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Introduction: Novel targeted therapies and monoclonal antibodies can be combined with allogeneic stem cell transplantation (allo-SCT) at different time-points: 1) before the transplant to reduce tumour burden, 2) as part of the conditioning in place of or in addition to conventional agents 3) after the transplant to allow long-term disease control. Areas covered: This review focuses on the current integration of new drugs with allo-SCT for the treatment of major hematological malignancies for which allo-SCT has been a widely-adopted therapy. Expert opinion: After having been used as single agent salvage treatments in relapsed patients after allo-SCT or in combination with donor lymphocyte infusions, many new drugs have also been safely employed before allo-SCT as a bridge to transplantation or after it as planned consolidation/maintenance. This era of new drugs has opened new important opportunities to ‘smartly’ combine ‘targeted drugs and cell therapies’ in new treatment paradigms that may lead to higher cure rates or longer disease control in patients with hematological malignancies.

We analyzed the outcome of 537 adolescents (age 12-18 years) with idiopathic aplastic anemia included in the database of the Severe Aplastic Anemia Working Party of the European Group for Blood and Marrow Transplantation comparing: i) matched family donor hematopoietic stem cell transplantation performed as first-line treatment with ii) front-line immunosuppressive therapy not followed by subsequent transplant given for failure and with iii) hematopoietic stem cell transplantation performed after failed front-line immunosuppressive therapy. Overall survival was 86% in the matched family donor hematopoietic stem cell transplantation group, 90% in patients given front-line immunosuppressive alone (those who did not fail this treatment and who did not receive subsequent rescue with hematopoietic stem cell transplantation) and 78% in subjects who underwent hematopoietic stem cell transplantation post failed front-line immunosuppressive therapy (P=0.14). Event-free survival in the same groups was respectively 83%, 64% and 71% (P=0.04). Cumulative incidence of rejection was 8% in matched family donor hematopoietic stem cell transplantation and 9% in transplants post failed front-line immunosuppression (P=0.62). Cumulative incidence of acute graft-versus-host disease was 12% in matched family donor transplants and 18% in transplants post failed immunosuppression (P=0.18). Chronic graft-versus-host disease was higher in matched family donor hematopoietic stem cell transplantation (8%) than in transplants post failed immunosuppressive therapy (20%) (P=0.0009). Cumulative incidence of post-therapy malignancies was 0.7% in matched family donor transplantations, 7% in transplantations post failed immunosuppression and 21% after front-line immunosuppression (P=0.0017). In the whole cohort, under multivariate analysis, the diagnosis to treatment interval of two months or under positively affected overall survival whereas up-front immunosuppression alone (with no subsequent rescue transplants) negatively affected event-free survival. In transplanted patients an interval from diagnosis to treatment of 2 months or under, bone marrow as source of cells and first-line matched family donor transplants provided a significant advantage in overall and event-free survival. Aplastic anemia in adolescents has a very good outcome. If a matched family donor is available, hematopoietic stem cell transplantation using bone marrow cells is the first choice treatment. If such a donor is not available, immunosuppressive treatment may still be an acceptable second choice, also because, in case of failure, hematopoietic stem cell transplantation is a very good rescue option.

INTRODUCTION Diamond-Blackfan Anemia (DBA) is a congenital pure red cell aplasia, secondary to ribosomal protein genetic defects that usually presents within the first year of age and can be associated with congenital abnormalities and increased risk of cancer. Some patients are successfully treated with steroids but most of them remain transfusion-dependent. Stem Cell Transplantation (SCT) represents the only curative option for this disease, however data from literature is scarce and limited to a very small number of cases. In this retrospective study we describe the outcome of SCT in patients with DBA reported in the EBMT data base. PATIENTS AND METHODS The study was conducted on behalf of Severe Aplastic Anemia Working Party of the EBMT and was based on data of patients affected with DBA who underwent SCT and registered in the EBMT Data Base. Clinical information of the disease and details on transplant procedures and follow-up were collected by a specific form distributed to Centres participating in the study. RESULTS Between 1985-2016, 106 patients (60 males-46 females) aged 6.8 yo (range 1-32) underwent SCT from matched sibling donor (58, 57%), unrelated donor (37, 36%) or from other donors (7, 7%) using bone marrow (73, 69%), peripheral blood (21, 20%) or cord blood (12, 11%) as cell source. 91 patients (86%) received a myeloblative regimen which included Busulfan in 66 cases. 86% (80-93%) of patients engrafted by day 28. Median days to neutrophils and platelets engraftment were 18 and 36, respectively. EFS and OS at 36 months were 81% (74-89%) and 84% (77-91%), respectively. OS was significantly higher (p=0.01) in patients CONCLUSION To the best of our knowledge, this is the largest reported cohort of patients transplanted for DBA and having a long follow-up. The very good OS of patients undergoing transplant from both sibling and unrelated donors and the rather low rate of cGvHD confirms that this procedure can be considered an alternative option for transfusion-dependent patients Disclosures Bader: Riemser, Neovii: Research Funding; Medac: Patents & Royalties, Research Funding; Amgen (Brasil), Novartis: Consultancy, Speakers Bureau; Celgene: Consultancy. Risitano:Alexion: Honoraria, Research Funding, Speakers Bureau; Achillion: Research Funding; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Biocryst: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Amyndas: Consultancy; Samsung: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Amyndas: Consultancy; Biocryst: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Research Funding; Ra Pharma: Research Funding; Alnylam: Research Funding; Alnylam: Research Funding; Achillion: Research Funding. Peffault de Latour:Pfizer: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding.

Background: PNH is a rare, acquired blood disorder. A somatic mutation in the PIGA gene of one or more hematopoietic stem cells generates a clone of abnormal erythrocytes (RBCs) that lack alternative pathway (AP) regulatory complement proteins CD55 and CD59. This leads to uncontrolled complement activation on affected RBCs and intravascular hemolysis (IVH). Standard of care is C5 inhibition to prevent membrane attack complex (MAC) formation. Despite preventing MAC-mediated IVH, many patients experience extravascular hemolysis (EVH). Despite C5 inhibition, ~ 70% of patients remain anemic and >1/3 were transfused > 1 time in the prior 12 months. Factor D (FD), a serine protease, catalyzes complement factor B cleavage, allowing formation of AP C3 convertase. By inhibiting FD, oral danicopan blocks C3 convertase formation, the control point for AP activation and amplification of all pathways. This leads to inhibition of C3 cleavage, C3 fragment deposition, terminal pathway activation and MAC formation. Thus, danicopan can control both IVH and EVH therefore, making FD a promising target. Aim: Demonstrate that danicopan is a potential treatment for PNH patients with an inadequate response to C5 inhibition. Methods: Data are presented for this Ph 2, dose-finding, proof of concept trial of danicopan in patients with an inadequate response to eculizumab who were transfusion dependent. Additional criteria in Table 1. In addition to the current eculizumab regimen, danicopan starting doses were 100-150 mg TID, with dose escalation up to 200 mg TID, based on clinical and biochemical response, at protocol defined time points (Figure 1). The primary endpoint was change in Hgb at Treatment Week (TW) 24. Secondary efficacy parameters included transfusion needs, effect on lactate dehydrogenase (LDH), and an exploratory an endpoint of Functional Assessment of Chronic Illness Therapy (FACIT) FATIGUE scores. General safety, tolerability, and PK/PD of danicopan were measured. After TW 24, patients entered a long-term extension. Results: Twelve patients received at least one dose of danicopan. One patient discontinued after 2 doses, due to a serious adverse event of worsening of an underlying condition (pulmonary hypertension/edema), considered unlikely related to danicopan. This patient is excluded from this analysis. Eleven patients continue to receive treatment. Dose titrations are shown in Table 2. Benefits were observed in multiple laboratory markers of PNH, shown in Table 3. Hgb improved in all patients, with a mean Hgb gain of 2.6 g/dL at 24 Weeks of treatment (n=4). Meaningful improvement in FACIT Fatigue scores were reported, with a mean increase of 8 points relative to the baseline on eculizumab. A 3-point change is clinically meaningful on this scale. Transfusion needs dramatically reduced, with one patient receiving one transfusion during the trial, as compared to 34 transfusions (58 units) in 10 patients, in the 6 months prior to screening. One patient (who does not accept blood products due to religious objections and also has hereditary elliptocytosis) had a baseline Hgb=5 g/dL with a 3.5 g/dL improvement at TW24 and significant improvement in fatigue. C3 fragment deposition was inhibited; reticulocytes, total/ direct bilirubin and LDH returned to normal range. Baseline C5 inhibition (classical pathway activity) was essentially inhibited (mean=1 [range 60-144]). Three of 11 patients received > 900 mg eculizumab, making PK a less likely reason for inadequate response. Danicopan has been well tolerated; 96% of treatment emergent adverse events (TEAEs) were mild to moderate in severity and no discontinuations due to TEAEs. Two patients had Grade 3 TEAEs which resolved and continued in the trial. Conclusion: Proof of concept is established with danicopan, an oral, small molecule FD inhibitor in the treatment of PNH in combination with eculizumab. Meaningful improvements in Hgb, transfusion needs, FACIT-FATIGUE and other parameters of interest were achieved. These clinically significant improvements demonstrate that further benefit can be achieved by blocking the alternative pathway at FD with danicopan, in combination with C5 inhibition. This benefit is likely due to the prevention of C3-mediated EVH, in addition to control of IVH. Danicopan targets an unmet need in PNH and will be further evaluated in a pivotal trial in combination with standard of care C5 inhibition. Disclosures Kulasekararaj: Achillion: Consultancy; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Akari: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alnylam: Membership on an entity's Board of Directors or advisory committees. Risitano:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amyndas: Consultancy; Achillion: Research Funding; Amyndas: Consultancy; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Alexion: Honoraria, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Alnylam: Research Funding; Achillion: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ra Pharma: Research Funding; Biocryst: Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Biocryst: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Research Funding; Samsung: Membership on an entity's Board of Directors or advisory committees; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maciejewski:Novartis: Consultancy; Alexion: Consultancy. Notaro:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: letture fees. Browett:Janssen: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Beigene: Research Funding; AbbVie: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding. Lee:Alexion: Consultancy, Honoraria, Research Funding; Achillion: Research Funding. Huang:Achillion: Employment, Equity Ownership. Geffner:Achillion: Employment, Equity Ownership. Brodsky:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: Grant funding; Achillion: Research Funding.

Introduction: Hematopoiesis is severely reduced in severe aplastic anemia (SAA) probably due to immunological destruction of hematopoietic stem and progenitor cells. Bone marrow transplantation is preferred as first-line therapy in patients younger than 40 years with a matched related sibling donor. For those who are not candidate for transplant, immunosuppressive therapy with horse antithymocyte globulin (hATG) plus cyclosporine (CsA) remains the standard of care. However, hATG was discontinued in most Asian, South American, and European countries with only rabbit ATG (rATG) available. rATG is a more potent immunosuppressant which has associated with a worst hematological response and survival at 6 months in prospective studies. Using rATG in first-line therapy appears to add little to what has been observed with CSA alone. Alemtuzumab (ALZ), a humanized anti-CD52 monoclonal antibody is also active in AA due to its lymphocytotoxic properties. The use of ALZ in monotherapy has shown an overall response rate (ORR) comparable to that of r-ATG (37% and 33%, respectively) when applied as salvage (following hATG failure) in a randomized study. Other experiences which combined ALZ and CsA led to a 58% ORR in SAA patients after a cumulative dose of 103 mg of subcutaneous (SC) ALZ. Here we describe a multicenter retrospective analysis of SC ALZ in association with CsA for patients with AA. Methods: We retrospectively analyzed all patients who received outpatient SC ALZ for the treatment of aplastic anemia in 2 centers: Universidade Federal de Sao Paulo (Sao Paulo-BRAZIL) and Leeds Teaching Hospitals, UK from March 2009 until March 2019. In Sao Paulo, alemtuzumab total dose was 103 mg in an escalating dose of 3-10-30-30-30 mg, except for three elderly patients who received a total dose of 73. In Leeds-UK, total dose varied from 120 mg to 150 mg. The primary outcome was overall response rate (ORR) at six months. Median follow up was 31 months (range 4-110 months). Results: We identified 35 treatments with SC ALZ in 32 AA patients of which 78% had SAA or very severe aplastic anemia. Calcineurin inhibitors were used in association with ALZ in 80% of cases (cyclosporine in 26 cases, tacrolimus in 2 cases). Median age was 44 years (range: 18-79), and nineteen patients (59%) were male. Seventeen patients (53%) were treatment-naïve, and six patients (19%) had one prior line of therapy. Nine patients (28%) were relapsed/refractory, and received ALZ after at least two lines of prior therapy. Seventeen patients (53%) presented a PNH clone at diagnosis. Median time between diagnosis and ALZ treatment was 6 months (range 1-293). No treatment-related serious adverse events were observed. Infectious complications were infrequent: there was only one case of successfully treated CMV reactivation and one case of fatal EBV-related infection. ORR at 6 months was 57% (complete response: 11%, partial response: 46%), with corresponding cumulative incidence of response of 47% at 6 months and 62% at 1 year (figure 1). ORR was 69% in treatment-naïve patients younger than 60 years. Overall survival was 72% at 2 years. Adverse events were of low grade and infectious events were infrequent. Conclusion: Subcutaneous alemtuzumab appears to be a feasible, effective and safe alternative to hATG in patients with AA requiring immunosuppressive treatment, especially in centers with limited access to hATG. Disclosures Hill: Apellis: Honoraria; Roche: Honoraria; Akari: Honoraria; Alexion: Honoraria; Bioverativ: Honoraria; Novartis: Honoraria; Regeneron: Honoraria; Ra Pharma: Honoraria. Munir:AbbVie: Honoraria; Alexion: Honoraria; Gilead: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Roche: Honoraria; Morphosys: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sunesis: Consultancy; Pharmacyclics: Other: TBC; Acerta: Membership on an entity's Board of Directors or advisory committees. Hillmen:Roche: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses, Research Funding; Pharmacyclics LLC, an AbbVie Company: Consultancy, Honoraria, Research Funding; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses, Research Funding; Apellis: Research Funding; Gilead: Research Funding. Risitano:Achillion: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Biocryst: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Amyndas: Consultancy; Ra Pharma: Research Funding; Alnylam: Research Funding; Achillion: Research Funding; Alexion: Honoraria, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Alnylam: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Biocryst: Membership on an entity's Board of Directors or advisory committees; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amyndas: Consultancy; Ra Pharma: Research Funding; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau. Scheinberg:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer,: Speakers Bureau; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau.

Background: In PNH, a somatic mutation in the PIGA gene of one or a few hematopoietic stem cells generates a clone of abnormal erythrocytes which lack two key alternative pathway (AP) regulatory proteins, CD55 and CD59, leading to uncontrolled complement activation on affected erythrocytes (RBCs) and membrane attack complex (MAC)-mediated lysis. Factor D (FD), a serine protease, catalyzes the cleavage of complement factor B into Ba and Bb, which allows for the formation of the AP C3 convertase. By inhibiting FD, danicopan, an oral small molecule FD inhibitor, blocks C3 convertase formation, the control point for AP activation as well as the amplification of all pathways. This leads to inhibition of C3 cleavage, C3 fragment deposition, terminal pathway activation and MAC formation. Therefore, FD is a promising target in diseases of excess activation of the AP, such as PNH. Aims: Correlation between complement activity biomarkers and clinical/laboratory efficacy of danicopan, administered as monotherapy, in the treatment of PNH. Given that danicopan is potential first in class AP inhibitor for the treatment of PNH, it is important to investigate changes and relationships among relevant biomarkers of the AP to better understand the mechanism of action of danicopan in vivo. Methods: Danicopan starting doses ranged from 100-150 mg Q8H, with subsequent dose escalation based on clinical and biochemical response to doses as high as 200 mg Q8H. Danicopan plasma concentrations were determined by liquid chromatography/tandem mass spectrometry (LC/MS-MS). Danicopan pharmacodynamics (PD) were determined by measuring serum AP activity (AP Wieslab). Plasma Bb concentration, serum FD concentration, serum complement C3 and C4 concentrations, and serum classical pathway (CP) activity were also measured. C3 fragment deposition on RBCs was measured by flow cytometry with FITC conjugated anti- human C3d antibody. Results: Clinical results were previously presented, Table 1. Pharmacokinetic (PK) and PD analysis of danicopan concentration and AP activity measured ex vivo showed a clear correlation: ≤10% AP activity was seen for 6 hours following dosing with some recovery in AP activity prior to the next danicopan dose, Figure 1. Mean baseline Bb levels in this untreated PNH population were elevated relative to the reference range, demonstrating ongoing AP activity. Bb levels decreased into normal range with danicopan, demonstrating decreased in vivo C3 convertase formation, Figure 2. A strong positive correlation existed between Bb concentration and LDH level, demonstrating Bb as a reliable biomarker of AP activation in vivo during therapeutic complement inhibition in PNH. Strong correlations were also observed between danicopan concentration with Bb concentration (positive), AP activity (negative) and LDH level (positive), validating the role of danicopan in the changes of these endpoints. As expected, serum FD concentration was unchanged throughout treatment as was CP activity, which was measured with an in vitro assay designed to exclude any AP contribution. Notably, in contrast with C5 inhibition, C3 fragment deposition was not observed ( Conclusion: Previously reported proof of concept study with oral danicopan has demonstrated that upstream complement inhibition at the level of FD can prevent MAC-mediated intravascular hemolysis even in the absence of terminal pathway blockade (i.e., C5 inhibitor). Notably, improvements in multiple clinical endpoints occurred with danicopan monotherapy where optimal AP inhibition was not achieved throughout the dosing period. The correlation analysis among danicopan concentration, AP activity, Bb concentration, LDH level and C3 fragment deposition demonstrated that meaningful inhibition of AP activity leads to sustained control of intravascular hemolysis, without emergence of C3-mediated extravascular hemolysis. Near complete and sustained AP inhibition may be achieved with a second-generation oral FD inhibitor that is under development, possessing higher potency and improved PK profile, allowing for twice daily dosing with a mean value of >95% inhibition of AP at steady state. Disclosures Risitano: Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding; Alexion: Honoraria, Research Funding, Speakers Bureau; Ra Pharma: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Biocryst: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Research Funding; Alexion: Honoraria, Research Funding, Speakers Bureau; Biocryst: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding; Amyndas: Consultancy; Alnylam: Research Funding; Amyndas: Consultancy; Alnylam: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lee:Achillion: Research Funding; Alexion: Consultancy, Honoraria, Research Funding. Notaro:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: letture fees. Brodsky:Alexion: Membership on an entity's Board of Directors or advisory committees, Other: Grant funding; Achillion: Research Funding. Kulasekararaj:Akari: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Achillion: Consultancy; Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Alnylam: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Alexion: Consultancy; Novartis: Consultancy. Huang:Achillion: Employment, Equity Ownership. Geffner:Achillion: Employment, Equity Ownership. Browett:AbbVie: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Merck: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding; Amgen: Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Research Funding.

Introduction Eculizumab (Ecu) is the standard treatment for paroxysmal nocturnal hemoglobinuria (PNH), as it results in sustained control of intravascular hemolysis and prevention of thrombosis, and significant improvement of long-term survival. However, the hematological benefits remain heterogeneous among patients, and most response analyses are restricted to transfusion independence. In the era of new anti-complement therapies, we found appropriate to investigate in detail the quality of hematological response of Ecu-treated PNH patients. Methods We have recently proposed (Risitano et al, Frontiers in Immunology 2019) a classification of hematological response to anti-complement therapies based on hemoglobin (Hb) levels and laboratory markers of hemolysis. Six distinct categories were determined (Table 1): i. complete response (no transfusion requirement with normal stable Hb and no evidence of hemolysis); ii. major response (no transfusion requirement with normal Hb but evidence of residual intravascular or extravascular hemolysis); iii. good response (no transfusion requirement with persistent chronic mild anemia); iv. partial response (persistent chronic moderate anemia and/or with occasional red blood cell transfusions); v. minor response (3-6 red blood cell transfusions every 6 months); vi. no response (>6 blood cell transfusions every 6 months). Here we have retrospectively exploited this classification to assess hematological response in a large cohort of 93 PNH patients evaluated in six international reference PNH centers (Paris, Naples, London, Florence, São Paulo, and Ribeirão Preto). Results Forty-two patients were male and 51 were female; the median age at diagnosis (treatment starting) was 36 years (range, 12 to 74 years). All the patients have started Ecu treatment for hemolytic PNH; 57% of patients had classic PNH, whereas 30% and 13% had aplastic anemia (AA)/PNH or intermediate PNH (Peffault de Latour et al, Blood 2006), respectively. Two-thirds of patients had been receiving red blood cell (RBC) transfusion before starting Ecu; the mean treatment duration was 97 months (range 6-179). The majority of patients (87%) received Ecu at the standard schedule of 900 mg every 2 weeks; due to chronic, residual, intravascular hemolysis 13% were treated with higher doses (1200 mg) or with shorter administration interval (10-12 days). Hematological responses were evaluated early during the treatment course (at 6 and 12 months), and then later during the last 6 months of treatment (Figure 1). After 6 (n=80) and 12 months (n=79) of Ecu, respectively, rates of hematological response were as follows: complete response 9% and 10%, major response 4% and 6%, good response 34% and 47%, partial response 34% and 28%, minor response 14% and 6% and no response 6% and 3%. In 83 patients treated for more than 18 months late response rates also were evaluated; most patients responded and the rates of hematological response during the last 6 months of treatment were as follows: complete response, 11%; major response, 6%; good response, 41%; partial response, 29%; minor response, 7% and no response, 6%. Breakthrough episodes were recorded in 21% of patients (equally split between pharmacokinetic and pharmacodynamic ones); thromboembolic events and AA were seen in 2% and 3% of patients, respectively. These data suggest that, albeit individual and unpredictable, the extent of the hematological benefit of Ecu in most PNH patient can be determined quite early in the treatment course, after a minimum of 12 months. Conclusion This is the first attempt to classify hematological response to Ecu based on Hb levels and residual hemolysis. The proposed classification allows the identification of distinct patient subgroups with different medical needs, as well as of meaningful clinical goals for future anti-complement therapies. In our cohort, about 60% of patients are well controlled with Ecu, even if less than 20% of patients reach normal Hb values; about 40% of patients remain anemic, and this represents a goal for future therapies in PNH. This classification is helpful to characterize more precisely the clinical response, which should be assessed after 1 year of treatment. Even if it should be validated in independent, larger cohorts, this classification represents a useful tool to design future trials, and to compare the clinical results of the novel anti-complement agents in development for PNH. Disclosures Notaro: Alexion: Membership on an entity's Board of Directors or advisory committees, Other: letture fees. Scheinberg:Pfizer,: Speakers Bureau; Celgene: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Kulasekararaj:Alexion: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene Corporation: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Achilleon: Consultancy; Ra Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Akari Therapeutics: Consultancy. Risitano:Biocryst: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Research Funding; Samsung: Membership on an entity's Board of Directors or advisory committees; Achillion: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Ra Pharma: Research Funding; Alnylam: Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alnylam: Research Funding; Amyndas: Consultancy; Achillion: Research Funding; Amyndas: Consultancy; Biocryst: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Apellis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion: Honoraria, Research Funding, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees. Peffault de Latour:Pfizer: Consultancy, Honoraria, Research Funding; Alexion: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding.

a b s t r a c t The IC50 of TKIs is significantly increased when BCR-ABL+ K562 cell line is cultured in stroma conditioned media produced by BM mesenchymal cells. In particular, while the Imatinib IC50 in the stromal co- cultures was well above the in vivo through levels of the drug, the IC50s of second generation TKIs were still below their through levels. Moreover, we provide a formal comparison of the synergy between first and second generation TKIs with the JAK inhibitor Ruxolitinib to overcome BM stroma related TKI resistance. Taken together, our data provide a rationale for the therapeutic combination of TKIs and Ruxolitinib with the aim to eradicate primary BCR-ABL+ cells homed in BM niches.

The outcome of patients suffering from acquired idiopathic aplastic anemia (IAA) has progressively improved in the past decades thanks to a better management of the two key treatment options—namely hematopoietic stem cell transplantation and immunosuppression. Nevertheless, a number of different therapies have been investigated aiming to further improve the results of the standard treatment of IAA. Here we focus on the emerging therapies in the context of nontransplant treatment of IAA. The list includes different attempts to intensify the standard immunosuppressive treatment, as well as, novel agents that have been proven beneficial independently from and/or in combination with immunosuppression. While most attempts to improve current immunosuppressive regimens resulted in limited clinical benefit, the introduction of the thrombopoietin-mimetic agent, eltrombopag, showed promising results both as rescue and front-line treatment (the latter in combination with immunosuppression). Different clinical trials are currently ongoing to confirm these data and to establish the actual role of eltrombopag in the treatment algorithm of IAA.

In this issue of Blood , Zaimoku et al demonstrate that the functional loss of the HLA-B4002 allele is common in aplastic anemia (AA) patients, suggesting that this allele plays a major role in the immune attack underlying the pathophysiology of this disease. 1

The treatment of paroxysmal nocturnal hemoglobinuria (PNH) has dramatically changed since the availability of the first clinical complement inhibitor eculizumab. Eculizumab treatment has been proven terrifically effective in controlling intravascular hemolysis of PNH; however, unmet clinical needs are emerging, starting with the possible occurrence of C3-mediated extravascular hemolysis. Indeed, scientific and commercial reasons have triggered the development of a number of novel anticomplement agents. Different strategies are currently under investigation, which may target distinct steps and pathways of the complement system. Indeed, starting from the anti-C5 therapy that is already in the clinic, novel approaches are investigating the possibility of intercepting the complement cascade upstream, at the level of C3 and C3 convertase or of other initiating factors. Here we will review the majority of these strategies, focusing on available data in PNH and/or other preclinical models. The possible relevance for clinical application in PNH will be highlighted, based on well-established and emerging knowledge of molecular complement derangement in PNH.

The diagnosis of aplastic anemia (AA) is defined as the association of pancytopenia with persistent and unexplained reduced marrow hematopoietic cellularity in the absence of dysplastic features. The presence of a significant PNH clone (1% or more) in 30–40% of the patients, at diagnosis or during evolution, illustrates the underlying autoimmune process. The pathophysiology of this classic association is described in this chapter. Except the higher risk of thrombosis, patients with AA and a PNH clone should be managed the same way as patients with idiopathic anemia without a clone. Sibling transplantation is mandatory for patients aged less than 40 years when a matched family donor is available while immunosuppression using the association of horse antithymocyte globulin and cyclosporine is the reference treatment for other patients. In some very rare cases, a significant intravascular hemolysis is associated, which may justify associating a treatment by eculizumab.

Idiopathic aplastic anemia (AA) is a rare disorder of acquired bone marrow failure, characterized by peripheral blood cytopenias resulting from a hypocellular marrow, often as a result of an immune insult. Significant advances have been made to characterize the dysregulated immune system and responses to understand the aetiopathogenesis of AA, that in turn have unraveled the genetic predisposition markers (host) and the causal role of environmental factors, and more importantly the interplay between them in a proportion of these cases. National registries and retrospective studies have identified a marked variation in disease incidence amongst certain ethnic populations and geographical regions where polymorphism for various human leukocyte antigen (HLA) alleles have subsequently been found. Idiosyncratic reaction of the bone marrow to certain drugs and toxins have led to better pharmacovigilance practices, better monitoring and the judicious use of these agents in clinical practice. AA following viral infections including posthepatitis AA, other autoimmune disorders, pregnancy and following vaccination all highlight a disturbed immune milieu in the body towards disease predisposition. Autoimmunity remains at the core of AA, but we highlight important epidemiological influences that give context and better understanding of the disordered nature of this autoimmune disease.

The most important advance in the management of paroxysmal nocturnal hemoglobinuria (PNH) has been the introduction of eculizumab, a human monoclonal antibody against the C5 component of complement, that abrogates intravascular hemolysis by preventing the formation of the complement membrane attack complex. Complement blockade by eculizumab has proven to be clinically effective in hemolytic PNH, reducing the transfusion requirement in most of patients. However, in almost all PNH patients on eculizumab, a proportion of PNH red cells become coated with C3 and thus a potential target of phagocytosis by macrophages. This phagocytosis results in a variable degree of extravascular hemolysis that in some patients may limit the clinical benefit from eculizumab: in fact, at least one in four of transfusion-dependent PNH patients remain transfusion dependent even on eculizumab, and these patients may also have a high risk to develop iron overload. Unfortunately, there are not yet established treatments for the cases in which this extravascular hemolysis became clinically relevant: it is possible that new strategies for complement blockade could overcome this condition.

Introduction Fanconi anemia (FA) is an inherited bone marrow failure syndrome (IBMFS) due to a DNA repair mechanism defect. FA generally leads to marrow failure and/or leukemia-myelodysplasia in the first decades of life. Hematopoietic stem cell transplantation (HCT), using conditioning regimens adapted for the increased sensitivity for chemo- and radiotherapy induced damage, is the only curative therapy. The Severe Aplastic Anemia Working Party recently showed greatly improved HCT results, especially if patients were transplanted at a young age (Peffault de la Tour et al, Blood 2013). However, some patients are diagnosed at an adult age or are not transplanted at a young age for varying reasons. Data on HCT results in adult FA patients are very limited. We report here the final results of a retrospective study on transplant results in FA patients over 18 years of age at time of HCT. Results Both EBMT as well as non-EBMT centres contributed to this study, involving one hundred ninety nine patients, transplanted between 1991-2014. Median age of FA diagnosis was 16 years (ranging from birth up to the age of 48 years).In twenty nine cases (76% of evaluable patients) the genotype was A, in four it was C (11%). Median age at transplant was 23 years (range 18-48). Thirty seven patients (19%) aged more than 30 at time of HCT. Median time from diagnosis to transplant was 7 years (range1-14), also depending on donor availability (median 2 years for sibling donors, 9 years for an unrelated donor, up to 13 years for other/mismatched related donors).Indication for transplant was clonal disease in fifty four patients, 46 % of evaluable cases (14 leukemia/ 40 MDS). In ninety one patients (46%) the donor was an identical sibling, while forty seven (24%) received a matched unrelated donor, and forty patients (20%) a mismatched unrelated HCT. Stem cell source was bone marrow in 115 cases (58%), peripheral blood stem cells in 72 (36%) and cord blood in 11 cases (6%). The conditioning regimen contained cyclophosphamide in 173 cases (96%), fludarabin in 116 (64%), busulfan in 33 (18%) and low dose TBI in fifty five patients (29%). Engraftment occurred in one hundred fifty nine patients (82%, 95%CI 76-87).In eightteen patients there was a primary graft failure, in ten a secondary. Acute GvHD 2-4 occurred in thirty nine patients (22%, 95%CI 16-28). cGvHD was present in forty four patients (Cumulative incidence (CI) 26% at 96 months, 95%CI 20-33). CI of non relapse mortality at 96 months: 56% (95%CI 47-63). CI of secondary malignancies: 19 of 131 evaluable patients (17%) at 96 months post-HCT (95%CI10-25). Probability of overall survival at 96 months was 34% (95%CI 27-43). Main causes of death included infection (n=44, 37%), GvHD (n=29, 24%), organ damage (n=15, 12%) and secondary malignancies (n=12, 10%). In multivariate analysis identical sibling donor availability and the use of fludarabin in the conditioning regimen were statistically significantly associated with better chance of survival (p Conclusion. This study reports on the largest dataset of adult patients with FA undergoing HCT. We confirm that adult age has a negative impact on HCT outcome, compared to younger patients with FA undergoing transplantation. The use of fludarabin in the conditioning regimen has a positive effect on outcome. HCT should be considered for every patient with FA at an early age, if deemed necessary. International efforts are necessary to treat adult FA patients in need of HCT in clinical trials, especially when only less well matched donors are available. Disclosures Peffault De La Tour: PFIZER: Consultancy, Honoraria, Research Funding; NOVARTIS: Consultancy, Honoraria, Research Funding; ALEXION: Consultancy, Honoraria, Research Funding. Niederwieser:Amgen: Speakers Bureau; Novartis Oncology Europe: Research Funding, Speakers Bureau. Michallet:Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Astellas Pharma: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Genzyme: Consultancy, Honoraria. Risitano:Novartis: Research Funding; Alnylam: Research Funding; Rapharma: Research Funding; Alexion Pharmaceuticals: Other: lecture fees, Research Funding.

Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.

Optimal management of aplastic anemia (AA) is not confined to immediate diagnosis, early decision making and timely initiation of major treatment strategies (immunosuppression or SCT) but also involves supportive treatment as a crucial part of patient care. Patients are threatened by complications of cytopenia. Here, we summarize current recommendations for prevention and early treatment of fungal, bacterial and viral infections, transfusion strategy and iron chelation and assess the evidence basis. In fact, many recommendations for patients with AA are not based on randomized studies in AA itself, but they are deduced from other conditions with similar severity of cytopenia. Prevention and treatment of complications like hemorrhage, bacterial and fungal infections and of secondary events like alloimmunization to blood products and iron overload have a significant impact on the prognosis of AA patients and need to be carefully observed in daily practice. More controlled studies on supportive care should be performed in this rare disease.

Paroxysmal nocturnal haemoglobinuria (PNH) is a haematopoietic disorder characterized by expansion of phosphatidylinositol glycan-A-defective progenitor(s). Immune-dependent mechanisms, likely involving a deranged T cell-dependent autoimmune response, have been consistently associated with the selection/dominance of PNH precursors. Natural killer (NK) lymphocytes might participate in PNH pathogenesis, but their role is still controversial. NK activity is dependent on the balance between activating and inhibiting signals. Key component in such regulatory network is represented by killer immunoglobulin-like receptors (KIR). KIR are also involved in the regulation of adaptive cytotoxic T cell response and associated with autoimmunity. This study investigated on the frequency of KIR genes and their known human leukocyte antigen (HLA) ligands in 53 PNH Italian patients. We observed increased frequency of genotypes characterized by ≤2 activating KIR as well as by the presence of an inhibitory/activating gene ratio ≥3.5. In addition, an increased matching between KIR-3DL1 and its ligand HLA-Bw4 was found. These genotypes might be associated with lower NK-dependent recognition of stress-related self molecules; this is conceivable with the hypothesis that an increased availability of specific T cell targets, not cleared by NK cells, could be involved in PNH pathogenesis. These data may provide new insights into autoimmune PNH pathogenesis.

Although recent advances in therapy offer the promise for improving survival in patients with severe aplastic anemia (SAA), the small size of the patient population, lack of a mechanism in North America for longitudinal follow-up of patients and inadequate cooperation among hematologists, scientists, and transplant physicians remain obstacles to conducting large studies that would advance the field. In order to address this issue, the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) convened a group of international experts in March 2010 to define the most important questions in the basic science, immunosuppressive therapy (IST), and bone marrow transplantation (BMT) of SAA and propose initiatives to facilitate clinical and biologic research. Key conclusions of the working group were: 1) new patients should obtain accurate, expert diagnosis and early identification of biological risk; 2) a population-based SAA outcomes registry should be established in North America to collect data on patients longitudinally from diagnosis through and after treatment; 3) a repository of biologic samples linked to the clinical data in the outcomes registry should be developed; 4) innovative approaches to unrelated donor BMT that decrease graft vs. host disease are needed, and 5) alternative donor transplantation approaches for patients lacking HLA-matched unrelated donors must be improved. A partnership of BMT, IST and basic science researchers will develop initiatives and partner with advocacy and funding organizations in order to address these challenges. Collaboration with similar study groups in Europe and Asia will be pursued.

Introduction The prospective randomized study on treatment of 192 severe aplastic anemia (SAA) patients with ATG and Cyclosporine (CSA) with and without G-CSF showed that G-CSF added to ATG/CSA decreases the rate of early infection episodes and days of hospitalization in very SAA (vSAA) patients, but has no significant impact on overall survival (OS), event free survival (EFS), relapse, or death rates (Blood, 2011;17:4434). The number of secondary MDS/AML was low, however with a short observation time. Now, 16 years after initiation of the study, a follow-up was planned to evaluate long-term outcome, comparing patients with and without G-CSF. Patients and Methods A total of 192 patients with newly diagnosed SAA, not eligible for stem cell transplantation (SCT) were entered into this prospective randomized multi-center study to receive ATG/CSA with (49.5%) or without G-CSF (50.5%). In 2011, 44 of the 192 patients had died. For the present study the follow-up of the 148 patients alive at time of first publication were requested. There were 49% males (49% G-CSF; 48% no G-CSF), 36% with vSAA (32% G-CSF; 41% no G-CSF). The median age at randomization was 46 years (2-80), 47 (2-80) for the G-CSF and 44 (7-80) for the non-G-CSF group. The median follow-up using reverse KM method was 11.7 years (10.9-12.5). Results Among the 110 survivors (17 missing), 71 (65%) were in CR, 33 (30%) in PR and 6 not in remission (5%), without any difference between the G-CSF and non-G-CSF group (P=0.523). At last follow-up 65 (34%) of the patients have died. Causes of death were infection (26), bleeding (3), SAA unspecified (3), MDS/AML (4), solid cancer (3), transplant related mortality (8), cardiovascular/aging (7), or unspecified (11). There was no difference in the causes of death between patients treated with or without G-CSF. OS at 15 years was 57±12% for the G-CSF and 63±12% for the non-G-CSF group (P=0.927). EFS, including SCT, relapse, non-response at day 120, second MDS/AML, PNH or death as an event, was 24±10% for the G-CSF, and 23±10% for the non-G-CSF group (P=0.367). Nine patients developed florid or morphological signs of MDS/AML, 9 clonal cytogenetic anomaly only, 7 a solid cancer, 18 clinical PNH, 8 avascular osteonecrosis, and 12 chronic kidney disease (No difference between patients treated with or without G-CSF). Cumulative incidence (CI) at 15 years of MDS/AML (isolated cytogenetic anomalies not included) was 5.0±2% (G-CSF) and 7.3±3% (no G-CSF), respectively (P=0.693); for clinical PNH it was 10.1±5% and 13.3±7% (P=0.499), for relapse of responding patients at day 120, 29.8±22% and 25.1±17% (P=0.545), and for chronic kidney failure 16%±12% and 13%±12% (P=0.513), respectively. Forty patients needed a second line immunosuppressive therapy (IST) for relapse (17), refractory disease (8), cyclosporine dependence (6) or isolated cytopenia (9) (G-CSF 26; no G-CSF 16; P=0.291); 16 patients needed a third line IST for relapse (5), refractory disease (7), isolated cytopenia (4) (G-CSF 12; no G-CSF 4; P=0.647). Twenty-eight patients were treated with allogeneic SCT in second or subsequent line (G-CSF 12; no G-CSF 16). CI at 15 years of SCT (competing risk, death without SCT) was 14±8% (G-CSF) and 22%±10% (no G-CSF), respectively (P=0.380). OS at 10 years since SCT was 46±24%. The most important risk factors for patients treated with ATG/CSA with or without G-CSF were age and severity of the disease at randomization: OS at 15 years was 89±12% (60 years of age), respectively (P>0.001), and 64±5% and 52±7% for patients with SAA and vSAA, respectively (P=0.021). There was no difference between patients treated with or without G-CSF. Finally, the lack of neutrophil response by day 30, which was significant at first evaluation, is still associated with borderline lower survival (46.6±14% versus 67.1±9%; P=0.058). Conclusion: Long-term outcome of SAA patients treated with ATG/CSA was not influenced by supplementing G-CSF in term of OS, EFS, death rates, relapse, PNH, secondary MDS/AML, solid cancer and non-malignant late complications. Due to the pre-cancer nature of the disease and its long-lasting treatment, patients treated with IST are at risk for a number of malignant and non-malignant late complications. It is somewhat disappointing that in this careful followed cohort less than 25% of patients are alive and event-free 10-15 years after initial treatment. Disclosures Peffault De Latour: Pfizer Inc.: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding; Novartis: Consultancy, Honoraria, Research Funding. Hoechsmann:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Schrezenmeier:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Kulasekararaj:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support . Bader:Neovii: Research Funding; Cellgene: Consultancy; Riemser: Research Funding; Medac: Patents & Royalties, Research Funding; Novartis: Consultancy, Speakers Bureau. Risitano:Pfizer Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amyndas Pharmaceuticals: Consultancy; Alnylam Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Ra Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Background/Objective: Breakthrough hemolysis (BTH) is the return of hemolytic disease activity during treatment with complement C5 inhibitors for paroxysmal nocturnal hemoglobinuria (PNH). BTH may be associated with inadequate C5 inhibition or complement activating conditions (eg, infection). Despite reports that up to 19% of patients (pts) receiving eculizumab may experience BTH, there is no commonly accepted definition of BTH. The definition of BTH was derived from literature review and expert consensus. BTH was defined as ≥1 new or worsening symptom/sign of intravascular hemolysis (fatigue, hemoglobinuria, abdominal pain, dyspnea, anemia [hemoglobin Methods: Two phase 3, randomized, open-label, noninferiority, multicenter studies (study 301, NCT02946463; study 302, NCT03056040) included pts ≥18 years of age with confirmed PNH diagnoses. Pts in study 301 were naive to C5 inhibitor therapy, with LDH ≥1.5 xULN and ≥1 sign/symptom of PNH at screening. Pts in study 302 were stable on eculizumab treatment for ≥6 months, with LDH ≤1.5 xULN at screening. Pts received weight-based dosing of ravulizumab q8w or the approved eculizumab dose (900 mg q2w) for 183 days. Higher eculizumab doses were prohibited. Results: In study 301, 246 pts received ravulizumab (n=125) or eculizumab (n=121); in study 302, 195 pts received ravulizumab (n=97) or eculizumab (n=98). Ravulizumab was associated with fewer episodes of BTH than eculizumab in both studies. In study 301, 5 pts (4.0%) experienced BTH in the ravulizumab group vs 13 pts (10.7%) in the eculizumab group; between-group difference (95% confidence interval [CI]) was −6.7% (−14.21%, 0.18%; P=0.0558 for superiority). In the eculizumab group, 7/15 events were temporally associated with elevations in free C5 (C5 ≥0.5 μg/mL), suggesting inadequate C5 inhibition. No BTH events in the ravulizumab group were associated with inadequate C5 inhibition. Among the BTH events in the ravulizumab group, the majority, 4/5 events, were associated with infection. In the eculizumab group, infection was associated with 6/15 BTH events, including 2 infections in pts with inadequate C5 inhibition. In study 302, no pts in the ravulizumab group experienced BTH vs 5 pts (5.1%) in the eculizumab group (1 pt had 3 BTH events, requiring hospitalization and study discontinuation for lack of efficacy); the between-group difference (95% CI) was −5.1% (−18.99%, 8.89%). Four of 7 BTH events in the eculizumab group were associated with concomitant elevations in free C5. Infection was associated with 3/7 BTH events, including 1 infection in a pt with inadequate C5 inhibition (Table). There was no clear pattern for timing of BTH events. All BTH episodes in the eculizumab groups were reported at the q2w study visits associated with pharmacokinetic (PK) troughs. All BTH episodes in the ravulizumab groups were reported at the q2w study visits, but not necessarily at q8w PK troughs. Conclusions: No BTH events in the ravulizumab group were associated with elevations in free C5. In contrast, pts treated with eculizumab 900 mg q2w experienced multiple BTH events with elevations in free C5 suggesting suboptimal C5 control. Similar numbers of pts receiving ravulizumab or eculizumab experienced infection-related BTH, possibly due to proximal complement activation. Differences in BTH rates for ravulizumab vs eculizumab may be due to improved pharmacodynamics (C5 inhibition throughout the dosing interval) and the optimized ravulizumab dosing regimen. Figure. Figure. Disclosures Peffault De Latour: Novartis: Consultancy, Honoraria, Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding. Rottinghaus:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Röth:Roche: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Bioverativ: Consultancy, Honoraria. Risitano:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Alnylam Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amyndas Pharmaceuticals: Consultancy; Pfizer Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Weitz:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Hillmen:Novartis: Research Funding; Alexion Pharmaceuticals, Inc: Consultancy, Honoraria; Pharmacyclics: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding. Maciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Apellis Pharmaceuticals: Consultancy; Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy. Szer:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support , Research Funding. Lee:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Kulasekararaj:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support . Volles:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Damokosh:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Ortiz:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Shafner:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Hill:Bioverativ: Consultancy, Honoraria; Akari: Consultancy, Honoraria; Ra Pharma: Consultancy, Honoraria; Regeneron: Consultancy, Honoraria; Apellis: Consultancy, Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Schrezenmeier:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding.

INTRODUCTION Fanconi anemia (FA) is an inherited bone marrow failure syndrome that carries a high risk of transformation to myelodysplasia (MDS) and acute leukemia. Hematopoietic stem cell transplantation (HSCT) is used to treat FA patients in clonal evolution. The roles of chemotherapy before HSCT and the intensity of conditioning regimens in transformed FA patients are controversial, because of the high sensitivity of FA patients to DNA-damaging agents increases the risk of severe toxicities, the duration of aplasia, the risk of infective complications thus limiting the possibility to administer full dose cytoreductive treatments. The sequence chemotherapy-HSCT has been reported by some groups (MehtaPA et al Pediatr Blood Cancer 2007, Talbot A et al Haematologica 2014, Mitchell R et al Br J Haematol 2014, Ayas M et al J Clin Oncol 2013), but conclusive information is lacking because of small number of patients described without a risk factors' analysis. The aim of this retrospective study is to report the outcome of a large cohort of transformed FA who underwent allo-HSCT and to define the factors that may impact on its outcome. PATIENTS AND METHODS The study was conducted on behalf of the Severe Aplastic Anemia (SAAWP) and Chronic Malignancies Working Parties (CMWP) of the EBMT and was based on data of patients who underwent allo-HSCT between 1999-2016 for transformed FA, defined as a diagnosis of FA in presence of any hematological malignancies or cytogenetic abnormalities, registered in the EBMT Data Base. Clinical and biological information of the disease and details on transplant procedures and outcome were collected by a specific form distributed to Centres participating in the study. RESULTS Data of 71 patients (35 males-36 females) affected by transformed FA (42 MDS, 25 AL, 4 with cytogenetic abnormalities but without blasts) undergoing allo-HSCT were collected from 25 Centres . A matched related donor (MRD) was used in 31% of cases, an unrelated donor (UD) in 56.3% and a mismatched related donor (MMRD) in 12.7%. Bone marrow was the main source of cells (54.3%) followed by cord blood (22.9%), peripheral blood (21.4%) and bone marrow plus peripheral blood (1.4%). The median age at allo-HSCT was 12.7 years (range 9.3-23.4). Thirty seven (52.1%) patients received a chemotherapy before HSCT. Pre-HSCT status of malignancy in available patients was complete remission (CR) in 24% (n = 12/50) and an active disease (no-CR) in 76 % patients (n = 38/50). The conditioning regimen included total body irradiation (TBI) in 37 (52.1%) (radiation dose: ≤ 4.0 Gy in 30; > 4.0 Gy in 7), busulphan (BUS) in 16 (22.6%), no-TBI nor BUS in 18 (25.3%). Median follow-up was 93.7 months (71-110.6). GvHD prophylaxis and transplants' details are summarized in Table 1. All patients engrafted. Median time for neutrophils was 17 days (14-23) and it was 25 days (23-42) for platelets. The 2-and 5-year overall survival (OS) probability were 54% (41-66%) and 45% (32-57%) respectively; the 2- and 5-year event-free survival (EFS) (events being death, relapse and graft loss) 52% (40-65%) and 45% (32-58%). The cumulative incidence of relapse were 15% (7-24%) and 21% (11-31%), , of non-relapse mortality (NRM) were 37% (25-49%) and 39% (27-51%) respectively at 2 and 5-year. Most frequent causes of death were GvHD (33.3%), infections (23.3%) and relapse of malignancy (16.7%). Patients transplanted in CR, (neither blasts, nor major dysplastic features) and from matched related donor had a significantly better outcome (5-year OS: CR 83% (62-100%) vs no-CR 36% (19-52%) [p 0.01], MRD 60% (37-83%) vs UD 47% (31-64%) vs MMRD 12% (0-35%) [p 0.03]; 5-year EFS: CR 83% (62-100%) vs no-CR 34% (17-51%) [p 0.01], MRD 61% (38-83%) vs UD 48% (31-64%) vs MMRD 12% (0-35%) [p 0.02]; 5-year NRM: CR 0% vs no-CR 44% (27-61%) [p 0.007]) vs those engrafted in no-CR and from no-MRD. (Fig 1 a, b, c). No other tested variable (therapy before transplant and conditioning regimen) significantly affected the outcome. CONCLUSION This study on large cohort of FA patients transplanted because of transformation shows that allo-HSCT from MRD has a better outcome and that CR from malignancy before transplant appears to be a major determinant for a favorable outcome. Disclosures Peffault De Latour: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding; Pfizer Inc.: Consultancy, Honoraria, Research Funding. Ganser:Novartis: Membership on an entity's Board of Directors or advisory committees. Risitano:Pfizer Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alnylam Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Ra Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amyndas Pharmaceuticals: Consultancy; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Background/Objective: In 2 large, phase 3 clinical studies, ravulizumab (ALXN1210), an innovative C5 inhibitor administered every 8 weeks, achieved noninferiority to eculizumab in complement inhibitor-naive and -experienced patients (pts) with paroxysmal nocturnal hemoglobinuria (PNH). The objective of this analysis was to characterize ravulizumab pharmacokinetics (PK) and pharmacodynamics (PD). Potential PD differences between ravulizumab and eculizumab were also examined to explore a possible mechanistic explanation for the robust clinical results observed in the phase 3 studies. The consistent ravulizumab vs eculizumab results across all primary and secondary clinical endpoints may be attributable to more complete inhibition of free C5 (defined as serum free C5 level Methods: Two phase 3, randomized, open-label, noninferiority studies (NCT02946463, study 301; NCT030560040, study 302) included pts ≥18 years of age with confirmed PNH. Pts in study 301 were complement inhibitor-naive, with lactate dehydrogenase (LDH) levels ≥1.5 times upper limit of normal (xULN), and ≥1 PNH-related sign/symptom at screening. Pts in study 302 were clinically stable after having been treated with eculizumab for ≥6 months, with LDH levels ≤1.5 xULN at screening. Pts received weight-based dosing regimens of ravulizumab q8w or eculizumab 900 mg q2w in accordance with PNH labeling for 183 days. Study protocols did not allow dose escalation. Coprimary efficacy endpoints in study 301 were transfusion avoidance and LDH normalization; the primary endpoint in study 302 was percent change from baseline in LDH. Serum samples for PK/PD analyses were collected on study days 1, 8, 15, 22, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, and 183. PK/PD outcomes included serum drug concentrations as well as serum free complement protein C5 and total C5. Free C5 was quantified in ravulizumab-treated pts utilizing a Gyros-based fluorescence assay, and in eculizumab-treated pts using an electrochemiluminescence ligand binding assay. Noncompartmental PK parameters were assessed for ravulizumab. Results: In study 301, 246 pts received study drug (ravulizumab, n=125; eculizumab, n=121); 195 received study drug in study 302 (ravulizumab, n=97; eculizumab, n=98). Ravulizumab met the primary objective of statistically significant noninferiority compared with eculizumab for all primary and key secondary endpoints in both studies. The PK profile of ravulizumab was similar in both studies (and when stratified by weight-based dosing [data not shown]), with Tmax of approximately 2.5 hours (Table 1). Following population PK modeling, mean (SD) post hoc terminal elimination half-life in all 222 pts was 49.7 (8.9) days. Ravulizumab steady-state therapeutic concentrations were rapidly achieved following the first dose and were sustained throughout the 183-day treatment period in both studies. Complete suppression of free C5 was attained by the end of first ravulizumab infusion (mean serum free C5 concentrations Conclusions: In 2 large, phase 3 studies of PNH pts who were either naive to or receiving prior complement inhibitor therapy, ravulizumab q8w led to immediate, complete, and sustained complement C5 inhibition in all pts, whereas the effect of eculizumab q2w was less consistent. In pts treated with ravulizumab, free C5 suppression below the free C5 threshold was associated with complete inhibition of intravascular hemolysis, providing a mechanistic basis for the consistency of the point estimates for all endpoints. Disclosures Ortiz: Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Risitano:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alnylam Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Ra Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Pfizer Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amyndas Pharmaceuticals: Consultancy. Hillmen:Celgene: Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Research Funding; F. Hoffmann-La Roche Ltd: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Alexion Pharmaceuticals, Inc: Consultancy, Honoraria; Acerta: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding; Gilead Sciences, Inc.: Honoraria, Research Funding. Kulagin:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Pradhan:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Rottinghaus:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Aguzzi:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Wells:Alexion Pharmaceuticals, Inc.: Honoraria, Other: Travel Support , Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria. Szer:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support , Research Funding.

Introduction Ravulizumab, an innovative complement C5 inhibitor given every 8 weeks (q8w), was recently demonstrated in a large phase 3 study of patients (pts) with paroxysmal nocturnal hemoglobinuria (PNH) and naïve to complement inhibitor therapy to be non-inferior to eculizumab given every 2 weeks (q2w) across all endpoints that measured different aspects of PNH disease (transfusion avoidance [TA], lactate dehydrogenase normalization [LDH-N], percent LDH reduction, breakthrough hemolysis [BTH], Functional Assessment of Chronic Illness Therapy [FACIT]-Fatigue, and hemoglobin stabilization [HGB-S]) (Lee JW et al. EHA Learning Center, Jun 17, 2018; LB2603). PNH pts currently receiving eculizumab may experience treatment burden associated with the q2w dosing regimen. With the development of ravulizumab, PNH pts on prior eculizumab therapy can potentially switch to ravulizumab without interruption of treatment. The primary objective of this study was to assess the noninferiority of ravulizumab compared to eculizumab in adult PNH pts who were clinically stable after having been treated with eculizumab for at least 6 months. Methods In this phase 3, open-label, multicenter study (NCT03056040), adult pts with a documented diagnosis of PNH who were treated with labelled-dose eculizumab for >6 months having LDH levels ≤1.5 times the upper limit of normal at screening were randomly assigned 1:1 to continue eculizumab or switch to ravulizumab. Pts randomized to ravulizumab received weight-based loading [day 1]/maintenance doses [day 15 and q8w thereafter]: ≥40 to Results Of 208 pts screened, 197 pts were randomized (1:1) to ravulizumab or eculizumab, 195 received treatment (ravulizumab, n=97; eculizumab, n=98), and 191 pts completed 183 days of treatment. Pt demographics and baseline clinical characteristics were similar between treatment groups. On average, pts had received prior eculizumab therapy for 5.8 years. All pts received all planned infusions of study medication. Ravulizumab was statistically significantly noninferior to eculizumab for the primary and all key secondary endpoints, with all point estimates favoring ravulizumab: percentage change in LDH levels (difference of -9.21% [95% CI: -18.84, 0.42]), BTH (difference of -5.1 [95% CI -18.89, 8.89]), change in FACIT-Fatigue score (difference of 1.47 [-0.21, 3.15]), TA (difference of 5.5 [95% CI -4.27, 15.68]), and HGB-S (difference of 1.4 [-10.41, 13.31]) (Fig A, Fig B). Superiority testing of the primary endpoint (percentage change in LDH) did not reach statistical significance (P=0.0583 vs P Conclusion Results of this large phase 3 study show that ravulizumab achieved noninferiority compared with eculizumab in pts with PNH on stable eculizumab therapy. Pts can be safely and effectively switched from eculizumab given q2w to ravulizumab given q8w. In pts previously stable on eculizumab, no pts switched to ravulizumab experienced BTH vs 5 pts on eculizumab during the 26-week treatment period. This may be related to optimized ravulizumab dosing compared to eculizumab, resulting in complete and sustained C5 inhibition for all pts. Figure. Figure. Disclosures Kulasekararaj: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support . Hill:Apellis: Consultancy, Honoraria; Bioverativ: Consultancy, Honoraria; Regeneron: Consultancy, Honoraria; Akari: Consultancy, Honoraria; Ra Pharma: Consultancy, Honoraria; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Rottinghaus:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Wells:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Alexion Pharmaceuticals, Inc.: Honoraria, Other: Travel Support , Research Funding. Gonzalez-Fernandez:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Gaya:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Lee:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Ojeda Gutierrez:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Szer:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support , Research Funding. Risitano:Pfizer Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amyndas Pharmaceuticals: Consultancy; Alnylam Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding. Nakao:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria; Novartis: Honoraria. Bachman:Alexion Pharmaceuticals, Inc.: Equity Ownership, Other: Former employee. Shafner:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Damokosh:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Ortiz:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Röth:Pfizer: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Bioverativ: Consultancy, Honoraria; Amgen: Research Funding. Peffault De Latour:Pfizer Inc.: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amgen Inc.: Research Funding.

Background/Objective: Ravulizumab (ALXN1210) is an innovative C5 inhibitor with high C5 affinity and a half-life approximately 4 times longer than eculizumab. In the largest interventional clinical study of C5 inhibitor-naive patients with paroxysmal nocturnal hemoglobinuria (PNH) to date, ravulizumab administered every 8 weeks (q8w) was shown to be noninferior to eculizumab (every 2 weeks [q2w]) across 2 coprimary endpoints and 4 key secondary endpoints, due to its ability to provide immediate, complete, and sustained inhibition of complement C5 (Lee et al, EHA-2018). Because PNH is a rare disease, prospective evaluation of new agents for the treatment of this disease, with the exception of ravulizumab, has been limited to small populations. As a result, assessing the effect of treatment in patients with unique disease characteristics and gaining a full understanding of the benefit-to-risk profile of new therapies, has been challenging. The objective of this protocol-specified analysis was to evaluate the relative efficacy of ravulizumab and eculizumab in adult patients with PNH based upon baseline hemolysis level, transfusion history, and demographics. Methods: In this phase 3, randomized, open-label, noninferiority, multicenter study (NCT02946463), patients ≥18 years of age with confirmed diagnosis of PNH, naive to complement inhibitor therapy, with lactate dehydrogenase (LDH) level ≥1.5 times upper limit of normal (xULN), and ≥1 PNH-related sign/symptom at screening received ravulizumab or eculizumab over a 183 day study period. Coprimary efficacy endpoints were transfusion avoidance and LDH normalization. Secondary endpoints were percent change in LDH, change in Functional Assessment of Chronic Illness Therapy (FACIT)-Fatigue score, proportion of patients with breakthrough hemolysis, and proportion with stabilized hemoglobin levels. Subgroup analyses were performed on each of these endpoints, based on transfusion history (0, 1-14, or >14 units of packed red blood cells [pRBCs] in the year prior to the first dose of study drug), screening LDH levels (1.5- Results: In total, 246 patients from countries across the Asia-Pacific region (n=124), Europe (n=91), North America (n=9), and South America (n=22) were randomized to ravulizumab (n=125) or eculizumab (n=121). For transfusion avoidance, the point estimates for 15/17 subgroups favored ravulizumab. The treatment effect favored ravulizumab irrespective of prior pRBC transfusion history and baseline LDH levels, as well as nearly all other demographic subgroups assessed (Panel A). With respect to LDH normalization, the point estimates for 11/17 subgroups favored ravulizumab. The treatment effect numerically favored ravulizumab among patients who received 1-14 and >14 pRBCs in the 12 months before randomization, both baseline LDH subgroups, and most of the demographic subgroups (Panel B). No sensitive subgroups were identified for either of the coprimary endpoints. Similarly, point estimates favored ravulizumab in the majority of patient subgroups across each of the key secondary endpoints, including: 8/17 of the subgroups on the assessment of percent change in LDH; 12/17 subgroups on the assessment of change in FACIT-Fatigue score; 13/17 subgroups on the assessment of breakthrough hemolysis; and 13/17 subgroups on the assessment of hemoglobin stabilization. Conclusions: Results of this prespecified analysis revealed point estimates favoring ravulizumab for the majority of the diverse subgroups of PNH patients analyzed. The consistency of these data across subgroups confirms the robustness of the results of this study. The benefit of ravulizumab in terms of transfusion avoidance and LDH normalization was retained across all subgroups. These results provide evidence supporting the use of ravulizumab for the treatment of complement inhibitor-naive patients with PNH regardless of transfusion history, baseline hemolysis level, sex, age, race, or geographic region. Figure. Figure. Disclosures Weitz: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Kulagin:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria. Nakao:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria; Novartis: Honoraria; Kyowa Hakko Kirin Co., Ltd.: Honoraria. Szer:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Other: Travel Support , Research Funding. Rottinghaus:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Volles:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Damokosh:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Aguzzi:Alexion Pharmaceuticals, Inc.: Employment, Equity Ownership. Larratt:Alexion Pharmaceuticals, Inc.: Honoraria, Research Funding. Risitano:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alnylam Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ra Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer Inc.: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amyndas Pharmaceuticals: Consultancy.

In Paroxysmal nocturnal haemoglobinuria (PNH), pregnancy is associated with increased maternal and foetal complications to such an extent that the condition has been considered relatively contra-indicated in PNH. Eculizumab has revolutionized the treatment of PNH. We evaluate its use in pregnancy to date. We report on seven patients exposed to eculizumab at different stages of pregnancy including the first two patients to receive the drug from conception to delivery. There was no evidence of complement blockade from cord blood samples taken at delivery. Eculizumab appears safe to use in this setting and is likely to prevent many of the complications usually observed.

To evaluate efficacy, safety, changes in biological features, and quality of life (QoL) in low-risk anemic patients with MDS treated with darbepoetin alfa (DPO), 41 patients received DPO 150 microg weekly for 24 weeks. The dose was increased to 300 microg weekly in non-responsive patients. During treatment, 10/17 (59%) transfusion-dependent (TD) and 13/23 (56%) transfusion-free (TF) patients responded. In TF patients, Hb increased from 9.2 +/- 0.9 g/dL to 10.3 +/- 1.4 g/dL by 24 weeks (p = 0.004). The mean response duration was 22 weeks (95% CI: 19.7-24.0) in TF patients compared with 15.1 weeks (95% CI: 13.3-17.5) in TD patients. Response to treatment was associated with increases in QoL. Decreases in the percentage of apoptotic progenitor cells (p = 0.007) and CD34+ cells (p = 0.005) were observed. These results confirm previous studies demonstrating the safety and efficacy of DPO in anemic patients with MDS. Biological changes and improvement in QoL were associated with response. Adequate dosing is to be determined.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by the expansion of a P/G-A mutated hematopoietic stem cell. An immune-mediated origin has been suggested for this disease. Because HLA genes represent a susceptibility factor for autoimmunity, we investigated HLA genotype in 42 Italian PNH patients compared with 301 control subjects of the same ethnic origin. A significantly increased frequency of the HLA class I alleles A*0201 (p < 0.05), B*1402 (p < 0.001), and Cw*0802 (p < 0.005), and of the HLA class II DRB1*1501 (p < 0.01) with the Linked DQB1*0602 (p

Clinical Trial, Phase III; Journal Article; Multicenter Study; Randomized Controlled Trial; Research Support, Non-U.S. Gov't; BACKGROUND Chronic graft-versus-host disease (GVHD) is the leading cause of later illness and death after allogeneic hematopoietic stem-cell transplantation. We hypothesized that the inclusion of antihuman T-lymphocyte immune globulin (ATG) in a myeloablative conditioning regimen for patients with acute leukemia would result in a significant reduction in chronic GVHD 2 years after allogeneic peripheral-blood stem-cell transplantation from an HLA-identical sibling. METHODS We conducted a prospective, multicenter, open-label, randomized phase 3 study of ATG as part of a conditioning regimen. A total of 168 patients were enrolled at 27 centers. Patients were randomly assigned in a 1:1 ratio to receive ATG or not receive ATG, with stratification according to center and risk of disease. RESULTS After a median follow-up of 24 months, the cumulative incidence of chronic GVHD was 32.2% (95% confidence interval [CI], 22.1 to 46.7) in the ATG group and 68.7% (95% CI, 58.4 to 80.7) in the non-ATG group (P

During malarial anemia, 20 uninfected red blood cells (RBCs) are destroyed for every RBC infected by Plasmodium falciparum (Pf). Increasing evidence indicates an important role for complement in destruction of uninfected RBCs. Products of RBC lysis induced by Pf, including the digestive vacuole and hematin, activate complement and promote C3 fragment deposition on uninfected RBCs. C3-opsonized cells are then subject to extravascular destruction mediated by fixed tissue macrophages which express receptors for C3 fragments. The Compstatin family of cyclic peptides blocks complement activation at the C3 cleavage step, and is under investigation for treatment of complement-mediated diseases. We demonstrate, that under a variety of stringent conditions, second-generation Compstatin analogue Cp40 completely blocks hematin-mediated deposition of C3 fragments on naïve RBCs. Our findings indicate that prophylactic provision of Compstatin for malaria-infected individuals at increased risk for anemia may provide a safe and inexpensive treatment to prevent or substantially reduce malarial anemia.

Hemolysis and hemoglobinemia contribute to serious clinical sequelae in hemolytic disorders. In paroxysmal nocturnal hemoglobinuria (PNH) patients, hemolysis can contribute to thromboembolism (TE), the most feared complication in PNH, and the leading cause of disease-related deaths. We evaluated whether long-term treatment with the complement inhibitor eculizumab reduces the rate of TE in patients with PNH. Clinical trial participants included all patients in the 3 eculizumab PNH clinical studies, which recruited patients between 2002 and 2005 (n = 195); patients from these studies continued treatment in the current multinational open-label extension study. Thromboembolism rate with eculizumab treatment was compared with the pretreatment rate in the same patients. The TE event rate with eculizumab treatment was 1.07 events/100 patient-years compared with 7.37 events/100 patient-years (P < .001) prior to eculizumab treatment (relative reduction, 85%; absolute reduction, 6.3 TE events/100 patient-years). With equalization of the duration of exposure before and during treatment for each patient, TE events were reduced from 39 events before eculizumab to 3 events during eculizumab (P < .001). The TE event rate in antithrombotic-treated patients (n = 103) was reduced from 10.61 to 0.62 events/100 patient-years with eculizumab treatment (P < .001). These results show that eculizumab treatment reduces the risk of clinical thromboembolism in patients with PNH. This study is registered at http://clinicaltrials.gov (study ID no. NCT00122317).

Hematopoietic stem cells (HSCs) are responsible for the production of mature blood cells in bone marrow; peripheral pancytopenia is a common clinical presentation resulting from several different conditions, including hematological or extra-hematological diseases (mostly cancers) affecting the marrow function, as well as primary failure of hematopoiesis. Primary bone marrow failure syndromes are a heterogeneous group of diseases with specific pathogenic mechanisms, which share a profound impairment of the hematopoietic stem cell pool resulting in global or selective marrow aplasia. Constitutional marrow failure syndromes are conditions caused by intrinsic defects of HSCs; they are due to inherited germline mutations accounting for specific phenotypes, and often involve also organs and systems other than hematopoiesis. By contrast, in acquired marrow failure syndromes hematopoietic stem cells are thought to be intrinsically normal, but subjected to an extrinsic damage affecting their hematopoietic function. Direct toxicity by chemicals or radiation, as well as association with viruses and other infectious agents, can be sometimes demonstrated. In idiopathic Aplastic Anemia (AA) immunological mechanisms play a pivotal role in damaging the hematopoietic compartment, resulting in a depletion of the hematopoietic stem cell pool. Clinical and experimental evidences support the presence of a T cell-mediated immune attack, as confirmed by clonally expanded lymphocytes, even if the target antigens are still undefined. However, this simple model has to be integrated with recent data showing that, even in presence of an extrinsic damage, preexisting mutations or polymorphisms of genes may constitute a genetic propensity to develop marrow failure. Other recent data suggest that similar antigen-driven immune mechanisms may be involved in marrow failure associated with lymphoproliferative or autoimmune disorders characterized by clonal expansion of T lymphocytes, such as Large Granular Lymphocyte leukemia. In this wide spectrum, a unique and intriguing condition is Paroxysmal Nocturnal Hemoglobinuria (PNH); even in presence of a somatic mutation of the PIG-A gene carried by one or more HSCs and their progeny, the typical marrow failure in PNH is likely due to pathogenic mechanisms similar to those involved in AA, and not to the intrinsic abnormality conferred to the clonal population by the PIG-A mutation. The study of hematopoietic stem cell function in marrow failure syndromes provides hints for specific molecular pathways disturbed in many diseases of hematopoietic and non-hematopoietic stem cells. Beyond the specific interest of investigators involved in the field of these rare diseases, marrow failure syndromes represent a model that provides intriguing insight into quantity and function of normal hematopoietic stem cells, improving our knowledge on stem cell biology.

We explored the feasibility of unrelated donor haematopoietic stem cell transplant (HSCT) upfront without prior immunosuppressive therapy (IST) in paediatric idiopathic severe aplastic anaemia (SAA). This cohort was then compared to matched historical controls who had undergone first-line therapy with a matched sibling/family donor (MSD) HSCT (n = 87) or IST with horse antithymocyte globulin and ciclosporin (n = 58) or second-line therapy with unrelated donor HSCT post-failed IST (n = 24). The 2-year overall survival in the upfront cohort was 96 ± 4% compared to 91 ± 3% in the MSD controls (P = 0·30) and 94 ± 3% in the IST controls (P = 0·68) and 74 ± 9% in the unrelated donor HSCT post-IST failure controls (P = 0·02).The 2-year event-free survival in the upfront cohort was 92 ± 5% compared to 87 ± 4% in MSD controls (P = 0·37), 40 ± 7% in IST controls (P = 0·0001) and 74 ± 9% in the unrelated donor HSCT post-IST failure controls (n = 24) (P = 0·02). Outcomes for upfront-unrelated donor HSCT in paediatric idiopathic SAA were similar to MSD HSCT and superior to IST and unrelated donor HSCT post-IST failure. Front-line therapy with matched unrelated donor HSCT is a novel treatment approach and could be considered as first-line therapy in selected paediatric patients who lack a MSD. © 2015 John Wiley & Sons Ltd.

Contains fulltext : 152153.pdf (Publisher’s version ) (Open Access) BACKGROUND: Eculizumab, a humanized monoclonal antibody against complement protein C5 that inhibits terminal complement activation, has been shown to prevent complications of paroxysmal nocturnal hemoglobinuria (PNH) and improve quality of life and overall survival, but data on the use of eculizumab in women during pregnancy are scarce. METHODS: We designed a questionnaire to solicit data on pregnancies in women with PNH and sent it to the members of the International PNH Interest Group and to the physicians participating in the International PNH Registry. We assessed the safety and efficacy of eculizumab in pregnant patients with PNH by examining the birth and developmental records of the children born and adverse events in the mothers. RESULTS: Of the 94 questionnaires that were sent out, 75 were returned, representing a response rate of 80%. Data on 75 pregnancies in 61 women with PNH were evaluated. There were no maternal deaths and three fetal deaths (4%). Six miscarriages (8%) occurred during the first trimester. Requirements for transfusion of red cells increased during pregnancy, from a mean of 0.14 units per month in the 6 months before pregnancy to 0.92 units per month during pregnancy. Platelet transfusions were given in 16 pregnancies. In 54% of pregnancies that progressed past the first trimester, the dose or the frequency of use of eculizumab had to be increased. Low-molecular-weight heparin was used in 88% of the pregnancies. Ten hemorrhagic events and 2 thrombotic events were documented; both thrombotic events occurred during the postpartum period. A total of 22 births (29%) were premature. Twenty cord-blood samples were examined for the presence of eculizumab; the drug was detected in 7 of the samples. A total of 25 babies were breast-fed, and in 10 of these cases, breast milk was examined for the presence of eculizumab; the drug was not detected in any of the 10 breast-milk samples. CONCLUSIONS: Eculizumab provided benefit for women with PNH during pregnancy, as evidenced by a high rate of fetal survival and a low rate of maternal complications. (ClinicalTrials.gov number, NCT01374360.).

The availability of anticomplement therapies has been a major achievement for medicine in the last decade. Indeed, eculizumab has changed the treatment paradigm of paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome and promises to do the same in several other human complement-mediated diseases. Nowadays, a 10-year experience has also taught us that there are some pitfalls that represent a challenge to improve the current anticomplement treatment. Most of these observations come from paroxysmal nocturnal hemoglobinuria, where unmet clinical needs are emerging, triggering the attention of several investigators and pharmaceutical companies.

Bone loss is a common complication after allogeneic stem cell transplantation. Osteoprotegerin (OPG) plays a critical role in bone remodeling by neutralizing the effect of receptor activator of nuclear factor-kappaB ligand (RANKL) on differentiation and activation of osteoclasts. We investigated OPG and RANKL in serum and marrow plasma in transplanted patients.In 36 patients and 36 controls, the relationships among bone mineral density, circulating OPG, RANKL, interferon-gamma, and interleukin-6 levels were investigated; in addition, OPG and RANKL were measured in marrow plasma and in conditioned medium of long-term cultures of marrow mesenchymal-derived osteogenic cells.Lumbar and femoral bone mineral density were lower in patients than in controls (P0.01). Serum OPG (sOPG) and interferon-gamma were significantly higher in patients than in controls (P0.05). Patients' interferon-gamma correlated with sOPG levels (r=0.4; P=0.03). Interleukin-6 did not differ between patients and controls. By contrast, OPG levels were lower in patients than in controls in marrow plasma (P0.001) and in conditioned media after one (P=0.035) and three months (P=0.003) of culture of marrow mesenchymal-derived osteogenic cells. RANKL was similar in patients and controls. The OPG/RANKL ratio "in situ" was significantly lower in patients than in controls (P0.05). There was no correlation between sOPG and marrow OPG, RANKL levels, densitometric values, and chronic graft-versus-host disease.Our findings suggest that after allogeneic stem cell transplantation: 1) sOPG bear no relationship with OPG in the bone marrow; 2) increased sOPG can be the result of its enhanced production in extra bone tissues triggered by inflammatory cytokines; 3) low bone marrow OPG levels may be partly related to the persistent quantitative and qualitative deficit of osteoblastic precursors; and 4) reduced OPG/RANKL ratio in bone microenvironment may increase bone remodeling by promoting bone resorption.

T-cell large granular lymphocyte (T-LGL) leukemia is a clonal lymphoproliferation of cytotoxic T cells (CTLs) associated with cytopenias. T-LGL proliferation seems to be triggered/sustained by antigenic drive; it is likely that hematopoietic progenitors are the targets in this process. The antigen-specific portion of the T-cell receptor (TCR), the variable beta (VB)–chain complementarity-determining region 3 (CDR3), can serve as a molecular signature (clonotype) of a T-cell clone. We hypothesized that clonal CTL proliferation develops not randomly but in the context of an autoimmune response. We identified the clonotypic sequence of T-LGL clones in 60 patients, including 56 with known T-LGL and 4 with unspecified neutropenia. Our method also allowed for the measurement of clonal frequencies; a decrease in or loss of the pathogenic clonotype and restoration of the TCR repertoire was found after hematologic remission. We identified 2 patients with identical immunodominant CDR3 sequence. Moreover, we found similarity between multiple immunodominant clonotypes and codominant as well as a nonexpanded, “supporting” clonotypes. The data suggest a nonrandom clonal selection in T-LGL, possibly driven by a common antigen. In contrast, the physiologic clonal CTL repertoire is highly diverse and we were not able to detect any significant clonal sharing in 26 healthy controls.

The primary therapeutic approach to acquired aplastic anemia (AA) in older adults differs from the primary approach used in children and younger adults because in the former group, the results of allogeneic bone marrow transplantation (BMT) are less favorable. With increasing age of the patients, immunosuppressive therapy with antithymocyte globulin (ATG) and cyclosporine (CsA) constitutes the primary treatment option and may be better than BMT. There are very few clinical clues as to the selection of patients likely to respond to immunosuppression. Repeated ATG/CsA cycles are often used as salvage regimens, but in refractory patients BMT may be the best treatment option, as the prognosis for non-responders is poor without definitive treatment. Conservative therapy such as intense immunosuppression is associated with a high relapse rate but does not impact the survival and overall prognosis. The inability to eliminate autoimmune T cell clones using current therapeutic strategies suggests that prolonged immunosuppressive maintenance therapy may be needed for a substantial proportion of patients. Late clonal complications of conservatively treated patients include evolution to myelodysplasia and paroxysmal nocturnal hemoglobinuria and may develop in 20% of the patients. However, BMT also has several sequelae including an increased frequency of solid tumors. Novel immunosuppressive and immunomodulatory agents and constantly improving results of allogeneic BMT will further improve the survival rate of adult patients with AA.

Stem cell transplantation (SCT) constitutes a major challenge to the immune system. Long-term impairment of immunity against various common infectious stimuli leads to increased susceptibility to infectious diseases; in contrast, an immune response against the recipient may cause the devastating graft-versus-host disease (GvHD). Recovery of the immune system (both qualitative and quantitative) after SCT is perhaps the most important factor in determining the clinical outcome. Consequently, immune reconstitution has been extensively studied using different approaches, including quantitative analysis of immune cells as well as their phenotypic characterization. Analysis of diversity and clonality is an important tool in determining competence of the immune system, assuming that a broad diversity assures efficient response to different stimuli and clonal dominance reflects ongoing, potentially relevant immune responses. Detailed analysis of the immune repertoire through the flow cytometric and molecular study of the T cell receptor repertoire has been applied to gain quantitative and qualitative insights about the T cell immune competence and responsiveness. After SCT, a contraction of the T cell pool and a reduction in T cell receptor diversity is clearly associated with clinical immunodeficiency. Reconstitution of the immune system is often characterized by dominance of oligoclonal T cell populations, reflecting specific antigen-driven immune responses. Detailed characterization of T lymphocytes by T cell receptor analysis is possible, and may lead to the identification of individual clones involved in specific immune reactions, such as alloresponses in GvHD, the closely related graft-versus-leukemia effect and opportunistic viral agents such as CMV or EBV.

SummaryWe analysed endothelial cell membrane microparticles (ECMP) in theperipheral blood of patients with paroxysmal nocturnal haemoglobinuria(PNH) (n ¼ 9), aplastic anaemia (AA) (n ¼ 10), sickle cell disease (SCD)(n ¼ 8), and healthy donors (HD) (n ¼ 11). There was no clinicallymanifested thrombosis in the PNH or AA group, except one curedthrombophlebitis (PNH), while all SCD patients had a history of vaso-occlusive crises. We used three-colour flow cytometry with blood cell-specificantibodies and antibodies to endothelial antigens CD105 and CD144.Phosphatidylserine-positive microparticles were detected using the annexinV-binding (AVB) assay. The population of CD105+AVB+ ECMP wassignificantly (P

Objective Similar immune mechanisms have been suggested to operate in aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH), and the presence of PNH clones in AA may indicate that an immune reaction directed against hematopoietic stem cells may be responsible for the immune selection pressure leading to PNH evolution. We previously described expansions of selective cytotoxic T-lymphocyte (CTL) clones in AA patients. Materials and methods We applied a molecular analysis of the T-cell receptor repertoire to study the characteristics of CTL response in patients with various forms of PNH. Immunodominant T-cell clones were detected using combined flow cytometric and molecular analysis of the variable beta (VB) chain and CDR3 representation, followed by determination of the frequency of individual CDR3 clonotypes. Clonotypic polymerase chain reaction (PCR) was performed to establish clonotypic utilization pattern. Results In patients with a past history of AA, and when subgrouped by current blood counts as "hypoproliferative" PNH patients (in contrast to purely hemolytic form of PNH), more pronounced skewing of VB family utilization was found, consistent with T-cell responses involving several immunodominant CTL clones. Sequences of the PNH-derived clonotypes were used to design PCR-based assays for the utilization analysis of individual clones in PNH patients. The clonotypic distribution pattern established by this PCR method indicated that immunodominant T-cell specificities were shared between some patients but also may be found at low frequencies in controls. Conclusion Analysis of the CDR3 sequence pattern as a marker for expanded immunodominant clonotypes may have an application in the study of T-cell responses in PNH.

Profound cytopenia involving all blood lineages, a hallmark of aplastic anemia (AA), can result in devastating morbidity and high mortality. Although various etiologies and distinct pathophysiologic mechanisms may be involved, a profound defect in the stem cell compartment is a unifying feature in most patients with AA. As a stem cell disease, AA is very instructive and provides insights into the function and quantity of normal hematopoietic stem cells and their ability to regenerate. Pathophysiologically, understanding of AA may reveal mechanisms as to the evolution of other related bone marrow failure syndromes such as paroxysmal nocturnal hemoglobinuria and myelodysplasia-clonal diseases of hematopoiesis associated with defective stem cells. Conversely, constitutional forms of AA occurring in association with Fanconi anemia and dyskeratosis congenita demonstrate the role of specific genes and pathways in the dysfunction of the stem cells leading to the failure of the stem cell compartment. The acquired mechanisms resulting in depletion of stem cells in AA may involve fundamental pathways such as apoptosis and senescence as well as exhaustion of proliferative capacity or excessive differentiation. Inherent in the paucity of the bone marrow in AA, the study of the stem cells in AA has been very difficult due to their natural rarity and disease-specific contraction of the stem cell pool. Despite these scientific challenges, laboratory studies and systematic clinical observation provide valuable information of significance beyond its specific application to AA.

SUMMARYIn recent years, a pathophysiological role for T cells in immune thrombocytopenia (ITP) has been established. We applied cDNA size distribution analysis of the T cell receptor (TCR) β-variable (VB) complementarity-determining region 3 (CDR3) in order to investigate T cell repertoire diversity among immune thrombocytopenia patients who had either responded or not responded to splenectomy, and compared them to normal controls. ITP patients who had had a durable platelet response to splenectomy showed a mean 2·8 ± 2·1 abnormal CDR3 size patterns per patient, similar to healthy volunteers (2·9 ± 2·0 abnormal CDR3 size patterns). In contrast, patients unresponsive to splenectomy demonstrated evidence of significantly more clonal T cell expansions than patients who had responded to splenectomy or controls (11·3 ± 3·3 abnormal CDR3 size patterns per patient; P

In this issue of Blood, Peffault de Latour et al describe ex vivo measurements of complement activity in paroxysmal nocturnal hemoglobinuria (PNH) patients on eculizumab treatment. This is the first systematic pharmacodynamic (PD) study of eculizumab in PNH patients which shows that CH50 is a promising biomarker of therapeutic complement blockade.

The clinical management of paroxysmal nocturnal hemoglobinuria (PNH), a rare but life-threatening hematologic disease, has fundamentally improved with the introduction of a therapeutic that prevents complement-mediated intravascular hemolysis. However, a considerable fraction of PNH patients show insufficient treatment response and remain transfusion dependent. Because the current treatment only prevents C5-induced lysis but not upstream C3 activation, it has been speculated that ongoing opsonization with C3 fragments leads to recognition and phagocytosis of PNH erythrocytes by immune cells. Here, for the first time, we provide experimental evidence for such extravascular hemolysis and demonstrate that PNH erythrocytes from anti-C5-treated patients are phagocytosed by activated monocytes in vitro. Importantly, we show that this uptake can be mediated by the end-stage opsonin C3dg, which is not traditionally considered a phagocytic marker, via interaction with complement receptor 3 (CR3). Interaction studies confirmed that C3dg itself can act as a ligand for the binding domain of CR3. The degree of C3dg-mediated erythrophagocytosis in samples from different PNH patients correlated well with the individual level of C3dg opsonization. This finding may guide future treatment options for PNH but also has potential implications for the description and management of other complement-mediated diseases.

This study analysed the outcome of 563 Aplastic Anaemia (AA) children aged 0-12 years reported to the Severe Aplastic Anaemia Working Party database of the European Society for Blood and Marrow Transplantation, according to treatment received. Overall survival (OS) after upfront human leucocyte antigen-matched family donor (MFD) haematopoietic stem cell transplantation (HSCT) or immunosuppressive treatment (IST) was 91% vs. 87% (P 0·18). Event-free survival (EFS) after upfront MFD HSCT or IST was 87% vs. 33% (P 0·001). Ninety-one of 167 patients (55%) failed front-line IST and underwent rescue HSCT. The OS of this rescue group was 83% compared with 91% for upfront MFD HSCT patients and 97% for those who did not fail IST up-front (P 0·017). Rejection was 2% for MFD HSCT and HSCT post-IST failure (P 0·73). Acute graft-versus-host disease (GVHD) grade II-IV was 8% in MFD graft vs. 25% for HSCT post-IST failure (P < 0·0001). Chronic GVHD was 6% in MFD HSCT vs. 20% in HSCT post-IST failure (P < 0·0001). MFD HSCT is an excellent therapy for children with AA. IST has a high failure rate, but remains a reasonable first-line choice if MFD HSCT is not available because high OS enables access to HSCT, which is a very good rescue option.