Paroxysmal Nocturnal Hemoglobinuria Patients Research Articles

Advances in research on renal injury in paroxysmal nocturnal.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disease. Clinical manifestations include intravascular hemolysis, renal dysfunction, fatigue, jaundice, pulmonary hypertension, and so on. Renal injury, as a clinical feature of PNH, is difficult to diagnose and is one of the causes of death in patients with PNH. This article reviews the progress in research on PNH combined with renal injury,to improve clinicians' understanding of renal injury in PNH patients, define and judge staging in a timely and accurate manner, enable patients to receive timely and appropriate treatment, and reduce mortality.

The histone demethylase JMJD1C regulates CPS1 expression and promotes the proliferation of paroxysmal nocturnal haemoglobinuria clones through cell metabolic reprogramming.

Paroxysmal nocturnal haemoglobinuria (PNH) is a disorder resulting from erythrocyte membrane deficiencies caused by PIG-A gene mutations. While current treatments alleviate symptoms, they fail to address the underlying cause of the disease-the pathogenic PNH clones. In this study, we found that the expression of carbamoyl phosphate synthetase 1 (CPS1) was downregulated in PNH clones, and the level of CPS1 was negatively correlated with the proportion of PNH clones. Using PIG-A knockout K562 (K562 KO) cells, we demonstrated that CPS1 knockdown increased cell proliferation and altered cell metabolism, suggesting that CPS1 participates in PNH clonal proliferation through metabolic reprogramming. Furthermore, we observed an increase in the expression levels of the histone demethylase JMJD1C in PNH clones, and JMJD1C expression was negatively correlated with CPS1 expression. Knocking down JMJD1C in K562 KO cells upregulated CPS1 and H3K36me3 expression, decreased cell proliferation and increased cell apoptosis. Chromatin immunoprecipitation analysis further demonstrated that H3K36me3 regulated CPS1 expression. Finally, we demonstrated that histone demethylase inhibitor JIB-04 can suppressed K562 KO cell proliferation and reduced the proportion of PNH clones in PNH mice. In conclusion, aberrant regulation of the JMJD1C-H3K36me3-CPS1 axis contributes to PNH clonal proliferation. Targeting JMJD1C with a specific inhibitor unveils a potential strategy for treating PNH patients.

A case of allogeneic peripheral blood stem cell in a paroxysmal nocturnal hemoglobinuria patient from a COVID-positive donor: Managing the unpreventable

Abstract: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired disease caused by clonal expansion of one or more hematopoietic stem cell lines due to a somatic mutation of the phosphatidylinositol glycan anchor gene. Peripheral blood stem cell (PBSC) harvest is a high-risk procedure and the major concern was regarding the development of COVID infection in the recipient. Herein, we describe an inevitable case of PBSC harvest in a PNH patient who was already started on myeloablation and where the donor became positive 1 day before the harvest. Considering the risk–benefit ratio and the need for elaborate arrangements, the initial plan for bone marrow harvesting was changed to PBSC, and hence, the harvest was performed in a COVID-designated unit. No features of graft versus host disease (GVHD) were observed in the patient. The patient was examined for signs and symptoms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and there was no evidence of any infection after the procedure. The weekly analysis of the same was done for 1 month after PBSC and the SARS-CoV-2 status remained negative. The authors opine that PBSC from SARS-CoV-2 can be performed safely in some unpreventable situations.

Detection of GPI-deficient Cells in Paroxysmal Nocturnal Hemoglobinuria Patients Using Flow Cytometry: Current Status in Korea and Proposal for Testing Guidelines

Detection of GPI-deficient Cells in Paroxysmal Nocturnal Hemoglobinuria Patients Using Flow Cytometry: Current Status in Korea and Proposal for Testing Guidelines

Evaluation of the impact of two C5 genetic variants on C5-eculizumab complex stability at the molecular level

Complement C5 is the target of the monoclonal antibody eculizumab, used in complement dysregulating disorders, like the rare disease Paroxysmal Nocturnal Hemoglobinuria (PNH). PNH is an acquired hematopoietic stem cell condition characterized by aberrant destruction of erythrocytes, chronic hemolytic anemia, and thromboembolism propensity. C5 is a protein component of the complement system which is part of the immune system of the body and plays a prominent role in the destruction of red blood cells, misidentifying them as a threat. This work describes the application of molecular dynamics simulations to the study of the underlying interactions between complement C5 and eculizumab. This study also reveals the importance of single nucleotide polymorphisms on C5 protein concerning the effective inhibition of the mAB, involving the mechanistic events taking place at the interface spots of the complex. The predicted conformational change in the C5 Arg885/His/Cys mutation has implications on the protein’s interaction with eculizumab, compromising their compatibility. The acquired insights into the conformational changes, dynamics, flexibility, and interactions shed light on the knowledge of the function of this biomolecule providing answers about the poor response to the treatment in PNH patient carriers of the mutations. By investigating the intricate dynamics, significant connections between C5 and eculizumab can be uncovered. Such insights may aid in the creation of novel compounds or lead to the enhancement of eculizumab’s efficacy. Communicated by Ramaswamy H. Sarma

Eculizumab in patients with paroxysmal nocturnal hemoglobinuria: a real-world study in China

Objective: To evaluate the efficacy and safety of eculizumab in the treatment of paroxysmal nocturnal hemoglobinuria (PNH) in China. Methods: Data from PNH patients who received at least 3 months of full-dose eculizumab and were followed for at least 3 months between December 2022 and July 2023 were retrospectively collected. We evaluated changes in clinical and laboratory parameters after 1, 2, 3, and 6 months of eculizumab treatment. The rates of breakthrough hemolysis (BTH), extravascular hemolysis (EVH), and the occurrence of adverse reactions were also monitored. Results: The study included nine patients, six males and three females, with a median age of 54 (28-69) years. 5 of the patients had classic PNH, while 4 had PNH/AA. The number of episodes of hemoglobinuria was 5 (1-25) per month before eculizumab. 4 patients required blood transfusion, 5 had thrombosis and one had renal impairment before eculizumab. The median time to eculizumab was 6 (3-7) months and the followup period was 3 (3-6) months after treatment. The number of episodes of hemoglobinuria following eculizumab was 0 (0-1). During the followup period, no additional thrombotic events occurred. LDH at any time after eculizumab was lower than at baseline, and some patients' HGB increased. All transfused patients became transfusion-independent after receiving eculizumab. The FACIT-Fatigue score improved by an average of 17.3 points following treatment. 2 patients developed BTH and improved with symptomatic treatment. There were three adverse events that caused mild symptoms. There are no serious adverse events or deaths. Conclusion: Eculizumab can effectively control the hemolytic-related symptoms of PNH in China, reducing the need for blood transfusions to some extent, while also demonstrating a higher safety profile.

Treating paroxysmal nocturnal hemoglobinuria with humanized IgG 2/4 kappa antibody: understanding the ravulizumab structure

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease caused by somatic mutations in hematopoietic stem cells. As molecular studies are essential for a better understanding of new drugs, tools such as the international ImMunoGeneTics information system® are powerful approaches to better understanding the molecular conformations, arrangements, and orientations of two-dimensional (2D) structures of PNH treatments. This review aims to characterize the structure of Ravulizumab, describe its drug design process, and show its amino acid sequence through IGMT Colliers de Perles. Methods: The IMGT/mAb-DB interface was used to evaluate therapeutic antibodies and to clarify their immunological applications and pharmacological effects. Results: Ravulizumab binds to C5 and blocks its activation by complement pathway convertases, thereby preventing the release of the proinflammatory C5a and activation of the terminal pathway. The healthcare cost of PNH patients decreased because smaller doses of RAVUL could promote full complement blockage, maintaining the therapeutic effect. Choosing the epitope, hinge, and Fc region is crucial to balance efficacy and undesirable effects. The better intermolecular interactions of RAVUL with binding sites are due to its nanostructure design. Conclusion: These results bridge the gap between linear amino acid sequences and 3D structures. It may guide the development of new drugs for rare diseases.

Kidney R2* Mapping for Noninvasive Evaluation of Iron Overload in Paroxysmal Nocturnal Hemoglobinuria.

The kidney iron deposition can cause kidney damage and renal insufficiency in paroxysmal nocturnal hemoglobinuria (PNH) patients. Assessment of iron deposition in the kidney is essential for the early diagnosis of renal damage in PNH patients. The purpose of this study was to evaluate kidney R2* (T2* reciprocals) values in PNH patients using the iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL-IQ). Two radiologists measured the R2* values of the renal cortex in 14 PNH patients and 13 healthy volunteers using IDEAL-IQ. Lactate dehydrogenase (LDH), a reliable marker of intravascular hemolysis, was also measured in all participants. The kidney R2* values were significantly higher in PNH patients compared with those in healthy volunteers (P < 0.001). High inter-operator reproducibility of the measurements was also acquired using IDEAL-IQ. LDH levels were also significantly higher in PNH patients compared with those in healthy volunteers (P < 0.001). Kidney R2* values strongly correlated with LDH levels in PNH patients. IDEAL-IQ has a possibility of becoming a useful method for the noninvasive evaluation of renal iron overload in PNH patients.

Treatment Patterns and Healthcare Resource Utilization of Patients With Paroxysmal Nocturnal Hemoglobinuria: A Retrospective Claims Data Analysis.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired hematologic disorder commonly treated with complement inhibitors such as eculizumab, ravulizumab, and pegcetacoplan. This study aims to describe treatment patterns, healthcare resource utilization, and cost for newly diagnosed PNH patients in 2 large, health insurance claims databases: MarketScan and Optum. Among the 271 patients meeting the inclusion criteria in MarketScan, 57.9% were female, and the average age was 46.6 years. Among these newly diagnosed patients, 25.1% (n = 68) of patients received a PNH-specific pharmacologic treatment, and the average time from diagnosis to treatment was 4.7 months. The medication possession ratio was 97.0%, but discontinuation was common (58.8%). The average per-patient-per-month costs were $18,978, driven by pharmacy and infusion ($11,182), outpatient ($4086), and inpatient ($3318) costs. Despite the availability of multiple treatments, 39.9% of patients had an inpatient stay, and 50.9% had an emergency department visit. Better care management and the introduction of new treatment options are needed to address delays between diagnosis and treatment, and high rates of hospitalization and emergency department use among patients with PNH.

Efficacy and Safety of Single Agent Proximal Complement Inhibitors in Paroxysmal Nocturnal Hemoglobinuria (PNH) Patients with Residual Anemia Despite of Adequate Terminal Complement Inhibitor Use

Background: Introduction of C5 inhibitors is a major advancement in the treatment of Paroxysmal Nocturnal Hemoglobinuria (PNH) patients. First Eculizumab and then Ravulizumab significantly improved anemia by controlling intravascular hemolysis (IVH), reduced thrombosis risk, improved quality of life and prolonged survival. However, a significant proportion of PNH patients remain anemic with transfusion dependence despite maximal C5 inhibition due to C3 mediated residual extravascular hemolysis (EVH). Recently two randomized, open label, controlled trials with proximal complement inhibitors were studied in this patient population comparing against C5 inhibitors as single agents with excellent results. Here we compare efficacy and safety data of proximal complement inhibitors pegcetacoplan (C3 inhibitor), Iptacopan (factor b inhibitor) based on published or presented data. Methods: We compared the efficacy and safety data of randomized portion of PEGASUS and APPLY-PNH trials. PEGASUS trial is a phase 3, randomized, open label, active-comparator controlled trial with Pegcetacoplan (a pegylated pentadecapeptide that targets C3) compared efficacy and safety against eculizumab in PNH patients with Hb &amp;lt;10.5 despite of treatment with Eculizumab (Hillerman, et al NEJM 2021;384). Primary end point of the study was mean change in Hb level from baseline to week 16. APPLY-PNH is an open label randomized, multicenter phase 3 trial compared Iptacopan 200 mg BID (oral factor b inhibitor) monotherapy with standard of care C5 inhibition therapy. (Regis P. de Latour et al. Blood (2022) 140 (Suppl 2): LBA-2). Primary end points of the trial were Hematological response defined as an increase in Hb of ≥ 2 g/dL from baseline and Hb ≥ 12 g/dL in absence of transfusions. Secondary end points included number of clinical and hematological markers of hemolysis and safety. Results: Summarized in table 1. PEGASUS trial included 80 patients with 41 in pegcetacoplan arm and 39 in Eculizumab arm. APPLY-PNH included 97 patients with 62 in Iptacopan arm compared to 35 in standard of care anti C5 therapy. Majority of baseline characteristics were similar between two studies. Mean age in both studies was around 50 years and close to 2/3rd of patients in each study were females. Median duration of prior C5 therapy was between 3-4 years in both studies. More than half of patients in both studies required PRBC transfusions in preceding months prior to enrollment. Baseline mean Hb was between 8-9 g/dL, LDH between 250-300 U/L, mean absolute retic count (ARC) 190-200x109/L. Both studies showed superiority in change from baseline Hb with +2.69 g/dL in PEGASUS trial with all available data regardless of transfusion events and +3.63 in APPLY-PNH trial. Both studies showed either noninferiority or superiority in freedom from transfusions and change in ARC. Interestingly, LDH change was not significant in both studies. Change from baseline fatigue FACIT-score was numerically better but not tested in PEGASUS trial and was superior in APPLY-PNH. No major vascular events or serious infections by encapsulated bacteria were reported in either study. Breakthrough hemolysis was reported in 4 out of 40 patients in PEGASUS trial that led to treatment discontinuation in 3 patients and 2 out of 62 patients in APPLY-PNH trial, however none of them required treatment discontinuation. Conclusions: PEGASUS and APPLY-PNH trials showed that in patients with persistent anemia despite C5 inhibitor therapy, proximal complement inhibition with either pegcetacoplan (C3 inhibitor) or Iptacopan (factor b inhibitor) as a single agent showed significant improvement in Hemoglobin with higher rate of transfusion independence with control of extra vascular hemolysis while maintaining intravascular hemolysis control with no serious infections. These studies represent another major advancement in the management of PNH patients. Long term follow up is needed regarding safety and to identify better ways to manage breakthrough hemolysis in patients on proximal complement inhibitors.

Characterization of Hematopoietic Stem and Progenitor Cells of PNH Patients Using Single-Cell RNA Sequencing Analysis

The etiology of paroxysmal nocturnal hemoglobinuria (PNH) is somatic PIGA mutations in hematopoietic stem and progenitor cells (HSPCs), and affected PNH-type cells lack cell surface expression of the glycosylphosphatidylinositol-anchored proteins (GPI-APs). In PNH, GPI-AP deficient cells clonally expand and occupy a large proportion of mature cells. Several hypotheses have been proposed to explain clonal expansion of PNH-type HSPCs, including reduced sensitivity to apoptosis and escape from immune-mediated cytotoxicity. However, definitive mechanisms remain unclear. To further understand PNH pathophysiology at high multi-dimensional resolution, we performed single-cell RNA sequencing (scRNA-seq) of bone marrow (BM) cells from PNH patients. Fresh BM samples were obtained from eight PNH patients (male, n = 2, female, n = 6; median age, 51 [range, 21-67] years), who had large PNH-type granulocyte populations and stable clone sizes (median, 45 [range, 16-99] %), and five age- and sex-matched healthy donors (HDs). PNH-type and wild-type (WT) lineage-CD34+HSPCs were enriched from bone marrow mononuclear cells (BMMNCs) by flow cytometric sorting. Sorted cells were processed through the Chromium Controller (10x genomics, Pleasanton, CA, USA). scRNA-Seq libraries were constructed using the Chromium Single Cell 3' Reagent Kits (10x genomics) according to the manufacturer's instructions. The constructed libraries were sequenced with the NovaSeq 6000 system (Illumina, San Diego, CA, USA). We obtained 81,470 high-quality cells from PNH patients and 56,545 cells from HDs for further analysis. Cells were clustered with Seurat and visualized in UMAP. Cell clusters were assigned to 6 HSPC subsets based on expression of canonical marker genes (Figure 1A). To investigate compositional changes in BMMNCs between PNH patients and HDs, we performed differential abundance analysis with Milo and identified a prominent decrease in hematopoietic stem cells (HSCs) and lymphoid progenitors, as compared to HDs (Figure 1B). We then evaluated differential abundance between PNH-type and WT HSPCs in PNH patients, identifying no significant differences in each HSPC subsets (Figure 1B). These data were consistent with flow cytometric profiling of BMMNCs, similar proportions of PNH-type cells in each HSPC subsets in individual patients despite with profound decrease in PNH-type lymphocytes (Figure 1C). We next sought to investigate skewed gene sets in PNH-type HSPCs using gene set enrichment analysis. Comparison between PNH-type and WT HSPCs identified upregulation of inflammatory signaling pathways and downregulation of the cell-cycling pathways in PNH-type HSPCs, as compared to WT HSPCs (Figure 1D). We made comparisons within individual subsets. In HSCs, genes involved in IFN-γ response and oxidative phosphorylation were upregulated in PNH-type HSCs, while cell-cycling genes (ie. E2F targets and G2M checkpoint) were not dysregulated, as compared to WT HSCs. In summary,no significant skewing in lineage differentiation of PNH-type HSPCs was observed, suggesting PNH-type lymphocytes were negatively selected during the maturation process in lymphoid tissues. Upregulation of the inflammatory and immune response pathways in PNH-type HSPCs indicated a proinflammatory phenotype of PNH-type HSPCs. Upregulation of cell cycle-related pathways was not observed in PNH-type HSCs compared to WT HSCs, consistent with stable PNH clone sizes in our cohort of PNH patients.

Paradoxical Findings in Patients with Paroxysmal Nocturnal Hemoglobinuria Treated with C5 Inhibitors: Suboptimal Disease Control Perceived As Satisfactory

Introduction: The introduction of C5 inhibitors (C5i) for the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH) has resulted in lower morbidity and mortality rates. However up to 86% of patients still experience anemia and fatigue in 63% of them which may impact their quality of life (QoL). The SATURNO study aimed to assess PNH patient and physician satisfaction with C5i treatment, along with the impact of the disease and clinical outcomes. Methods: A multicenter, observational, cross-sectional study conducted in PNH patients receiving C5i treatment for at least 3 months in Spain. Key clinical and laboratory data were collected, including red blood cells (RBC) transfusions, hemolytic crisis, hemoglobin (Hb), haptoglobin (Hp), lactate dehydrogenase (LDH), absolute reticulocyte count (ARC) and bilirubin. Patient satisfaction was assessed using the Treatment Satisfaction Questionnaire for Medication (TSQM) and Visual Analog Scale (VAS). Physician satisfaction was measured using the TSQM general item and VAS. Fatigue and QoL were evaluated using the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) and the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30), respectively. The Work Productivity and Activity Impairment questionnaire (WPAI) was also used. Results: A total of 56 patients and 13 physicians participated in the study. Median (IQR) age of patients was 54.0 (44.5; 63.5) years, 35.7% were female and median (IQR) duration of treatment with C5i was 6.6 (3.3; 11.6) years. Overall, 94.6% of patients and 90.6% of physicians reported to be satisfied, very satisfied, or extremely satisfied with the treatment. Mean (SD) VAS score for patient satisfaction was 9.0 (1.2) and for physicians 7.9 (1.7) out of 10. Mean (SD) EORTC QLQ-C30 score was 71.7 (21.7) out of 100. Mean (SD) FACIT-F score was 39.7 (10.6) out of 52 and fatigue was reported by 55.2% of patients. Clinical and laboratory parameters showed a significant proportion of patients exhibiting abnormal values. Hb and Hp were below the normal ranges in 67.9% and 65.1% of patients, respectively, whereas elevated LDH, ARC and indirect bilirubin were observed in 50.0%, 83.0% and 75.9% of them, respectively. In the last 12 months, 30.9% of patients had received at least one RBC transfusion, with a mean (SD) number of 8.6 (8.4) RBC transfusions, and 33.9% experienced at least one hemolytic crisis with a mean (SD) of 4.9 (9.4). Work productivity assessment revealed that 60.7% of patients were currently unemployed, a mean (SD) percentage of absenteeism of 5.7% (12.4) and presenteeism of 23.8% (26.0). Overall mean (SD) percentage of work productivity impairment was 25.6% (27.3). Activity impairment was reported by 36.4% (28.3) of patients. Conclusions: The SATURNO study showed that although patients and physicians reported satisfaction with C5i treatment, this might be challenged. A substantial number of PNH patients treated with C5i still experience anemia, fatigue and abnormal laboratory values, indicative of ongoing hemolysis and suboptimal control. Moreover, patients with PNH had affected work productivity and a proportion of them were experiencing activity impairment. These findings highlight the need for comprehensive management strategies to address the impact of the disease on patients with PNH receiving C5i treatment.

Insights from Trajectories of PIGA-Mutated Clones in Paroxysmal Nocturnal Hemoglobinuria

Background: Patients with paroxysmal nocturnal hemoglobinuria (PNH) exhibit clonal expansion of hematopoietic cells deficient in glycosylphosphatidylinositol-anchored proteins (GPI-APs). Mutations in PIGA gene, involved in the biosynthesis of GPI-APs, result in the loss of GPI-APs from hematopoietic cells. However, PIGA mutations alone are insufficient to drive this clonal expansion. The pathogenesis of clonal expansion in PNH remains elusive, while several hypotheses or factors have been proposed, including immune attacks with concomitant aplastic anemia (AA) or additional mutations offering intrinsic growth advantage. Recently high-sensitivity flow cytometry and next-generation sequencing have allowed us to evaluate the clonal dynamics and architecture more precisely. The method has the potential to clarify the trajectories of PIGA mutated clones before the significant clonal expansion or the diagnosis, thereby to gain insight into the pathogenesis of the clonal expansion in PNH. Methods: To estimate the trajectories of PIGA-mutated clones, we constructed phylogenetic trees using whole genome sequencing (WGS) data of single cell-derived colonies of bone marrow samples from two PNH patients (53 colonies for Case 1 and 75 for Case 2). We used the targeted-sequencing and exome sequencing to detect driver mutations after isolating GPI-deficient and GPI-positive cells using flow cytometry. Results: Case 1 was diagnosed with AA at the age of 28. He developed hemoglobinuria at the age of 40 and was diagnosed with PNH at the age of 45. Bone marrow and peripheral blood samples were collected when he was 47 years old. The PNH clone sizes in granulocytes and red blood cells were 88.7% and 46.1%, respectively. Targeted-sequencing revealed six PIGA mutations in GPI-deficient cells and three BCOR mutations in GPI-positive cells. Case 2 was diagnosed with AA at the age of 33 and with PNH at the age of 34. Bone marrow and peripheral blood samples were collected when she was 59 years old. The PNH clone sizes in granulocytes and red blood cells were 98.6% and 96.3%, respectively. We previously identified chromosomal abnormalities in chr12, with the breakpoint located in an HMGA2 gene (Inoue et al. Blood. 2006). Targeted-sequencing detected no other driver mutations than single PIGA mutation and the HMGA2 mutation.In Case 1, Phylogenetic tree analysis constructed with WGS of single cell-derived colonies revealed multiple PIGA-mutated and BCOR-mutated clones that expanded independently of each other. All BCOR-mutated clones were identified in the GPI-positive population. In Case 2, all PIGA-mutated clones harbored the HMGA2 mutation, while no GPI-positive populations acquired the HMGA2 mutation.In both cases, the numbers of passenger single nucleotide variants per year were higher in PIGA-mutated clones than in PIGA-unmutated clones (16 vs. 11 mutations/year for Case 1 and 14 vs. 12 for Case 2). Assuming that variant acquisition rates per cell division are similar in both clones, the result suggests that PIGA-mutated clones had growth advantage against PIGA-unmutated clones.The acquisition of PIGA mutations was estimated to have occurred more than ten years prior to the PNH diagnosis. In Case 1, following the AA diagnosis, both PIGA-mutated clones and BCOR-mutated clones started expansion simultaneously, which suggests an association with immune attacks. Interestingly, even wild type clones expanded after AA diagnosis, suggesting bottle-neck effects of post-AA hematopoietic recovery. In Case 2, PIGA-mutated clones began expanding more than ten years before the AA diagnosis. Either a subclinical immune attack that might have existed before the diagnosis of clinical AA or the concomitant HMGA2 mutation conferred survival advantage for PIGA-mutated clones in Case 2. Conclusions: This estimation of the trajectories of the PIGA-mutated clone indicates that the pathogenesis of clonal expansion exhibits heterogeneity between two PNH patients.

OMS906, a Novel Alternative Pathway MASP-3 Inhibitor, Normalizes Hemoglobin Levels and Increases Clone Size in Treatment-Naïve PNH Patients

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a life-threatening disorder characterized by hemolytic anemia in combination with various degrees of marrow failure. PNH is driven by dysregulation of the complement system where the absence of CD55 and CD59 cell surface proteins leads to red blood cell (RBC) lysis. Untreated PNH can lead to debilitating anemia, thrombosis, fatigue, and increased mortality. Terminal complement inhibition attenuates intravascular hemolysis (IVH) but inevitably leads to extravascular hemolysis (EVH). Proximal and alternative pathway (AP) inhibition block both IVH and EVH, as demonstrated with C3, Factor B, and Factor D inhibitors. Mannan-binding lectin-associated serine protease-3 (MASP-3) activates pro-Factor D, thus activating the AP upstream of Factor D. OMS906 is a highly selective humanized mAb that binds to and inhibits MASP-3. In a Phase 1 study in healthy subjects, OMS906 was well tolerated and provided substantial MASP-3 inhibition. In the present study, safety and efficacy of OMS906 were evaluated in complement-inhibitor treatment-naïve patients with PNH. Methods: This is an ongoing, single-arm, open-label, Phase 1b proof-of-concept clinical trial (NCT05889299; EudraCT 2022-002450-22) evaluating safety, pharmacokinetics, pharmacodynamics, and preliminary efficacy of OMS906 in adults with PNH. Eligibility criteria include: confirmed PNH diagnosis by flow cytometry (RBC clone size &amp;gt;10%); complement-inhibitor treatment-naïve or inadequate response to C5 inhibitors; and baseline hemoglobin (Hgb) &amp;lt;10.5 g/dL. Patients received 5 mg/kg subcutaneous (SC) OMS906 every 4 weeks, the lowest exposure cohort in this study. Primary endpoints are safety and tolerability. Secondary endpoints include changes from baseline in Hgb and lactate dehydrogenase (LDH) levels, RBC clone size, and absolute reticulocyte count; achievement of Hgb ≥12 g/dL or an increase of ≥2 g/dL; and transfusion independence. Planned enrollment was approximately 10 patients. Results: This pre-specified interim analysis included data available as of July 20, 2023. All patients (N=11) included in this analysis were complement-inhibitor treatment-naïve (55% female; mean age 41 years [range 27-72]). The majority of patients (64%) received RBC transfusions in the 12 months prior to OMS906 treatment. Co-existing conditions included iron deficiency (n=6), chronic renal failure (n=3), aplastic anemia (n=2), and myelodysplastic syndrome (MDS; n=2). At baseline (N=11), mean Hgb was 7.0 g/dL (median 6.4 g/dL, range 3.9-10.4 g/dL) and LDH was 1835 U/L (median 1831 U/L, range 905-3480). To date, all patients have received ≥2 doses of OMS906 with 4 patients having received ≥6 doses. OMS906 was well tolerated. The most common adverse events (AEs) in ≥20% of patients were increased thrombocytopenia (36%), neutropenia (27%), and itching (27%). No patients experienced clinical breakthrough hemolysis. There were no major adverse vascular events, treatment-related serious AEs, discontinuations, or deaths. Following initiation of OMS906, mean Hgb increased from baseline by 3.1 g/dL ( P&amp;lt;0.001) at 4 weeks (N=11), and by 9.5 g/dL ( P=0.016) at the latest timepoint of 24 weeks (n=3) [Figure 1]. Among patients receiving ≥2 doses, all but 3 experienced gender-specific normalization of Hgb levels. The remaining patients had MDS (n=2) or marrow failure (n=1). No patients required transfusions following OMS906 treatment. Mean LDH levels decreased from baseline by 1483 U/L (&amp;gt;80%; P&amp;lt;0.001) at 4 weeks (N=11) and by 1939 U/L ( P=0.004) at 24 weeks (n=3). Three patients had increases in LDH, suggesting initiation of hemolysis at the end of a dosing period. Mean absolute reticulocyte counts decreased from baseline by 87,000-122,000 ×10 9/L at all timepoints. Mean PNH RBC clone size increased by up to 38.6% (n=6; P=0.016) versus baseline. Conclusion: In this interim analysis, MASP-3 inhibitor OMS906 was well tolerated with no safety signals of concern. Once-monthly SC OMS906 resulted in clinically meaningful, beneficial effects on Hgb, LDH, and RBC clone size in treatment-naïve PNH patients. OMS906 dose escalation guided by occurrence of subclinical hemolysis is being evaluated with the goal of achieving quarterly dosing.

Neutrophil Functionalities in Patients with Paroxysmal Nocturnal Haemoglobinuria Are Not Affected By Complement C5- Inhibition

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare, acquired haematological stem cell disorder characterized by haemolysis, thrombophilia, and cytopenia. PNH is caused by a somatic mutation in the phosphatidylinositol glycan class A (PIG-A) gene at the level of pluripotent, hematopoietic stem cells leading to a lack of GPI anchored cell surface proteins, such as complement-inactivating proteins, (e.g., CD55, CD59) on stem cells, polymorphonuclear neutrophil granulocytes (PMN), erythrocytes, and platelets. Subsequently, uncontrolled complement overactivity leads to the systemic complications. Phenotypic mosaicism is a distinct characteristic of PNH. For diagnostic purposes, the absence of the GPI-anchor on granulocytes and monocytes in the flow cytometry is considered the gold standard. However, the exact consequences of GPI deficiency in PMNs and monocytes have not been studied sufficiently yet. Since the implementation of complement inhibitors, the primary cause of morbidity and mortality in PNH has been significantly reduced. Despite this, the tendency towards thrombosis, as well as the consequences of the deficiency of GPI-linked proteins on PMNs and monocytes, remain largely unclear. Therefore, the main objective of this project is to gain a deeper understanding of the impact of PNH-PMNs and monocytes on thrombo-inflammation in PNH. Specifically, the impact of complement inhibitors on these factors has been compared. Blood from PNH patients (n=14) with a median clone size of 74 % were compared with an age- and sex-matched control group (n =20). Various cellular markers indicating a state of activation (CD11b, TREM1, CD64) or showing platelet adherence (CD42), were measured using flow cytometry. Unmutated PMNs and monocytes from PNH patients (PMN) served as intrinsic control, GPI positive PMNs (WT-PMN (PNH)) and monocytes from a control group served as extrinsic control. We compared unstimulated PNH and WT cells for their basal phenotype, and then functional changes upon stimulation (phorbol 12-myristate 13-acetate (PMA)). Our data present a contrary perspective to our initial assumptions, indicating that PNH-PMNs have a direct impact on unmutated PMNs, resulting in the generation of a thrombogenic state and increased levels of activation. The unmutated GPI-positive PMNs and monocytes exhibit a significantly stronger direct aggregation with platelets compared to GPI-deficient clones. It is hypothesized that the PNH clones influence the activity and aggregation propensity of the remaining unmutated granulocytes through additional cellular mechanisms. In a subgroup analysis, the impact of complement factor 5 inhibitors (C5i) on the aforementioned factors was investigated detecting no significant differences in terms of PMN activation or platelet aggregation. Thus, it can be hypothesized that thrombosis and hyper-inflammation are not solely driven by the PNH clones directly and modulated C5 inhibition. Additional cellular mechanisms and signaling pathways appear to play an important role, and these aspects have not been adequately addressed by the current therapy.

Efficacy of Parallel and Crossover Analysis As Well As Pharmacokinetic Similarity Were Confirmed between ABP 959 and Eculizumab Reference Product in Patients with PNH

Introduction: ABP 959, a biosimilar to eculizumab reference product (RP), binds to the human complement component 5 protein to inhibit terminal complement activation. Eculizumab RP is approved for the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome, generalized myasthenia gravis, and neuromyelitis optical spectrum disorder. Regulatory approval of biosimilars is based on a totality of evidence approach which includes demonstration of similarity in analytical and non-clinical characteristics, and in comparative clinical studies. Analytical and non-clinical similarities of ABP 959 with eculizumab RP and clinical pharmacokinetics (PK)/pharmacodynamics (PD) and safety similarities in healthy adults have been previously reported. Similarity of the primary efficacy and safety results from the parallel and crossover comparisons in patients with PNH have also been previously reported. Here, we report together the primary efficacy results from both the parallel and the cross-over comparisons along with additional PK analyses in patients with PNH to further support the demonstration of similarity of ABP 959 with eculizumab RP. Methods: A multicenter, randomized, double-blind, active-controlled, 2-period crossover study evaluated the clinical similarity of ABP 959 compared with eculizumab RP in adult patients with PNH. Patients were randomized 1:1 to receive each investigational product (900 mg of ABP 959 or eculizumab RP IV q14d) in 1 of 2 treatment sequences (ABP 959/eculizumab RP or eculizumab RP/ABP 959). Primary efficacy determined by hemolysis, measured by lactate dehydrogenase (LDH), was analyzed for both the parallel and crossover comparisons. The parallel comparison endpoint was measured at week 27 and clinical similarity was assessed by comparing the 1-sided 97.5% upper confidence interval (CI) limit for the geometric least squares (LS) mean ratio of LDH between ABP 959 treatment group and eculizumab RP treatment group with a non-inferiority (NI) margin of 2.873. The crossover comparison endpoint was measured by the time-adjusted area under the effect curve (AUEC) of LDH from weeks 13 to 27, 39 to 53, and 65 to 79. The similarity of efficacy between treatment groups was assessed by comparing the 2-sided 90% CI for the geometric mean ratio (GMR) of the time-adjusted AUEC of LDH with a similarity margin of (0.77, 1.30). The total and unbound PK area under the curve (AUC) of ABP 959 and eculizumab RP from week 13 to week 15 and trough PK were identified as secondary endpoints in this study. Results: Forty-two patients (20 in ABP 959/eculizumab RP group; 22 in eculizumab RP/ABP 959 group) were randomized across 25 centers. Similarity of efficacy was established in both the parallel and crossover comparisons. In the parallel comparison, the ratio of the geometric LS means of LDH at week 27 (ABP 959 vs eculizumab RP) was 1.0628, with a 1-sided 97.5% upper CI of 1.1576 was contained within the NI margin of 2.873. In the crossover comparison, a point estimate of the GMR of time-adjusted AUEC of LDH (ABP 959 vs eculizumab RP) of 0.9812 with a 2-sided 90% CI of (0.9403-1.0239) was contained within the prespecified margin of similarity. The GMR (90% CI) for the total and unbound PK AUC from week 13 to week 15 was 0.9122 (0.7586, 1.0968) and 0.9508 (0.7454, 1.2130), respectively. Geometric mean values for trough total and unbound concentrations of ABP 959 and eculizumab RP were similar between the treatment groups at all time points tested over the entire study. Conclusions: Similarity in clinical efficacy in both parallel and crossover comparisons as determined by hemolysis in patients with PNH was established between ABP 959 and eculizumab RP. Additionally, analyses of serum total and unbound PK concentrations for patients with PNH further demonstrated PK similarity between ABP 959 and eculizumab RP. The results of this study, along with previously demonstrated analytical, non-clinical, clinical PK/PD in healthy adults, and efficacy and safety evaluations in PNH patients, further support a demonstration of no clinically meaningful differences between ABP 959 and eculizumab RP.

The Change of Complement Deposition after Complement Inhibitor Treatment in Paroxysmal Nocturnal Hemoglobinuria Patients

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disease in which abnormal complement activation results in hemolysis and thromboembolic events (TEE). Complement inhibitors (CI) have been proved to be able to suppress intravascular hemolysis (IVH) and reduce the risk of TEE. Few studies were available on the changes of complement deposition after CI and their relationship with efficacy. Methods: PNH patients treated with full dose eculizumab, crovalimab or iptacopan at Peking Union Medical Colleague Hospital from Oct 2021 to Jul 2023 were enrolled.Patients' clinical information and blood samples were collected after 0, 1, 3, 6, 9 and 12 months of treatment. The deposition of C5b-9, C3, C4b and factor B (FB) on peripheral red blood cells (RBC), white blood cells (WBC) and platelets (PLT) was detected by double-color staining flow cytometric assay. The efficacy of each CI was correlated with complement deposition levels on three blood lineages. Results: 23 PNH patients were enrolled. Among them, 13 patients were treated with C5 inhibitors (4 with eculizumab and 9 with crovalimab), 10 were treated with iptacopan (FB inhibitor). For patients treated with eculizumab, C5b-9+/RBC and C5b-9+/WBC significantly decreased after 3, 6 and 12 months of treatment(P&amp;lt;0.05); however, C3+/WBC and C3+/PLT significantly increased after 12 months (P&amp;lt;0.05). For covalimab-treated patients, C5b-9+/RBC significantly decreased after 6 and 12 months (P&amp;lt;0.05), while C3+/RBC, C3+/WBC, C4b+/RBC and C4b+/WBC increased (P&amp;lt;0.05) after 6 months. For those treated with iptacopan, after 1, 3, 6, 9 and 12 months, the deposition of C5b-9 on all three blood lineages significantly decreased (P&amp;lt;0.05), meanwhile, C3 and FB on all three lineages decreased (P&amp;lt;0.05); moreover, C4b+/WBC and C4b+/PLT decreased after 3, 6, 9 and 12 months (P&amp;lt;0.05). C5b-9+/RBC and C5b-9+/WBC were significantly associated with hemoglobin improvement after eculizumab treatment (P=0.004 and 0.018, respectively). C5b-9+/RBC was positively correlated with bilirubin (P=0.023) and C3+/RBC was positively correlated with absolute reticulocyte counts (Ret, P=0.001) after crovalimab treatment. After iptacopan treatment, C3+/RBC was positively correlated with Ret (P=0.010), C4b+/RBC was positively correlated with Ret (P=0.032) and lactose dehydrogenase (LDH, P=0.003), and FB+/RBC was positively correlated with LDH. Conclusion: Complement inhibitors could effectively reduce C5b-9 deposition. Unlike C5 inhibitors, iptacopan also reduced the deposition of C3, C4b, and FB. C5b-9+/RBC may be correlated with the efficacy of inhibiting intravascular hemolysis. Upstream complement deposition is probably associated with breakthrough hemolysis or extravascular hemolysis. 【Key words】Paroxysmal nocturnal hemoglobinuria, complement inhibitor, complement deposition, flow cytometric assay, hemolysis

Efficacy of SB12 (Eculizumab Biosimilar) in Asian and Non-Asian Patients with Paroxysmal Nocturnal Hemoglobinuria: Subgroup Analysis of a Global Phase III Randomized Controlled Trial

Background SB12 is a humanized monoclonal antibody biosimilar to reference eculizumab (ECU) which received marketing authorization in Europe in May 2023 after a comprehensive biosimilarity demonstration including non-clinical and clinical assessments. The pivotal SB12 Phase III study demonstrated equivalent clinical efficacy by evaluating lactate dehydrogenase (LDH) and comparable safety, pharmacokinetics, pharmacodynamics, and immunogenicity between SB12 and ECU in patients with paroxysmal nocturnal hemoglobinuria (PNH) (Jun Ho Jang et al., eJHaem 2022). Objective To analyze the efficacy of SB12 in Asian and non-Asian patients with PNH from the pivotal phase III study. Methods A total of 50 adult patients with a confirmed diagnosis of PNH and ≥ 1.5 upper limit of normal range (ULN) of LDH without previous exposure to a complement inhibitor (naïve) were randomized. All patients provided written informed consents and were randomized (1:1) to treatment sequence I (TS1: SB12 to ECU, n=25) or II (TS2: ECU to SB12, n=25), to receive 600 mg of SB12 or ECU intravenously every week for first 4 weeks (initial phase) and 900 mg for the fifth week, followed by 900 mg every 2 weeks thereafter (maintenance phase). Patients were switched to ECU or SB12 at Week 26, and switched treatment was provided until Week 50. The primary endpoints included LDH level at Week 26 and time-adjusted area of under the effect curve (AUEC) of LDH from Week 14 to Week 26 and Week 40 to Week 52. Equivalence was declared for LDH level at Week 26 if the two-sided 95% confidence interval (CI) of the mean difference in between SB12 and ECU lied within the pre-defined equivalence margin of [−1.2 × ULN, 1.2 × ULN] = [−337.2, 337.2], where ULN = 281 U/L. In addition, equivalence was declared for time-adjusted AUEC of LDH if the two-sided 90% CI of the ratio of geometric means between SB12 and ECU lied within the pre-defined equivalence margin of [0.77, 1.29]. Secondary endpoints included the LDH profile over time. Overall, post-hoc analysis in Asian and Non-Asian (based on race) patients who received either ECU or SB12 during the study period was conducted for the aforementioned efficacy endpoints. Results Asian patients were 27 (54.0%) of total 50. Overall, 15 (60%) Asian patients and 10 (40%) Non-Asian patients were randomized to TS1 and 12 (48%) Asian patients and 13 (52%) Non-Asian patients were randomized to TS2. Baseline demographic and disease characteristic were comparable between the two treatment sequences, with no significant differences between the treatment sequences by race between Asian and Non-Asian patients. The 95% CI of mean difference in LDH level at Week 26 between SB12 and ECU in Asian (SB12 − ECU: -12.02, 95% CI [−126.87, 102.83]) and Non-Asian patients (SB12 − ECU: 76.12, 95% CI [−56.75, 208.99]) lied within the pre-defined equivalence margins (Table). The 90% CI of ratio of time-adjusted AUEC of LDH between SB12 and ECU in Asian patients (SB12/ECU: 1.01, 90% CI [0.92, 1.10]) lied within the pre-defined equivalence margin and was also comparable in Non-Asian patients (SB12/ECU: 1.15, 90% CI [0.90, 1.46]). The overall LDH profile during the study period was also comparable between Asian and Non-Asian patients. No patient developed anti-drug antibodies during the study period and no patient discontinued due to lack of efficacy. While the limited sample size suggests for caution, the comparable efficacy results by Asian and Non-Asian subgroups are also comparable to previously published results in the overall population (Jun Ho Jang et al., eJHaem 2022), with no significant differences identified. Conclusion SB12 showed comparable efficacy to ECU in complement-inhibitor naïve PNH patients in both Asian and Non-Asian subgroups. No patient developed anti-drug antibodies nor discontinued due to lack of efficacy during the study period.

Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Treated with Pegcetacoplan: Real Life Experience

Introduction: C5 inhibitors (C5i) treatment has dramatically improved the management of intravascular hemolysis (IVH) in PNH patients. However, a significant proportion of patients remain anemic due to suboptimal control of IVH and the development of extravascular hemolysis (EVH), consequence of C5i treatment. Pegcetacoplan (PEG) is the first proximal inhibitor of the complement system, through the inhibition of C3, allowing to control both IVH and EVH. PEG has recently been marketed in Spain for use in adult PNH patients who remain anemic after treatment with an C5i for at least 3 months. The objective of this work is to analyze the response of the first patients treated with PEG in real life in Spain. Patients and methods: clinical and analytical variables were collected from a series of 10 patients diagnosed with PNH who received treatment with PEG through an early access program. PEG was self-administered subcutaneously at 1080 mg twice a week. See Table 1. Transfusion needs and the adverse effects that occurred were also collected. Results: median (range) duration of treatment with PEG at the time of the analysis was 4 (2-8) months. Hemoglobin (Hb) values increased a median of 2.4 g/dL after PEG treatment. This improvement was rapidly observed since the beginning of the treatment (median increase at 1 week was 1.3 g/dL and at 2 weeks 2.4 g/dL). All patients reached Hb levels of &amp;gt;8 g/dL, 80% &amp;gt;10 g/dL and 40% &amp;gt;12 g/dL. An increased in Hb &amp;gt;2 g/dL was observed in 60% of the patients. Median values of parameters of hemolysis (absolute reticulocyte counts, bilirubin, LDH) became normal. See Table 2. The direct antiglobulin test turned into negative in all positive cases pre-PEG (4 out of 4). No patient suffered from acute hemolytic events, thrombosis, or required PRBC transfusion once PEG had been initiated. On a 5-point scale (0: nothing; 5: very much), subjective improvement of patient quality of life and physician satisfaction were 4 (3-5), and 5 (3-5) [median (range)]. PEG-related adverse events were just reported in one patient (10%): mild post-infusion nausea. At the end of the analysis, all patients continue on PEG. Conclusions: the results observed in the first patients treated with PEG in real life in Spain, although with a limited follow-up, confirm those reported in phase 3 clinical trials, with significant improvement in the Hb levels and hemolysis parameters, which results in transfusion independence. PEG was also shown to act quickly from the start of treatment, to significantly improve quality of life, and to present an excellent safety profile. PEG, the first approved proximal complement inhibitor, can therefore be considered as a relevant therapeutic option in the current management of PNH.

Quality of Life Analysis in Patients with AA Using the QLQ-AA/PNH-54 at Initial Diagnosis of AA - a Registry Based Analysis

Objectives: Aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are ultra-rare, acquired bone marrow failure syndromes (BMFS) with need for long-term care and high morbidity despite recent therapeutic advances. There is a lack of disease-specific assessment tools of quality of life (QoL) in AA/PNH patients. So far, QoL has mostly been estimated according to surrogate parameters (e.g. blood counts, transfusion dependence or toxicity of treatment in AA patients) or has been assessed with tools specifically designed for cancer patients (e.g. EORTC Quality of Life Questionnaire QLQ-C30) or with tools focusing on fatigue (e.g. Functional Assessment of Chronic Illness Therapy, FACIT-Fatigue, PNH patients). Therefore, the QLQ-AA/PNH-54 has been developed to allow a patient tailored QoL assessment in patients with AA or PNH. Here, we present the first descriptive data of the QLQ-AA/PNH-54 of 74 AA patients at the timepoint of initial diagnosis collected between 2018 and 2020 (prior to the SARS-CoV-2 pandemic). Methods: The QLQ-AA/PNH-54 was designed, psychometrically validated, and is currently finally validated for PNH within the IPIG (international PNH interest group)-registry. Parallel to this process, the tool was implemented within the Aachen AA/BMF registry as an instrument for real-life QoL evaluation in AA patients. In total, 54 QoL items regarding physical functioning (21 items), psychological aspects (26 items) and health care related issues (7 items) are comprised. Patients rank the occurrence of each item within the last 14 days on a scale between 0 and 3 (0 = did not occur, 1 = low, 2 = moderate, 3 = severe). Results: 74 AA patients (37 female/ 37 male, mean/median/range age 38.42/ 35.47/ 11-73 years) were included. Patients were diagnosed with nsAA (n=11), sAA (n=23), vsAA (n=18), AA NOS (n=20) and AA/PNH-Overlap (n=2), respectively. Broken down into different functional scales, the most frequently and highest rated scales concerned psychosocial aspects (&amp;gt; 60 % of the patients scored 1-3; &amp;gt; 30% of the patients scored 3). Patients were especially burdened by worries and uncertainties with regards to their future, activity planning security and need for cautiousness in everyday life. Tiredness was the major symptom reported in relation to physical functioning. Patients often feared deterioration of their blood results as the most important health care-related issue. In comparison, low occurrence of pain, orthostatic dysregulation, susceptibility for infections, dyspnea, sleep disturbance and tendency to bleed was reported regarding physical functioning (scored 0 by over 40 to over 50 % of the patients). Similarly, low scores were reported for impairment of activities of daily life in terms of completion of household tasks and hobbies or loss of physical attraction. Conclusion: QoL of AA patients at initial diagnosis was mainly impaired by tiredness and a high psychological burden. Interestingly, complications frequently feared and highlighted by treating physicians had a minor impact on patients' QoL. These results emphasize the importance of further QoL analysis from patient view and recommend to especially focus on the psychological burden of AA patients in patient communication at initial diagnosis.