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.

Complement inhibition in paroxysmal nocturnal hemoglobinuria: From biology to therapy.

Complement inhibitors are the mainstay of paroxysmal nocturnal hemoglobinuria (PNH) treatment. The anti-C5 monoclonal antibody eculizumab was the first treatment to improve hemolysis, thrombotic risk, and survival in PNH although at the price of a life-long intravenous fortnightly drug. Additionally, suboptimal response may occur in up to 2/3 of patients with persistent anemia due to incomplete control of intravascular hemolysis, development of upstream C3-mediated extravascular hemolysis (EVH), or concomitant bone marrow failure. Ravulizumab, a longer half-life anti-C5 developed from eculizumab, administered every 8 weeks, improved patient convenience, and reduced pharmacokinetic breakthrough hemolysis (BTH) by establishing more stable anti-C5 concentrations. More recently, several other anti-C5 compounds (crovalimab, pozelimab, tesidolumab, cemdisiran, zilucoplan, and coversin) are on study in clinical trials. Upstream inhibition of complement cascade was also explored with the anti-C3 pegcetacoplan, and with the alternative pathway inhibitors iptacopan (anti-factor B) and danicopan (anti-factor D). These drugs efficiently target EVH and are able to improve anemia and transfusion need in suboptimal responders to anti-C5. The route and schedule of administration (twice weekly subcutaneously for pegcetacoplan and twice or thrice oral daily dosing for iptacopan and danicopan, respectively) are very convenient but pose novel issues regarding adherence. Additionally, both anti-C5 and upstream inhibitors do not resolve the unmet need of pharmacodynamic BTH events due to complement amplifying conditions such as infections, traumas, and surgery. In this review, we will recapitulate PNH physiopathology, clinical presentation, and diagnosis and describe available and developing drugs that will lead to a precision medicine approach for this rare though heterogenous disease.

Deciphering cytopenias in internal medicine: a single-center observational study.

Cytopenia is a common finding in patients admitted to internal medicine wards and the clinical workup may be long and time-consuming. In this single-center observational study, we analyzed a series of 151 inpatients who received hematologist referral due to cytopenia observed during hospital admission. Patients were mainly elderly (median 71 years, 15-96) and 87% had at least one comorbidity. Anemia was the most common cytopenia (91%), followed by thrombocytopenia (51%), and neutropenia (22%); 73 (48%) patients had a bicytopenia and 5 (3%) pancytopenia. Cytopenias were mainly severe, 66% of cases required RBC transfusions, and 21% platelet pools. During a median hospital stay of 15 days (1-166), 53 subjects (35%) received a hematologic discharge diagnosis, whilst the two-thirds had secondary cytopenia mainly due to associated comorbidities. Only about 34% of 2,728 diagnostic tests performed (including laboratory, imaging, and histology) clearly informed the discharge diagnosis in this heterogenous setting. Specifically, bone-marrow evaluation indicated in 46 (30%) patients, was diagnostic in 32 (69.6%). Eleven percent of patients died due to progression of the oncohematologic disease (29%), sepsis (24%), and solid tumor progression (24%). In conclusion, cytopenias in the internal medicine setting are mainly severe, more frequently secondary to associated comorbidities (2/3 of patients) and deserve proper workup before second/third-level tests (immune-hematological assays and CT scan or PET and bone-marrow evaluation, respectively).

Current use of androgens in bone marrow failure disorders: a report from the Severe Aplastic Anemia Working Party of the European Society for Blood and Marrow Transplantation.

Androgens represent the historical therapeutic backbone of bone marrow failure (BMF) syndromes. However, their role has rarely been analyzed in a prospective setting, and systematic and long-term data regarding their usage, effectiveness and toxicity in both acquired and inherited BMF are currently unavailable. Here, taking advantage of a unique disease-specific international dataset, we retrospectively analyzed the largest cohort so far of BMF patients who received androgens before or in the absence of an allogeneic hematopoietic cell transplantation (HCT), re-evaluating their current use in these disorders. We identified 274 patients across 82 European Society for Blood and Marrow Transplantation (EBMT) affiliated centers: 193 with acquired (median age 32 years) and 81 with inherited (median age 8 years) BMF. With a median duration of androgen treatment of 5.6 and 20 months, respectively, complete and partial remission rates at 3 months were 6% and 29% in acquired and 8% and 29% in inherited disorders. Five-year overall survival and failure-free survival (FFS) were respectively 63% and 23% in acquired and 78% and 14% in inherited BMF. Androgen initiation after second-line treatments for acquired BMF, and after >12 months post diagnosis for inherited BMF were identified as factors associated with improved FFS in multivariable analysis. Androgen use was associated with a manageable incidence of organ-specific toxicity, and low rates of solid and hematologic malignancies. Sub-analysis of transplant-related outcomes after exposure to these compounds showed probabilities of survival and complications similar to other transplanted BMF cohorts. This study delivers a unique opportunity to track androgen use in BMF syndromes and represents the basis for general recommendations on this category of therapeutics on behalf of the Severe Aplastic Anemia Working Party of the EBMT.

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.

Deciphering treatment patterns in non-severe/moderate aplastic anemia: an international observational study.

Non-severe aplastic anemia is a rare bone marrow failure disorder characterized by variable degrees and combination of cytopenias, with limited data on management and outcome. We describe a large multicentric series of 259 patients, focusing on clinical and molecular features, treatment, evolution, and survival. The majority required treatment with cyclosporine (CyA) alone (N = 84) or in combination with anti-thymocyte globulin (ATG,44) or eltrombopag (20), eltrombopag alone (10), or others (25) including androgens. Similar outcomes were observed across different strategies, with a 6-month overall response rate of 73% for CyA, 74% for ATG plus CyA, 68% for CyA plus eltrombopag, 87% for eltrombopag, and 79% for others. Notably, 56 patients (39%), mainly receiving CyA plus eltrombopag, achieved a trilineage response (p = 0.02). Progression to myeloid neoplasms was limited (8%) and not related to mutational status. Hemolytic PNH developed in 10% of cases, being predicted by detection of small clones at diagnosis. Survival was negatively impacted by age, male gender, LDH, platelets/erythrocyte transfusion need, and somatic mutations by NGS, and positively by higher neutrophils at diagnosis, PNH clones, and trilineage response at 6 and 12 months. Multivariable analysis confirmed the detrimental role of age and the favorable association with PNH clone and trilineage response at 6 months.

Androgen use in bone marrow failures and myeloid neoplasms: Mechanisms of action and a systematic review of clinical data.

Despite recent advancements, treatment of cytopenia due to bone marrow failures (BMF) and myeloid neoplasms remains challenging. Androgens promote renewal and maturation of blood cells and may be beneficial in these forms. Here we report a systematic review of androgens use as single agent in hematologic conditions. Forty-six studies, mainly retrospective with various androgen types and doses, were included: 12 on acquired aplastic anemia (AA), 11 on inherited BMF, 17 on myelodysplastic syndromes (MDS), and 7 on myelofibrosis. Responses ranged from 50 to 70% in inherited BMF, 40-50% in acquired AA and MDS, while very limited evidence emerged for myelofibrosis. In acquired AA, response was associated with presence of non-severe disease; in MDS androgens were more effective on thrombocytopenia or mild to moderate anemia, whilst limited benefit was observed for transfusion dependent anemia. Toxicity profile mainly consisted of virilization and liver enzyme elevation, whilst the risk of leukemic evolution remains controversial.

Eltrombopag for Low-Risk Myelodysplastic Syndromes With Thrombocytopenia: Interim Results of a Phase II, Randomized, Placebo-Controlled Clinical Trial (EQOL-MDS).

PURPOSE: In myelodysplastic syndromes (MDS), severe thrombocytopenia is associated with poor prognosis. This multicenter trial presents the second-part long-term efficacy and safety results of eltrombopag in patients with low-risk MDS and severe thrombocytopenia. METHODS: In this single-blind, randomized, placebo-controlled, phase-II trial of adult patients with International Prognostic Scoring System low- or intermediate-1-risk MDS, patients with a stable platelet (PLT) count (<30 × 103/mm3) received eltrombopag or placebo until disease progression. Primary end points were duration of PLT response (PLT-R; calculated from the time of PLT-R to date of loss of PLT-R, defined as bleeding/PLT count <30 × 103/mm3 or last date in observation) and long-term safety and tolerability. Secondary end points included incidence and severity of bleeding, PLT transfusions, quality of life, leukemia-free survival, progression-free survival, overall survival and pharmacokinetics. RESULTS: From 2011 to 2021, of 325 patients screened, 169 patients were randomly assigned oral eltrombopag (N = 112) or placebo (N = 57) at a starting dose of 50 mg once daily to maximum of 300 mg. PLT-R, with 25-week follow-up (IQR, 14-68) occurred in 47/111 (42.3%) eltrombopag patients versus 6/54 (11.1%) in placebo (odds ratio, 5.9; 95% CI, 2.3 to 14.9; P < .001). In eltrombopag patients, 12/47 (25.5%) lost the PLT-R, with cumulative thrombocytopenia relapse-free survival at 60 months of 63.6% (95% CI, 46.0 to 81.2). Clinically significant bleeding (WHO bleeding score ≥ 2) occurred less frequently in the eltrombopag arm than in the placebo group (incidence rate ratio, 0.54; 95% CI, 0.38 to 0.75; P = .0002). Although no difference in the frequency of grade 1-2 adverse events (AEs) was observed, a higher proportion of eltrombopag patients experienced grade 3-4 AEs (χ2 = 9.5, P = .002). AML evolution and/or disease progression occurred in 17% (for both) of eltrombopag and placebo patients with no difference in survival times. CONCLUSION: Eltrombopag was effective and relatively safe in low-risk MDS with severe thrombocytopenia. This trial is registered with ClinicalTrials.gov identifier: NCT02912208 and EU Clinical Trials Register: EudraCT No. 2010-022890-33.

Strategies to overcome the diagnostic challenges of autoimmune hemolytic anemias.

INTRODUCTION: The direct antiglobulin test (DAT) or Coombs test is the cornerstone of the diagnosis of autoimmune hemolytic anemia (AIHA). It can be performed by several methods with different sensitivity and specificity and enables the distinction of warm, cold, and mixed forms, which require different therapies. AREAS COVERED: The review describes the different DAT methods, including the tube test with monospecific antisera, microcolumn and solid phase methods that are routinely accessible in most laboratories. Additional investigations include the use of cold washes and low ionic salt solutions, the identification of auto-Ab specificity and thermal range, the study of the eluate, and the Donath-Landsteiner test, available in most reference laboratories. Experimental techniques are the dual-DAT, flow cytometry, ELISA, immuno-radiometric assay, and mitogen-stimulated DAT, which may help the diagnosis of DAT-negative AIHAs, a clinical challenge with delayed diagnosis and possible improper therapy. Further diagnostic challenges include the correct interpretation of hemolytic markers, the infectious and thrombotic complications, and the possible underlying conditions (lymphoproliferative disorders, immunodeficiencies, neoplasms, transplants, and drugs). EXPERT OPINION: These diagnostic challenges may be overcome by a 'hub' and 'spoke' organization among laboratories, a clinical validation of experimental techniques, and a continuous dialogue between clinicians and immune-hematologic laboratory experts.

Vaccinations in hematological patients in the era of target therapies: Lesson learnt from SARS-CoV-2.

Novel targeting agents for hematologic diseases often exert on- or off-target immunomodulatory effects, possibly impacting on response to anti-SARS-CoV-2 vaccinations and other vaccines. Agents that primarily affect B cells, particularly anti-CD20 monoclonal antibodies (MoAbs), Bruton tyrosine kinase inhibitors, and anti-CD19 chimeric antigen T-cells, have the strongest impact on seroconversion. JAK2, BCL-2 inhibitors and hypomethylating agents may hamper immunity but show a less prominent effect on humoral response to vaccines. Conversely, vaccine efficacy seems not impaired by anti-myeloma agents such as proteasome inhibitors and immunomodulatory agents, although lower seroconversion rates are observed with anti-CD38 and anti-BCMA MoAbs. Complement inhibitors for complement-mediated hematologic diseases and immunosuppressants used in aplastic anemia do not generally affect seroconversion rate, but the extent of the immune response is reduced under steroids or anti-thymocyte globulin. Vaccination is recommended prior to treatment or as far as possible from anti-CD20 MoAb (at least 6 months). No clearcut indications for interrupting continuous treatment emerged, and booster doses significantly improved seroconversion. Cellular immune response appeared preserved in several settings.

Luspatercept in combination with recombinant erythropoietin in patients with myelodysplastic syndrome with ring sideroblasts: Stimulating early and late-stage erythropoiesis.

Patients with myelodysplastic syndromes and ring sideroblasts (MDS RS) are clinically characterized by severe anemia and transfusion need. Erythropoiesis-stimulating agents (ESAs), which stimulate hemoglobin production and early maturation of erythroid precursors, are effective only in a portion of patients and for limited time. Luspatercept, an inhibitor of the TGF-beta pathway, is beneficial in unblocking late-stage erythropoiesis and has been approved for MDS RS patients failing or not-candidate to ESAs. ESAs and/or luspatercept failure represents an unmet clinical need and most patients become life-long transfusion dependent. Here, we describe the clinical combination of luspatercept with ESAs (subcutaneous epoetin alpha 40-80 000 IU/week) in seven MDS RS patients. Two patients had ESAs as pre-existing therapy, while five were re-challenged with ESAs as add-on treatment due to luspatercept failure. Three patients achieved hematologic improvement, and one became transfusion independent. No adverse events were noted. This is the first clinical evidence that stimulating both early and late-stage erythropoiesis may offer a further option for this challenging patient population.

Use of steroids in the management of low-risk myelodysplastic syndromes with autoimmune features.

BACKGROUND: The boundaries between myelodysplastic syndromes (MDS) and immune-mediated cytopenias are often difficult to establish and both conditions may benefit from immunosuppressive therapy. The optimal timing and doses of immunosuppressants are largely unknown. MATERIALS AND METHODS: We systematically evaluated a retrospective cohort of 79 patients with low-risk MDS tested for anti-erythrocyte or anti-platelet autoantibodies to assess their frequency and the efficacy of immunosuppression, particularly with steroids. RESULTS: We found autoantibody positivity in 43% of cases and overt autoimmune diseases in 18%, including autoimmune hemolytic anemia, immune thromboctyopenia, and Evans syndrome. Steroid treatment improved cytopenia in about half of patients, with 26% achieving a complete recovery lasting for a median of 12 months. Better responses were observed in anemic patients with anti-erythrocyte autoantibodies than in those with anti-platelet autoantibodies, and the combination with recombinant erythropoietin (7/10) had a possible synergistic effect. Steroid doses were heterogeneous depending on the clinical intent (i.e., anti-inflammatory, immunosuppressive, anabolizing). Patients treated with a dose of 1 mg/kg day of prednisone for overt autoimmune cytopenia showed high rates of complete responses (60%). DISCUSSION: This observation suggests a trial with a short course (2-3 weeks) of standard steroid doses to ascertain efficacy and properly silence the autoimmune pathogenic mechanism. Steroid-related adverse events (16% of cases) should be monitored carefully in this elderly, frail population. In conclusion, features of autoimmunity are present in more than two-thirds of low-risk MDS patients and a trial with prednisone 0.5-1 mg/kg day for 2-3 weeks, with proper monitoring of adverse events, may be useful to improve cytopenias in selected cases.

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.

Novel Therapies for Unmet Clinical Needs in Myelodysplastic Syndromes.

Myelodysplastic syndromes (MDS) are a very heterogeneous disease, with extremely variable clinical features and outcomes. Current management relies on risk stratification based on IPSS and IPSS-R, which categorizes patients into low (LR-) and high-risk (HR-) MDS. Therapeutic strategies in LR-MDS patients mainly consist of erythropoiesis stimulating agents (ESAs), transfusion support, and luspatercept or lenalidomide for selected patients. Current unmet needs include the limited options available after treatment failure, and the consequent transfusion burden with several hospital admissions and poor quality of life. Therapeutic approaches in HR-MDS patients are aimed at changing the natural course of the disease and hypometylating agents (HMA) are the first choice. The only potentially curative treatment is allogeneic stem cell transplant (allo-HCT), restricted to a minority of young and fit candidates. Patients unfit for or those that relapse after the abovementioned options harbor an adverse prognosis, with limited overall survival and frequent leukemic evolution. Recent advances in genetic mutations and intracellular pathways that are relevant for MDS pathogenesis are improving disease risk stratification and highlighting therapeutic targets addressed by novel agents. Several drugs are under evaluation for LR and HR patients, which differ by their mechanism of action, reported efficacy, and phase of development. This review analyzes the current unmet clinical needs for MDS patients and provides a critical overview of the novel agents under development in this setting.