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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.).

The present study focused on the desensitization process of the H(2) receptor in U937 cells and the recovery of the cyclic AMP (cAMP) response. Treatment of U937 leukemic cells with the H(2) histamine receptor agonists (+/-)-N(1)-[3-(3, 4-difluorophenyl)-3-(pyridin-2-yl)propyl]-N(2)-[3-(1H-imidazol-4-yl)p ropyl]guanidine (BU-E-75) and amthamine produced a rapid desensitization characterized by decreased cAMP production (T(1/2) = 20 min). Pretreatment with 10 microM BU-E-75 did not induce modifications in the responses to prostaglandin E(2), isoproterenol, or forskolin. H(2) receptor desensitization was not affected by protein kinase A and C inhibitors, but was reduced drastically by Zn(2+) and heparin, known to act as inhibitors of G protein-coupled receptor kinases. Recovery studies of the cAMP response showed that cAMP levels reached 50% of the initial values within 5 hr. Furthermore, desensitization produced an important decrease in the basal level of this cyclic nucleotide. The minimal value was observed 12 hr later, and corresponded to approximately 1.3% of the initial basal level (7.5 vs 0.1 pmol/10(6) cells). This result could be explained by an increase in phosphodiesterase activity following 10 microM BU-E-75 treatment. When cells were exposed for 2 hr to an H(2) agonist, binding assays showed no modification in the number of H(2) receptors; internalization began just after 8 hr. Although the initial desensitization seems to involve G protein-coupled receptor kinases, results indicate that additional mechanisms of regulation were triggered by the H(2) agonists. Fil: Lemos Legnazzi, Bibiana. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Shayo, Carina Claudia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Monczor, Federico. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Martín, María E.. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Fernández, Natalia Brenda. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Brodsky, Andres. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Baldi, Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Davio, Carlos Alberto. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

The present study examines the effects of forskolin on U937 cell differentiation. We recently reported that dibutyryl cAMP (dbcAMP), but not cAMP-elevating agents such as histamine, promotes U937 cell differentiation. cAMP production elicited by stimulation of histamine H2 receptors showed a rapid, homologous desensitization, which might explain the dissimilar responses to histamine and dbcAMP. Forskolin induced an increase in cAMP levels in a concentration-dependent manner (EC50=30 microM) for an extended period of at least 24 h. Forskolin but not histamine (up to 100 microM), also inhibited cell growth in a dose-dependent fashion (EC50=22 microM). After 3 days of incubation, 75 microM forskolin induced U937 cell differentiation as judged by an increased rate of reduction of nitrobluetetrazolium (mean+/-S.E.M.: 21.3+/-6.6% in treated cells vs. 3.2+/-1.9% in the control group, P0.001) and an augmented chemotactic response to complement 5a (C5a) (33.2+/-5.9% in forskolin-treated vs. 0.34+/-0.12% in control cells, P0.01). Furthermore, c-Myc levels decreased following forskolin treatment, while the histamine H2 receptor agonist dimaprit had no effect. We conclude that forskolin induces U937 cell differentiation through a sustained rise in cAMP levels.

We examined the effects of histamine and its agonists on the expression of the c-fos and c-myc proto-oncogenes at the transcriptional and translational levels in the human promonocytic U937 cell line. Histamine transiently increased cAMP and c-fos expression through H2 receptors. Dibutyryl cAMP also increased c-fos mRNA and protein, and levels remained elevated even after 12 hr of treatment. Dose-dependence studies using histamine and dimaprit showed that the EC50 values for cAMP production and c-fos increase were similar, suggesting that cAMP might be involved in c-fos induction via H2 receptors. Furthermore, studies carried out using H7, a protein kinase A/protein kinase C inhibitor, blocked c-fos induction, whereas no effect was observed with bisindolylmaleimide, a specific protein kinase C inhibitor. No modification of c-myc expression could be detected on treatment with histamine or its analogues. Nevertheless, dibutyryl cAMP induced a down-regulation of the levels of this proto-oncogene. In addition, dibutyryl cAMP inhibited cell growth in a dose-dependent manner, whereas histamine failed to affect proliferation and differentiation of U937 cells. Cells pretreated with dimaprit showed a decrease in the cAMP response to subsequent addition of H2 agonists, whereas the cAMP response to prostaglandin E2 remained unaltered. This homologous mechanism of H2 receptor desensitization was time dependent. These results indicate that histamine activates several mechanisms involved in the induction of differentiation, such as cAMP and c-fos production, but fails to promote differentiation of U937 cells, apparently due to the rapid desensitization of H2 receptors.

Introduction: Early reduction of BCR-ABL transcript level has been associated with improved outcomes in CML treatment. Inability to achieve early molecular response(MR) at 3 months (M3>10%) is considered a predictor factor for unfavourable outcome. However, the kinetics of BCR-ABL transcript level reduction measured at early time points have shown to be an independent predictor of response.The aim of this analysis was to determine whether the "M3-M6" status is critical to categorize CML patients (pts) focusing in high-risk group. Method: Molecular monitoring was performed in all pts prior treatment (M0), at months 3 (M3), 6 (M6), 12 (M12) and every 6 months thereafter, applying Q-PCR method according international recommendations. Results of BCR-ABL1 transcript level were reported on the international scale as IS-BCR-ABL %. Optimal responses: M3≤10%, M6≤1%, M12≤0,1%. Deep responses (MR4.0): ≤0,01% or undetectable/10.000 ABL copies. Results: A total of 70 CML pts were included, median age 49 (19-82), female 39%. First line treatment: sustained branded 81% and generic 19% TKIs. Imatinib 59%, Dasatinib and Nilotinib 41%. Sokal risk score: low (L) 51%, intermediate (In) and high (H) 49%. Optimal responses at molecular milestones: 75% at M3, 72% at M6, 61% at M12 and 53% pts achieved MR4.0. Event-free survival (EFS) was evaluated according to time point M3: M3≤10% group had significantly better EFS compared with the M3>10% (96% vs 70%; P=0.028). M3-M6 status defined 4 groups of pts: M3≤10%-M6≤1%, M3≤10%-M6>1%, M3>10%-M6≤1%, M3>10%-M6>1%. Molecular response evolution by M3-M6 status is described in Table 1. EFS stratified by groups according to combined M3-M6 responses showed significant differences: 92% for group 1, 87% for group 2, 68% for group 3, 54% for group 4. (P=0.002). M6 time point was shown to be critical in 32 high-risk pts (H+In): 17 pts with M6 ≤1% showed significant differences in MR4.0 achievement compared to 15 pts with M6 >1% (82% vs 27% P=0.02). Better EFS was observed in this high-risk group under branded vs generic TKIs treatment (97% vs 54% P=0.04). Statistical differences in deep responses and MMR at M12 were observed between branded and generic TKIs independently of Sokal risk (P=0.06, P=0.02). Conclusions: M3≤10% pts showed a favourable evolution with better EFS than M3>10% group. However not all patients with M31%. In pts with M3 >10% and optimal response at M6 also showed higher MR4.0 rate. Our study supports that M6 is a crucial endpoint to predict MMR at M12 and deep responses in CML pts.Pts with M3≤10% without optimal response at M6 (>1%) had a worse evolution than those slow responders who showed M3>10% and M6≤1%.High-risk pts are still a challenge, observing better outcomes in those under branded TKIs treatment. The M3-M6 status would be a prognostic marker of responses and EFS in chronic phase CML pts treated with TKIs. Our data support the critical role of M6 response in non-optimal M3>10% pts and intermediate and high risk Sokal score. Treatment adherence is mandatory for achieving and sustaining optimal responses. This multicentric Argentine study, reinforces the importance of clinical follow-up and molecular monitoring under IS standardization at early time points. Education on early molecular monitoring with adequate resources must continue to be an objective in our region. Table 1 Table 1. Disclosures Pavlovsky: Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Speakers Bureau. Moiraghi:Novartis: Speakers Bureau; Bristol: Speakers Bureau. Varela:Novartis: Speakers Bureau; Bristol: Speakers Bureau. Enrico:Novartis: Honoraria, Patents & Royalties; Bristol Myers squib: Speakers Bureau. Brodsky:International PNH Registry: Other: -; Alexion Pharma Argentina: Speakers Bureau. Pavlovsky:Novartis: Speakers Bureau; Janssen: Speakers Bureau; Bristol Myers Squib: Speakers Bureau.

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.

I have read the very interesting and informative article of Hoover et al,[1][1] which may open new avenues in treating imatinib-refractory and imatinib-sensitive chronic myeloid leukemia (CML). However, when testing the synergic potential of STI + farnesyl transferase inhibitor (FTI) combination on

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.