Paroxysmal Nocturnal Hemoglobinuria Red Blood Cells Research Articles

Navigating the Complement Pathway to Optimize PNH Treatment with Pegcetacoplan and Other Currently Approved Complement Inhibitors.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare and potentially life-threatening hematologic disorder caused by a somatic mutation in a relevant portion of hematopoietic stem cells. Mutation of the phosphatidylinositol glycan biosynthesis class A (PIGA) gene prevents the expression of cell-surface proteins, including the complement regulatory proteins CD55 and CD59. With decreased or a lack of CD55 and CD59 expression on their membranes, PNH red blood cells become susceptible to complement-mediated hemolysis (symptoms of which include anemia, dysphagia, abdominal pain, and fatigue), leading to thrombosis. State-of-the-art PNH treatments act by inhibiting the dysregulated complement at distinct points in the activation pathway: late at the C5 level (C5 inhibitors, eculizumab, ravulizumab, and crovalimab), centrally at the C3 level (C3/C3b inhibitors and pegcetacoplan), and early at the initiation and amplification of the alternative pathway (factor B inhibitor, iptacopan; factor D inhibitor, danicopan). Through their differing mechanisms of action, these treatments elicit varying profiles of disease control and offer valuable insights into the molecular underpinnings of PNH. This narrative review provides an overview of the mechanisms of action of the six complement inhibitors currently approved for PNH, with a focus on the C3/C3b-targeted therapy, pegcetacoplan.

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 >10%); complement-inhibitor treatment-naïve or inadequate response to C5 inhibitors; and baseline hemoglobin (Hgb) <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<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 (>80%; P<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.

Ahemolytic PNH: Clinical Features of a Distinct Phenotype of Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem/progenitor cell disorder caused by PIGA mutations. All hematopoietic lineages derived from the affected clone exhibit the PNH phenotype [deficiency of glycosyl phosphatidylinositol linked membrane proteins (GPI-APs)]. Diagnosis is made by flow cytometric analysis of red blood cells (RBC), granulocytes, and monocytes. The percentage of granulocytes and monocytes with absent expression of GPI-APs is a measure of the size of the PNH clone. Typically, the percentage of GPI-AP deficient RBCs is somewhat less than that of granulocytes and monocytes due to selective destruction by complement-mediated intravascular hemolysis, manifested by high LDH, bilirubin, reticulocyte count and low haptoglobin. Herein, we report five patients with large PNH clones based on percentage of GPI-AP deficient neutrophils and monocytes, but with absent or near absent PNH RBCs and no biochemical evidence of hemolysis. We call this unique disease subtype, ahemolytic PNH. Methods: The medical records of PNH patients (174 patients with PNH granulocytes >20%) at the University of Texas Southwestern, Cleveland Clinic Foundation, and National Institutes of Health from 2000 to 2022 were examined. Information pertaining to their clinical, laboratory, and molecular attributes (NGS sequencing) was collected. Five patients with granulocyte clones >50% and RBC clones <5% were identified (Table). Results: Patient 1 is a 63-year-old male with a remote history of non-severe aplastic anemia (AA) diagnosed at 11 years old. The first PNH flow cytometry, obtained at age 61 years, showed 0.02% type II PNH RBC, 0.5% type III PNH RBC, 96% PNH monocyte and 97% PNH granulocytes (Table). There was no biochemical evidence of hemolysis at the time of diagnosis or in the preceding 5 years. Patient 2 is a 50-year-old male who presented with pancytopenia and was diagnosed with acute myeloid leukemia with myelodysplastic-related mutations. Flow cytometry showed 85% PNH monocytes and 59% PNH with no detectable PNH RBCs and no biochemical evidence of hemolysis at diagnosis or in 3 years of follow-up (Table). Patient 3 is a 73-year-old female who presented with pancytopenia and marrow aplasia with unremarkable morphology, and a PNH granulocyte clone of 93.6% with 1.3% type III PNH RBCs and 1.3% and 0.5% type II PNH RBCs, with no evidence of hemolysis (Table). Patient 4 is a 21-year-old female who presented with Budd-Chiari syndrome. Flow cytometry showed 73% PNH granulocytes with 3.6% type III PNH RBCs and 1.8% type II PNH RBCs. There was no biochemical evidence of hemolysis, but because of thrombosis, she was treated complement inhibitor therapy (Table). Patient 5 is a 61-year-old female with history of severe aplastic anemia treated with immunosuppression (IST) and eltrombopag. PNH flow cytometry showed PNH granulocytes of 92.4% and 2.1% PNH type II RBCs 2.1% post IST treatment with no biochemical evidence of hemolysis (Table). Conclusion: Herein, we present five patients with a distinct PNH phenotype that we call ahemolytic PNH. Except for the absence or near absence of PNH erythrocytes, this entity shares clinical and pathophysiological features with classic PNH, including thrombophilia (Budd-Chiari Syndrome, Table). The basis of this phenotype is speculative but may be determined by the genotype of the affected HSPC in which the somatic mutation of PIGA first occurred. According to this hypothesis, somatic mutations other than PIGA skew differentiation toward granulocyte/monocyte lineages. The International PNH Interest Group recognizes the following three categories of PNH: classic PNH, PNH in the setting of another define bone marrow abnormality, and subclinical PNH. Like ahemolytic PNH, subclinical PNH has no biochemical evidence of hemolysis. In this case, however, the absence of hemolysis is due to the small size of the PNH clone (median clone size <1%) as determined by flow cytometry analysis of peripheral blood granulocytes and monocytes. We propose that ahemolytic PNH, defined as patients having less than <5% PNH red cells and >50% PNH granulocytes/monocytes, be recognized as a distinct category of PNH.

Danicopan As Add-on Therapy to Ravulizumab or Eculizumab Versus Placebo in Patients with Paroxysmal Nocturnal Hemoglobinuria and Clinically Significant Extravascular Hemolysis: Phase 3 Long-Term Data

Background C5 inhibitors eculizumab (Ecu) and ravulizumab (Rav) have transformed the natural history of paroxysmal nocturnal hemoglobinuria (PNH). Ecu resulted in patient (pt) survival comparable to that of the general population over 20 y of follow-up under real-world conditions. Overall survival rate at 6 years for pts treated with Rav was 98.4%. Where available, Rav is the standard of care for PNH, inhibiting terminal complement activation to prevent intravascular hemolysis (IVH) and thrombosis. Of pts with PNH treated with Rav/Ecu, 10-20% experience clinically significant extravascular hemolysis (cs-EVH). Efficacy and safety of the first-in-class oral factor D inhibitor danicopan (Dan; ALXN2040) as add-on treatment to Rav or Ecu for pts with PNH and cs-EVH were assessed in a phase 3, randomized, double-blind, placebo (Pbo)-controlled superiority clinical trial (ALPHA, NCT04469465). 12-wk data (double-blind treatment period [TP] 1) showing superiority of Dan vs Pbo on primary and key secondary endpoints were previously reported and represent the final analysis set for the trial. Open-label 24-wk (TP2) and ongoing long-term extension (LTE) data are presented. Methods Pts (≥18 y) with PNH and cs-EVH (hemoglobin [Hgb] ≤9.5 g/dL; absolute reticulocyte count [ARC] ≥120×10 9/L) on Rav/Ecu >6 mos were randomized double-blind 2:1 to Dan or Pbo add-on therapy for 12 wks (TP1). At wk 12, Pbo arm pts switched to Dan (Pbo-Dan) and Dan arm pts continued Dan (Dan-Dan) for another 12 wks (TP2), followed by a 1-y LTE in which all pts received Dan add-on therapy. The initial Dan dose of 150 mg 3 times daily (TID) could be escalated to 200 mg TID based on clinical response at investigator discretion. Primary endpoint was change from baseline (CFB) at wk 12 in Hgb. Other secondary endpoints: proportions of pts with Hgb increase ≥2 g/dL in absence of transfusion and with transfusion avoidance through wk 24; CFB in Hgb at wk 24; and CFB in ARC, lactate dehydrogenase (LDH), and C3 fragment deposition on PNH red blood cells at wks 12 and 24. Safety assessments included treatment-emergent adverse events (TEAEs) and laboratory abnormalities throughout the study. Results As of 20 September 2022, 86 pts were randomized; 60 completed TP2 (Dan n=40; Pbo n=20). Baseline characteristics were similar between arms ( Table). At wk 24 ( Fig), mean Hgb level was maintained in the Dan-Dan arm and increased from wk 12 in Pbo-Dan arm. ARC ( Fig) and other secondary endpoints were maintained in the Dan-Dan arm and improved in Pbo-Dan arm ( Table) at wk 24. The proportion of pts with Hgb increase of ≥2 g/dL in the absence of transfusion was maintained in the Dan-Dan arm and improved in the Pbo-Dan arm from wk 12 (Dan, 59.5%; Pbo, 0%) to wk 24 (Dan-Dan 46.3%; Pbo-Dan, 35.0%). Transfusion avoidance was maintained in the Dan-Dan arm and increased in Dan-Pbo arm from wk 12 (Dan, 83.3%; Pbo, 38.1%) to wk 24 (Dan-Dan 78.0%; Pbo-Dan, 90.0%). Mean LDH levels were maintained from wk 12 to 24 ( Table) and were near normal (<1.5×ULN) in both arms. Transfusions decreased in the Pbo-Dan arm from wk 12 (Pbo; mean [SD], 2.2 [2.3]) through 24 wks (Pbo-Dan; mean [SD], 0.1 [0.5]). The safety analysis included the n=80 pts exposed to Dan during the trial. At wk 24, study drug compliance was 98.8% (10.35) in the Dan-Dan arm and 98.3% (3.95) in Pbo-Dan arm. Escalation to 200 mg TID occurred for 41/57 (71.9%) pts in the Dan-Dan arm and 14/23 (60.9%) in Pbo-Dan arm. There were no deaths, meningococcal infections, or discontinuations due to hemolysis. Through data cut-off, 90% (72/80; 464 events) of pts had ≥1 TEAE after exposure to Dan. Serious AEs related to Dan were reported by 2 pts (gastrointestinal disorders/increased blood bilirubin; headache). 6 events in 4 pts led to withdrawal of study drug. 4 events were reported as breakthrough hemolysis (BTH) based on investigator discretion. Only 1 AE was associated with LDH >2×ULN (actual value 2.2×ULN) and potentially met the BTH definition used in other clinical studies. This AE was related to a complement-amplifying condition, COVID-19. This pt continued the study and BTH was resolved. Conclusions Danicopan as add-on to Rav or Ecu significantly improves Hgb and ARC levels and reduces the need for transfusion by addressing cs-EVH while maintaining control of IVH through 48 wks of treatment. Danicopan demonstrated a favorable benefit-risk profile with no deaths, meningococcal infections, or discontinuations due to hemolysis.

Dose-Exposure-Response Relationships of Biomarkers and Efficacy Measures with Iptacopan, a Complement Factor B Inhibitor, in Patients (pts) with Paroxysmal Nocturnal Hemoglobinuria (PNH) with or without Concomitant Anti-C5 Therapy

Background: Standard of care treatment for pts with PNH targets C5 in the complement pathway; however, residual intravascular hemolysis (IVH) and emerging C3-mediated extravascular hemolysis (EVH) can still occur. In two Phase II trials (X2201 [NCT03439839] and X2204 [NCT03896152]), the oral, selective, reversible complement factor B inhibitor iptacopan provided hematologic benefit in pts with PNH who had active hemolysis, both as add-on therapy in anti-C5-treated pts and monotherapy in anti-C5-naïve pts (Risitano et al. Lancet Haematol 2021; Jang et al. Blood Adv 2022). Using data from these two studies, this integrated analysis aimed to evaluate dose-exposure-response relationships of biomarkers and efficacy measures to support dose selection for iptacopan. Methods: X2201 and X2204 were open-label studies that enrolled pts with PNH who had active hemolysis, despite treatment with anti-C5 therapy, or in the absence of prior anti-C5 therapy, respectively. In X2201, pts with lactate dehydrogenase (LDH) ≥1.5 x upper limit of normal (ULN) received iptacopan 200 mg twice daily (bid; Cohort 1) and pts with LDH ≥1.25 x ULN and hemoglobin (Hb) <10.5 g/dL received iptacopan 50 mg bid (Cohort 2). Pts in Cohort 2 could have their dose increased to 200 mg bid if hemolysis persisted. Both cohorts received iptacopan as add-on therapy to eculizumab; eculizumab treatment could be adjusted or withdrawn after 26 weeks (wks) at the investigator's discretion. In X2204, pts with LDH ≥1.5 x ULN and Hb <10.5 g/dL who were naïve to anti-C5 treatment were randomized to receive iptacopan 25 mg bid for 4 wks followed by iptacopan 100 mg bid for 8 wks (Cohort 1), or iptacopan 50 mg bid for 4 wks followed by iptacopan 200 mg bid for 8 wks (Cohort 2). To quantify the exposure-response relationship, trough concentrations of iptacopan (Ctrough) were correlated with LDH, Hb, factor Bb fragments, Wieslab® alternative complement pathway activity assay data, C3 fragment deposition on red blood cells (RBCs) and PNH RBC clone size using sigmoid maximum effect (Emax) models. Analyses were performed after pts had received iptacopan at the same dose for 4 wks. Results: A total of 29 pts with PNH were enrolled (X2201, N=16; X2204, N=13). The pharmacokinetic parameters of iptacopan were in line with previous reports in these two studies (Risitano et al. Lancet Haematol 2021; Jang et al.Blood Adv 2022) and were similar between the two study populations. In the dose range 25-200 mg bid, increases in dose resulted in a less than proportional increase in exposure, probably because of on-target binding to the highly abundant plasma factor B. Age and sex did not significantly impact exposure; the impact of ethnicity was not assessed because it was confounded by the study populations (15/16 pts in X2201 were Caucasian; all pts in X2204 were Asian). When assessing LDH, Hb, factor Bb fragments and Wieslab® assay data, 95% of the maximum response was reached with Ctrough >700 ng/mL, >650 ng/mL, >500 ng/mL and >750 ng/mL (EC95), respectively. C3 deposition was only informative in X2201; in X2204, C3 deposition was negligible at baseline and throughout the study because the pts were anti-C5-naïve. Complete abrogation of C3 deposition was reached at all dose levels in X2201 (Ctrough >600 ng/mL). PNH RBC cell clone size increased in both studies, but no clear dose-dependent relationship was observed. Accounting for interpatient variability in exposure, iptacopan 200 mg bid was identified as the optimum regimen to achieve Ctrough >750 ng/mL in most pts. Conclusions: Results from this pooled dose-exposure-response analysis of the X2201 and X2204 studies indicate that iptacopan 200 mg bid is the optimum dose to achieve Ctrough >750 ng/mL and consequently to provide maximal disease control via alternative complement pathway inhibition. The dose-exposure-response relationship was similar between pts regardless of whether they had a partial response to C5 inhibition (X2201) or were anti-C5-naïve (X2204), supporting the use of iptacopan 200 mg bid in both populations. Based on clinical efficacy and safety data from these two studies (Risitano et al. Lancet Haematol 2021; Jang et al. Blood Adv 2022) and supported by data from this integrated dose-exposure-response analysis, two ongoing Phase III trials are investigating iptacopan monotherapy at a dose of 200 mg bid in anti-C5 treated (APPLY-PNH; NCT04558918) and anti-C5-naïve (APPOINT-PNH; NCT04820530) pts with PNH.

Elucidation of Autoinflammatory Mechanism in Pigt-PNH

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder caused by somatic mutations in PIGA gene (PIGA-PNH). PNH red blood cells are susceptible to complement-mediated intravascular hemolysis because of a loss of the glycosylphosphatidyl-inositol (GPI) anchored proteins including complement regulatory proteins. Recently, a PNH patient caused by the PIGT mutations was reported (PIGT-PNH) and had unusual symptoms similar to autoinflammatory syndrome. Whereas PIGA is essential for the first step in the biosynthesis of GPI anchors, PIGT is involved in transfer of GPI to proteins; thus its defect causes accumulation of free GPI, non-protein linked GPI, in affected cells. It is likely that the free GPI accumulated in affected cells together with activated complement contribute to inflammasome activation. Here, we aimed to investigate whether free GPI together with complement activation can induce inflammasome activation in human monocytic cells in PIGT-PNH.We generated THP-1 human monocytic cells in which PIGT or PIGA gene was deleted using the CRISPR/Cas9 system. Each knockout cell was rescued with a corresponding wild-type cDNA. Cells were differentiated into macrophages under RPMI-1640 medium containing PMA (100 ng/ml, 3h) in the presence of 10% fetal bovine serum. For induction of inflammasome activation, both of the priming signal and the stimulating signal are necessary. PMA-differentiated cells were primed for 5hr with Pam3CSK4 (200 ng/ml), which specifically activates the Toll-like receptor 2 (TLR 2), followed by ATP stimulation (5 mM, 1h), or cells were stimulated by acidified serum (21:1 of 0.4M HCl) alone, which activates alternative complement pathway. Heat-inactivated (56°C for 30 min) serum was used as a negative control. ELISA kits were used to measure the interleukin (IL) -1β levels in supernatants according to the manufacturers' instructions. Total RNA was extracted from cultured cells and quantitative Real-Time PCR (qRT-PCR) was performed for mRNA level of IL-1β and NLRP3.Results are expressed as means ± SD of three independent experiments. Priming of PMA-differentiated WT, PIGT deficient (PIGTKO), and PIGA deficient (PIGAKO) THP-1 cells with Pam3CSK4, followed by ATP stimulation induced similar levels of IL-1β secretion (167.9±61.5, 144.3±19.7, 200.3±48.8 pg/1.6x105 cells). In contrast, when cells were stimulated with acidified serum alone, PIGTKO cells significantly enhanced IL-1β secretion compared to WT and PIGAKO cells, in which PIGTKO cells showed about 50- and 2-fold increase, respectively (23.7±2.2, 1221.8±91.6, 568.2±101 pg/1.6x105 cells; P<0.01 PIGTKO compered to PIGAKO). This elevated levels of IL-1β were decreased comparably to WT cells by the treatments with heat-inactivated acidified serum or anti-C5 monoclonal antibody, indicating IL-1β secretion was enhanced due to complement activation in PIGTKO and PIGAKO cells. PIGT and PIGA rescued cells showed similar levels of IL-1β to WT cells. Interestingly, qPCR revealed that PMA treatment alone induced transcription of proIL-1β and NLRP3 and further incubation of PIGTKO cells with acidified serum showed similar elevation in transcription compered to WT and PIGAKO cells.Thus, our data demonstrated that complement activation works as both a priming signal and a stimulating signal for inflammasome activation in PIGAKO and PIGTKO THP-1 cells. The facts that the PIGT-PNH patients, but not PIGA-PNH patients, showed autoinflammatory symptoms and that PIGTKO THP-1 secreted higher level of L-1β than PIGAKO THP-1 cells upon stimulation with acidified serum, indicate that the free GPI accumulated in PIGTKO cells contribute to inflammasome activation together with complement activation. DisclosuresOsato:Alexion Pharmaceuticals: Honoraria, Research Funding. Nishimura:Alexion Pharma GK: Honoraria, Research Funding. Ueda:Alexion Pharma GK: Honoraria, Research Funding. Kinoshita:Alexion Pharmaceuticals: Honoraria. Kanakura:Kyowa Hakko Kirin: Research Funding; Fujimotoseiyaku: Research Funding; Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding; Toyama Chemical: Research Funding; Bristol Myers: Research Funding; Eisai: Research Funding; Astellas: Research Funding; Pfizer: Research Funding; Nippon Shinyaku: Research Funding; Shionogi: Research Funding; Chugai Pharmaceutical: Research Funding.

BCX9930, a Potent, Selective, Oral Factor D Inhibitor, Demonstrates Proof-of-Concept As Monotherapy in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

BCX9930, a Potent, Selective, Oral Factor D Inhibitor, Demonstrates Proof-of-Concept As Monotherapy in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Reduced red blood cell surface level of Factor H as a mechanism underlying paroxysmal nocturnal hemoglobinuria.

The absence of the cell-surface complement inhibitors CD55 and CD59 is considered the mechanism underlying the complement-mediated destruction of affected red blood cells (RBCs) in paroxysmal nocturnal hemoglobinuria (PNH) patients, but Factor H (FH), a fluid-phase complement inhibitor, has also been proposed to be involved. However, the status of FH on the PNH patient RBC surface is unclear and its precise role in PNH pathogenesis remains to be further defined. In this study, we identified significantly lower levels of surface-bound FH on the affected CD59- RBCs than on the unaffected CD59+ RBCs. Although this reduction in surface-bound FH on PNH RBCs was accompanied by decreased surface sialic acid levels, the enzymatic removal of sialic acids from these RBCs did not significantly affect the levels of surface-bound FH. We further observed higher surface levels of FH on the C3b/iC3b/high RBCs than on C3b/iC3blow RBCs within the affected PNH RBCs of patients treated with eculizumab. Finally, we determined that enhanced surface levels of FH on CD55/CD59-deficient RBCs from mice and PNH patients protected against complement-mediated hemolysis. Taken together, our results suggest that a reduced surface level of FH is another important mechanism underlying the pathogenesis of PNH.

A review of the treatment landscape in paroxysmal nocturnal haemoglobinuria: where are we now and where are we going?

Review of current and future treatments for patients with Paroxysmal Nocturnal Haemoglobinuria What is Paroxysmal Nocturnal Haemoglobinuria? Paroxysmal nocturnal haemoglobinuria (PNH) is a very rare disease. It arises from PNH stem cells in the bone marrow. In a normal bone marrow these are inactive; however, if there has been a problem in the bone marrow, the PNH stem cells can expand and make PNH red blood cells, white blood cells and platelets. The problem with these cells is that they lack the cell surface markers that usually protect them. Red blood cells are broken down in the circulation rather than the spleen, which gives rise to PNH symptoms such as abdominal pain, difficulty swallowing, erectile dysfunction and red or black urine (known as haemoglobinuria). The white blood cells and platelets are 'stickier' increasing the risk of blood clots. Previously life expectancy was reduced as there were limited treatment options available. What was the aim of this review? To provide an overview of current and future treatment options for PNH Which treatments are available? • Eculizumab is an treatment given through a vein (intravenous) every week for 5 weeks then every 2 weeks after this, and has been available for 13 years, improving life expectancy to near normal.• Ravulizumab is a newer intravenous treatment similar to eculizumab but is given every 8 weeks instead of every 2 weeks. In clinical studies it was comparable with eculizumab.• Future Treatments - There is new research looking at different methods of treatment delivery, including injections under the skin (subcutaneous) that patients can give themselves, treatments taken by mouth (oral) or a combination of an intravenous and oral treatment for those patients who are not optimally controlled on eculizumab or ravulizumab. What does this mean? PNH is now treatable. For years, the only drug available was eculizumab, but now different targets and drug trials are available. Ravulizumab is currently the only second licenced product available, in USA and Europe, there are other medications active in clinical trials. Why is this important? The benefit for patients, from treatment every 2 weeks to every 8 weeks is likely to be improved further with the development of these new treatments, providing patients with improved disease control and independence.As we move into an era of more patient-friendly treatment options, the PNH community both physicians and patients look forward to new developments as discussed in this article.

CD71 improves delineation of PNH type III, PNH type II, and normal immature RBCS in patients with paroxysmal nocturnal hemoglobinuria.

The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.

Eculizumab treatment: stochastic occurrence of C3 binding to individual PNH erythrocytes

BackgroundC5 blockade by eculizumab prevents complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH). However, C3-bound PNH red blood cells (RBCs), arising in almost all treated patients, may undergo extravascular hemolysis reducing clinical benefits. Despite the uniform deficiency of CD55 and of CD59, there are always two distinct populations of PNH RBCs, with (C3+) and without (C3−) C3 binding.MethodsTo investigate this paradox, the phenomenon has been modeled in vitro by incubating RBCs from eculizumab untreated PNH patients with compatible sera containing eculizumab, and by assessing the C3 binding after activation of complement alternative pathway.ResultsWhen RBCs from untreated patients were exposed in vitro to activated complement in the context of C5-blockade, there was the prompt appearance of a distinct C3+ PNH RBC population whose size increased with time and also with the rate of complement activation. Eventually, all PNH RBCs become C3+ to the same extent, without differences between old and young (reticulocytes) PNH RBCs.ConclusionsThis study indicates that the distinct (C3+ and C3−) PNH RBC populations are not intrinsically different; rather, they result from a stochastic all-or-nothing phenomenon linked to the time-dependent cumulative probability of each individual PNH red cell to be exposed to levels of complement activation able to trigger C3 binding. These findings may envision novel approaches to reduce C3 opsonization and the subsequent extravascular hemolysis in PNH patients on eculizumab.

Development of Paroxysmal Nocturnal Hemoglobinuria in Children with Aplastic Anemia

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic stem cells resulting from a somatic mutation in the PIGA gene. PNH frequently manifests in association with aplastic anemia (AA), in which PIGA mutations are believed to enable escape from the immune-mediated destruction by pathogenic T cells. Recent studies using next-generation sequencing have revealed that frequent somatic PIGA mutationsin AA patients are associated with a better response to IST and prognosis (Yoshizato et al N Engl J Med. 2015; 373: 35-47). However, clinical PNH is a progressive and life-threatening disease driven by chronic hemolysis that leads to thrombosis, renal impairment, poor quality of life, and death. Large studies in adults have reported that clinical PNH developed in 10%-25% of AA patients; however; the frequency of clinical PNH in children with AA has rarely been described. Here we aimed to elucidate the pathological link between PNH and AA in children.Methods: In total, 57 children (35 boys and 22 girls) diagnosed with acquired AA at our hospital between 1992 and 2010 were retrospectively studied. Patients who underwent hematopoietic stem cell transplantation as first-line treatment within 1 year after AA diagnosis and those with clinical PNH at AA diagnosis were excluded. Flow cytometry (FCM) was used to detect PNH CD13+/CD55−/CD59− granulocytes and PNH glycophorin A+/CD55−/CD59− red blood cells (RBCs). Clinical PNH was defined as the presence of intravascular hemolysis and ≥5% PNH granulocytes or PNH RBCs. Minor PNH clones were defined as those with >0.005% PNH granulocytes or >0.010% PNH RBCs. We performed targeted sequencing of bone marrow samples from patients with clinical PNH that were obtained at 2 time points: at AA diagnosis and after PNH development. The panel of 184 genes for targeted sequencing included most of the genes known to be mutated in inherited bone marrow failure syndromes and myeloid cancers, as well as PIGA.Results: The median patient age at AA diagnosis was 9.3 (1.2-17.8) years, and the median follow-up period was 123 (2-228) months. A total of 43 patients were screened for PNH clones by FCM after AA diagnosis, and 21 of these with minor PNH clones were identified. The median percentages of PNH granulocytes and PNH RBCs were 0.001% (0.000%-4.785%) and 0.000% (0.000%-3.829%), respectively. During follow-up, 5 patients developed clinical PNH after adolescence (15-22 years of age). The median time between AA diagnosis and PNH development was 4.9 (3.3-7.9) years. All clinical PNH patients were treated with IST for AA, and complete and partial response after 6 months were achieved in 1 and 4 patients, respectively. Gross hemoglobinuria was present in all clinical PNH patients, but thrombosis was not observed. The size of PNH clones varied greatly among patients: PNH granulocytes and PNH RBCs were 42.96% (10.04%-59.50%) and 48.87% (15.02%-90.80%), respectively. Oral cyclosporine A and intravenous eculizumab were administered to 3 and 1 patients, respectively; all patients showed sustained response as indicated by improvement in gross hemoglobinuria and normal blood counts after treatment. The remaining 1 patient underwent bone marrow transplantation from the HLA-identical mother and was alive without any complications. Overall, the 10-year probability of developing clinical PNH was 10.2% (95%CI, 3.6-20.7). Among 43 patients screened for PNH clones at AA diagnosis, the 10-year cumulative clinical PNH incidence was significantly higher in patients with minor PNH clones than in those without minor PNH clones at AA diagnosis [29% (95% CI, 10%-51%) vs. 0% (95% CI, 0%-0%); p = 0.015]. Among all clinical PNH patients, a total of 8 somatic PIGA mutations were detected (missense, 2; splice site, 2; and frameshift, 4). However, PIGA mutations were not detected at AA diagnosis even in patients who subsequently developed clinical PNH.Conclusion: In our cohort, the percentage of patients who eventually developed clinical PNH was comparable to that reported in adults in a previous study. Furthermore, the current study showed that the presence of minor PNH clones at AA diagnosis was a risk factor for the subsequent development of clinical PNH, although the clones were not detected by targeted sequencing. Thus, pediatric AA patients with PNH clones at AA diagnosis should undergo long-term periodic monitoring for potential clinical PNH development. DisclosuresKojima:SANOFI: Honoraria, Research Funding.

CD59 Deficiency Is Critical for C3 Binding on Red Blood Cells of Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) during Anti-C5 Treatment (eculizumab)

C5-blockade with eculizumab prevents complement-mediated intravascular hemolysis in PNH patients and its clinical consequences. However, a distinct population of PNH red blood cells bound with C3 fragments appears in almost all treated patients. This C3 binding results in extravascular hemolysis that in some patients reduces the clinical benefit from eculizumab. In each PNH patients on eculizumab there are always two distinct populations of PNH red blood cells, one with (C3+) and one without (C3-) C3 binding. This phenomenon is somehow paradoxical since the glycosylphosphatidylinositol (GPI)-linked complement regulators, CD55 and CD59, are uniformly deficient on the surface of PNH red cells.To investigate this phenomenon, we have modeled in vitro the C3 binding in the context of C5 blockade by incubating red blood cells from PNH patients with AB0-matched sera from patients on eculizumab. Complement alternative pathway has been activated by mild acidification (in presence of Mg/EGTA to prevent the activation of complement classical pathway) and C3 binding has been assessed by flow cytometry at serial time points. In these experimental conditions a fraction of PNH red blood cells, similar to what happens in vivo, become promptly C3+ and its size increases with the time: from 9.4±2.7% after 5 minutes to 21.2±9.5% after 24 hours.The membrane defects of PNH cells suggested that the deficiency of CD55, which regulates the formation and accelerates the dissociation of C3 convertases, should be responsible for C3 binding to PNH red blood cells in presence of eculizumab (Parker CJ. Hematology Am Soc Hematol Educ Program. 2011;2011:21-29).In order to verify experimentally this hypothesis we have inactivated CD55 or CD59 on normal red blood cells by using blocking monoclonal antibodies (moAb - listed in the figure legend), and we have tested them in vitro upon activation of complement alternative pathway by mild acidification in presence or absence of C5 blockade.We found that CD55 inactivation on normal red blood cells results neither in hemolysis (without C5 blockade) nor in any C3 binding (with C5 blockade). As expected without C5 blockade CD59-inactivated normal red blood cells undergo hemolysis but, surprisingly, we found that in presence of C5 blockade they become bound with C3 fragments (Figure 1), just as it occurs in vivo in PNH patients on eculizumab. The simultaneous inactivation of both CD55 and CD59 further increased the level of C3 binding. Thus, at variance with the starting hypothesis, the deficiency of CD59, not that of CD55, plays the major role in C3 binding to PNH red cells of patients on eculizumab.Therapeutic C5 blockade in PNH patients has unmasked a novel function of CD59: in addition to prevent MAC formation, it plays a central role also in the regulation of C3 activation on cell surface through molecular mechanisms not elucidated yet. It remains to be established the physiological role, if any, of this novel function of CD59 and whether it play a role in determining the pleomorphic clinical features of the congenital CD59 deficiency. Finally, these findings may lead to investigate innovative approaches to reduce C3 binding and extravascular hemolysis in PNH patients on eculizumab and, in a broader context, to modulate complement activity. [Display omitted] DisclosuresRisitano:Novartis: Research Funding; Alexion Pharmaceuticals: Other: lecture fees, Research Funding; Rapharma: Research Funding; Alnylam: Research Funding.

Persisting Hyperbilirubinemia in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Chronically Treated with Eculizumab: The Role of Hepatocanalicular Transport Variants

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is caused by an acquired mutation in the phosphatidylinositol (GPI)-anchors. The lack of GPI-anchored proteins on the surface of affected red blood cells (RBCs) results in complement mediated chronic intravascular hemolysis. The monoclonal anti-C5 antibody eculizumab protects PNH RBCs by blocking the terminal complement cascade. Eculizumab treated PNH patients show a dramatic decrease of LDH and bilirubin, however few patients remain hyperbilirubinemic, eventually indicating an inadequate response. Hepatocanalicular transporters are rate-limiting in the process of bile formation, and mutations in the genes encoding these transporters cause intrahepatic cholestasis. PATIENTS and METHODS: Mutation analysis regarding hepatobiliary transporter and nuclear receptor defects (ABCB4, ABCB11, ATP8B1, NR1H4) were performed in eight [75% (6/8) male; mean age, 40 years (range 25 - 70 years)] out of the currently 141 patients with PNH/-clone at our department due to a persistent (isolated) increase in total serum bilirubin (median 4.5 mg/dL; range 3.0 - 8.5) during chronic treatment with eculizumab (LDH: median 274 U/L; range 172 - 836) and normal or slightly increased aminotransferases. Clinical symptoms resembled those of hyperbilirubinemia including jaundice. Median clone size of GPI-deficient granulocytes (FLAER) was 98.1% (range 79.0 - 99.5%), with a median observation time of 56.4 months (range 10.6 - 82.7 months). All patients were treated according to the current German PNH guidelines. RESULTS: Both homozygous and heterozygous procholestatic variants in the ABCB4, ABCB11 and ATP8B1 genes, encoding canalicular transporters, were identified in the studied patients, and all patients carried the common ABCB4 c.787 A&gt;T polymorphism (Table 1). None of the patients were tested positive for the common mutation c.-1g&gt;t of the central bile acid sensor NR1H4. Table 1. Hepatobiliary transporter gene variants in PNH patients (n=8) Variant Pat. 1 Pat. 2 Pat. 3 Pat. 4 Pat. 5 Pat. 6 Pat. 7 Pat. 8 ABCB4 c.504T&gt;C (rs1202283) HOM HET HET HOM neg. neg. HOM neg. ABCB4 c.787A&gt;T (rs2109505) HOM HOM HOM HOM HOM HOM HOM HET ABCB11 p.A444V (rs2287622) HOM HET neg. HET neg. neg. HET HOM ABCB11 c.3084A&gt;G (rs497692) HET HET HET neg. HOM HET HOM HET ATP8B1 p.R952Q (rs12968116) HOM neg. HET HET neg. HET neg. neg. NR1H4 (FXR) c.-1 g&gt;t (rs56163822) neg. neg. neg. neg. neg. neg. neg. neg. Pat.: Patient; HOM: homozygous; HET: heterozygous; neg.: negative Routine sonographic follow-up examinations were performed in 75% of the patients (6/8) ruling out post-hepatic cholestasis. In three patients (pat. 2, 3 and 4), a cholecystectomy due to symptomatic cholelithiasis had been performed prior to diagnosis of PNH, whereas in one patient the diagnosis of a persistent, asymptomatic cholelithiasis (pat. 6) was made. A chronic (active) or recently acquired viral hepatitis was ruled out in all patients. Lab parameters were consistent with an isolated persistent increase in total serum bilirubin, despite normal (7/8) or slightly elevated transaminases (1/8; pat. 6) throughout observation time. SUMMARY/CONCLUSIONS: This is the first report of mutations in hepatobiliary transporter genes predisposing to intrahepatic cholestasis in PNH patients. Therefore, persisting hyperbilirubinemia in PNH patients chronically treated with eculizumab might not be due to an insufficient response to eculizumab but due to bile acid transporter variants and ongoing extravascular hemolysis. Patients should be tested for and possible therapeutic agents established in the management include ursodeoxycholic acid, promoting bile flow, and rifampicin, accelerating hepatic detoxification and excretion of bilirubin and bile acids. Our data warrant further studies concerning the role of hepatobiliary transporter variants in PNH patients in order to determine their clinical significance. Disclosures Dührsen: Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Alexion Pharmaceuticals: Honoraria, Research Funding. Röth:Geron: Research Funding; Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

High-Sensitivity Detection of PNH Red Blood Cells, Red Cell Precursors, and White Blood Cells.

Flow cytometry is the method of choice to 'diagnose' paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used. Careful selection and validation of antibody conjugates has allowed the development of reagent cocktails suitable for detection of PNH RBCs, CD71+ reticulocytes, and WBCs in clinical/sub-clinical PNH samples. A CD235a-FITC/CD59-PE assay was developed capable of detecting Type III PNH RBCs at 0.01% sensitivity. A protocol targeting immature CD71+ RBCs can detect PNH reticulocytes at similar sensitivity. Four-color FLAER-based neutrophil and monocyte assays were developed to detect PNH phenotypes at a level of 0.01% and 0.04% sensitivity, respectively. For instrumentation with five or more PMTs, a single-tube 5-color FLAER/CD157-based assay to simultaneously detect PNH neutrophils and monocytes is described. Using these standardized approaches, results have demonstrated good intra- and inter-laboratory performance characteristics even in laboratories with little prior experience performing PNH testing.

Clinical and Immunological Characterisation of Coversin, a Novel Small Protein Inhibitor of Complement C5 with Potential As a Therapeutic Agent in PNH and Other Complement Mediated Disorders

▪Coversin is a clinical stage recombinant protein molecule (16.7kDa) derived from a salivary molecule from the Ornithodros moubata tick where it assists the parasite to feed without provoking a host immunological response. It prevents activation of complement C5 and binds leukotriene B4 (LTB4) with high affinity. It is not an antibody and can therefore be manufactured in prokaryotic cells such as E. coli leading to potential savings in manufacturing costs. As its mode of action on C5 is similar to that of the monoclonal antibody eculizumab (Soliris®), it is considered to be a promising alternative therapeutic approach to thrombotic and hemolytic diseases mediated by complement activation including atypical hemolytic uremic syndrome (aHUS), catastrophic anti-phospholipid syndrome (APS) and paroxysmal nocturnal hemoglobinuria (PNH).In order to test this hypothesis, experiments were undertaken to determine whether Coversin could inhibit the hemolysis of PNH red cells in vitro using a modified Ham test combined with visualisation of the results by flow cytometric measurement of CD59 expression. Initial experiments showed maximum inhibition of hemolysis compared to control occurred at a concentration of approximately 10mcg/mL. Further experiments using PNH red blood cells also demonstrated the effectiveness of Coversin in blocking in vitro hemolysis of PNH red cells that had both type III (complete GPI-deficiency) and type II (partial GPI deficiency) red cells and also the deposition and accumulation of C3d on both types of PNH red cells. In a comparative experiment Coversin 10mcg/mL was found to be as effective as eculizumab 50mcg/mL, a molar equivalent dose.As Coversin is a non-humanised xenologous protein a mouse study was performed in order to assess immunogenicity and its potential to induce the formation of neutralising antibodies. Daily repeat subcutaneous doses of Coversin (0.57mg/kg) or vehicle were administered to 5 groups of 6 BALB/c mice and blood samples taken after 7, 14, and 28 days administration with an additional group where samples were taken 14 days following cessation of 28 days dosing. Immunoblots showed that by Day 14 33% of mice had developed low titre IgG antibodies to Coversin. By 28 days this had risen to 75% of mice receiving the active compound. Using CH50 lytic assays the antibodies were shown to be non-neutralising and no animals exhibited clinical signs of allergy or injection site reactions. The group of 6 mice that received vehicle showed no evidence of antibodies at 28 days.A heterozygous C5 mutation leading to a p.Arg885His polymorphism and interfering with the binding of eculizumab was recently reported in Japanese PNH patients and this was associated with a poor response to therapy. This was subsequently found to affect 3.5% of the general Japanese and Han Chinese population. A similar polymorphism has now been shown in a 4 year old Caucasian child with persistent thrombocytopenia following a stem cell transplant for chronic granulomatous disease. Prolonged eculizumab treatment was ineffective leading to discovery of the mutation. Serum from the patient was spiked with ascending concentrations of Coversin and CH50 activity was measured by ELISA. This produced a dose response curve corresponding to that found in serum from normal subjects. Coversin, which binds to a slightly different site on the C5 molecule, seems therefore to be unaffected by the polymorphism and to achieve superior inhibition of C5 activation compared to eculizumab.A Phase I, single ascending dose clinical trial was performed in 24 healthy volunteers. In the top dose cohort total blockade of complement C5 was achieved following subcutaneous injection at the predicted therapeutic loading dose of 0.57mg/kg. Activity remained below 50% for 48 hours suggesting that dosing once a day is feasible at steady state (see figure). The drug was well tolerated and no drug-related side effects were seen. Phase II clinical trials in PNH and other thrombotic micro-angiopathies (TMAs) are planned.Coversin appears to be a promising alternative to eculizumab for patients with PNH and other TMAs including those with polymorphisms which interfere with optimal binding of eculizumab to complement C5. [Display omitted] DisclosuresWeston-Davies:Volution Immuno Pharmaceuticals (VIP) SA: Consultancy. Nunn:Volution Immuno Pharm: Consultancy. Prudo:Volution Immuno Pharmaceuticals (VIP) SA: Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Rapidly evolving skin manifestations due to progressive thrombosis in a patient with paroxysmal nocturnal haemoglobinuria resolved with prompt initiation of eculizumab

Funding sources: none. Conflicts of interests: P.M. has sat on advisory boards for Alexion. Dear Editor, Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal disorder of the haematopoietic stem cell. PNH arises from mutations in the PIGA gene, which encodes glycosylphosphatidylinositol (GPI)‐anchored proteins. Consequently, these mutations result in a deficiency of GPI‐anchored proteins in cell membranes.1 This deficiency causes PNH red blood cells to be susceptible to complement‐induced intravascular haemolysis.2 However, PNH is also characterized by a high rate of thrombosis.3 4 Venous and arterial thrombosis can be the first symptom of PNH. PNH‐induced thrombosis often occurs at unusual sites and may progress in days to weeks.5 Moreover, thromboembolic complications and consequences are the leading cause of morbidity and death in PNH.4 The pathogenesis of thrombosis in PNH is complex and not completely elucidated at this time.3 The clinical picture of PNH is highly variable and depends on the PNH clone size, factors of complement activation and whether or not there is an underlying bone marrow disease. Complications of PNH occur spontaneously or can be triggered by bacterial or viral infections, for example by highly contagious parvovirus B19.6 7 Cutaneous manifestations of PNH comprise rapidly evolving purpura, haemorrhagic bullae and ulcerations presumably due to necrosis, secondary to intravascular clotting.8 9 10

Perisurgical induction of eculizumab in a patient with paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by complement (C')-induced lysis of PNH red blood cells (RBCs), which are deficient in the expression of CD55 and CD59. Surgery is one of the major clinical situations that trigger hemolytic attack and thrombosis in PNH. We describe here a case of 64-year-old man with classic PNH complicated by early-stage gastric cancer requiring distal gastrectomy under general anesthesia. We administered humanized monoclonal anti-C5 antibody (eculizumab; Soliris) for a limited period (600 mg, once a week × four times) perisurgically. Eculizumab effectively inhibited the C' system and the patient underwent a curative distal gastrectomy without significant surgery-triggered hemolytic attack. Although discontinuation of eculizumab induced mild hemolysis 2 weeks after the last administration, it was treated conservatively without thrombotic complication. Limited-term induction of eculizumab could be an option for PNH patients with transient and anticipated high risks, with careful preparation for the discontinuation-related risks afterwards.

Intra‐ and interlaboratory variability of paroxysmal nocturnal hemoglobinuria testing by flow cytometry following the 2012 Practical Guidelines for high‐sensitivity paroxysmal nocturnal hemoglobinuria testing

Sutherland et al. recently published the Practical Guidelines for high-sensitivity detection of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM), containing concise protocols for PNH testing. Using this approach, we studied the intra- and interlaboratory variability observed in a multicenter study in which fresh blood samples containing three clinically relevant PNH clone sizes within the granulocytic, monocytic, and red blood cell (RBC) populations were shipped to each participating center. Coefficients of variation (CVs) for precision/reproducibility analysis ranged from 0.01%/0.02% to 0.48%/0.45% (big clone), from 0.69%/1.52% to 4.24%/5.80% (small-intermediate clone), from 1.47%/3.91% to 15.01% /17.83% (minor clone) for PNH white blood cells (WBCs) and from 0.24%/0.48% to 1.76%/1.83% (big clone), from 0.80%/1.14% to 2.39%/4.45% (small-intermediate clone), from 1.09%/3.36% to 10.54%/10.23% (minor clone) for PNH RBCs, respectively. Linear regression analysis showed excellent performance correlation between centers (r > 0.99), Wilcoxon rank test revealed no statistically significant differences for PNH granulocytes, monocytes, and RBCs (P > 0.05%), Bland-Altman analysis demonstrated good performance agreement for all target PNH clones (mean bias ranging from -1.47 to 0.71). Our results demonstrate very good intra- and interlaboratory performance characteristics for both precision and reproducibility analyses and excellent correlation and agreement between centers for all target PNH clone sizes. Our data confirm the reliability and robustness of the recently published Practical Guidelines approach for high sensitivity PNH testing by flow cytometry and suggest that such an approach represents an excellent basis for standardization of PNH testing by flow cytometry.

CH50 Activity Correlates with Residual Intravascular Hemolysis in Pts with Paroxysmal Nocturnal Hemoglobinuria Treated by Eculizumab

AbstractAbstract 3193 Background:Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by intravascular hemolysis, which is effectively controlled with eculizumab. However, in some cases, unexplained upper limit lactate dehydrogenase levels (LDH), as well as low haptoglobin levels are not unusual under treatment, suggesting residual low-level hemolysis. C3-mediated clearance of PNH red blood cells has been recently suggested in patients (pts) under eculizumab (Risitano Blood 2010). We hypothesized residual hemolysis may also be due to incomplete C5 blockage under treatment. CH50 activitiy (residual functional C5 activity) as well as C3 deposition on PNH red blood cells were assessed among eculizumab-treated pts. We also examined the hypothesis that mutations in complement genes are implicated in residual hemolytic process. Patients and Methods:From October 2010 to February 2012, 22 pts (7 male, median age 42 years range 21 to 72) with hemolytic PNH treated with eculizumab (900 mg intravenously every 14±2 days) were prospectively followed systematically every 2 weeks. Before each Eculizumab infusion, clinical data (abdominal pain, thrombosis events and transfusion requirements) as well as complete blood count, LDH, bilirubin levels and reticulocytes were systematically collected. Concomitantly, CH50 using sensibilized sheep red blood cells, C3d deposition on red blood cells using flow cytometry as well as C3 and C4 circulating levels were also studied. Complete C5 blockage was defined by a CH50 activity ≤10% (as for pts with hereditary complete C5 deficiency). All pts included in the study have at least 6 months follow-up under treatment. Only measurements performed after at least 6 months of treatment were considered. Residual intravascular hemolysis was defined by upper limit LDH level before eculizumab injection. CH50 was analyzed with a longitudinal tobit regression model accounting for repeated sampling and the limit of detection. The model was fit in a Bayesian framework, so no p-values are presented. Results:Before starting eculizumab, 21 pts were transfusion-dependent with a mean of 7 red blood cell (RBC) transfusions per year and 9 pts had a significant PNH-related complication (9 thrombosis and 7 with a previous history of aplastic anemia). During the study period, 6 pts received at least one transfusion (mean of 3 RBC transfusions per year) and 1 patient presented a deep vein thrombosis. All pts were analyzed for CH50 activity (412 samples; median 19 per patient; range, 4 to 31). Overall, CH50 measurements showed substantial variability for most pts (Figure 1). Residual CH50 activity (>10%) was significantly associated with higher LDH levels whereas pts who were still transfused (as well as pts with lower hemoglobin level and higher reticulocytes counts) tended to have higher CH50. Type III PNH red blood cell C3d deposition (assessed in all but 1 patient; 277 samples; median 14 per patient; range, 2 to 21) was found in all pts evaluated during the study period (Fig. 1). However, we did not find any correlation between C3 opsonization and clinical or biological signs of hemolysis. The association between CH50 and higher C3 deposition was weak (on average −0.8% CH50 per 10%) more C3 deposition (95%CI −1.7 to 0.2) (Fig. 1). Those results were confirmed in the subset of 15 patients with pure hemolytic PNH (no history of aplastic anemia). C3 and C4 circulating levels were in the normal range during the study period in all but one patient (pts#15, Fig. 1), who carry a complement factor H (CFH) mutation, leading to a quantitative Factor H deficiency. Systematic screening was positive for 2 other pts (CFH, pts#1; C3 mutation, pts#14) but with no phenotypical consequence. Conclusion:Our results confirm the excellent overall clinical and biological response of hemolytic PNH pts to eculizumab. We found that unblocked CH50 activity, reflecting the residual C5 activity, was significantly associated with residual intravascular hemolysis while the selective C3d deposition of PNH red cells (found in all pts under treatment) was not. We also identified mutations in C3 and factor H genes. Whether incomplete C5 blockade is due to low residual level of eculizumab, complement mutations or polymorphisms, or other mechanisms are under investigation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.