PNH Patients Research Articles

Health-related quality of life and influencing factors of patients with paroxysmal nocturnal hemoglobinuria in China

BackgroundParoxysmal nocturnal hemoglobinuria (PNH) is a rare blood disorder, leading to various complications and impairments in patients’ health-related quality of life (HRQOL). Limited research has been conducted to evaluate the HRQOL of Chinese patients with PNH. Understanding the HRQOL in this specific population is crucial for providing effective healthcare interventions and improving patient’ health outcomes. This study aimed to assess HRQOL of Chinese patients with PNH, and identify key determinants.MethodsA cross-sectional study was conducted during 2022 to recruit patients with PNH in China. The study population was recruited from PNH China, one of the largest public welfare PNH patient mutual aid organization in China. Data were collected via an online questionnaire including the EQ-5D-5L (5L), and social-demographic and clinical characteristics. Descriptive statistics were employed to summarize the characteristics of the participants and their HRQOL. Multiple linear and logistic regression analyses were adopted to explore key factors affecting HRQOL.ResultsA total of 329 valid questionnaires were collected. The mean (SD) age of the patients was 35.3 (10.0) years, with 52.3% of them being male. The patients reported more problems in Anxiety/Depression (81.5%) and Pain/Discomfort (69.9%) dimensions compared to the other three 5L dimensions. The mean (SD) of 5L health utility score (HUS) and EQ-VAS score were 0.76 (0.21) and 62.61 (19.20), respectively. According to multiple linear regression, initial symptoms (i.e., Anemia [fatigue, tachycardia, shortness of breath, headache] and back pain) and complication of thrombosis were significant influencing factors affecting 5L HUS. Total personal income of the past year, initial symptom of hemoglobinuria and complication of thrombosis were significantly influencing factors of VAS score. Social-demographic and clinical characteristics, such as gender, income, and thrombosis, were also found to be significantly related to certain 5L health problems as well.ConclusionOur study manifested the HRQOL of PNH patients in China was markedly compromised, especially in two mental-health related dimensions, and revealed several socio-demographic and clinical factors of their HRQOL. These findings could be used as empirical evidence for enhancing the HRQOL of PNH patients in China.

No Benefits from Glucocorticoid Maintenance Therapy in Patients with PNH

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, complement-mediated hemolytic anemia resulting from a somatic phosphatidylinositol glycan class A ( PIGA) mutation which bugs the biosynthesis of glycosylphosphatidylinositol (GPI). The lack of GPI-anchored proteins, especially CD55 and CD59 molecules, prompts blood cells to be prone to complement attack and lysis. Targeted on the terminal complement inhibitor has been recommended as a first-line treatment, such as eculizumab (C5 inhibitors), which not only controls hemolytic anemia, reduces thrombosis and blood transfusion, but also improves the staging status of chronic kidney disease in PNH patients. Complement inhibitors are not yet widely administrated by Chinese PNH patients, so glucocorticoids are still mostly used for the first-line treatment. However, the efficacy of glucocorticoid maintenance therapy in PNH is uncertain. Methods We conducted a prospective self-controlled comparison of 40 PNH patients with high disease activity (HDA), collecting anemia, hemolysis, thrombosis and renal function indicators at baseline and 1st, 3rd and 6th month after treatment in the Chinese Eastern Collaboration Group of Anemia (CECGA). All enrolled patients were administrated with prednisone 1.0 mg/kg/d at first and then adjusted the dosage according to the situation. The clinical trial registration number is ChiCTR2100050945. Results A total of 40 patients with PNH were enrolled in this study, 21 (52.5%, 21/40) were male, 19 (47.5%, 19/40) were female and the median age was 33.5 years (range 18-69 years). 29 of 40 (72.5%) presented classical PNH and 11 patients (27.5%) were diagnosed as PNH associated with bone marrow disease (BMD)-10 were aplastic anemia and 1 was myelodysplastic syndrome. Four cases detected single-site thrombosis and one had multi-site thrombosis. The most common locations were portal vein thrombus (2/6, 33.33%) and lower extremity intermuscular thrombus (2/6, 33.33%), others included mesentery vein, hepatic vein, splenic vein and intracranial microthrombus. The laboratory indexes at baseline were presented in Table 1. Compared to the baseline (Table 2), at 1 st, 3 rd, and 6 th month after treatment, both hemoglobin and red blood cell (RBC) have improved, but hemoglobin and RBC didn't keep rising, and showed no difference when comparing 3 rd month with 1 st month and 6 th month with 3 rd month. 19 patients (19/40, 47.5%) required blood transfusion before treatment, and 11 patients (11/40, 27.5%) needed transfusion at 1 st month ( P=0.021). Then 13 patients (13/40, 32.5%) at 3 rd month, 15 patients (15/40, 37.5%) at 6 th month were transfusion dependent ( P=0.109; P=0.388). After 6-month treatment, 3 cases of severe anemia (Hb < 30 g/L) at baseline were still severe anemia (3/7, 42.86%), and 4 cases (4/7, 57.14%) were upgraded to moderate anemia (60 g/L ≤ Hb ≤ 90 g/L). Among the original moderate anemia, 20 (20/31, 64.52%) cases were still moderate anemia, 6 (6/31, 19.35%) cases decreased to severe anemia and 5 (5/31, 16.13%) cases were improved to mild anemia (Hb > 90 g/L). 1 (1/2, 50%) patient of mild anemia originally maintained mid anemia and one (1/2, 50%) patient decreased to moderate anemia. The levels of LDH, indirect bilirubin (IBIL) and reticulocyte (Ret) were not changed significantly at 1 st, 3 rd, 6 th month after treatment. At 1 st, 3 rd, 6 th month, only 1, 2 and 2 patients achieved hemolytic control (LDH < 1.5 ULN). The d-dimer and glomerular filtration rate (GFR) also were indicated no difference. At 1 st, 3 rd, 6 th month, 3 (3/11, 27.27%), 4 (4/11, 36.36%), 6 (6/11, 54.54%) patients were back to normal range, 1 (1/11, 9.09%), 2 (2/11, 18.18%), 4 (4/11, 36.36%) patient was newly elevated (D-dimer > 0.55 mg/L) separately. At baseline 2 patients' renal function was severely reduced (GFR＜60ml/min/1.73m 2), and at 1 st, 3 rd, 6 th month, 1, 1, 2 patients had declined respectively. Discussions and Conclusions The use of prednisone in PNH patients with HDA may initially improve anemia, but the improvement in anemia may also be due to self-remission after paroxysmal episodes of PNH itself, or transfusion. The maintenance therapy with prednisone cannot control hemolysis, improve thrombosis and renal function damage. Long-term glucocorticoid maintenance therapy for PNH is not recommended.

KP104, a Bifunctional C5 Antibody/Factor H Fusion Protein, Effectively Controls Both Intravascular and Extravascular Hemolysis: Interim Results from a Phase 2 Study in Complement Inhibitor-Naïve PNH Patients

Background Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and life-threatening hematologic disease characterized by intravascular and extravascular hemolysis (IVH and EVH) mediated by complement activation through both the terminal and alternative pathways (TP and AP). The current treatment options for PNH are limited to addressing only a single complement pathway, resulting in either unresolved EVH with TP inhibitors or potentially exposing patients to the risk of life-threatening breakthrough hemolysis with AP inhibitors (Notaro et al. N Engl J Med 2022;387:160-6). KP104 is an innovative fusion protein comprised of a humanized anti-C5 mAb and the functional domain of complement regulator factor H to inhibit both TP and AP activation. We report here the interim results from a phase 2 study of KP104 in complement inhibitor-naïve PNH patients. Methods This open-label Phase 2 clinical trial (NCT05476887) aims to assess the efficacy, safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of KP104 in patients diagnosed with PNH who have not received prior complement inhibitors. The primary objectives of the study are to evaluate key clinical markers including lactate dehydrogenase (LDH) and hemoglobin (Hgb) levels, transfusion requirements, and FACIT-fatigue scores. The first part of the study consists of three cohorts, with an enrollment of 6 patients per cohort. The study includes an initial treatment period of 12 or 13 weeks of KP104 dosing regimen, followed by a 9-month extension treatment period. The specific dosing regimens for each cohort are as follows: Cohort 1: 1,200 mg IV (Day 1) + 720 mg SC QW (starting Day 8) Cohort 2: 2,400 mg IV (Day 1) + 1,920 mg SC Q2W (starting Day 8) Cohort 3: 3,600 mg IV (Day 1) + 2,880 mg SC Q2W (starting Day 8) Results Interim results include data from 15 patients who completed 16/17 weeks of treatment (6 patients each from Cohort 1 and 2, and 3 from Cohort 3), as of the data cutoff on July 12 th, 2023. Among these patients (4 males and 11 females, mean (±SD) age 33(±12) years, a median of 3 transfusions were required 12 months prior to the study and 9 patients were previously diagnosed with aplastic anemia. Baseline mean (SD) Hgb and LDH levels were 7.0 (±1.5) g/dL and 1,824 (±512) U/L, respectively. By week 16/17, all 15 patients demonstrated positive clinical improvements. Mean (SD) Hgb levels increased by 4.9 (±1.7) g/dL, 5.8 (±1.6) g/dL, and 5.8 (±2.8) g/dL over baseline, and mean (SD) LDH levels reduced by 88.0 (±3.67) %, 83.5 (±7.45) %, and 89.5 (±4.05) % over baseline for Cohort 1, 2 and 3, respectively. Additionally, 9/15 (60%) patients achieved hemoglobin levels ≥12 g/dL (3/6, 5/6 and 1/3 for Cohort 1, 2 and 3, respectively), and all 15 patients had LDH levels below 1.5xULN. None of the 15 patients required transfusions or encountered any thromboembolic events after receiving KP104 treatment. Positive changes were observed in absolute reticulocyte count and bilirubin levels, along with significant enhancements in FACIT-fatigue scores seen in all 3 cohorts, with mean (SD) increases of 13.8 (±7.91), 12.8 (±9.15), and 9.3 (±10.69) for Cohorts 1, 2, and 3, respectively. Dose-dependent reductions in two PD biomarkers, C3b deposition and free C5 levels, further confirmed KP104's dual mechanism of AP and TP inhibition. KP104 was well tolerated, with no serious adverse events or treatment-emergent adverse events (TEAEs) that led to drug discontinuation or study withdrawal. 10/15 (67%) patients reported at least one mild or moderate TEAE, with no observed dose dependency. The most frequently reported TEAEs included transient injection site induration, headache, and COVID-19 infection, all of which were promptly or duly resolved. A single occurrence of clinical breakthrough hemolysis, observed in the lowest dose cohort and concurrent with an episode of gastroenteritis, was quickly resolved after an extra dose. Conclusion The interim results of this phase 2 study demonstrate the safety and efficacy of KP104 in effectively controlling IVH and EVH in complement inhibitor-naïve PNH patients. The findings provide compelling clinical evidence that a bifunctional inhibitor targeting the AP and TP can properly address the current treatment gaps associated with single complement pathway inhibitors, potentially eliminating the need for cumbersome and costly combination therapies (Notaro et al. N Engl J Med 2022;387:160-6).

Allogeneic Hematopoietic Stem Cell Transplantation for Classical Paroxysmal Nocturnal Hemoglobinuria

Classical paroxysmal nocturnal hemoglobinuria (cPNH) is a rare acquired clonal hematopoietic cell disease. Eculizumab is effective in PNH patients, though, it must be administered indefinitely. As the technology of allogeneic hematopoietic stem cell transplantation (allo-HSCT) improves in recent years, what is the position of HSCT in treatment of cPNH? This study discusses the efficacy and safety of allo-HSCT in cPNH. The clinical data of 5 cPNH patients who underwent allo-HSCT from 2019 to 2022 were collected. All patients were diagnosed cPNH according to guidelines from International PNH Interest Group. Indications for HSCT were transfusion-dependent and nonavailability of eculizumab. The cohort was all male, with a median age of 26 years (range, 26 to 46 years) at the time of transplantation. All patients had documented hemolysis and no thrombosis during monitoring. The median time from diagnosis to allo-HSCT was 5.5 years (range, 3.6 to 18 years). The median PNH granulocyte clone size was 96.3% (range, 90% to 99.7%), and the median lactate dehydrogenase (LDH) level was 2224IU/L (8.896-fold of the upper limit of normal). All patients were detected bone marrow hyperplasia by trephine biopsy. The stem cell source came from 4 haploidentical donors and 1 HLA-identical sibling donor. Among the 5 patients, 3 patients underwent myeloablative conditioning (MAC) and 2 patients underwent reduced-intensity conditioning (RIC) treatment. None of the patients experienced primary implantation failure. Neutrophil implantation time was 15 (13-21) days, and platelet implantation time was 24 (13-60) days. 3 patients developed grade II acute graft-versus-host disease (aGVHD). No patients developed grade Ⅲ-Ⅳ aGVHD. 2 patients developed localized chronic graft-versus-host disease (cGVHD) involving lung and skin respectively, and no patients developed extensive cGVHD. PNH clones turned negative in all patients after 2 months (range, 1 to 3 months) of transplantation. At a median follow-up of 16 (6-34) months, 1 patient died of relapse and infection. The remaining 4 patients survived, of which 2 patients had discontinued all drugs. cPNH has its own unique clinical features. Allo-HSCT could cure cPNH patients who can withdrawal from treatment of all medication. Haploidentical donor HSCT is an acceptable choice for cPNH, and RIC constitutes a valid option.

Interventions in cerebrovascular emergencies among patients with Paroxysmal nocturnal haemoglobinuria - A word of caution

IntroductionParoxysmal nocturnal haemoglobinuria (PNH) is a clonal hematopoietic disorder, where there is deficiency of glycosylphosphatidylinositol (GPI) anchored proteins in the cell membrane, leading to increased complement sensitivity of red blood cells, intravascular hemolysis and vascular inflammation. Arterial and venous strokes in patients with PNH are a rarity posing significant diagnostic and therapeutic challenges. We report our experience with management of PNH patients with cerebrovascular emergencies. MethodsWe report 2 patients with PNH, one who was previously diagnosed with PNH and had arterial stroke, the other had an index presentation of cerebral venous sinus thrombosis (CVT) and was subsequently diagnosed with PNH. We also present the systematic review of literature reporting similar cases, highlighting the challenges in management. ResultsBoth patients presented to our centre with cerebrovascular emergency. The first patient was a diagnosed with PNH, and presented with left hemispheric infarction caused by thrombosis of middle cerebral artery. He was thrombolysed and underwent mechanical thrombectomy, which was unsuccessful in view of repeated re – thrombosis of the vessel. The patient survived with significant disability. The second patient had severe cerebral venous sinus thrombosis with large right hemispheric hemorrhagic venous infarction. She underwent emergency decompressive hemicraniectomy complicated by massive blood loss and disseminated intravascular coagulation. She subsequently had recurrent life threatening intracranial bleed secondary to platelet transfusions, thrombocytopenia, and use of contrast agents. She progressed to develop Budd Chiari syndrome and was initiated on Eculuzimab. She became transfusion independent, however remained in minimally conscious state and succumbed to sepsis. ConclusionsManagement of arterial and venous strokes is complex in patients with PNH. Invasive procedures and platelet transfusions are to be avoided in acute thrombosis, till robust evidence is available establishing the safety of the same in patients with PNH. Eculuzimab is a promising option, but far from reach for patients in developing countries.

Vascular Diseases in PNH

PNH is a condition in which uncontrolled complement activity leads to systemic complications, principally through intravascular hemolysis and platelet activation. It arises through a somatic mutation of the phosphatidylinositol glycan A (PIG-A) gene in bone marrow stem cells, 1,2 resulting in disruption to glycosylphosphatidylinositol (GPI) biosynthesis, 3. Among the deficient proteins are the complement regulatory proteinsCD55 andCD59, resulting in increased complement sensitivity of PNH cells, intravascular hemolysis, promotion of inflammatory mediators, and systemic hemoglobin release4 . Patients with PNH can present with multisystemic clinical manifestations due to intravascular hemolysis, thrombosis and bone marrow failure5 . Symptoms are therefore often non-specific, ranging from loss of vision (due to retinal thrombosis), headache and nausea/vomiting (due to cerebral thrombosis), pulmonary hypertension (due to pulmonary embolism), anaemia, through to pain and swelling in the lower extremities (due to deep vein thrombosis), renal failure and other symptoms affecting different systems6 . Thromboembolism is the most common cause of mortality in patients with PNH and accounts for approximately 40% to 67% of deaths of which the cause is known. Further, 29% to 44% of patients with PNH have been reported to have at least 1 thromboembolic event during the course of their disease, although the reason(s) a thrombotic event may suddenly occur remains an enigma7,8,9. Platelet activation, complementmediated hemolysis, impaired nitric oxide (NO) bioavailability, impairment of the fibrinolytic system, and inflammatory mediators are all proposed mechanisms and thought to be responsible for the increased thrombotic risk in patients with PNH. Multiple factors are likely to contribute to any one thrombotic event in patients with PNH. 10 Therapeutic strategies include terminal complement blockade and bone marrow transplantation. Eculizumab, a monoclonal antibody complement inhibitor, is highly effective and the only licensed therapy for PNH.11 The therapeutic anti-C5 antibody eculizumab (Soliris, Alexion) has proven effective in controlling intravascular hemolysis in vivo, leading to remarkable clinical benefit in a majority of PNH patients.12,13 Yet, persistent C3 activation occurring during eculizumab treatment may lead to progressive deposition of C3 fragments on affected erythrocytes and subsequent C3-mediated extravascular hemolysis, possibly limiting the hematologic benefit of anti-C5 treatment.14,15 Thus, upstream inhibition of the complement cascade seems an appropriate strategy to improve the results of current complement-targeted treatment.16,17

Associations of OPRM1 A118G Gene Polymorphism with Pain Sensitivity and Opioid Dosage of Patients with Postherpetic Neuralgia

This study aimed to explore the associations of OPRM1 A118G gene polymorphism with pain sensitivity and opioid dosage of patients with postherpetic neuralgia (PHN). According to the 3-month follow-up results, 103 PNH patients were enrolled as a PHN group, and 112 without PHN after herpes zoster were included as a non-PHN group. The factors affecting PHN occurrence were subjected to one-way analysis of variance and multivariate logistic regression analysis. The frequency distribution of GG, AG and AA genotypes in both PHN and non-PHN groups conformed to the Hardy-Weinberg equilibrium law (P>0.05). Initial treatment duration of more than 72 hours, severe acute pain and genotype GG were independent risk factors for PHN. Genotype GG exhibited significantly increased distribution frequency in mild, moderate and severe PHN groups. The 48-hour opioid dosage was highest in the patients with genotype GG (P<0.05). OPRM1 A118G gene polymorphism causes individual differences in the pharmacodynamics of opioids in PHN patients.

Upregulation of Checkpoint Ligand Programmed Death-Ligand 1 in Patients with Paroxysmal Nocturnal Hemoglobinuria Explained by Proximal Complement Activation.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hemolytic disease driven by impaired complement regulation. Mutations in genes encoding the enzymes that build the GPI anchors are causative, with somatic mutations in the PIG-A gene occurring most frequently. As a result, the important membrane-bound complement regulators CD55 and CD59 are missing on the affected hematopoietic stem cells and their progeny, rendering those cells vulnerable to complement attack. Immune escape mechanisms sparing affected PNH stem cells from removal are suspected in the PNH pathogenesis, but molecular mechanisms have not been elucidated. We hypothesized that exuberant complement activity in PNH results in enhanced immune checkpoint interactions, providing a molecular basis for the potential immune escape in PNH. In a series of PNH patients, we found increased expression levels of the checkpoint ligand programmed death-ligand 1 (PD-L1) on granulocytes and monocytes, as well as in the plasma of PNH patients. Mechanistically, we demonstrate that complement activation leading to the decoration of particles/cells with C3- and/or C4-opsonins increased PD-L1 expression on neutrophils and monocytes as shown for different in vitro models of classical or alternative pathway activation. We further establish in vitro that complement inhibition at the level of C3, but not C5, inhibits the alternative pathway-mediated upregulation of PD-L1 and show by means of soluble PD-L1 that this observation translates into the clinical situation when PNH patients are treated with either C3 or C5 inhibitors. Together, the presented data show that the checkpoint ligand PD-L1 is increased in PNH patients, which correlates with proximal complement activation.

Clinical observation of low-dose combination chemotherapy in refractory/recurrent paroxysmal nocturnal hemoglobinuria patients: A single-center retrospective analysis.

BackgroundWe performed a retrospective analysis to investigate the clinical characteristics and therapeutic strategies of 20 refractory/recurrent PNH patients, including the clinical efficacy of chemotherapy treatment, safety, and survival.MethodsThe clinical data of 20 classic PNH patients who were refractory/recurrent or had glucocorticoid dependence in our hospital were analyzed, including clinical manifestations, laboratory examinations, treatment efficacy, and survival.ResultsSeventeen patients had a marked improvement in anemia after chemotherapy, 14 patients acquired blood transfusion independence, and the Hb of 3 patients increased to normal levels. Although 6 patients still needed blood transfusion, the transfusion interval was significantly prolonged. The percentages of LDH, TBIL, and RET, which are indicators of hemolysis, were significantly lower than those before chemotherapy. The dosage of adrenal glucocorticoids was reduced by more than half compared with that before chemotherapy.ConclusionsChemotherapy can reduce PNH clones, promote normal hematopoiesis, and control hemolytic attack. It is a promising and widely used therapeutic method.

Total Oral Anticoagulation for Classic PNH As Primary Prophylaxis Strategy in a Complement Inhibitor Restricted Scenario: A Retrospective Analysis

Introduction.Paroxysmal nocturnal hemoglobinuria (PNH) is associated with thrombosis and bone marrow failure. Thrombosis is the leading cause of death and morbidity, with 29-44% of patients presenting at least one thrombotic episode during their disease's natural course. Treatment with complement inhibitors is a strategy associated with reducing up to 80% of thrombosis risk, and improving survival of PNH patients. However, the high cost of these therapies limited their access in Low and Middle-Income Countries (LMIC).Mexico, like other LMIC, experience low access to complement inhibitors. Hence, we established a primary thromboprophylaxis strategy in classic PNH. The primary objective of the present study is to analyze thrombosis-free survival and all-cause mortality in patients with classic PNH subjected to this prophylaxis strategy compared to historical controls.Methods.Single-center, retrospective, case-control study. We included patients treated from 1991 to 2021, diagnosed with classic PNH (clone size ≥50% determined by flow cytometry). We defined the treatment group as patients who received total oral anticoagulation as primary prophylaxis. We excluded patients who had presented thrombosis prior to classic PNH diagnosis, sub-clinic PNH, bone marrow failure, platelets <50x10 3/µ, and patients who failed to maintain a therapeutic (2 to 3) INR value three months after starting total oral anticoagulation with vitamin K antagonists (VKA). We collected data regarding clone type (II, III, or mixed), clone size, LDH values (<1.5 vs ≥1.5 x ULN), platelet count, thrombosis occurrence, thrombosis site, bleeding cases and their severity according to the international society on thrombosis and haemostasis (ISTH) classification, and causes of death.Results.We found 44 patients with classic PNH diagnosed from 1990 to 2021. Median age at diagnosis was 39 years (28 -51), 50% were female, median LDH value 1669 U/L (926-2,241), and median clone size 95% (80-99). Clone type III was most prevalent, with 61.36% cases, followed by mixed type II-III with 38.64%; we found no type II clone patients. Median follow-up was 21.03 years. Four patients developed a site of thrombosis; three cases (75%) presented simultaneous venous and arterial thrombosis, and one (25%) venous only. The most common site for thrombosis was the portal vein (n=3), followed by supra-hepatic veins (n=1).A total 19 patients (43.2%) received primary prophylaxis with total oral anticoagulation, 17 (89.5%) used VKA and 2 (10.5%) direct oral anticoagulants. We found no differences regarding age, LDH value, clone size and clone type between the treatment and control groups. There were no cases of thrombosis in the treatment group, while 5 patients (20%) in the control group developed thrombosis at some point (p=0.038, Table 1). Thrombosis-free survival was not reached in the treatment group, vs 12.78 years (IC95%,6.19-19.37) in the control group (p=0.026, Figure 1). Bleeding episodes occurred in 15.8% (n=3) of the treatment group patients, all of them minor according to the ISTH classification.The global survival was 85% at 15 years in the anticoagulation group vs 74% at 15 years in the control group, (p=0.071, figure 2). Mortality was 15.8% (n=3) in the treatment group vs 28.0% (n=7) in the control group, p=0.338. The only cause of death in the treatment group was progression to aplastic anemia. Causes of death in the control group were thrombosis (28.6%, n=2), aplastic anemia (28.6%, n=2), acute leukemia (14.2%, n=1), sepsis (14.2%, n=1) and digestive tract bleeding (14.2%, n=1).Discussion and conclusions.Thrombosis is the most frequent cause of morbidity and mortality in classic PNH patients. In 2003, Hall et al. found that primary prophylaxis with full-dose oral anticoagulation drastically reduced the thrombotic risk in these patients (0 vs. 36.5%, p=0.01). Meanwhile, complement inhibitor has become the mainstay therapy, thromboprophylaxis with oral anticoagulation has virtually stopped in countries where this therapy is accessible. The present study replicated the Hall et al. results, and demonstrated that primary thromboprophylaxis is safe and effective in reducing the risk of thrombosis. In LMIC, where access to complement inhibitors is limited, the use of thromboprophylaxis could abate thrombosis and should be taken in consideration in all patients. Hence, the clonal progression would become the leading cause of death. [Display omitted] DisclosuresApodaca Chavez: Abbvie: Speakers Bureau; Asofarma: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Rangel-Patiño: Abbvie: Speakers Bureau; Bristol: Consultancy. Demichelis: Jazz: Consultancy; Novartis: Consultancy, Research Funding, Speakers Bureau; ASH: Research Funding; Astellas: Consultancy; Gilead: Consultancy; Abbvie: Consultancy, Speakers Bureau; Bristol/Celgene: Consultancy, Speakers Bureau; AMGEN: Consultancy, Speakers Bureau.

Spectrum of Molecular Modes of Immune Escape in Idiopathic Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

The pathogenesis of idiopathic aplastic anemia (IAA) involves a human leukocyte antigen (HLA)-restricted T-cell autoreactivity against unknown antigens preferentially distributed on early hematopoietic stem and progenitor cells (HSPCs). Genetically acquired GPI-anchor and HLA deficiency have been both linked to clonal immune evasion from T-cell pressure. We hypothesized that, in analogy to anti-tumor adaptive immune evasion, pathophysiology of immune escape in IAA originates together with a broader dysfunction of antigen presentation/processing machinery and immune regulatory proteins, beyond HLA molecules, as an effect of immune pressure under T-cell attack. This initial immune reaction would produce up-modulation of these pathways, ultimately promoting the acquisition of mutations and expansion of immune resistant clones.To test this hypothesis, we first performed single-cell RNAseq analysis in HSPCs in IAA patients at disease manifestation, 1 which showed signatures of dysfunction of antigen presentation machinery, with up-regulation of most of the HLA molecules, proteasome subunits and endoplasmic reticulum related organelle transporters. Strikingly, DRB1 was among the top 3 genes upregulated in IAA patients compared to controls (q-values 1.23E-35; Fig.1A), underscoring the etiological impact that antigen presentation via this locus has in the initiation of autoimmune process. Mild upregulation was also seen in DQB1 and B loci (q-values 4.7E-07 and 2.1E-10, respectively).We then studied molecular escape mechanisms by genotyping 204 IAA and PNH patients, with either a targeted or whole genome sequencing (WGS) platform. By application of a newly in-house developed bioinformatic pipeline, we detected somatic aberrations in HLA region involving both class I and II alleles in 36% of IAA patients including point mutations, frameshift insertions or deletions and copy number variations inducing allelic loss. B*14:02 and A*02:01 emerged as the most commonly mutated class I alleles with a few hotspot mutations identified, particularly in exon 1 (c.19C>T, p.R7X, Fig.1B,C), confirming previous reports. 2,3 In class II, DQB1 and DPA1 loci were frequently targeted by fine mutational events, while more complex allelic loss phenomena interested prevalently DRB1 and DQB1 loci. Those aberrations were identified at diagnosis (35%), during disease follow-up (33%) or at the time of malignant evolution (27%), with higher clonal size in specimens collected during the course of the disease (median VAF 3% [2-27%] at diagnosis, 8% [2-98%] at follow-up, and 2.2% [2.0-6.1%] at evolution). Of 41 patients with at least one HLA aberration and characterized with an extended genotypic study, only 6 harbored also >1 somatic myeloid mutation (14%), versus 30/90 (33%) not affected by somatic hits in HLA (p=.026; Fig.1D). HLA aberrant cases also showed lower number of somatic myeloid mutations (OR=0.44; p=.0262) with driver hits rarely present (Fig.1E). In terms of PIGA mutations, an increased PIGA mosaicism was observed in the HLA mutant group, underlying that both processes have similar pathophysiologic origin as a product of the immune selection pressure (OR: 1.55 [95%CI 1.1-4.2], p=.0201). We then investigated, through WGS of 53 patients, the presence of somatic mutations in other immune genes which could be triggered by immune pressure. Hence, in 47% of the cases we were able to find pathogenic or likely pathogenic hits in genes encompassing proteasome complex, vesicle trafficking, transactivators and interferon regulatory factors, including CREBBP, TAP1, CIITA, PSMC5, PSMB4 and IRF9 (Fig.1F), whose pathogenicity was computationally assessed through recently implemented somatic classifiers. 4 Those hits were not mutually exclusive neither with HLA nor with PIGA mutations, however their VAF was significantly lower compared to concurrent HLA and PIGA lesions, underscoring their lower driver potential within the immune escape environment compared to PNH and HLA-lacking clones.Altogether our results describe the diversity of molecular and immune events taking place in IAA and PNH. Our study suggests that following initial immune insult, clonal architecture of residual hematopoiesis can be dominated by multiple modes of immune escape, agonistically participating to a mechanism of “adaptive” clonal recovery, likely in opposition to the “maladaptive” malignant progression. [Display omitted] DisclosuresMaciejewski: Alexion: Consultancy; Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.

CT-118: PH3 Study of Efficacy and Safety of Iptacopan (LNP023), an Oral Complement Factor B Inhibitor, in Patients with Paroxysmal Nocturnal Hemoglobinuria and Residual Anemia Despite Anti-C5 Antibody Treatment

Context The standard of care in PNH is anti-C5 monoclonal antibody (MAb) treatment with eculizumab (ECU) or ravulizumab (RAV), but hematological responses can be heterogenous. Iptacopan (LNP023) is a first-in-class, oral, small-molecule, selective and reversible complement factor B inhibitor that inhibits both intra- and extravascular hemolysis. In an ongoing Ph2 study (NCT03439839), iptacopan add-on treatment demonstrated improvements in hematological response and biomarkers of disease activity in adult PNH patients with active hemolysis despite ECU treatment. Objective APPLY-PNH (NCT04558918) will assess efficacy and safety of iptacopan monotherapy in adult PNH patients with residual anemia despite prior anti-C5 MAb treatment by evaluating its superiority to anti-C5 MAb treatment. Design The study comprises an ≤8-wk screening period; 24-wk, randomized, open label, active controlled, parallel group treatment period; and 24-wk treatment extension period. Patients (N≈91) are randomized (8:5) to iptacopan monotherapy 200mg orally BID (seamless switch from prior treatment) or intravenous ECU/RAV (continue prior treatment). Patients are stratified based on prior anti-C5 treatment and transfusion history. On completion of Wk 24 visit, patients may enter 24-wk iptacopan treatment extension. Eligible patients must have confirmed diagnosis of PNH (RBCs and WBCs [granulocyte/monocyte] clone size ≥10%) and residual anemia (mean Hb Conclusions This study will be the first head-to-head comparison of iptacopan vs an anti-C5 MAb in PNH patients. Study sponsored by Novartis Pharmaceuticals Corporation.

Costs higher for PNH patients receiving eculizumab plus blood transfusions in the USA

Costs higher for PNH patients receiving eculizumab plus blood transfusions in the USA

Effects of Eculizumab Treatment on the Quality of Life in Patients with Paroxysmal Nocturnal Hemoglobinuria Treated in Japan

Introduction: PNH is a hematopoietic stem cell disorder with the expansion of a PIGA mutated clone(s). PNH clones are deficient in the membrane expression of GPI-anchored proteins including CD55 and CD59, which leave erythrocytes susceptible to complement attack, resulting in intravascular hemolysis. Many symptoms experienced by PNH patients such as fatigue, dyspnea, and stomachache have been found to be associated with serum free hemoglobin (Hb) released from erythrocytes by intravascular hemolysis (JAMA. 2005;293(13):1653-1662). Eculizumab (Soliris®), a humanized monoclonal antibody against Complement Factor C5, has shown dramatic effect in blocking intravascular hemolysis. Previous reports have revealed that eculizumab has not only improved anemia but also patients' quality of life (QoL) (N Engl J Med. 2006; 355: 1233-1243, Blood. 2008; 111: 1840-1847, Int J Hematol. 2011; 93: 36-46). However, factors which may be associated with improvement of QoL have yet to be fully understood. To clarify the clinical significance of QoL improvement in PNH patients treated with eculizumab, we analyzed the change in QoL in 54 PNH patients treated with eculizumab based on the data from the post-marketing surveillance (PMS) of eculizumab in Japan.Methods: All PNH patients treated with eculizumab have been registered in PMS based on the revised Pharmaceutical Affairs Law in Japan since June 2010. Of 491 patients registered as at March 2017, QoL questionnaires were obtained from 225 patients. 54 patients who received both baseline and one-year assessment of QoL were analyzed in this study. QoL was assessed using Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-Fatigue) and European Organization for Research and Treatment of Cancer Quality of Life Questionnaire (EORTC QLQ-C30) instruments. Summary statistics were obtained from the figures of QoL scoring systems and the lab values, and paired Student's t-test was used for the change of the values in one year from the baseline. Canonical correlation analysis was performed among QoL variates (consisted of 16 items) and lab data variates (LDH, Hb, and platelets).Results: There was no bias in the baseline characteristics between the total (491) and the analyzed (54) patients. One-year administration of eculizumab improved serum LDH level (mean: from 1435.3 to 289.5 U/L), Hb level (mean: from 8.22 to 9.46 g/dL), and QoL in most of their items in comparison with the baseline. In particular, significant improvement of EORTC QLQ-C30 was shown in the items of fatigue, dyspnea, physical function, and global health status. Canonical correlation analysis revealed a high correlation between QoL variates and lab data variates (Canonical R=0.8393, p=0.004). Of the lab data variates, LDH (Canonical correlation coefficient: -0.91) and Hb (0.76) showed strong correlations with QoL improvement as expected. Interestingly, QoL improvement was independent of patient's baseline characteristics such as the coexistence of aplastic anemia and/or myelodysplastic syndrome and the degree of lactate dehydrogenase (LDH) (Fig. 1, 2).Conclusion: In this study, QoL improvement following treatment with eculizumab was statistically strongly correlated to the degree of hemolysis, which is consistent with the previous reports. Herein we also demonstrated, for the first time, that the degree of improvement was independent of the baseline serum LDH level before eculizumab treatment and of the presence or absence of bone marrow failure. Those PNH patients without eculizumab treatment due to mild hemolysis might benefit from eculizumab treatment regardless of the presence of bone marrow failure syndromes. [Display omitted] DisclosuresUeda:Alexion Pharma GK: Honoraria, Research Funding. Obara:Alexion Pharma GK: Honoraria, Research Funding. Yonemura:Alexion Pharma GK: Honoraria, Research Funding. Noji:Alexion Pharma GK: Honoraria, Research Funding. Matsuda:Alexion Pharma G.K.: Employment. Akiyama:Alexion Pharma G.K.: Employment. Chiba:Nippon Shinyaku: Honoraria, Research Funding. Kawaguchi:Alexion Pharma G.K.: Honoraria; Novartis,Pfizer: Consultancy, Honoraria. Shichishima:Alexion Pharma GK: Honoraria. Okamoto:Bristol-Myers K.K: Honoraria; Pfizer Japan: Speakers Bureau; Alexion Pharma G.K.: Honoraria; Astellas: Honoraria; Pfizer: Research Funding; Chugai Pharmaceutical Co.: Research Funding; Novartis: Research Funding; Bristol-Myers K.K: Research Funding. Nishimura:Alexion Pharma GK: Honoraria, Research Funding. Kanakura:Fujimotoseiyaku: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Kyowa Hakko Kirin: Research Funding; Astellas: Research Funding; Eisai: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding; Nippon Shinyaku: Research Funding; Pfizer: Research Funding; Bristol Myers: Research Funding; Toyama Chemical: Research Funding. Ninomiya:Alexion Pharma GK: Honoraria.

Paroxysmal Nocturnal Hemoglobinuria Results from Initial Clonal PIG-a Mosaicism

The pathogenesis of PNH involves alteration of at least one HSC clone, characterized by a somatic mutation in the PIG-A gene. The resulting phenotype appears to offer a growth advantage over other unaffected HSCs, yet the failure of PIG-A mutant clones to expand in animal models suggests that additional factors are necessary for the development of PNH. The association of PNH with aplastic anemia (AA) is evidence of selection pressure by immune attack, but secondary hits in additional genes may also be clonally acquired to render PNH clones competitive irrespective of immune pressure. With the advent of NGS, clonal architecture analysis has been revolutionized. Yet, despite increased resolution, NGS is hampered by the presence of normal/non-clonal cells at varying frequencies in the sequenced sample, often decreasing the detection threshold and precluding recognition of subclonal mosaicism.To study these issues in PNH, 133 patients with PNH (N=33), AA (N=33), and AA/PNH (N=67) were sequenced using deep targeted NGS covering all exons of the PIG-A gene. All patients had a detectable PNH clone by flow cytometry, with the mean WBC clone size of 0.22% in AA, 27.4% in AA/PNH, and 77.1% in PNH. 223 PIG-A mutations were identified in this cohort; 35% were frameshift mutations, 34% missense, 14% splice site, 13% nonsense, and 3% non-frameshift. In unpurified blood, PIG-A mutations were found in 27/33 (82%) of PNH patients, 36/67 (54%) of AA/PNH, and 2/33 (6%) of AA, with a clear association between PNH clone size and the ability to detect mutations by NGS (P<0.0001). In DNA extracted from cells flow sorted by FLAER negativity, detection rates were 26/29 (90%), with an average of nearly 3 mutations per patient, indicating that mechanisms such as microdeletions may affect as many as 10% of PNH or AA/PNH patients and that clonal mosaicism is common. Our results also show that indeed type III and II PNH cells are derived from 2 distinct clones, each with a distinct mutation.To examine disease pathogenesis, we studied 47 patients longitudinally using both PNH flow and deep sequencing. Serial testing illustrates possible modes of evolution: mutations can fluctuate in the contribution to clonal burden over time with the variant allelic frequency of some mutations increasing while others decrease. Linear regression of the number of mutations vs . time from diagnosis suggests that in AA/PNH (N=29), the number of mutant clones detected increases over time (P=.012) while in classic PNH (N=18) the trend is reversed, as the number of mutations detected decreases (P<.0001) and a dominant clone emerges to supply a majority of blood cells.Traditionally, PNH has been considered a monoclonal disease in which a PIG-A mutant HSC clone can outcompete normal hematopoiesis within the context of permissive conditions. Using deep NGS, our results clearly indicate that while a single clone ultimately may dominate, PNH begins as oligoclonal process with the eventual emergence of the most competitive HSC clone(s) populating hematopoietic space. Initially, an environment that “favors” a state of GPI-anchor deficiency thereby allows multiple clones to evolve. Over time, the most “fit” PNH HSC clones dominate, resulting in monoclonality. In addition, our results confirm the presence of distinct mutations with corresponding phenotype through sequencing of sorted type II and type III PNH cells. Clonal drift may also be observed, likely through a newly acquired advantage of one clone within the available pool of GPI-deficient PIGA mutant clones. Our previous work indicated that acquisition of additional enabling mutations results in a more complex clonal architecture comparable to that seen in MDS, an intrinsic mechanism of the selection process that involves mutations in genes altered in MDS. Additional intrinsic hits also explain patient-specific kinetics of PNH evolution and apparent independence of established PNH from the effect of immunosuppression. Thus, while immune selection may provide initial conditions favorable for oligoclonal PNH evolution, once pressure is relieved, intrinsic factors can lead to PNH monoclonality. Alternative mechanisms could include loss of dominance by exhaustion, senescence, or telomere shortening.In sum, our study clearly demonstrates initial polyclonality early in the pathogenesis of PNH with selection of dominant clones during disease duration. DisclosuresMakishima:Yasuda Medical Foundation: Research Funding.

Assessment of Eculizumab and C5 Levels in PNH Treatment: Molar Ratio of Eculizumab to C5 Level Effectively Predicts Ongoing Dosage Changes in a Cohort of 50 Patients Treated with Eculizumab

IntroductionEculizumab is highly effective in preventing the intravascular haemolysis and long-term complications of PNH. However a significant proportion (>20%) of patients in the Leeds cohort require a dose in excess of the standard 900mg Q2W to achieve and maintain efficacy. It is, therefore, important to determine the circulating concentration of both the drug and its target, complement component C5, throughout treatment to accurately inform dosing and to minimise the risk of a potentially serious breakthrough event.MethodsSerum samples were collected prior to scheduled treatment and were analysed for trough levels of eculizumab by bridge ELISA (Cambridge Biomedical, MA), and subsequently for both eculizumab levels and C5 levels by mass spectrometry and immunonephelometry, respectively (Mayo Clinic, MN). Eculizumab levels determined by the two techniques were compared for a total of 54 patient samples, and C5 levels were determined in these sample, as well as in 20 normal controls. For 7 patients multiple samples collected over the treatment course were analysed to determine the concentration of drug, target and the molar ratio between the two over a period of up to 24 months. Clinical data maintained by the UK PNH National Service (Leeds, UK) was correlated with these findings.ResultsELISA measurements for eculizumab levels identified 35/50 (70%) patients below the target trough level of 100ug/ml; mass spectrometry indicated that only 7/50 (14%) patients were below this level. All 7 patients below 100ug/ml by mass spectrometry were also reported as below this limit by ELISA. There was some correlation between the two tests with respect to overall position within the range of values reported (r2 = 0.65), but absolute values differed significantly. This is expected, the C5 ELISA assay is only able to detect free eculizumab while the mass spectrometry assay can detect total eculizumab.The median C5 level in healthy controls was 205ug/ml (range:128-305ug/ml) and fell within the expected normal range (106-263ug/ml); C5 levels for PNH patients receiving eculizumab were elevated in all patients, with a median of 403ug/ml (range:241-655ug/ml).For the 7 patients with multiple samples: 5/7 had increasing circulating C5 over time whereas 2/7 had decreasing C5 (though only one was reduced to the high end of the normal range: 241ug/ml)The eculizumab levels of 3/4 four patients on standard dose decreased over time (all falling below the clinically significant cut-off of 100ug/ml), 1 was unchanged. All patients receiving a higher dose (1200mg) showed an increased eculizumab level in excess of 100ug/ml.A molar ratio of C5:eculizumab < 2 is required for adequate protection from intravascular haemolysis, as there are two C5 binding sites per eculizumab molecule. Four patients with a ratio >2.0 on the standard dose (900mg) had the ratio reduced to 2.0 or less following an increase to 1200mg and have remained on this dose. One patient had a ratio that decreased from 3.7 to 2.6 following dose increase (to 1200mg), and remains on this dose. Two patients on standard dose across the sampling time showed ratios of 4.0 and 2.4 and were subsequently given increased doses (1200mg).ConclusionThe two methods used here for measuring eculizumab and C5 levels returned values in line with previous studies highlighting the increase in C5 levels associated with eculizumab treatment. Whilst eculizumab levels alone are a reasonable predictor of efficacy, the molar ratio between eculizumab and C5 correlated well with the clinical decision to increase dosage and should be tested in larger, prospective studies to assess its potential to inform clinical decisions regarding dose escalation. [Display omitted] DisclosuresRicardo:Ra Pharmaceuticals: Employment. Hoarty:Ra Pharmaceuticals: Employment. Arnold:Alexion Pharmaceuticals, Inc.: Honoraria. Munir:AbbVie: Honoraria; Roche: Honoraria; Janssen: Honoraria; Gilled: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria. Griffin:Alexion Pharmaceuticals, Inc.: Honoraria. Hill:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Hillmen:Pharmacyclics LLC, an AbbVie Company: Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria, Research Funding; GSK: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Celgene: Research Funding.

Characterization of Fasciculation Potentials (FPs) in Amyotrophic Lateral Sclerosis (ALS) and Peripheral Nerve Hyperexcitability Syndromes (PNH).

This study is aimed at investigating the features of fasciculation potentials (FPs) in amyotrophic lateral sclerosis (ALS) and peripheral nerve hyperexcitability syndromes (PNH). Needle electrophysiologic examination (EMG) was performed for 5-15 muscles in the ALS and PNH patients. The spontaneous activity of fasciculations and fibrillations/sharp-waves (fibs-sw) was recorded. The distribution, firing frequency, and waveform parameters of FPs in muscles were calculated and compared. In total, 361 muscles in ALS patients and 124 muscles in PNH patients were examined, with the FP detection rates of 45.1% and 53.2%. Moreover, the ALS patients with the upper limb onset had the highest FP detection rate. Fasciculations occurred more frequently in the upper limbs than in the lower limbs in ALS and PNH. The detection rate of fibs-sw in the bulbar muscle was relatively low, which could be elevated when combining fibs-sw and FPs. Benign FPs in PNH were of smaller amplitude, shorter duration, and fewer phases/turns, compared with malignant FPs in ALS. The FP area in PNH was significantly smaller than that in ALS. The incidence of polyphasic FPs in ALS was distinctly greater than that in PNH. The firing frequency of FPs in PNH was higher than that in ALS. There was no significant difference in the amplitude, duration, phases and turns, and area of FPs between groups with and without fibs-sw in the muscles of normal strength in ALS. Conclusively, it is necessary to detect the FPs in the thoracic and bulbar muscles of patients suspected having ALS. FP parameters in ALS are significantly different from PNH.

Advances in research for pathogenesis of paroxysmal nocturnal hemoglobinuria

Paroxysmal Nocturnal hemoglobinuria, PNH is usually caused by the somatic mutation of X-linked PIGA gene followed by the clonal expansion of the GPI (glycosylphosphatidylinositol) anchor defective hematopoietic stem cell clone. There are two hypotheses for the mechanism of clonal expansion, one is selection theory, in which GPI deficient cells escape from attacks of cytotoxic cells, and another is benign tumor theory in which GPI deficient cells get the additional mutations and acquire proliferative nature. Recently, we identified two types of PNH patients caused by the biallelic mutation of PIGT on chromosome 20 and PIGB on chromosome 15. Both PNH clones had the germ-line mutation in one allele and another allele was somatically mutated in a hematopoietic stem cell. Both somatic mutations were loss of heterozygosity (LOH), deletion in PIGT-PNH and copy neutral LOH (CN-LOH) in PIGB-PNH. These PNH patients had typical PNH symptoms, but they have in addition auto-inflammatory features. Unlike in PIGA-PNH cells, GPI is synthesized in PIGT-PNH cells and, since its attachment to proteins is blocked, free GPI is expressed on the cell surface. Similarly, in PIGB-PNH cells, GPI intermediates are accumulated and expressed on the cell surface. Those GPIs together with complement activation cause the inflammasome activation.

Phase 3 Study of Danicopan, an Oral Complement Factor D Inhibitor, As Add-on Therapy to a C5 Inhibitor in Patients with Paroxysmal Nocturnal Hemoglobinuria with Clinically Evident Extravascular Hemolysis

Background: PNH is a rare, life-threatening disease caused by uncontrolled terminal pathway activation leading to intravascular hemolysis (IVH). The C5 inhibitors eculizumab (ECU) and ravulizumab (RAV) prevent IVH by inhibiting terminal complement; however, some C5 inhibitor-treated patients may experience persistent anemia due to extravascular hemolysis (EVH) caused by C3 fragment deposition and opsonization driven by constitutive activation of the alternative pathway (AP). Inhibition of factor D (FD), the rate limiting enzyme of the AP, acts on the complement cascade upstream of C3. Danicopan (ALXN2040, ACH-4471) is a first-in-class oral small molecule FD inhibitor. In vitro studies with RBCs collected from PNH patients have shown that danicopan not only inhibited hemolysis but also prevented deposition of C3 fragments on PNH RBCs. In a 24-week Phase 2 study of ECU-treated PNH patients with transfusion-dependent anemia (hemoglobin [Hgb] &amp;lt;10 g/dL), danicopan add-on resulted in clinically significant improvements in Hgb, near transfusion-independence, improvements in FACIT-Fatigue scores, and was generally well-tolerated; 96% of treatment-emergent adverse events were mild-to-moderate in severity and did not result in discontinuation. The purpose of this randomized, double-blind, pivotal Phase 3 trial (NCT04469465; EudraCT 2019-003829-18) is to evaluate the efficacy of oral danicopan add-on therapy in PNH patients with clinically evident EVH (CE-EVH) on an approved C5 inhibitor. Study Design and Methods: This study consists of a 12-week double-blind placebo-controlled treatment period 1 followed by a 12-week danicopan+C5 inhibitor treatment period 2 and a long-term extension up to 1-year. Patients (target enrollment, N=84) will be randomized to danicopan or matched placebo TID in a 2:1 ratio for the 12-week treatment period 1. Patients randomized to placebo for treatment period 1 will switch to danicopan at week 12 (Figure). Eligible adult patients must be receiving a stable regimen of ECU or RAV (no change in drug/dose/interval for ≥24 weeks), and have CE-EVH, defined by anemia (Hgb ≤9.5 g/dL), absolute reticulocyte count ≥120 x 109/L, and ≥1 transfusion within 6 months before study entry. The starting dose of danicopan is 150 mg TID. Patients with alanine aminotransferase (ALT) or direct bilirubin values &amp;gt;1.5 × upper limit of normal (ULN) will start at 100 mg TID. Doses may be escalated in 50-mg increments, with ≥4 weeks between escalations, to a maximum of 200 mg TID based on safety and clinical effect at protocol-specified time points. Exclusion criteria include major organ transplant or hematopoietic stem cell transplantation (HSCT), aplastic anemia requiring HSCT, complement deficiency, or ALT &amp;gt;2 × ULN. The primary efficacy endpoint is change in Hgb at week 12. Secondary endpoints are proportion of patients not requiring a transfusion through week 12, change from baseline in FACIT-Fatigue scores, and change from baseline in absolute reticulocyte count at week 12. Other secondary endpoints (at weeks 12 and 24) include RBC units transfused, Hgb stabilization, laboratory markers (including bilirubin, LDH, and PNH clone size), and patient-reported outcomes (EQ-5D-3L, EORTC-QLQ-C30, WPAI, and health resource utilization). Primary and secondary efficacy analyses will be performed on the intent-to-treat population; safety analyses will include all patients who received ≥1 dose of study drug. Danicopan has the potential to be the first oral PNH therapy, and offers an opportunity to enhance the well-characterized efficacy of C5 inhibitors without compromising safety. This Phase 3, pivotal trial will be the largest clinical evaluation of danicopan to date. More importantly, the trial has the potential to generate robust data to demonstrate the efficacy and safety of add-on, oral danicopan to C5 inhibitor therapy in PNH patients with clinically evident hemolysis. Figure Disclosures Kulasekararaj: Alexion Pharmaceuticals Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees. Risitano:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alnylam: Research Funding; Alexion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Jazz: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Amyndas: Consultancy; RA pharma: Research Funding; Biocryst: Membership on an entity's Board of Directors or advisory committees; Apellis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Achillion: Membership on an entity's Board of Directors or advisory committees; Pfizer: Speakers Bureau. Lee:Alexion Pharmaceuticals Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Huang:Alexion: Current Employment, Current equity holder in publicly-traded company. Nishimura:Alexion: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Consultancy, Other: All authors received editorial support for this abstract, furnished by Scott Battle, funded by F. Hoffmann-La Roche Ltd, Basel, Switzerland. ; Chugai: Consultancy. Ramirez-Santiago:Alexion: Current Employment, Current equity holder in publicly-traded company.

Immunogenomics of Paroxysmal Nocturnal Hemoglobinuria: A Model of Immune Escape

Unlike leukemic driver mutations, PIGA mutations produce an escape phenotype in the context of immune-mediated bone marrow failure such as aplastic anemia (AA). Another way to create clinical advantage will be to disable HLA-mediated cytotoxic T cell recognition. Determinants of cytotoxic T cell response might include some accessories glycosylphosphoinositol (GPI)-linked moieties but the main stimulus is likely to be provided by HLA-presented antigenic peptides. Somatic hits in HLA genomic region (microdeletions, uniparental disomies [UPDs] of HLA locus on 6p and later mutations) have been previously assessed in AA patients [1-3]. Mechanistic analogy to immune-privileged GPI-anchor protein deficiency in PNH due to PIGA mutations[4] or deletion[5] of PIGA locus are obvious. We stipulate that HLA mutations may contribute to the intrinsic expansion of PNH clones under immune pressure being: i) additive to the effects of PIGA mutations in creating immune escape or ii) redundant and thus less frequent in PNH clones as in patients without PIGA mutation. Using a deep targeted-sequencing panel covering HLA classical loci, and applying an in-house newly developed pipeline for the study of the HLA region (AbstractID#142501), we detected class I/II HLA somatic mutations of 10 patients with PNH. An integrative mutational analysis of PIGA and myeloid genes was then performed in order to comprehensively evaluate the role of HLA somatic hits within the scenario of PNH clonal evolution. At the time of this submission HLA sequencing was completed for a total of 35 patients but full analysis is available for the first 10 cases. Overall, of these 10 PNH patients 20 samples were analyzed from sorted GPI(+) and GPI(-) myeloid fractions (mean purity &amp;gt;95%). Median age at diagnosis was 36 years (11-66) while median PNH granulocyte clone size at time of sampling was 76% (5.11-99). A total of 41 PIGA mutations (Fig.1A) were detected solely in the GPI(-) fraction (mean VAF 58%), with 8 patients harboring clonal mosaicism as previously described.[6] Six somatic mutations of HLA class I (N=3, Fig.1B) and class II (N=3, Fig.1C) loci were found in 4 patients (67% detected on GPI(+) and 33% on GPI(-) fraction) at a low VAF (mean 3.36%). All these events were insertions or deletion of one or more bases. Class I mutations were located in intron 5, exon 3 and 3' untranslated regions (UTR). Class II were found instead in exon 2 (N=2) and intron 4. A functional and topographical annotation based on IPD-IMGT/HLA database suggested that exonic mutations were disruptive, impairing the bio-functionality of antigen presentation site. The detected intronic mutations instead impair HLA moiety assembling within cellular membrane, possibly altering splicing of the transmembrane domain. Moreover, a computational prediction of the regulatory domains involved in the 3'UTR aberration, showed a possible involvement of the miRNA has-miR-4524a-3p binding site, potentially affecting HLA post-transcriptional regulation. Of note, in 1 patient (UPN 9, Fig.1D) we did not find any PIGA, PIGT or HLA mutation. Finally, myeloid gene mutations analysis revealed the presence of a subclonal ASXL1 mutation in 1/10 patients in the GPI(-) fraction. Of note, this patient (UPN 1) had older age and showed 12 different somatic PIGA hits. This finding is probably explicable with the scenario of PIGA as the initial ancestral event accompanied by secondary mutations previously shown by our group as occurring in 10% of PNH cases in the course of disease evolution.[7-8] In summary, somatic HLA class I/II mutations can be found in patients with PNH. HLA mutations can occur in GPI(+) cells in subclonal fashion but also in GPI(-) cells. The latter clonal mosaicism indicates that various mechanisms of immune escape may play a role. Subclonal HLA mutations may impact the immune pressure on PNH clone dynamics, reflecting an alternative immune escape pathway in patients without PNH clone. (Fig.1E) In addition, detection of occasional "myeloid" hits suggests that various modes of PNH clone maintenance and expansion may be operative. We will present at ASH analysis of a full cohort of these patients including properly powered clinical correlations. Figure 1 Disclosures Maciejewski: Novartis, Roche: Consultancy, Honoraria; Alexion, BMS: Speakers Bureau.