Terminal Complement Activation Research Articles

Diagnostic and risk criteria for HSCT-associated thrombotic microangiopathy: a study in children and young adults

AbstractTransplant-associated thrombotic microangiopathy (TMA) leads to generalized endothelial dysfunction that can progress to multiorgan injury, and severe cases are associated with poor outcomes after hematopoietic stem cell transplantation (HSCT). Identifying patients at highest risk for severe disease is challenging. We prospectively evaluated 100 consecutive HSCT recipients to determine the incidence of moderate and severe TMA and factors associated with poor overall outcomes. Thirty-nine subjects (39%) met previously published criteria for TMA. Subjects with TMA had a significantly higher nonrelapse mortality (43.6% vs 7.8%, P < .0001) at 1 year post-HSCT compared with those without TMA. Elevated lactate dehydrogenase, proteinuria on routine urinalysis, and hypertension were the earliest markers of TMA. Proteinuria (>30 mg/dL) and evidence of terminal complement activation (elevated sC5b-9) in the blood at the time of TMA diagnosis were associated with very poor survival (<20% at 1 year), whereas all TMA subjects without proteinuria and a normal sC5b-9 serum concentration survived (P < .01). Based on these prospective observations, we conclude that severe TMA occurred in 18% of HSCT recipients in our cohort and propose an algorithm to identify the highest-risk patients who might benefit from prompt clinical interventions.

Biomarkers of terminal complement activation confirm the diagnosis of aHUS and differentiate aHUS from TTP

AbstractAtypical hemolytic uremic syndrome (aHUS) is characterized by dysregulated complement activity, the development of a thrombotic microangiopathy (TMA), and widespread end organ injury. aHUS remains a clinical diagnosis without an objective laboratory test to confirm the diagnosis. We performed a retrospective analysis of 103 patients enrolled in the Ohio State University TTP/aHUS Registry presenting with an acute TMA. Nineteen patients were clinically categorized as aHUS based on the following criteria: (1) platelet count <100 × 109/L, (2) serum creatinine >2.25 mg/dL, and (3) a disintegrin and metalloprotease with thrombospondin type 1 motif, 13 (ADAMTS13) activity >10%. Sixteen of 19 patients were treated with plasma exchange (PEX) therapy, with 6/16 (38%) responding to PEX. Nine patients were treated with eculizumab with 7/9 (78%) responding to therapy. In contrast to thrombotic thrombocytopenic purpura (TTP) patients, no aHUS patients demonstrated ultralarge von Willebrand factor multimers at presentation. Median markers of generalized complement activation (C3a), alternative pathway (Bb), classical/lectin pathway (C4d), and terminal complement activation (C5a and C5b-9) were increased in the plasma of these 19 patients. Compared with a cohort of ADAMTS13-deficient TTP patients (n = 38), C5a and C5-9 were significantly higher in the 19 patients clinically characterized as aHUS, suggesting that pretreatment measurements of complement biomarkers C5a and C5b-9 may confirm the diagnosis of aHUS and differentiate it from TTP.

Spectrum of complement-mediated thrombotic microangiopathies: pathogenetic insights identifying novel treatment approaches.

Thrombotic microangiopathy (TMA) is a rare but severe disorder characterized by endothelial cell activation and thrombus formation. It manifests with the triad of hemolytic anemia, thrombocytopenia, and organ failure. Prompt diagnosis and treatment initiation are crucial for long-term outcome. TMA often manifests subsequent to infectious events, of which (enterohemorrhagic) Escherichia coli is the most frequently reported. TMA also occurs on the background of genetic/autoimmune defects in the complement system (atypical hemolytic uremic syndrome [aHUS]) and underlying conditions, such as pregnancy, transplantation, drugs, other glomerulopathies, vasculitides, or metabolic defects. Complement activation or defects in its regulation have now been described in an increasing number of acquired diseases with TMA. Coinciding with this expanding spectrum of complement-mediated diseases, the question arises which patients might benefit from a complement-targeted therapy. Success of therapy depends on the individual contribution of complement activation in disease pathogenesis. The advent of eculizumab, a monoclonal antibody that blocks terminal complement activation, has markedly improved outcome and quality of life in patients with aHUS. This review discusses the contribution of complement and highlights its complex interaction with inflammation, coagulation, and the endothelium. Treatment experiences focusing on eculizumab therapy are discussed in detail across the emerging spectrum of complement-mediated thrombotic microangiopathies.

Complement activation products C5a and sC5b-9 are associated with low-grade inflammation and endothelial dysfunction, but not with atherosclerosis in a cross-sectional analysis: The CODAM study

Low-grade inflammation is involved in endothelial dysfunction, atherosclerosis and cardiovascular disease (CVD) [ [1] Libby P. Inflammation in atherosclerosis. Arterioscler Thromb Vasc Biol. 2012; 32: 2045-2051 Crossref PubMed Scopus (1468) Google Scholar ]. The complement system is an ancient inducer of inflammation, which generates various soluble and membrane-bound factors upon activation [ [2] Ricklin D. Hajishengallis G. Yang K. Lambris J.D. Complement: a key system for immune surveillance and homeostasis. Nat Immunol. 2010; 11: 785-797 Crossref PubMed Scopus (2367) Google Scholar ]. Complement C5a and soluble C5b-9 (sC5b-9) are products of the final step of the complement activation cascade (=terminal complement activation). The anaphylatoxin C5a is a soluble mediator, which can activate immune and endothelial cells [ [3] Guo R.F. Ward P.A. Role of C5a in inflammatory responses. Annu Rev Immunol. 2005; 23: 821-852 Crossref PubMed Scopus (717) Google Scholar ]. sC5b-9 is the circulating form of C5b-9, also called membrane-attack complex, which has been shown to affect endothelial cells in vitro [ [4] Bossi F. Fischetti F. Pellis V. et al. Platelet-activating factor and kinin-dependent vascular leakage as a novel functional activity of the soluble terminal complement complex. J Immunol. 2004; 173: 6921-6927 Crossref PubMed Scopus (81) Google Scholar ]. In humans, C5a and sC5b-9 are increased in acute inflammatory situations and in acute cardiovascular events, where they are thought to mediate ischemia–reperfusion injury [ [5] Arumugam T.V. Magnus T. Woodruff T.M. et al. Complement mediators in ischemia–reperfusion injury. Clin Chim Acta. 2006; 374: 33-45 Crossref PubMed Scopus (103) Google Scholar ]. Indeed, in patients with acute or advanced CVD, C5a and sC5b-9 have been associated with adverse cardiovascular outcomes [ 6 Speidl W.S. Exner M. Amighi J. et al. Complement component C5a predicts future cardiovascular events in patients with advanced atherosclerosis. Eur Heart J. 2005; 26: 2294-2299 Crossref PubMed Scopus (110) Google Scholar , 7 Speidl W.S. Katsaros K.M. Kastl S.P. et al. Coronary late lumen loss of drug eluting stents is associated with increased serum levels of the complement components C3a and C5a. Atherosclerosis. 2010; 208: 285-289 Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar , 8 Mellbin L.G. Bjerre M. Thiel S. Hansen T.K. Complement activation and prognosis in patients with type 2 diabetes and myocardial infarction: a report from the DIGAMI 2 trial. Diabetes Care. 2012; 35: 911-917 Crossref PubMed Scopus (47) Google Scholar , 9 Lindberg S. Pedersen S.H. Mogelvang R. et al. Soluble form of membrane attack complex independently predicts mortality and cardiovascular events in patients with ST-elevation myocardial infarction treated with primary percutaneous coronary intervention. Am Heart J. 2012; 164: 786-792 Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar ]. However, it is currently unknown whether C5a and sC5b-9 are also increased in earlier, less advanced stages of the cardiovascular disease process. Therefore, we investigated the associations of plasma C5a and sC5b-9 with systemic low-grade inflammation, endothelial dysfunction, markers of atherosclerosis and with prevalent CVD in a cohort of middle-aged individuals with a moderately increased cardiometabolic risk.

Atypical HUS associated with severe, unexpected antibody‐mediated rejection post kidney transplant

We present a case of an unsensitized patient with end-stage kidney disease secondary to atypical haemolytic uremic syndrome (aHUS) with mutations in CD46/MCP and CFH who developed severe, intractable antibody-mediated rejection (ABMR) unresponsive to therapy post kidney transplantation. There were no haematological features of thrombotic microangiopathy. The patient received standard induction therapy and after an initial fall in serum creatinine, severe ABMR developed in the setting of urosepsis. Despite maximal therapy with thymoglobulin, plasma exchange and methylprednisolone, rapid graft loss resulted and transplant nephrectomy was performed. Luminex at 4 weeks showed a new DSA and when repeated after nephrectomy showed antibodies to each of the 5 mismatched antigens with high MFI. The rate of recurrence of disease in patients with aHUS referred for transplantation is 50% and is associated with a high rate of graft loss. It is dependent in part on the nature of the mutation with circulating factors CFH and CFI more likely to cause recurrent disease than MCP which is highly expressed in the kidney. There is increasing interest in the role of complement in the development and propagation of ABMR via terminal complement activation. This case suggesting that dysregulation of the alternative complement pathway within the transplant kidney may have contributed to the severe AMR. Very little is known about the impact of complement dysregulation and the development of anti HLA antibodies however the strength of HLA antibody formation was prominent in this case.

Complement modulation in solid-organ transplantation

The complement system is a major constituent of the innate immune system. It has a critical role in defense against pathogens but dysregulation of complement activation may lead to tissue injury and modulate the adaptive immune response. In organ transplantation, local complement activation is involved in hyper-acute rejection and antibody-mediated rejection. This last decade, interest in complement activation has increased due to new insights into the pathophysiology of antibody-mediated rejection, but also since the availability of news drugs that target terminal complement activation. In this review, we discuss our current understanding of how local complement activation induces acute and chronic graft injury, and review recent advances in clinical trials that block complement activation using the anti-C5 monoclonal antibody, eculizumab. Finally, we discuss how complement-targeted therapy may be integrated into our current immunosuppressive regimen and what type of patient will benefit most from this therapy.

Current treatment of atypical hemolytic uremic syndrome.

Tremendous advances have been made in understanding the pathogenesis of atypical Hemolytic Uremic Syndrome (aHUS), an extremely rare disease. Insights into the molecular biology of aHUS resulted in rapid advances in treatment with eculizumab (Soliris(®), Alexion Pharmaceuticals Inc.). Historically, aHUS was associated with very high rates of mortality and morbidity. Prior therapies included plasma therapy and/or liver transplantation. Although often life saving, these were imperfect and had many complications. We review the conditions included under the rubric of aHUS: S. pneumoniae HUS (SpHUS), inborn errors of metabolism, and disorders of complement regulation, emphasizing their differences and similarities. We focus on the clinical features, diagnosis, and pathogenesis, and treatment of aHUS that results from mutations in genes encoding alternative complement regulators, SpHUS and HUS associated with inborn errors of metabolism. Mutations in complement genes, or antibodies to their protein products, result in unregulated activity of the alternate complement pathway, endothelial injury, and thrombotic microangiopathy (TMA). Eculizumab is a humanized monoclonal antibody that inhibits the production of the terminal complement components C5a and the membrane attack complex (C5b-9) by binding to complement protein C5a. This blocks the proinflammatory and cytolytic effects of terminal complement activation. Eculizumab use has been reported in many case reports, and retrospective and prospective clinical trials in aHUS. There have been few serious side effects and no reports of tachphylaxis or drug resistance. The results are very encouraging and eculizumab is now recognized as the treatment of choice for aHUS.

Role for terminal complement activation in amyotrophic lateral sclerosis disease progression

Lobsiger et al. (1) recently demonstrated that superoxide dismutase 1 (SOD1) transgenic mice deficient in complement components C1q and C3 do not have extended survival, concluding that global complement activation does not affect overall disease in amyotrophic lateral sclerosis (ALS). Complement activation has long been implicated in the pathogenesis of ALS, with numerous clinical and animal studies demonstrating strong complement factor up-regulation, including C1q and C3, in regions of motor neuron death (2). Intriguingly, in the study by Lobsiger et al. C3- (and C1q-) deleted SOD1 mice did not show alterations in survival or motor neuron estimates, and this is interpreted by the authors as complement activation not contributing to ALS mice neurotoxicity. Indeed, in figure S1 of their article, Lobsiger et al. present three C3-dependent pathways of complement activation (classic, lectin, and alternative). However, one point missing in this report, and perhaps overlooked by the casual reader, is that there is now a well-described fourth pathway of complement activation, termed the “extrinsic pathway,” which can bypass the traditional upstream activation pathways that are reliant on complement factor C3.

Alternative and Terminal Complement Pathway Biomarkers At Presentation More Precisely Define The Clinical Diagnosis Of aHUS

IntroductionRecent studies demonstrating the effectiveness of eculizumab in the treatment of atypical hemolytic uremic syndrome (aHUS) have increased the need for more rapid and objective diagnostic tests to help confirm the diagnosis of aHUS over other thrombotic microangiopathies (TMA). MethodsWe performed a retrospective study of all patients enrolled in the Ohio State University TTP/aHUS Registry since 2003. From this cohort of patients diagnosed with an acute TMA (platelets<100 x 109/ with schistocytes present, and without an alternative explanation), patients with a pretreatment serum creatinine >2.25 mg/dL and measureable ADAMTS13 activity (>10%) were analyzed. The clinical and demographic data for these 19 patients are shown in Table 1. Banked plasma samples from these study subjects were used to measure complement activation factors Bb (alternative pathway (AP)), C4d (classical pathway), C3a, (generalized complement activation), C5a, and C5b-9 complex (terminal complement pathway (TCP)). These complement biomarker data were analyzed in the context of a documented mutation in complement regulatory proteins and response to therapy with PEX and/or eculizumab. Response was defined as a normal platelet count and improved serum creatinine (at 6 months) independent of PEX therapy or with continued eculizumab therapy.Table 1Median Demographic and Clinical Data at PresentationAge (range)Sex (M/F)Race (AA/C)DialysisPlatelets (150-400x109)Creatinine (< 1.1 mg/dL)LDH (<190 U/L)N=1947 (20-69)3/163/1614/19 (74%)46 (8-93)4.39 (2.38-12.61)1094 (140-3548) ResultsThe median complement biomarker data at presentation are shown in Table 2. Complement activation products are generally sensitive biomarkers for aHUS patients. Notably, elevation of the AP (Bb) and the TCP (C5b-9) biomarkers were more prominent and were seen in 16/19 and 19/19 patients respectively. This is in contrast to previously published data from acquired TTP patients from our group that demonstrated less pronounced activation of the AP and TCP at presentation (accepted for publication, JTH). Similar levels of alternative, classical and terminal complement activation were seen among patients regardless of mutation status or response to PEX. In a preliminary analysis using a one sample T-test model, (Bb) activation was shown to be significantly lower in the non-responder to eculizumab compared to the 8 responding patients. TCP activation was also lower in the non-responder but did not show statistical significance. ConclusionsComplement biomarkers of the alternative and terminal complement pathway may more precisely define the diagnosis of aHUS. These hypothesis generating data suggest that pre-treatment complement biomarkers provide valuable information in the diagnosis of aHUS and could be useful in the prediction of response to eculizumab Disclosures:No relevant conflicts of interest to declare.

“Red Cell Complement Loading In PNH Patients On Eculizumab Is Associated With a C3 Polymorphism Which Influences C3 Function, Predicts For Increased Extravascular Hemolysis and Provides a Rationale For C3 Inhibition.”

IntroductionParoxysmal nocturnal hemoglobinuria (PNH) is an acquired bone marrow disorder characterised by intravascular hemolysis and hemoglobinuria, potentially life-threatening thrombosis and an association with aplastic anemia. Most of the clinical features and complications of PNH are due to the unopposed activity of complement due to the absence of CD59 and CD55, two key regulators of complement. Eculizumab prevents the cleavage of C5 complement thereby preventing terminal complement activity and protecting PNH cells from lysis. The inhibition of C5 preserves the early part of complement pathway and leads to the build up of C3 on the PNH red cells, perhaps in part due to their lack of CD55. The majority of PNH patients receiving eculizumab have evidence of extravascular haemolysis that can be clinically significant, including with anemia, hyperbilirubinemia and in some a continued requirement for transfusions. This extravascular hemolysis in thought to be due to the C3 loading of PNH red cells. MethodsWe report the C3-loading of the PNH red cells from 119 patients treated with eculizumab and correlate this with hemoglobin, LDH, bilirubin, reticulocytes and transfusions. We have studied genetic polymorphisms that affect both C3 and FCγR. We have genotyped 46 eculizumab patients for a functional mutation in the C3 gene (rs2230199). The two alleles of this gene can be distinguished by the presence or absence of a HindIII restriction site that distinguishes the electophoretically slow (arg80) from the electrophoretically fast (gly80) allotype. The fast (C3F) allotype allele of this snp is associated with a range of disorders including age-related macular degeneration, IgA nephropathy, systemic vasculitis and partial lipodystrophy. APL-1 is a small cyclic peptide that binds to and inhibits the activation of complement C3. APL-2 is a large conjugate of APL-1 with enhanced bioactivity and a long systemic half-life. APL-1 and APL-2 molecules as well as other complement inhibitors were studied for lysis of red cells and C3 loading in vitro in a modified Ham test in which flow cytometry was used to identify non-lysed cells. ResultsOut of the 119 Eculizumab treated patients, 55 (46.2%) required at least one transfusion on treatment. 110 patients had C3 detectable by flow cytometry on their PNH red cells (mean of 19.8%; range: <0.1 to 64.6%). C3-loading was not seen on the normal red cells from the same patients on treatment nor on the PNH red cells in patients not receiving eculizumab. The mean LDH (735IU/l) and reticulocyte count (193 x 109/l) were not statistically significantly different for the transfused group compared to the non-transfused group (518 and 163 respectively). Mean PNH C3 and RBC C3 did not differ stastistically between the transfused and non-transfused groups (26.20 Vs 24.78 PNH C3;15.96 vs 15.09 RBC C3 respectively).We studied one functional polymorphism in the Fcγ receptor but this showed no correlation with haemolytic parameters. Conversely, for the C3 polymorphism eculizumab-treated patients homozygous for the slow (C3S) allele had a significantly higher degree of C3 loading on PNH red blood cells with C3S/C3S having a mean of 33.7% C3 loaded PNH red cells (n=26), C3S/C3F 19.0% (n=19) and C3F/C3F 12.8% (n=3)(all P<0.01). Homozygote C3S also had increased reticulocytes (P<0.01) and bilirubin (P<0.01). The C3S allele has previously been shown to be more efficient in a haemolysis assay using sheep erythrocytes. This polymorphism appears to explain much of the variation in C3 loading between different patients with PNH.In vitro experiments show that inhibitors of C5, such as eculizumab, protect the PNH red cells from lysis but lead to a very rapid deposition of C3 on the PNH red cells. However both APL1 and APL2 demonstrated similar protection of PNH red cell lysis with virtually no C3 loading on PNH red cells. ConclusionA significant proportion of patients on eculizumab experience extravascular haemolysis with the majority of patients developing C3 loading. We show for the first time that a functional polymorphism in the C3 gene is associated parameters of hemolysis. The S(low) allele alters the function of C3 protein and appears to be associated with extravascular haemolysis in patients with PNH. The C3 inhibitors, APL-1 and APL-2, protect PNH red cells and prevent C3 loading in vitro and if safe to be given chronically would be expected to reduce extravascular hemolysis significantly. Disclosures:Hill:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kelly:Alexion: Honoraria. Richards:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees. Hillmen:Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Use of eculizumab and plasma exchange in successful combined liver–kidney transplantation in a case of atypical HUS associated with complement factor H mutation

Atypical hemolytic uremic syndrome (aHUS) evolves into end-stage renal failure in nearly half of affected patients and is associated with defective regulation of the alternative complement pathway. Patients with a complement factor H (CFH) mutation have a 30-100% risk of graft loss due to aHUS recurrence or graft thrombosis. Since CFH is produced predominantly by the liver, combined liver-kidney transplant is a curative treatment option. One major unexpected risk includes liver failure secondary to uncontrolled complement activation. We report a successful combined liver-kidney transplantation with perioperative plasma exchange and use of the humanized anti-C5 monoclonal antibody eculizumab. An 11-month-old female presented with oliguric renal failure after 3 weeks of flu-like symptoms in the absence of diarrhea. Following the identification of Escherichia coli 0157:H7 in her stool, she was discharged home on peritoneal dialysis with a diagnosis of Shiga toxin-associated HUS. Three months later, she developed severe anemia, thrombocytopenia, and neurological involvement. aHUS was diagnosed and confirmed, and genetic testing revealed a mutation in CFH SCR20. Once donor organs became available, she received preoperative plasma exchange followed by eculizumab infusion with intra-operative fresh frozen plasma prior to combined liver-kidney transplant. At 19 months post-transplant, she continues to have excellent allograft and liver function without signs of disease recurrence. Perioperative use of eculizumab in conjunction with plasma exchange during simultaneous liver-kidney transplant can be used to inhibit terminal complement activity, thereby optimizing successful transplantation by reducing the risk of graft thrombosis.

Eculizumab in an anephric patient with atypical haemolytic uraemic syndrome and advanced vascular lesions

Atypical haemolytic uraemic syndrome (aHUS) is associated with dysfunction of the alternative pathway of complement. Disease activity subsides as renal failure progresses but recurs upon renal transplantation, indicating that viable renal tissue contributes to disease activity. We present evidence of cerebrovascular occlusive disease indicating that vascular injury may occur in the absence of kidneys. A currently 12-year-old girl developed renal failure at the age of 20 months. She underwent bilateral nephrectomy and renal transplantation but lost the transplant due to recurrences. She was on haemodialysis for 7 years. At 10 years of age she developed a transient ischaemic attack. Imaging, genetic investigation and mutation characterization were performed. Imaging demonstrated occlusion and stenosis of the carotid arteries. Two complement mutations, a novel mutation in factor B and a previously described mutation in factor I, and the H3-factor H haplotype, were identified. The factor B mutation, L433S, did not induce excessive complement activation in vitro. Measurement of C3 degradation products indicated ongoing complement activation. In spite of the patient being anephric, treatment was initiated with eculizumab, a humanized anti-C5 antibody that blocks terminal complement activation. She underwent a successful kidney transplant 9 months later and has not developed a recurrence or progression of vascular stenosis 1 year later. The course of disease in this patient with aHUS suggests that complement-mediated vascular injury may occur in the total absence of renal tissue and overt recurrences. To our knowledge, this is the first description of eculizumab treatment in an anephric aHUS patient.

Long-term efficacy and safety of eculizumab in Japanese patients with PNH: AEGIS trial

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, progressive hematopoietic stem cell disorder characterized by chronic complement-mediated hemolysis leading to life-threatening complications and early mortality. Eculizumab, a humanized anti-C5 monoclonal antibody, inhibits terminal complement activation, reduces hemolysis, decreases the risk of thrombosis, and improves renal function and quality of life in PNH patients. The long-term efficacy and safety of eculizumab in Japanese patients were assessed in a 2-year extension to a 12-week, open-label study (AEGIS). Eculizumab treatment led to an immediate and sustained reduction in intravascular hemolysis (P < 0.001) and red blood cell transfusions (P = 0.0016) compared with baseline levels. There were no reports of thromboembolism during eculizumab treatment. The majority of patients had stable (56 %) or improved (41 %) renal function and an improved quality of life (P = 0.015), with sustained reductions in fatigue and dyspnea. Eculizumab was well tolerated; no deaths or serious hemolytic events were reported, and the rate of infections declined over time. There were no significant differences in the response to eculizumab in patients with or without bone marrow dysfunction. These results demonstrate that eculizumab is an effective, well-tolerated long-term treatment for Japanese PNH patients and leads to continued amelioration of some hemolytic complications.

Delayed Erythroid and Platelet Response to Eculizumab in Paroxysmal Nocturnal Haemoglobinuria – A Case Report and Literature Review

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder of haemopoiesis characterised by haemolytic anaemia, thrombophilia and variable cytopaenias. Complement-mediated blood cell damage leads to the main clinical features of PNH, including anaemia, haemoglobinuria, other haemolysis-related symptoms, thrombosis, and thrombocytopaenia. The treatment of PNH has remained supportive until the development of the first complement inhibitor, eculizumab. This antibody efficiently blocks terminal complement activity, quickly halting intravascular haemolysis. However, both the time course and the magnitude of erythroid and platelet responses to this drug are highly variable. Here, we report a case illustrating both delayed erythroid and platelet responses to eculizumab, and review mechanisms and therapeutic options for partial responses.

Soluble C5b-9, a marker for terminal complement activation, is not associated with markers of atherosclerosis or prevalent cardiovascular disease: The CODAM study

Soluble C5b-9, a marker for terminal complement activation, is not associated with markers of atherosclerosis or prevalent cardiovascular disease: The CODAM study

Long‐term safety and efficacy of sustained eculizumab treatment in patients with paroxysmal nocturnal haemoglobinuria

Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by chronic, uncontrolled complement activation resulting in elevated intravascular haemolysis and morbidities, including fatigue, dyspnoea, abdominal pain, pulmonary hypertension, thrombotic events (TEs) and chronic kidney disease (CKD). The long-term safety and efficacy of eculizumab, a humanized monoclonal antibody that inhibits terminal complement activation, was investigated in 195 patients over 66 months. Four patient deaths were reported, all unrelated to treatment, resulting in a 3-year survival estimate of 97·6%. All patients showed a reduction in lactate dehydrogenase levels, which was sustained over the course of treatment (median reduction of 86·9% at 36 months), reflecting inhibition of chronic haemolysis. TEs decreased by 81·8%, with 96·4% of patients remaining free of TEs. Patients also showed a time-dependent improvement in renal function: 93·1% of patients exhibited improvement or stabilization in CKD score at 36 months. Transfusion independence increased by 90·0% from baseline, with the number of red blood cell units transfused decreasing by 54·7%. Eculizumab was well tolerated, with no evidence of cumulative toxicity and a decreasing occurrence of adverse events over time. Eculizumab has a substantial impact on the symptoms and complications of PNH and results a significant improvement in patient survival.

Acute But Transient Release of Terminal Complement Complex After Reperfusion in Clinical Kidney Transplantation

Ischemia/reperfusion (I/R) injury has a major impact on kidney graft function and survival. Animal studies have suggested a role for complement activation in mediating I/R injury; however, results are not unambiguous. Whether complement activation is involved in clinical I/R injury in humans is still unclear. In the present study, we assessed the formation and release of C5b-9 during early reperfusion in clinical kidney transplantation in living donor, brain-dead donor, and cardiac dead donor kidney transplantation. By arteriovenous measurements and histologic studies, local terminal complement activation in the reperfused kidney was assessed. There was no release of soluble C5b-9 (sC5b-9) from living donor kidneys, nor was there a release of C5a. In contrast, instantly after reperfusion, there was a significant but transient venous release of sC5b-9 from the reperfused kidney graft in brain-dead donor and cardiac dead donor kidney transplantation. This short-term activation of the terminal complement cascade in deceased-donor kidney transplantation was not reflected by renal tissue deposition of C5b-9 in biopsies taken 45 min after reperfusion. This systematic study in human kidney transplantation shows an acute but nonsustained sC5b-9 release on reperfusion in deceased-donor kidney transplantation. This instantaneous, intravascular terminal complement activation may be induced by intravascular cellular debris and hypoxic or injured endothelium.

Post-partum atypical haemolytic-uraemic syndrome treated with eculizumab: terminal complement activity assessment in clinical practice.

Atypical haemolytic-uraemic syndrome (aHUS) is a disease of uncontrolled complement alternative pathway activation. Eculizumab induces terminal complement blockade (TCB), and is safe and effective for aHUS treatment as evidenced by its recent approval by the US Food and Drug Administration and the European Commission. A 26-year-old woman was admitted 1 week after her first childbirth for aHUS with creatinine 6.2 mg/dL (550 µmol/L), haemoglobin 3.6 g/dL (36 g/L), lactate dehydrogenase 2155 U/L (upper normal limit 248), 9% schizocytes and platelet count 49 × 103/µL (49 × 109/L). She had a medical history of Crohn's disease 5 years earlier and a skin melanoma 3 years earlier. Family history was remarkable with her father's brother presenting two renal graft losses related to aHUS despite plasma exchange (PE). The patient and her uncle both carried combined heterozygous mutations in genes coding for Factor H (p.Lys1186Thr) and Factor I (p.Ile322Thr). Daily PE was started with 60 mL/kg fresh frozen plasma substitution for 80 kg body weight with an initial positive haematological response without improvement of renal function requiring haemodialysis. Due to family history and suspected genetic background that was confirmed later, the patient was switched to eculizumab [1–3], 900 mg on Day 3 post-admission (PA). Placental retention was subsequently diagnosed and treated but a second 900 mg eculizumab infusion 10 days-PA was not effective in controlling aHUS (decreasing platelet count). Daily PE was re-started without supplemental eculizumab. During eculizumab treatment, complement activity measured with a validated sensitized sheep erythrocytes lysis assay (CH50) is expected to be <10% as observed in hereditary C5 deficiency [4]. We considered that complete TCB would be achieved for a serum CAE (complement activation enzyme immunosorbent assay, Diasorin®) [5] value <0.5 U/mL, (normal range 63–145 U/mL), corresponding to CH50 <10%. Patient serum CAE was 77 U/mL before the first eculizumab administration. Following the first three eculizumab administrations, complete TCB was maintained for a maximum of 4 days. On Day 39-PA, because of a continuous drop in platelet count from 161 to 81 × 103/µL despite intensive PE and as other causes of thrombocytopenia than aHUS were ruled out, eculizumab was resumed at a higher dose (1200 mg) and administrated whenever CAE activity increased over 0.5 U/mL. Under complete and sustained TCB, the outcome was rapidly positive (Figure 1): blood count normalized and the patient recovered normal renal function [serum creatinine stabilized to 0.8 mg/dL(75 µmol/L) without proteinuria after 6 months-PA] on the approved eculizumab dosing regimen. Far from aHUS trigger, eculizumab was tapered from 18 months-PA, and the patient had no signs of aHUS at the last follow-up 2 months after interrupting eculizumab. Fig. 1. Treatment regimen and clinical course of a patient with complement factor H and complement factor I gene mutations presenting post-partum (aHUS). Time is represented from the day of admission that corresponded to the first PE. Patient was switched to ... This is the first case of post-partum aHUS treated with eculizumab and the first description of TCB functional assessment to guide eculizumab dose and frequency of administration so as to obtain permanent complete complement blockade. This pragmatic therapeutic option seems to be adequate to rescue acute phases of aHUS, when the expected improvement does not occur under the recommended dose regimen of eculizumab. The degree of uncontrolled complement activation likely is influenced by the genetic background and the magnitude of the biological stress such as post-partum status [2, 6].

Eculizumab in Paroxysmal Nocturnal Hemoglobinuria (PNH): A Report of All 153 Patients Treated in the UK

Abstract 3472Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal stem cell disorder arising on the background of bone marrow failure and resulting in hemolytic anemia, thrombosis, pulmonary hypertension (PHT) and chronic kidney disease (CKD) through uncontrolled complement activation. Approximately half of patients treated with supportive therapies alone die as a result of their PNH. Eculizumab blocks C5 and thereby terminal complement activation. Complications are therefore prevented with reduction in intravascular hemolysis, transfusion requirements, thromboses, pulmonary pressures and renal impairment all associated with significantly improved quality of life. The UK has a nationally commissioned PNH service led by 2 Centres, Leeds and King’s. Between May 2002 - April 2012, a total of 153 patients (76 male; 77 female) were treated with eculizumab for PNH in the UK for a mean of 42 mths (0.4 – 119). Patients are treated if they have 1) transfusion-dependent hemolysis or 2) independent of transfusions, either i) thrombosis, ii) CKD, iii) PHT, iv) pregnancy or v) LDH>1.5× upper limit normal with anemia and symptoms including fatigue, dysphagia, dyspnea or abdominal pain due to PNH. Median age at PNH diagnosis was 34 yrs (range 12–80) and at initiation of eculizumab was 42 yrs (range 14–84). There were 28 thrombotic episodes in 15/65 (23%) patients anticoagulated prior to commencing eculizumab therapy. Nine patients have died - 3 due to progression of their underlying bone marrow failure to MDS/AML, one died immediately after BMT and the remaining 5 deaths are not directly related to PNH. Seven patients discontinued eculizumab - one had predominant aplastic anemia (AA), 2 had spontaneous remissions of the PNH clone, 3 commenced for a pregnancy discontinuing after and one who had a successful transplant for very severe AA. One hundred and thirty-seven patients remain on eculizumab. There were 36 thrombotic episodes in 22 patients in the 12 months prior to eculizumab and 3 thromboses in 3 patients in the most recent 12 months on therapy (1 Budd-Chiari during complement blockade breakthrough caused by infection, 1 CVA during reversal of coumadin overanticoagulation, 1 TIA/lacunar infarct thought to be due to diabetic small vessel disease) (P<0.05). Of the 22 patients who had a thrombosis within 12 months prior to starting eculizumab there were no further thrombotic episodes once on eculizumab. Importantly, primary and secondary prophylaxis with warfarin has been safely stopped in 43 and 4 patients respectively, with no thrombotic sequelae. Ten patients had not required a transfusion before receiving eculizumab. Of the 117 patients transfused in the 12 months before receiving eculizumab and on therapy for the most recent 12 months, 77 (66%) became transfusion independent. Of the 40 patients still needing transfusions, there was a significant (P<0.05) reduction in the number of units transfused from a median of 26 units 12 months before therapy to 8 units in the most recent 12 months on therapy. There were 3 cases of meningococcal septicaemia out of 153 patients treated (0.6 cases/100 patient yrs on therapy) with none in the last 2 yrs since routine penicillin prophylaxis was instituted. All were promptly and effectively managed and remain on eculizumab therapy. The survival of UK PNH patients on eculizumab was compared with age and sex matched controls (Fig 1) and demonstrates improved survival compared with historical controls. Survival after 10 years is slightly inferior to controls with causes of death related to bone marrow failure and not hemolysis or thrombosis. Conclusions:the total UK cohort provide more than 10 years experience of eculizumab for PNH and shows a) continuing safety and efficacy of eculizumab with persistent significant improvement in symptoms and quality of life b) no evidence of tolerance or refractoriness c) significant reduction in thrombosis risk on eculizumab therapy and safety in discontinuing primary anticoagulation d) significant reduction in the need for transfusions e) progression to MDS/AML as expected for the underlying bone marrow failure and not considered related to eculizumab and f) significant improvement in survival for PNH patients receiving eculizumab. This cohort of all PNH patients treated with eculizumab in the UK demonstrates the impact of eculizumab in the quality of life, reduction in complications and thereby improved survival for patients. [Display omitted] Disclosures:Hill:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Kelly:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Gandhi:Alexion Pharmaceuticals, Inc.: Research Funding. Mitchell:Alexion Pharmaceuticals, Inc: Honoraria. Elebute:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Arnold:Alexion Pharmaceuticals, Inc.: Honoraria. Marsh:Alexion Pharmaceuticals, Inc.: Honoraria. Hillmen:Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Eculizumab (ECU) in Atypical Hemolytic Uremic Syndrome (aHUS) Patients with Progressing Thrombotic Microangiopathy (TMA): 2-Year Data.

AbstractAbstract 2084 Introduction:aHUS is a rare, life-threatening genetic disease marked by chronic, uncontrolled complement activation leading to TMA and subsequent kidney and vital organ damage. 33%–40% patients (pts) die or progress to end-stage renal disease after the first clinical manifestation. In a phase 2 extension trial (64-week [wk] median ECU duration), pts receiving the terminal complement inhibitor ECU had a highly significant increase in platelet count (98×109/L; P=0.0001)—a measure of TMA improvement—and 4 pts (80%) discontinued prior dialysis by data cutoff. Here we report 2-year (y) results. Methods:Pts ≥12 y with aHUS and progressing TMA enrolled in an open-label, phase 2 trial with long-term extension. Pts had ≥4 plasma exchange/infusion (PE/PI) sessions in the wk before screening. ECU was administered at 900 mg/wk × 4 wk, 1200 mg at wk 5 and 1200 mg q2 wk thereafter. Results:The 26-wk study enrolled 17 pts, and 13 entered the extension phase. Data analysis was on the intention-to-treat population (n=17). Median time from diagnosis to screening was 10 months (range 0.3–236). Four pts (24%) had no identified complement regulatory factor mutation. Seven pts (41%) had prior renal transplant. All pts had estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m2 prior to ECU. Median ECU duration was 100 wk (range 2–145).Suppression of terminal complement activity was sustained at data cutoff. Significant platelet count improvement from baseline was seen at the 2-y data cutoff (P<0.0001; Table), with 15 pts (88%) achieving and maintaining a platelet count ≥150×109/L. Hematologic normalizationa was achieved by 88% of pts for an average duration of 629 days (d). Fifteen pts (88%) achieved TMA event-free status,b and TMA intervention ratec decreased from a median 0.88 (range 0.04–1.59) prior to ECU to 0 (range 0–0.31) during ECU treatment. Significant improvements in eGFR were observed throughout the study period (P=0.0008; Table). Earlier intervention with ECU was associated with greater eGFR improvement (P<0.01). Results of a 3-piece linear model demonstrated a flat slope for eGFR change from d −42 to 0, followed by a steep positive slope from d 0 to 28 (P<0.0001), then a continuous increase from d 28 to 860 (P=0.03). Improvements in other renal endpoints, including serum creatinine, proteinuria, and chronic kidney disease (CKD) stage, were observed with ongoing ECU. Significant improvements in quality of life (QOL) were observed. Pts received benefit regardless of the presence or absence of an identified complement mutation. Treatment with ECU was generally well tolerated, with 1 report of a serious adverse event (SAE; hypertension) deemed possibly drug related, which resolved with no change in ECU dosing. Two pts discontinued treatment due to worsening renal function unrelated to ECU. Conclusions:Long-term ECU therapy inhibited complement-mediated TMA and resulted in significant and continuous time-dependent improvements in renal function. At a median duration of almost 2 y, all pts entering the extension phase remain alive and report better QOL. The low rate of SAEs is consistent with the known safety profile of ECU. These results highlight the ongoing benefit of earlier intervention and long-term treatment with ECU in aHUS.TableChange from baseline in key outcomes from 26 wk and 100 wk of ECU treatment (N=17)Key OutcomesWk 26Median 100 WkMean platelet count change (primary), ×109/L (95% CI)73 (40–105)88 (63–112)P=0.0001P<0.0001 (wk 96)Hematologic normalization (primary),a n (%)13 (76)15 (88)TMA event-free status,b n (%)15 (88)15 (88)eGFR increase of ≥15 mL/min/1.73 m2, n (%)8 (47)10 (59)CKD improvement ≥1 stage, n (%)10 (59)12 (71)Serum creatinine decrease ≥25%, n (%)11 (65)13 (76)Mean eGFR change, mL/min/1.73 m2 (95% CI)31 (17, 45)32 (15, 49)dP=0.0001P<0.0008 (wk 96)Proteinuria decrease ≥1 grade,e n/N12/157/9 (wk 96)Mean health-related QOL change,f0.32 (0.27, 0.38)0.33 (0.30, 0.36)EuroQOL 5D score (95% CI)P<0.0001P=0.0001 (wk 96)CI=confidence interval.cPE/PI or new dialysis events/pt/d.aNormal platelet and lactate dehydrogenase levels, ≥2 consecutive measurements, ≥4 wk apart.b≥12 wk of stable platelet count, no PE/PI and no new dialysis.dECU eliminated need for dialysis in 4/5 pts receiving dialysis at baseline. An additional pt initiated dialysis at wk 64 (eGFR, 19 mL/min/1.73 m2 at screening).eEvaluable pts.fClinically meaningful threshold ≥0.6. Disclosures:Greenbaum:Alexion Pharmaceuticals: Consultancy, Honoraria, Research Funding, Speakers Bureau. Legendre:Alexion Pharmaceuticals: Speakers Bureau. Babu:Alexion Pharmaceuticals: Honoraria, Research Funding. Cohen:Alexion Pharmaceuticals Inc.: Research Funding. Bedrosian:Alexion Pharmaceuticals: Employment. Loirat:Alexion Pharmaceuticals: Coordinator of Alexion trials of eculizumab in atypical HUS for France. Honoraria for conferences. Other.