Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients: A proof-of-concept study.

Glycolytic activity and in vitro effect of the pyruvate kinase activator AG-946 in red blood cells from low-risk myelodysplastic syndromes patients. Data showed decreased glycolytic activity in red blood cells of 2/3 of patients with lower-risk MDS. These results highlight a potential effect of the PK activator in this setting.

Transfusions in autoimmune hemolytic anemias: Frequency and clinical significance of alloimmunization.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) may be associated with transfusion reactions and risk of alloimmunization. OBJECTIVES: To evaluate the transfusion policy and rate of alloimmunization and its clinical significance in AIHA. METHODS: Data from 305 AIHA patients followed at a reference hematologic Center in Milan, Italy from 1997 to 2022 were retrospectively/prospectively collected (NCT05931718). RESULTS: Overall, 33% patients required transfusions with a response rate of 83% and eight transfusion reactions (7%), none hemolytic. Alloantibodies were detected in 19% of patients, being associated with higher transfusion burden (p = 0.01), lower Hb increase post-transfusion (p = 0.05), and transfusion reactions (p = 0.04). Along decades, the rate of RBC transfusions decreased from 53% to 20% and that of alloimmunization dropped from 30% to 6% likely due to the adoption of prestorage leukoreduction, the use of more restrictive Hb thresholds, and the implementation of molecular typing. CONCLUSIONS: Severe symptomatic AIHA may be safely transfused provided appropriate matching of patients and donors.

Recombinant erythropoietin in autoimmune hemolytic anemia with inadequate bone marrow response: a prospective analysis.

ABSTRACT: Up to 30% of patients with autoimmune hemolytic anemia (AIHA) show inadequate bone marrow (BM) compensatory response with inappropriately low levels of reticulocytes and endogenous erythropoietin. Ineffective BM compensation is associated with more severe anemia, transfusion need, and hospital admission, and treatment with recombinant erythropoietin (rEPO) may be beneficial. Here, we prospectively analyzed the efficacy and safety of rEPO in a single-center cohort of 47 patients with AIHA with inadequate reticulocytosis and endogenous erythropoietin at baseline. Epoetin alpha 40 000 international units per week were administered subcutaneously until hemoglobin (Hb) >11 g/dL and then tapered off. Overall response was 55% at 15 days, 74% at 1 month, 74% at 3 months, 80% at 6 months, and 91% at 12 months. Consistently, Hb values significantly increased from baseline to each subsequent time point (P<.001) with a median increase of +1.4, +2.4, +3.4, +3.8, and +4.4 g/dL, respectively. Transfusion needs reduced from 30% to <10% at 15 days and thereafter (P < .001). Concomitant medications included prednisone or methylprednisolone (N = 40, stable since >2 weeks from enrollment), mycophenolate mofetil (N = 1, ongoing since >3 months from enrollment), and rituximab (N = 7 patients with cold agglutinin disease from day 8). No association between concomitant medications and response to rEPO was found. Treatment was generally safe without rEPO-related severe adverse events. The comparison with an AIHA population not treated with rEPO showed a significant benefit of rEPO at 15 days and 1 month on response and Hb increase. These data support the use of rEPO as an add on to standard immunosuppression in AIHA with inadequate BM compensation. This trial was registered at www.clinicaltrials.gov as #NCT05931718.

Cytokine polymorphisms in patients with autoimmune hemolytic anemia.

Autoimmune hemolytic anemia (AIHA) is due to autoantibodies with or without complement activation and involves cellular and cytokine dysregulation. Here, we investigated cytokine single-nucleotide polymorphisms (SNPs) of TNF-α, TGF-ß1, IL-10, IL-6, and IFN-γ, along with their serum levels. The former were related to hematological parameters, therapy, and clinical outcome. The study included 123 consecutive patients with primary AIHA [77 warm AIHA and 46 cold agglutinin disease (CAD)], followed up for a median of 49 months. Results show that the allelic frequency of TNF-α -308 G/A polymorphisms was significantly lower in patients versus controls. Moreover, the genotypic frequency of TNF-α -308G/A and TGF-ß gene codon 25 G/C genotypes was significantly lower in patients versus controls. Considering cytokine SNP genotypes associated with different gene expression levels, TNF-α high gene expression was significantly more frequent in patients, TGF-ß and IL-10 high gene expression was higher in patients with more severe anemia, and TGF-ß high gene expression was higher in patients with active disease. Considering treatment, TNF-α and TGF-ß high gene expression was more frequent in multitreated patients and particularly in CAD. It may be speculated that this genetic predisposition to a stronger inflammatory response may result in a greater immune dysregulation and in a relapsed/refractory disease. Regarding cytokine serum levels, TNF-α and TGF-ß were significantly lower, and IL-10 and IL-6 were significantly higher in patients versus controls, underlying the complex interplay between genetic background and disease features.

Evaluation of the main regulators of systemic iron homeostasis in pyruvate kinase deficiency.

Iron homeostasis and dyserythropoiesis are poorly investigated in pyruvate kinase deficiency (PKD), the most common glycolytic defect of erythrocytes. Herein, we studied the main regulators of iron balance and erythropoiesis, as soluble transferrin receptor (sTfR), hepcidin, erythroferrone (ERFE), and erythropoietin (EPO), in a cohort of 41 PKD patients, compared with 42 affected by congenital dyserythropoietic anemia type II (CDAII) and 50 with hereditary spherocytosis (HS). PKD patients showed intermediate values of hepcidin and ERFE between CDAII and HS, and clear negative correlations between log-transformed hepcidin and log-EPO (Person's r correlation coefficient = - 0.34), log-hepcidin and log-ERFE (r = - 0.47), and log-hepcidin and sTfR (r = - 0.44). sTfR was significantly higher in PKD; EPO levels were similar in PKD and CDAII, both higher than in HS. Finally, genotype-phenotype correlation in PKD showed that more severe patients, carrying non-missense/non-missense genotypes, had lower hepcidin and increased ERFE, EPO, and sTFR compared with the others (missense/missense and missense/non-missense), suggesting a higher rate of ineffective erythropoiesis. We herein investigated the main regulators of systemic iron homeostasis in the largest cohort of PKD patients described so far, opening new perspectives on the molecular basis and therapeutic approaches of this disease.

Case report: Transfusion independence and abolition of extravascular hemolysis in a PNH patient treated with pegcetacoplan.

More than half of patients with paroxysmal nocturnal hemoglobinuria (PNH) treated with complement fraction C5 inhibitors experience residual anemia and hemolysis. This is partly due to the persistent activation of the complement cascade upstream C5, resulting in C3 deposition on PNH erythrocytes and extravascular hemolysis in the reticuloendothelial system. Pegcetacoplan is the first proximal C3 inhibitor to be approved for PNH basing on favorable efficacy and safety data in both naïve and eculizumab treated PNH. Here we report the first Italian patient treated with pegcetacoplan in a named patient program. The patient suffered from hemolytic PNH associated with CALR+ myeloproliferative neoplasm and was heavily transfusion dependent despite eculizumab therapy. Treatment with pegcetacoplan induced a dramatic improvement in Hb, along with normalization of unconjugated bilirubin and reticulocytes, as markers of extravascular hemolysis. Sequential laboratory workup showed the disappearance of C3 deposition on erythrocytes by direct anti-globulin test, the increase of PNH clone on erythrocytes, and a peculiar right shift of the ektacytometry curve. The drug was well tolerated, and the patient reported a significant improvement in his quality of life. Overall, pegcetacoplan appears a safe and effective option "ready to use" in the clinic for patients with PNH and suboptimal response to anti-C5 agents.

Effect of primary lesions in cytoskeleton proteins on red cell membrane stability in patients with hereditary spherocytosis.

We investigated by targeted next generation sequencing the genetic bases of hereditary spherocytosis in 25 patients and compared the molecular results with the biochemical lesion of RBC membrane obtained by SDS-PAGE analysis. The HS diagnosis was based on available guidelines for diagnosis of congenital hemolytic anemia, and patients were selected because of atypical clinical presentation or intra-family variability, or because presented discrepancies between laboratory investigation and biochemical findings. In all patients but 5 we identified pathogenic variants in SPTA1, SPTB, ANK1, SLC4A1, EPB42 genes able to justify the clinical phenotype. Interestingly, a correspondence between the biochemical lesion and the molecular defect was identified in only 11/25 cases, mostly with band 3 deficiency due to SLC4A1 mutations. Most of the mutations in SPTB and ANK1 gene didn't hesitate in abnormalities of RBC membrane protein; conversely, in two cases the molecular lesion didn't correspond to the biochemical defect, suggesting that a mutation in a specific cytoskeleton protein may result in a more complex RBC membrane damage or suffering. Finally, in two cases the HS diagnosis was maintained despite absence of both protein defect and molecular lesion, basing on clinical and family history, and on presence of clear laboratory markers of HS. The study revealed complex relationships between the primary molecular lesion and the final effect in the RBC membrane cytoskeleton, and further underlines the concept that there is not a unique approach to the diagnosis of HS.

Intravascular hemolysis and multitreatment predict thrombosis in patients with autoimmune hemolytic anemia.

BACKGROUND: Thrombosis may complicate autoimmune hemolytic anemia (AIHA), but its predictors are still lacking, and no clear-cut indications for anticoagulant prophylaxis are available. OBJECTIVES: To characterize frequency and severity of thromboses in AIHA patients and identify risk factors for thrombosis that may advise primary anticoagulant prophylaxis. PATIENTS/METHODS: A total of 287 consecutive AIHA patients diagnosed and followed from 1978 at a tertiary Italian center were retrospectively studied; 174 of them were prospectively evaluated from January 2020 until December 2021. AIHA relapse, thrombosis occurrence, and primary anticoagulant prophylaxis were evaluated. RESULTS: Thirty-three AIHA patients (11.4%) experienced thrombosis, 70% of whom hospitalized. The cumulative thrombosis incidence was higher in patients with lactate dehydrogenase (LDH) ≥ 1.5 (hazard ratio [HR] 3.22), in those experiencing infections (HR 3.57), receiving transfusions (HR 3.06), rituximab (HR 3.3), or cyclophosphamide (HR 2.67). By multivariable analysis, LDH, transfusions, rituximab, and cyclophosphamide treatment emerged as independent factors associated with thrombosis. Among 174 patients prospectively followed in the past 2 years, we observed 70 acute hemolytic episodes in 45 patients; 33/45 displayed LDH ≥1.5 × upper limit of normal, and 17 received anticoagulant prophylaxis with low molecular weight heparin for a median of 70 days (30-300). In those receiving prophylaxis no thrombotic complications occurred, whereas five thrombotic episodes were registered in the remaining 16 cases. CONCLUSIONS: Thrombosis was observed in about 11% of AIHA patients, mainly grade 3, and associated with intravascular hemolysis, need of transfusions, multitreatment, and infections, advising primary anticoagulant prophylaxis in these settings.

Efficacy and Immunomodulating Properties of Eltrombopag in Aplastic Anemia following Autologous Stem Cell Transplant: Case Report and Review of the Literature.

Thrombopoietin receptor agonists (TPO-RA) are currently indicated for the treatment of chronic immune thrombocytopenia and relapsed refractory aplastic anemia. However, the off-label use of these drugs is more and more frequent, including in the setting of aplasia secondary to chemotherapy and hemopoietic stem cell transplant (SCT). Growing evidence suggests that mechanisms of action of TPO-RA go beyond the TPO-receptor stimulation and point at the immunomodulating properties of these drugs. Here, we present a case of prolonged bone marrow aplasia secondary to autologous SCT treated with eltrombopag. We describe the clinical efficacy and the immunomodulating effect of this drug on inflammatory cytokine profile and bone marrow histology. Furthermore, we provide a review of the most recent literature highlighting the efficacy and safety of TPO-RA after SCT and chemotherapy for hematologic conditions.

Low-Risk Myelodysplastic Syndrome Revisited: Morphological, Autoimmune, and Molecular Features as Predictors of Outcome in a Single Center Experience.

Low-risk myelodysplastic syndromes (LR-MDS) are a very heterogeneous disease, with extremely variable clinical features and outcome. Therapeutic strategies are still limited and mainly consist of erythropoiesis-stimulating agents (ESAs) and transfusion support. The contribution of molecular lesions and of autoimmune phenomena to pathogenesis and clinical course, including leukemic evolution, is a field of open investigation. We analyzed data from a cohort of 226 patients with LR-MDS followed at our center in the last 20 years, focusing on morphological, immunological (antiplatelets and anti-erythrocyte autoantibodies, anti-erythroblast antibodies), and molecular features. Hypoplastic bone marrow was found in 7% of the cases correlating with younger age, deeper cytopenia, lower dysplasia, and worse response to ESAs. A marker of autoimmunity was observed in 46% of the tested cases, who were younger, were less frequent dysplastic changes, and responded better to ESAs and steroids. Finally, 68% of the tested cases displayed at least one somatic mutation, most commonly SF3B1, TET2, ASXL1, and SRSF2, associated with older age, presence of neutropenia, and lower response to ESAs. Leukemic evolution (2.2%) was associated with presence of somatic mutations, and survival was favorably related to response to ESAs and transfusion independence. Overall, granular evaluation and re-evaluation are pivotal in LR-MDS patients to optimize clinical management.

Changing trends of splenectomy in hereditary spherocytosis: The experience of a reference Centre in the last 40 years.

We describe the clinical/haematological characteristics of 446 patients with hereditary spherocytosis diagnosed in the last 40 years in a reference centre. The frequency of splenectomy decreased over time (44% before 1990 to 7% in 2011-2020), notwithstanding a confirmed good efficacy. Age at splenectomy progressively increased (63% in children before 1990 to 88% in patients aged ≥20 years in 2011-2020). Our real-life experience showed that even a fraction of patients in the trait/mild categories (19/92, 21%) were splenectomised, whilst 30/78 (38%) in the moderate/severe groups were not. Overall, these data pinpoint to the increasing awareness about post-splenectomy thromboses and infections.

Evans syndrome in adults: an observational multicenter study.

Evans syndrome (ES) is a rare condition, defined as the presence of 2 autoimmune cytopenias, most frequently autoimmune hemolytic anemia and immune thrombocytopenia (ITP) and rarely autoimmune neutropenia. ES can be classified as primary or secondary to various conditions, including lymphoproliferative disorders, other systemic autoimmune diseases, and primary immunodeficiencies, particularly in children. In adult ES, little is known about clinical features, disease associations, and outcomes. In this retrospective international study, we analyzed 116 adult patients followed at 13 European tertiary centers, focusing on treatment requirements, occurrence of complications, and death. ES was secondary to or associated with underlying conditions in 24 cases (21%), mainly other autoimmune diseases and hematologic neoplasms. Bleeding occurred in 42% of patients, mainly low grade and at ITP onset. Almost all patients received first-line treatment (steroids with or without intravenous immunoglobulin), and 23% needed early additional therapy for primary refractoriness. Additional therapy lines included rituximab, splenectomy, immunosuppressants, thrombopoietin receptor agonists, and others, with response rates >80%. However, a remarkable number of relapses occurred, requiring ≥3 therapy lines in 54% of cases. Infections and thrombotic complications occurred in 33% and 21% of patients, respectively, mainly grade ≥3, and correlated with the number of therapy lines. In addition to age, other factors negatively affecting survival were severe anemia at onset and occurrence of relapse, infection, and thrombosis. These data show that adult ES is often severe and marked by a relapsing clinical course and potentially fatal complications, pinpointing the need for high clinical awareness, prompt therapy, and anti-infectious/anti-thrombotic prophylaxis.

Mechanistic ion channel interactions in red cells of patients with Gárdos channelopathy.

In patients with Gárdos channelopathy (p.R352H), an increased concentration of intracellular Ca2+ was previously reported. This is a surprising finding because the Gárdos channel (KCa3.1) is a K+ channel. Here, we confirm the increased intracellular Ca2+ for patients with the KCa3.1 mutation p.S314P. Furthermore, we provide the concept of KCa3.1 activity resulting in a flickering of red blood cell (RBC) membranepotential, which activates the CaV2.1 channel allowing Ca2+ to enter the RBC. Activity of the nonselective cation channel Piezo1 modulates the aforementioned interplay in away that a closed Piezo1 is in favor of the KCa3.1-CaV2.1 interaction. In contrast, Piezo1 openings compromise the membrane potential flickering, thus limiting the activity of CaV2.1. With the compound NS309, we mimic a gain-of-function mutation of KCa3.1. Assessing the RBC Ca2+ response by Fluo-4-based flow cytometry and by measuring the membrane potential using the Macey-Bennekou-Egée method, we provide data that support the concept of the KCa3.1/CaV2.1/Piezo1 interplay as a partial explanation for an increased number of high Ca2+ RBCs. With the pharmacological inhibition of KCa3.1 (TRAM34 and Senicapoc), CaV2.1 (ω-agatoxin TK), and Piezo1 (GsMTx-4), we could project the NS309 behavior of healthy RBCs to the RBCs of Gárdos channelopathy patients.

Targeted Next Generation Sequencing and Diagnosis of Congenital Hemolytic Anemias: A Three Years Experience Monocentric Study.

Congenital hemolytic anemias (CHAs) are heterogeneous and rare disorders caused by alterations in structure, membrane transport, metabolism, or red blood cell production. The pathophysiology of these diseases, in particular the rarest, is often poorly understood, and easy-to-apply tools for diagnosis, clinical management, and patient stratification are still lacking. We report the 3-years monocentric experience with a 43 genes targeted Next Generation Sequencing (t-NGS) panel in diagnosis of CHAs; 122 patients from 105 unrelated families were investigated and the results compared with conventional laboratory pathway. Patients were divided in two groups: 1) cases diagnosed with hematologic investigations to be confirmed at molecular level, and 2) patients with unexplained anemia after extensive hematologic investigation. The overall sensitivity of t-NGS was 74 and 35% for families of groups 1 and 2, respectively. Inside this cohort of patients we identified 26 new pathogenic variants confirmed by functional evidence. The implementation of laboratory work-up with t-NGS increased the number of diagnoses in cases with unexplained anemia; cytoskeleton defects are well detected by conventional tools, deserving t-NGS to atypical cases; the diagnosis of Gardos channelopathy, some enzyme deficiencies, familial siterosterolemia, X-linked defects in females and other rare and ultra-rare diseases definitely benefits of t-NGS approaches.

The Immunomodulatory Effect and Clinical Efficacy of Daratumumab in a Patient With Cold Agglutinin Disease.

Daratumumab is a monoclonal antibody directed against the transmembrane glycoprotein CD38 expressed on plasma cells and lymphoplasmocytes, with a proven efficacy in multiple myeloma. Here we show its clinical efficacy in a patient with cold agglutinin disease (CAD) relapsed after multiple lines of therapy. CAD is caused by cold reactive autoantibodies that induce complement mediated hemolysis and peripheral circulatory symptoms. The disease is also characterized by the presence of monoclonal IgM gammopathy and of a lymphoid bone marrow infiltration that benefits from B-cell targeting therapies (i.e., rituximab) but also from plasma cell directed therapies, such as proteasome inhibitors. In the patient described, we also show that daratumumab therapy influenced the dynamics of several immunoregulatory cytokine levels (IL-6, IL-10, IL-17, IFN-γ, TNF-α, TGF-ß) indicating an immunomodulatory effect of the drug beyond plasma cell depletion. In addition, we provide a literature review on the use of daratumumab in autoimmune conditions, including multi-treated and refractory patients with autoimmune hemolytic anemia (both CAD and warm forms), Evans syndrome (association of autoimmune hemolytic anemia and immune thrombocytopenia) and non-hematologic autoimmune diseases, such as systemic lupus erythematosus and rheumatoid arthritis.