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Modulation of neutrophil recruitment and function is crucial for targeting inflammatory cells to sites of infection to combat invading pathogens while, at the same time, limiting host tissue injury or autoimmunity. The underlying mechanisms regulating recruitment of neutrophils, 1 of the most abundant inflammatory cells, have gained increasing interest over the years. The previously described classical recruitment cascade of leukocytes has been extended to include capturing, rolling, adhesion, crawling, and transmigration, as well as a reverse-transmigration step that is crucial for balancing immune defense and control of remote organ endothelial leakage. Current developments in the field emphasize the importance of cellular interplay, tissue environmental cues, circadian rhythmicity, detection of neutrophil phenotypes, differential chemokine sensing, and contribution of distinct signaling components to receptor activation and integrin conformations. The use of therapeutics modulating neutrophil activation responses, as well as mutations causing dysfunctional neutrophil receptors and impaired signaling cascades, have been defined in translational animal models. Human correlates of such mutations result in increased susceptibility to infections or organ damage. This review focuses on current advances in the understanding of the regulation of neutrophil recruitment and functionality and translational implications of current discoveries in the field with a focus on acute inflammation and sepsis.

Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B-cell lymphocytosis (MBL), a CLL precursor state with a prevalence of up to 12% in aged individuals; however, the duration of MBL and the mechanisms of its evolution to CLL remain largely unknown. In this study, we sequenced the B-cell receptor (BcR) immunoglobulin heavy chain (IGH) gene repertoire of 124 patients with CLL and 118 matched controls in blood samples taken up to 22 years prior to diagnosis. Significant skewing in the BcR IGH gene repertoire was detected in the majority of patients, even before the occurrence of lymphocytosis and irrespective of the clonotypic IGH variable gene somatic hypermutation status. Furthermore, we identified dominant clonotypes belonging to major stereotyped subsets associated with poor prognosis up to 16 years before diagnosis in 14 patients with CLL. In 22 patients with longitudinal samples, the skewing of the BcR IGH gene repertoire increased significantly over time to diagnosis or remained stable at high levels. For 14 of 16 patients with available samples at diagnosis, the CLL clonotype was already present in the prediagnostic samples. Overall, our data indicate that the preclinical phase of CLL could be longer than previously thought, even in adverse-prognostic cases.

Chronic obstructive pulmonary disease (COPD) is associated with age and smoking, but other determinants of the disease are incompletely understood. Clonal hematopoiesis of indeterminate potential (CHIP) is a common, age-related state in which somatic mutations in clonal blood populations induce aberrant inflammatory responses. Patients with CHIP have an elevated risk for cardiovascular disease, but the association of CHIP with COPD remains unclear. We analyzed whole-genome sequencing and whole-exome sequencing data to detect CHIP in 48 835 patients, of whom 8444 had moderate to very severe COPD, from four separate cohorts with COPD phenotyping and smoking history. We measured emphysema in murine models in which Tet2 was deleted in hematopoietic cells. In the COPDGene cohort, individuals with CHIP had risks of moderate-to-severe, severe, or very severe COPD that were 1.6 (adjusted 95% confidence interval [CI], 1.1-2.2) and 2.2 (adjusted 95% CI, 1.5-3.2) times greater than those for noncarriers. These findings were consistently observed in three additional cohorts and meta-analyses of all patients. CHIP was also associated with decreased FEV1% predicted in the COPDGene cohort (mean between-group differences, −5.7%; adjusted 95% CI, −8.8% to −2.6%), a finding replicated in additional cohorts. Smoke exposure was associated with a small but significant increased risk of having CHIP (odds ratio, 1.03 per 10 pack-years; 95% CI, 1.01-1.05 per 10 pack-years) in the meta-analysis of all patients. Inactivation of Tet2 in mouse hematopoietic cells exacerbated the development of emphysema and inflammation in models of cigarette smoke exposure. Somatic mutations in blood cells are associated with the development and severity of COPD, independent of age and cumulative smoke exposure.

Observation is the current standard of care for patients with early-stage asymptomatic chronic lymphocytic leukemia (CLL), as chemotherapy-based interventions have failed to prolong survival. We hypothesized that early intervention with ibrutinib would be well tolerated and lead to superior disease control in a subgroup of early-stage patients with CLL. The phase 3, double-blind, placebo-controlled CLL12 trial randomly assigned asymptomatic, treatment-naïve Binet stage A CLL patients at increased risk of progression in a 1:1 ratio to receive ibrutinib (n = 182) or placebo (n = 181) at a dose of 420 mg daily. At a median follow-up of 31 months, the study met its primary endpoint by significantly improving event-free survival in the ibrutinib group (median, not reached vs 47.8 months; hazard ratio = 0.25; 95% confidence interval = 0.14-0.43, P < .0001). Compared with placebo, ibrutinib did not increase overall toxicity, yielding similar incidence and severity of adverse events (AEs). The most common serious AEs were atrial fibrillation, pneumonia, and rash in the ibrutinib group, and basal cell carcinoma, pneumonia, and myocardial infarction in the placebo group. Ibrutinib-associated risk for bleeding (33.5%) was decreased by prohibiting the use of oral anticoagulants through an amendment of the study protocol and by avoiding CYP3A4 drug–drug interactions. Ibrutinib confirms efficacy in CLL patients at an early stage with an increased risk of progression. However, the results do not justify changing the current standard of “watch and wait.” This trial was registered at www.clinicaltrials.gov as #NCT02863718.

Coagulation protease, factor VIIa (FVIIa), binds to endothelial cell protein C receptor (EPCR) and induces anti-inflammatory and endothelial barrier protective responses via protease-activated receptor-1 (PAR1)–mediated, biased signaling. Our recent studies had shown that the FVIIa-EPCR-PAR1 axis induces the release of extracellular vesicles (EVs) from endothelial cells. In the present study, we investigated the mechanism of FVIIa release of endothelial EVs (EEVs) and the contribution of FVIIa-released EEVs to anti-inflammatory and vascular barrier protective effects, in both in vitro and in vivo models. Multiple signaling pathways regulated FVIIa release of EVs from endothelial cells, but the ROCK-dependent pathway appeared to be a major mechanism. FVIIa-released EEVs were enriched with anti-inflammatory microRNAs (miRs), mostly miR10a. FVIIa-released EEVs were taken up readily by monocytes/macrophages and endothelial cells. The uptake of FVIIa-released EEVs by monocytes conferred anti-inflammatory phenotype to monocytes, whereas EEV uptake by endothelial cells resulted in barrier protection. In additional experiments, EEV-mediated delivery of miR10a to monocytes downregulated the expression of TAK1 and activation of the NF-κB–mediated inflammatory pathway. In in vivo experiments, administration of FVIIa-released EEVs to wild-type mice attenuated LPS-induced increased inflammatory cytokines in plasma and vascular leakage into vital tissues. The incorporation of anti-miR10a into FVIIa-released EEVs diminished the ability of FVIIa-released EEVs to confer cytoprotective effects. Administration of the ROCK inhibitor Y27632, which significantly inhibits FVIIa release of EEVs into the circulation, to mice attenuated the cytoprotective effects of FVIIa. Overall, our study revealed novel insights into how FVIIa induces cytoprotective effects and communicates with various cell types.

Sickle cell disease (SCD) is characterized by hemolytic anemia, which can trigger oxidative stress, inflammation, and tissue injury that contribute to disease complications. Bone marrow mesenchymal stromal cells (MSCs) tightly regulate hematopoietic stem cell (HSC) homeostasis in health and disease, but their functionality in SCD remains unclear. We identified for the first time that murine SCD MSCs have altered gene signatures, reduced stem cell properties, and increased oxidative stress, due in part to hemolysis. Murine SCD MSCs had lower HSC maintenance ability in vitro and in vivo, as manifested by increased HSC mobilization and decreased HSC engraftment after transplant. Activation of Toll-like receptor-4 through p65 in MSCs further contributed to MSC dysfunction. Transfusions led to an improved MSC and HSC oxidative state in SCD mice. Improving the regulation between MSCs and HSCs has vital implications for enhancing clinical HSC transplantation and gene therapy outcomes and for identification of new molecular targets for alleviating SCD complications.

Acute graft-versus-host disease (GVHD) is a major life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). The classical target organs of acute GVHD include the intestines, liver, and skin. The damage of these organs is relatively easy to detect for the clinician as diarrhea, increased bilirubin, and rash. However, there is increasing evidence that other organs, where the acute damage is less apparent or more difficult to distinguish from drug toxicity, such as the central nervous system, lungs, ovaries and testis, thymus, bone marrow, and kidney, can be target organs of acute GVHD. Here, we review current evidence for nonclassical manifestations of acute GVHD in rodent models and in patients and discuss them in the context of novel emerging therapies for GVHD. A better understanding of the involvement of nonclassical GVHD target organs may help to improve patient outcomes after allo-HCT.

MYD88 and CXCR4 mutations are common in Waldenström macroglobulinemia (WM). Mutated CXCR4 (CXCR4Mut) impacts BTK-inhibitor response. We conducted a phase 1 trial of the CXCR4-antagonist ulocuplumab with ibrutinib in this first-ever study to target CXCR4Mut in WM. Ibrutinib was initiated at 420 mg/d with cycle 1 and continued until intolerance or progression; ulocuplumab was given cycles 1 to 6, with a 3 + 3 dose-escalation design. Each cycle was 4 weeks. Thirteen symptomatic patients, of whom 9 were treatment-naive patients were enrolled. Twelve were evaluable for response. At best response, their median serum immunoglobulin M declined from 5574 to 1114 mg/dL; bone marrow disease decreased from 65% to 10%, and hemoglobin increased from 10.1 to 14.2 g/dL (P < .001). The major and VGPR response rates were 100% and 33%, respectively, with VGPRs observed at lower ulocuplumab dose cohorts. Median times to minor and major responses were 0.9 and 1.2 months, respectively. With a median follow-up of 22.4 months, the estimated 2-year progression-free survival was 90%. The most frequent recurring grade ≥2 adverse events included reversible thrombocytopenia, rash, and skin infections. Ulocuplumab dose-escalation did not impact adverse events. The study demonstrates the feasibility of combining a CXCR4-antagonist with ibrutinib and provides support for the development of CXCR4-antagonists for CXCR4Mut WM. This trial was registered at www.clinicaltrials.gov as #NCT03225716.

Gene therapy as a potential cure for sickle cell disease (SCD) has long been pursued, given that this hemoglobin (Hb) disorder results from a single point mutation. Advances in genomic sequencing have increased the understanding of Hb regulation, and discoveries of molecular tools for genome modification of hematopoietic stem cells have made gene therapy for SCD possible. Gene-addition strategies using gene transfer vectors have been optimized over the past few decades to increase expression of normal or antisickling globins as strategies to ameliorate SCD. Many hurdles had to be addressed before clinical translation, including collecting sufficient stem cells for gene modification, increasing expression of transferred genes to a therapeutic level, and conditioning patients in a safe manner that enabled adequate engraftment of gene-modified cells. The discovery of genome editors that make precise modifications has further advanced the safety and efficacy of gene therapy, and a rapid movement to clinical trial has undoubtedly been supported by lessons learned from optimizing gene-addition strategies. Current gene therapies being tested in clinical trial require significant infrastructure and expertise, given that cells must be harvested from and chemotherapy administered to patients who often have significant organ dysfunction and that gene-modification takes place ex vivo in specialized facilities. For these therapies to realize their full potential, they would have to be portable, safe, and efficient, to make an in vivo–based approach attractive. In addition, adequate resources for SCD screening and access to standardized care are critically important for gene therapy to be a viable treatment option for SCD.

Purpose Intolerance is the most common reason for kinase inhibitor (KI) discontinuation in CLL. Umbralisib a novel, highly selective PI3Kδ/CK1e inhibitor, is active and well tolerated in CLL patients. This phase 2 trial evaluated umbralisib in CLL patients who are intolerant to prior BTK or PI3K inhibitor therapy. Patients and methods In this phase 2 trial (NCT02742090), umbralisib was initiated at 800 mg oral daily in CLL patients requiring therapy per investigator discretion who were intolerant to prior BTK or PI3K inhibitor therapy, until progression or toxicity. Primary endpoint was progression-free survival (PFS). Secondary endpoints included time to treatment failure and umbralisib safety profile. DNA isolated from buccal swabs was genotyped for polymorphisms in CYP3A4, CYP3A5 and CYP2D6. Results Fifty-one patients were enrolled (44 BTKi and 7 PI3Kδi intolerant). Median age was 70 years (range 48-96), median of 2 prior lines of therapy (1-7), 24% had del17p and/or TP53 mutation, and 65% were IGHV unmutated. Most common AEs leading to prior KI discontinuation were rash (27%), arthralgia (18%), and atrial fibrillation (16%). Median progression free survival (PFS) was 23.5 months (95% CI 13.1-not estimable). 58% of patients were on umbralisib for a longer duration than prior KI. Most common (≥5%) grade ≥3 AEs on umbralisib (all causality) were neutropenia (18%), leukocytosis (14%), thrombocytopenia (12%), pneumonia (12%), and diarrhea (8%). Six patients (12%) discontinued umbralisib due to an AE. Eight patients (16%) had dose reductions and were successfully re-challenged. Conclusions Umbralisib is safe and effective in this BTK and alternate PI3K inhibitor intolerant CLL population. These are the first prospective data to confirm that switching from a BTK or alternate PI3K inhibitor to umbralisib can result in durable, well tolerated responses.

Vaccination using the adenoviral vector COVID-19 vaccine ChAdOx1 nCoV-19 (AstraZeneca) has been associated with rare vaccine-induced immune thrombotic thrombocytopenia (VITT). Affected patients test strongly positive in platelet factor 4 (PF4)/polyanion enzyme immunoassays (EIAs), and serum-induced platelet activation is maximal in the presence of PF4. We determined the frequency of anti-PF4/polyanion antibodies in healthy vaccinees and assessed whether PF4/polyanion EIA+ sera exhibit platelet-activating properties after vaccination with ChAdOx1 nCoV-19 (n = 138) or BNT162b2 (BioNTech/Pfizer; n = 143). In total, 19 of 281 participants tested positive for anti-PF4/polyanion antibodies postvaccination (All: 6.8% [95% confidence interval (CI), 4.4-10.3]; BNT162b2: 5.6% [95% CI, 2.9-10.7]; ChAdOx1 nCoV-19: 8.0% [95% CI, 4.5% to 13.7%]). Optical densities were mostly low (between 0.5 and 1.0 units; reference range

Polycythemia and pulmonary hypertension are 2 human diseases for which better therapies are needed. Upregulation of hypoxia-inducible factor-2α (HIF-2α) and its target genes, erythropoietin (EPO) and endothelin-1, causes polycythemia and pulmonary hypertension in patients with Chuvash polycythemia who are homozygous for the R200W mutation in the von Hippel Lindau (VHL) gene and in a murine mouse model of Chuvash polycythemia that bears the same homozygous VhlR200W mutation. Moreover, the aged VhlR200W mice developed pulmonary fibrosis, most likely due to the increased expression of Cxcl-12, another Hif-2α target. Patients with mutations in iron regulatory protein 1 (IRP1) also develop polycythemia, and Irp1-knockout (Irp1-KO) mice exhibit polycythemia, pulmonary hypertension, and cardiac fibrosis attributable to translational derepression of Hif-2α, and the resultant high expression of the Hif-2α targets EPO, endothelin-1, and Cxcl-12. In this study, we inactivated Hif-2α with the second-generation allosteric HIF-2α inhibitor MK-6482 in VhlR200W, Irp1-KO, and double-mutant VhlR200W;Irp1-KO mice. MK-6482 treatment decreased EPO production and reversed polycythemia in all 3 mouse models. Drug treatment also decreased right ventricular pressure and mitigated pulmonary hypertension in VhlR200W, Irp1-KO, and VhlR200W;Irp1-KO mice to near normal wild-type levels and normalized the movement of the cardiac interventricular septum in VhlR200Wmice. MK-6482 treatment reduced the increased expression of Cxcl-12, which, in association with CXCR4, mediates fibrocyte influx into the lungs, potentially causing pulmonary fibrosis. Our results suggest that oral intake of MK-6482 could represent a new approach to treatment of patients with polycythemia, pulmonary hypertension, pulmonary fibrosis, and complications caused by elevated expression of HIF-2α.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with thrombotic complications in adults, but the incidence of COVID-19-related thrombosis in children and adolescents is unclear. Most children with acute COVID-19 have mild disease, but coagulopathy has been associated with multisystem inflammatory syndrome in children (MIS-C), a postinfectious complication. We conducted a multicenter retrospective cohort study to determine the incidence of thrombosis in children hospitalized with COVID-19 or MIS-C and evaluate associated risk factors. We classified patients into 1 of 3 groups for analysis: COVID-19, MIS-C, or asymptomatic SARS-CoV-2. Among a total of 853 admissions (COVID-19, n = 426; MIS-C, n = 138; and asymptomatic SARS-CoV-2, n = 289) in 814 patients, there were 20 patients with thrombotic events (TEs; including 1 stroke). Patients with MIS-C had the highest incidence (9 [6.5%] of 138) vs COVID-19 (9 [2.1%] of 426) or asymptomatic SARS-CoV-2 (2 [0.7%] of 289). In patients with COVID-19 or MIS-C, a majority of TEs (89%) occurred in patients age ≥12 years. Patients age ≥12 years with MIS-C had the highest rate of thrombosis at 19% (9 of 48). Notably, 71% of TEs that were not present on admission occurred despite thromboprophylaxis. Multivariable analysis identified the following as significantly associated with thrombosis: age ≥12 years, cancer, presence of a central venous catheter, and MIS-C. In patients with COVID-19 or MIS-C, hospital mortality was 2.3% (13 of 564), but it was 28% (5 of 18) in patients with TEs. Our findings may help inform pediatric thromboprophylaxis strategies.

Friedreich ataxia (FRDA) is a frequent autosomal recessive disease caused by a GAA repeat expansion in the FXN gene encoding frataxin, a mitochondrial protein involved in iron-sulfur cluster (ISC) biogenesis. Resulting frataxin deficiency affects ISC-containing proteins and causes iron to accumulate in the brain and heart of FRDA patients. Here we report on abnormal cellular iron homeostasis in FRDA fibroblasts inducing a massive iron overload in cytosol and mitochondria. We observe membrane transferrin receptor 1 (TfR1) accumulation, increased TfR1 endocytosis, and delayed Tf recycling, ascribing this to impaired TfR1 palmitoylation. Frataxin deficiency is shown to reduce coenzyme A (CoA) availability for TfR1 palmitoylation. Finally, we demonstrate that artesunate, CoA, and dichloroacetate improve TfR1 palmitoylation and decrease iron overload, paving the road for evidence-based therapeutic strategies at the actionable level of TfR1 palmitoylation in FRDA.

Krabbe disease is a rare neurodegenerative disorder caused by a deficiency in galactocerebrosidase. The only effective treatment is hematopoietic stem cell transplantation (HSCT). Approximately 85% of Krabbe disease cases are the infantile subtypes, among which ∼20% are late infantile. Prior studies have demonstrated that HSCT is effective for early-infantile patients (0-6 months of age) who undergo transplantation while asymptomatic, compared with those receiving transplants while symptomatic. However, no studies evaluated the efficacy of HSCT for late-infantile patients (6-36 months). In this prospective, longitudinal study, patients were evaluated at a single site according to a standardized protocol. Survival analysis was performed using the Kaplan-Meier method. Differences between groups were estimated using mixed regression models to account for within-person repeated measures. Nineteen late-infantile patients underwent HSCT (March 1997 to January 2020). Compared with untreated patients, transplant recipients had a longer survival probability and improved cognitive and language function. Gross and fine motor development were most affected, with variable results. Asymptomatic patients benefitted the most from transplantation, with normal to near-normal development in all domains and some gross motor delays. Among symptomatic patients, those with disease onset at >12 months of age had better cognitive outcomes than untreated patients. Those with disease onset at ≤12 months were comparable to untreated patients. We found that HSCT prolonged the lifespan and improved the functional abilities of late-infantile patients with Krabbe disease, particularly those who underwent transplantation before onset of symptoms. In addition, our findings support prior literature that reclassifies late-infantile Krabbe disease to be symptom onset at 12 to 36 months of age.

Philadelphia chromosome–negative myeloproliferative neoplasms (MPNs), polycythemia vera, essential thrombocythemia, and primary myelofibrosis, are hematopoietic stem cell disorders that are defined by activating mutations in signal transduction pathways and are characterized clinically by the overproduction of platelets, red blood cells, and neutrophils, significant burden of disease-specific symptoms, and high rates of vascular events. The focus of this review is to critically reevaluate the clinical burden of thrombosis in MPNs, to review the clinical associations among clonal hematopoiesis, JAK2V617F burden, inflammation, and thrombosis, and to provide insights into novel primary and secondary thrombosis-prevention strategies.

Hereditary hemorrhagic telangiectasia (HHT) management is evolving because of the emergence and development of antiangiogenic therapies to eliminate bleeding telangiectasias and achieve hemostasis. This progress is reflected in recent clinical recommendations published in the Second International Guidelines for the Diagnosis and Treatment of HHT, in which systemic therapies including antiangiogenics and antifibrinolytics are now recommended as standard treatment options for bleeding. This review highlights the new recommendations especially relevant to hematologists in managing bleeding, anticoagulation, and anemia in patients with HHT.

Neutrophils are critical mediators of host defense in pathogen-induced and sterile inflammation. Excessive neutrophil activation has been associated with increased host pathology through collateral organ damage. The beneficial aspects of neutrophil activation, particularly in sterile inflammation, are less well defined. We observed accumulation of nuclear debris in the lungs of neutropenic mice exposed to acid-induced injury compared with wild type. Size analysis of DNA debris showed that neutropenic mice were unable to degrade extracellular DNA fragments. In addition, we found that neutrophils are able to differentially express DNA-degrading and repair-associated genes and proteins. Once neutrophils are at sites of lung inflammation, they are able to phagocytose and degrade extracellular DNA. This neutrophil-dependent DNA degradation occurs in a MyD88-dependent pathway. The increased DNA debris in neutropenic mice was associated with dysregulated alveolar repair and the phenotype is rescued by intratracheal administration of DNase I. Thus, we show a novel mechanism as part of the inflammatory response, in which neutrophils engulf and degrade extracellular DNA fragments and allow for optimal organ repair.

Ciraparantag, an anticoagulant reversal agent, is a small molecule specifically designed to bind noncovalently by charge-charge interaction to unfractionated heparin and low-molecular-weight heparin. It shows binding characteristics that are similar to those of direct oral anticoagulants (DOACs). A dynamic light-scattering methodology was used to demonstrate ciraparantag’s binding to the heparins and DOACs and its lack of binding to a variety of proteins including coagulation factors and commonly used drugs. Ciraparantag reaches maximum concentration within minutes after IV administration with a half-life of 12 to 19 minutes. It is primarily hydrolyzed by serum peptidases into 2 metabolites, neither of which has substantial activity. Ciraparantag and its metabolites are recovered almost entirely in the urine. In animal models of bleeding (rat tail transection and liver laceration), a single IV dose of ciraparantag given at peak concentrations of the anticoagulant, but before the bleeding injury, significantly reduced the blood loss. Ciraparantag, given after the bleeding injury, also significantly reduced blood loss. It appears to have substantial ability to reduce blood loss in animal models in which a variety of anticoagulants are used and has potential as a useful DOAC reversal agent.

Ferroportin (FPN), the only known cellular iron exporter, plays a central role in intracellular and systemic iron homeostasis, and its levels are regulated by hepcidin. Using genetic screens and models, Jiang and colleagues identified an E3 ubiquitin ligase, RNF217, that promotes the degradation of FPN and that, if deleted, interferes with iron homeostasis in a cell type– dependent manner. Further, they identified the iron-responsive demethylase TET1 as a key upstream regulator of RNF217. These data further refine our understanding of iron homeostasis and how it can be manipulated., Key Points RNF217 functions as a novel E3 ubiquitin ligase, mediating the degradation of the iron exporter FPN and regulating iron homeostasis.Rnf217 expression is upregulated by Tet1-mediated demethylation; loss of Tet1 stabilizes FPN and impairs the response to iron overload., Visual Abstract, Ferroportin (FPN), the body’s sole iron exporter, is essential for maintaining systemic iron homeostasis. In response to either increased iron or inflammation, hepatocyte-secreted hepcidin binds to FPN, inducing its internalization and subsequent degradation. However, the E3 ubiquitin ligase that underlies FPN degradation has not been identified. Here, we report the identification and characterization of a novel mechanism involving the RNF217-mediated degradation of FPN. A combination of 2 different E3 screens revealed that the Rnf217 gene is a target of Tet1, mediating the ubiquitination and subsequent degradation of FPN. Interestingly, loss of Tet1 expression causes an accumulation of FPN and an impaired response to iron overload, manifested by increased iron accumulation in the liver together with decreased iron in the spleen and duodenum. Moreover, we found that the degradation and ubiquitination of FPN could be attenuated by mutating RNF217. Finally, using 2 conditional knockout mouse lines, we found that knocking out Rnf217 in macrophages increases splenic iron export by stabilizing FPN, whereas knocking out Rnf217 in intestinal cells appears to increase iron absorption. These findings suggest that the Tet1-RNF217-FPN axis regulates iron homeostasis, revealing new therapeutic targets for FPN-related diseases.

T-cell responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been described in recovered patients, and may be important for immunity following infection and vaccination as well as for the development of an adoptive immunotherapy for the treatment of immunocompromised individuals. In this report, we demonstrate that SARS-CoV-2–specific T cells can be expanded from convalescent donors and recognize immunodominant viral epitopes in conserved regions of membrane, spike, and nucleocapsid. Following in vitro expansion using a good manufacturing practice-compliant methodology (designed to allow the rapid translation of this novel SARS-CoV-2 T-cell therapy to the clinic), membrane, spike, and nucleocapsid peptides elicited interferon-γ production, in 27 (59%), 12 (26%), and 10 (22%) convalescent donors (respectively), as well as in 2 of 15 unexposed controls. We identified multiple polyfunctional CD4-restricted T-cell epitopes within a highly conserved region of membrane protein, which induced polyfunctional T-cell responses, which may be critical for the development of effective vaccine and T-cell therapies. Hence, our study shows that SARS-CoV-2 directed T-cell immunotherapy targeting structural proteins, most importantly membrane protein, should be feasible for the prevention or early treatment of SARS-CoV-2 infection in immunocompromised patients with blood disorders or after bone marrow transplantation to achieve antiviral control while mitigating uncontrolled inflammation., Key Points • Coronavirus-specific polyfunctional T cells can be expanded from convalescent individuals for use for patients after bone marrow transplant. • SARS-CoV-2 T-cell products target structural viral proteins, including commonly recognized regions in the C terminus of membrane protein.

Introduction: The outbreak of a novel infection, COVID-19, has greatly impacted the well-being of individuals worldwide. Persons with sickle cell disease (SCD) constitute a vulnerable population, subject to health disparities, who may have worse outcomes from COVID-19. Within the United States, New York has a large population of patients with SCD. Here, we analyze the clinical course and outcomes of SCD patients with COVID-19 who were admitted to a community teaching hospital in Brooklyn, NY. Methods: We conducted a retrospective chart review of adult patients with SCD hospitalized with laboratory- confirmed COVID-19. Electronic health records were reviewed to identify patients and analyze their clinical course. Clinical characteristics, laboratory and radiology data were assessed. Rates of acute chest syndrome (ACS), acute kidney injury (AKI) and venous thromboembolism (VTE) were determined. ACS was defined by the presence of fever and/or respiratory symptoms accompanied by a new pulmonary infiltrate on chest Xray. Data on use of blood transfusion, treatments, length of stay and mortality were collected. Results: Between March 1 to June 30, 2020, 53 adults with SCD were hospitalized at our institution. Of these, 13 patients had COVID-19 infection. The mean (±SD) age of the COVID-19 patients was 34±10 years (range, 22 to 50) with 54% being female. Seven patients (54%) were Hb SS, and 6 patients (46%) were Hb SC. Comorbid conditions included Diabetes Mellitus (1 patient), SLE (1), End-stage renal disease (1), prior VTE (4) and Avascular necrosis of hip (3). Four patients were on hydroxyurea. Clinical, laboratory and radiological findings are summarized in Table 1. While all the Hb SS patients presented with vaso-occlusive crisis, 4 of the 6 patients with Hb SC did not have symptoms of pain crisis. Chest pain and cough were the most common symptoms at presentation. During the hospital stay, 12 patients (92%) had at least one febrile episode >38°C, with 77% having recurrent fevers above 38.5°C. Eleven patients (85%) met criteria for ACS. Seventy-seven percent of all patients required supplemental oxygen. Nine patients (69%) were transfused, with 4 patients undergoing exchange transfusion. Sixty-seven percent of the transfused patients were transfused within 48 hours of admission. No patients required intubation or mechanical ventilation and none were admitted to the intensive care unit (ICU). Five patients (38.5%) received hydroxychloroquine while 84.6% were treated with antibiotics. No patient received remdesivir. Three patients (23%) developed AKI: of these, one patient required acute hemodialysis, the other two cases were mild with peak creatinine less than 2.0 mg/dl. Ninety-two percent of patients received prophylactic anticoagulation with either unfractionated heparin, enoxaparin or fondaparinux. One patient who did not receive an anticoagulant due to thrombocytopenia developed an acute deep vein thrombosis which was also catheter-related. Of note, during the initial phase of the pandemic standard dosing of prophylactic anticoagulants were used but in the later months, some patients received higher prophylactic doses in keeping with hospital protocol. The median length of hospital stay was 9.4 days (interquartile range, 8.1 to 13.3). There were no deaths - all patients were discharged home. Summary: Panepinto et al (Emerg Infect Dis.) reported a mortality of 7% in 178 SCD patients with COVID-19 in the United States. Other published reports have detailed more favorable outcomes (Arlet et al, Lancet and Appiah-Kubi et al, Br J Haematol.). In this small retrospective analysis of hospitalized SCD patients, there was no mortality. Acute chest syndrome was the most common complication observed. VTE and severe AKI were infrequent. Blood transfusion was performed in the majority of patients (69%); two thirds of the patients transfused received blood within 48 hours of hospitalization. There were no ICU admissions and no use of mechanical ventilation indicative perhaps of less severe COVID-19 disease. This may have been due to the young age of the cohort. Early use of blood transfusion may have been a factor in reducing disease severity and improving outcomes. The best approach to managing these patients is unclear. We advocate for the development and dissemination of evidence-based guidelines to manage SCD patients with COVID-19 to reduce morbidity and mortality in this at-risk population. Disclosures No relevant conflicts of interest to declare.

The SARS-CoV-2 (COVID-19) outbreak is upending current life and generating much anxiety and uncertainty. The effects of home confinement, social isolation, cancelled schools, closed businesses, and negative economic impacts have had serious consequences. Hematologic patients (HP) are a subset of highly vulnerable population with increased risk of developing severe COVID-19 symptoms due to their immunocompromised status. These risks have been augmented during the COVID outbreak because of deviations from current standards of care, e.g., reduced visits, treatment supply and access to routine exams. This study investigated the impact of the current pandemic on HP assessing demographics, medical information, mental health and caregiver practical management. In collaboration with AIL (Italian Association against Leukemia) and CNR (National Research Council), a survey was generated and distributed to Italian HP. The general population (GP) were used as controls. The assessment used the DASS-21 questionnaire, a self-reported, 21 item screening instrument that provides independent measures of depression, stress and anxiety with recommended severity thresholds subscales. The survey was self-administered between April and August, 2020. The questionnaires' reliability was verified based on an analysis of its internal consistency using Cronbach's alpha. As of 30 June 2020, 1113 HP and 1125 GP completed the survey from 20 Italian Regions. The two population groups were homogeneous by age, gender and distribution and included regions at both high (CHP) and low (CLP) prevalence of infections at the time of the survey. HP and GP median age was 50 years (range: HP 11- 93; GP 13-85). 61% HP and 68% GP were female; the rest were male. The year of diagnosis of hematological disease ranged from 1965 to 2020; 21.9% had chronic myeloid leukemia, followed by Hodgkin (15.7%) and non-Hodgkin (15.9%) lymphomas, chronic myeloproliferative neoplasms (15.9%), multiple myeloma (8.9%), chronic lymphocytic leukemia (4.9%), acute myeloid (5.6%) and lymphoblastic (3.25%) leukemias, other (7%). 1071 HP and 1125 GP responded about their occupation as follows: employed full/part time (38.7% HP , 47.7% GP), retired (19.2% HP,10.8% GP), freelancer (9.2% HP, 12.2% GP), unemployed (6.2% HP, 3.4% GP) , students (4.6% HP, 3.2% GP), company executive (4.1% HP, 4.8% GP), manager (2% HP, 4.7 % GP), cooperative member (0.7% HP , 0.7% GP), housewife (8.5% HP 4.8% GP), other (6.8% HP,7.7% GP). During the pandemic 63.7% HP didn't work and 36.3% did work compared to the GP group (33.8% didn't; 66.2 % did). Where specified, the reasons for not working were: layoffs (10.9% HP, 15.9% GP), lack of work (8.1%HP, 15.6 % GP), vacation/ parental leave (4.4% HP, 3.5% GP), reduced business activity for economic reasons (2.2% HP, 3.3% GP), occasional work (2.1% HP, 5.7% GP), seasonal employment, (0.6% HP,1.1 % GP). 625/1073 HP (58.2%) were in active treatment. Of these, 40.1% were in Day Hospital, 56.4% were outpatient; and 3.5% inpatients. The remaining HP were off therapy (448/1073; 41.8%). 1105 HP and 1127 GP responded to the DASS questionnaire. Extremely severe depression was found in 12.9% HP vs 7% GP; 18.1% HP expressed severe anxiety vs 9.6% GP and extremely severe stress was present in 7.1% HP and 5.3% GP. The Cronbach's alpha coefficient for the internal homogeneity of the questionnaire was 0.95, confirming the cohorts correctness (0.8 or greater, indicates a very good level of reliability). Providing care to HP during the pandemic has been challenging for both patients and doctors. Restrictions on visits and lab/instrumental exams, reduced equipment supply and a paucity of personal protection equipment (PPEs) for health care providers and patients have penalized normal routine care. As a result, 38.2% HP postponed or did not attend scheduled therapy and >50% had difficulty obtaining PPEs through normal sources; 57% in CHP and 36% in CLP regions had to buy them themselves. To our knowledge this is the first report of the impact of the pandemic on psychological distress, work consequences and illness management in HP. Most of HP in this study are outpatients in active treatment. Interesting data emerged from job losses, which is more common among HP. The DASS-21 instrument revealed higher anxiety and depression levels in HP. Detailed results including longitudinal analysis and high versus low geographic prevalence of COVID-19 infections will be presented. Figure 1 Disclosures Abruzzese: Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bms: Honoraria.

Background: Since lowering the hemoglobin (Hb) and hematocrit (Hct) diagnostic thresholds for polycythemia vera (PV) to 165 g/L and 49% in men and 160 g/L and 48% in women respectively, by the World Health Organization (WHO) in 2016 (Blood. 2016;127:2391), hematologists have witnessed an increase in referrals for erythrocytosis, a significant proportion of which are found to have secondary erythrocytosis (SE). This has yielded novel challenges as comprehensive data on SE and reports comparing SE vs PV are scarce. Further, data regarding thrombosis in SE have been inconsistent (Clin Appl Thromb Hemost. 2013;19:363; J Clin Anesth. 1991;3:99; Ann Hematol. 2016;95:233). Methods: This multicentric retrospective study included PV patients enrolled in a pan-provincial registry of the chronic myeloid leukemia and myeloproliferative neoplasms Quebec Research Group (GQR LMC-NMP). SE patients were recruited from the Maisonneuve-Rosemont Hospital, Montreal, Quebec, Canada. PV diagnosis was per WHO 2016 criteria (Blood. 2016;127:2391). SE was defined as sustained elevation in Hb and/or Hct above WHO thresholds for PV with negative JAK2V617F testing and non subnormal serum erythropoietin (Epo) levels. Epo levels were drawn in therapy-naïve patients within 3 months of diagnosis using a standard immune-enzymatic assay (reference 3-30 mIU/mL). Endogenous erythroid colony assays used standard methods. JAK2 mutation screen and risk-stratification for PV were according to convention. Standard statistical methods were used to assess variables across i) SE and PV groups, and ii) subjects having experienced a thrombotic event vs no thrombosis. JMP® Pro 13.0.0 software was used for all analyses (SAS Institute, Cary, NC, USA). Results: Among 102 informative cases of erythrocytosis, 36 (35%) were SE and 66 (65%) were PV. Median age was significantly lower in the SE (57 years old; range 19-76) vs PV group (63.5 years old; range 20-89) (p=0.005), with a preponderance of males (75% in SE vs 45% PV; p=0.004). Median serum Epo levels were normal in the SE cohort (10.3 mIU/mL; range The most prevalent etiology of erythrocytosis in the SE cohort was idiopathic, reported in 50% of cases (n=18), followed by smoking/chronic obstructive pulmonary disease in 22.2% (n=8), smoking plus additional factors in 8.3% (n=3), sleep apnea, polycystic kidney disease, or a combination of the latter two in 5.6% each (n=2 each) and post kidney transplant in 2.7% (n=1) (Table 1). Nearly as many SE (n=11, 31%) as PV patients (n=26, 39%) were exposed to bone marrow sampling (p=0.37) and SE patients were frequently managed with phlebotomy (42% vs 76% PV, p=0.0007) and aspirin therapy (56% vs 93% PV, p SE and PV populations had similar rates of thrombosis at/prior to diagnosis including events in 9 (25%) SE and 19 (29%) PV patients (p=0.68), both predominantly arterial (n=7 and n=16 respectively; p=0.58) (Table 2). History of thrombosis at any time in both groups clustered significantly with older age (p=0.001), hypertension (p Conclusions: SE patients are significantly younger, more likely to be male, active smokers, and obese, with normal-high Epo, normal LDH, and exceedingly rare leukocytosis and thrombocytosis, discriminating them phenotypically from PV patients. Importantly, rates of thromboembolic events prior to/at diagnosis are comparable in SE vs PV, suggesting that SE may not be benign from a thrombosis standpoint. Classic cardiovascular risk factors significantly cluster with thrombosis risk, emphasizing the importance of controlling these variables in both cohorts. Finally, a significant portion of SE patients are subject to bone marrow sampling and, though controversial, treatment with phlebotomy and aspirin, highlighting the need for formal studies to guide management in this population. Disclosures Szuber: Novartis: Honoraria. Busque:BMS: Honoraria; Pfizer: Honoraria; Novartis: Honoraria. Assouline:Takeda: Research Funding; BeiGene: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; AstraZeneca: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding. Olney:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

INTRODUCTION: Adult T-cell leukemia/lymphoma (ATLL) is an aggressive peripheral T-cell neoplasm caused by the Human T-cell Leukemia Virus Type 1 (HTLV-1). HTLV-1 infects up to 10 million people worldwide and is most endemic in Southwestern Japan, Western Africa, the Caribbean basin and South America. Cutaneous signs of ATLL are varied and may consist of macules (M), plaques (P), multiple papules (MP), tumoral nodules (TN), erythroderma (E) or mixed-lesions (≥2 predominant lesions, ML). M and P forms are believed to carry a better prognosis. However, data on cutaneous presentation of ATLL remains scarce. Herein, we report cases of ATLL with cutaneous involvement diagnosed in 4 Latin American countries over the last 3 decades. METHODS: We retrospectively analyzed patients (pts) diagnosed with ATLL between January 1995 and December 2019. ATLL subtypes were classified according to the Shimoyama criteria into acute, lymphomatous, chronic and smoldering. Primary cutaneous tumoral (PCT) variant was classified according to the 2019 International Revised ATLL Consensus. We designed 2 cohorts: the first, ATLL pts with cutaneous involvement, and the second, matched cases without cutaneous involvement. We determined the type of skin lesion as well as the survival associated with the various types of skin lesions. We compared the frequency of clinical features using Fisher's exact test. Treatment response was assessed according to Tsukasaki et al. (JCO 2009) criteria. To be classified as complete response (CR), partial response, and stable disease, these had to persist for a period of at least 4 weeks. We analyzed survival data according to ATLL subtype, cutaneous involvement status, and type of skin lesion using the Kaplan-Meier method and Log rank test. RESULTS: A total of 169 pts with ATLL were identified; 63 had cutaneous involvement and 106 did not. Clinical features are shown in Table 1. In both groups the median age was 57 years with a female predominance. Cutaneous involvement was most frequently found in acute (41%) and lymphomatous (37%) ATLL pts. The E (24%) and P (22 %) types were the most frequent skin lesions. Disease stage, presence of B symptoms, hypercalcemia, ECOG ≥2, elevated LDH, and IPI/ PIT score were not different among groups. Table 2 and Table 3 summarize the first-line therapy used and response rates. The use of first-line zidovudine plus interferon alpha (AZT-IFN), regardless of the type of skin lesion, resulted in relatively high response rates [overall response (OR) 100%, n=8; CR 62.5%] as compared to multi agent-chemotherapy (OR 33.3%, n=12). Overall, the presence of cutaneous involvement was associated with better overall survival (OS) compared to non-cutaneous involvement (aHR 0.55 [95% CI: 0.37-0.82], p CONCLUSION: In Latin American pts with aggressive ATLL, cutaneous involvement appears to be associated with better survival compared to non-cutaneous involvement. PCT subtype, an ATLL variant characterized by isolated skin lesions with no organ involvement and poor outcome, appeared to have a better prognosis compared to acute and lymphomatous ATLL forms. P and MP skin lesions were both associated with better survival. Hypercalcemia was found as an independent prognostic factor for survival in pts with cutaneous involvement. Finally, AZT-IFN appears to be reasonable first-line option for aggressive ATLL subtypes with cutaneous involvement regardless of the type of skin lesion at diagnosis, based on the relatively high response rates observed in this subset; further investigation in randomized clinical trials is needed. Disclosures Peña: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sandoz: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Amgen: Speakers Bureau; BindingSite: Research Funding. Idrobo:Takeda: Honoraria, Speakers Bureau; Tecnofarma: Honoraria, Speakers Bureau; Abbvie: Honoraria, Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Amgen: Honoraria, Speakers Bureau. Altamirano:Hospital Nacional Guillermo Almenara Irigoyen: Other: Servicio de Hematologia. Perini:Abbvie: Speakers Bureau; Janssen: Honoraria, Speakers Bureau; Takeda: Honoraria. Castillo:Pharmacyclics: Consultancy, Research Funding; Beigene: Consultancy, Research Funding; Abbvie: Research Funding; Kymera: Consultancy; TG Therapeutics: Research Funding; Janssen: Consultancy, Research Funding. Ramos:NIH: Research Funding. Villela:amgen: Speakers Bureau; Roche: Other: advisory board, Speakers Bureau.

Patients with hereditary rare bleeding disorders (RBDs) present with diverse hemorrhagic symptoms. Correlation between factor activity levels and clinical bleeding severity is poor for most RBDs. Threshold factor activity levels have been previously described in relation to bleeding severity but have not yet been validated. The Rare Bleeding Disorders in the Netherlands (RBiN) study is a nationwide cross-sectional study of patients registered in all 6 Dutch Haemophilia Treatment Centers with a known RBD and who are age 1 to 99 years. Bleeding scores were determined, and laboratory and clinical data were extracted from patient files. In all, 263 patients were included, of whom 202 (77%) attended the scheduled study visit. The median International Society of Thrombosis and Haemostasis (ISTH) bleeding assessment tool (BAT) score was 9. Correlations between baseline factor activity levels and ISTH BAT scores were strong for deficiencies in factor II (FII) (r = –0.792) and FX (r = –0.838) and were moderate for deficiencies of fibrinogen (r = –0.683), FV (r = –0.623), FVII (r = –0.516), FXIII (r = –0.516), and α2-antiplasmin (r = –0.594). There was no correlation for FXI deficiency (r = –0.218). The RBD BAT identified more women (94% vs 83%) and children (100% vs 71%) with an RBD than the ISTH BAT did. Importantly, 48% of patients had more severe bleeding than predicted for their baseline factor activity level. In addition, 34% of patients were predicted to be asymptomatic, but they actually had grade 2 (31%) or 3 (3%) bleeding. Bleeding severity in patients with RBDs is more pronounced than previously anticipated. The previously determined threshold factor activity levels to ensure no (spontaneous) bleeding in patients with an RBD are inaccurate. This trial was registered at www.clinicaltrials.gov as #NCT03347591.

The diagnosis of chronic mountain sickness (CMS) is based on a score including 7 clinical features (breathlessness, sleep disturbance, cyanosis, venous dilatation, paresthesia, headache, and tinnitus) in the setting of extreme erythrocytosis. Examining individuals in La Rinconada, Peru, the highest city in the world, the authors demonstrated that CMS at extreme altitude is not linked to elevation of hemoglobin, since CMS+ and CMS− individuals had similar levels of erythrocytosis.

Haploinsufficiency of GATA2 caused by heterozygous loss-of-function mutations is associated with cytopenias and predisposition to myelodysplasia and AML with other variable extrahematopoietic manifestions, including lymphedema, pulmonary alveolar proteinosis, and hearing loss. The authors report on 2 siblings with the disorder whose father was asymptomatic because of an acquired missense mutation in the affected allele that was restricted to hematopoietic cells; surprisingly, he also had no extrahematopoietic complications.

Hematopoietic stem cell (HSC) aging correlates with an increasing risk of myeloproliferative disease and immunosenescence. In this study, we show that aging-related inflammation promotes HSC aging through tumor necrosis factor-α (TNF-α)→ERK→ETS1→interleukin27Ra (IL27Ra) pathway. TNF-α, a well-known biomarker of inflammation, increases during aging and induces the expression of IL27Ra on HSCs via ERK-ETS1 signaling. Deletion of IL27Ra rescues the functional decline and myeloid bias of HSCs and also reverses the inhibitory effect of TNF-α on HSCs. Aged IL27Ra−/− mice had a reduced proportion of myeloid-biased HSCs and did not display the biased myeloid differentiation that occurs in aged wild-type mice. IL27Ra+ HSCs exhibit impaired reconstitution capacity and myeloid-bias compared with IL27Ra− HSCs and serve as a myeloid-recovery pool upon inflammatory insult. Inflammation-related genes were enriched in IL27Ra+ HSCs and this enrichment increases with aging. Our study demonstrates that age-induced IL27Ra signaling impairs HSCs and raises the possibility that interfering with IL27Ra signaling can counter the physiologically deleterious effect of aging on hematopoietic capacity.

Aging is associated with significant changes in the hematopoietic system, including increased inflammation, impaired hematopoietic stem cell (HSC) function, and increased incidence of myeloid malignancy. Inflammation of aging (“inflammaging”) has been proposed as a driver of age-related changes in HSC function and myeloid malignancy, but mechanisms linking these phenomena remain poorly defined. We identified loss of miR-146a as driving aging-associated inflammation in AML patients. miR-146a expression declined in old wild-type mice, and loss of miR-146a promoted premature HSC aging and inflammation in young miR-146a–null mice, preceding development of aging-associated myeloid malignancy. Using single-cell assays of HSC quiescence, stemness, differentiation potential, and epigenetic state to probe HSC function and population structure, we found that loss of miR-146a depleted a subpopulation of primitive, quiescent HSCs. DNA methylation and transcriptome profiling implicated NF-κB, IL6, and TNF as potential drivers of HSC dysfunction, activating an inflammatory signaling relay promoting IL6 and TNF secretion from mature miR-146a−/− myeloid and lymphoid cells. Reducing inflammation by targeting Il6 or Tnf was sufficient to restore single-cell measures of miR-146a−/− HSC function and subpopulation structure and reduced the incidence of hematological malignancy in miR-146a−/− mice. miR-146a−/− HSCs exhibited enhanced sensitivity to IL6 stimulation, indicating that loss of miR-146a affects HSC function via both cell-extrinsic inflammatory signals and increased cell-intrinsic sensitivity to inflammation. Thus, loss of miR-146a regulates cell-extrinsic and -intrinsic mechanisms linking HSC inflammaging to the development of myeloid malignancy.

Polyclonal anti-D (Rh immune globulin [RhIg]) therapy has mitigated hemolytic disease of the newborn over the past half century, although breakthrough anti-D alloimmunization still occurs in some treated females. We hypothesized that antiviral responses may impact the efficacy of immunoprophylaxis therapy in a type 1 interferon (IFN)-dependent manner and tested this hypothesis in a murine model of KEL alloimmunization. Polyclonal anti-KEL immunoprophylaxis (KELIg) was administered to wild-type or knockout mice in the presence or absence of polyinosinic-polycytidilic acid (poly[I:C]), followed by the transfusion of murine red blood cells (RBCs) expressing the human KEL glycoprotein. Anti-KEL alloimmunization, serum cytokines, and consumption of the transfused RBCs were evaluated longitudinally. In some experiments, recipients were treated with type 1 IFN (IFN-α/β). Recipient treatment with poly(I:C) led to breakthrough anti-KEL alloimmunization despite KELIg administration. Recipient CD4+ T cells were not required for immunoprophylaxis efficacy at baseline, and modulation of the KEL glycoprotein antigen occurred to the same extent in the presence or absence of recipient inflammation. Under conditions where breakthrough anti-KEL alloimmunization occurred, KEL RBC consumption by inflammatory monocytes and serum monocyte chemoattractant protein-1 and interleukin-6 were significantly increased. Poly(I:C) or type I IFN administration was sufficient to cause breakthrough alloimmunization, with poly(I:C) inducing alloimmunization even in the absence of recipient type I IFN receptors. A better understanding of how recipient antiviral responses lead to breakthrough alloimmunization despite immunoprophylaxis may have translational relevance to instances of RhIg failure that occur in humans.

Vaso-occlusive crisis (VOC) is the primary cause of morbidity and hospitalization in sickle cell disease (SCD); however, only 4 therapies (hydroxyurea, l-glutamine, crizanlizumab, and voxeletor) are currently approved in SCD. These agents limit the duration, severity, and frequency of crises. Activation of coagulation is a hallmark of SCD. Studies in animal models of SCD have shown that coagulation contributes to the chronic inflammation and end-organ damage associated with the disease; however, it is unknown whether coagulation directly contributes to the microvascular stasis that causes VOC. Herein, we demonstrate that inhibition of tissue factor (TF) and the downstream coagulation proteases factor Xa and thrombin significantly attenuates heme-induced microvascular stasis in mouse models of VOC. Pharmacologic inhibition of the principal thrombin receptor, protease activated receptor-1 (PAR-1), as well as deficiency of PAR-1 in all nonhematopoietic cells, also reduces stasis in sickle mice. PAR-1 deficiency was associated with reduced endothelial von Willebrand factor expression, which has been shown to mediate microvascular stasis. In addition, TF inhibition reduces lung vaso-occlusion in sickle mice mediated by arteriolar neutrophil-platelet microemboli. In sum, these results suggest that prophylactic anticoagulation might attenuate the incidence of VOC.

There are unresolved questions regarding the association between persistent leukocytosis and risk of thrombosis and disease evolution in polycythemia vera (PV), as much of the published literature on the topic does not appropriately use repeated-measures data or time-dependent modeling to answer these questions. To address this knowledge gap, we analyzed a retrospective database of 520 PV patients seen at 10 academic institutions across the United States. Taking hematologic laboratory data at ∼3-month intervals (or as available) for all patients for duration of follow-up, we used group-based trajectory modeling to identify latent clusters of patients who follow distinct trajectories with regard to their leukocyte, hematocrit, and platelet counts over time. We then tested the association between trajectory membership and hazard of 2 major outcomes: thrombosis and disease evolution to myelofibrosis, myelodysplastic syndrome, or acute myeloid leukemia. Controlling for relevant covariates, we found that persistently elevated leukocyte trajectories were not associated with the hazard of a thrombotic event (P = .4163), but were significantly associated with increased hazard of disease evolution in an ascending stepwise manner (overall P = .0002). In addition, we found that neither hematocrit nor platelet count was significantly associated with the hazard of thrombosis or disease evolution.

Daratumumab has shown promising first results in systemic amyloid light-chain (AL) amyloidosis. We analyzed a consecutive series of 168 patients with advanced AL receiving either daratumumab/dexamethasone (DD, n = 106) or daratumumab/bortezomib/dexamethasone (DVD, n = 62). DD achieved a remission rate (RR) of 64% and a very good hematologic remission (VGHR) rate of 48% after 3 months. Median hematologic event-free survival (hemEFS) was 11.8 months and median overall survival (OS) was 25.6 months. DVD achieved a 66% RR and a 55% VGHR rate. Median hemEFS was 19.1 months and median OS had not been reached. Cardiac organ responses were noted in 22% with DD and 26% with DVD after 6 months. Infectious complications were common (Common Terminology Criteria [CTC] grade 3/4: DD 16%, DVD 18%) and likely related to a high rate of lymphocytopenia (CTC grade 3/4: DD 20%, DVD 17%). On univariable analysis, hyperdiploidy and gain 1q21 conferred an adverse factor for OS and hemEFS with DD, whereas translocation t(11;14) was associated with a better hemEFS. N-terminal prohormone of brain natriuretic peptide >8500 ng/L could not be overcome for survival with each regimen. Multivariable Cox regression analysis revealed plasma cell dyscrasia (difference between serum free light chains [dFLC]) >180 mg/L as an overall strong negative prognostic factor. Additionally, nephrotic-range albuminuria with an albumin-to-creatinine-ratio (ACR) >220 mg/mmol was a significantly adverse factor for hemEFS (hazard ratio, 2.1 and 3.1) with DD and DVD. Daratumumab salvage therapy produced good results and remission rates challenging any therapy in advanced AL. Outcome is adversely influenced by the activity of the underlying plasma cell dyscrasia (dFLC) and nephrotic-range albuminuria (ACR).

Recently, erythropoietin (EPO) was identified as regulator of fibroblast growth factor 23 (FGF23). Proteolytic cleavage of biologically active intact FGF23 (iFGF23) results in the formation of C-terminal fragments (cFGF23). An increase in cFGF23 relative to iFGF23 suppresses FGF receptor signaling by competitive inhibition. EPO lowers the i:cFGF23 ratio, thereby overcoming iFGF23-mediated suppression of erythropoiesis. We investigated EPO-FGF23 signaling and levels of erythroferrone (ERFE) in 90 patients with hereditary hemolytic anemia (www.trialregister.nl [NL5189]). We show, for the first time, the importance of EPO-FGF23 signaling in hereditary hemolytic anemia: there was a clear correlation between total FGF23 and EPO levels (r = +0.64; 95% confidence interval [CI], 0.09-0.89), which persisted after adjustment for iron load, inflammation, and kidney function. There was no correlation between iFGF23 and EPO. Data are consistent with a low i:cFGF23 ratio. Therefore, as expected, we report a correlation between EPO and ERFE in a diverse set of hereditary hemolytic anemias (r = +0.47; 95% CI, 0.14-0.69). There was no association between ERFE and total FGF23 or iFGF23, which suggests that ERFE does not contribute to the connection between FGF23 and EPO. These findings open a new area of research and might provide potentially new druggable targets with the opportunity to ameliorate ineffective erythropoiesis and the development of disease complications in hereditary hemolytic anemias.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome describing patients with severe systemic hyperinflammation. Characteristic features include unremitting fever, cytopenias, hepatosplenomegaly, and elevation of typical HLH biomarkers. Patients can develop hepatitis, coagulopathy, liver failure, central nervous system involvement, multiorgan failure, and other manifestations. The syndrome has a high mortality rate. More and more, it is recognized that while HLH can be appropriately used as a broad summary diagnosis, many pediatric patients actually suffer from an expanding spectrum of genetic diseases that can be complicated by the syndrome of HLH. Classic genetic diseases in which HLH is a typical and common manifestation include pathogenic changes in familial HLH genes (PRF1, UNC13D, STXBP2, and STX11), several granule/pigment abnormality genes (RAB27A, LYST, and AP3B1), X-linked lymphoproliferative disease genes (SH2D1A and XIAP), and others such as NLRC4, CDC42, and the Epstein-Barr virus susceptibility diseases. There are many other genetic diseases in which HLH is an infrequent complication of the disorder as opposed to a prominent manifestation of the disease caused directly by the genetic defect, including other primary immune deficiencies and inborn errors of metabolism. HLH can also occur in patients with underlying rheumatologic or autoinflammatory disorders and is usually designated macrophage activation syndrome in those settings. Additionally, HLH can develop in patients during infections or malignancies without a known (or as-yet-identified) genetic predisposition. This article will attempt to summarize current concepts in the pediatric HLH field as well as offer a practical diagnostic and treatment overview.

Castleman disease (CD) describes a group of at least 4 disorders that share a spectrum of characteristic histopathological features but have a wide range of etiologies, presentations, treatments, and outcomes. CD includes unicentric CD (UCD) and multicentric CD (MCD), the latter of which is divided into idiopathic MCD (iMCD), human herpes virus-8 (HHV8)-associated MCD (HHV8-MCD), and polyneuropathy, organomegaly, endocrinopathy, monoclonal plasma cell disorder, skin changes (POEMS)-associated MCD (POEMS-MCD). iMCD can be further subclassified into iMCD–thrombocytopenia, ascites, reticulin fibrosis, renal dysfunction, organomegaly (iMCD-TAFRO) or iMCD–not otherwise specified (iMCD-NOS). Advances in diagnosis, classification, pathogenesis, and therapy are substantial since the original description of UCD by Benjamin Castleman in 1954. The advent of effective retroviral therapy and use of rituximab in HHV8-MCD have improved outcomes in HHV8-MCD. Anti–interleukin-6–directed therapies are highly effective in many iMCD patients, but additional therapies are required for refractory cases. Much of the recent progress has been coordinated by the Castleman Disease Collaborative Network (CDCN), and further progress will be made by continued engagement of physicians, scientists, and patients. Progress can also be facilitated by encouraging patients to self-enroll in the CDCN’s ACCELERATE natural history registry (#NCT02817997; www.CDCN.org/ACCELERATE).

Gastrointestinal (GI) tract involvement is the major cause of morbidity and mortality in acute graft-versus-host disease (GVHD), and pathological damage is largely attributable to inflammatory cytokine production. Recently, granulocyte-macrophage colony stimulating factor (GM-CSF) has been identified as a cytokine that mediates inflammation in the GI tract, but the transcriptional program that governs GM-CSF production and the mechanism by which GM-CSF links adaptive to innate immunity within this tissue site have not been defined. In the current study, we identified Bhlhe40 as a key transcriptional regulator that governs GM-CSF production by CD4+ T cells and mediates pathological damage in the GI tract during GVHD. In addition, we observed that GM-CSF was not regulated by either interleukin 6 (IL-6) or IL-23, which are both potent inducers of GVHD-induced colonic pathology, indicating that GM-CSF constitutes a nonredundant inflammatory pathway in the GI tract. Mechanistically, GM-CSF had no adverse effect on regulatory T-cell reconstitution, but linked adaptive to innate immunity by enhancing the activation of donor-derived dendritic cells in the colon and subsequent accumulation of these cells in the mLNs. In addition, GM-CSF promoted indirect alloantigen presentation, resulting in the accumulation of donor-derived T cells with a proinflammatory cytokine phenotype in the colon. Thus, Bhlhe40+ GM-CSF+ CD4+ T cells constitute a colitogenic T-cell population that promotes indirect alloantigen presentation and pathological damage within the GI tract, positioning GM-CSF as a key regulator of GVHD in the colon and a potential therapeutic target for amelioration of this disease.

Dysregulated platelet functions contribute to the development and progression of ischemic stroke. Utilizing mice with a platelet-specific deletion of cyclophilin D (CypD), a mediator of necrosis, we found that platelet necrosis regulates tissue damage and outcomes during ischemic stroke in vivo. Mice with loss of CypD in platelets (CypDplt-/-mice) exhibited significantly enhanced cerebral blood flow, improved neurological and motor functions, and reduced ischemic stroke infarct volume after cerebral ischemia-reperfusion injury. These effects were attributable, at least in part, to platelet-neutrophil interactions. Twenty-four hours after stroke, significantly more circulating platelet-neutrophil aggregates (PNAs) were found in CypDplt+/+ mice. Underscoring the role of platelet necrosis in PNA formation, we observed a significant number of phosphatidylserine (PS)+ platelets in PNAs in CypDplt+/+ mice. In contrast, significantly fewer platelets in PNAs were PS+ in CypDplt-/- counterparts. Accordingly, mice with CypD-deficient platelets had fewer neutrophils and PNAs recruited to their brain following stroke relative to wild-type counterparts. Neutrophil depletion in wild-type mice conferred protection from ischemic stroke to a similar degree as observed in mice with CypD-deficient platelets. Neutrophil depletion in CypDplt-/- mice did not further reduce infarct size. Transmission electron microscopy of ex vivo-formed PNAs revealed a propensity of necrotic platelets to interact with neutrophils. These results suggest that necrotic platelets interact with neutrophils to exacerbate brain injury during ischemic stroke. Because inhibiting platelet necrosis does not compromise hemostasis, targeting platelet CypD may be a potential therapeutic strategy to limit brain damage following ischemic stroke. ispartof: BLOOD vol:135 issue:6 pages:429-440 ispartof: location:United States status: published

Sickle cell disease (SCD) leads to significant morbidity and early mortality, and hematopoietic cell transplantation (HCT) is the only widely available cure, with impacts seen on SCD-related organ dysfunction. Outcomes are excellent following matched-related donor (MRD) HCT, leading to significantly expanded application of this treatment over the past decade. The majority of SCD patients lack an MRD, but outcomes following alternative donor HCT continue to improve on clinical trials. Within this framework, we aim to provide our perspective on how to apply research findings to clinical practice, for an individual patient. We also emphasize that the preparation of SCD recipients for HCT and supporting them through HCT have special nuances that require awareness and close attention. Through the use of clinical vignettes, we provide our perpsective on the complex decision-making process in HCT for SCD as well as recommendations for the evaluation and support of these patients through HCT.

Introduction Immunomodulatory drugs (IMiDs), particularly lenalidomide, are associated with adverse skin reactions most commonly rash, xeroderma, and pruritus. While multiple myeloma disproportionately impacts black patients, the clinical trials used for registration of these medications predominantly enrolled white patients. The incidence and severity of skin pigment changes in black patients are therefore not known. Hyperpigmentation can be a highly visible and distressing side effect leading to noncompliance. Methods This retrospective study evaluated all patients treated with thalidomide, lenalidomide or pomalidomide from January 2013 to March 2020 across all indications at Boston Medical Center. An internally developed survey consisting of 14-questions was mailed to all patients identified through prescriptions written in the electronic medical record (Epic). The survey questions included ethnicity and race identification and whether skin changes, particularly hyperpigmentation, occurred during treatment. For those patients with skin changes, follow-up questions asked about the nature of the skin change, pattern, location and duration. Distress related to skin changes was reported on a scale of 1 to 10; 1 being minimal distress and 10 being maximal distress. Photographs were requested (if available) before, during and after IMiD therapy. Results A total of 214 patients were identified who were prescribed thalidomide (4, 1.9%), lenalidomide (204, 95.3%) or pomalidomide (81, 37.9%). Of the 214 surveys, 106 (49.5%) were completed and all of the responses were included in statistical analysis. Of the 106 patients, 49 (46.2%) identified as black/African American and 57 (53.8%) reported being non-black (Asian, Hispanic or Latino, American Indian/Alaskan Native, White, Native Hawaiian/Other Pacific Islander or other). Skin changes were reported by 27 (25.5%) of the patients who completed surveys. Consistency and description of the skin changes can be found in Table 1. Hyperpigmentation (skin darkening), specifically, was reported by 20 (40.8%) of the black/African American patients and 2 (3.5%) of the non-black patients. The most commonly reported location of hyperpigmentation was on the palms and soles (15, 68.2%), forearms (7, 31.8%) and face (6, 27.3%). Onset began within 3 months of starting therapy in 10 (45.5%) of these patients. Of the 11 patients who are no longer on IMiD therapy, 4 (36.6%) had full resolution of skin changes, 5 (45.5%) noted partial resolution, and 2 (18.2%) had no improvement. The reported distress score ranged from 1-9 (median 5). Photographs of typical skin hyperpigmentation will be included at conference presentation. Conclusion Skin hyperpigmentation due to IMiD therapy is commonly seen in black/African American patients. Specifically, hyperpigmentation is 11.6 times more likely to occur in black/African American patients as compared to all other races. In these patients, hyperpigmentation is most noticeable on the palms, face and soles of the feet. These changes typically occur early in the course of therapy and do not completely reverse, even long after drug cessation. It is important for providers to discuss the possibility of these skin changes when using IMiDs in this patient population. Download : Download high-res image (378KB) Download : Download full-size image Disclosures Blevins: Epizyme: Other: Focus Group. Hughes: Karyopharm: Speakers Bureau; Abbvie: Speakers Bureau; Amgen: Speakers Bureau; Rigel: Other: advisory board. Sarosiek: Spectrum: Research Funding. Sanchorawala: UpToDate: Patents & Royalties; Abbvie: Other: advisory board; Proclara: Other: advisory board; Caleum: Other: advisory board; Regeneron: Other: advisory board; Oncopeptide: Research Funding; Caelum: Research Funding; Prothena: Research Funding; Celgene: Research Funding; Takeda: Research Funding; Janssen: Research Funding. Sloan: Abbvie: Consultancy; Stemline: Consultancy.

The newly identified 13-series (T-series) resolvins (RvTs) regulate phagocyte functions and accelerate resolution of infectious inflammation. Because severe acute respiratory syndrome coronavirus 2 elicits uncontrolled inflammation involving neutrophil extracellular traps (NETs), we tested whether stereochemically defined RvTs regulate NET formation. Using microfluidic devices capturing NETs in phorbol 12-myristate 13-acetate–stimulated human whole blood, the RvTs (RvT1-RvT4; 2.5 nM each) potently reduced NETs. With interleukin-1β–stimulated human neutrophils, each RvT dose and time dependently decreased NETosis, conveying ∼50% potencies at 10 nM, compared with a known NETosis inhibitor (10 μM). In a murine Staphylococcus aureus infection, RvTs (50 ng each) limited neutrophil infiltration, bacterial titers, and NETs. In addition, each RvT enhanced NET uptake by human macrophages; RvT2 was the most potent of the four RvTs, giving a >50% increase in NET-phagocytosis. As part of the intracellular signaling mechanism, RvT2 increased cyclic adenosine monophosphate and phospho–AMP-activated protein kinase (AMPK) within human macrophages, and RvT2-stimulated NET uptake was abolished by protein kinase A and AMPK inhibition. RvT2 also stimulated NET clearance by mouse macrophages in vivo. Together, these results provide evidence for novel pro-resolving functions of RvTs, namely reducing NETosis and enhancing macrophage NET clearance via a cyclic adenosine monophosphate–protein kinase A–AMPK axis. Thus, RvTs open opportunities for regulating NET-mediated collateral tissue damage during infection as well as monitoring NETs.

The study aimed to compare the efficacy and safety of all-trans retinoic acid (ATRA) plus low-dose rituximab (LD-RTX) with LD-RTX monotherapy in corticosteroid-resistant or relapsed immune thrombocytopenia (ITP) patients. Recruited patients were randomized at a ratio of 2:1 into 2 groups: 112 patients received LD-RTX plus ATRA, and 56 patients received LD-RTX monotherapy. Overall response (OR), defined as achieving a platelet count of ≥30 × 109/L confirmed on ≥2 separate occasions (≥7 days apart), at least a doubling of the baseline platelet count without any other ITP-specific treatment, and the absence of bleeding within 1 year after enrollment, was observed in more patients in the LD-RTX plus ATRA group (80%) than in the LD-RTX monotherapy group (59%) (between-group difference, 0.22; 95% CI, 0.07-0.36). Sustained response (SR), defined as maintenance of a platelet count >30 × 109/L, an absence of bleeding, and no requirement for any other ITP-specific treatment for 6 consecutive months after achievement of OR during 1 year following enrollment, was achieved by 68 (61%) patients in the combination group and 23 (41%) patients in the monotherapy group (between-group difference, 0.20; 95% CI, 0.04-0.35). The 2 most common adverse events (AEs) for the combination group were dry skin and headache or dizziness. Our findings demonstrated that ATRA plus LD-RTX significantly increased the overall and sustained response, indicating a promising treatment option for corticosteroid-resistant or relapsed adult ITP. This study is registered at www.clinicaltrials.gov as #NCT03304288.

Background: Isocitrate dehydrogenase enzymes catalyze the conversion of isocitrate to alpha-ketoglutarate. Mutated IDH enzymes generate the "oncometabolite" 2-hydroxyglutarate (2-HG), which inhibits TET2 function. IDH2 mutations have been reported in 9% to 19% of acute myeloid leukemia (AML) cases. Inhibition of the mutant IDH2 enzyme led to decreased 2-HG levels and induced myeloid differentiation in IDH2 mutant AML. Enasidenib, a small-molecule inhibitor of mutant IDH2, was approved in 2017, after a successful phase I/II trial showing an overall response rate (ORR) of 40.3% in relapsed/refractory (R/R) disease, with 19.3% of patients achieving complete remission (CR). A Phase III trial is currently ongoing. It targets the mutant IDH2 variants R140Q, R172S, and R172K; at an approved dose of 100 mg oral daily dose. At higher doses the drug was less tolerated, however, few subjects received dose modification at the 650 mg daily dose group; these events did not qualify as a dose-limiting toxicity (DLT). In this descriptive study, we are reporting a case series of R/R AML, with an IDH2 mutation, that are treated with escalated dose enasidenib. Method: This case series is based on retrospective observations of patients with R/R IDH2 mutant AML since January 2017, who are treated with enasidenib. We are reporting two cases treated with an escalated dose of 200 mg daily. We included patients with intermediate to poor-risk AML, who received at least one prior line of therapy, started on standard dose enasidenib (100 mg daily) for a minimum of 6 months and followed until disease relapse or death. We excluded patients who are primarily resistant to enasidenib (AML risk stratification and response evaluation by ELN-Leukemia NET. IDH2 mutations were identified by a local diagnostic laboratory that is regulated under CLIA through PCR which is validated to detect 10% or more mutant allele frequency. To quantify IDH2 variant frequency, confirmatory testing was performed via ARUP next-generation sequencing panel (NGS). Results: Here we report the descriptive outcomes of 2 cases from a single institution, who were treated with escalated dose enasidenib (200 mg oral daily) for R/R AML with an IDH2 mutation. The first case describes a 65-year-old female diagnosed with poor-risk AML in the setting of pancytopenia with bone marrow evaluation consistent with the background of erythroid and granulocytic dysplasia, with normal karyotype analysis. The patient has treated with induction chemotherapy and 2 cycles consolidation that was complicated with bacterial infections, prolonged hospitalization, and profound deconditioning led to stopping treatment. After 18 months of surveillance, repeat bone marrow evaluation for new-onset pancytopenia revealed relapsed AML with evidence of IDH2 mutation at codon 140, with a variant frequency of 36.9% by NGS. The case was started on enasidenib at 100 mg oral daily dose. After 6 months of therapy, the patient continued to be cytopenic, repeated bone marrow evaluation showed residual AML with IDH2 variant frequency of 27.6%. The decision was made to increase the enasidenib dose to 200 mg daily. With follow up for another 5 months on the escalated dose, the patient achieved hematologic complete response (hCR) with normalization of peripheral platelet count and ANC. [Figures 1-2] The patient maintained hCR for another 5 months on 200 mg enasidenib daily until she died from COVID-19 infection. The second case describes a 59-year-old female with poor-risk AML in the setting of leukocytosis, with bone marrow evaluation consistent with the background of megakaryocytic dysplasia and karyotype analysis positive for monosomy 7. The patient was treated with induction chemotherapy and failed to respond. Repeat bone marrow evaluation showed primary refractory disease with positive IDH2 mutation at codon 140 with a variant frequency of 40.7%. Treatment with 100 mg enasidenib was started. The patient maintained a partial response for 6 months, then peripheral blasts counts started to gradually increase. The decision was made to increase enasidenib dose to 200 mg daily, which improved peripheral blasts within one month of escalated dose therapy. [Figure 3] The patient maintained a response to therapy for another 3 months before she relapsed. Conclusion: Our limited data showed that initial response to standard dose enasidenib could potentially be optimized by dose escalation to 200 mg daily. Disclosures No relevant conflicts of interest to declare.

Preclinical Rationale: Myelofibrosis (MF) is a chronic myeloproliferative neoplasm for which there are limited therapies. TGFβ plays a pivotal role in the pathobiology of MF by not only promoting bone marrow fibrosis (BMF) and collagen deposition, but also by enhancing the dormancy of normal but not MF hematopoietic stem cells (HSCs). TGFβ has also previously been reported to inhibit normal megakaryocyte (MK) production (Bruno et al Blood 1998). TGFβ1 promotes the synthesis of collagen by normal human mesenchymal stromal cells (MSCs) and activates the TGFβ receptor I/SMAD pathway as well as non-canonical TGFβ pathways. We generated MKs from MF subject mononuclear cells (MNCs) and showed that they elaborated significantly greater levels of TGFβ1 than TGFβ2/3 TGFβ1 treatment reduced the numbers of hematopoietic colonies generated by normal but not MF MNCs. Treatment of MSCs with AVID200, a potent TGFβ1/3 protein trap, significantly decreased MSC proliferation, phosphorylation of SMAD2, and collagen expression. Robust expression of pSMAD2 was observed in the absence of exogenous TGFβ in normal donor or MF-MKs, Addition of AVID200 to -MKs decreased pSMAD2 without affecting total SMAD2/3, indicating that AVID200 blocks the effects of autocrine TGFβ produced by MKs and led to increased numbers of MKs. Moreover, treatment of primary MF MNCs with AVID200 led to increased numbers of progenitor cells with wild type JAK2 and a reduction of mutated colonies. AVID200 blocked TGFβ1-induced p57Kip2 expression and SMAD2 activation by MF MNCs allowing the normal progenitor cells to preferentially cycle, proliferate, and form hematopoietic colonies. Clinical Trial Design: Based on these findings, a phase 1 trial of AVID200 is ongoing in INT-2/high risk MF subjects resistant or intolerant to ruxolitinib; baseline platelet count of ≥ 25 x 109/L, and grade 2/3 BMF. Subjects received intravenous AVID200 (Lots A and B) in dose cohorts of 180 mg/m2 (A), 550 mg/m2 (A), 180 mg/m2 (B) on Day 1 of a 21 day cycle. Cohorts of 3 subjects with a target toxicity rate of 30% were enrolled to estimate the maximum tolerated dose (MTD). A modified toxicity probability interval design was used. Response was assessed by IWG/ELN criteria after 6 cycles of AVID200. Subjects attaining at least a CI or SD with a decrease in BMF by ≥1 grade, continued AVID200. Clinical Trial Results: 10 subjects were enrolled (1 withdrew before receiving treatment) and 9 were treated with AVID200 and were evaluable for DLT assessment [Table1]. Median time after ruxolitinib discontinuation was 3.5 months (0.5-12.2). No DLTs were observed. Grade 3/4 AEs (regardless of attribution) were observed in 6 (66.7%) subjects. Grade 3/4 non-hematologic AEs observed were epistaxis (1, 11.1%), extraocular muscle paresis (1, 11.1%), fatigue (1, 11.1%) and rash (1, 11.1%). Grade 3/4 hematologic AEs were anemia (3, 33.3%) and thrombocytopenia (2, 22.2%) [Table 2]. The median number of cycles received was 5.7 (range 0 - 12). 5 subjects received 6+ cycles and were evaluable. CI occurred in 2 subjects [anemia, spleen and TSS (n=1); TSS (n=1)] 1 of which is still being treated, 2 subjects had SD, 1 subject with 21% blasts prior to study treatment had progressive MPN-BP. 4 subjects failed to reach response evaluation after 6 cycles, 2 had PD due to increasing splenomegaly, 1 subject received an allogeneic transplant and 1 is still being treated [Cycle 2]. The median platelet count at baseline was 114 (range: 42-290) and 159 after cycle 6 [Figure 1]. Maximum changes in platelets from baseline was +64% [range -73%, 169%] in all subjects. 7 subjects had an increase in platelets from baseline during treatment. 2 subjects normalized their platelet count from thrombocytopenic levels. The effect of AVID200 on BMF is currently being examined. 2 subjects remain on treatment. Conclusions: AVID200 a TGFβ1/3 protein trap is well tolerated in advanced MF subjects. Clinical responses were observed at the 550 mg dose and the expansion efficacy cohorts at doses 2 and 3 are enrolling 12 additional subjects. Furthermore, AVID200 therapy improved thrombocytopenia in MF subjects which may be due to AVID200 inhibiting the effects of TGFβ1 on normal MKpoiesis. Updated subject safety and efficacy data along with correlative data will be presented. Disclosures Mascarenhas: Celgene, Prelude, Galecto, Promedior, Geron, Constellation, and Incyte: Consultancy; Incyte, Kartos, Roche, Promedior, Merck, Merus, Arog, CTI Biopharma, Janssen, and PharmaEssentia: Other: Research funding (institution). Kuykendall:Blueprint Medicines: Research Funding; BMS: Research Funding; Incyte: Research Funding; Novartis: Research Funding. Komrokji:Jazz: Honoraria, Speakers Bureau; Abbvie: Honoraria; Agios: Speakers Bureau; BMS: Honoraria, Speakers Bureau; Geron: Honoraria; Incyte: Honoraria; Acceleron: Honoraria; Novartis: Honoraria. Gerds:Gilead Sciences: Research Funding; Imago Biosciences: Research Funding; Sierra Oncology: Research Funding; Celgene: Consultancy, Research Funding; Roche/Genentech: Research Funding; CTI Biopharma: Consultancy, Research Funding; Apexx Oncology: Consultancy; AstraZeneca/MedImmune: Consultancy; Pfizer: Research Funding; Incyte Corporation: Consultancy, Research Funding. Migliaccio:Novartis: Research Funding. O'Connor-McCourt:Forbius: Current Employment. Tremblay:Forius: Current Employment. Nadler:Forbius: Consultancy; Nadler Pharma Associates: Current Employment; Symphogen: Consultancy; Iksuda Therapeutics: Consultancy; Tessa Therapeutics: Consultancy. Mesa:Celgene: Research Funding; Genetech: Research Funding; Samus: Research Funding; Promedior: Research Funding; CTI: Research Funding; LaJolla Pharma: Consultancy; Incyte: Research Funding; Sierra Onc: Consultancy; Abbvie: Research Funding; Novartis: Consultancy. Hoffman:Forbius: Consultancy; Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Dompe: Research Funding; Protagonist: Consultancy.