Kurzfassung: Not available.

Kurzfassung: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a heterogeneous disease characterized by complement-mediated hemolysis and an extremely high risk of thrombosis. Eculizumab, a monoclonal antibody to complement factor C5, effectively blocks hemolysis and reduces thrombotic risk. The mechanism of thrombosis in PNH, and particularly the possible contribution of PNH neutrophils, is still unknown. Recently, neutrophil activation in form of neutrophil extracellular traps (NETs) characterized by the exposure of nucleosomes and neutrophil proteases on a meshwork of DNA was described. NETs are highly procoagulant. Whether they also play a role in the pathogenesis of thrombosis in PNH is not known. Here, we have assessed markers of neutrophil activation and NET formation in untreated PNH patients, and in PNH patients before and after starting eculizumab treatment. Methods Plasma markers of neutrophil activation such as elastase-α1-antitrypsin (EA) complexes and circulating nucleosomes as a marker of NET formation were measured by ELISA in a cohort of 51 patients with a median PNH clone size of 63% (range 5-100%) and 17 healthy controls. Median time (range) since diagnosis in patients with and without a history of thrombosis (n=12 and 39, respectively) was 8 (1-33) and 2 (0.2-24) years. The median (range) interval between the most recent thrombotic event and the time of sampling was 7.5 (0-240) months. In 20 of 51 PNH patients (clone size 83 (50-100)%), levels immediately before and at various time points during eculizumab treatment (1 hour, 2 hours, 1 week, 4 weeks and ≥ 12 weeks after the first dose) were compared. Results In the total cohort, nucleosomes and EA complexes were not significantly different between PNH patients and healthy controls. However, levels of circulating nucleosomes were significantly higher in PNH patients with a history of thrombosis (n = 12, median 16, range 7-264 U/ml) than in PNH patients without (n=39; median 6, range 6-35 U/ml, p 〈 0.001) and healthy controls (n=17, median 6, range 6-23 U/ml, p 〈 0.05). Levels of EA complexes did not differ between PNH patients with or without a history of thrombosis and controls. No correlation was found between PNH granulocyte clone size and nucleosome or EA complex levels. In 20 PNH patients with an indication for treatment with eculizumab, based upon clinical characteristics and clone size, baseline levels (median and range) of nucleosomes and EA complexes were 10 (6-264) U/ml and 27.5 (9-263) ng/ml respectively and did not differ from those in controls (8.1 (6-22.7) U/ml and 24.8 (15.8-44.2) ng/ml). Interestingly EA and nucleosome levels significantly decreased, starting as early as 1 hour after the first dose of eculizumab (p=0.0038 and p=0.002, respectively). EA complexes were still significantly decreased at ≥12 weeks (p 0.0023), whereas this was not the case for nucleosomes. When comparing patients with and without a history of thrombosis, similar as in the total cohort, baseline nucleosome levels (median, range) were significantly higher in patients with a history of thrombosis (n=9; 25 (6-264) U/ml) than in patients without (n=11; 6 (6-101) U/ml; p=0.035), whereas EA complexes were comparable. Immediately after commencement of eculizumab treatment, EA complexes significantly decreased in both groups (p 〈 0.05), whereas for nucleosomes this was only the case in the thrombosis group (p=0.023). Conclusion Similar levels of EA complexes in PNH patients and healthy controls argue against marked neutrophil activation in PNH. However, significantly elevated nucleosomes in PNH patients with a history of thrombosis may suggest low-grade NET formation. The prompt and persistent decrease in EA complex levels upon commencement of eculizumab treatment may point at a role of complement in neutrophil activation. The subtle effects of eculizumab on nucleosome levels in the thrombosis group may suggest an inhibitory role of eculizumab on NET formation in PNH patients. The clinical implications of our findings remain to be studied. Disclosures: Muus: Alexion Pharmaceuticals: Membership on an entity’s Board of Directors or advisory committees.

Kurzfassung: Abstract 3480 Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a chronic and life-threatening hematopoietic stem cell disorder characterized by uncontrolled complement-mediated hemolysis. PNH, in large part due to chronic hemolysis and platelet hyperactivation, is associated with thromboembolism (TE), one of the leading causes of disease mortality. Eculizumab, a monoclonal antibody that inhibits terminal complement activation, has been shown in clinical trials to reduce hemolysis and the incidence of TE. The International PNH Registry provides the opportunity to understand from real world experience the impact of eculizumab on TE reduction in PNH patients. Aim: To assess the risk factors for TE and mortality in PNH patients enrolled in the Registry and to assess the effectiveness of eculizumab in reducing PNH-associated TEs. Methods: Patients are eligible for the Registry if they have a detectable PNH clone, regardless of disease severity, comorbidities, or treatments (past, current or planned). As of June 30, 2012, there were 1547 patients enrolled from 25 countries on 5 continents. Patients were excluded from analysis if they were missing key demographic variables or dates of eculizumab use, or did not yet have follow-up information. The cumulative incidence of TE was determined using competing risks methods to take into account bone marrow transplantation and death, while Kaplan-Meier methods were used for the cumulative incidence of mortality. Risk factors for TE and mortality were explored using a Cox proportional hazards model with stepwise selection (the significance level was relaxed to P=0.20 due to the small number of events for analysis). Variables examined in the models included: ethnicity; prior TEs, bone marrow disorders, impaired renal function, impaired hepatic function (IHF), abdominal pain, dysphagia, dyspnea, easy bruising/bleeding, fatigue, headache, hemoglobinuria, Karnofsky performance score, granulocyte clone size and lactate dehydrogenase (LDH) at enrollment, red blood cell (RBC) transfusions 6 months prior to enrollment as a marker for hemolysis, and treatments after enrollment (eculizumab and warfarin/heparin). Results: The mean age of the 1047 patients eligible for analysis was 45 years; 537 patients (51.3%) were female and 868 were Caucasian (82.9%). Anti-coagulants (heparin/warfarin) were used by 28% of patients and eculizumab was used by 51% during follow-up (18% used both). During a mean (SD) follow-up of 22.5 (18.4) months, 16 patients had a TE and 51 were deceased. Patients taking eculizumab during follow-up had a cumulative incidence of TE at 1 year of 0.41% and 1.35% at 2 years, while patients not taking eculizumab had TE incidence of 1.70% and 2.61% at 1 and 2 years, respectively. In the multivariate Cox model, the greatest associations with TE were RBC transfusions in the 6 months before enrollment (hazard ratio [HR]=9.61), history of IHF (HR=4.78), dyspnea (HR=2.42) and headache (HR=2.33) at enrollment. While controlling for these variables, eculizumab had a significant protective effect (HR=0.23, 95% CI = 0.08–0.66). The cumulative incidence of mortality in eculizumab-treated patients was 2.31% and 4.21% at 1 and 2 years, while in untreated patients it was 4.40% and 7.01%, respectively. In the multivariate model of mortality, the greatest associations were age 60+ years (HR=4.72), Karnofsky score 〈 80 (HR=2.34), fatigue at enrollment (HR=1.94), and recent RBC transfusion (HR=1.75). While controlling for these variable, eculizumab had a significant protective effect (HR=0.41, 95% CI = 0.23–0.73). Conclusions: This analysis of a large international cohort of ‘real world’ patients with PNH showed that eculizumab is associated with a significantly reduced risk of TE and mortality, consistent with prior research. Recent RBC transfusion, a surrogate marker for hemolysis, was associated with increased risk of TE and mortality. Several symptoms and hepatic dysfunction also showed increased risks for these outcomes. As might be expected, older age and low performance status were associated with mortality. These data should be interpreted within the context of a contemporary cohort of PNH patients who may or may not be treated (with either eculizumab and/or anticoagulation). These analyses are limited due to small number of TE and mortality outcomes. Disclosures: Muus: Alexion Pharmaceuticals : Sat on advisory board of Alexion Pharmaceuticals. Other. Urbano-Ispizua:Alexion Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees. Maciejewski:NIH: Research Funding; Aplastic Anemia & MDS International Foundation: Research Funding. Kanakura:Shire: Consultancy. Rosse:Alexion Pharmaceuticals, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees. Khursigara:Alexion Pharmaceuticals, Inc: Employment. Bedrosian:Alexion Pharmaceuticals: Employment, Equity Ownership. Hillmen:Alexion Pharmaceuticals, Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Kurzfassung: Abstract 257 The AML-12 randomized phase III trial of EORTC-LG and GIMEMA assessed the efficacy and toxicity of HD-AraC (3 g/sqm/12 hrs for 4 days) combined with daunorubicin (50 mg/sqm/d for 3 days) and etoposide (50 mg/sqm/d for 5 days) vs SD-AraC (100 mg/sqm/d for 10 days) with the same drugs, in previously untreated AML 〈 61 year old patients (APL excluded). Patients (pts) in complete remission (CR) had to receive consolidation consisting of AraC (500 mg/sqm/12 hrs for 6 days) and daunorubicin. Subsequently an allogeneic (allo-SCT) or autologous stem cell transplantation (auto-SCT) was planned according to donor availability and age. A 2nd randomization had to be done after evaluation of consolidation in pts without a donor: auto-SCT followed or not by low dose IL-2. The aim of the trial was to detect an 8% treatment difference (from 35% to 43%) in the 5-yr overall survival (OS) rate, corresponding to a hazard ratio (HR) of 0.80 (alpha=5%, power=95%); secondary endpoints were response to induction, toxicity, disease-free survival (DFS) from CR. Randomization was performed centrally; the 1st randomization was stratified for age ( 〈 46 vs 〉 45 yrs), performance status, WBC and center. Intent-to-treat analysis was done. From 9/1999 till 1/2008, 2005 pts from 68 centers were randomized. Due to insufficient reporting, 3 centers, who recruited 63 patients, have been excluded from the analysis. The remaining 1942 pts (872 pts entered by EORTC-LG and 1070 by GIMEMA) 969 were randomized in SD-AraC and 973 in HD-AraC arm; among them 25 and 28, respectively, were ineligible, but kept in the analysis. Both arms were comparable with respect to gender, age (median=45 yrs), disease history, initial leukocyte count, PS, FAB and cytogenetics. At a median follow up of 6 yrs, 1114 pts had died. Results: After 1 or 2 courses of induction, CR was achieved in 1430 pts (73.6%); 684 (71.9%) pts (SD-AraC group) vrs 746 (78.7%) pts (HD-AraC group): p=0.002. Resistance was documented in 173 (18.2%) vrs 123 (13%), and death during induction in 85 (8.9%) vrs 71 (7.5%) pts, respectively. Induction toxicity profile and grade was similar in the 2 arms except for conjunctivitis grade 3: HD-AraC 〉 SD-AraC. CR rates for pts 〈 46 yrs were 74.7% (SD-AraC) and 81.4% (HD-AraC) and for pts 〉 45 yrs 66.4% (SD-AraC) and 71.8% (HD-AraC). 634 pts (SD-AraC and 686 (HD-AraC) received a consolidation cycle. Among 765 CR-pts 〈 46 yrs 284 pts had an HLA identical sibling ( 〈 46D) and 481 did not or had not been typed ( 〈 46NoD). Among 665 CR-pts 〉 45 yrs 225 pts had an HLA identical sibling ( 〉 45D) and 440 did not or had not been typed ( 〉 45NoD). In the 〈 46D group 211 underwent an allo-SCT and 11 an auto-SCT. In the 〈 46NoD group 274 underwent an auto-SCT and 29 a MUD-SCT; in the 〉 45D group 147 underwent an allo-SCT and 14 an auto-SCT. In the 〉 45NoD group 244 underwent an auto-SCT and 12 a MUD-SCT. Comparisons of treatments arm regarding OS from randomization, DFS and Survival (S) from CR are indicated in the Table. The impact of age on the treatment difference regarding OS was almost significant (p=0.06). In pts 〉 45 who reached CR, the decrease in the relapse rate in the HD-Ara-C arm vrs SD-Ara-C arm (45.5% vs 49.4%) was counterbalanced by an increase in the death in CR rate (18.4% vs 13.2%). Conclusion: The final evaluation of the EORTC-GIMEMA AML-12 trial shows that, with a median follow-up of 6 years, HD-AraC in the induction treatment leads to a significantly higher CR rate than SD-Ara-C and results in improvement in overall survival but only in pts under the age of 46 years. Disclosures: Muus: Amgen: Membership on an entity's Board of Directors or advisory committees. Beksac:Janssen Cilag: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau.

Kurzfassung: In acute promyelocytic leukemia (APL), differentiation therapy with all-trans retinoic acid (ATRA) and/or arsenic trioxide can induce a differentiation syndrome (DS) with massive pulmonary infiltration of differentiating leukemic cells. Because chemokines are implicated in migration and extravasation of leukemic cells, chemokines might play a role in DS. ATRA stimulation of the APL cell line NB4 induced expression of multiple CC-chemokines (CCLs) and their receptors ( 〉 19-fold), resulting in increased chemokine levels and chemotaxis. Induction of CCL2 and CCL24 was directly mediated by ligand-activated retinoic acid receptors. In primary leukemia cells derived from APL patients at diagnosis, ATRA induced chemokine production as well. Furthermore, in plasma of an APL patient with DS, we observed chemokine induction, suggesting that chemokines might be important in DS. Dexamethasone, which efficiently reduces pulmonary chemokine production, did not inhibit chemokine induction in APL cells. Finally, chemokine production was also induced by arsenic trioxide as single agent or in combination with ATRA. We propose that differentiation therapy may induce chemokine production in the lung and in APL cells, which both trigger migration of leukemic cells. Because dexamethasone does not efficiently reduce leukemic chemokine production, pulmonary infiltration of leukemic cells may induce an uncontrollable hyperinflammatory reaction in the lung.

Kurzfassung: Introduction: Studies of children with paroxysmal nocturnal hemoglobinuria (PNH) are scarce and include a very limited number of patients. The objective of this analysis was to describe characteristics of PNH at enrollment for the largest available registry of pediatric patients, and to compare demographic and clinical characteristics with those of adult patients. Methods: The International PNH Registry is a prospective, multi-center worldwide, observational study of patients with a PNH clone of 0.01-100%. Data are collected from patient medical records at the time of Registry enrollment and every six months thereafter. Adult patients were ³18 years of age at enrollment and disease start and pediatric patients were 〈 18 years at enrollment. Demographics and clinical parameters in patients untreated with eculizumab at enrollment for the two age cohorts were compared using the Wilcoxon-Mann-Whitney test for medians and PearsonÕs chi-square for frequencies. The rate of thrombotic events (TE) between disease start (defined as the earliest reported PNH symptoms, granulocyte clone, or PNH diagnosis) and enrollment was calculated per 100 person-years. Results: As of March 2, 2015, a total of 2,184 patients were eligible for analysis: 94 (4.3%) pediatric patients and 2,090 (95.7%) adult patients. Median age (range) at enrollment was 14.0 years (3-17) in pediatrics and 45.5 years (18-100) in adults; median disease duration was 0.7 years and 2.1 years, respectively (p 〈 0.001). More pediatric than adult patients had a PNH clone of 〈 10% and severe cytopenia (Table). Pediatric patients had lower percent of reticulocytes compared with adults (2.1% vs. 2.6%, respectively; p=0.015). History of aplastic or hypoplastic anemia was more frequent in pediatric than adult patients (76.5% vs 54.4%, respectively; p 〈 0.001). History of TE and any major adverse vascular event was less frequent in pediatrics (2.1% vs 8.7%; p=0.025, and 4.3% vs. 14.4%; p=0.005). The rate of TE between disease start and enrollment was lower in pediatric patients, but not statistically significant: 1.4 per 100 person-years (95%CI 0.2-5.2) compared to adult patients (2.3 per 100 person-years (95%CI 2.0-2.6). More pediatric patients than adults had abdominal pain at enrollment. Conclusions: The International PNH Registry provides the largest available pediatric cohort of patients with a PNH clone to characterize this understudied population and demonstrate an important disease burden. Pediatric patients were more likely to have smaller PNH clones and a higher component of aplastic/hypoplastic anemia. Pediatric patients had fewer vascular events. These findings may reflect the natural evolution of the disease and can be useful in the clinical management of PNH. Table 1. Clinical Characteristics at Enrollment of Pediatric and Adult Patients with PNH Pediatric(n=94) Adult(n=2,090) P-value Clone size (percent GPI-deficient granulocytes), n (%) 〈 10% 10 to 〈 50% ³50% 47 (55.3) 16 (18.8) 22 (25.9) 550 (38.3) 322 (22.4) 565 (39.3) 0.006* Cytopenia status, n (%)None (neutrophils ³ 1.5 x 109/L and platelets ³100 x 109/L) Moderate (neutrophils 〈 1.5 x 109/L or platelets 〈 100 x 109/L) Severe (neutrophils 〈 0.5 x 109/L or platelets 〈 20x109/L) 22 (29.3) 28 (37.3) 25 (33.3) 735 (42.2) 784 (45.1) 221 (12.7) 〈 0.001* Percent reticulocytesMedian (Q1, Q3) 2.1 (1.1, 3.5) 2.6 (1.6, 4.6) 0.015 Hemolytic status, n (%)Hemolytic (LDH ³1.5 x ULN and/or reticulocytes ³60 x 109/L) Not hemolytic (LDH 〈 1.5 x ULN and reticulocytes 〈 60 x 109/L) 33 (58.9) 23 (41.1) 1,038 (65.3) 551 (34.7) NS LDH Ratio, n (%) 〈 1.5 x ULN³1.5 x ULN 30 (58.8) 21 (41.2) 684 (47.0) 770 (53.0) NS History of TE, n (%)Yes No 2 (2.1) 92 (97.9) 181 (8.7) 1,902 (91.3) 0.025 Rate of TENumber of TE, n Person-years (disease start to enrollment) Rate/100 person-years (95% CI) 2 139.9 1.4 (0.2-5.2) 255 11,119.8 2.3 (2.0-2.6) NS History of MAVE, n (%) 4 (4.3) 300 (14.4) 0.005 GPI, glycosylphosphatidylinositol; LDH, lactate dehydrogenase; MAVE, major adverse vascular event; TE, thrombotic event; ULN, upper limit of normal *P-values for clone size and cytopenia status represent overall comparison of categories. Disclosures Muus: Alexion Pharmaceuticals: Honoraria. Schrezenmeier:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Almeida:Celgene: Consultancy; Novartis: Consultancy; Bristol Meyer Squibb: Speakers Bureau; Shire: Speakers Bureau. Wilson:Alexion Pharmaceuticals: Employment. Ware:Bayer Pharmaceuticals: Consultancy; Biomedomics: Research Funding; Eli Lilly: Other: DSMB membership; Bristol Myers Squibb: Research Funding.

Kurzfassung: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired clonal stem cell disorder. Pregnancy in patients with PNH results in an increase in maternal and fetal complications and a consequent increase in maternal and fetal mortality. Maternal complications include thrombosis, cytopenias and infections while fetal complications relate to preterm delivery (de Guibert et al, 2011). Eculizumab is a humanized monoclonal antibody that blocks terminal complement activation, preventing intravascular hemolysis and its consequences. In PNH it reduces the need for transfusions, protects against thrombosis, and may improve long-term survival. We previously showed a benefit in using eculizumab in pregnancy for patients with PNH in a limited number of cases (Kelly et al, BJH 2011). This study was of 70 pregnancies in 57 women. Patients were identified through physician willingness to participate as well as through the Global PNH Registry. A specific questionnaire was sent to physicians. Local IRBs approved the study. Across the 70 pregnancies, 41 women were on eculizumab before conceiving and remained on the drug during pregnancy, 28 women started eculizumab in either the second or third trimester and 1 woman stopped eculizumab at 12 weeks gestation. The median age at PNH diagnosis was 23 years (range 13-36). The median age at the start of each pregnancy was 29 years (range 18-40). Nineteen women (33%) had a prior documented history of aplastic anemia and 8 (14%) had a prior thrombosis. The median PNH granulocyte clone around the start of pregnancy was assessed in 51 cases and was 94.3% (range 24.3-100). There were 62 live births, 2 stillbirths and 6 first trimester miscarriages. There were 3 twin pregnancies. In 1 of these, there was a fetal death due to intrauterine growth retardation (IUGR). Anticoagulation (AC) with low molecular weight heparin (LMWH) was used in 60 pregnancies (86%) and with fondaparinux in 1 case: therapeutic doses were used in 26 pregnancies, prophylactic doses in 28 pregnancies and an intermediate dose in a further 7. Aspirin was used in 4 pregnancies. Folic acid and oral iron supplements were used in 62 (89%) and 26 (41%) pregnancies, respectively. Transfusion requirements increased in pregnancy from a mean of 0.13 units per month in the 6 months before conception to a mean of 0.94 units per month whilst pregnant. This returned to pre-pregnancy levels after delivery. No platelet transfusions were needed in the 6 months before being pregnant compared with 99 platelet transfusions during the pregnancies. Higher doses, or more frequent dosing, of eculizumab was used to treat recurrent intravascular hemolysis in over half the pregnancies (52%) that progressed past the first trimester. Eculizumab was stopped in 10 instances after the postpartum period, 9 due to funding issues and 1 as the patient underwent a bone marrow transplant. Two patients who stopped eculizumab 12 weeks after delivery experienced a thrombosis. The first experienced a mesenteric thrombosis 4 weeks after stopping eculizumab whilst on therapeutic dose of LMWH and the second suffered a Budd-Chiari 8 weeks after stopping eculizumab. Both were recommenced on eculizumab immediately. The mean reduction in platelet count from the start of pregnancy to delivery was 37 x 109/l. There were 10 significant bleeding episodes: 1 recurrent epistaxis, 1 antepartum bleed and 8 postpartum bleeds. Two patients experienced a postpartum thrombosis whilst on eculizumab, 1 a deep vein thrombosis and the other a mesenteric thrombosis, likely precipitated by a plasma infusion. Nineteen births were premature (31%), mainly due to pre-eclampsia (6 cases) and IUGR (5 cases). Four babies had complications due to prematurity: 1 had toxic megacolon and required a temporary ileostomy, and the other 3 had initial growth retardation due to prematurity. Twenty-five babies were breast fed and in 10 cases, breast milk was tested for eculizumab but no drug was detected. Eculizumab was not detected in 13 cord blood samples and was found at very low levels in a further 7 samples (11.8-21.2µg/ml). In conclusion, eculizumab appears safe to use in pregnancy in PNH and does not appear to cross the placenta in significant quantities to block complement or to be excreted in breast milk. Higher doses may be required later in pregnancy to prevent hemolysis. Overall pregnancy outcomes in this group are better than historical controls with supportive therapies alone without eculizumab. Disclosures Kelly: Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Höchsmann:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Kulasekararaj:Alexion Pharmaceuticals: Speakers Bureau. Röth:Alexion Pharmaceuticals: Speakers Bureau. Weitz:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Hill:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Risitano:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Patriquin:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Muus:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Turner:ICON Clinical Research: Employment. Schrezenmeier:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Szer:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Peffault de Latour:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Kurzfassung: Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoietic disorders, characterized by ineffective hematopoiesis resulting in cytopenias and a highly variable clinical course. Cytogenetics are routinely used as one of the diagnostic, prognostic and therapeutic markers in the clinical management of MDS (Greenberg et al. Blood 120:2454,2012). Although karyotyping is generally considered as the gold standard in the cytogenetic characterization of MDS, 40-60% of the patients exhibit a normal karyotype. Intrinsically, the resolution of karyotyping is limited by its capacity to detect only those copy number changes that are microscopically visible (5-10 Mb in size). In contrast, microarray-based genomic profiling analyses allow a genome-wide detection of copy number alterations (CNAs), down to 100 kb in size, and regions of copy neutral loss of heterozygosity (CNLOH). Such analyses also overcome some of the other major limitations of karyotyping such as low success rate due to inadequate metaphase yield and/or poor banding quality. We have compared karyotyping and fluorescence in situ hybridization (FISH) with microarray-based genomic profiling with respect to the detection yield for genetic abnormalities in bone marrow samples from lower risk MDS patients. We used the HOVON89 study-cohort, a prospective phase II randomized multicenter study to assess the efficacy of lenalidomide with or without erythropoietin and granulocyte-colony stimulating factor in patients with low/intermediate-1 risk MDS; www.trialregister.nl; NTR1825; EudraCT nr.: 2008-002195-10. Inclusion target of the study is 200 low/intermediate-1 risk MDS patients (134 enrolled, inclusion ongoing). Data regarding cytogenetics, FISH and microarray were obtained in a fully blinded fashion for 68 MDS patients. For microarray-based genomic profiling we used the recently launched high resolution CytoScan HD Array (Affymetrix) platform. The following interpretation criteria were applied: (i) the threshold for CNAs was set at 〉 5 Mb, (ii) inclusion of 〈 5 Mb CNAs segments that coincide with known cancer genes as reported on www.sanger.ac.uk and (iii) the threshold for CNLOH was set at 〉 10 Mb and to telomere. Karyotyping and interphase FISH were performed using standard cytogenetic methods. In all 4 patients where karyotyping revealed no metaphases interphase FISH was performed. Thirty-six of the 68 (53%) MDS patients had an abnormal microarray profile. Of interest, in 13 of these 36 patients no abnormalities were observed by karyotyping and/or FISH. All these abnormalities observed by microarray only, involved focal 〈 5 Mb CNAs (containing e.g. the TET2, RUNX1 and DNMT3A genes) and regions of CNLOH (coinciding with 1p, 2p, 4q, 7q, 11p, 11q, 12q, 14q and 18q), which are all out of the scope of karyotyping and FISH. All CNAs identified by karyotyping and FISH were also observed by microarray-based genomic profiling, including a case with loss of 5q in 5% of the cells and loss of 7q in 9% of the cells as observed by interphase FISH and another case with loss of 5q in 3 of 20 the analyzed metaphases by karyotyping. These observations demonstrate the high sensitivity of the CytoScan HD array platform for the identification of CNAs in (small) subclones. As expected balanced translocations such as t(3;3)(q21;q26) and t(2;14)(q37;q22) present in 2 of the patients in our cohort were not identified by microarray-based genomic profiling. This study will be extended to all patients to be included in the HOVON89 trial. In conclusion, we demonstrate that in the present cohort of 68 patients, microarray-based genomic profiling allows the identification of almost all copy number abnormalities also observed by karyotyping and FISH. In addition, we show that microarray-based genomic profiling allows the detection of potential prognostic relevant abnormalities (focal CNAs and CNLOH) which would have remained undetected by karyotyping and FISH. The predictive and/or prognostic value of these novel CNAs and CNLOH will be evaluated within the ongoing prospective clinical HOVON89 trial in lower risk MDS. Disclosures: No relevant conflicts of interest to declare.

Kurzfassung: Abstract 2235 Background: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disease characterized by hemolysis due to an acquired mutation in the X-linked PIG-A gene in the hematopoietic stem cell (HSC). This leads to a clone of hematopoietic cells with deficient expression of glycosyl phosphatidyl inositol (GPI) anchored proteins at the cell membrane. The clinical evolution of PNH arises through clonal expansion of PIG-A mutated HSC which is insufficiently explained by the PIG-A mutation alone. Hypothetically, clonal expansion could result from autoreactive T cells selectively attacking normal HSC, whereas GPI deficient HSC are unharmed. Methods and Results: we investigated the presence of potentially autoreactive T cells in peripheral blood of patients with PNH (n = 39) by flow cytometry. We compared T cell subset frequencies and absolute numbers with healthy controls (n = 25) using Mann-Whitney U test. In PNH patients, T cells expressing the NK cell marker CD56 were significantly elevated, both in percentage (p 〈 0.001) and in absolute numbers (p 〈 0.01). Furthermore, the frequency of T cells expressing the activating NK cell receptors (NKRs) NKG2D (p 〈 0.01), NKG2C (p 〈 0.01), and KIR2DS4 (p = 0.01) was significantly increased. KIR2DS4+, NKG2C+ and NKG2D+ T cells mainly consist of highly differentiated effector memory CD45RA+ T cells (TEMRA) (KIR2DS4: median 90%, range 70–96%, NKG2C: median 83%, range 49–99%, NKG2D: median 40%, range 38–66%). A highly variable proportion of these T cell populations consists of γδ T cells (KIR2DS4: median 28%, range 6–72%, NKG2C: median 36%, range 3–75%, NKG2D: median 11%, range 7–40%). By 10 color flow cytometry, we examined NKR coexpression patterns. KIR2DS4+ and NKG2C+ T cells mainly coexpress either only NKG2D (KIR2DS4+ T cells: median 24%, range 2 – 74%, NKG2C+ T cells: median 21%, range 3–71%), or a combination of NKG2D, NKG2C and CD158b1/b2,j, but not inhibitory NKG2A (KIR2DS4+ T cells: median 16%, range 1 – 55%, NKG2C+ T cells: median 20%, range 1–60%). In contrast, NKG2D+ T cells generally do not express any other NKRs tested (median 77%, range 53–84%). NKG2D+ KIR2DS4+ cytotoxic T lymphocyte (CTL) lines isolated from PNH patient peripheral blood and bone marrow display high cytolytic activity towards CD34+ hematopoietic progenitor cell lines and MHC class I deficient K562 cells, suggesting T cell receptor independent cytolytic activity. These NKR+ CTL lines are capable of differentially lysing GPI+ and GPI- hematopoietic cell lines, however not in all cell line models and CTL lines. This suggests that multiple factors, as for example the highly activated status of in vitro cultured CTLs, influence whether or not GPI dependent lysis occurs. Conclusion: The increased frequency of T cells expressing activating NK cell receptors KIR2DS4, NKG2C, and NKG2D, with a CD8+ effector-memory phenotype and differences in cytotoxicity towards GPI+ and GPI- hematopoietic cell lines suggests that these T cell populations may be involved in bone marrow failure and expansion of PNH clones. Disclosures: Muus: Alexion: member of advisory board.

Kurzfassung: Abstract 1885 Background: Thrombocytopenia is a significant clinical problem in MDS, found in approximately 35% to 65% of pts, and is an independent risk factor for survival. Romiplostim increases platelet production by binding to and activating the thrombopoietin receptor. MDS pts who completed a romiplostim clinical study could continue romiplostim treatment in an open-label extension study. Interim results from this ongoing study are reported to provide updated safety and efficacy information on the effects of continued, long-term romiplostim treatment in pts with MDS. Methods: MDS pts who had completed a prior romiplostim study and had platelets ≤50 × 109/L with no evidence of disease progression were eligible. Pts enrolled into the extension study from 3 prior studies with the following study designs: (1) romiplostim only for up to 52 weeks [Kantarjian JCO 2009], (2) romiplostim or placebo plus decitabine for ≥4 cycles [Greenberg ASH 2009], and (3) romiplostim or placebo plus lenalidomide for ≥4 cycles [Lyons ASH 2009] . The primary endpoint was adverse event (AE) incidence; secondary endpoints were incidence of bleeding events, platelet transfusions, and platelet response durations. On the basis of previous dosing, pts received romiplostim at 250, 500, 750, 1000, or 1500 mcg weekly, with subsequent adjustments based on platelet count. If no response was observed after 4 weeks at 1000 mcg/week, treatment was discontinued. Results: As of July 2, 2010, 40 pts had enrolled: 28 from 72 previously treated with romiplostim only, 7 from 29 previously treated with decitabine plus romiplostim or placebo, and 5 from 39 previously treated with lenalidomide plus romiplostim. In the extension study, 3 pts continued receiving decitabine and 3 pts continued receiving lenalidomide. Twenty-eight pts (70%) were male, the median age was 72 (Q1-Q3: 64–77) years, median baseline platelets were 31 × 109/L (Q1-Q3: 19–45 × 109/L), 11 pts (28%) had platelets 〈 20 × 109/L. At the end of their prior study, IPSS status was low (19 pts), int-1 (15), int-2 (1), or missing (5), and MDS subtypes at diagnosis were RA (11 pts), RCMD (10), MDS-U (6), RAEB-1 (8), MDS del 5q (2), RAEB-2 (1), RCMD-RS (1), and RARS (1). Twenty-one pts (53%) had bleeding events during the year before entering this study. Median duration of treatment in the extension study was 50 weeks (range: 6–134 weeks), with a median average weekly dose of 729 mcg (Q1-Q3: 593–893 mcg). The incidence of all AEs during 12-week study periods ranged from 60% to 92% and did not increase with time on study. Most AEs were mild-to-moderate; the most common being epistaxis (40%), fatigue (33%), and cough (30%). No neutralizing antibodies to romiplostim or endogenous TPO were detected. No pts died during the study; one pt died 3 months after withdrawing from the study (causality was uncertain). Peripheral blasts were increased in 2 pts (MDS-U and RA at baseline) and resolved in both cases after discontinuation of romiplostim. Three cases of progression to AML were reported, corresponding to an annual on-study event rate of 5.9% (95% CI: 1.9% to 18.3%). These pts were IPSS-risk low or int-1 at baseline and had a WHO classification of RAEB-1 or RCMD. During this study, they received 750 mcg romiplostim for 6, 49, and 36 weeks. Bone marrow blasts were 39%, 65%, and 40% at AML diagnosis. Thirty pts (75%) reported ≥1 bleeding event(s), with 17 pts (43%) reporting ≥1 clinically significant bleeding event(s) [CTCAE grade ≥3, serious AE, or any bleeding AE requiring intervention]. The proportion of pts experiencing a significant bleeding event decreased from 23% (9/40) during weeks 1–12 to 0% (0/21) during weeks 48–60. Similarly, the proportion of pts receiving a platelet transfusion decreased from 28% (11/40) during weeks 1–12 to 0% (0/21) during weeks 48–60. From week 3 onwards, the median platelet count was ≥ 50 × 109/L (Figure). Thirty-four pts (85%) had a platelet response (per IWG 2006). The median duration of platelet response was 41 weeks (Q1-Q3: 15–87 weeks). Conclusion: In this study, long-term treatment of MDS pts with romiplostim demonstrated a toxicity profile consistent with published data and disease progression was consistent with expected rates. Most pts achieved a sustained platelet response while receiving romiplostim. Disclosures: Fenaux: Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen Cilag: Honoraria, Research Funding; ROCHE: Honoraria, Research Funding; AMGEN: Honoraria, Research Funding; GSK: Honoraria, Research Funding; Merck: Honoraria, Research Funding; Cephalon: Honoraria, Research Funding. Off Label Use: Romiplostim is approved for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenia (ITP). Kantarjian:Amgen Inc.: Research Funding. Lyons:Amgen Inc.: Consultancy, Honoraria, Research Funding, Speakers Bureau. Larson:Amgen Inc.: Research Funding. Sekeres:Celgene: Consultancy, Honoraria, Speakers Bureau; Seattle Genetics: Consultancy. Becker:Amgen Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Muus:Celgene: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Jia:Amgen Inc.: Employment, Equity Ownership. Yang:Amgen Inc: Employment, Equity Ownership.