Analyses of the FranceCoag cohort support differences in immunogenicity among one plasma-derived and two recombinant factor VIII brands in boys with severe hemophilia A.

Around one third of boys with severe hemophilia A develop inhibitors (neutralizing antibodies) against their therapeutic factor VIII product. This adverse effect may result in more life-threatening bleeding, disability, impaired quality of life, and costly care. We compared the incidence of inhibitors in boys treated with the three factor VIII products most used in France: one plasma-derived (Factane) and two recombinant products (Advate and Kogenate Bayer). A previously untreated cohort of patients was created in 1994 to investigate risk factors for inhibitor development. We selected boys with severe hemophilia A (factor VIII <1 IU/dL) first treated with one of the three factor VIII products studied. Details of product infusions, inhibitor assays and main fixed and time-varying inhibitor risk factors were recorded for the first 75 exposure days. Three outcomes (all inhibitors, high-titer inhibitors and subsequently treated inhibitors) were analyzed by univariate and multivariate Cox models. We studied 395 boys first treated between 2001 and 2016 (131, 137, and 127 with Factane, Advate, and Kogenate Bayer, respectively). Clinically significant inhibitors were diagnosed in 121 patients (70 high-titer). The incidence of high-titer inhibitors was significantly associated with the factor VIII product received (P=0.005): the cumulative incidence at 75 exposure days was 12.7% (95% CI: 7.7-20.6) with Factane, 20.4% (95% CI: 14.0-29.1) with Advate, and 31.6% (95% CI: 23.5-41.7) with Kogenate Bayer. The low inhibitor incidence observed with Factane is concordant with recent findings from the SIPPET randomized trial. These consistent results from observational and experimental studies should lead to improved care for previously untreated patients and cost savings for healthcare systems worldwide.

Bleeding risk assessment in hemophilia A carriers from Dakar, Senegal.

: Hemophilia A carriers have an abnormal X chromosome with a molecular abnormality of FVIII gene. These carriers, long considered to be free of bleeding risk, could have the same symptoms as mild hemophiliacs. This study aim to assess bleeding risk of hemophilia A carriers monitored at the Clinical Hematology Department of Dakar. This is a prospective study of a period of 6 months including 22 hemophilia A carriers aged between 8 and 48 years. Hemophilia carriers were recruited using the genealogical tree of hemophiliacs followed in the service. Their diagnosis was carried out by long range PCR and Sanger sequencing method searching the molecular abnormality responsible for hemophilia in their family. Bleeding risk was determined using a questionnaire consisting of different bleeding symptoms quoted from -1 to 4 according to the severity. Total of different values allow to determine the bleeding score which was pathological if it was greater than or equal to 1. Medium age was 22.5 years (8-48) (SD = 9.28). Four hemophilia A carriers (18.1%) presented bleeding symptoms and had a bleeding score at least 1 (P = 0.02). Menorrhagia was predominant (13.6%) followed by epistaxis (9%), gingivorrhagia (9%), and prolonged bleeding after tooth extraction (9%). Factor VIII level was lower in hemophilia carriers who presented bleeding (42 ±â€Š8.61 UI/l) versus hemophilia carriers without bleeding (100 ±â€Š50.95 UI/l) (P = 0.001). There was no significant correlation between bleeding occurrence and age (P = 0.81), activated patial thromboplastin time value (P = 0.97) and FVIII/Von Willebrand Factor ratio (P = 0.12). One in five hemophilia carriers presented bleeding and the questionnaire was effective to identify hemophilia carriers who had a risk of bleeding.

Long-term results of ankle arthrodesis in children and adolescents with haemophilia.

INTRODUCTION: Ankle arthrodesis in patients with haemophilia is a joint-non-preserving treatment option often resulting in substantial pain relief and termination of haemarthrosis by osseous consolidating of the joint. However, limited data are available regarding ankle arthrodesis in young patients. AIM: The aim of this study was to evaluate the long-term clinical and radiographic results with a minimum follow-up of ten years in children with haemophilia who underwent ankle arthrodesis. METHODS: We retrospectively reviewed the results of ankle fusions performed in young haemophiliacs in our department between 1980 and 2006. The Méary procedure was used for arthrodesis, performed on patients with closed growth plates. Only one patient had no fixation, due to being only six years old. The modified American Orthopaedic Foot & Ankle Society (AOFAS) hindfoot score was calculated at last follow-up, and standard radiographic evaluations were performed. RESULTS: A total of 22 ankles were analysed from 17 patients. The mean follow-up was 19.7 ± 8 years. The mean age of the patients was 15.5 (6-23) years at the time of index surgery. There were no intra-operative or peri-operative complications related to ankle arthrodesis. The mean modified AOFAS score at last follow-up was 83 ± 10.5. Radiographic assessment demonstrated solid osseous fusion at the arthrodesis site, with no axis deformities. Two patients developed secondary subtalar arthrosis, treated by subtalar arthrodesis six years after initial ankle arthrodesis in one case. CONCLUSION: In our study, tibiotalar arthrodesis in young patients with haemophilia resulted in good long-term functional outcome with a low surgery-related complication rate.

A national French noninterventional study to assess the long-term safety and efficacy of reformulated nonacog alfa.

BACKGROUND: Nonacog alfa, the recombinant Factor IX (F IX) used for the treatment of hemophilia B, was approved in Europe in 1998. A reformulated version was approved for European use in 2007. STUDY DESIGN AND METHODS: This postmarketing study, as recommended by the risk management plan, was conducted to confirm the safety of reformulated nonacog alfa in a usual care setting in France. This open-label, noninterventional, prospective, longitudinal postmarketing study comprised 19 French hemophilia centers. Patients with hemophilia B receiving reformulated nonacog alfa for prophylaxis or on-demand treatment were followed up on usual care schedule. RESULTS: A total of 58 subjects were enrolled, of whom 29 (50%) were less than 18 years of age. Hemophilia was severe (baseline F IX activity < 1%) in 47 (81%) patients. All subjects except one were already treated with reformulated nonacog alfa before enrollment. One subject was receiving reformulated nonacog alfa as immune tolerance induction at time of enrollment. At enrollment, treatment regimen was mainly prophylactic in subjects less than 18 years and on-demand in subjects 18 years or older. Median duration of follow-up in the survey was 3.3 (2.3-3.8) years. The median annualized bleeding rate was 3.9 (1.5-5.2) for prophylaxis regimen and 12.2 (3.9-22.1) for on-demand regimen. One subject, a previously untreated patient, developed F IX inhibitors during follow-up. No allergic reaction, no blood cell agglutination, no lack of efficacy or recovery, and no thrombotic events were reported. CONCLUSION: Reformulated nonacog alfa was shown to be safe in a usual care setting.

LIM kinase/cofilin dysregulation promotes macrothrombocytopenia in severe von Willebrand disease-type 2B.

von Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein's multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine Vwf gene and in a patient bearing this mutation. We provide evidence of a profound defect in megakaryocyte (MK) function since: (a) the extent of proplatelet formation was drastically decreased in 2B MKs, with thick proplatelet extensions and large swellings; and (b) 2B MKs presented actin disorganization that was controlled by upregulation of the RhoA/LIM kinase (LIMK)/cofilin pathway. In vitro and in vivo inhibition of the LIMK/cofilin signaling pathway rescued actin turnover and restored normal proplatelet formation, platelet count, and platelet size. These data indicate, to our knowledge for the first time, that the severe macrothrombocytopenia in VWD-type 2B p.V1316M is due to an MK dysfunction that originates from a constitutive activation of the RhoA/LIMK/cofilin pathway and actin disorganization. This suggests a potentially new function of vWF during platelet formation that involves regulation of actin dynamics.

Notes from the Field: Fatal Infection Associated with Equine Exposure - King County, Washington, 2016.

On March 17, 2016, Public Health-Seattle & King County in Washington was notified of two persons who received a diagnosis of Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) infections. S. zooepidemicus is a zoonotic pathogen that rarely causes human illness and is usually associated with consuming unpasteurized dairy products or with direct horse contact (1). In horses, S. zooepidemicus is a commensal bacterium that can cause respiratory, wound, and uterine infections (2). The health department investigated to determine the magnitude of the outbreak, identify risk factors, and offer recommendations.

Apoptotic Platelet Events Are Not Observed in Severe von Willebrand Disease-Type 2B Mutation p.V1316M.

Thrombocytopenia and increased platelet clearance observed in von Willebrand disease-type 2B (VWD-2B) may be explained by platelet apoptosis triggered by the constitutive binding of VWF to its receptor, glycoprotein Ib (GPIb). Apoptosis was assessed in platelets from two patients with a severe VWD-2B mutation VWF/p.V1316M and from mice transiently expressing VWF/p.V1316M. We now report that the VWD-2B mutation VWF/p.V1316M which binds spontaneously to its receptor GPIbα does not induce apoptosis. In 2 unrelated patients (P1 and P2) exhibiting different VWF plasma levels (70% and 36%, respectively, compared with normal pooled human plasma given as 100%), inner transmembrane depolarization of mitochondria, characteristic of apoptotic events was undetectable in platelets, whether washed or in whole blood. No or a moderate phosphatidyl serine (PS) exposure as measured by annexin-V staining was observed for P1 and P2, respectively. Expression of pro-apoptotic proteins Bak and Bax, and caspase-3 activity were similar to control platelets. In the VWD-2B mouse model expressing high levels of mVWF/p.V1316M (423%), similar to what is found in inflammatory pathologies, no significant difference was observed between mice expressing mVWF/WT and mVWF/p.V1316M. These results strongly argue against apoptosis as a mechanism for the thrombocytopenia of severe VWD-2B exhibiting the VWF/p.V1316M mutation.

EQOFIX: a combined economic and quality-of-life study of hemophilia B treatments in France.

BACKGROUND: EQOFIX is a medicoeconomic study that analyzed the health-related quality of life (HRQoL) and costs of care of the moderate and severe forms of hemophilia B, treated on demand or by prophylaxis with either plasma-derived Factor IX (pdFIX) or recombinant FIX (rFIX). STUDY DESIGN AND METHODS: The primary objectives were evaluations of the impact of hemophilia B on HRQoL and of the costs associated with its management. The secondary objectives were evaluations of the clinical efficacy and costs of care of pdFIX and rFIX. In this observational study we included and followed for 1 year severe and moderate hemophilia B patients without inhibitor. HRQoL was evaluated through generic and disease-specific questionnaires. Information on the health resources consumed was collected every 3 months. RESULTS: The EQOFIX cohort was composed of 155 patients, including 51 children and 104 adults, with 114 having severe disease and 41 having moderate disease. The regimens were prophylactic for 61 and on demand for 94. Altogether, 78 were treated with rFIX and 77 with pdFIX. There was no difference in the QoL between the pdFIX and rFIX treatments. The extra cost of prophylaxis was €22,605 per bleeding event prevented. The consumption of FIX was 1.4-fold higher for the patients treated with rFIX than for the patients treated with pdFIX. CONCLUSION: Our findings in a cohort composed of 25% of the French population of moderate and severe hemophilia B patients show, with similar clinical and HRQoL results, that treatment with rFIX is more expensive than treatment with pdFIX.

Recombinant factor VIII products and inhibitor development in previously untreated boys with severe hemophilia A.

Six recombinant factor VIII (rFVIII) products have been marketed worldwide. In 2013, the Research of Determinants of Inhibitor Development (RODIN) study group reported an unexpectedly high risk of inhibitor development with a second-generation full-length rFVIII (Product D) in previously untreated patients (PUPs) with severe hemophilia A (HA). In 1994, French public health authorities established a prospective cohort to monitor hemophilia treatment safety. A PUP subgroup was designed to investigate inhibitor risk factors. We analyzed this subcohort in view of the RODIN findings. After excluding 50 patients who participated in the RODIN study, the primary analysis focused on 303 boys with severe HA first treated with a rFVIII product. A clinically significant inhibitor was detected in 114 boys (37.6%). The inhibitor incidence was higher with Product D vs the most widely used rFVIII product (adjusted hazard ratio [aHR], 1.55; 95% confidence interval [CI], 0.97-2.49). Similar results were found for high-titer inhibitors and in 10 sensitivity analyses. No heterogeneity was observed between RODIN and our results. Combined aHRs were 1.58 (95% CI, 1.17-2.14) for all inhibitors and 1.70 (95% CI, 1.15-2.52) for high-titer inhibitors. Our results confirm the higher immunogenicity of Product D vs other rFVIII products in PUPs with severe HA.

Development of inhibitory antibodies to therapeutic factor VIII in severe hemophilia A is associated with microsatellite polymorphisms in the HMOX1 promoter.

Induction of heme oxygenase-1, a stress-inducible enzyme with anti-inflammatory activity, reduces the immunogenicity of therapeutic factor VIII in experimental hemophilia A. In humans, heme oxygenase-1 expression is modulated by polymorphisms in the promoter of the heme oxygenase-1-encoding gene (HMOX1). We investigated the relationship between polymorphisms in the HMOX1 promoter and factor VIII inhibitor development in severe hemophilia A. We performed a case-control study on 99 inhibitor-positive patients and 263 patients who did not develop inhibitors within the first 150 cumulative days of exposure to therapeutic factor VIII. Direct sequencing and DNA fragment analysis were used to study (GT)n polymorphism and single nucleotide polymorphisms located at -1135 and -413 in the promoter of HMOX1. We assessed associations between the individual allele frequencies or genotypes, and inhibitor development. Our results demonstrate that inhibitor-positive patients had a higher frequency of alleles with large (GT)n repeats (L: n≥30), which are associated with lesser heme oxygenase-1 expression (odds ratio 2.31; 95% confidence interval 1.46-3.66; P<0.001]. Six genotypes (L/L, L/M, L/S, M/M, M/S and S/S) of (GT)n repeats were identified (S: n<21; M: 21≤n<30). The genotype group including L alleles (L/L, L/M and L/S) was statistically more frequent among inhibitor-positive than inhibitor-negative patients, as compared to the other genotypes (33.3% versus 17.1%) (odds ratio 2.21, 95% confidence interval 1.30-3.76; P<0.01). To our knowledge, this is the first association identified between HMOX1 promoter polymorphism and development of anti-drug antibodies. Our study paves the way towards modulation of the endogenous anti-inflammatory machinery of hemophilia patients to reduce the risk of inhibitor development.

Absence of collagen-induced platelet activation caused by compound heterozygous GPVI mutations.

The glycoprotein VI (GPVI)/FcRgamma complex is a key receptor for platelet activation by collagen. We describe, for the first time, 2 genetic abnormalities in one patient. This 10-year-old girl presented ecchymoses since infancy, a prolonged bleeding time despite a normal platelet count and no antiplatelet antibodies. Collagen-induced platelet activation was null, whereas GPVI quantification by flow cytometry evidenced an incomplete deficiency. Immunoblotting showed an abnormal migration of residual GPVI, and no FcRgamma defect. GPVI DNA sequencing revealed (1) an R38C mutation in exon 3 of one allele and (2) an insertion of 5 nucleotides in exon 4 of the other allele, leading to a premature nonsense codon and absence of the corresponding mRNA. Introduction of the R38C mutation into recombinant GPVI-Fc resulted in abnormal protein migration and a loss of collagen binding. Thus, this composite genetic GPVI deficiency and dysfunction cause absence of platelet responses to collagen and a mild bleeding phenotype.

Characteristics, mechanisms of action, and epitope mapping of anti-factor VIII antibodies.

The development of anti-factor VIII (FVIII) antibodies (Abs), also called inhibitors, is currently one of the most serious complications arising during the treatment of hemophilia A patients. Improved prevention and eradication of these Abs remain a challenge both for clinicians and scientists. Numerous studies in the literature have reported on their epitope specificity, on their mechanism of FVIII inactivation, as well as on the methods used for their detection. In this review, we summarize the current knowledge on the nature (isotypes, kinetic properties), epitope properties, and mechanisms of action of anti-FVIII Abs. Furthermore, we present methods for detection and epitope characterization of anti-FVIII Abs with emphasis on the Luminex technique susceptible to facilitate the monitoring of changes in the epitope specificity of these Abs.

Successful conservative treatment of traumatic subacute subdural haematomas in neonates with haemophilia A.

PURPOSE: The aim of this report is to enlighten the role of an early diagnosis and treatment of haemophilia A during the management of an intracranial haemorrhage as well as to discuss the efficiency of a subdural transcoronal puncture compared to a craniectomy as surgical treatment of the haematoma. BACKGROUND: Haemophilia A constitutes a well-known risk factor for intracranial bleeding. However, it has been rarely described as a cause of subdural haematoma in neonates. Management of subdural haematomas in haemophilic patients is still debated. MATERIALS AND METHODS: We report two cases of infants with subacute subdural haematoma. The first of them had a familial history of haemophilia A. In the second subject, the diagnosis was obtained during the etiological workup of an intracranial haematoma. Both infants were successfully treated with transcoronal puncture of the subacute component of the haematoma and factor VIII infusion. CONCLUSIONS: Transcranial punctures associated to infusion of factor VIII should be considered an alternative surgical option in the management of subdural haematomas in neonates with haemophilia A.

Influence of the type of factor VIII concentrate on the incidence of factor VIII inhibitors in previously untreated patients with severe hemophilia A.

Inhibitor development is the major treatment complication in children with severe hemophilia A. It is not clear whether the risk of inhibitors is higher with recombinant factor VIII or with plasma-derived factor VIII. We used multivariate analysis to compare 2 cohorts of previously untreated patients (PUPs) with severe hemophilia A: 62 patients treated with the same brand of high-purity plasma-derived FVIII (pFVIII) containing von Willebrand factor (VWF) and 86 patients treated with full-length recombinant FVIII (rFVIII). In addition to the usual end points (all inhibitors, high inhibitors), we also examined a third end point (high inhibitors and/or immune tolerance induction). The risk of inhibitor development was higher in patients treated with rFVIII than in patients treated with pFVIII, regardless of other risk factors (F8 genotype; nonwhite origin; history of inhibitors in patients with a family history of hemophilia; age at first FVIII infusion). The adjusted relative risk (RRa) for inhibitor development with rFVIII versus pFVIII was 2.4 (all inhibitors), 2.6 (high inhibitors), and 3.2 (high inhibitors and/or immune tolerance induction), respectively, depending on the end point (above). The pathophysiology of this large effect must be understood in order to improve the characteristics of recombinant products and to reduce the incidence of inhibitors to FVIII.

Full-length sucrose-formulated recombinant factor VIII for treatment of previously untreated or minimally treated young children with severe haemophilia A: results of an international clinical investigation.

The safety and efficacy of a full-length sucrose-formulated recombinant factor VIII product (rFVIII-FS; Kogenate FS; Kogenate Bayer) was evaluated in previously untreated (PUPs) and minimally treated (MTP) patients with severe haemophilia A (FVIII <2%). Patients (37 PUPs; 24 MTPs) aged 0.1-25.7 months were treated with rFVIII-FS for a cumulative of 9,141 exposure days (EDs), median 114 EDs (range 4-478), on prophylactic or on-demand therapy. Eighty-nine percent of all treated bleeding episodes were successfully treated with 1 (74%) or 2 (15%) infusions. Clinical response to first infusion for each bleeding episode was rated as 'excellent' in 58%, or 'good' in 33%, of all cases. Recombinant FVIII-FS was used in 27 surgical procedures, mainly catheter implantations, which were all conducted without bleeding complications. FVIII recovery mean values (approximately 2%/kg/IU) were as expected for any licensed FVIII concentrate. FVIII neutralizing antibody formation was 15% (9/60). Aside from inhibitor formation, three adverse events were rated as 'at least possibly drug-related' for a total drug-related adverse event rate of 0.14%. No viral seroconversions were observed. Overall, excellent safety and efficacy were demonstrated with rFVIII-FS for therapy of young children with severe haemophilia A.

The safety and efficacy of recombinant human blood coagulation factor IX in previously untreated patients with severe or moderately severe hemophilia B.

This international clinical trial evaluated the safety and efficacy of recombinant factor IX (rFIX) in previously untreated patients (PUPs) with severe or moderately severe hemophilia B (FIX activity, < or = 3 IU/dL). Sixty-three PUPs aged younger than 1 month to 14 years received rFIX (median treatment duration, 37 months; range, 4-64 months). Mean rFIX recovery (0.68 +/- 0.27 IU/dL per IU/kg) remained constant over 5 years and was similar in infants (1 month to < 2 years) and children (2 to < 12 years). Fifty-four PUPs used rFIX (median dose, 62.7 IU/kg per infusion; range, 8.2-292 IU/kg) to treat 997 hemorrhages. Bleeding was well controlled, with 75% of hemorrhages requiring only one rFIX infusion. Response to rFIX was "excellent" or "good" in 94% of cases. Effective hemostasis was achieved in 32 PUPs receiving rFIX for routine prophylaxis, with 91% of prophylaxis responses rated "excellent." rFIX administered for 30 surgical procedures in 23 PUPs achieved hemostasis for all rated procedures. Five patients experienced allergic-type manifestations, including 2 (3%) patients who developed FIX inhibitors (both > 5 BU/dL). rFIX was well tolerated, with no associated thrombotic events or evidence of viral transmission. These data indicate that rFIX is a safe and effective treatment for PUPs with hemophilia B.

Effects of trimethoprim-sulfadiazine on tear production and the fluctuations of Schirmer tear test values in horses.

The objectives of this study were to observe the effects of trimethoprim-sulfadiazine on equine tear production and to determine normal fluctuations in Schirmer tear test (STT) values in horses. A randomized, placebo-controlled, blinded clinical trial measuring STT values in 15 horses over an 8-week period was performed. The treatment group (eight horses) received 30 mg/kg trimethoprim-sulfadiazine orally once a day and the control group (seven horses) received placebo (flour) at the same time. All horses were housed outdoors throughout the study. Schirmer tear test values were measured at 0, 2, 4, 6 and 8 weeks, and 4 weeks after discontinuation of treatment. There were no significant differences in tear production between the treated and control groups. Fluctuations in STT were observed and may result from individual and environmental variations. Trimethoprim-sulfadiazine did not decrease tear production in the horses in this study. Horses normally experience periodic fluctuations in STT values.

Effects of trimethoprim-sulfadiazine on thyroid function of horses.

Trimethoprim-sulfadiazine was administered to horses in a randomized, placebo controlled study to determine the effects of potentiated sulfonamides on thyroid function in normal horses. The treatment group included eight horses that received trimethoprim-sulfadiazine mixed with molasses orally at 30 mg/kg once daily for eight weeks. The control group included 8 horses that received an oral placebo (flour mixed with molasses) once daily for the same period. Thyroid function was evaluated prior to initiation of treatment and after 8 weeks of treatment. Serum concentrations of total and free triiodothyronine (T3), total and free thyroxine (T4), and thyroid stimulating hormone (TSH) were determined at rest and after a thyrotropin-releasing hormone (TRH) stimulation test. There was no detectable difference between treatment and control groups.