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Background: Etranacogene dezaparvovec is an investigational gene therapy for hemophilia B (HB) comprising an adeno-associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) gene with a liver specific promoter. In a Phase 2b study, a single dose of etranacogene dezaparvovec provided mean FIX activity of 41.0% sustained at 1yr post-dose in 3 participants (pts). Although most gene therapy clinical studies exclude pts with pre-existing neutralizing antibodies (NAbs) to the capsid serotype, early clinical studies and nonhuman primate data suggest that generally prevalent titers of anti-AAV5 NAbs may not preclude successful transduction with etranacogene dezaparvovec. Aims: A Phase 3, Health Outcomes with Padua gene; Evaluation in Hemophilia B (HOPE-B; NCT03569891) was established to further assess efficacy and safety of etranacogene dezaparvovec in adults with HB with a wide range of pre-existing NAbs to AAV5. Here we report outcomes at 26 weeks (wks). Methods: HOPE-B is a Phase 3, open-label, single-dose, single-arm, multi-national trial in adult males with severe or moderate-severe HB (FIX≤2%). All pts received routine FIX prophylaxis prior to study. Pts were not excluded based on pre-existing NAbs to AAV5. Pts entered a prospective lead-in period of at least 6 months during which bleeding/factor use was monitored, then received a single intravenous dose of etranacogene dezaparvovec (2x1013 gc/kg). Pts will be followed for 5yrs. Primary endpoints comprised FIX activity (one stage) at 26 and 52wks after dosing and 52wk annualized bleeding rate. For pts with no clean post-treatment FIX samples (≥10d post exogenous FIX), factor activity was imputed as baseline value based on historic disease severity. Secondary endpoints include factor replacement use, adverse events (AEs), and reactive use of corticosteroids. Results: 75 pts were screened, of whom 67 entered lead-in. 54 pts were dosed (44 severe, 10 moderately severe HB) and completed 26wks of follow-up. Mean age (±SD) was 41.5 (15.8) yrs. 38/54 pts (70.4%) had bleeds (n=123) during the lead-in despite prophylaxis, and 23/54 (42.6%) had NAbs to AAV5 at baseline (max titer: 3212.3). Following treatment, FIX activity increased rapidly to a mean (SD; min,max) of 37.2% (±19.6; 1.0, 97.1) at wk26, representing a mean (SD; min,max) change from baseline of 36.0% (±19.7; 0, 96.1 p Conclusions: The first co-primary endpoint of this study was met. This is the first report of a Phase 3 study in HB and the largest gene therapy trial cohort to date. Following a single dose of etranacogene dezaparvovec, FIX activity increased, without the need for prophylactic immunosuppression, into the mild-to-normal range at 26wks in pts with severe/moderately severe HB. Importantly, this included pts with titers of pre-existing anti-AAV5 NAbs. Pts were able to discontinue prophylaxis and bleeding was abolished in the majority. The safety profile was consistent with early phase AAV5 studies and together these data support a favorable safety and efficacy profile for etranacogene dezaparvovec Disclosures Pipe: HEMA Biologics: Consultancy, Other; Catalyst Biosciences: Consultancy; CSL Behring: Consultancy; ApcinteX: Consultancy; Bayer: Consultancy, Other: Contracted Research; BioMarin: Consultancy, Other: Contracted Research; Takeda: Consultancy; uniQure: Consultancy, Other; Siemens: Other; Pfizer: Consultancy; Freeline Therapeutics: Consultancy, Other: Contracted Research; Novo Nordisk: Consultancy, Other: Contracted Research; Roche/Genentech: Consultancy, Other: Contracted Research; Sangamo Therapeutics: Consultancy; Sanofi Genzyme: Consultancy, Other; Spark Therapeutics: Consultancy. Recht:CSL Behring: Consultancy, Other: personal fees; Genentech: Consultancy, Other: personal fees, Research Funding; Pfizer: Consultancy, Other: personal fees, Research Funding; BioMarin: Research Funding; Takeda: Consultancy, Other: personal fees, Research Funding; uniQure: Consultancy, Other: personal fees, Research Funding; Novo Nordisk: Consultancy, Other: personal fees, Research Funding; Spark: Research Funding; Bayer: Research Funding; Grifols: Research Funding; Hema Biologics: Consultancy, Research Funding; LFB: Research Funding; Octapharma: Research Funding; Catalyst Biosciences: Consultancy; Kedrion: Consultancy; Sanofi: Consultancy, Research Funding. Key:Uniqure: Consultancy; Grifols: Research Funding; Takeda: Research Funding; Novo Nordisk: Other: Chair of Grants Committee. Leebeek:Shire/Takeda: Research Funding; uniQure: Consultancy; Shire/Takeda: Consultancy; BioMarin: Consultancy; SOBI: Other: Travel grant; Roche: Other: DSMB member for a study; CSL Behring: Research Funding. Castaman:Bayer, Roche, Sobi, Grifols, Novo Nordick, Werfen, Kedrion: Consultancy, Honoraria, Speakers Bureau; CSL Behring, Pfizer, Sobi: Research Funding; Ablynx, Alexion, Bayer, Takeda, CSL Behring, Novo Nordisk, Pfizer, Roche,Sanofi, SOBI, uniQure: Membership on an entity's Board of Directors or advisory committees. Lattimore:uniQure: Other: Study Steering Committee member. Van Der Valk:Baxalta: Research Funding. Peerlinck:Bayer: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding; NovoNordisk: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Roche: Research Funding; Sobi: Consultancy; Takeda: Consultancy, Research Funding. Coppens:Roche: Research Funding; Portola/Alexion: Research Funding; Sanquin Blood Supply: Research Funding; uniQure: Research Funding; NovoNordisk: Consultancy; Pfizer: Consultancy; Sobi: Consultancy; Medcon International: Consultancy; MEDtalks: Consultancy; Bayer: Consultancy, Research Funding; CSL Behring: Consultancy, Research Funding; Daiichi Sankyo: Research Funding. O'Connell:uniQure: Consultancy; F. Hoffmann-La Roche Ltd, Novo Nordisk, SOBI: Speakers Bureau; SOBI: Research Funding. Pasi:Sanofi: Honoraria, Other: Personal fees and nonfinancial support; honoraria as member of scientific advisory boards and symposia, Research Funding; BioMarin: Consultancy, Honoraria, Other: Grants, personal fees, and nonfinancial support; honoraria as member of scientific advisory boards and symposia, Research Funding; uniQure: Other: Grants and nonfinancial support , Research Funding; ApcinteX: Consultancy, Other: Personal fees ; Takeda: Consultancy, Honoraria, Other: Personal fees; honoraria as member of scientific advisory boards and symposia ; Biotest: Consultancy, Honoraria, Other: Personal fees and nonfinancial support; honoraria as member of scientific advisory boards and symposia; Catalyst Biosciences: Consultancy, Other: Personal fees and nonfinancial support; honoraria as member of scientific advisory boards and symposia; Novo Nordisk: Honoraria, Other: Personal fees and nonfinancial support; honoraria as member of scientific advisory boards and symposia ; Octapharma: Honoraria, Other: Personal fees and nonfinancial support; honoraria as member of scientific advisory boards and symposia , Speakers Bureau; Roche: Honoraria, Other; Sobi: Consultancy, Honoraria, Other; Tremeau: Consultancy. Kampmann:Uniqure: Research Funding, Speakers Bureau; Shire Pharmaceuticals: Speakers Bureau. Meijer:Pfizer: Research Funding; Sanquin: Speakers Bureau; Bayer: Speakers Bureau; Sanquin: Research Funding; Bayer: Research Funding; Boehringer Ingelheim: Speakers Bureau; BMS: Speakers Bureau; Aspen: Speakers Bureau; Uniqure: Consultancy. von Drygalski:Biomarin: Consultancy; Bioverativ/Sanofi Genzyme: Consultancy; NovoNordisk: Consultancy; Pfizer: Consultancy; uniQure: Consultancy; Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees. Young:Genentech/Roche, Grifols, and Takeda: Research Funding; BioMarin, Freeline, Genentech/Roche, Grifols, Kedrion, Novo Nordisk, Sanofi Genzyme, Spark, Takeda, and UniQure: Honoraria; Bayer, CSL Behring, Freeline, UniQure: Consultancy. Hermans:WFH: Other; EAHAD: Other; LFB: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau; CAF-DCF: Consultancy, Speakers Bureau; Biogen: Consultancy, Speakers Bureau; Sobi: Consultancy, Research Funding, Speakers Bureau; Shire, a Takeda company: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Octapharma: Consultancy, Speakers Bureau; Kedrion: Speakers Bureau. Astermark:Bayer, CSL Behring, Novo Nordisk, Octapharma, Roche, Sobi, Spark, Takeda, uniQure: Consultancy; uniQure: Research Funding. Klamroth:Bayer: Consultancy, Research Funding, Speakers Bureau; Biomarin: Consultancy, Research Funding, Speakers Bureau; CSL Behring: Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Research Funding, Speakers Bureau; Octapharma: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau; Roche/Chugai: Consultancy, Speakers Bureau; Takeda/Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Consultancy, Speakers Bureau; Biotest: Speakers Bureau; LEO: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Lemons:uniQure: Research Funding. Visweshwar:Biogen Idec: Membership on an entity's Board of Directors or advisory committees. Escobar:Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genentech, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees; National Hemophilia Foundation: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biomarin, Genetech/Roche, CSL Behring, Kedrion, Magellan Healthcare: Honoraria. Gomez:Global Blood Therapeutics: Speakers Bureau. Kruse-Jarres:CSL Behring, Genentech, Inc., Spark: Research Funding; Biomarin, Chugai Pharmaceutical Co., CSL Behring, CRISPR Therapeutics, Genentech, Inc.: Honoraria; F. Hoffmann-La Roche Ltd: Speakers Bureau; Biomarin, Chugai Pharmaceutical Co., CSL Behring, CRISPR Therapeutics, Genentech, Inc.: Consultancy. Kotowski:uniQure: Research Funding. Quon:Orthopaedic Institute for Children: Current Employment; Bayer: Honoraria; Biomarin: Honoraria, Speakers Bureau; Bioverativ/Sanofi: Honoraria, Speakers Bureau; Genentech, Inc./F. Hoffmann-La Roche Ltd: Honoraria, Speakers Bureau; Novo Nordisk: Honoraria, Speakers Bureau; Octapharma: Honoraria; Shire/Takeda: Speakers Bureau. Wang:Bayer: Honoraria; Takeda: Honoraria; Genentech: Honoraria; Biomarin: Honoraria; CSL Behring: Honoraria; Bioverativ Inc: Honoraria; Catalyst Biologics: Consultancy; NovoNordisk: Consultancy; Hema biologics / LFB: Consultancy. Wheeler:Takeda: Membership on an entity's Board of Directors or advisory committees; uniQure: Membership on an entity's Board of Directors or advisory committees; Biomarin: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees. Sawyer:uniQure: Current Employment, Current equity holder in publicly-traded company. Verweij:uniQure: Current Employment. Colletta:uniQure: Current Employment. Bajma:uniQure: Current Employment. Gut:uniQure: Current Employment. Miesbach:Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biomarin: Consultancy, Membership on an entity's Board of Directors or advisory committees; Freeline: Consultancy, Membership on an entity's Board of Directors or advisory committees; LFB: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Consultancy; Sanofi: Consultancy, Membership on an entity's Board of Directors or advisory committees; uniQure: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biotest: Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: Etranacogene dezaparvovec is an investigational gene therapy

This systematic review was designed to provide more precise effect estimates of inhibitor development for the various types of F8 gene mutations in patients with severe hemophilia A. The primary outcome was inhibitor development and the secondary outcome was high-titer-inhibitor development. A systematic literature search was performed to include cohort studies published in peer-reviewed journals with data on inhibitor incidences in the various F8 gene mutation types and a mutation detection rate of at least 80%. Pooled odds ratios (ORs) of inhibitor development for different types of F8 gene mutations were calculated with intron 22 inversion as the reference. Data were included from 30 studies on 5383 patients, including 1029 inhibitor patients. The inhibitor risk in large deletions and nonsense mutations was higher than in intron 22 inversions (pooled OR = 3.6, 95% confidence interval [95% CI], 2.3-5.7 and OR = 1.4, 95% CI, 1.1-1.8, respectively), the risk in intron 1 inversions and splice-site mutations was equal (pooled OR = 0.9; 95% CI, 0.6-1.5 and OR = 1.0; 95% CI, 0.6-1.5), and the risk in small deletions/insertions and missense mutations was lower (pooled OR = 0.5; 95% CI, 0.4-0.6 and OR = 0.3; 95% CI, 0.2-0.4, respectively). The relative risks for developing high titer inhibitors were similar.

The pathogenesis of inhibitory antibodies has been the focus of major scientific interest over the last decades, and several studies on underlying immune mechanisms and risk factors for formation of these antibodies have been performed with the aim of improving the ability to both predict and prevent their appearance. It seems clear that the decisive factors for the immune response to the deficient factor are multiple and involve components of both a constitutional and therapy-related nature. A scientific concern and obstacle for research in the area of hemophilia is the relatively small cohorts available for studies and the resulting risk of confounded and biased results. Careful interpretation of data is recommended to avoid treatment decisions based on a weak scientific platform. This review will summarize current concepts of the underlying immunological mechanisms and risk factors for development of inhibitory antibodies in patients with hemophilia A and discuss how these findings may be interpreted and influence our clinical management of patients.

The aim of the Malmö International Brother Study (MIBS) is to evaluate host genetic factors associated with the development of inhibitory antibodies in patients with hemophilia. Factor VIII gene mutations and genetic polymorphisms of the IL1beta, IL4, and IL10 genes, known to influence antibody production in autoimmune diseases, were analyzed in 164 patients (124 with severe, 26 with moderate, and 14 with mild disease) in 78 unrelated families with hemophilia A. Seventy-seven (47%) patients in 54 families had a history of inhibitors (57 high responding, 20 low responding). Inversions were found in 36 families (75 patients). There was no association between the development of inhibitor and the IL1beta Taq I RFLP alleles in exon 5 or the –590 C/T single nucleotide polymorphism (SNP) in the promoter region of IL4. There was, however, a strong association between an allele with 134 bp in one of the CA repeat microsatellites, IL10G, located in the promoter region of the IL10 gene, and the development of inhibitor (odds ratio [OR], 4.4; 95% confidence interval [95% CI], 2.1-9.5; P < .001). The association was consistent in the subgroup of families with severe hemophilia and inversions. IL10 is the first gene located outside the causative factor VIII gene mutation to be associated with inhibitor development.

Studies of determinants of development of inhibitory Abs to factor VIII in people with hemophilia A indicate a complex process involving multiple factors. The Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort was formed to extend our understanding of the genetic background of risk. The study group contains 833 subjects from 3 independent cohorts: brother pairs and singletons with and without a history of inhibitors, as well as 104 brother pairs discordant for inhibitor status. Using an Illumina iSelect platform, 13 331 single-nucleotide polymorphisms from 1081 genes, primarily immune response and immune modifier genes, were typed. Each cohort was analyzed separately with results combined using a meta-analytic technique. After adjustment for potential confounders, 53 single-nucleotide polymorphisms were found to be significant predictors of inhibitor status using the criteria of odds ratios in the same direction in all cohorts or allowing for a 20% interval around an odds ratio = 1 in 1 of the 3 and significant in at least 2. Of the 53 markers, 13 had meta P.001. Eight of the 53 were significant predictors among the discordant pairs. Results support the complexity of the immune response and encourage further research with the goal of understanding the pathways involved.

The development of inhibitory antibodies to factor VIII is a serious complication of hemophilia. FEIBA (factor VIII inhibitor-bypassing activity), an activated prothrombin complex concentrate (aPCC), and NovoSeven, recombinant factor VIIa (rFVIIa), are used as hemostatic bypassing agents in treating patients with inhibitors. The FENOC study was designed to test equivalence of the products in the treatment of ankle, knee, and elbow joint bleeding. A prospective, open-label, randomized, crossover, equivalency design was used. The parameters of interest were the percentage of patients who reported efficacy in response to FEIBA and the percentage that reported efficacy in response to NovoSeven. A difference in these percentages of no more than 15% was determined to be a clinically acceptable magnitude for equivalence of the 2 products. The primary outcome was evaluation 6 hours after treatment. Data for 96 bleeding episodes contributed by 48 participants were analyzed. The criterion for declaring the 2 products equivalent at 6 hours was not met; however, the confidence interval of the difference in percentages of efficacy reported for each product only slightly exceeded the 15% boundary (−11.4%-15.7%), P = .059. FEIBA and NovoSeven appear to exhibit a similar effect on joint bleeds, although the efficacy between products is rated differently by a substantial proportion of patients. This trial was registered at www.clinicaltrials.gov as #NCT00166309.

WOS: 000242309800025, PubMed ID: 16926287, The HLA class I/II alleles and the tumor necrosis factor alpha (TNFA) locus are closely linked in the MHC complex. We have characterized the causative factor VIII mutation, HLA alleles as well as 4 polymorphisms (-827C > T, -308G > A, -238A > G, and 670A > G) in the TNFA gene in 164 patients (124 severe, 26 moderate, and 14 mild) in 78 families with hemophilia A enrolled in the Malmo International Brother Study (MIBS). Inhibitors were identified in 77.8% of patients with a single haplotype (Hap 2) and 72.7% of the patients with the TNFA -308 A/A genotype within this haplotype compared with 39.70/6 for TNFA -308 G/G patients and 46.9% for TNFA -308 G/A heterozygotes (OR 4.0; 95% CI, 1.4-11.5; P = .008). The association between the -308 A/A genotype and inhibitors was enhanced in subgroups of patients with severe hemophilia (OR 19.2; 95% CI 2.4-156.5; P < .001) and with inversions (n = 75; OR, 11.8; 95% CI, 1.3-105.1; P = .013). Associations were found for the HLA A26 and B44 alleles, but these were not consistent in the subgroup analysis. Our data imply that the TNFA -308G > A polymorphism within Hap 2 is a useful marker and potential modulator of the immune response to replacement therapy in patients with hemophilia.

3481 Poster Board III-418 Hereditary FVII deficiency (FVIID) is a rare congenital bleeding disorder with an estimated prevalence of symptomatic individuals of 1:500,000. In the “Greifswald Registry FVII Deficiency” molecular defects of more than 1000 FVII deficient patients were described. By direct sequencing of the F7 genes in congenital FVIID revealed 146 different F7 gene mutations including 25 different mutations (18% of all) in the naturally-occurring acceptor or donor splice sites (Tab.1) were identified. In seven FVIID patients from Sweden and Germany the novel lesion g.IVS5+78G>A - downstream of the naturally-occurring donor splice sites of exon 5 - was identified. This variation was detected heterozygous in FVIID patients with FVII: C levels of 15%, 27%, 31%, 40% and 65%, and FVII: Ag levels between 25-50%. In two compound heterozygous patients with FVII: C levels of 1% und FVII: Ag levels of 2% and 3% respectively, one well-known causative FVII mutation is combined with the novel lesion g.IVS5+78G>A. The influence of this novel F7 gene variation on splicing was investigated by RT-PCR analysis and in vitro expression studies using exon-trap vector constructs. The total RNA was isolated from peripheral leucocytes and analyzed by one step RT-PCR and sequencing. Fragments of exon 5 and a part of the flanking intron 5 region (g.7679 –g.8073) were amplified of patients' DNA and cloned into the exon trap-vector pET01. Different vector constructs containing minigenes of the wild type (g.IVS5+78G) or mutant form (g.IVS5+78A) and the corresponding minigenes with an “optimized” naturally-occurring donor splice site in position +5 respectively were transfected into HEK293 cells. The expressed RNA was isolated and characterized. Consensus Values (CV) for all donor splice sites were calculated using a splice site detection tool according Shapiro and Senapathy (1987). The RT-PCR analysis in patients indicate that the novel variation g.IVS5+78G>A in intron 5 created an aberrant splice site in position 79bp downstream of exon 5 even though the naturally-occurring donor-splice-site of exon 5 is not abolished. An insertion of 79bp of intron 5 into the mRNA leads to a frame shift and predicts a premature termination 11 codons past the last unaltered codon. Minigenes include the naturally-occurring splice site and the variation g.IVS5+78A used exclusively the aberrant splice position 79bp downstream of exon 5 whereas wild type minigenes with the naturally-occurring splice site and the wild type form g.IVS5+78G produced normally spliced mRNA. In a following experiment the “naturally-occurring splice site” of exon 5 was optimized by the additional substitution g.IVS5+5C>G which increased their CV from 76.6 to 90.9 compared to the CV of the novel mutant g.IVS5+78A of 80.3. In presence of both mutations (g.IVS5+5G and g.IVS5+78A) only normal spliced mRNA was expressed of this minigene. In this construct the mutation g.IVS5+78G>A was without importance for the mRNA splicing. The results of the in vitro experiments demonstrated, that the Consensus Values (CV) seems to be an important factor for the selection of donor splice sites in the F7 gene. In the “Greifswald Registry FVII Deficiency” 26 different splice site variations in F7 gene were identified (Tab. 1). The atypical splice site variation g.IVS5+78G>A, +78bp downstream of exon 5 was present in 7 FVIID patients from Sweden and Germany in different genotypes. This novel F7 gene mutation g.IVS5+78G>A creates an aberrant splice site in position +79 of intron 5 and predicts premature termination. RNA analysis and expression studies demonstrated, that this novel F7 gene lesion is a type I mutation with low FVII:C and FVII: Ag levels and is the basis defect in 7 FVIID patients of the “Greifswald Registry FVII Deficiency”. | Intron | Acceptor splice site | Intron | Donor splice site | |:------:| --------------------- | ------ | ------------------ | | 1b | g.IVS1b-11G>A | 1a | g.IVS1a+5G>A | | 1b | [*][1]g.IVS1b8del14bp | 1a | [*][1]g.IVS1a+6T>G | | 1b | [*][1]g.IVS1b-3C>G | 1a | [*][1]g.IVS1a+8C>T | | 2 | [*][1]g.IVS2-3C>G | 2 | g.IVS2+1G>A | | 3 | g.IVS3-1G>A | 2 | [*][1]g.IVS2+1G>T | | 3 | [*][1]g-IVS3-1G>T | 2 | [*][1]g.IVS2+1G>C | | 4 | [*][1]g.IVS4-7T>G | 2 | [*][1]g.IVS2+1delG | | 7 | [*][1]g.IVS7-10T>C | 2 | g.IVS2+5G>T | | 7 | [*][1]g.IVS7-3C>G | 3 | [*][1]g.IVS3+1G>T | | 7 | [*][1]g.IVS7-1G>A | 4 | g.IVS4+1G>A | | | | 5 | [*][1]g.IVS5+78G>A | | | | 6 | [*][1]g.IVS6+1G>A | | | | 6 | g.IVS6+1G>T | | | | 6 | [*][1]g.IVS6+3A>G | | | | 7 | [*][1]g.IVS7+1G>A | | | | 7 | g.IVS7+3_6 del4bp | * [↵][2]* novel mutations (HGMD Factor 7 Database, 2009 / ) Tab. 1 26 different intronic F7 gene mutations analysed in FVII deficiency patients of the “Greifswald Registry FVII Deficiency” Disclosures: No relevant conflicts of interest to declare. [1]: #fn-1 [2]: #xref-fn-1-1

Abstract 115 Background: Treatment with lenalidomide in patients with low risk MDS with deletion del(5q) has resulted in a high proportion of transfusion independence and also cytogenetic responses. The aim of this study was to evaluate the effect of lenalidomide in high risk MDS and AML patients with del(5q) or monosomy 5 not eligible for induction treatment, primary cases as well as patients relapsing/refractory to intensive treatment. Methods: This investigator-initiated prospective phase II study assessed the effects of continuous lenalidomide treatment during 16 weeks, in increasing doses from 10 to 30 mg, with 8 weeks on the highest level. Dose modifications due to hematologic toxicity were performed according to the clinical judgment of the treating physician. The primary objective was cytogenetic response, assessed by FISH on bone marrow (BM) smears at inclusion and after 8 and 16 weeks. Clinical and morphological BM response was evaluated every 4 weeks. Results: 25 patients were included from October 2007 to July 2009, 22 of which are so far evaluable. Overall, the patient cohort included several patients with very advanced disease and two patients died before treatment was started. Fourteen MDS patients and 11 AML patients have been included. Twenty-two of the patients had 5q- (9 of these had a complex karyotype and 5 had del(5q)+1) and 3 patients had monosomy 5 (all complex karyotype). The median time of treatment was 13 weeks (range 0–16 weeks). The reason for early termination in the patients who started lenalidomide treatment was progressive disease (3) and adverse events (10). Six of 7 patients who completed the study (16 weeks of treatment) had a response: Two MDS patients with del(5q-)+1, and del(5q)+2, respectively, achieved a major cytogenetic response (≥50 % reduction), in one of these patients the blast count decreased from 9.5% to Conclusion: Lenalidomide as a single agent, but in higher doses than those applied to low-risk MDS patients resulted in cytogenetic and/or morphological bone marrow response in 30% of patients with high-risk MDS or AML with del(5q) or monosomy 5. We considered this a promising response rate, considering the high frequency of very advanced patients, and complex karyotypes. The main serious adverse event was infections. Future combinations with cytostatic or hypometylating agents may further improve these results. Disclosures: Möllgård: Celgene: Research Funding. Hellström-Lindberg:Celgene: Research Funding.

Abstract 217 Introduction: Both genetic and environmental factors have been implicated as potential risk factors for the development of inhibitory factor VIII (FVIII) antibodies. Previous studies suggest that genetic factors are of major importance. The causative FVIII mutation likely sets the stage for inhibitor risk, with other genetic markers important in determining the final outcome. Data suggest that the process of inhibitor development is complex, involving a variety of immune regulatory genes, several of which have the potential to modify risk. Through a collaboration among three multi-center studies: the Hemophilia Inhibitor Genetics Study (HIGS), the Malmö International Brother Study (MIBS), and the Hemophilia Growth and Development Study (HGDS), a combined cohort was formed to conduct an association study to test the hypothesis that antibody development to FVIII is mediated by immune response genes. Methods: The study includes clinical and laboratory data for 680 people with hemophilia A. Participants from Europe and North America account for 43% and 57% of the population, respectively. Eighty-five percent have severe (0.05 – 0.4 IU/mL) hemophilia. The cohort is predominately Caucasian, 81.0%, with 6.2% of African heritage, 8.8% Hispanic, and the remaining 4% of other races and ethnicities. Forty-nine percent have a current, or history of, an inhibitor ≥1 BU. Using the Illumina iSelect platform, 14,626 single nucleotide polymorphisms (SNPs) from 1,081 candidate genes were genotyped. These included immune response and immune modifier genes: cytokines and their receptors, chemokines and their receptors, and pathway genes involved in inflammatory and immune responses. Analyses were completed among the total group and the subgroup with severe hemophilia to identify SNPs associated with inhibitor status. The models were adjusted for population admixture, severity of hemophilia, type of mutation (high vs. low risk), year of birth, and geographic region. Meta analyses were used to obtain single odds ratios (OR) and p-values for the three cohorts. Results: 13,952 of the 14,626 (95.4%) SNPs were successfully genotyped. One hundred fourteen were associated with inhibitor status at the p Conclusions: Our findings suggest that functional pathways involved in a variety of cellular processes will be important in inhibitor development, but these results warrant further study and replication in similarly powered case-control or cohort studies. Disclosures: Ewenstein: Baxter Healthcare: Employment. Spotts:Baxter Healthcare: Employment.

Patients with high-risk myelodysplastic syndromes (MDS) or acute myeloid leukaemia (AML) secondary to MDS may reach complete remission (CR) following intensive chemotherapy but the CR-duration is usually very short. Promoter hypermethylation of e.g. tumour suppressor genes is considered to be important in cancerogenesis and a negative risk factor for survival in patients with MDS. We designed a prospective clinical trial to assess the impact of methylation status on the outcome of induction chemotherapy and to evaluate the effect of maintenance treatment with 5-azacytidine (aza) on CR duration. Sixty patients with a median age of 68 years (54–83) with high risk MDS (n=23) or AML (n=37) following MDS were treated with standard doses of daunorubicin and ara-C. Standard prognostic variables, and methylation status of the P15ink4b (P15), E-cadherin (CDH) and Hypermethylated in Cancer 1 (HIC) genes were analyzed before treatment. Patients in CR were given low dose aza subcutaneously for 5 days/4 weeks until relapse. The CR rate was 40%; significantly lower in patients with high white blood cell counts (P=0.03) and high CD34 expression on bone marrow cells (P=0.02). While P15 status alone was not associated with CR rate (P=0.25) patients with CDH methylation showed a lower CR rate (P=0.008). Moreover, no patient with hypermethylation of all three genes achieved CR (P=0.03). CDH methylation retained its prognostic value in the multivariate analysis. Hypermethylation was associated with increased CD34 expression but not with blast count or cytogenetic risk group. Median duration of CR was 13 months (2 to +37) and 7/ 23 patients (30%) had a CR duration >20 months. Three of four patients with trisomy 8 had CR >20 months. The overall survival of patients reaching CR was 17 months (6 to +40). Methylation status before treatment did not correlate with CR duration. We demonstrate for the first time a significant impact of methylation status on the outcome of conventional chemotherapy in high-risk MDS and MDS-AML and propose that this variable should be further evaluated in prospective studies. CR duration is promising, with a substantial portion of patients showing durable responses.

To optimise bleeding control in severe haemophiliacs on prophylaxis, treatment should be individualised, considering patient-related and external determinants. Independent effects of both determinants were assessed using data from Advate pre-licensure trials. Data from 148 patients with FVIII 1–6y 10–17y 18–65y N=39 N=39 N=37 |------------- Median (P25, P75) ----------------| Follow up (days) 396 (269, 455) 294 (265, 336) 305 (268, 341) Adherence to prophylactic dose (% of infusions) 100 (75,100) 99 (81,100) 99 (89,100) Adherence to frequency of prophylaxis (% of time) 86 (70,98) 78 (62,83) 70 (50,87) FVIII half life (hrs) 9.3 (8.0,10.7) 10.4 (9.3,12.4) 11.6 (10.4,13.6) Time below 1% FVIII (hrs/wk) 29 (8,45) 20 (9,30) 12(6,26) Joint bleeds (%) 0 (0,33) 50 (14,88) 72 (24,100) Bleeds in Jun–Aug (%) 25 (0,44) 29 (0,50) 33 (8,55)

BACKGROUND: The development of inhibitory antibodies to factor VIII is a serious complication in haemophilia. Two haemostatic agents with different bypassing mechanisms have been used in the treatment of patients with inhibitors: activated prothrombin complex concentrate (aPCC) and recombinant factor VIIa (rFVIIa). To date, published analyses on costs and outcomes in inhibitor treatment have been based on comparatively small samples and on expert opinion. Existing comparisons have used decision-analytic modelling. DATA: The FENOC study used a prospective, crossover design where each patient used activated prothrombin complex concentrate (FEIBA®) at one bleed and recombinant factor VIIa (NovoSeven®) at another bleed. The order of bypassing agents was randomised. The resource use and haemostatic efficacy was recorded at 2, 6, 12, 24, 36 and 48 hours following treatment. Time until bleeding had stopped was also evaluated. Forty-eight pairs (96 bleeding episodes) were included in the analysis. METHODS: Two types of health-economic analysis were performed: cost analysis identifying the determinants of cost using regression methods, and cost-effectiveness analysis to relate resource use to three measures of outcome; patient perception of treatment efficacy (defined as effective and/or partially effective), whether the bleeding had stopped and reduction in patient-perceived pain on a Visual Analogue Scale from start of treatment. Confidence intervals (CI) of the incremental cost-effectiveness ratios were calculated by bootstrap. The sensitivity of results were tested using the different relative prices in the US (Red Book AWP) and Sweden (national negotiated prices from the Swedish Pharmaceutical Benefits Board). RESULTS: Key determinants of cost were prescribed dose per kg bodyweight, weighing relatively more than persons of the same age and treatment in addition to protocol. FEIBA® cost on average less (p CONCLUSIONS: Health-economic analysis from the FENOC study indicated that FEIBA® was a dominant strategy having on average lower cost and slightly higher percentages of patient-stated efficacy. The large variation at the individual level of the two bypassing agents’ effect on reduction of pain supports 1) decisions that take the individual patient’s experience into account, 2) decisions that make trade-offs between cost and reduction in pain rather than focusing on cost only, 3) further research on the underlying causes of differences in reduction of pain.

The utility of pharmacokinetic (PK) measurements in clinical hemophilia practice remains to be proven. To address this issue data from 152 patients with severe hemophilia A (FVIII 12 yrs with more than one PK measurement, there was a low within patient variance in respect to between patient variance for clearance, MRT and AUC. This suggests that these parameters are more reliably measured for individual patients. Association of PK parameters with age and observed weight/ideal weight ratio was assessed using regression analysis. Median elimination t½ in patients was significantly shorter in children ages 1–6 yrs versus patients ≥ 12. Approximately 20% of this overall difference was due to fewer sampling timepoints in children but the remainder may reflect true biological differences; t½ increased by 0.41 h·y−1 (CI, 0.08–0.74) in children between 1–6 yrs and remained stable in older subjects. The relationship between PK parameters and age and ideal weight/weight ratio differed according to age [table - signs represent the direction of the change (increase, +; decrease, −); numbers are the associated P values for regression on age and weight/ideal weight for each parameter]. In children ages 1–6 yrs, both t½ and MRT of rAHF-PFM increased significantly with age. In subjects ≥ 12 years old, however, recovery, AUC/dose, rates of clearance and Vss increased significantly with both age and increasing weight/ideal weight. Differences noted in the PK parameters and age and body habitus in the two groups may reflect physical differences such as proportionately larger liver size and weight-adjusted plasma volume in children versus adults but may also reflect other yet undefined age-related biological differences. Initial analysis of bleed event data showed a different rate of bleeding on certain days of the week in children ages 1–6 but not in older subjects. Whether this is due to dosing practices, difference in PK or other factors is currently being investigated. These results have important implications for clinicians managing patients with hemophilia, and treatment regimens tailored to individual PK parameters are more likely to allow a more cost effective use of FVIII. In particular, dosing schedules based on the ratio of actual to ideal weight rather than weight appears to be appropriate. The implications of these PK findings on bleeding events whilst on prophylactic FVIII replacement therapy will be further analysed. Age 1–6 (N=52) Age ≥ 12 (N=100) Pharmacokinetic Parameter Age Weight/Ideal Weight Age Weight/Ideal Weight Recovery − 0.60 − 0.07 + 0.01 +

The development of inhibitory antibodies is a severe and costly complication to replacement therapy occurring in 10–15% of patients with hemophilia A, and the aim of the Malmo International Brother Study (MIBS) is to evaluate host genetic factors associated with this adverse effect of treatment. In the present study, factor VIII mutations, HLA genotypes and polymorphisms of the interleukin IL-1beta, IL-4 and IL-10 genes known to influence antibody production in autoimmune diseases, were analyzed in 164 patients with hemophilia A (120 severe, 30 moderate and 14 mild) belonging to 78 unrelated families. Seventy-seven (47.0%) of the subjects had a history of inhibitors (57 high-responding, 20 low-responding) in 54 unrelated families (34 discordant, 20 concordant siblings). In 24 families, no inhibitor was reported in any of the siblings. Seventy-five patients (45.7%) in 36 families had an inversion. In this group, 40 patients (53.3%) in 28 families had inhibitors (17 concordant, 11 discordant). Weak associations between inhibitor development and the HLA alleles A26 and B44 were found. No association was found with the IL-1beta Taq 1 RFLP alleles in exon 5, and the −590 C/T SNP in the promoter region of IL-4. There was however, a strong association between an allele with 134 bp in one of the CA repeat microsatellites, IL-10G, located in the promoter region of the IL-10 gene, and development of inhibitor. Allele 134 was found in 32 (41.6%) of the patients with inhibitors compared with 12 (13.8%) of the inhibitor negative patients (p

The occurrence of inhibitors in severe hemophilia A can make replacement with factor VIII concentrates insufficient for treatment of acute hemorrhages. An alternative is use of bypassing agents. The FEIBA® NovoSeven® Comparative Study (FENOC) is a randomized multicenter equivalence trial conducted in Europe and North America to study the efficacy of two bypassing agents. The usual framework of testing for differences between effectiveness of two treatments is not the focus in an equivalency trial. Instead, one tests that there is no clinically significant difference between treatments. In FENOC, the goal was to show that FEIBA® and NovoSeven® differed by less than 15% in efficacy. Subjects age 2+ with factor VIII deficiency, a history of inhibitor and need for bypassing agents were eligible. An episode of ankle, elbow, or knee bleeding was treated with one dose of FEIBA® and two doses of NovoSeven® with crossover between the options for the following episode. Subjects reported evaluation of the hemostatic effect 2, 6, 12, 24, 36, and 48 hours after treatment. The primary endpoint was hemostatic effect at 6 hours. Differences in pain, rated using the visual analog scale (VAS), and subject’s evaluation of when bleeding had stopped were secondary endpoints. The target sample size was 60 pairs of treatments. Data were analyzed using a modified version of McNemar’s test proposed by Lee and Lusher (1991). The equivalence hypothesis was tested and an associated confidence interval was calculated. If the confidence interval was within plus or minus 15% (−15%, 15%) significance was declared at the 0.05 level.Sixty-six subjects in 25 sites were enrolled. Twelve withdrew prior to treatment, 2 had 1 treatment, and 4 had unresolved queries at study close. Forty-eight subjects completed both treatments. Results of the intent-to-treat analysis are reported. The median age was 27. Approximately 43% had knee bleeds, 32% elbow, and 20% ankle. The remaining 5% had bleeding episodes involving more than one joint (n=2) or hip (n=2) or shoulder (n=1) joints. One serious adverse event not related to study or treatment was reported. At 6 hours after treatment 80.9% of recipients of FEIBA® and 78.7% of recipients of NovoSeven® reported the treatment was effective. The confidence interval for the treatment difference was −11.4% to 15.7%, (p=0.059). About 32% of the recipients had effective relief with one treatment but not the other. The mean within pair difference in VAS scores at 6 hours (FEIBA®-NovoSeven®) was −2.3 mm. At 6 hours 76.1% of those treated with FEIBA® reported that bleeding had stopped compared to 65.2% of those treated with NovoSeven®. The confidence interval was −2.7%, 24.5% so we could not conclude that the difference between the two products was less than 15%. However, the treatments were determined to be equivalent with respect to subjects’ evaluation of cessation of bleeding at 24 and 48 hours. FEIBA® and NovoSeven® may have similar efficacy in the treatment of acute hemorrhages in patients with inhibitors, although this was not consistently demonstrated in the FENOC study. There appears to be variation among patients in response to each product.

Malignant hematologic disorders may be associated with coagulopathy causing bleeding or thromboembolic complications. Severe infections as well as chemotherapy may augment this risk by the action of inflammatory mediators and the release of proteases. We evaluated the hemostatic process in 10 patients with acute myeloid leukemia (no acute promyelocytic leukemia) before the start of induction therapy with IDA-C and daily during the following two weeks of treatment. Various hemostatic parameters were studied, including the complex between activated protein C and the protein C inhibitor (APC-PCI) with a newly developed method, thrombin generation, protein C, prothrombin fragment 1+2 (F1+2), antithrombin and factor VIII. The APC-PCI concentration was relatively high in all patients at start of treatment (mean 0.81 ± SEM 0.27 μg/L; normal range 0.07–0.26 μg/L) reflecting an activation of the coagulation system, and further increased during the initial phase of chemotherapy reaching the highest level on Day 3 to 5 of 1.67±0.53 μg/L (p=0.0047). The concentration of the complex then declined during the following 10 days to 0.10±0.03 μg/L on Day 14, i.e. to a concentration significantly lower than that at start of treatment (p=0.005). The concentration of F1+2 varied in a similar way with a concentration of 2.17±0.51 nM at start of treatment and an increase on Day 3–5 to 4.17±0.93 nM (p=0.005) followed by a decline to 1.09±0.22 nM on Day 14. Thrombin generation was relatively high in the initial phase of chemotherapy (319.0±44.5 nM) and then declined to 154.6±17.2 nM on Day 14 (p=0.008). The protein C activity was relatively low at start (0.70±0.05 IU/mL) indicating a state of consumption, but showed a trend towards normalization during treatment reaching an average level of 0.78±0.05 IU/mL for all patients on Day 14 (p=0.103). The factor VIII level was high and relatively stable during the two weeks of treatment with a range of 1.73±0.16 to 1.89±0.17 IU/mL, indicating a reactive state. The level of antithrombin was normal at start of treatment (1.00±0.05 IU/mL) and throughout the study period. Our data show that acute myeloid leukemia may be associated with a significant coagulopathy and that the activity in the coagulation process is increased during the initial phase of chemotherapy. This should be considered in patients at start of treatment, in particular, in patients with septicemia and/or signs of disseminated intravascular coagulation.