Robert A. Brodsky

HLA-Haploidentical Bone Marrow Transplantation for Hematologic Malignancies Using Nonmyeloablative Conditioning and High-Dose, Posttransplantation Cyclophosphamide

We evaluated the safety and efficacy of high-dose, posttransplantation cyclophosphamide (Cy) to prevent graft rejection and graft-versus-host disease (GVHD) after outpatient nonmyeloablative conditioning and T cell-replete bone marrow transplantation from partially HLA-mismatched (haploidentical) related donors. Patients with advanced hematologic malignancies (n = 67) or paroxysmal nocturnal hemoglobinuria (n = 1) received Cy 50 mg/kg i.v. on day 3 (n = 28) or on days 3 and 4 (n = 40) after transplantation. The median times to neutrophil (>500/microL) and platelet recovery (>20,000/microL) were 15 and 24 days, respectively. Graft failure occurred in 9 of 66 (13%) evaluable patients, and was fatal in 1. The cumulative incidences of grades II-IV and grades III-IV acute (aGVHD) by day 200 were 34% and 6%, respectively. There was a trend toward a lower risk of extensive chronic GVHD (cGVHD) among recipients of 2 versus 1 dose of posttransplantation Cy (P = .05), the only difference between these groups. The cumulative incidences of nonrelapse mortality (NRM) and relapse at 1 year were 15% and 51%, respectively. Actuarial overall survival (OS) and event-free survival (EFS) at 2 years after transplantation were 36% and 26%, respectively. Patients with lymphoid malignancies had an improved EFS compared to those with myelogenous malignancies (P = .02). Nonmyeloablative HLA-haploidentical BMT with posttransplantation Cy is associated with acceptable rates of fatal graft failure and severe aGVHD or cGVHD.

Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria

The complement system provides critical immunoprotective and immunoregulatory functions but uncontrolled complement activation can lead to severe pathology. In the rare hemolytic disease paroxysmal nocturnal hemoglobinuria (PNH), somatic mutations result in a deficiency of glycosylphosphatidylinositol-linked surface proteins, including the terminal complement inhibitor CD59, on hematopoietic stem cells. In a dysfunctional bone marrow background, these mutated progenitor blood cells expand and populate the periphery. Deficiency of CD59 on PNH red blood cells results in chronic complement-mediated intravascular hemolysis, a process central to the morbidity and mortality of PNH. A recently developed, humanized monoclonal antibody directed against complement component C5, eculizumab (Soliris; Alexion Pharmaceuticals Inc., Cheshire, CT, USA), blocks the proinflammatory and cytolytic effects of terminal complement activation. The recent approval of eculizumab as a first-in-class complement inhibitor for the treatment of PNH validates the concept of complement inhibition as an effective therapy and provides rationale for investigation of other indications in which complement plays a role.

Butyrate Greatly Enhances Derivation of Human Induced Pluripotent Stem Cells by Promoting Epigenetic Remodeling and the Expression of Pluripotency-Associated Genes

We report here that butyrate, a naturally occurring fatty acid commonly used as a nutritional supplement and differentiation agent, greatly enhances the efficiency of induced pluripotent stem (iPS) cell derivation from human adult or fetal fibroblasts. After transient butyrate treatment, the iPS cell derivation efficiency is enhanced by 15‐ to 51‐fold using either retroviral or piggyBac transposon vectors expressing 4 to 5 reprogramming genes. Butyrate stimulation is more remarkable (>100‐ to 200‐fold) on reprogramming in the absence of either KLF4 or MYC transgene. Butyrate treatment did not negatively affect properties of iPS cell lines established by either 3 or 4 retroviral vectors or a single piggyBac DNA transposon vector. These characterized iPS cell lines, including those derived from an adult patient with sickle cell disease by either the piggyBac or retroviral vectors, show normal karyotypes and pluripotency. To gain insights into the underlying mechanisms of butyrate stimulation, we conducted genome‐wide gene expression and promoter DNA methylation microarrays and other epigenetic analyses on established iPS cells and cells from intermediate stages of the reprogramming process. By days 6 to 12 during reprogramming, butyrate treatment enhanced histone H3 acetylation, promoter DNA demethylation, and the expression of endogenous pluripotency‐associated genes, including DPPA2, whose overexpression partially substitutes for butyrate stimulation. Thus, butyrate as a cell permeable small molecule provides a simple tool to further investigate molecular mechanisms of cellular reprogramming. Moreover, butyrate stimulation provides an efficient method for reprogramming various human adult somatic cells, including cells from patients that are more refractory to reprogramming. STEM CELLS 2010;28:713–72028:713–720

Cyclophosphamide and cancer: golden anniversary

Cyclophosphamide remains one of the most successful and widely utilized antineoplastic drugs. Moreover, it is also a potent immunosuppressive agent and the most commonly used drug in blood and marrow transplantation (BMT). It was initially synthesized to selectively target cancer cells, although the hypothesized mechanism of tumor specificity (activation by cancer cell phosphamidases) transpired to be irrelevant to its activity. Nevertheless, cyclophosphamides unique metabolism and inactivation by aldehyde dehydrogenase is responsible for its distinct cytotoxic properties. Differential cellular expression of aldehyde dehydrogenase has an effect on the anticancer therapeutic index and immunosuppressive properties of cyclophosphamide. This Review highlights the chemistry, pharmacology, clinical toxic effects and current clinical applications of cyclophosphamide in cancer and autoimmune disorders. We also discuss the development of high-dose cyclophosphamide for BMT and the treatment of autoimmune diseases.

Immunoablative High-Dose Cyclophosphamide without Stem-Cell Rescue for Refractory, Severe Autoimmune Disease

High-dose cytotoxic therapy followed by autologous stem-cell transplantation has been proposed as a novel treatment for severe autoimmune disease [1, 2]. This approach was prompted by autoimmune animal models that demonstrated marked improvement or complete eradication of autoimmune disease after syngeneic marrow transplantation [3, 4]. In addition, allogeneic marrow transplantation (performed chiefly for aplastic anemia) has been reported to eradicate concurrent autoimmune disease [5, 6]. Allogeneic marrow transplantation is not routinely used to treat autoimmune disease because of substantial associated morbidity and mortality. Although interest in the use of high-dose cytotoxic therapy followed by autologous stem-cell transplantation to treat autoimmune disease is increasing, disease progresses or relapses early in many patients [7, 8]. It is unclear whether reappearance of the disease after autologous transplantation results from failure of high-dose therapy to eradicate autoaggressive lymphocytes, reinfusion of autoaggressive lymphocytes with the autograft, or renewed challenge from the autoantigen [7, 8]. However, the success of syngeneic transplantation in animal models and allogeneic transplantation in patients with autoimmune diseases suggests that high-dose cytotoxic therapy may be sufficient to eradicate autoaggressive lymphocytes [8]. We previously found that the immunoablative doses of cyclophosphamide used for transplantation can induce durable, complete remission (median follow-up > 10 years) without stem-cell rescue in most patients with severe aplastic anemia [9]. Because most cases of aplastic anemia result from immune suppression of hematopoiesis [10], high-dose cyclophosphamide without the addition of other cytotoxic immunosuppressive agents seems to ablate the autoaggressive lymphocytes. We also reported that high-dose cyclophosphamide spared hematopoietic stem cells because full hematopoietic recovery occurred [9]. Hematopoietic stem cells express high levels of aldehyde dehydrogenase, an enzyme responsible for cellular resistance to cyclophosphamide, and are therefore resistant to the cytotoxic effects of cyclophosphamide [11, 12]. We investigated the efficacy of high-dose cyclophosphamide without stem-cell rescue in patients with various severe autoimmune diseases. Methods Treatment Schedule Our study was approved by the institutional review boards of Johns Hopkins University and Hahnemann University. After giving informed consent, eight patients (Table 1 and Table 3) with refractory autoimmune disorders received cyclophosphamide (50 mg/kg of body weight per day) intravenously for 4 consecutive days. Granulocyte colony-stimulating factor therapy (5 g/kg per day) was started 6 days after the last dose of cyclophosphamide and was continued until the absolute neutrophil count reached 109 cells/L. Inclusion in the study required failure of two previous therapies. Patients were excluded if their cardiac ejection fraction was less than 0.45, their serum creatinine level was greater than 176.8 mol/L, or they were older than 70 years of age. Red blood cell transfusions were administered to patients with a hematocrit less than 0.25, and platelet transfusions were given to patients with platelet counts less than 20 109 cells/L or clinically significant bleeding. Complete remission required the absence of any clinical or serologic evidence of disease. Complete remission from lupus nephritis was defined as fewer than 10 dysmorphic erythrocytes per high-powered field, absence of cellular casts, and excretion of less than 1 g of protein per day without doubling of the serum creatinine level [13]. For patients with systemic lupus erythematosus, daily activity indices [14] were measured at 3-month intervals. Table 1. Patient Characteristics and Response to High-Dose Cyclophosphamide Table 3. Table 1. Continued Selected Case Reports Patient 1 was a 64-year-old man with a 35-year history of rheumatoid arthritis treated with prednisone and gold. The Felty syndrome had been diagnosed 3 years earlier when the patient developed a perirectal abscess and profound neutropenia (neutrophil count < 0.2 109 cells/L). Examination of bone marrow showed hypercellularity with myeloid maturation arrest. The patient was treated with myeloid growth factors and steroids but showed no response. He required frequent hospitalizations for recurrent infections. Before the patient received high-dose cyclophosphamide, he was positive for antineutrophil antibodies, the neutrophil count was 0.1 109 cells/L, the rheumatoid factor level was elevated, complement levels were depressed, and the Karnofsky score [15] was 40%. The patient tolerated high-dose cyclophosphamide well and had few side effects other than alopecia; he achieved a neutrophil count greater than 0.5 109 cells/L by day 15, and infections (perirectal abscess, pneumonia, and sinusitis) that were present at the time of treatment resolved. Two units of red blood cells and five platelet transfusions were required. The patient is in complete remission 21 months after treatment and has normal peripheral blood counts, has normal complement levels, and is negative for antineutrophil antibodies. He has not been receiving any immunosuppressive agents for more than 15 months. Patient 6 was a 23-year-old woman in whom lupus was diagnosed at 12 years of age after she presented with the Raynaud phenomenon and stomatitis. She later developed severe proteinuria, hyperlipidemia, polyarthralgia, and an extensive skin rash. Renal biopsy performed 4 years before initiation of high-dose cyclophosphamide therapy showed membranous nephropathy. The patient required hospitalization for lupus flares three to four times per year despite treatment with methylprednisolone (4 mg/d), hydroxychloroquine (400 mg/d), azathioprine (150 mg/d), and pulse-dose cyclophosphamide. Before high-dose cyclophosphamide therapy began, the hematocrit was 0.27, the leukocyte count was 2.8 109 cells/L, the platelet count was 278 109 cells/L, and the erythrocyte sedimentation rate was 104 mm/h. Anti-DNA antibodies were present at a titer of 1:320, the C3 level was 0.41 g/L, and the 24-hour urine protein level was 2 g. The patient tolerated high-dose cyclophosphamide well; side effects were alopecia and febrile neutropenia. A neutrophil count greater than 0.5 109 cells/L was reached on day 18, and only six units of red blood cells and three platelet transfusions were needed. The patient is in continuous complete remission 12 months after treatment; the erythrocyte sedimentation rate is 20 mm/h, no anti-DNA antibodies are present, the C3 level is 1.49 g/L, and the 24-hour urine protein level is 86 mg. Immunosuppressive therapy has been tapered to 1 mg of prednisone daily. Results High-dose cyclophosphamide was well tolerated and was associated with rapid hematologic recovery in all eight patients despite their poor medical condition at time of treatment. Four patients were hospitalized for complications of their autoimmune disease, and four patients were being treated for active infections at the time of cyclophosphamide therapy; the median Karnofsky score was 40% (range, 20% to 70%). The median time to achievement of a neutrophil count greater than 0.5 109 cells/L was 17 days (range, 11 to 22 days), and the median time to the last platelet transfusion was 16 days (range, 12 to 33 days). All patients experienced complete alopecia, and six patients required antibiotics for febrile neutropenia. No patient developed hemorrhagic cystitis or mucositis. Patients 2 and 4 eventually died of complications of autoimmune disease. Patient 2, who was treated for autoimmune hemolytic anemia, died of complications of immune thrombocytopenic purpura, which was not present when she was treated with high-dose cyclophosphamide. Autoimmune hemolytic anemia remained in complete remission until the patients death, 16 months after cyclophosphamide therapy. Patient 4 achieved brief remission of immune thrombocytopenic purpura and died of her disease 8 months later. Six patients remain alive, and five (patients 1, 3, 6, 7, and 8) have no symptomatic manifestations of their disease. In addition, four patients have no laboratory or clinical evidence of disease (Table 2). Patient 3, who has the Evans syndrome, shows continued improvement in blood counts; prednisone therapy is being tapered to 10 mg every other day. Patient 3 has been independent of transfusion for more than 10 months and has a normal hemoglobin level and a platelet count of 66 109 cells/L. One of the patients with lupus achieved complete remission; the other still has clinical and serologic evidence of the disease but continues to improve 14 months after treatment. Patient 7, who has the Felty syndrome, is in complete remission 3 months after cyclophosphamide therapy. Patient 10, who has chronic inflammatory demyelinating polyneuropathy, had progressive upper- and lower-extremity paralysis and was unable to walk. Plasmapheresis, intravenous immunoglobulin, and pulse-dose cyclophosphamide therapy had proven ineffective. Three months after therapy with high-dose cyclophosphamide, he has no neurologic manifestations, is not receiving immunosuppressive therapy, and can walk normally. Table 2. Laboratory Results* Discussion Most immunoablative therapy for severe autoimmune disease uses autologous stem-cell rescue after high-dose therapy with cyclophosphamide in combination with other immunosuppressive agents [8]. Although our study was small and the follow-up was relatively short, the results indicate that high-dose cyclophosphamide alone can be effective therapy for some patients with severe autoimmune disease. In addition, our study confirms that high-dose cyclophosphamide (50 mg/kg per day for 4 days) spares hematopoietic stem cells; the kinetics of bone marrow recovery after high-dose cyclophosphamide therapy without stem-cell rescue are similar to those of engraftment after autol

Whole-Genome Sequencing Analysis Reveals High Specificity of CRISPR/Cas9 and TALEN-Based Genome Editing in Human iPSCs

Human iPSCs provide renewable cell sources for human biology and disease research and the potential for developing gene and cell therapy. Realization of this potential will rely in part on our ability to precisely edit or engineer the human genome in an efficient way. Recent developments in designer endonuclease technologies such as zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas9 endonuclease have provided ways to significantly improve genome editing efficiency in human iPSCs. These endonucleases make a double-stranded break (DSB) at a predetermined DNA sequence and trigger natural DNA repair processes such as nonhomologous end joining (NHEJ) or homologous recombination (HR) with a donor DNA template. Among these existing approaches, RNA-guided CRISPR/Cas9 is the most user-friendly and versatile system, and it has been applied in both animal models and cell lines (Cong et al., 2013; Hsu et al., 2014; Mali et al., 2013). The most commonly used system consists of a single polypeptide endonuclease Cas9 complexed with a single guide RNA (gRNA) that provides complementarity to 20-nucleotide target DNA sequence. However, the specificity and efficiency of this approach in human iPSCs have not been studied in detail (Cong et al., 2013; Ding et al., 2013; Mali et al., 2013; Yang et al., 2013). Some analyses using cancer cell lines reported higher-than-expected levels of off-target mutagenesis by Cas9-gRNAs (Fu et al., 2013; Hsu et al., 2013), raising concerns about the practical applicability of this approach in therapeutic contexts. Some recent studies, including one on human adult stem cells, showed a minimal level of off-target effects by CRISPR/Cas9 (Schwank et al., 2013). However, these existing analyses of off-target effects and mutational load in gene-corrected stem cells have been restricted to checking predicted off target sites and are therefore limited in scope. To assess the value of this type of gene editing approach for therapeutic applications, it is critical to rigorously examine whether it is possible to generate gene-edited cell lines with minimal mutational load. To this end, we have conducted whole-genome sequencing of four iPSC clones successfully targeted at the AAVS1 locus, a “safe harbor” in the human genome that is used for stable transgene expression in a variety of contexts. To generate the lines, we used an integration-free human iPSC line, BC1, whose genomic integrity has been characterized in detail by next-generation sequencing (Cheng et al., 2012) and targeted a GFP expression cassette into the AAVS1 site with either a previously reported Cas9-gRNA combination or a pair of improved heterodimeric TALENs (Mali et al., 2013; Yan et al., 2013) (Table S1 and Supplemental Experimental Procedures available online). Twenty days after transfection of the donor plasmid and either the TALENs or Cas9-gRNA into BC1, we harvested four clones with confirmed targeted integration (hCas9-C4, hCas9-C16, TALEN-C3, and TALEN-C6; Table S1 and Supplemental Experimental Procedures) and the parental BC1 iPSCs for whole-genome sequencing. The sequencing reads, ranging from 83 Gbps to 100 Gbps from each targeted clone, were first aligned to the human hg19 reference genome to enable identification of single-nucleotide variants (SNVs) and small indels (Table S1). Our analysis identified ≥4.2 million SNVs and ≥500,000 indels in each genome (Table S1) in comparison to the hg19 reference genome, suggesting that it is a rigorous data set that covers the genome in sufficient depth to detect sequence variants. The “germ-line” variants (present in BC1 parental iPSCs and different from hg19) were readily detectable in each targeted cell line (80%%–88%), indicating that the sensitivity of variant detection in our analysis is high (Table S1). The variations from each targeted clone were then compared to the BC1 parental iPSCs to enable the generation of a list of potential variations arising during the gene editing process, which we then confirmed using genomic PCR and Sanger sequencing. We confirmed 62 out of 69 SNVs tested for an overall confirmation rate of 90%, and based on that we estimate that the total SNVs in the four iPSC clones range between 217 and 281 and that the total indels range between 7 and 12 (Table S1). Overall the genomic variation levels in TALEN- and Cas9-targeted groups were comparable. One important consideration is how many of these detected SNVs and indels were the results of off-target mutagenesis by the engineered endonucleases. To address this question, we generated a list of 3,665 (Cas9) and 238 (TALEN) putative off-target positions by using the EMBOSS fuzznuc software package. Each candidate SNV and indel was compared to this list and none of them are within a potential off-target region (Table S1), consistent with previous analyses looking at predicted off-target sites. Our analysis also shows that each SNV and indel is unique and that none of them occurred in more than one cell line. The absence of recurring mutations and the fact that none of the mutations resides in any putative off-target site by bioinformatic prediction strongly suggest that these mutations were randomly accumulated during regular cell expansion and are not direct results of off-target activities by Cas9 or TALENs. Our results from whole-genome sequencing analysis of Cas9- and TALEN-targeted human iPSC clones demonstrate that these engineered endonucleases provide efficient genome-editing tools with high specificity. It remains to be clarified whether the higher off-target rates observed in cancer cell lines are due to the overexpression of gRNAs and Cas9 protein and/or due to exacerbated and faulty DNA repair in these cell types. The higher specificity observed in human iPSCs, combined with the rapid development of next-generation sequencing technology, makes it possible to characterize and isolate high quality genome-edited stem cell clones with minimal mutational load. The guiding principle established with human iPSCs will likely be applicable to other types of stem cells and come with improvements in gene transfer and targeting efficiencies. Our current study of gene targeting in human iPSCs will help to establish better models for human biology and disease research and to provide proof-of-principle for future gene therapy.

HLA-haploidentical bone marrow transplantation with posttransplant cyclophosphamide expands the donor pool for patients with sickle cell disease

Allogeneic marrow transplantation can cure sickle cell disease; however, HLA-matched donors are difficult to find, and the toxicities of myeloablative conditioning are prohibitive for most adults with this disease. We developed a nonmyeloablative bone marrow transplantation platform using related, including HLA-haploidentical, donors for patients with sickle cell disease. The regimen consisted of antithymocyte globulin, fludarabine, cyclophosphamide, and total body irradiation, and graft-versus-host disease prophylaxis with posttransplantation high-dose cyclophosphamide, mycophenolate mofetil, and tacrolimus or sirolimus. After screening 19 patients, we transplanted 17, 14 from HLA-haploidentical and 3 from HLA-matched related donors. Eleven patients engrafted durably. With a median follow-up of 711 days (minimal follow up 224 days), 10 patients are asymptomatic, and 6 patients are off immunosupression. Only 1 patient developed skin-only acute graft-versus-host disease that resolved without any therapy; no mortality was seen. Nonmyeloablative conditioning with posttransplantation high-dose cyclophosphamide expands the donor pool, making marrow transplantation feasible for most patients with sickle cell disease, and is associated with a low risk of complications, even with haploidentical related donors. Graft failure, 43% in haploidentical pairs, remains a major obstacle but may be acceptable in a fraction of patients if the majority can be cured without serious toxicities.

High-dose cyclophosphamide as single-agent, short-course prophylaxis of graft-versus-host disease

Because of its potent immunosuppressive yet stem cell-sparing activity, high-dose cyclophosphamide was tested as sole prophylaxis of graft-versus-host disease (GVHD) after myeloablative allogeneic bone marrow transplantation (alloBMT). We treated 117 patients (median age, 50 years; range, 21-66 years) with advanced hematologic malignancies; 78 had human leukocyte antigen (HLA)-matched related donors and 39 had HLA-matched unrelated donors. All patients received conventional myeloablation with busulfan/cyclophosphamide (BuCy) and T cell-replete bone marrow followed by 50 mg/kg/d of cyclophosphamide on days 3 and 4 after transplantation. The incidences of acute grades II through IV and grades III through IV GVHD for all patients were 43% and 10%, respectively. The nonrelapse mortality at day 100 and 2 years after transplantation were 9% and 17%, respectively. The actuarial overall survival and event-free survivals at 2 years after transplantation were 55% and 39%, respectively, for all patients and 63% and 54%, respectively, for patients who underwent transplantation while in remission. With a median follow-up of 26.3 months among surviving patients, the cumulative incidence of chronic GVHD is 10%. These results suggest that high-dose posttransplantation cyclophosphamide is an effective single-agent prophylaxis of acute and chronic GVHD after BuCy conditioning and HLA-matched BMT (clinicaltrials.gov no. NCT00134017).

Natural history of paroxysmal nocturnal haemoglobinuria using modern diagnostic assays

Paroxysmal nocturnal haemoglobinuria (PNH) is an uncommon, acquired disorder of blood cells caused by mutation of the phosphatidylinositol glycan class A (PIG‐A) gene. The disease often manifests with haemoglobinuria, peripheral blood cytopenias, and venous thrombosis. The natural history of PNH has been documented in retrospective series; but there has only been one study that correlated the more sensitive and specific flow cytometric assays that have become available in the last decade with severe symptoms associated with PNH. In a retrospective analysis of 49 consecutive patients with PNH evaluated at Johns Hopkins, large PNH clones were associated with an increased risk for thrombosis as well as haemoglobinuria, abdominal pain, oesophageal spasm, and impotence. Of the 14 (29%) patients that developed thrombosis, nine died; six of these from complications related to thromboses. According to logistic regression modelling, for a 10% change in PNH clone size, the odds ratio for risk of thrombosis was estimated to be 1·64. No patient with <61% PNH granulocytes developed a thrombosis, whereas 12 of 22 patients (54·5%) with ≥61% PNH granulocytes manifested with thrombosis. These data not only confirm that the size of the PNH clone correlates with the risk for thrombosis, but they also suggest a correlation of PNH clone size to more symptomatic PNH.

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.