The prospective Hemophilia Inhibitor PUP Study reveals distinct antibody signatures prior to FVIII inhibitor development.

Preventing factor VIII (FVIII) inhibitors following replacement therapies with FVIII products in patients with hemophilia A remains an unmet medical need. Better understanding of the early events of evolving FVIII inhibitors is essential for risk identification and the design of novel strategies to prevent inhibitor development. The Hemophilia Inhibitor Previously Untreated Patients (PUPs) Study (HIPS; www.clinicaltrials.gov #NCT01652027) is the first prospective cohort study to evaluate comprehensive changes in the immune system during the first 50 exposure days (EDs) to FVIII in patients with severe hemophilia A. HIPS participants were enrolled prior to their first exposure to FVIII or blood products ("true PUPs") and were evaluated for different immunological and clinical parameters at specified time points during their first 50 EDs to a single source of recombinant FVIII. Longitudinal antibody data resulting from this study indicate that there are 4 subgroups of patients expressing distinct signatures of FVIII-binding antibodies. Subgroup 1 did not develop any detectable FVIII-binding immunoglobulin G (IgG) antibodies. Subgroup 2 developed nonneutralizing, FVIII-binding IgG1 antibodies, but other FVIII-binding IgG subclasses were not observed. Subgroup 3 developed transient FVIII inhibitors associated with FVIII-binding IgG1 antibodies, similar to subgroup 2. Subgroup 4 developed persistent FVIII inhibitors associated with an initial development of high-affinity, FVIII-binding IgG1 antibodies, followed by IgG3 and IgG4 antibodies. Appearance of FVIII-binding IgG3 was always associated with persistent FVIII inhibitors and the subsequent development of FVIII-binding IgG4. Some of the antibody signatures identified in HIPS could serve as candidates for early biomarkers of FVIII inhibitor development.

The future of bypassing agents for hemophilia with inhibitors in the era of novel agents.

Bypassing agents are presently the standard of care for the treatment of bleeding episodes in patients with hemophilia and high-titer inhibitors and are also used for bleed prevention. Only two bypassing agents are available to patients, and these products trace their lineage to the 1970s (activated prothrombin complex concentrates) and the 1980s (recombinant factor VIIa). Given the limited repertoire of available products, clinicians have relied on experience, empirical observation, registry data and individualized care to improve clinical outcomes on a case-by-case basis. Research over the past two decades has culminated in a greatly improved understanding of human coagulation; resulting from this, new products have been developed that offer treatment options and mechanisms of actions that differ from current bypassing agents. The most advanced in clinical development is emicizumab, a bispecific antibody that mimics the function of FVIIIa in the intrinsic Xase complex and is indicated for once-weekly or every-other-week prophylactic dosing in inhibitor patients. Other non-traditional products in clinical development include fitusiran and antibodies directed against tissue factor pathway inhibitor. As non-factor-based therapies become more widely utilized over time, the use of bypassing agents may be expected to decrease; however, bypassing agents will remain essential for the foreseeable future. As such, clinical development of bypassing agents continues, with some products (e.g. eptacog beta) under regulatory review. In this review we examine the optimal use of bypassing agents and their mechanism of action. We also discuss newer products and how these might theoretically be administered in conjunction with traditional bypassing agents.

Optimizing bleed prevention throughout the lifespan: Womb to Tomb.

The focus of care providers, patients and families is the ability to tailor care for persons with haemophilia (PWH) across the lifespan. Care requires knowledge of the bleeding disorder and age-related complications, risk of therapeutic interventions, and evaluation of individual characteristics that contribute to outcomes. The ultimate goal is to live a normal life without the burden of bleeding, for PWH and carriers. A wide range of therapeutic options is required to achieve personalized care. Over the last decade, substantial therapeutic advantages have been achieved in the treatment of haemophilia that include the development of a robust array of factor concentrates, novel haemostatic agents, and increased knowledge and awareness of disease associated outcomes and risk factors. Significant strides on the road to accessible gene therapy have been realized. This increased range of therapeutic modalities provides options for development and implementation of care plans for each patient at each stage of life that are more flexible compared to prior care regimens. Paradigms for management of haemophilia are changing. As a community, we must work together to use these resources wisely, to learn from outcomes with new therapies and diagnostic tools, to assure all patients can achieve improved care and outcomes regardless of disease state or country of origin.

Limit of detection and threshold for positivity of the Centers for Disease Control and Prevention assay for factor VIII inhibitors.

Essentials Immunologic methods detect factor VIII (FVIII) antibodies in some inhibitor-negative specimens. Specimens were tested by modified Nijmegen-Bethesda assay (NBA) and fluorescence immunoassay. The NBA with preanalytical heat inactivation detects FVIII inhibitors down to 0.2 NBU. IgG4 frequency validates the established threshold for positivity of ≥ 0.5 NBU for this NBA. SUMMARY: Background The Bethesda assay for measurement of factor VIII inhibitors called for quantification of positive inhibitors by using dilutions producing 25-75% residual activity (RA), corresponding to 0.4-2.0 Bethesda units, with the use of 'more sensitive methods' for samples with RA closer to 100% being recommended. The Nijmegen modification (Nijmegen-Bethesda assay [NBA]) changed the reagents used but not these calculations. Some specimens negative by the NBA have been shown to have FVIII antibodies detectable with sensitive immunologic methods. Objective To examine the performance at very low inhibitor titers of the Centers for Disease Control and Prevention (CDC)-modified NBA (CDC-NBA), which includes preanalytic heat inactivation to liberate bound anti-FVIII antibodies. Methods Specimens with known inhibitors were tested with the CDC-NBA. IgG4 anti-FVIII antibodies were measured by fluorescence immunoassay (FLI). Results Diluted inhibitors showed linearity below 0.4 Nijmegen-Bethesda units (NBU). With four statistical methods, the limit of detection of the CDC-NBA was determined to be 0.2 NBU. IgG4 anti-FVIII antibodies, which correlate most strongly with functional inhibitors, were present at rates above the background rate of healthy controls in specimens with titers ≥ 0.2 NBU and showed an increase in frequency from 14.3% at 0.4 NBU to 67% at the established threshold for positivity of 0.5 NBU. Conclusions The CDC-NBA can detect inhibitors down to 0.2 NBU. The FLI, which is more sensitive, demonstrates anti-FVIII IgG4 in some patients with negative (< 0.5) NBU. The sharp increase in IgG4 frequency between 0.4 and 0.5 NBU validates the established threshold for positivity of ≥ 0.5 NBU for the CDC-NBA, supporting the need for method-specific thresholds.

Clinical and laboratory phenotype variability in type 2M von Willebrand disease.

Essentials The pathophysiology of type 2M von Willebrand disease (VWD) is poorly understood. Sequence variations in type 2M VWD subjects were characterized. A high degree of clinical and laboratory variability exists within type 2M VWD variants. Some type 2M variants may share features of type 2A VWD. SUMMARY: Background von Willebrand factor (VWF) is a multimeric coagulation factor that tethers platelets to injured subendothelium. Type 2M von Willebrand disease (VWD) is characterized by a qualitative defect in VWF with preserved multimer distribution. Objectives Through the Zimmerman Program for the Molecular and Clinical Biology for VWD, five VWF sequence variations were studied in subjects diagnosed with type 2M VWD. Methods Bleeding phenotype was assessed using the ISTH bleeding assessment tool. Full-length VWF gene sequencing was performed for each subject. Each variant was placed into a recombinant VWF vector using site-directed mutagenesis and expressed in HEK293T cells as homozygous or heterozygous VWF. Variant expression, collagen binding and platelet GPIbα binding were studied through ELISA assays. Multimer analysis was performed by gel electrophoresis. Results Bleeding scores were elevated for all subjects except for the p.P1162L and p.R1374C variants. Although all had reduced VWF ristocetin cofactor activity/VWF antigen ratios on plasma testing, recombinant VWF did not show a classic type 2M phenotype for any of the five variants. Homozygous expression of variants p.D1283Y, p.R1349C, p.R1374C and p.I1453N was consistent with type 2A VWD, although all had normal expression as heterozygous recombinant VWF. Variant p.P1162L had normal VWF expression and function, consistent with the lack of bleeding symptoms. Conclusions Although originally classified as type 2M VWD, these homozygous recombinant VWF variants do not fulfill complete 2M VWD diagnostic criteria. A better classification schema and improved testing for putative type 2M variants is needed in order to effectively diagnose and treat affected patients.

Treatment of bleeding episodes with recombinant factor VIII Fc fusion protein in A-LONG study subjects with severe haemophilia A.

INTRODUCTION: The Phase 3 A-LONG study demonstrated the safety and efficacy of rFVIIIFc for the control and prevention of bleeding episodes in severe haemophilia A. AIM: To describe the treatment of bleeding episodes with rFVIIIFc in the A-LONG study. METHODS: A-LONG subjects (<1 IU dL-1 endogenous FVIII) were treated with individualized prophylaxis (Arm 1), weekly prophylaxis (Arm 2) or episodic treatment (Arm 3). Information recorded for each bleeding episode included type, location and dose to treat the episode. RESULTS: During A-LONG, 757 bleeding episodes occurred during the efficacy period; the majority [456 (60%)] occurred in Arm 3 (episodic treatment). Of 93 subjects in the prophylaxis arms who entered the study with target joints, 43 (60%) in Arm 1 and 11 (52%) in Arm 2 did not experience a target joint bleed. Overall, 98% of bleeding episodes (and 98% of bleeds involving a target joint) resolved with one or two infusions; the median dose per infusion to treat a bleed was 27 IU kg-1 (27 IU kg-1 for target joints). Using population pharmacokinetic simulations, FVIII activity levels were predicted to be below the upper limit of normal (150 IU dL-1 ) in most patients in the event that rFVIIIFc is used to treat a bleeding episode in close proximity to a prophylactic dose. CONCLUSIONS: These findings demonstrate the efficacy of rFVIIIFc for the treatment of acute bleeding episodes in subjects with severe haemophilia A, regardless of treatment regimen.

Complications of haemophilia in babies (first two years of life): a report from the Centers for Disease Control and Prevention Universal Data Collection System.

AIM: To describe the prevalence and complications in babies ≤2 years with haemophilia. METHODS: We used a standardized collection tool to obtain consented data on eligible babies aged ≤2 years with haemophilia enrolled in the Centers for Disease Control and Prevention Universal Data Collection System surveillance project at US Hemophilia Treatment Centers (HTCs). RESULTS: Of 547 babies, 82% had haemophilia A, and 70% were diagnosed within one month of birth. Diagnosis was prompted by known maternal carrier status (40%), positive family history (23%), bleeding (35%) and unknown 2%; 81% bled during the first two years. The most common events were bleeding (circumcision, soft tissue, oral bleeding) and head injury. There were 46 episodes of intracranial haemorrhage (ICH) in 37 babies (7%): 18 spontaneous, 14 delivery related, 11 traumatic, 2 procedure related and 1 unknown cause. Of the 176 central venous access devices (CVADs) in 148 (27%) babies, there were 137 ports, 22 surgically inserted central catheters and 20 peripherally inserted central catheters. Ports had the lowest complication rates. Inhibitors occurred in 109 (20%) babies who experienced higher rates of ICH (14% vs. 5%; P = 0.002), CVAD placement (61% vs. 19%; P < 0.001) and CVAD complications (44% vs. 26%; P < 0.001). The most common replacement therapy was recombinant clotting factor concentrates. CONCLUSION: Bleeding events in haemophilic babies ≤2 years were common; no detectable difference in the rates of ICH by the mode of delivery was noted. Neonatal factor exposure did not affect the inhibitor rates. Minor head trauma, soft tissue and oropharyngeal bleeding were the leading indications for treatment.

Integrated analysis of safety data from 12 clinical interventional studies of plasma- and albumin-free recombinant factor VIII (rAHF-PFM) in haemophilia A.

INTRODUCTION: Antihaemophilic factor (recombinant), plasma/albumin-free method (rAHF-PFM) is a human recombinant full-length factor VIII (FVIII) approved worldwide for the control and prevention of bleeding episodes, routine prophylaxis and perioperative management in adults and children with haemophilia A. AIM: To evaluate rAHF-PFM safety [including adverse events (AEs) and inhibitor incidence] from 12 interventional studies spanning >10 years. METHODS: The study population comprised 418 treated patients (median age = 18.7 years) with FVIII levels ≤2% of normal, including 55 previously untreated or minimally treated patients (PUPs/MTPs) from all rAHF-PFM phase I-IV studies, excluding observational safety studies. RESULTS: Most AEs were non-serious; only 93 AEs in 45 patients (10.8%) were related to rAHF-PFM. A total of 106 serious AEs (SAEs) occurred in 69 patients (16.5%); the most common were FVIII inhibitors (4.1%), device-related infection (1.0%) and pyrexia (0.7%). The 17 SAEs considered related to treatment consisted of FVIII inhibitors in 1 previously treated patient (PTP) (≤5 Bethesda Units [BU]) and 16 PUPs/MTPs [7/55 high titre (>5 BU), 12.7%; 9/55 low titre (≤5 BU), 16.4%]. Overall, the incidence of FVIII inhibitors was 0.36% in PTPs and 29.1% in PUPs/MTPs. No deaths or cases of hypersensitivity related to rAHF-PFM occurred. CONCLUSION: This integrated safety analysis evaluated the safety and tolerability of rAHF-PFM in children and adults with moderately severe or severe haemophilia A in all interventional studies completed to date. It was important to review consolidated evidence as some AEs are rare. There were no new safety signals in a wide variety of clinical settings.

Recombinant factor VIII Fc fusion protein: extended-interval dosing maintains low bleeding rates and correlates with von Willebrand factor levels.

BACKGROUND: Routine prophylaxis with replacement factor VIII (FVIII) - the standard of care for severe hemophilia A - often requires frequent intravenous infusions (three or four times weekly). An FVIII molecule with an extended half-life could reduce infusion frequency. The A-LONG study established the safety, efficacy and prolonged pharmacokinetics of recombinant FVIII Fc fusion protein (rFVIIIFc) in previously treated adolescents and adults with severe hemophilia A. OBJECTIVE: In this post hoc analysis, we investigated the relationship between subjects' prestudy (FVIII) and on-study (rFVIIIFc) regimens. METHODS: We analyzed two subgroups of subjects: prior prophylaxis and on-study individualized prophylaxis (n = 80), and prior episodic treatment and on-study weekly prophylaxis (n = 16). Subjects' prestudy dosing regimens and bleeding rates were compared with their final rFVIIIFc regimens and annualized bleeding rates (ABRs) in the last 3 months on-study. Dosing regimen simulations based on population pharmacokinetics models for rFVIII and rFVIIIFc were performed. RESULTS: As compared with their prestudy regimen, 79 of 80 (98.8%) subjects on individualized rFVIIIFc prophylaxis decreased their infusion frequency. Overall ABRs were low, with comparable factor consumption. Longer dosing intervals, including 5-day dosing, were associated with higher baseline von Willebrand factor antigen levels. Simulated dosing regimens predicted a greater proportion of subjects with steady-state FVIII activity trough levels of ≥ 1 IU dL(-1) (1%) with rFVIIIFc than with equivalent rFVIII regimens. CONCLUSION: These results suggest that patients on rFVIIIFc prophylaxis can reduce their infusion frequency as compared with their prior FVIII regimen while maintaining low bleeding rates, affording more patients trough levels of ≥ 1 IU dL(-1) than with rFVIII products requiring more frequent dosing regimens.

The obstetric, gynaecological and fertility implications of homozygous PAI-1 deficiency: single-centre experience.

Complete plasminogen activator inhibitor type 1 (PAI-1) deficiency is an exceedingly rare autosomal recessive bleeding disorder previously identified and reported in a large Old Order Amish (OOA) kindred in Indiana [Fay et al. Blood 1997; 90: 204]. Mouse models suggest that proteolysis via the plasminogen activator/plasmin system plays a crucial role in reproduction including degradation of the follicular wall during ovulation, fertilization, embryo implantation and embryogenesis [Leonardsson et al., Proc Natl Acad Sci USA 1995; 92: 12446]. We report the obstetric, gynaecological and fertility histories of OOA individuals with homozygous PAI-1 deficiency. In this family, there are 10 affected members identified to date ranging in age between 10 and 32 years, including seven female patients and three male patients. To date, two women have achieved pregnancies without difficulty; however, they experienced antenatal bleeding and preterm labour. The early initiation and continuation of antifibrinolytic agents, Epsilon-aminocaproic acid or tranexamic acid, during the pregnancy and in the postpartum period, was believed to be successful in preventing major bleeding complications in our patients with complete PAI-1 deficiency.

Safety of recombinant activated factor VII (rFVIIa) in patients with congenital haemophilia with inhibitors: overall rFVIIa exposure and intervals following high (>240 µg kg⁻¹) rFVIIa doses across clinical trials and registries.

Recombinant activated factor VII (rFVIIa) is indicated for treatment of bleeding in congenital haemophilia with inhibitors (CHwI) using 90 µg kg(-1) every 2-3 h (EU and US) or a single 270 µg kg(-1) dose (EU only) with ~90% efficacy reported for both regimens. Dosing of rFVIIa varies, and home treatment makes assessment of frequency of doses >90 µg kg(-1), the intervals before additional treatment, and the risk for thromboembolic events (TEs) more difficult. This post hoc analysis assessed the safety and distribution of rFVIIa dosing in CHwI and the impact of >240 µg kg(-1) dosing on subsequent bypassing agent (BPA) dosing interval and frequency. Data regarding on-demand or prophylactic rFVIIa dosing, TE incidence and subsequent BPA dosing after high rFVIIa doses were compiled from multiple sources incorporating safety surveillance. A total of 61 734 rFVIIa doses were reported in 481 patients treated for 3947 bleeds and for 43 135 prophylaxis days. Over half (52%) exceeded 120 µg kg(-1), 37% exceeded 160 µg kg(-1) and 15% exceeded 240 µg kg(-1). Subsequent doses of BPA(s) were administered after 38% of initial and 49% of any rFVIIa dose >240 µg kg(-1), and were most frequently administered ≥24 h after initial (40%) or any (53%) doses >240 µg kg(-1). No TEs were reported. The findings of this analysis show that rFVIIa doses >90 µg kg(-1) are utilized for 'real-world' treatment of children and adults. When additional BPA was administered following an rFVIIa dose >240 µg kg(-1), reported intervals were prolonged, often ≥24 h. No safety issues were identified in the 61,734 doses analysed.

Comparison of clot-based, chromogenic and fluorescence assays for measurement of factor VIII inhibitors in the US Hemophilia Inhibitor Research Study.

BACKGROUND: Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety and assessment of population trends. METHODS: Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA) and a novel fluorescence immunoassay (FLI). RESULTS: NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU) < 0.5 and positive on 42/42 specimens (100%) with NBU ≥ 2.0 and 43/80 specimens (53.8%) with NBU 0.5-1.9. Among specimens with positive NBA and negative CBA, 58.1% were FLI negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0-1.9 NBU specimens and 43.1% and 50.0% of 0.5-0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P = 0.004). Among 21 new inhibitors detected by NBA, five (23.8%) with 0.7-1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5-1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%). CONCLUSIONS: FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5-1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays.

Variability in platelet- and collagen-binding defects in type 2M von Willebrand disease.

Type 2M von Willebrand disease (VWD) includes qualitative defects in von Willebrand factor (VWF) function, with normal multimer distribution but a defect in VWF activity with respect to platelet or collagen binding. We characterized novel VWF gene mutations found in type 2M VWD subjects enrolled in the Zimmerman Program for the Molecular and Clinical Biology of VWD. Subjects were enrolled based on a pre-existing diagnosis of type 2M VWD. Testing included full-length gene sequencing, VWF antigen (VWF:Ag), VWF ristocetin cofactor activity (VWF:RCo), VWF collagen binding and multimer distribution. Recombinant VWF variants were synthesized using site-directed mutagenesis and expressed in HEK293T cells. Platelet binding was measured by flow cytometry with fixed platelets and ELISA with recombinant glycoprotein Ibα (GPIbα). Four novel VWF A1 domain mutations were found in individuals with type 2M VWD: S1358N, S1387I, S1394F and Q1402P. All subjects had a history of bleeding, VWF:RCo < 40 IU dL(-1) , VWF:RCo/VWF:Ag ratios <0.6 and normal multimer distribution. No defect in expression, secretion, or multimerization was found for any of the mutations. All showed decreased binding to intact platelets, and decreased or absent binding to a mutant GPIbα construct with spontaneous VWF binding. 1387I had decreased binding to all collagen types tested. 1402P had reduced binding exclusively to type VI collagen. Type 2M VWD is a heterogeneous category comprised of both collagen- and platelet-binding defects. Understanding the precise defect for each mutation may ultimately lead to better diagnosis and treatment.

F8 haplotype and inhibitor risk: results from the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort.

Ancestral background, specifically African descent, confers higher risk for development of inhibitory antibodies to factor VIII (FVIII) in haemophilia A. It has been suggested that differences in the distribution of FVIII gene (F8) haplotypes, and mismatch between endogenous F8 haplotypes and those comprising products used for treatment could contribute to risk. Data from the Hemophilia Inhibitor Genetics Study (HIGS) Combined Cohort were used to determine the association between F8 haplotype 3 (H3) vs. haplotypes 1 and 2 (H1 + H2) and inhibitor risk among individuals of genetically determined African descent. Other variables known to affect inhibitor risk including type of F8 mutation and human leucocyte antigen (HLA) were included in the analysis. A second research question regarding risk related to mismatch in endogenous F8 haplotype and recombinant FVIII products used for treatment was addressed. Haplotype 3 was associated with higher inhibitor risk among those genetically identified (N = 49) as of African ancestry, but the association did not remain significant after adjustment for F8 mutation type and the HLA variables. Among subjects of all racial ancestries enrolled in HIGS who reported early use of recombinant products (N = 223), mismatch in endogenous haplotype and the FVIII proteins constituting the products used did not confer greater risk for inhibitor development. Haplotype 3 was not an independent predictor of inhibitor risk. Furthermore, our findings did not support a higher risk of inhibitors in the presence of a haplotype mismatch between the FVIII molecule infused and that of the individual.