Risk factors for antibody formation in children with hemophilia: methodological aspects and clinical characteristics of the HEMFIL cohort study.

Up to 35% of patients with hemophilia A and 5% with hemophilia B develop neutralizing antibodies which can inhibit the therapeutic activity of factor replacement (inhibitors). Despite the clinical relevance of antifactor VIII and IX neutralizing antibodies, there is still a major gap on the knowledge of risk factors for their development. Furthermore, most of the studies on risk factors for inhibitor development come from Caucasian and Afro-American populations. The HEMFIL is a Brazilian prospective cohort study of previously untreated children with hemophilia, which primary aim is to identify new risk factors related to inhibitor development. This manuscript aims at describing the study design and its methodology. After the diagnosis, children are followed up to 75 exposure days or to inhibitor development. Standardized forms and blood samples are collected to describe clinical characteristics and to perform the measurement of immunological and genetic biomarkers at three time points; Inclusion time (T0), at inhibitor development or at 75 exposure days without inhibitors (T1) and after immune tolerance induction for patients in whom it is indicated and performed (T2). Currently, 120 children have been included, of whom, 95 have completed the follow-up. For severe/moderately severe hemophilia A, the cumulative incidence of inhibitors at 75 exposure days was 35% (95% confidence interval, 26-46%). The inclusion of additional patients and a longer follow-up will allow the analysis of risk factors for inhibitor development.

BAX 335 hemophilia B gene therapy clinical trial results: potential impact of CpG sequences on gene expression.

Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus serotype 8 (AAV8)-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetic variables, effects on FIX activity, and immune responses for dosed participants. Eight adult male participants (aged 20-69 years; range FIX activity, 0.5% to 2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events, all considered unrelated to BAX 335. No serious adverse event led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity was reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0% to 58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ∼20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5 to 11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses, including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. This trial was registered at www.clinicaltrials.gov as #NCT01687608.

Role of Small Intestine and Gut Microbiome in Plant-Based Oral Tolerance for Hemophilia.

Fusion proteins, which consist of factor VIII or factor IX and the transmucosal carrier cholera toxin subunit B, expressed in chloroplasts and bioencapsulated within plant cells, initiate tolerogenic immune responses in the intestine when administered orally. This approach induces regulatory T cells (Treg), which suppress inhibitory antibody formation directed at hemophilia proteins induced by intravenous replacement therapy in hemophilia A and B mice. Further analyses of Treg CD4+ lymphocyte sub-populations in hemophilia B mice reveal a marked increase in the frequency of CD4+CD25-FoxP3-LAP+ T cells (but not of CD4+CD25+FoxP3+ T cells) in the lamina propria of the small but not large intestine. The adoptive transfer of very small numbers of CD4+CD25-LAP+ Treg isolated from the spleen of tolerized mice was superior in suppression of antibodies directed against FIX when compared to CD4+CD25+ T cells. Thus, tolerance induction by oral delivery of antigens bioencapsulated in plant cells occurs via the unique immune system of the small intestine, and suppression of antibody formation is primarily carried out by induced latency-associated peptide (LAP) expressing Treg that likely migrate to the spleen. Tolerogenic antigen presentation in the small intestine requires partial enzymatic degradation of plant cell wall by commensal bacteria in order to release the antigen. Microbiome analysis of hemophilia B mice showed marked differences between small and large intestine. Remarkably, bacterial species known to produce a broad spectrum of enzymes involved in degradation of plant cell wall components were found in the small intestine, in particular in the duodenum. These were highly distinct from populations of cell wall degrading bacteria found in the large intestine. Therefore, FIX antigen presentation and Treg induction by the immune system of the small intestine relies on activity of a distinct microbiome that can potentially be augmented to further enhance this approach.

Allergy and inhibitors in hemophilia - a rare complication with potential novel solutions.

INTRODUCTION: Hemophilia is a rare bleeding disorder caused by a deficiency of the plasma coagulation factors VIII and IX (hemophilia A [HA] and hemophilia B [HB], respectively). Replacement therapy with clotting factor concentrates is the mainstay of treatment. Unlike in patients with HB, anaphylaxis in patients with HA is extremely rare. METHODS: A retrospective study of prospectively collected data on patients with hemophilia who experienced anaphylaxis was conducted in our center. Demographic and clinical data were collected, and laboratory workups that included thrombin generation were conducted. RESULTS: Our first patient underwent successful immune tolerance induction (ITI) following the administration of rituximab. The second patient was transitioned to emicizumab. The third patient receives recombinant activated VIIa (rFVIIa) on demand. Thrombin generation was performed following current medical management protocols for supporting hemostasis. DISCUSSION: Our case series illustrates the difficulty in managing patients with anaphylaxis to replacement therapy. In the era of novel therapies, such as emicizumab, the management of HA patients who experience anaphylaxis to replacement therapy is becoming easier and may obviate the need for ITI. Current treatment strategies for HB patients with such anaphylaxis, however, are limited to rFVIIa, and it continues to pose a challenge.

Perioperative safety and haematostatic efficacy of a new bypassing agent pd-FVIIa/FX (Byclot) in haemophilia patients with high-responding type inhibitors.

: The novel agent pd-FVIIa/FX is a 1 : 10 protein weight mixture of activated factor VII (FVIIa) and factor X (FX) derived from donated blood plasma. A phase III clinical trial of pd-FVIIa/FX revealed high efficacy for bleeding episodes in haemophilia patients with inhibitors. However, up to now, only one case of this new agent being used for surgery had been reported. The objective of this study is to evaluate the perioperative haemostatic efficacy and safety of pd-FVIIa/FX in haemophilia patients with inhibitors. We retrospectively reviewed 25 operation charts from 14 haemophilia patients with high-responding inhibitors using pd-FVIIa/FX during the perioperative period. Efficacy was evaluated by attending physicians and results divided into four groups (excellent, good, fair, and poor). The operation chart was provided by nine Japanese medical institutes with expertise in haemophilia management. Out of the total of 25 surgical procedures, 44% (11/25) were classified as major surgery and the remainders were minor surgeries. In all of the surgeries but one, rFVIIa and/or APCC were administered in combination or sequential method. In all cases except one, the haemostatic efficiency rate was judged as excellent or good by treating physicians for an overall efficacy rate of 96%. No thrombotic adverse effects were reported. This study's results suggest that both combination and sequential therapy of pd-FVIIa/FX and other bypassing agents are well tolerated and effective for the control of perioperative bleeding in haemophilia patients with high-responding inhibitors.

Perioperative laboratory monitoring in congenital haemophilia patients with inhibitors: a systematic literature review.

: Although the use of clotting factor concentrates is the mainstay of haemophilia care, the development of inhibitors complicates disease management. Perioperative management of patients with inhibitors is therefore a challenge. A systematic literature review was performed to identify literature reporting on the perioperative monitoring and management of haemophilia. MEDLINE, Embase and Cochrane databases were searched from database inception to 26 March 2018. Recent congress proceedings were also searched. Titles and abstracts, then full texts, were screened for relevance by two reviewers. Quality of included studies was assessed using the Critical Appraisal Skills Programme checklist. Of the 2033 individual entries identified, 86 articles met the inclusion criteria. The identified studies were screened again to find articles reporting perioperative laboratory monitoring in patients with congenital haemophilia A or B, resulting in 24 articles undergoing data extraction. Routine perioperative assay monitoring practices were the most commonly reported (n = 20/24); thrombin generation assay was the least commonly reported (n = 2/24). Other monitoring practices described were factor VII and factor VIII coagulation activity (n = 8/24, n = 5/24, respectively), and thromboelastography or rotational thromboelastometry assessments (n = 3/24). The impact of monitoring on treatment decisions was, however, rarely reported. In conclusion, many methods of perioperative monitoring of haemophilia patients with inhibitors have been identified in this review, yet there is a lack of reporting in larger scale cohort studies. More detailed reporting on the impact of monitoring outcomes on treatment decisions is also needed to share best practice, particularly as new therapeutic agents emerge.

Diagnosis and treatment of hemophilia.

Hemophilia A and B are inherited bleeding disorders characterized by deficiency or dysfunction of coagulation protein factors VIII and IX, respectively. Recurrent joint and muscle bleeds are the major clinical manifestations. Replacement therapy with clotting factors, either at the time of bleeding or as part of a prophylactic regimen, is adapted to individual patient needs. The major complication of therapy is the development of neutralizing antibodies. In response, researchers have developed novel agents to both reduce the treatment burden and prevent bleeding regardless of the presence of inhibitors. Another new development, gene therapy, has the potential for a definitive cure. This review summarizes the pathophysiology, clinical presentation, diagnosis, and treatment of hemophilia, as well as information regarding neutralizing antibodies, immune tolerance induction, novel agents, and gene therapy.

Development of an inhibitor in a child with severe hemophilia B: treatment with immunosuppression and an extended desensitization protocol.

: Development of neutralizing alloantibodies in hemophilia B is a less common, but clinically challenging phenomenon. Novel therapeutics for hemophilia B have recently been developed and reports of clinical experience with these agents outside of clinical trials are needed. We report development of an inhibitor in a child with severe hemophilia B, and subsequent immune tolerance induction using an extended desensitization protocol with the addition of immunosuppression. This case highlights successful management of a rare complication in a rare bleeding disorder and the need for additional investigation into this infrequent and clinically challenging occurrence.

Phenotypic characterization of haemophilia B - Understanding the underlying biology of coagulation factor IX.

Haemophilia B is a recessive, X-linked bleeding disorder due to inherited deficiency in vitamin K-dependent coagulation factor IX (FIX). FIX activity levels, as a basis for the definition of disease severity, do not clearly correlate with bleeding phenotype, likely due to the multiple steps regulating coagulation. Timely, with the availability of extended half-life products and successful steps in gene therapy, haemophilia B therapy is in an active developmental phase. Therefore, increased knowledge of the factors contributing to the variation of haemostatic and clinical outcome and response to therapy is welcomed. FIX acts at the crossroads of both the extrinsic and intrinsic pathways, and on the platelet procoagulant membrane at the site of vascular injury, and therefore, FIX biology is targeted for multiple effectors and regulators. The synthesis, cellular and molecular interactions, and elimination routes of FIX are not as well studied as for FVIII. The specific roles of magnesium in both platelet adhesion and FIX activation, and of vascular collagen at the haemostatic site of platelet adhesion and FIX residence are of particular interest. Biochemical and translational research on these issues should improve our understanding of the mechanisms involved, leading to the development of relevant assays that measure both haemostasis and treatment response. The latter is becoming increasingly important in the new era of haemophilia management and ultimately may lead to improved treatment strategies individually tailored to a patient's needs and cost-efficiency.

Radiosynovectomy in haemophilia.

Radiosynovectomy (RS) is a simple, effective and safe procedure for the control of haemophilic synovitis that causes repetitive haemarthrosis. It must be done after confirming clinically (hard and painless mass on palpation) and by ultrasonography the existence of synovitis in a joint with recurrent haemarthrosis. RS should be the first invasive option (instead of arthroscopic synovectomy) for treatment of chronic synovitis. The technique is highly cost effective in comparison to arthroscopic synovectomy. The indication for RS is the presence of repeated haemarthroses associated with synovitis (confirmed clinically and by imaging techniques) that cannot be controlled by means of haematological treatment. No increase in the risk of cancer has been published and the dose of radiation utilized in RS is minimal. In haemophilic patients with recurrent haemarthrosis, RS should be performed under factor coverage as soon as possible, once the existence of synovitis has been confirmed by ultrasonography. RS should really be considered as a useful adjunctive procedure to the primary intervention, which is intensive replacement therapy.

Complexity of immune responses to AAV transgene products - Example of factor IX.

After two decades of research, in vivo gene transfer with adeno-associated viral (AAV) vectors has now resulted in successful treatments and even cures for several human diseases. However, the potential for immune responses against the therapeutic gene products remains one of the concerns as this approach is broadened to more patients, diverse diseases, and target organs. Immune responses following gene transfer of coagulation factor IX (FIX) for the treatment of the bleeding disorder hemophilia B has been extensively investigated in multiple animal models. Findings from these studies have not only influenced clinical trial design but have broader implications for other diseases. The impact of vector design and dose, as well as target organ/route of administration on humoral and cellular immune responses are reviewed. Furthermore, the potential for tolerance induction by hepatic gene transfer or combination with immune modulation is discussed.

Inhibitors in haemophilia A and B: Management of bleeds, inhibitor eradication and strategies for difficult-to-treat patients.

The standard therapy for patients with haemophilia is prophylactic treatment with replacement factor VIII (FVIII) or factor IX (FIX). Patients who develop inhibitors against FVIII/FIX face an increased risk of bleeding, and the likelihood of early development of progressive arthropathy, alongside higher treatment-related costs. Bypassing agents can be used to prevent and control bleeding, as well as the recently licensed prophylaxis, emicizumab, but their efficacy is less predictable than that of factor replacement therapy. Antibody eradication, by way of immune tolerance induction (ITI), is still the preferred management strategy for treating patients with inhibitors. This approach is successful in most patients, but some are difficult to tolerise and/or are unresponsive to ITI, and they represent the most complicated patients to treat. However, there are limited clinical data and guidelines available to help guide physicians in formulating the next treatment steps in these patients. This review summarises currently available treatment options for patients with inhibitors, focussing on ITI regimens and those ITI strategies that may be used in difficult-to-treat patients. Some alternative, non-ITI approaches for inhibitor management, are also proposed.

Intracranial Hemorrhage in Children With Inherited Bleeding Disorders: A Single Center Study in China.

BACKGROUND: Intracranial hemorrhage (ICH) is a life-threatening condition in children. Inherited bleeding disorders (IBD) have high risk of ICH. AIM: This single center study aims to identify the incidence, risk factors, and neurological outcome of ICH in children who suffer from IBD. METHODS: From 2005 to 2017, 241 children with IBDs from Nanfang hospital, Department of Pediatrics, were evaluated. The ICH episodes were identified by medical history, general physical examination, detailed neurological examination, and computed tomographic or magnetic resonance imaging examination. The risk factors, location of ICH, management strategies, and outcome were noted. RESULTS: ICH was confirmed in 54/241 (22.4%) children with IBD among them 52/54 (96.2%) (95% confidence interval [CI], 91.1%-99.9%) were hemophilia A and hemophilia B patients. The overall risk of ICH among children with IBD was 22.4% (95% CI, 17.2%-27.8%). The median age of ICH was 30 months (0 to 204) and 18/54 (33.3%) (95% CI, 20.3%-46.3%) children had an ICH in the first year of life. Twenty-eight of 52 (53.8%) hemophilic children with ICH were assessed for inhibitor of FVIII and FIX. Nine of 28 (32%) hemophilic children with inhibitor developed the ICH. Six of 52 (11.5%) (95% CI, 2.6%-20.5%) hemophilic children had multiple episodes of ICH in which 4 were inhibitor positive. Thirteen of 54 (24%) (95% CI, 12.3%-35.9%) had positive family history of IBD. Twenty-two (36%) (95% CI, 23.7%-48.5%) of 61 ICH episodes were caused by trauma and 39 (63.9%) (95% CI, 51.5%-76.3%) were nontrauma related. Subdural hematoma was most frequently observed. Mortality risk from ICH in children with IBD was 5/54 (9.2%) (95% CI, 1.3%-17.2%). Eleven (22.4%) (95% CI, 10.3%-34.6%) of 49 survivors had known neurological squeal, whereas 38 (77.5%) (95% CI, 65.4%-89.7%) had no documented evidence of neurological impairment. CONCLUSIONS: Hemophilia is the most common IBD and most frequently associated with ICH. Risk and consequences of ICH in IBD were high during the first year of life while in older children better outcome may be expected. The optimal management of ICH depends on immediate recognition and prompt replacement therapy.

Tolerance induction in hemophilia: innovation and accomplishments.

PURPOSE OF REVIEW: Hemophilia is an X-linked blood coagulation genetic disorder, which can cause significant disability. Replacement therapy for coagulation factor VIII (hemophilia A) or factor IX (hemophilia B) may result in the development of high-affinity alloantibodies ('inhibitors') to the replacement therapy, thus making it ineffective. Therefore, there is interest in directing immunological responses towards tolerance to infused factors. RECENT FINDINGS: In this review, we will discuss latest advancements in the development of potentially less immunogenic replacement clotting factors, optimization of current tolerance induction protocols (ITI), preclinical and clinical data of pharmacological immune modulation, hepatic gene therapy, and the rapidly advancing field of cell therapies. We will also evaluate publications reporting data from preclinical studies on oral tolerance induction using chloroplast-transgenic (transplastomic) plants. SUMMARY: Until now, no clinical prophylactic immune modulatory protocol exists to prevent inhibitor formation to infused clotting factors. Recent innovative technologies provide hope for improved eradication and perhaps even prevention of inhibitors.

The socioeconomic burden of patients affected by hemophilia with inhibitors.

Hemophilia is associated with a high financial burden on individuals, healthcare systems, and society. The development of inhibitors significantly increases the socioeconomic burden of the diseases. This study aimed to review and describe the burden of hemophilia with inhibitors, providing a reference scenario to assess the impact of new products in the real word. Two systematic literature reviews were performed to collect data on (i) health economics and (ii) health-related quality of life evidences in hemophilic patients with inhibitors. The costs associated with patients with hemophilia and inhibitors are more than 3 times greater than the costs incurred in those without inhibitors, with an annual cost per patient that can be higher than €1 000 000. The costs of bypassing agents account for the large majority of the total healthcare direct costs for hemophilia treatment. The quality of life is more compromised in patients with hemophilia and inhibitors compared to those without inhibitors, in particular the physical domains, whereas mental domains were comparable to that of the general population. The development of an inhibitor has a high impact on costs and quality of life. New treatments have the potential to change positively the management and socioeconomic burden of hemophilia with inhibitors.

Methodologies for data collection in congenital haemophilia with inhibitors (CHwI): critical assessment of the literature and lessons learned from recombinant factor VIIa.

AIMS: To systematically review the effectiveness of on-demand treatment with recombinant coagulation factor VIIa (rFVIIa) in congenital haemophilia with inhibitors and, if feasible, perform a meta-analysis of the data. MATERIALS AND METHODS: Publications from Embase® , MEDLINE® , MEDLINE® In-Process and the Cochrane Central Register of Controlled Trials were searched. Selected publications were reviewed for inclusion by two independent expert reviewers. Discrepancies were reconciled by a third independent reviewer. Data from selected studies were extracted using a predefined grid to ensure uniform and comparable results were captured. RESULTS: A systematic search (cut-off date of 2 May 2016) identified 20 studies (13 observational; seven randomized controlled trials). All studies were of sufficient quality to include in this analysis and comprised 1221 participants, with 5981 bleeds in 746 individuals treated with rFVIIa. Haemostatic overall effectiveness of the individual studies identified ranged from 68% to 100% at ≤12 hours, 86% to 96% at 13-24 hours and 76% to 99% at 24-48 hours with rFVIIa <100 µg/kg, with similar rates reported for the ≥250 µg/kg dose. However, heterogeneity between the studies precluded pooling of results. CONCLUSIONS: Data from the individual studies confirmed that rFVIIa is an effective therapy for the on-demand treatment of bleeds in congenital haemophilia with inhibitors. However, the high levels of heterogeneity between studies precluded pooling of results for a valid, reliable or precise summary measure. There remains a need to implement standardized clinical definitions and measurements for the effectiveness and safety of haemophilia therapies in future clinical trials.