Hemolysis In Paroxysmal Nocturnal Hemoglobinuria Patients Research Articles

The Effect of Respiratory Viral Infections on Breakthrough Hemolysis in Patients with Paroxysmal Nocturnal Hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by hemolysis and thrombosis and is associated with significant morbidity and mortality. Although complement inhibitors have significantly changed the outcomes in PNH patients, breakthrough hemolysis (BTH) may still occur as a response to stress factors such as pregnancy, surgery, and infections. Despite the well-described association between bacterial infections and hemolysis in PNH patients, little is known about the effect of respiratory viruses on triggering hemolytic episodes. This is the first study, to our knowledge, addressing this question. We retrospectively analyzed 34 patients with PNH disease between 2016 and 2018, who were on eculizumab treatment and who presented with respiratory symptoms and were subsequently tested for 10 respiratory viruses (influenza A, influenza B, parainfluenza, respiratory syncytial virus, adenovirus, rhinovirus, and human metapneumovirus). NTS+ patients had higher inflammatory markers, with the majority requiring antibiotics. Acute hemolysis, along with a significant drop in hemoglobin, was noted in the NTS+ group, with three of them requiring a top-up transfusion and two requiring an extra dose of eculizumab. Furthermore, the time from the last eculizumab dose was longer in the NTS+ patients who had BTH, than those who did not. Our data indicate that respiratory virus infections pose a significant risk for BTH in PNH patients on complement inhibitor treatment, underlining the need for regular screening and close monitoring of patients with respiratory symptoms. Furthermore, it implies a higher risk for patients who are not established on complement inhibitors, suggesting the necessity for greater vigilance in these patients.

The complement alternative pathway in paroxysmal nocturnal hemoglobinuria: From a pathogenic mechanism to a therapeutic target.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal, not malignant, hematological disease characterized by intravascular hemolysis, thrombophilia and bone marrow failure. While this latter presentation is due to a T-cell mediated auto-immune disorder resembling acquired aplastic anemia, the first two clinical presentations are largely driven by the complement pathway. Indeed, PNH is characterized by a broad impairment of complement regulation on affected cells, which is due to the lack of the complement regulators CD55 and CD59. The deficiency of these two proteins from PNH blood cells is due to the somatic mutation in the phosphatidylinositol N-acetylglucosaminyltransferase subunit A gene causing the disease, which impairs the surface expression of all proteins linked via the glycosylphosphatidylinositol anchor. The lack of the complement regulators CD55 and CD59 on PNH erythrocytes accounts for the hallmark of PNH, which is the chronic, complement-mediated intravascular hemolysis. This hemolysis results from the impaired regulation of the alternative pathway upstream in the complement cascade, as well as of the downstream terminal pathway. PNH represented the first indication for the development of anti-complement agents, and the therapeutic interception of the complement cascade at the level of C5 led to remarkable changes in the natural history of the disease. Nevertheless, the clinical use of an inhibitor of the terminal pathway highlighted the broader derangement of complement regulation in PNH, shedding light on the pivotal role of the complement alternative pathway. Here we review the current understanding of the role of the alternative pathway in PNH, including the emergence of C3-mediated extravascular hemolysis in PNH patients on anti-C5 therapies. These observations provide the rationale for the development of novel complement inhibitors for the treatment of PNH. Recent preclinical and clinical data on proximal complement inhibitors intercepting the alternative pathway with the aim of improving the treatment of PNH are discussed, together with their clinical implications which are animating a lively debate in the scientific community.

Different Levels of Incomplete Terminal Pathway Inhibition by Eculizumab and the Clinical Response of PNH Patients.

Background: Eculizumab blocks the lytic complement pathway by inhibiting C5 and has become the standard of care for certain complement-mediated diseases. Previously, we have shown that strong complement activation in vitro overrides the C5 inhibition by Eculizumab, which accounts for residual terminal pathway activity.Results: Here we show that the levels of residual hemolysis in ex vivo assays differ markedly (up to 3.4-fold) across sera collected from different paroxysmal nocturnal hemoglobinuria (PNH) patients on Eculizumab treatment. This large variability of residual activity was also found in sera of healthy donors, thus cross-validating the findings in patients. While PNH patients with residual lytic activities of 11–30% exhibited hemolysis levels around the upper limit of normal (i.e., plasma LDH of ~250 u/L), as expected for PNH patients on Eculizumab therapy, we found sustained and markedly increased LDH levels of around 400 u/L for the patient with the highest residual activity of 37%. Furthermore, the clinical history of nine out of 14 PNH patients showed intravascular breakthrough hemolysis at the time of documented infections despite ample amounts of administered Eculizumab and/or experimentally determined excess over C5.Conclusion: The occurrence of extraordinary high levels of residual terminal pathway activity in PNH patients receiving Eculizumab is rare, but can impair the suppression of hemolysis. The commonly observed low levels of residual terminal pathway activity seen for most PNH patients can exacerbate during severe infections and, thus, can cause pharmacodynamic breakthrough hemolysis in PNH patients treated with Eculizumab.

Current Status and Optimal Management of Eculizumab Poor-Responders Due to C5 Polymorphisms

(Introduction) Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare but life-threatening stem cell disease caused by expansion of PIGA mutated clone(s). PIGA mutation abolishes the expression of GPI-anchored proteins on the cell surface including CD55 and CD59 which protect red blood cells from complement attack, resulting in complement-mediated destruction of PNH erythrocytes. Eculizumab (Ecu) effectively ameliorates the intravascular hemolysis in PNH patients by blocking complement C5 in the terminal pathway. We previously reported that 3-4 % of the Japanese patients did not respond to Ecu due to C5 polymorphism c.2654G>A (predicting p.Arg885His) and one Argentina patient with similar but different polymorphism c.2653C>T (predicting p.Arg885Cys) (N Engl J Med 370:632, 2014). Since then, we have received various consults regarding poor response cases and requests for analysis from around the world. Here, we summarize the latest series of analyses in C5 polymorphisms and propose optimal management based on these findings. (Method) Once poor response to Ecu, defined as sustained high serum LDH, was suspected with sustained high serum LDH level, peripheral blood samples with clinical data were sent to our institute after obtaining patients' informed consent. DNA was extracted from the samples, and the hot spot of C5 polymorphisms at exon 21 was sequenced by Sanger method. If no polymorphism was identified, all 41 exons of C5 were sequenced. (Results) At the initial publication (2014), 11 cases of c.2654G>A were identified out of 345 PNH patients (3.2%). As of July, 2018, a total of 22 patients were identified among roughly 600 patients treated with Ecu in Japan (3.7%). To determine the distribution of the polymorphism, a DNA panel containing 120 Han Chinese persons were previously screened, and one had the polymorphism. The same C5 polymorphism was newly identified in one among 89 patients treated with Ecu in Korea. A similar polymorphism c.2653C>T (predicting p.Arg885Cys) was also previously identified in an Argentina patient. Another polymorphism c.2653C>A (predicting p.Arg885Ser) was identified in a Dutch patient. Furthermore, a novel mutation c.2422 G>A (predicting p.Val 808 Ile) was found in a poor responder in Israel, and currently under functional analyses. (Discussion) In PNH patients treated with Ecu, serum LDH level usually drops importantly after the first loading dose, and mostly comes to upper limit of normal range after second or third loading dose. If the serum LDH level remains high, poor response should be considered. CH50 is usually not detected in Ecu responsive patients, so monitoring of CH50 level is critical to evaluate the responsiveness to Ecu. Once poor response is suspected, hot spot of exon 21 should initially be sequenced, and then the whole 41 exons of C5 may need to be sequenced. c.2654G>A was found among Han Chinese in our previous study, so it is reasonable that a Korean case had the same polymorphism considering the geography. Surprisingly, a Caucasian case with the same polymorphism has been reported (Blood Advances 1:1254, 2017) in addition to the Dutch case, underlining the importance of poor responsiveness due to polymorphisms (p.Arg885) even outside of Asia. We previously reported that Coversin, a C5 inhibitor derived from tick saliva protein, blocked hemolysis in vitro using the serum from the patients with c.2654G>A, and it was reported that post-transplant thrombotic microangiopathy was successfully treated with Coversin for the patient with the polymorphism (Blood Advances 1:1254, 2017). C5 inhibitors targeting a different epitope or having different mechanisms from Ecu as well as other upcoming complement inhibitors targeting Factor D or C3 are expected to benefit patients with C5 polymorphism and resistance to Ecu. Further analyses and clinical trials may pave the way to a second generation anti-complement drug to treat PNH patients. [Display omitted] DisclosuresUeda:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Muus:Akari Therapeutics: Consultancy. Lee:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Nishimura:Chugai Pharmaceuticals: Consultancy, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Kanakura:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding.

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.

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.

Successful management of orthopedic operations requiring general anesthesia in a PNH patient after introduction of eculizumab

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal hematopoietic stem cell disorder that presents with hemolytic anemia, thrombosis, and bone marrow failure. Stressors such as infection and pregnancy have been known to exacerbate hemolysis in PNH patients. Surgery can also trigger prominent complement activation and is an important risk factor for hemolysis. Furthermore, the postoperative thrombosis risk is high. Eculizumab, which is a humanized monoclonal antibody against C5, suppresses hemolysis and prevents thrombosis, and thus improves quality of life for PNH patients. However, few reports have focused on eculizumab-treated PNH patients undergoing surgery. We report a 79-year-old PNH patient receiving eculizumab treatment who underwent three consecutive orthopedic surgeries requiring general anesthesia. Perioperative management was carried out routinely, as in non-PNH patients, and no postoperative complications developed. Surgery was formerly considered to be a high risk event for PNH patients, but this case raises the possibility that even elderly PNH patients may undergo surgery safely when maintained on eculizumab treatment.

Complement Activation in Paroxysmal Nocturnal Hemoglobinuria (PNH) Causes Oxidative Damage Which May Affect Response to Eculizumab

PNH is a rare acquired clonal disorder of the hematopoietic stem cell, characterized by a somatic mutation that inactivates the X-linked PIGA gene: this in turn results in deficiency on the cell surface of all proteins anchored by the glycosylphosphatidylinositol (GPI) molecule. Two of these proteins,CD55 andCD59, are complement regulators and their deficiency is responsible for the susceptibility of red cells (RBCs) from the mutant clone to lysis by activated complement. Since oxidative damage is another well-known mechanism of hemolysis (as in G6PD deficient red cells), we have investigated whether this plays a role also in PNH.To this end, we have carried out experiments on RBCs from healthy donors and on PNH-like RBCs (obtained in vitro from the same donors through the use of anti-CD55 and anti-CD59 blocking moAb). After exposure to AB0-compatible serum (in which thecomplement alternative pathway was activated by mild acidification) all PNH-like (but not normal) RBCs were lysed. In parallel experiments in which complement was blocked by eculizumab (ECU) - a moAb that binds to the complement component C5 and controls intravascular hemolysis in PNH patients - we measured the levels of reactive oxygen species (ROS) by the dichlorofluorescin diacetate assay. We found no significant difference of ROS levels between normal RBCs and PNH-like RBCs. We next tested in a similar way G6PD-deficient RBCs, because these are known to be exquisitely sensitive to oxidative damage. We found that ROS levels were significantly higher in the G6PD deficient RBCs that have been made PNH-like (Fig. 1). Thus, complement activation on the surface of PNH-like RBCs results in the production of ROS that can be demonstrated when C5-blockade prevents complement-mediated lysis of RBCs.The notion that G6PD deficiency can interact with PNH was strongly corroborated by the clinical observation of a 40yo woman from Sardinia (Italy) with a 2 years history of pancytopenia, who then developed florid hemolytic PNH: she had anemia with normal granulocyte and platelet counts, dark urine, high reticulocytosis, LDH up to 5x upper normal level, 95% GPI-negative granulocytes. When the patient was started on ECU. LDH levels promptly returned to normal, PNH RBCs rose from 20% (before ECU) to 42%, but reticulocyte count (~250x109/L) and blood transfusion requirement remained high (10 units in the last year). 39% of the GPI-negative RBCs had bound C3 fragments The peripheral blood smear revealed marked macro-anisocytosis, poikilocytosis, spherocytes, and hemighosts: a picture consistent with oxidative damage as seen in G6PD deficient patients during a hemolytic attack. The RBC G6PD activity was about one-half of normal (5 IU/g Hb), and DNA analysis revealed heterozygosity for the G6PD Mediterranean (Med) mutation. By mRNA sequence analysis we found that the GPI-negative clone expressed only the G6PD Med allele, suggesting that the PIG-A mutation took place in a stem cell in which the normal G6PD gene was on the inactive X-chromosome (G6PD, like PIG-A, is on the X chromosome); therefore, all the patients’ PNH RBCs were also all G6PD deficient.We have previously shown that the clinical expression of PNH can be influenced by inherited factors: specifically, a polymorphism of the complement receptor 1 (CR1) gene correlates with the blood transfusion requirement of patients on ECU (Rondelli et al, Haematologica 2014). However, the patient here reported was homozygous for the more favorable allele ofCR1. Instead, in keeping with our experimental data, the poor response to ECU seen in this patient results probably from a unique interaction, within the same population of RBCs, between the acquired PNH abnormality and her inherited G6PD deficiencyThis type of interaction is novel and it seems to have pharmacogenetic implications. Indeed, on its own G6PD deficiency affects mildly the clinical expression of PNH, because complement activation causes RBC lysis regardless; however, paradoxically, when the lysis of PNH RBCs is prevented by C5 blockade, complement activation results in oxidative damage, with which PNH G6PD deficient RBCs are unable to cope.Except for one case previously reported by Oni et al (Blood 1970), this is the first detailed study of PNH associated with G6PD deficiency. Since in some parts of the world the frequency of G6PD deficiency can be as high as 30% or more, we expect that more cases of this association will be discovered in the future. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Eculizumab effect on the hemostatic state in patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a hypercoagulable state associated with acute hemolysis. Eculizumab is used to reduce the intensity of intravascular hemolysis in PNH patients. The hemostatic status of three patients with PNH was assessed during eculizumab treatment by D-dimer assay and the global assays: thromboelastography (TEG), thrombin generation test (TGТ), and thrombodynamics (TD). In the state of hemolytic crisis before the therapy D-dimer concentration was increased in two patients accompanied by hypercoagulation changes in TEG parameter angle (α). TD parameter the clot growth velocity (V) revealed hypercoagulability while TGT parameter ETP was within the normal range in all patients. The lactate dehydrogenase (LDH) activity decreased during the 8months of eculizumab therapy. The physical health was improved, the frequency of hemolytic crisis decreased. Patients periodically exhibited hypercoagulable state: the mean values α=38±11° (with normal range 20-40°), ETP=1311±442nM·min (with normal range 800-1560nM·min), V=31±4μm/min (with normal range 20-29μm/min). During the eculizumab therapy two patients had the repeated clinical manifestation of acute hemolytic crisis, the parameters of the global tests were increased compared to the previous measurement. The global hemostasis tests TEG, TGT and TD revealed hypercoagulability in patients with PNH during eculizumab therapy.

A Rare Genetic Polymorphism In C5 Confers Poor Response To The Anti-C5 Monoclonal Antibody Eculizumab In 11 Japanese Patients With PNH

Eculizumab is a humanized monoclonal antibody targeting the terminal complement protein C5 and inhibiting terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). In the Japanese AEGIS PNH-eculizumab study, 2 poor-responders were identified out of 29 cases. Currently, more than 300 patients have been treated with eculizumab, and a total of 11 poor-responders were identified all of whom are Japanese.To clarify the mechanism of difference in the responsiveness of eculizumab, blood samples from poor and good responders were analyzed after obtaining informed consent. Approval for these studies was obtained from the institutional review boards at each study site taking care of patients as well as from Osaka University. The levels of lactate dehydrogenase in these two patients were markedly elevated before eculizumab treatment, and were not decreased during the 12 weeks AEGIS study. From the pharmacokinetic analysis, peak and trough levels of eculizumab during the study were well above the minimal level required to completely inhibit complement-mediated hemolysis in PNH patients. The pharmacodynamics of eculizumab were determined by measuring the capacity of the patients' serum to lyse chicken erythrocytes in a standard hemolytic assay. Serum samples analysed from these two patients failed over the entire treatment period, to show a suppression of hemolysis, prompting further study of the effect of exogenous eculizumab on the hemolytic activity of patient pre-drug sera. Eculizumab up to 2000μg/mL did not block hemolytic activity in the sera of either poor-responder. However, hemolytic activity both in the two poor-responders and in control patient was blocked completely using a different anti-C5 antibody (N19/8) at 50μg/mL. Therefore, the DNA of C5 from Japanese PNH patients with a good or poor response to eculizumab was sequenced, and a single missense C5 heterozygous mutation at exon 21, c.2654G>A, which predicts p.Arg885His, was found in all of the 11 poor responders identified to date, but not in any of the responders. Among about 300 Japanese patients treated with eculizumab, 11 patients (about 3.7%) have been identified as poor responders. A similar prevalence (3.5%) was seen in healthy volunteers, since we determined that 10 out of 288 Japanese healthy volunteers have the same mutation. This polymorphism was also identified in 1 out of 120 China Han healthy volunteers, but not in 100 persons of British ancestry living in England and Scotland, and not in 90 persons of Mexican ancestry in Los Angels.To close the genotype-phenotype loop, electrophoretically pure recombinant C5 (rC5) and rC5 mutant (rC5m) containing c.2654G>A were generated and functionally compared in various in vitro experiments. As a preliminary experiment, we confirmed that natural C5, rC5, and rC5m restored classical pathway lysis equivalently when added to C5-depleted serum. Eculizumab did not block classical pathway lysis reconstituted with rC5m but did block rC5 and nC5-dependent lysis. By contrast, as observed with patient sera, N19/8 inhibited lysis reconstituted with nC5, rC5, and rC5m. Finally, while eculizumab bound nanomolar concentrations of rC5 using surface plasmon resonance, with clear association and dissociation phases, there was no detectable binding with rC5m in the same assay up to the highest concentration (1 µM) of eculizumab examined.A single missense C5 heterozygous mutation, c.2654G>A, which predicts p.Arg885His, was commonly identified in poor-responders, but not in responders. This polymorphism had at least spread to other East Asian countries. After determining that the poor responders likely express both wild-type C5 and a structural variant C5, we then showed that the hemolytic activity supported by this structural variant in vitro, like the effects on patient sera, was not blocked by eculizumab but was fully blocked by N19/8, and that the variant was incapable of binding eculizumab. Collectively, these data are consistent with the hypothesis that the functional capacity of the mutant C5 together with its inability to bind to and undergo blockade by eculizumab fully account for the poor response in patients carrying this mutation.(JN and MY contributed equally to this work) Disclosures:Nishimura:Alexion Pharmaceuticals, Inc.: Research Funding, Speakers Bureau. Yamamoto:Alexion Pharm: Research Funding. Ohyashiki:Alexion: Research Funding. Noji:Alexion Pharmaceuticals: Honoraria. Shichishima:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hase:Alexion Phama: Employment, Equity Ownership. Lan:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Johnson:Alexion Pharmaceuticals, Inc.: Employment. Tamburini:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership, Patent inventor but do not receive royalties, Patent inventor but do not receive royalties Patents & Royalties. Kinoshita:Alexion: Honoraria. Kanakura:Alexion Pharmaceuticals: Research Funding, Speakers Bureau.

Genetic Polymorphism of the Complement Receptor-1 (CR1) Gene Correlates with the Clinical Response to Eculizumab of Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

AbstractAbstract 983Eculizumab, a monoclonal antibody that binds to the complement component C5, controls intravascular hemolysis in PNH patients and causes significant clinical improvement. However, the extent to which patients respond is variable in terms of hemoglobin levels, and some patients remain red blood cell (RBC) transfusion-dependent in spite of regular eculizumab treatment. We have hypothesized that genetic polymorphisms of complement-related genes might contribute to determining this variability of response to eculizumab. In order to test this hypothesis, we have analyzed polymorphic alleles of the Complement Receptor 1 (CR1) gene and of the Complement Component 3 (C3) gene. We have genotyped by standard methods (1) the HindIII restriction length polymorphism (RFLP) in the CR1 intron 27 (rs11118133 A>T) whose co-dominant alleles, H (common) and L (rare), are associated with high and low levels of CR1 expression on RBC, respectively; (2) the single nucleotide polymorphism rs2230199 C>G of the C3 gene, responsible for the allelic electrophoretic variants, S (slow, common allele) and F (fast, rare allele), that influence the complement alternative pathway activity.We have studied 72 patients with hemolytic PNH treated with eculizumab for at least 6 months. Patients with clear evidences of bone marrow failure (platelets ≤ 50,000/μL, neutrophils ≤1000/μL) have not been included in this study because this condition may affect the clinical response independently of the control of hemolysis. In order for our criterion for response to be stringent and objective we used blood transfusion requirement: patients who received no RBC transfusion have been defined as Responders; patients who received any RBC transfusion have been defined as Poor Responders.In this series of 72 hemolytic PNH patients on eculizumab the allelic frequency of the CR1 rare allele L was 0.31, and that of the C3 rare allele F was 0.19. We found no correlation between response and the C3 genotype (Chi square for trend: p=0.939). On the other hand the proportion of Poor Responders is significantly higher in patients who were heterozygotes (H/L) for the CR1 polymorphism, and even higher in those who were homozygous for the rare allele (L/L) of the CR1 HindIII RFLP polymorphism (Chi square for trend: p=0.016): see figure 1. [Display omitted] We do not know yet the precise mechanism whereby the CR1 genotype affects the response to eculizumab. CR1 enhances the decay of C3 and C5 convertase by binding to C3b and C4b and it plays a role in the clearance of immune complex and in phagocytosis. Thus, the variable levels of CR1 expression on RBC membrane associated with the HindIII RFLP polymorphism may affect the regulation of complement alternative pathway. Moreover, it is well established that in almost all PNH patients on eculizumab a significant fraction of GPI-negative RBCs are opsonized by C3 and therefore may undergo extravascular hemolysis. Thus, a possible explanation of our findings is that the lower levels of CR1 expression on RBCs associated with H/L and L/L genotypes are responsible for a more intense extravascular hemolysis in PNH patients on eculizumab.In conclusion, our data show that the CR1 HindIII polymorphism may predict the poor response to eculizumab in PNH patients and provide additional insight in the mechanisms responsible for the variable clinical effectiveness of complement blockade. Disclosures:Risitano:Alexion: Membership on an entity's Board of Directors or advisory committees, Research Funding. Peffault de Latour:Alexion: Consultancy. Iori:Alexion: invited speaker Other. Socie:Alexion: Consultancy. Luzzatto:Alexion: Consultancy. Notaro:Alexion: Consultancy.

Safety and efficacy of the terminal complement inhibitor eculizumab in Japanese patients with paroxysmal nocturnal hemoglobinuria: the AEGIS Clinical Trial

Paroxysmal nocturnal hemoglobinuria (PNH) is a progressive and life-threatening disease characterized by complement-mediated chronic hemolysis, resulting in serious life-threatening complications and early mortality. Eculizumab, a humanized anti-C5 monoclonal antibody that inhibits terminal complement activation, has been shown to reduce hemolysis in PNH patients. The pivotal open-label, 12-week phase II registration study (AEGIS) was designed to evaluate the efficacy and safety of eculizumab in Japanese patients with PNH. This trial achieved its primary endpoint of reducing intravascular hemolysis with high statistical significance. Twenty-seven of the 29 patients responded to eculizumab treatment, resulting in an 87% reduction in hemolysis (P<0.0001) and subsequent improvement in anemia (P=0.0003) despite reduction in transfusion requirements (P=0.006). Fatigue and dyspnea significantly improved within 1-2weeks of eculizumab treatment and the improvement was independent of changes in hemoglobin. Chronic kidney disease (CKD) was common (66%) and eculizumab treatment improved CKD in 41% of patients at 12weeks (P<0.001). Elevated thrombotic risk was evident in Japanese PNH patients and eculizumab treatment normalized D: -dimer levels in 45% of patients with elevated D: -dimers at baseline (P<0.001). The AEGIS results demonstrate that eculizumab is effective, safe and well tolerated in Japanese patients with PNH.

C3-Mediated Extravascular Hemolysis as Additional Mechanism of Disease in Paroxysmal Nocturnal Hemoglobinuria (PNH) Patients Treated by the Complent Inhibitor Eculizumab

PNH is a hematological disorder characterized by complement-mediated intravascular hemolysis due to lack on RBCs of the complement regulators CD55 and CD59 and subsequent activation of the membrane attack complex (MAC). Eculizumab (EC) is an anti-complement fraction 5 antibody which abolishes intravascular hemolysis in PNH patients, leading to reduction of transfusion requirement and of anemia. Although the effector complement pathway is completely blocked by eculizumab in all patients, response to this agent in terms of transfusion requirement and hemoglobin level varies considerably. We have investigated the notion that patients with suboptimal hematological response may suffer from residual hemolysis mediated by mechanisms other than intravascular hemolysis via MAC. After the initial observation of a positive C3d Coombs test in some PNH patients on EC, we extensively studies C3 coating by flow cytometry in 56 PNH patients (41 of them while receiving EC). We found that in all cases on EC treatment a significant proportion of RBCs were coated with complement fraction 3 (C3); by converse, in 28 untreated PNH patients we found no evidence of C3 on red cells. C3 coating was strictly limited to CD59-neg RBCs, as CD59+/C3+ RBCs (as those seen in cold agglutinine disease patients, positive control) were never found. C3 coating was quite different among EC-treated patients, and correlated with the PNH RBC population. The percentage of C3+ cells within the PNH RBC population (the only subjected to C3 coating) was quite different in individual patients (0.5–61.3%, median 22.6%) and substantially preserved over time. We compared the level of C3 coating with the hematological response: all the 41 EC-treated patients showed marked LDH reduction with a substantial improvement of anemia, leading to transfusion independence in 34/41 patients (83%) and stable resolution of anemia in 14 (34%, defined optimal responders). The optimal responders showed a lower percentage of C3+ cells in comparison to suboptimal responders (20.9±19.0 vs 32.2±17.8; p=0.04). Indeed, patients with lower C3 coating (below the median value of 22.6%) showed a significantly higher rate of optimal response (51% in comparison to 15% of those with coating above the median, p=0.01). C3 coating also correlated with the absolute reticulocyte count (p=0.03), clearly suggesting that C3 coating was associated with ongoing residual extravascular hemolysis in vivo, and with pre-treatment LDH level (p=0.001). To confirm the presence of extravascular hemolysis, in 3 index patients with suboptimal response reduced RBC half-life was demonstrated in vivo by 51Cr RBC survival study, which showed reduced RBC half-life and excess uptake in liver and spleen. One of these patients underwent video-laparoscopic splenectomy, which led to transfusion independence and significant increase in Hb level. These data demonstrate that C3 coating of PNH RBCs is a common phenomenon in PNH patients on EC; in addition, we provide evidence that this leads to C3-mediated extravascular hemolysis through the reticulo-endothelial system. This novel mechanism of disease may account for residual hemolysis and suboptimal clinical benefit in some EC treated PNH, paving the way for additional therapeutic strategies to optimize the hematological response to this agent.

Blockade of Intravascular Hemolysis in PNH with the Terminal Complement Inhibitor Eculizumab Unmasks Low-Level Hemolysis Potentially Occurring through C3 Opsonization.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia characterized by intravascular hemolysis, often resulting in the need for red blood cell (RBC) transfusions. PNH RBCs lack two complement regulatory molecules - CD59, a terminal complement inhibitor, and CD55, a C3 convertase inhibitor. Eculizumab, a humanized monoclonal antibody that inhibits terminal complement by binding to C5, effectively controls intravascular hemolysis as determined by a dramatic reduction in lactate dehydrogenase (LDH) to levels in or just above the normal range. Control of intravascular hemolysis in these patients led to a reduction in, or cessation of, RBC transfusions. During eculizumab treatment, a majority of patients demonstrate evidence of residual, low-level hemolysis; LDH levels remain slightly elevated, haptoglobin levels are low or undetectable, and bilirubin levels are above normal. We hypothesized that this low-level residual hemolysis may be due to clearance of PNH RBCs through a C3b-mediated mechanism. Therefore we investigated C3 deposition on RBC in PNH patients before and on eculizumab. A direct antiglobulin test (DAT) using monoclonal anti-C3d was positive in 29 out of 39 PNH patients on eculizumab. Of these 29 DAT-positive patients, who were all receiving transfusions, 25 had DAT testing prior to eculizumab therapy and only one of these was positive. DAT was negative in all of 8 normal volunteers. By two-color flow cytometric analysis with anti-CD59 and anti-C3, the majority of patients on eculizumab demonstrated three distinct RBC populations:CD59+/C3− (normal RBCs);CD59-/C3− (PNH RBCs without C3 coating); andCD59-/C3+ (PNH RBCs coated by C3).No CD59+/C3+ RBCs were observed. Of 21 DAT positive eculizumab treated patients tested, the median proportion of total RBCs that were C3b positive was 17.6%. 18 of 29 [62%] eculizumab patients with a positive DAT received at least one transfusion during eculizumab therapy compared with 1 of 10 [10%] for DAT negative patients (p=0.01), although even patients who did not become transfusion independent during eculizumab treatment showed a marked reduction in transfusion requirement. The median hemoglobin value for the 29 DAT positive eculizumab patients was 9.8 g/dL compared with 11.3 g/dL in the 10 DAT negative eculizumab patients (p= 0.08). No apparent relationship between LDH and DAT positivity was observed. It is proposed that resolution of intravascular hemolysis in PNH patients on eculizumab results in deposition of C3b on the surface of PNH RBCs which may explain, at least in part, the residual low level hemolysis occurring in some patients. This appears to be a previously undescribed mechanism of RBC clearance in PNH, most likely obscured by the rapidity of intravascular hemolysis in the absence of eculizumab therapy. Despite the low-level residual hemolysis, patients continue to receive significant benefit from eculizumab treatment.