our data suggest that dysregulated intestinal antibody responses against commensals contribute to the pathophysiology of cGVHD.

A recent Perspective in Blood suggested that previous evidence from over a decade ago established that a liberal rather than a restrictive blood transfusion strategy results in better outcomes in patients with anemia and either acute myocardial infarction or stable cardiovascular disease. Their premise was that physiological evidence, and a different interpretation of the Transfusion Requirements in Critical Care (TRICC) trial should have been sufficient to establish clinical practice. They also suggest that a more personalized approach to the administration of transfusions would have been made possible by including a usual-care arm in all transfusion trials. In this counterpoint Perspective, we describe how and why 2 discrete and common blood transfusion thresholds were selected in the TRICC, FOCUS, REALITY, and MINT trials. We explain why a usual-care arm would have been uninformative. We also propose that we still do not have evidence to provide firm transfusion recommendations in several specific subpopulations of patients, including those with stable atherosclerotic coronary artery disease. Finally, we provide our perspective on the state of existing evidence and on the clinical recommendations that should be adopted in practice.

No prospective study has evaluated the incidence of transplant-associated thrombotic microangiopathy (TA-TMA) in adult allogeneic hematopoietic cell transplant (HCT) recipients. The MIDAS (microangiopathy, endothelial damage in adults undergoing stem cell transplantation) Consortium conducted, to our knowledge, the first multicenter study to prospectively screen for TA-TMA. Longitudinal blood samples and detailed clinical data were collected weekly through day +100 and at months 5, 6, 9, and 12 from first allogeneic HCT recipients at 3 sites (The Ohio State University, Moffitt Cancer Center, and Roswell Park Comprehensive Cancer Center). Adjudication of TA-TMA diagnosis was reviewed in real time by 3 blinded independent reviewers. Incidence of TA-TMA was scored using 6 published criteria, as well as the center-reported diagnosis and MIDAS adjudication categorized as none, nonsevere, or severe TA-TMA. Incidence of severe TA-TMA by day +100 was similar across the 3 centers at 21.8%, with a median onset of 14.5 days in 239 patients. Incidence of nonrelapse mortality was 42% in severe compared with 8.4% in nonsevere and 5.8% in no-TMA groups (P < .001). Rise in serum creatinine as early as day +7 and occurrence of hypertension by day +14 after HCT were early indicators of severe TA-TMA. Our prospective study of systematic screening for TA-TMA identifies a higher incidence of a clinically impactful phenotype of TA-TMA than previously reported in adult HCT recipients. Our natural history study provides an essential foundation for urgently needed studies of TA-TMA prophylaxis and treatment in adults and suggests clinical value in our inexpensive screening strategy.

Orchestrating key homeostatic functions, mitochondria likely entail cancer vulnerabilities. Moreover, because of their bacterial ancestry, they can release potent immunogenic signals. In this study, we showed that the mitochondrial protease caseinolytic peptidase P (ClpP) is both a cell-intrinsic metabolic vulnerability and an actionable immunogenic trigger in multiple myeloma (MM). We found that ClpP messenger RNA is higher in bone marrow (BM)-purified malignant plasma cells than in normal or premalignant counterparts and that MM lines rank first in ClpP expression among human cancers. Moreover, we demonstrated that human MM cells are highly vulnerable to ClpP inhibition in vitro and in vivo. Surprisingly, MM cell dependence on ClpP was not accounted for by its acknowledged oxidative phosphorylation surveillance activity. Proteomic discovery of proteolytic targets, metabolomics, and metabolic tracing identified a critical control exerted by ClpP on ornithine aminotransferase abundance to sustain cytosolic biosynthesis of polyamines, which are essential for MM cells. Transcriptomics and targeted validation also revealed the activation of a cyclic GMP-AMP synthase (cGAS)-dependent type I interferon (IFN) response in ClpP-silenced MM cells, whose supernatants boosted dendritic cell activation and ability to stimulate IFN-γ production by T cells. In vivo, ClpP silencing reshaped the BM immune environment in immunocompetent mice by significantly expanding IFN-γ-producing CD4+ and CD8+ T cells and CD4+ T memory cells, while containing exhausted CD4+ T cells and myeloid-derived suppressor cells. Thus, ClpP is a newly identified addiction of MM cells whose inhibition not only exerts cell-intrinsic toxicity but also triggers otherwise indolent antitumoral immunity. Our findings yield a novel immunogenic chemotherapeutic framework with potential relevance to myeloma.

Previous reports have highlighted that some patients with low von Willebrand factor (VWF) with significant bleeding were diagnosed based on an isolated but persistent reduction in plasma VWF activity levels in the 30 to 50 IU/dL range. These patients had plasma VWF antigen (VWF:Ag) levels >50 IU/dL and thus had qualitative low VWF (low VWF-QL) rather than quantitative low VWF. Although the clinical importance of functional VWF defects in type 2 von Willebrand disease (VWD) is well recognized, the translational implications of mild functional defects in patients with low VWF-QL have not been defined. To address this clinically important question, we combined low VWF data sets from the low VWF in Ireland cohort and the low VWF in Erasmus MC studies. Overall, we observed that low VWF-QL was common and accounted for ∼50% of our combined low VWF cohort. Importantly, our findings demonstrated that many of these patients with mild isolated functional VWF defects in the 30 to 50 IU/dL range had significant bleeding phenotypes, although their plasma VWF:Ag levels were within the normal range. In addition, we further showed that low VWF-QL is a distinct clinicopathological entity compared to type 2 VWD. Finally, our studies highlighted that low VWF-QL is predominantly caused by abnormalities in VWF biosynthesis within endothelial cells that are occurring largely independent of identifiable pathological VWF sequence variants. Cumulatively, these novel observations have important clinical implications for the diagnosis and management of patients with mild functional VWF defects.

Modakafusp alfa is a first-in-class immunocytokine-directing interferon alfa to CD38+ cells. Our previous phase 1/2 trial identified 2 potential phase 2 doses of modakafusp alfa for patients with relapsed/refractory multiple myeloma (RRMM): 1.5 or 3 mg/kg every 4 weeks. The overall response rate (ORR) among 30 patients treated at 1.5 mg/kg was 43%. This phase 2 dose optimization study randomized 147 patients with triple-class refractory disease and ≥3 previous lines of therapy 1:1 to modakafusp alfa 120 mg (n = 71) or 240 mg (n = 75) every 4 weeks (fixed-dose equivalents of 1.5 and 3 mg/kg every 4 weeks). Patients had received a median of 6 previous lines of therapy; 66% were penta-exposed and 45% had previously been exposed to anti-B-cell maturation antigen (BCMA) therapy. Modakafusp alfa development was discontinued for strategic reasons by the sponsor and the study was terminated early. At median follow-up of 7.3 and 7.6 months in the 120- and 240-mg arms, ORRs were 32% and 41%, and median progression-free survival was 4.1 and 5.3 months, respectively. ORRs were higher in patients who had not received previous BCMA therapy (46% vs 29%). The most common treatment-related adverse events (TEAEs) in the 120- and 240-mg arms were thrombocytopenia (75% and 84%; grade ≥3, 55% and 61%; respectively) and neutropenia (68% and 73%; grade ≥3, 56% and 68%; respectively); 90% and 96% of patients, respectively, experienced grade ≥3 TEAEs; 39% and 44%, respectively, experienced serious TEAEs. Our results confirm the efficacy of single-agent modakafusp alfa for patients with RRMM. This trial was registered at www.clinicaltrials.gov as #NCT03215030.