PNH ABSTRACTS Authors 4393 Richard J Kelly, MBChB, PhD1*, Britta Höchsmann, MD2*, Austin Kulasekararaj3*, Sophie de Guibert, MD4*, Alexander Röth, MD5*, Ilene C Weitz, MD6, Elina Armstrong, MD, PhD7*, Anita Hill, MBChB, PhD1*, Antonio M. Risitano, MD, PhD8, Christopher J Patriquin, MD FRCPC9*, Louis Terriou, MD10*, Petra Muus, MD, PhD11, Michelle P Turner, MS in Biostatistics12*, Hubert Schrezenmeier, MD, PhD2*, Jeffrey Szer, MB BS FRACP13 and Regis Peffault de Latour, MD, PhD14* Title Eculizumab Treatment Improves Outcomes of Pregnancy in Patients with Paroxysmal Nocturnal Hemoglobinuria Abstract Introduction: 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). Methods: 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. Results: 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 prepregnancy 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). Conclusions: 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. Authors 256 Carlo Dufour, MD1, Marta Pillon, MD2*, Elisa Carraro, PHD3*, Neha Bhatnagar4*, Rob Wynn5*, Brenda Gibson6, Ajay J Vora7, Colin G Steward, MD, PhD8, Anna Maria Ewins, MD9*, Rachael E Hough, MD10*, Josu de la Fuente, PhD, FRCP, MRCPCH, FRCPath11, Mark Velangi12*, Paul Veys, FRCP, FRCPath13*, Persis J Amrolia14*, Roderick Skinner, PhD, MBChB, FRCPCH15*, Andrea Bacigalupo, MD16, Antonio M. Risitano, MD, PhD17, Gerard Socie, MD, PhD18, Regis Peffault de Latour, MD, PhD19,20*, Jakob Passweg, MD21, Alicia Rovo22, André Tichelli, MD23, Hubert Schrezenmeier, MD24,25, Britta Hochsmann, MD26*, Peter Bader27, Anja van Biezen28*, Judith C Marsh, MD29, Mahmoud D. Aljurf, MD30 and Sujiith Samarasinghe, MD31* Title Similar Outcome of Upfront Unrelated and Matched Sibling Donor Hematopoietic Stem Cell Transplantation in Idiopathic Aplastic Anaemia of Childhood and Adolescence: A Cohort Controlled Study on Behalf of the UK Paediatric BMT WP, of the PD WP and of the SAA WP of the EBMT Abstract The classical treatment algorythm of idiopathic severe aplastic anemia (SAA) forsees, if a sibling donor is not available in the family, a first line option represented by immunosuppressive therapy (IST) with ATG and Cyclosporine A (CsA). IST provides a very good survival rate but a fairly high rate of failures (no response, relapse, need for transplant). This is particularly important in childhood and adolescence where faulty hematopoiesis represents a relevant limitation of quality of life. The option of haematopoietic stem cell transplantation (HSCT) from matched unrelated donor (MUD) as front line treatment with no prior failed IST, has never been investigated so far. This study provides for the first time the outcome analysis of 29 consecutive SAA patients who received an upfront unrelated donor HSCT without prior IST, in 9 UK Centres and compares each of these patient with 3 matched controls from the data base of the SAAWP of the EBMT who, in a similar time-span, underwent Matched Sibling Donor (MSD) HSCT. Twenty four patients received HLA-A,-B,-C,-DQ-,-DRB1 matched MUD HSCTs, whilst 5 received single antigen/allele Mismatched Unrelated Donor (MMUD) grafts. The conditioning regimen was FCC (Fludarabine Cyclophosphamide and Alemtuzumab) with the addition of low dose (3cGy) TBI for the MMUD HSCTs. GVHD prophylaxis was with CsA +-Mycophenolate. The primary endpoints were overall survival (OS) and event free survival (EFS). Events were death, disease recurrence, second HSCT, clinical PNH and post-transplant malignancies. Complete remission (CR) was defined as Hb > 10 g/dl, neutrophil count > 1x 109/l and platelet count > 100x109/l. The 87 (3 for each MUD/MMUD HSCT) patients from the EBMT data base who underwent MSD HSCT as first line treatment were matched to the upfront MUD/MMUD cohort by age, gender, time from diagnosis to HSCT and source of stem cells. Their median follow-up was 1.6 years (range 0.01-9.39). In the upfront MUD/MMUD cohort there were 12 males (41%) and 17 females (59%). Median age at HSCT was 8.46 years (range 1.73-19.11), 72% was aged below and 28% above 12 years; median interval from diagnosis to HSCT was 0.39 years (range 0.19-1.35) with 72% receiving bone marrow and 28% peripheral blood stem cells. The median follow-up was 1.7 years (range 0.19-8.49). The median time to neutrophil recovery (>0.5 x 109/l) was 18 days (range 9-29) and the median time to platelet recovery (>50 x 109/l) was 19 days (range 10-40). The 100 day cumulative incidence (CI) of grade II-IV acute GVHD was 10 ± 6%; there was only one case of grade III-IV acute GVHD (frequency of 3.5%). The 1 year CI of chronic GVHD was 19 ± 8%. There were 5/29 cases (frequency of 17,2%), 4 in the MUD and 1 the MMUD subset. Chronic GVHD was limited in all cases and restricted to skin. There were only 2 events in this cohort; one primary graft failure following a HLA-A MMUD HSCT who has now successfully received a second HSCT and one death due to idiopathic pneumonia syndrome. No post-treatment malignancies occurred at last follow-up. The other 27 patients are in CR at last follow-up. The median interval diagnosis-neutrophil recovery was similar in MUD/MMUD (0.39 years; range 0.19-1.35) vs MSD transplants (0,31 years; range 0,1- 0,45) (p=0.93). The 2 year OS was 96%±4% in the upfront MUD/MMUD cohort vs 91%±3% of the MSD cohort (p=0.30). The 2 year EFS was 92%±5% in the upfront cohort vs 87%±4% in MSD (p=0.37) (Fig 1). The 2 year CI of rejection was 4%±4% in the MUD/MMUD vs .1%±1% in the MSD group (p=0.48). This is the first controlled study indicating that in idiopathic SAA of childhood and adolecence upfront MUD/MMUD HSCT using FCC as conditioning regimen, has similar outcome to MSD HSCT. MUD HSCT may be considered a feasible and successful treatment option when children lack a MSD and a MUD is readily available. Authors 1595 Hideyoshi Noji, MD, PhD1,2*, Tsutomu Shichishima, MD, PhD1,2,3, Chiharu Sugimori, MD, PhD2,4*, Naoshi Obara, MD, PhD2,5*, Kohei Hosokawa, MD, PhD2,6*, Shigeru Chiba, MD, PhD2,5, Yoshihiko Nakamura, MD, PhD2,7*, Kiyoshi Ando, MD, PhD2,7, Satoru Hayashi, MD, PhD2,8*, Yuji Yonemura, MD, PhD2,9, Tatsuya Kawaguchi, MD, PhD2,10, Haruhiko Ninomiya, MD, PhD2,11, Jun-ichi Nishimura, MD, PhD2,8, Yuzuru Kanakura, MD, PhD2,8 and Shinji Nakao, MD, PhD2,12 Title The Interim Analysis of the Optima (observation of GPI-anchored protein-deficient [PNH-type]) Cells in Japanese Patients with Bone Marrow Failure Syndrome and in Those Suspected of Having PNH) Study Abstract Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder derived from an acquired mutation of the phosphatidylinositol glycan class A (PIGA) gene in the hematopoietic stem cells which results in the expansion of glycosylphospatidylinositol-anchored protein (GPI-AP)-deficient (PNH-type) hematopoietic cells. PNH-type blood cells are also observed in patients with bone marrow failure (BMF). PNH is conventionally diagnosed when patients have >1% of GPI-AP-deficient erythrocytes and granulocytes determined by flow cytometry. Analyses with high resolution flow cytometry by several different groups have shown that patients with aplastic anemia (AA) or low-risk types of myelodysplastic syndromes (MDS) have small percentages of PNH-type erythrocytes, granulocytes, and/or other lineages of blood cells and that these patients respond better to immunosuppressive therapies compared with BMF patients lacking PNH-type cells. In order to determine the prevalence and clinical significance of PNH-type cells in BMF patients, we conducted a nationwide multi-center prospective observational investigation, the OPTIMA study. Methods: From July 2011, Japanese patients with PNH, AA, MDS or BMF of uncertain origin have been prospectively enrolled into the study. Six laboratories in different cities in Japan were assigned as regional analyzing centers and measured the percentages of PNH-type cells in the study population as well as collecting clinical and laboratory data. The high-resolution flow cytometry assessments used a liquid fluorescein-labeled proaerolysin (FLAER) method and a cocktail method with anti-CD55 and anti-CD59 antibodies for the detection of PNH-type granulocytes and erythrocytes, respectively. Periodic blind cross validation tests using a standard blood sample containing 0.01% PNH-type cells and a normal control were conducted to minimize inter-laboratory variations. From analysis of 68 healthy individuals >0.003% of PNH-type granulocytes and >0.005% of PNH-type erythrocytes were considered to be abnormal (Sugimori et al, Blood, 2006). Results: As of May 2014, flow cytometry data have been collected from 1685 patients and are included in this interim analysis. Of these patients, 65 (4%) were diagnosed with PNH, 523 (31%) with AA, 459 (27%) with MDS, and 638 (38%) with BMF of unknown etiology. Overall, 154 (9%) patients had ≥1% of both PNH-type erythrocytes and granulocytes: 63 (97%) patients with PNH; 57 (11%) with AA; 18 (4%) with MDS; and 16 (3%) with BMF of unknown etiology. In total, 545 (32%) patients had ≥0.005% PNH-type erythrocytes and ≥0.003% PNH-type granulocytes. These consisted of the followings; all 65 (100%) patients with PNH; 264 (51%) with AA; 76 (17%) with MDS; and 140 (22%) with BMF of unknown origin. Lactate dehydrogenase (LDH) levels ≥1.5 × upper limit of normal range were seen in 14/329 (4%) patients with 0.005-1% PNH-type erythrocytes, 23/62 (37%) patients with 1-10% PNH-type erythrocytes, and 69/71 (97%) patients with ≥10% PNH-type erythrocytes. Periodic blind validation tests revealed that inter-laboratory differences in absolute measurements of PNH-type cells were always within 0.02%. Conclusions: A high-resolution flow cytometry-based method, based on the Kanazawa method, that enables the detection of very low percentages of PNH-type cells was successfully transferred to 6 laboratories across Japan. Our results demonstrated that the proportion of patients identified as having small percentages of PNH-type cells differed depending on diagnosis (PNH, AA, MDS, or unknown BMF) and that elevated LDH levels (>1.5 x upper limits of normal range) were more frequently associated with higher percentages of PNH-type erythrocytes. Our findings suggest that the high resolution method is helpful as a diagnostic tool in BMF syndromes, including AA, MDS, and PNH, and may prove useful in understanding the pathophysiology of these disorders. Authors 4389 Marcus A.F. Corat, PhD1,2*, Jean-Yves Metais, Ph.D.1* and Cynthia E. Dunbar, MD3 Title Progress Towards Creation of a Rhesus Macaque Animal Model for PNH Disease Via Crispr/Cas9 Technology to Knock out the PIG-a Gene Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem/progenitor cell (HSPC) disease characterized by severe intravascular hemolysis, bone marrow failure, and propensity to thrombosis, causing early death in untreated patients. PNH has been linked to acquired somatic loss-of-function mutations in the X-linked PIGA gene in HSPC, with resultant disruption of the first step of the biosynthesis of GPI anchors and loss of cell-surface expression of GPI-linked proteins such as CD55 or CD59. PNH red cells are sensitive to complement-mediated lysis due to loss of these GPI-linked proteins. Even though the molecular mechanism of PNH has been well-characterized, the apparent clonal dominance of PIG-A mutant HSPC over residual unmutated HSPC is still poorly understood. Murine models for PNH generated via conditional knockout of PigA do not recapitulate the hemolysis, thrombotic phenotype, or clonal dominance. We took advantage of the newly-described CRISPR-Cas9 gene editing technology to disrupt the PIG-A gene via non-homologous end joining DNA repair and generate a relevant animal model for PNH, in order to investigate these important pathophysiologic questions in rhesus macaques, a species phylogenetically closely related to humans. We constructed a lentiviral vector expressing GFP, Cas9, and one of a series of five guide RNAs manually designed to target the rhesus macaque PIG-A gene at several sites within exon 2, where most human missense mutations are clustered. Two of these five gRNAs also 100% matched homologous sequences in the human PIG-A gene, while three of the gRNAs had 1-2 mismatches with the human sequence. We characterized the efficiency of each guide at the genotypic level using the SURVEYOR assay for locus disruption and direct sequencing, and at the phenotypic level by flow cytometry for GPI-linked proteins. We used human K562 or rhesus macaque FRhK-4 cell lines for proof of concept in vitro studies. The majority of human K562 cells transduced with vectors expressing guide RNAs targeting human PIG-A sequences in exon 2 gradually lost expression of cell surface CD55 and CD59, approximately 70% of double negative for CD55 and CD59 markers by 3 weeks following transduction, in contrast to K562 cells transduced with control vectors not expressing gRNA. It is notable that of the 5 guides tested in K562 cells, the 2 guides resulting in efficient knockdown of CD55 and CD59 were targeted to regions with 100% homology between the rhesus and the human sequence of PIG-A. The other 3 guides with only 1 or 2 mismatches to the human sequence were very inefficient at gene disruption in human cells, suggesting high fidelity and limited off-target effects The SURVEYOR assay confirmed disruption of the K562 PIG-A gene by these two gRNAs (see Figure). Upon sequencing, we demonstrated a variety of indels consisting of insertions or deletions of 1 to 40 nucleotides at the target site in the PIG-A gene. Analysis for off-target indels is ongoing. Similar experiments were carried out in the FRhK4 rhesus cell line, with the SURVEYOR assay demonstrating gene disruption with all five gRNA targeting sequences, all 100% homologous to the rhesus PIG-A gene targets (see Figure) . In FRhK-4 rhesus cells all the PIG-A gRNAs, knocked down CD55 and CD59 expression. gRNAs #4.1, #6.2 and #9.1 showed higher efficiency than the others, with approximately 78% of double negative cells for CD55 and CD59 markers 3 weeks following transduction. We have validated the lentiCRISPR-/Cas9 approach for use in rhesus cells for PIG-A knockout in vitro, and the guide-RNAs have shown effectiveness and specificity. We are in the process of implementing the technique in primary rhesus macaque CD34+ HSPC prior to proceeding to transplantation of these cells in an autologous rhesus model, allowing investigation of the mechanisms of clonal dominance and thrombosis. Authors Publication Only Igor Lisukov, Alexander Kulagin, Alexey Maschan, Elena Shilova, Kudrat Abdulkadyrov, Andrey Zaritskiy, Valentina Ivanova, Yulia Bogdanova, Svetlana Volkova, Andrey Gavrilenko, Tatyana Kaporskaya, Sergey Meresiy, Tatyana Schneider, Svetlana Yakupova, Ludmila Anchukova, Bulat Bakirov, Natalia Kosacheva, Tatiana Krinitsina, Viktoria Ustyantseva, Yuriy Shatokhin, Elena Vinogradova, Tatiana Kazankova, Alexander Rumyantsev, Boris Afanseyev Title Effect of Eculizumab on Physician-Reported Symptoms in the Russian Cohort of the Paroxysmal Nocturnal Hemoglobinuria (PNH) International Registry Abstract Background: Paroxysmal nocturnal hemoglobinuria is a rare clonal hematopoietic stem cell disease that can lead to life-threatening complications including thrombotic events (TE), chronic kidney disease (CKD) and pulmonary hypertension. In 2011 the international PNH Registry was implemented in the Russian PNH cohort to assess the natural history of PNH and the effects of treatment with eculizumab. Aims: We aim to evaluate the change from baseline to last follow-up in physician-reported symptoms, LDH and hemoglobin concentration in treated and untreated PNH patients. Design and methods: The international PNH Registry is a non-interventional, prospective, multicenter observational study. The study population of this analysis comprised the Russian cohort of the Registry: all patients had a confirmed diagnosis of PNH according to international guidelines. As per local guidelines, blood transfusion dependency and a history of TE were the main indications for treatment with eculizumab. Data are presented as descriptive statistics only. The reporting period includes time from baseline until the earliest of: death; bone marrow transplant; discontinuation from the Registry; discontinuation from eculizumab; last follow-up in the Registry. Results: As of June 2014, the international PNH Registry has enrolled 479 patients from Russia, 75 patients were ever-treated with eculizumab (15.7%), among whom 60 had available data. The median (range) duration of follow-up of patients after eculizumab treatment was 0.8 (-0.7 – 1.9) years and the median years (range) from disease onset to the start of eculizumab treatment was 8.1 (1 – 30) years. One patient discontinued treatment due to the disappearance of PNH clones and disease manifestations. Fifteen deaths were reported during the reporting period; all in patients never treated with eculizumab. Median (Q1, Q3) Hb concentrations at baseline in ever-treated and never-treated patients were 7.3 (6.3, 9.2) g/dL and 9.0 (7.2, 11.3) g/dL, respectively, and median (Q1, Q3) changes in Hb concentration from baseline to last follow up were 2.1 (1.2, 3.8) g/dL and 0.75 (-0.4, 2.5) g/dL, respectively. Median (Q1, Q3) LDH level at baseline in ever-treated and never-treated patients were 6.0 (3.6, 8.7) x the upper limit of normal (ULN) and 1.1 (0.8, 1.8) x ULN, respectively, and median (Q1, Q3) changes in LDH level from baseline to last follow up were -4.8 (-6.9, -1.9) and 0.0 (-0.2, 0.3) x ULN, respectively. Conclusion: This analysis represents the first report on longitudinal outcomes during eculizumab therapy in Russian patients included in the international PNH Registry and are considered essential for planning patients’ follow-up, including monitoring of therapeutic effects and prophylaxis against break-through hemolysis. Overall, the data show an improvement in physician-reported symptoms and reduced hemolysis, as measured by plasma LDH level, in patients treated for a relatively short period of time.