Predicting inhibitor development using a random peptide phage-display library approach in the SIPPET Cohort.

Inhibitor development is the most severe complication of hemophilia A care, and is associated with increased morbidity and mortality. The aim of this study was to use a novel IgG epitope mapping method to explore the factor VIII (FVIII)-specific epitope profile in the SIPPET cohort population and to develop an epitope-mapping based inhibitor prediction model. The population consisted of 122 previously untreated patients with severe hemophilia A that were followed-up for 50 days of exposure to FVIII or 3 years, whichever occurred first. Sampling was performed before FVIII treatment and at the end of the follow-up. The outcome was inhibitor development. The FVIII epitope repertoire was assessed by means of a novel random peptide phage-display assay. A LASSO regression model and a random forest model were fitted on post-treatment sample data and validated in pre-treatment sample data. The predictive performance of these models was assessed by the C-statistic and a calibration plot. We identified 27,775 peptides putatively directed against FVIII, which were used as input for the statistical models. The C-statistic of the LASSO and random forest models were good at 0.78 (95%CI: 0.69-0.86) and 0.80 (95%CI: 0.72-0.89). Model calibration of both models was moderately good. Two statistical models, developed on data from a novel random peptide phage display assay, were used to predict inhibitor development before exposure to exogenous FVIII. These models can be used to set up diagnostic tests that predict the risk of inhibitor development before starting treatment with FVIII.

Predictive parameters for spontaneous joint bleeding during emicizumab prophylaxis.

Emicizumab is approved for prophylaxis of patients with hemophilia A (HA). Despite its efficacy in reducing bleeding, a few patients on emicizumab still experience hemarthrosis, but no tool is yet available to identify those at higher risk of spontaneous joint bleeding. To evaluate whether laboratory measurements (global coagulation assays and emicizumab concentration) and/or arthropathy scores can distinguish patients at higher risk of spontaneous joint bleeding while on emicizumab prophylaxis. Thrombin generation assay (TGA) was assessed upon the addition of tissue factor and synthetic phospholipids. Non-activated thromboelastography (NATEM) was performed in citrated whole blood. Emicizumab concentrations were measured with a modified one-stage FVIII assay. The degree of hemophilic arthropathy was assessed with the Haemophilia Joint Health Score (HJHS) and Hemophilia Early Arthropathy Detection with Ultrasound score (HEAD-US). A Cox proportional hazards model was used to evaluate the association between variables and bleeding. The predictive power of these variables was investigated by ROC analysis. Forty HA patients with and without inhibitors on emicizumab prophylaxis were enrolled in an observational cohort study. Ten of 40 developed spontaneous joint bleeding. None of the lab parameters were able to distinguish patients at higher risk of spontaneous joint bleeding. ROC analysis showed that during emicizumab prophylaxis only the presence of synovitis and a higher HEAD-US score were associated with spontaneous joint bleeding (AUC 0.84). A greater degree of arthropathy and the presence of synovitis could help to predict the risk of spontaneous joint bleeding in HA patients on emicizumab prophylaxis.

Congenital fibrinogen disorders: a retrospective clinical and genetic analysis of the Prospective Rare Bleeding Disorders Database.

ABSTRACT: Congenital fibrinogen deficiency (CFD) is a rare bleeding disorder caused by mutations in FGA, FGB, and FGG. We sought to comprehensively characterize patients with CFD using PRO-RBDD (Prospective Rare Bleeding Disorders Database). Clinical phenotypes, laboratory, and genetic features were investigated using retrospective data from the PRO-RBDD. Patients were classified from asymptomatic to grade 3 based on their bleeding severity. In addition, FGA, FGB, and FGG were sequenced to find causative variants. A total of 166 CFD cases from 16 countries were included, of whom 123 (30 afibrinogenemia, 33 hypofibrinogenemia, 55 dysfibrinogenemia, and 5 hypodysfibrinogenemia) were well characterized. Considering the previously established factor activity and antigen level thresholds, bleeding severity was correctly identified in 58% of the cases. The rates of thrombotic events among afibrinogenemic and hypofibrinogenemic patients were relatively similar (11% and 10%, respectively) and surprisingly higher than in dysfibrinogenemic cases. The rate of spontaneous abortions among 68 pregnancies was 31%, including 86% in dysfibrinogenemic women and 14% with hypofibrinogenemia. Eighty-six patients received treatment (69 on-demand and/or 17 on prophylaxis), with fibrinogen concentrates being the most frequently used product. Genetic analysis was available for 91 cases and 41 distinct variants were identified. Hotspot variants (FGG, p.Arg301Cys/His and FGA, p.Arg35Cys/His) were present in 51% of dysfibrinogenemia. Obstetric complications were commonly observed in dysfibrinogenemia. This large multicenter study provided a comprehensive insight into the clinical, laboratory, and genetic history of patients with CFDs. We conclude that bleeding severity grades were in agreement with the established factor activity threshold in nearly half of the cases with quantitative defects.

Diagnostic utility of bleeding assessment tools in congenital fibrinogen deficiencies.

BACKGROUND: The assessment of clinical history is crucial before referring a patient for further laboratory testing. Bleeding assessment tools (BAT) are developed to standardize clinical evaluation. A small number of patients with congenital fibrinogen deficiencies (CFDs) have been evaluated with these tools without definitive results. AIMS: We compared the adequacy of the ISTH-BAT and the European network of rare bleeding disorders bleeding score system (EN-RBD-BSS) to identify patients with CFDs. The correlation between the two BATs and fibrinogen levels and patient clinical grade severity was further analyzed. METHODS: We included 100 Iranian patients with CFDs. Routine coagulation and fibrinogen-specific tests (fibrinogen antigen [Fg:Ag] and activity [Fg:C]) were performed. The ISTH-BAT and EN-RBD-BSS were used to assess the bleeding score (BS) of all patients. RESULTS: The ISTH-BAT and EN-RBD-BSS median (range) were 4 (0-16) and 2.21 (-1.49 to 6.71), with a statistically significant moderate correlation between the two systems (r = .597, P < .001). In patients with quantitative deficiencies (afibrinogenemia and hypofibrinogenemia), the correlation between Fg:C and the ISTH-BAT was moderately negative (r = -.4, P < .001), while the correlation between Fg:C and the EN-RBD-BSS was weakly negative (r = -.38, P < .001). Overall, 70% and 72% of patients with fibrinogen deficiencies were correctly identified by both the ISTH-BAT and EN-RBD-BSS, respectively. CONCLUSION: These results suggest that in addition to the ISTH-BAT, the EN-RBD-BSS may also be useful in identifying CFD patients. We found a significant level of sensitivity for detecting fibrinogen deficiency in the two BATs, and bleeding severity classification correctly identified severity grades in almost two-thirds of patients.

Building the foundation for a community-generated national research blueprint for inherited bleeding disorders: research priorities for ultra-rare inherited bleeding disorders.

BACKGROUND: Ultra-rare inherited bleeding disorders (BDs) present important challenges for generating a strong evidence foundation for optimal diagnosis and management. Without disorder-appropriate treatment, affected individuals potentially face life-threatening bleeding, delayed diagnosis, suboptimal management of invasive procedures, psychosocial distress, pain, and decreased quality-of-life. RESEARCH DESIGN AND METHODS: The National Hemophilia Foundation (NHF) and the American Thrombosis and Hemostasis Network identified the priorities of people with inherited BDs and their caregivers, through extensive inclusive community consultations, to inform a blueprint for future decades of research. Multidisciplinary expert Working Group (WG) 3 distilled highly feasible transformative ultra-rare inherited BD research opportunities from the community-identified priorities. RESULTS: WG3 identified three focus areas with the potential to advance the needs of all people with ultra-rare inherited BDs and scored the feasibility, impact, and risk of priority initiatives, including 13 in systems biology and mechanistic science; 2 in clinical research, data collection, and research infrastructure; and 5 in the regulatory process for novel therapeutics and required data collection. CONCLUSIONS: Centralization and expansion of expertise and resources, flexible innovative research and regulatory approaches, and inclusion of all people with ultra-rare inherited BDs and their health care professionals will be essential to capitalize on the opportunities outlined herein.

Living with an ultra-rare inherited bleeding disorder is challenging. Patients can feel alone and unsure of where to find support because their disorder is so rare. In this paper, a group of ultra-rare bleeding disorder experts, including doctors, researchers, regulators, patient advocates, and patients, identify the research that could best improve the lives of people with these disorders. They propose a national network of specialists who can help doctors, who may never have seen these disorders before, to find the right diagnosis faster. A centralized laboratory specialized in ultra-rare bleeding disorders could also improve diagnosis and do research studies. This would help us learn, for example, how symptoms change throughout a patient's life, how effective different treatments are, and what it is like for patients to live with these disorders. A second research priority is to better understand each individual disorder so that the best treatments can be chosen or developed. A pathway showing doctors which treatment options to try, in which order, would help them help their patients. The third research priority is to make it easier to study new treatments for ultra-rare bleeding disorders. This requires designing studies with very small numbers of participants, identifying meaningful outcomes to measure, and convincing pharmaceutical companies to invest in these studies. International agreement on these requirements would allow more patients to participate and benefit from the research. These top-priority research goals should greatly improve knowledge about, and diagnosis and treatment of, ultra-rare inherited bleeding disorders.

Performance of a clinical risk prediction model for inhibitor formation in severe haemophilia A.

BACKGROUND: There is a need to identify patients with haemophilia who have a very low or high risk of developing inhibitors. These patients could be candidates for personalized treatment strategies. AIMS: The aim of this study was to externally validate a previously published prediction model for inhibitor development and to develop a new prediction model that incorporates novel predictors. METHODS: The population consisted of 251 previously untreated or minimally treated patients with severe haemophilia A enrolled in the SIPPET study. The outcome was inhibitor formation. Model discrimination was measured using the C-statistic, and model calibration was assessed with a calibration plot. The new model was internally validated using bootstrap resampling. RESULTS: Firstly, the previously published prediction model was validated. It consisted of three variables: family history of inhibitor development, F8 gene mutation and intensity of first treatment with factor VIII (FVIII). The C-statistic was 0.53 (95% CI: 0.46-0.60), and calibration was limited. Furthermore, a new prediction model was developed that consisted of four predictors: F8 gene mutation, intensity of first treatment with FVIII, the presence of factor VIII non-neutralizing antibodies before treatment initiation and lastly FVIII product type (recombinant vs. plasma-derived). The C-statistic was 0.66 (95 CI: 0.57-0.75), and calibration was moderate. Using a model cut-off point of 10%, positive- and negative predictive values were 0.22 and 0.95, respectively. CONCLUSION: Performance of all prediction models was limited. However, the new model with all predictors may be useful for identifying a small number of patients with a low risk of inhibitor formation.

IgG subclasses as biomarkers for persistence of factor VIII inhibitors in previously untreated patients with severe haemophilia A.

We investigated longitudinally the behaviour of anti-factor VIII (anti-FVIII) IgG subclasses for 6 months from inhibitor development in 43 patients from the Survey of Inhibitors in Plasma-Products Exposed Toddlers (SIPPET) trial who developed persistent or transient inhibitors. We first analysed 43 patients within 60 days post inhibitor detection. Then, 14 of these 43 patients were studied at five time points over 6 months. Our study showed that during the first 60 days, the risk of inhibitor persistence increased with the concomitant presence of an increasing number of IgG subclasses. Over the 6-month period post inhibitor detection, only the IgG2 subclass could be considered a hallmark of inhibitor persistence.

No changes of parameters nor coagulation activation in healthy subjects vaccinated for SARS-Cov-2.

Background: Recent reports of thrombotic events after SARS-Cov-2 vaccination raised concern. However, modifications of the most common coagulation parameters after vaccination are unknown. Aims: We measured parameters of coagulation including (i) basic coagulation tests, (ii) procedures aimed to assess the ex-vivo potential capacity to generate thrombin and (iii) in vivo thrombin activity. We also assessed anti-platelet factor 4 (aPF4) with two methods. Design: Laboratory measurements were performed for a cohort of subjects (n = 30) before (baseline) and after (7 and 21days after first dose, and 14days after second dose) SARS-Cov-2 vaccination. Results: All subjects received the Pfizer-BioNTech vaccine, and none developed symptomatic thrombotic events during the study period. None of the parameters showed clinically relevant variations at different time-points before and after vaccination. Only platelet count showed a slight increase, and F1.2 and the thrombin generation parameters ETP and ETP-TM ratio, showed a small decline, at the last time-point after vaccination when compared to baseline. aPF4 was negative in all the subjects, except two, who were positive (one with the chemiluminescent and the other with the ELISA assay). Conclusions: The study shows no modifications of the coagulation parameters nor the presence of biochemical signs of coagulation activation following the administration of the Pfizer-BioNTech vaccine.

An international registry of patients with plasminogen deficiency (HISTORY).

Plasminogen deficiency is an ultra-rare multisystem disorder characterized by the development of fibrin-rich pseudomembranes on mucous membranes. Ligneous conjunctivitis, which can result in vision impairment or loss, is the most frequent symptom reported. Affected systems may also include the respiratory tract, oropharynx, female reproductive tract, gingiva, middle ear, renal collecting system, skin and central nervous system. Untreated, plasminogen deficiency may result in significant reduction in quality of life and morbidity with potential life-threatening complications. Non-specific therapies are inadequate and plasminogen concentrates are not commercially available. The current understanding of plasminogen deficiency and management of disease symptoms and its progression are based on case reports/series and two small clinical trials. To date there has never been a comprehensive, international study to examine the natural history or optimal therapeutic intervention; knowledge gaps include identification of contributing factors and triggers of disease manifestations, inability to predict disease course, and insufficient real-world data for use of therapeutics. We have created an international, observational study (HISTORY) in a large cohort of persons with plasminogen deficiency and first-degree family members to address these gaps and to advance knowledge and care. HISTORY will build upon the established relationship between the Indiana Hemophilia and Thrombosis Center and the Fondazione Angelo Bianchi Bonomi, IRCCS Ca' Granda Ospedale Maggiore Policlinico - University of Milan and will utilize a modified version of the Prospective Rare Bleeding Disorders Database (PRO-RBDD). A biorepository containing samples from subjects with plasminogen deficiency will be established. This article describes the rationale behind the study and efforts towards its goals.

Clinical and laboratory diagnosis of rare coagulation disorders (RCDs).

Rare coagulation disorders (RCDs) are a group of diseases due to coagulation factors deficiency leading to life-long bleeding diathesis. The diagnosis of RCDs is challenging due to the limited knowledge of these disorders and the large heterogeneity of their bleeding patterns. The clinical symptoms of RCDs are extremely diverse in terms of bleeding type, site, severity, age at onset, and duration. The strength of the association between clotting factor activity level in plasma and clinical symptoms is also variable within each RCD. The clinical evaluation of RCDs starts with a detailed collection of clinical history and has been facilitated by bleeding assessment tools, however their effectiveness in diagnosing RCDs requires further investigation. The following laboratory diagnosis of RCDs involves coagulation screening tests, including activated partial thromboplastin time, prothrombin time, and thrombin time. After ruling out the presence of an inhibitor by mixing studies, in case of abnormal results, the specific deficiency is identified by performing one-stage clotting assays using the specific factor-depleted plasmas as substrate. In fibrinogen and FXIII deficiencies coagulation screening tests are not informative, therefore additional tests are needed. Global assays have been developed and are thought to aid in patient management, however, they are not well standardized yet. In addition to outlining the principles of clinical and laboratory diagnosis, this review explores molecular basis of RCDs and laboratory techniques for genetic analysis, and discusses the importance and effectiveness of quality control programs to ensure standardized laboratory results.

Clinical and Laboratory Features of Patients with Acquired Thrombotic Thrombocytopenic Purpura: Fourteen Years of the Milan TTP Registry.

Acquired thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic microangiopathy caused by the immune-mediated severe deficiency of ADAMTS13. We hereby report the demographic and disease-related data of acquired TTP patients recorded in the Milan TTP Registry (www.ttpdatabase.org). We performed a cross-sectional study of 302 individuals enrolled in our registry for an acute episode of acquired TTP occurred between 2002 and 2015 (female 77%; median age at onset 40 years, interquartile range: 30-50). Twenty per cent of patients had concomitant autoimmune disorders. Among potential triggers of acute episodes, infections were the most prevalent (27%), followed by estroprogestinics use and pregnancy (5 and 4% of women, respectively). At presentation, systemic (72%), bleeding (68%) and neurological (43%) symptoms were the most frequent, whereas a lower prevalence of renal (18%) and cardiovascular (10%) signs and symptoms was observed. Almost all acute events were treated by plasma exchange and steroids, and 15% by rituximab. Exacerbation of acute TTP occurred in 15% of events. The TTP-related mortality was 5%. In survivors, the median number of plasma exchange procedures to remission was 9 (interquartile range: 6-14), longer for first events than relapses (median difference 3, 95% confidence interval: 2-4). Of 251 survivors of the first TTP episode with at least a 6-month follow-up, 55% had a relapse. In conclusion, acquired TTP is a severe disease with highly variable clinical presentation, usually requiring a long hospitalization. The Milan TTP Registry represents a powerful tool to improve our knowledge and management of acquired TTP.

Risk factors for inhibitor development in severe hemophilia a.

Although significant advances in hemophilia treatment have improved patient outcomes and quality of life, one of the greatest complications in severe hemophilia A is the development of anti-Factor VIII (FVIII) antibodies that inhibit FVIII activity in almost 30% of previously untreated patients (PUPs). Inhibitors make very difficult the management of patients and increase their morbidity and mortality reducing drastically their quality of life. Numerous studies have investigated the mechanisms leading to the development of FVIII inhibitors. However, the etiology of their onset is complex and not yet fully understood. Inhibitors develop from a multicausal immune response involving both genetic (unmodifiable) and environmental (modifiable) factors. F8 gene mutations are the most important genetic risk factor, with null mutations being associated with the highest risk of inhibitor development. Immune response genes (e.g. the human leukocyte antigen complex) and proteins (e.g. cytokines) were studied without any strong confirmation of their role in modulating of inhibitor development. Type of FVIII product is the most important modifiable risk factor. The plasma-derived products containing von Willebrand factor were recently suggested to determine a lower incidence of inhibitor development than recombinant products in PUPs, in the first 50 exposure days (EDs). Other environmental factors including, age at first treatment, treatment intensity and the danger signal effect (surgery, severe bleeds, vaccinations and infections) has also been postulated as an explanation for environment-related inhibitor risk. This review reports the current knowledge on genetic and environmental risk factors on inhibitor development in patients with severe hemophilia A.

Genetic risk stratification to reduce inhibitor development in the early treatment of hemophilia A: a SIPPET analysis.

A recent randomized trial, the Survey of Inhibitors in Plasma-Product Exposed Toddlers (SIPPET), showed a higher risk of inhibitor development with recombinant factor VIII (rFVIII) than plasma-derived concentrates (pdFVIII). We investigated whether risk stratification by F8 mutation identifies patients who do not suffer this deleterious effect of rFVIII. Among 235 randomized patients with severe hemophilia A previously untreated with FVIII concentrate, 197 with null mutations were classified as high risk and 38 with non-null mutations were classified as low risk. With pdFVIII, no inhibitors occurred in those with low genetic risk, whereas high-risk patients had a cumulative incidence of 31%. The risk among low- and high-risk patients did not differ much when they were treated with rFVIII (43% and 47%, respectively). This implies that patients with low genetic risk suffer disproportionate harm when treated with rFVIII (risk increment 43%), as also shown by the number needed to harm with rFVIII, which was 6.3 for genetically high-risk patients and only 2.3 for low-risk patients. Risk stratification by F8 mutation does not identify patients who can be safely treated with rFVIII, as relates to immunogenicity. This trial was registered at the European Clinical Trials Database (EudraCT) as #2009-011186-88 and at www.clinicaltrials.gov as #NCT01064284.

Nonneutralizing antibodies against factor VIII and risk of inhibitor development in severe hemophilia A.

The development of anti-factor VIII (FVIII) neutralizing antibodies (inhibitors) is the major complication in hemophilia A. Nonneutralizing antibodies (NNAs) have been detected in hemophilia patients and also in unaffected individuals. The aim of this study was to assess the prevalence of NNAs and to evaluate whether their presence is associated with the development of inhibitors in a cohort of previously untreated or minimally treated patients with hemophilia A; plasma samples of 237 patients with severe hemophilia A enrolled in the SIPPET trial were collected before any exposure to FVIII concentrates and analyzed for the presence of anti-FVIII NNAs. Patients were observed for the development of neutralizing antibodies. NNAs were found in 18 (7.6%) of 237 patients at screening, and there was a clear age gradient. Of those with NNAs, 7 patients subsequently developed an inhibitor for a cumulative incidence of 45.4% (95% confidence interval [CI], 19.5% to 71.3%); among the 219 patients without NNAs, 64 (29%) developed an inhibitor (cumulative incidence, 34.0%; 95% CI, 27.1%-40.9%). In Cox regression analyses, patients with NNAs at screening had an 83% higher incidence of inhibitor development than patients without NNAs (hazard ratio [HR], 1.83; 95% CI, 0.84-3.99). For high-titer inhibitors, the incidence rate had an almost threefold increase (HR, 2.74; 95% CI, 1.23-6.12). These associations did not materially change after adjustment. The presence of anti-FVIII NNAs in patients with severe hemophilia A who were not previously exposed to FVIII concentrates is associated with an increased incidence of inhibitors.

Establishment of a bleeding score as a diagnostic tool for patients with rare bleeding disorders.

INTRODUCTION: Bleeding manifestations among patients with rare bleeding disorders (RBDs) vary significantly between disorders and patients, even when affected with the same disorder. In response to the challenge represented by the clinical assessment of the presence and severity of bleeding symptoms, a number of bleeding score systems (BSSs) or bleeding assessment tools (BATs) were developed. The majority of these were specifically developed for patients with more common bleeding disorders than RBDs. Few RBDs patients were evaluated with these tools and without conclusive results. METHODS: A new BSS was developed using data retrieved from a large group of patients with RBDs enrolled in the EN-RBD database and from healthy subjects. These data included previous bleeding symptoms, frequency, spontaneity, extent, localization, and relationship to prophylaxis and acute treatment. The predictive power of this BSS was also compared with the ISTH-BAT and examined for the severity of RBDs based on coagulant factor activity. RESULTS: This BSS was able to differentiate patients with RBDs from healthy individuals with a bleeding score value of 1.5 having the highest sum of sensitivity (67.1%) and specificity (73.8%) in discriminating patients with RBD from those without. An easy-to-use calculation was also developed to assess the probability of having a RBD. Its comparison with the ISTH-BAT confirmed its utility. Finally, in RBDs patients, there was a significant negative correlation between BS and coagulant factor activity level, which was strongest for fibrinogen and FXIII deficiencies. CONCLUSION: The use of this quantitative method may represent a valuable support tool to clinicians.

Rare bleeding disorders: diagnosis and treatment.

Despite the worldwide prevalence of rare bleeding disorders (RBDs), knowledge of these conditions and their management is suboptimal; health care professionals often have little diagnostic and treatment experience with variable access to diagnostic modalities required for accurate identification. Therefore, patients often experience morbidity and mortality due to delayed diagnosis. As RBDs represent a small potential commercial market, few, if any, specific therapies exist for these conditions. As a result, affected individuals commonly face delayed diagnosis, incomplete laboratory evaluation, and limited treatment options. Standardization and customization of coagulation assays, full genome sequencing, and global clotting assays will significantly improve diagnosis of patients with RBDs. In addition, new therapeutic modalities, both recombinant and plasma derived, are emerging, at least in developed countries. Registries and clinical trials have demonstrated decreased bleeding and improved outcomes when patients are appropriately diagnosed and properly treated. Expansion and harmonization of international registries has been initiated to correlate genotype, laboratory, and clinical phenotypes including bleeding severity to improve the diagnosis and therapeutic approach. This review focuses on the latest advances in our understanding, diagnosis, and treatment of RBDs.