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Budd–Chiari syndrome (BCS) is a frequent thrombotic complication classically found in Western series of patients with paroxysmal nocturnal hemoglobinuria (PNH), with a high morbidity and mortality. Hematopoietic stem cell origin of liver endothelial cells and complement activation by enteric microbiota could explain the high frequency of this unusual thrombosis in PNH patients. However, Asian series of PNH patients show a much lower BCS prevalence, suggesting the existence of other unknown causative factors, genetic or environmental, that could explain this discrepancy. The finding of BCS is an indication to make a peripheral blood flow cytometry study to find PNH in Western patients, but this indication is not so clear in every Asian patient with BCS. Additional clinical findings, including other venous thrombosis, hemolysis, cytopenias, or renal iron overload in magnetic resonance imaging (MRI) studies of abdomen, may be required to increase the probability of PNH as the underlying thrombophilia in Asian cases. With the availability and success of eculizumab as the first complement blocker in PNH with thrombosis, a prompt diagnosis of PNH and immediate start of complement blockade plus anticoagulation are crucial for the prognosis and management of these patients. Preliminary results show that complement blockade markedly improves the results in every step of BCS treatment, preventing the complications of rethrombosis either with medical treatment, or with angioplasty, or with TIPS insertion, or with liver transplantation. Allogeneic bone marrow transplant has been relegated to the rare cases in which a syngeneic donor is available.

Eculizumab is a humanized monoclonal antibody that targets complement protein C5 and inhibits terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). The molecular basis for the poor response to eculizumab in a small population of Japanese patients is unclear.We assessed the sequences of the gene encoding C5 in patients with PNH who had either a good or poor response to eculizumab. We also evaluated the functional properties of C5 as it was encoded in these patients.Of 345 Japanese patients with PNH who received eculizumab, 11 patients had a poor response. All 11 had a single missense C5 heterozygous mutation, c.2654G → A, which predicts the polymorphism p.Arg885His. The prevalence of this mutation among the patients with PNH (3.2%) was similar to that among healthy Japanese persons (3.5%). This polymorphism was also identified in a Han Chinese population. A patient in Argentina of Asian ancestry who had a poor response had a very similar mutation, c.2653C → T, which predicts p.Arg885Cys. Nonmutant and mutant C5 both caused hemolysis in vitro, but only nonmutant C5 bound to and was blocked by eculizumab. In vitro hemolysis due to nonmutant and mutant C5 was completely blocked with the use of N19-8, a monoclonal antibody that binds to a different site on C5 than does eculizumab.The functional capacity of C5 variants with mutations at Arg885, together with their failure to undergo blockade by eculizumab, account for the poor response to this agent in patients who carry these mutations. (Funded by Alexion Pharmaceuticals and the Ministry of Health, Labor, and Welfare of Japan.).

INTRODUCTION: paroxysmal nocturnal hemoglobinuria (PNH) is a clonal, non malignant disease of hematopoiesis characterized by intravascular hemolysis, a variable depth of bone marrow failure and a marked thrombophilia. Classic therapeutic resources are supportive care and allogeneic hematopoietic stem cell transplantation. Eculizumab arrival to our country -in 2009- revolutionized PNH treatment. We report here our experience with a cohort of 33 patients treated with eculizumab. AIMS: to evaluate indications of eculizumab, hematologic response, delays to initiate treatment, interruptions and its consequences, and mortality in our patients. PATIENTS and METHODS: data of 33 patients with PNH who begun treatment with eculizumab between '01/09 and '12/14 were collected with a specific questionnaire. Qualitative data were analyzed as proportions. Quantitative data were evaluated as median (Md), range (R) and percentiles 25% and 75%, or mean (Mn) and 95% confidence interval (95% CI). Quantitative responses to treatment were evaluated with a non parametric test for paired data (Wilcoxon signed Rank test). A probability value RESULTS: cohort characteristics: Md age at diagnosis: 32 years old. R: 18 a 73. Eighteen were females and 15 males. Interval simptoms-PNH diagnosis Md: 284 days, R: 1 a 4,518. Granulocyte PNH clon size Md: 91%, R: 18 a 99%. Symptoms prevalence: fatigue: 90.6%, dyspnea: 71.9%, abdominal pain 56.2%, dysphagia 25%, erectil dysfunction: 20% (of males). Complications frequency: thrombosis 27.3%, impaired renal function 21.2%, pulmonary hypertension 3.1%. Severity criteria of PNH leading to eculizumab prescription: disabling symptoms 90.6%, steroid dependence 70%, transfusional requirement 54.5%, thrombosis 24.2%, impaired renal function 21.2%, severe dyspnea/ pulmonary hypertension 21.2%. Delay of eculizumab treatment (from medical prescription to first infusion) in 28 patients: Md: 196 days, R: 37 to 592. Hematologic responses to eculizumab: Mn LDH decline: 2,081 U/L (95% CI: 1,204 to 2,958) p=0.0003. Mn elevation of hemoglobin 1,5 g/dL (95% CI: 0.82 to 2.18) p=0.0005. Platelet counts increase in thrombocytopenic patients ( Eculizumab treatment interruptions were extremely frequent: 93.7% of patients suffered at least one or more treatment delay(s) ≥ 7 days. Only 2/33 patients did not have any treatment interruption. The mean cause of this treatment irregularities were delays in drug provision by the medical insurance (in 100% of affected patients). In only 10% of cases, lack of patient adherence was responsible for treatment interruptions. Even more, 25.8% of drug supplies were delayed for 7 days or more (99/384 provisions). Results of treatment interruptions were: transfusional requirement in 41.4% of cases, severe anemia in 34.5%, hospital admission in 27.6%, acute renal impairment in 16.7%, Budd-Chiari syndrome progression in one case, and probable cause of death in another one. Three patients died (9.1%). Causes were infection in 1, advanced colon cancer in 1, and multiorgan failure, probably related to visceral thrombosis, in the third case. CONCLUSIONS: this argentinian cohort shows that disabling symptoms, corticosteroids dependence and transfusional requirements are the most frequents causes to start eculizumab treatment in our patients. Eculizumab was effective to block hemolysis, to improve anemia and thrombocytopenia and to prevent new thrombotic events. However, eculizumab achievements were hindered in our experience by the delay to initiate treatment and the frequent interruptions it suffered, as it is usual in Latin America. These treatment delays/interruptions, uncommon in the medical literature, led to multiple and severe complications. Disclosures Brodsky: Alexion Pharmaceuticals: Consultancy, Speakers Bureau. Touliet:Alexion Pharmaceuticals: Speakers Bureau. Dinardo:Alexion Pharmaceuticals: Speakers Bureau. Blanca:Alexion Pharmaceuticals: Speakers Bureau.

Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal disease, caused by an inactivating mutation in the PIG-A gene in a hematopoietic stem cell. The PIG-A gene encodes an enzyme required for glycosylphosphatidylinositol (GPI) anchor synthesis. Its inactivation results in a deficiency of many plasma membrane GPI-anchored proteins -including CD55 and CD59, natural inhibitors of the complement cascade- in the involved stem cell and all its progeny (the PNH clone). Intravascular hemolysis, anemia, thrombosis, acute and chronic renal damage, pulmonary hypertension, abdominal pain, esophagic spasm, erectile dysfunction -among others manifestations- are consequences of complement mediated damage of the sensitive PNH blood cells. In 2007 both the FDA and the EMA approved eculizumab, a monoclonal chimeric antibody targeted against C5 fraction of complement, as the first specific treatment of complement mediated PNH manifestations. Flow cytometry (FC) is the gold standard method for diagnosis. The former diagnostic test -the Ham test- is based on the susceptibility of PHN red blood cells (RBC), when they are incubated with both normal and patient sera to lysis mediated by the alternative pathway of complement (APC). APC is activated, in the Ham test, through sera acidification. Despite its physiopathological value, Ham test has been replaced with flow cytometry to diagnose PNH due to a much higher sensitivity and reproducibility. Aims To evaluate the Ham test in PNH treated patients, to monitor the eculizumab-mediated blockade of APC. Patients and methods Ham test was used to monitor APC blockade in the patient serum, testing the ability of the acidified patient serum to lyse his or her own PNH-RBC. Eight patients were diagnosed as PNH by FC and were treated with eculizumab. Six had a good therapeutic response, with decreased levels of both, LDH and the serum total complement hemolytic capacity (CH50). Ham test, in these six patients, showed hemolysis when PNH-RBC were mixed with normal acidified serum but absence of hemolysis when the acidified serum of eculizumab treated patient was added to the PNH-RBC. This result was called “blockade profile” and shows the “ex vivo” APC blockade, confirming thus the eculizumab success. The remaining two patients showed a persistent positivity of the Ham test at day 14 of eculizumab administration (as PNH-RBC lysis continued taking place with both normal and patient acidified sera). One patient demonstrated break through hemolysis occurring near the end of eculizumab dosing period as indicated by increase in LDH. As LDH may increase due to other possible factors (ie hepatic lesions) the positive Ham test confirmed that intravascular hemolysis was taking place, possibly due to a shorter eculizumab half life. An increase of the eculizumab dose to 1,200 mg/14 days reinstated lower LDH levels and the blockade profile in the Ham test (Table). There has been a single patient treated with eculizumab where LDH did not reduce. There was a persistently positive Ham test, elevated LDH and free hemoglobin levels and normal CH50 values despite a dose of 1,200 mg of eculizumab every 14 days (Table). A genetic study found in this case a C5 mutation, which seems responsible of the lack of response to eculizumab. Conclusions In our experience, the Ham test has proved to be a useful and economic method to monitor the effectiveness of eculizumab treatment in cases with high LDH levels due to either a) other causes than intravascular hemolysis, or b) no responsive patients due to pharmacokinetic (inadequate eculizumab concentration) or pharmacodynamic causes. Disclosures: Brodsky: Alexion Pharmaceuticals: Consultancy, Speakers Bureau. Colin:Alexion Pharmaceuticals: Consultancy.

Abstract 5274 Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a chronic and life-threatening hematopoietic stem cell disorder characterized by deficiency of the GPI-anchored complement inhibitory proteins CD55 and CD59 on blood cells. The resulting uncontrolled complement activation is responsible for chronic hemolysis and can lead to serious clinical morbidities including thromboembolism (TE) and chronic kidney disease (CKD), which have been shown to increase risk of mortality. Patients may also experience debilitating quality-of-life (QoL) issues, including fatigue, shortness of breath, erectile dysfunction, and abdominal pain, attributed to chronic hemolysis and resultant nitric oxide scavenging by free hemoglobin. Although hematopoietic stem cell transplantation (HSCT) remains the only potentially curative option for PNH, the risk for substantial morbidities and mortality still exist. In patients with PNH undergoing HSCT, up to 45% die or develop acute or chronic graft-versus-host disease. Eculizumab, a first-in-class terminal complement inhibitor, is the only approved treatment for patients with PNH. By inhibiting terminal complement activity and chronic hemolysis, eculizumab reduces the incidence of TE, CKD, and transfusion requirements, improves anemia and QoL, and normalizes survival in patients with PNH. Aim: Report the benefits of eculizumab in a patient with PNH who relapsed after HSCT. Case Report: A 27-year-old woman presented in December 1993 with fever, diarrhea, hemoglobinuria, and acute renal failure requiring temporary hemodialysis (Table). She was subsequently diagnosed with PNH. In February 1995, allogeneic HSCT from an HLA-identical sibling donor was performed. In 2003, 8 years after successful engraftment, the patient relapsed and presented with hemoglobinuria, abdominal pain, corticosteroid dependence requiring 20 to 40 mg methylprednisone, and high transfusional requirements. In November 2007, she had a granulocyte clone size of 37.2% as determined by flow cytometry. In June 2009, she started eculizumab therapy which was associated with a rapid reduction in lactate dehydrogenase (LDH) from 4964U/L to 456U/L. She subsequently achieved resolution of asthenia, disabling fatigue, and abdominal pain, as well as transfusion independence and improvement in hemoglobin. In May 2010, she had a granulocyte clone size of 86.1% as determined by both CD55- and CD59-negative cells, and a granulocyte clone size of 98.7% as determined by CD16b-negative cells. Conclusions: The only potential cure for PNH—bone marrow transplantation—is associated with high risks of morbidity and mortality; therefore, for most patients the associated risks preclude this option. In this case study, we show that HSCT may not be curative in all patients and the PNH associated symptoms can arise after BMT. The potential benefits of eculizumab in this patient population should be considered in light of recent data that demonstrated normalized survival in PNH patients receiving long-term eculizumab treatment. Disclosures: Brodsky: Alexion Pharmaceuticals, Inc.: Consultancy, Speakers Bureau. Velez:Alexion Pharmaceuticals, Inc.: Consultancy.

A patient presenting with a cervix uteri epidermoid carcinoma had acute renal failure after treatment with cisplatin and bleomycin. She later developed fatal bleomycin-induced pneumonitis and Stevens-Johnson syndrome. This is a very rare association of adverse effects. The risk of causing a drug interaction thus increasing the severity of bleomycin undesired effects must be taken into account when a nephrotoxic drug is added to the former. The authors stress the importance of monitoring renal function under these conditions.