Risk Factors For Inhibitor Development Research Articles

Correlations of Plasma Cytokine Levels and Anti-FVIII Antibodies during Immune Tolerance Induction

IntroductionThe development of neutralizing anti-FVIII antibodies (inhibitors) with reduced or absent activity of substituted factor VIII (FVIII) remains the most serious complication of hemophilia A therapy (Kempton, 2014). Frequent and high doses of FVIII with or without bypassing products can reestablish immune tolerance in 60-70% of patients. Polymorphism in immune response genes including IL-10 and TNFa were identified as risk factors for inhibitor development (Astermark, 2006). Cross-sectional studies showed different cytokine profiles in patients with hemophilia, especially in those with history of an inhibitor (Oliveira, 2013). In this study cytokine profiles were monitored longitudinally during immune tolerance induction (ITI).Methods107 plasma samples from 18 patients were collected during the RES.I.S.T Experienced and Naive trial, which included patients with a poor prognosis for ITI success (Gringeri, 2007). We quantified 14 cytokines in each sample by using a Human High Sensitivity T-Cell Discovery Array 14-Plex (Eve Technologies Corp, Calgary, AB, Canada). ELISA based FVIII antibody assays were used for anti-FVIII IgG detection. FVIII inhibitor titers (Bethesda assay, BU) were measured and available for the analysis. The cut-off for a positive inhibitor was >0,6 BU mL-1. Bethesda titers (BUpos) between 0,6 - <5,0 were considered as low titer (BUlow), whereas BU ≥5 as high titer (BUhigh). Statistical analyses were performed using GraphPad Prism 7. Mann-Whitney U tests and Spearman correlation tests considered significant when P value <0.05.ResultsPlasma levels of TNFa (P=0,014) and IL-8 (P=0,048) were positively correlated with FVIII inhibitor titers. Negative correlation was found in levels of IL-10 (P=0,041), IL-12 (P=0,038) and IL-1B (P=0,026). When cytokine levels of plasma samples with detectable and undetectable FVIII inhibitor titers were compared, significant higher plasma levels of TNFa (median: 11,56pg/ml, 8,11pg/ml; P=0,016) and lower levels of IL-12 (median: 4,29 pg/ml, 6,25 pg/ml; P=0,047) and IL-23 (median: 1016 pg/ml, 1353 pg/ml; P=0,049) were measured in samples with detectable FVIII inhibitor (BUpos). Furthermore, TNFa levels were higher in BUlow (median: 10,83 pg/ml; P=0,047) as well as in BUhigh samples (median: 11,75 pg/ml; P=0,019), compared to BUneg (median: 8,11 pg/ml). Cytokine concentrations of IL-1B (median: 2,64 pg/ml, 3,77 pg/ml; P=0,023), IL-2 (median: 2,44 pg/ml, 2,97pg/ml; P=0,043) and IL-17 (median: 15,79 pg/ml, 19,42 pg/ml; P=0,036) were significantly lower in BUhigh plasma samples compared to BUneg. Additionally, plasma level of IL-10 correlated negatively with levels of anti-FVIII IgG (P=0.045).ConclusionThis is the first study of cytokine measurement in a longitudinal setting as well as during ITI in patients with hemophilia. FVIII inhibitors and anti-FVIII IgG antibodies were correlated to IL-10 and TNFa levels - of note, polymorphisms in the genes of these cytokines are a known risk factor for inhibitor development. Furthermore, IL-12, IL-17 and IL-23 levels were higher in samples with loss of FVIII Inhibitors. In addition to prediction of inhibitor development, cytokine profiles might serve as prognostic factors for ITI success and considering the emerging evidence of the IL-17-IL-23 immune axis in autoimmunity might also be promising therapeutic approaches for higher ITI success rates. DisclosuresEwing:Genentech: Honoraria; Shire: Honoraria; Bayer: Honoraria; Grifols: Honoraria; CSL Behring: Honoraria; Novo Nordisk: Honoraria; Hema Biologics: Honoraria; Biogen: Research Funding. Koenigs:Jansen: Research Funding; Gilead: Research Funding; Biotest: Research Funding, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Intersero: Research Funding; CSL Behring: Consultancy, Research Funding; EU (IMI, FP7): Research Funding; Sobi: Consultancy, Research Funding, Speakers Bureau; Shire: Consultancy, Research Funding; Novo Nordisk: Consultancy, Speakers Bureau; Bioverativ: Consultancy; Roche/Chugai: Consultancy.

Incidence of Inhibitors in a Previously Untreated Patients with Severe Hemophilia Α Cohort Treated with a Single Third-Generation Recombinant Factor VIII Concentrate

Introduction: One of the most important complications in the treatment of patients with hemophilia A is the formation of neutralizing antibodies (inhibitors) interfering in the coagulant activity of factor VIII (FVIII). The presence of inhibitor causes a direct impact on mortality and morbidity in these patients and considerably increases the cost of treatment. Among the non-genetic risk factors for inhibitor development, the influence of the type of factor concentrate used in replacement therapy (recombinant or plasma-derived) remains controversial. Thus, the evaluation of an additional population in the real world setting may contribute to elucidate this problem. Since August 2013, almost all previously untreated patients (PUPs) with hemophilia A in Brazil have been receiving exclusively the same third-generation recombinant FVIII (rFVIII) (Advate®, Shire).Objective: The aim of this study is to evaluate the immunogenicity of rFVIII (Advate®). In this context, we analyzed the occurrence of inhibitor among severe and moderately severe hemophilia A PUPs during the first 50 exposure days (EDs) to Advate®.Methods: This is an open-label, multicenter, prospective/retrospective, uncontrolled, observational study conducted in eight reference hemophilia treatment centers from distinct geographic areas in Brazil. The inclusion criteria were (a) diagnosis of severe or moderately severe hemophilia A (FVIII:C <2 IU/dL), (b) absence of previous exposure to other FVIII concentrates, except a maximum of 5 previous exposures to any blood components (whole blood, fresh-frozen plasma, packed red cells, platelets, or cryoprecipitate), and (c) exclusive treatment with Advate® until the 50th ED or until inhibitor development (primary endpoint). Positive inhibitor was defined as at least two consecutive plasma samples with Bethesda-Nijmegen assay results ≥0.60 BU/mL. Patients were considered as having low-titer inhibitors when peak titers were <5 BU/mL, and high-titer inhibitors if inhibitor titer was ≥5 BU/mL on at least one occasion. Any clinical information considered relevant for the risk of inhibitor development was analyzed when available, and included family history of inhibitor, F8 genotype, ethnicity (defined according physical traits and ancestry ethnic background in the last three generations), age at first rFVIII exposure, treatment regimens (prophylaxis or episodic), doses, occurrence of a severe bleeding episode, surgery, and use of FVIII concentrate simultaneously to infection or vaccination.Results: So far, 122 patients were enrolled, and 100 patients reached the 50th ED to rFVIII or developed inhibitor. Twenty-two are still on Advate® and have not achieved 50EDs (7 patients: 20 to 50EDs; 15 patients: <20ED). Overall, the median age at first exposure to Advate® was 11.9 months (interquartile range (IQR): 7.5-16.7), and most patients were African-descendants (48%), followed by Caucasians (45%). Positive inhibitor was detected in 35 of the 100 patients (35%), and 71% occurred during the first 20EDs. Most inhibitors were detected during prophylactic treatment (29 of 35; 82.9%). Twenty-five (25%) patients had high-titer inhibitor. Although not statistically significant, 19/48 (39.6%) of the African-descendants patients developed inhibitor, in contrast to 15/45 (33%) of the Caucasians. Interestingly, inhibitor was detected in only 1/7 (14.6%) of the patients with the indigenous background (native population). The influence of other risk factors, as severe bleeding episodes, presence of infection, surgery and history of blood transfusion, were not statistically significant.Conclusions: Overall inhibitor development in this cohort is consistent with results reported in other PUP studies with recombinant products. The majority of inhibitors developed during the first 20EDs. However, no other risk factor as intensive treatment was statistically significant, due to the small number of events observed. Although observational studies have limitations to assess the immunogenicity of FVIII products, our study contributes to this knowledge, since it evaluates a single third-generation rFVIII in a distinct population, with similar access to factor concentrate and same treatment regimen. DisclosuresPrezotti:Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bioverative: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Medina:Shire: Speakers Bureau. Ozelo:Novo Nordisk: Honoraria, Research Funding, Speakers Bureau; BioMarin: Honoraria, Speakers Bureau; Shire: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Research Funding, Speakers Bureau; Bioverativ: Honoraria, Research Funding; Grifols: Honoraria.

HLA-DRB1–factor VIII binding is a risk factor for inhibitor development in nonsevere hemophilia: a case-control study

AbstractDevelopment of anti-factor VIII (FVIII) inhibitory antibodies (inhibitors) is the most significant treatment complication of hemophilia A. Characteristics of the interaction between major histocompatibility complex (MHC) class II and FVIII peptides may influence FVIII antigen presentation to T cells and subsequent inhibitor development. We analyzed predicted HLA-DRB1, a subset of MHC class II, and FVIII peptide binding and its association with inhibitor development among subjects with nonsevere hemophilia A, including 20 cases (inhibitor titer ≥ 1.0 BU/mL on 2 occasions or on 1 occasion with subsequent immune tolerance induction) and 37 controls (who had received FVIII infusions and did not develop inhibitor). Using the MHC-II Binding Predictions Tool (https://www.iedb.org), the binding affinity and core binding were determined for endogenous FVIII (eFVIII) and treatment FVIII (tFVIII). A tFVIII peptide was considered novel if it was predicted to bind and present a surface to the T-cell receptor that was unique from that presented by eFVIII. Having >10 novel HLA-DRB1 allele–tFVIII peptide combinations was associated with inhibitor development (adjusted odds ratio, 4.1; 95% confidence interval, 1.1-15.0). Cases and controls with p.Arg612Cys and p.Arg2169His demonstrated a high level of novel HLA-DRB1–tFVIII peptide combinations. Assessing the likelihood that tFVIII is presented to T cells in a novel fashion may be useful for understanding and ultimately reducing the risk for inhibitor development among patients with nonsevere hemophilia, particularly those with F8 mutations other than p.Arg612Cys and p.Arg2169His.

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.

Anti-factor VIII antibodies in brothers with haemophilia A share similar characteristics.

The development of neutralizing antibodies (inhibitors) against coagulation factor VIII (FVIII) is currently the most serious complication for patients with haemophilia A undergoing FVIII replacement therapy. Several genetic factors have been acknowledged as risk factors for inhibitor development. To analyze the influence of genetic factors on the nature of the humoral immune response to FVIII in eight brother pairs with inhibitors. The domain specificity of FVIII-specific IgG was analysed by antibody binding to FVIII fragments and homologue-scanning mutagenesis (HSM). The FVIII-specific IgG subclasses were measured by direct ELISA. Of the 16 patient analysed with both methods, 12 had A2- and 13 had C2-specific IgG. The presence of A1-, A3- or C1-specific IgG was identified in nine of 14 patients analysed by HSM. IgG1, IgG2 and IgG4 subclasses contributed to the anti-FVIII IgG response, and the amount of FVIII-specific IgG1 (r = 0.66) and IgG4 (r = 0.69) correlated significantly with inhibitor titres. Patients with high concentrations of total anti-FVIII IgG (r = 0.69) or high inhibitor titres (r = 0.52) had a high proportion of FVIII-specific IgG4. Statistical analysis revealed trends/evidence that the subclass distribution (P = 0.0847) and domain specificity to HC/LC (P = 0.0883) and A2/C2 (P = 0.0011) of anti-FVIII IgG were more similar in brothers compared to unrelated subjects. Overall, our data provide a first hint that anti-FVIII IgG characteristics are comparable among haemophilic brothers with inhibitors. Whether genetic factors also influence the nature of patients' antibodies needs to be confirmed in a larger study population.

Inhibitor development in previously untreated patients with severe haemophilia A: a nationwide multicentre study in Finland.

Currently the most serious treatment complication of haemophilia is the inhibitor development (ID), i.e. neutralizing antibody development. This nationwide multicentre study in Finland evaluated the incidence and risk factors of ID in previously untreated patients (PUPs) with severe haemophilia A (FVIII:C < 0.01 IU mL(-1) ). We enrolled all PUPs (N = 62) born between June 1994 and May 2013 with at least 75 exposure days (EDs) to screen ID during follow-up extending to September 2013. Thirteen ID (21% of 62) occurred; 10 (16% of 62) with high titre. Fifty-one patients (82%) were on primary prophylaxis (regular prophylaxis before the age of 2 and before the first joint bleed) from the median age of 11.4 months, 90% via a central venous access device. The initial product was rFVIII in 63% and pd-FVIII in 37%, moreover in 24% pd-FVIII was switched to rFVIII concentrate during the 75 EDs. Non-transient inhibitors developed in 9/51 (17.6%; 13.7% high titre) children with primary and in 4/11 (36.4%; 27.3% high titre) patients with secondary prophylaxis (P = 0.24). Overall, 74% had a high-risk genotype similarly distributed among the prophylaxis groups. The history of a major bleed enhanced ID (aHR, 4.0; 95% CI, 1.2-13.7), whereas FVIII treatment intensity or source and early implantation of ports did not increase ID risk. The cumulative incidence of ID was low notwithstanding prevalent high-risk mutations. Despite patient-related risk factors, our management involving early intensive primary prophylaxis via ports helps to prevent bleeds and lower the incidence of inhibitors.

Antibody profile in Indian severe haemophilia A patients with and without FVIII inhibitors

Diagnosis and management of haemophilia patients with inhibitors is often tricky due to the heterogeneous nature of the antibodies with regard to their kinetics, as well as the co-existence of other interfering antibodies. Plasma samples from severe haemophilia A patients from India with and without FVIII inhibitors were analysed for the presence of possible co-existing antibodies such as lupus anticoagulants (LA), anti-cardiolipin antibodies (ACLA), anti-β2-glycoprotein-I (anti-β2-GP-I) antibodies, viral transfusion transmitted disease (HIV, HBsAg, HCV) related antibodies, anti-cyclic citrullinated peptides (anti-CCP), and anti-nuclear antibodies. A high incidence of LA and anti-HCV antibodies was detected in Indian haemophilia A patients similar to earlier reports. More importantly, a relatively high incidence of autoantibodies to nuclear antigens (18.62%) and anti-CCP antibodies (1.38%) associated with autoimmune disorders was also seen in these congenital haemophilia A patients with and without inhibitors. Knowledge on the antibody profile in these haemophilia patients especially in those with FVIII inhibitors along with correlation with the clinical manifestations and other risk factors for inhibitor development could possibly shed more light on the complex immune response in these patients.

Type and intensity of FVIII exposure on inhibitor development in PUPs with haemophilia A. A patient-level meta-analysis.

The impact of treatment-related factors on inhibitor development in previously untreated patients (PUPs) with haemophilia A is still debated. We present the results of a collaborative, individual patient data meta-analytic project. Eligible data sources were published cohorts of PUPs for which patient-level data were available. The exposures of interest were factor (F)VIII type (recombinant [rFVIII] vs plasma-derived [pdFVIII]) and treatment intensity (≥ vs < 150 IU/kg/week) at first treatment. Family history of inhibitors, F8 mutations, age, treatment regimen (on-demand vs prophylaxis), secular trend and surgery were analysed as putative confounders using different statistical approaches (multivariable Cox regression, propensity score analyses, CART). Analyses accounted for the multi-centre origin of the data. We included 761 consecutive, unselected PUPs with moderate to severe haemophilia A from 10 centres in Egypt, Germany, Israel and Italy. A total of 27 % of patients developed inhibitors; 40 % and 22 % of patients treated with rFVIII and pdFVIII (unadjusted HR 2.2, 95 % CI 1.6-2.9), respectively; 51 % and 24 % of patients receiving high- and low-intensity treatment (unadjusted HR 2.9, 95 % CI 2.0-4.2), respectively. In adjusted analyses, only treatment intensity remained an independent predictor; the effect of FVIII type was largely due to confounding, but with a significant interaction between FVIII type and treatment intensity. This patient-level meta-analysis confirms, across different statistical approaches, that high-intensity treatment is a strong risk factor for inhibitor development. The possible role of FVIII type in subgroups is suggested by the test for interactions but could not be proven because of the limited subgroups sample sizes.

Inhibitors incidence rate in Czech previously untreated patients with haemophilia A has not increased since introduction of recombinant factor VIII treatment in 2003.

Our objective was to assess the incidence of inhibitors development in Czech Republic since the introduction of recombinant factor VIII (rFVIII) and to look for the factors potentially influencing this parameter. It is to be expected that inhibitors risk may be increased after the introduction of recombinant products. Data of Czech National Haemophilia Programme registry entered from 2003 till 2013 were analysed. Both annual and absolute incidences of newly developed inhibitors in previously untreated patients (PUPs) were calculated. Bleeding and treatment data were also extracted, and association to the treatment regimen and development of inhibitors were analysed. Within the given period, we commenced 45 PUPs with haemophilia A on rFVIII and treated them for 137 treatment-years. Twenty-two of the PUPs had severe haemophilia A, being treated for 88 treatment-years. Treatment strategy for them was prophylaxis. Other PUPs were treated on demand. Median annual bleeding rate was 5 for boys with severe haemophilia, 3 for moderate haemophilia and 1 for mild form of the disease. No inhibitors developed in PUPs with moderate/mild haemophilia A. Annual inhibitor incidence rate in PUPs with severe haemophilia A treated with rFVIII was 56.8 per 1000 treatment-years. Absolute incidence was 22.7% (5/22). All inhibitors appeared within the first 50 exposure days. Comparing rFVIII-treated group with the control group treated under same/similar conditions with plasma-derived FVIII during the same follow-up period, we were not able to find significant difference in inhibitor development between these two groups. Our results support the finding that use of rFVIII is not a proven risk factor for inhibitor development in patients with haemophilia A.

Immune-related gene polymorphisms as risk factors for the development of Factor VIII inhibitors in hemophilia A patients: a sibling-pair association analysis (HUM1P.257)

Abstract Inhibitors are one of the most feared complications in hemophilia, occurring in 15-30% of individuals with severe forms of Hemophilia A Human Leukocyte Antigen (HLA) haplotype, and polymorphism of genes regulating the immune response have been indicated as risk factors for inhibitor development. However, it is still unclear the extent to which each of these genetic variants contribute to inhibitor formation as a function of the F8 gene mutation. Here, 96 patients clustered in 45 sibling-pairs and 2 sibling-triplets of which 15 are discordant and the remaining are concordant for inhibitors were considered in a case-control analysis. Next Generation Sequencing on patients’ genomic DNA was performed for detection of FVIII mutation, genotyping of HLA-DRB1 allele and for TNF-α, IL-10, CTLA4, DOCK2 and MAPK9. Our results demonstrate a higher frequency of DRB1*15 allele in inhibitor patients [odds ratio (OR) 2.57; P = 0.022]. In TNF-α, the A allele of the -308G&amp;gt;A polymorphism was found with higher frequency in the inhibitor cohort. This finding was ore evident for the homozygous A/A genotype (7.7% vs 1.8% in inhibitor patient group vs. non-inhibitor respectively, p=0.028). For IL-10, the -1082G allele was observed more frequently in patients with inhibitors (59% vs. 37.5%; P = 0.0037). Our data validate the hypothesis that specific genomic haplotypes affect the development of FVIII inhibitors and can support inhibitor prediction for prognostic and therapeutic applications.

Distribution of F8 Gene Mutations in 81 Inhibitor-Free, Severe Hemophilia a Patients with Full Exposure to FVIII

Introduction: Factor (F) VIII genotype has been shown to be a risk factor for inhibitor development, where different mutation types are associated with different degrees of risk (Oldenburg J, et al.Haemophilia 2006;12 Suppl 6:15–22). However, it is not clear why some patients with a specific mutation develop inhibitors while others with the same mutation do not, including those with high-risk defects such as null mutations. Patients with severe hemophilia A (HA) with low-risk mutations are thought to produce some functional or non-functional FVIII that is sufficient to induce immune tolerance (Oldenburg J, et al.Haemophilia 2002;8 Suppl 2:23–29). In the present study, the distribution of F8 gene mutations in 81 patients fully exposed to FVIII with severe HA and no inhibitors is compared to data on mutation distribution among severe HA patients with/without inhibitors.Materials & Methods: Turoctocog alfa (NovoEight®) is a new recombinant FVIII product with a truncated B-domain. Previously treated patients (PTPs) with severe HA participated in the pivotal trials guardian™1 (n=150; 12–65 yrs) or guardian™3 (n=63; <12 yrs), in which they received turoctocog alfa as prophylaxis and to treat bleeds. The majority continued in an ongoing extension trial, guardian™2. All patients ≥12 yrs had a history of ≥150 exposure days (EDs) to FVIII products and patients <12 yrs had ≥50 EDs. No patients had inhibitors prior to or after turoctocog alfa treatment. Distribution of mutation types has previously been reported in populations with HA that consisted of or included patients with inhibitors (Salviato R, et al. Haemophilia 2007;13:361–372; Gouw SC, et al.Blood 2012;119:2922–2934). Here we report mutation distribution in a sample of guardian™ patients with severe HA who had long been previously treated with FVIII protein and remained inhibitor free, and compared this distribution with that from 2 published studies (Oldenburg J, et al. 2006; Salviato R, et al. 2007).Results: Eighty-one patients enrolled in the guardian™ trials had F8 gene mutation analyzed. For 52 patients <12 yrs old, genotype was analyzed during guardian™3. For 29 patients ≥12 yrs old, genotype analysis was done prior to enrollment and obtained from medical records. In 77/81 patients, the F8 gene defect was identifiable. Distribution of gene mutations in guardian™, in a population with severe HA with inhibitors, and in a population with severe HA with/without inhibitors are compared in table 1. It is interesting that the overall distribution of mutations among the populations appears quite similar, pointing toward the multifactorial nature of inhibitor development.Null mutations (inversions, nonsense mutations, and large deletions) were present in 66% and 75% of patients with severe HA and severe HA with inhibitors, respectively (table 1). In guardian™, 57% (46/81) of patients had null mutations despite never having developed an inhibitor. The difference in distribution of null mutations in the 3 populations is mainly accounted for by large deletions and nonsense mutations, while distribution of inversions was similar. It should be noted that in guardian™, 10/11 patients with missense mutations had these located outside the C1/C2 domain, which reportedly has a lower frequency of inhibitors compared with missense mutations at the C1/C2 domain (Oldenburg J, et al. 2006).Conclusions: Eighty-one patients in the guardian™ trial with severe hemophilia A had their genotype analyzed and all of them were inhibitor-free over their lifetime. Interestingly, the prevalence of high-risk mutation types does not seem to differ markedly from previously published data in patients with inhibitors, probably indicating the relevance of other factors in inhibitor development.Table 1Distribution of F8 gene mutation typesguardian™Additional studiesguardian™ patients (N)Distribution (%) in guardian™ patients (severe HA without inhibitors)Distribution (%) in patients with severe HA and inhibitors (Salviato R, et al. 2007)*Distribution (%) in patients with severe HA with/without inhibitors (Oldenburg J, et al. 2006)Total, N818176753Intron 22 inversion3340.744.745.0Intron 1 inversion33.71.32.5Nonsense89.918.413.5Large deletion22.510.55.0Missense1113.65.214.5Small del/ins/dupl1619.810.516.0Splice site44.95.23.5Unidentified/ not enough information44.93.90.0Total81100.0100.0100.0*Only patients with severe HA included. DisclosuresMatytsina:Novo Nordisk A/S: Employment. Arai:Novo Nordisk Pharma, Ltd: Employment. Oldenburg:Baxter, Bayer, Biogen Idec, Biotest, CSL-Behring, Grifols, Novo Nordisk, Octapharma, Swedish Orphan Biovitrum and Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Factor VIII Gene Variants and Inhibitor Risk in African American Hemophilia a Patients

African American hemophilia A (HA) patients experience a higher incidence of neutralizing antibodies (“inhibitors”) against factor VIII (FVIII) vis-à-vis Caucasian patients. Non-HA-causing non-synonymous single-nucleotide polymorphisms (ns-SNPs) in the F8 gene encoding FVIII-484H, FVIII-1241E and FVIII-2238V are more prevalent in African Americans than in Caucasians. Currently available recombinant FVIII products have FVIII-R484, FVIII-D1241 or E1241, and FVIII-M2238. Therefore a number of HA patients are infused with FVIII products (plasma-derived or recombinant) having amino acid sequences that differ from the sequence encoded by their hemophilic F8 gene. This study tested the hypothesis that immune responses to sequences encoded by these ns-SNPs are a significant risk factor for inhibitor development in the African American HA community.As part of the Personalized Approaches to Therapies for Hemophilia (PATH) Study, blood samples were obtained from 175 African American and 198 Caucasian severe HA subjects, including several subjects with an intron-22 inversion mutation but moderate bleeding disorder. Questionnaires indicated which subjects had an inhibitor history, defined as ≥1 inhibitor titer measurements of >0.6 Bethesda units/ml. The F8 gene sequences at the three ns-SNPs and HLA-DRB1 genotypes were determined and PBMCs isolated from blood samples.To determine which HLA-DRB1 proteins are capable of presenting the sequences encoded by these F8 ns-SNPs, synthetic 20-mer peptides with these polymorphic sequences were synthesized and quantitative peptide-HLA-DRB1 binding assays carried out. Binding to the HLA-DRB1 peptide-binding groove is a prerequisite for presentation of potentially immunogenic sequences on antigen-presenting cells. These assays identified several HLA-DRB1 that bound with moderate to high affinity to each of the polymorphic sequences, however none of the peptides showed promiscuous HLA-DRB1 binding. Most of the 11 HLA-DRB1 proteins tested, which together comprise ~40% of HLA-DRB1 alleles in the African American population, did not bind to the polymorphic FVIII sequences.CD4 T cells from 1 (the only), 7 and 20 HA subjects who had been infused with FVIII products that were mismatched with sequences encoded by their endogenous F8 gene at FVIII-484, -1241 or -2238, respectively, and whose HLA-DRB1 proteins bound these sequences, were cultured with FVIII peptides containing both versions of these polymorphic sequences, in an attempt to identify HLA-restricted T-cell responses to “sequence-mismatched” FVIII. Positive control cultures consisted of parallel cultures of CD4 T cells with peptides corresponding to validated HLA-restricted epitopes in tetanus toxoid (TT) protein. Flow cytometry experiments then quantified the number of CD4 T cells that bound fluorescent HLA-DRB1 tetramers loaded with FVIII or TT peptides. Tetramer-positive cells were then sorted and cultured in attempts to isolate T-cell clones and polyclonal lines recognizing these sequences, as these would constitute unambiguous confirmation of T-cell responses to the peptide sequences. TT-specific clones were obtained routinely, as expected. The few cells positive for FVIII peptide-loaded tetramers did not produce T-cell clones or polyclonal T-cell lines, indicating the marginal tetramer-positive staining had been nonspecific.Among the 373 HA subjects, inhibitor risks associated with the most common F8 ns-SNPs in African Americans, FVIII-E1241 and FVIII-V2238, were: E1241/D1241, OR=1.3(0.8-2.0, p=0.3); V2238/M2238, OR=1.1(0.5-2.4, p=0.84); 95%CI). Interestingly, African Americans with a F8 intron-22 inversion mutation showed a significantly higher inhibitor incidence compared to Caucasians with the same mutation, OR=2.9(2.4 – 5.8, p=0.004), but this did not correlate with any of the 3 ns-SNPs.We conclude that immune responses to FVIII products mismatched with the FVIII sequences encoded by these three ns-SNPs are unlikely to contribute appreciably to the overall inhibitor incidence in African Americans with severe HA, but we emphasize that the at-risk population should be better defined through additional immunological investigations. The higher inhibitor incidence in African Americans with an intron-22 inversion mutation clearly warrants further investigation.On behalf of the PATH Study Investigators (NIH 1RC2 HL101851) DisclosuresFletcher:Biogen Idec: Research Funding. Pratt:Pfizer ASPIRE hemophilia research award: Research Funding; Grifols, Inc: Research Funding.

A public health approach to the prevention of inhibitors in hemophilia.

The development of an antibody in people with hemophilia to products used in the treatment and prevention of bleeding, also referred to as an inhibitor, is the most serious complication of hemophilia care today. CDC, together with healthcare providers, consumer organizations, hemophilia organizations, and federal partners, has developed a public health agenda to prevent the development of inhibitors. This paper describes a public health approach that combines a national surveillance program with epidemiologic, laboratory, and prevention research to address knowledge gaps in rates and risk factors for inhibitor development, and in knowledge and behaviors of patients and providers, in addition to screening and treatment practices.

Incidence and risk factors for inhibitor development in previously untreated severe haemophilia A patients born between 2005 and 2010.

The objective of this study was to evaluate the inhibitor development (ID) in previously untreated patients (PUPs) with severe haemophilia A (FVIII≤0.01IUmL(-1) ). All Canadian Haemophilia Treatment Centres completed a questionnaire on patients born between September 2005 and August 2010 and followed for up to 7years. Eligible patients had at least 20 exposure days (ED) or had developed an inhibitor. The odds ratio (OR) and 95% confidence intervals (95% CI) for risk factors to develop an inhibitor were estimated using unconditional logistic regression. A total of 99 haemophilia A PUPs were studied. Thirty-four (34%) developed an inhibitor (24/34 of high titre). Inhibitors developed in 25/63 (40%) patients with a high-risk mutation. ID was most frequent in Aboriginals (86%). Dose intensity (IUkg(-1) day(-1) X number of ED) at first exposure to factor VIII (FVIII) was associated with a crude OR increase of 1.10 (95% CI: 0.99-1.23) with each increase of 100 dose-intensity units. Haemarthrosis and intracranial bleeding as the indication for first exposure to FVIII concentrate were associated with a crude OR for ID of 7.63 (95% CI: 2.14-27.17) and 5.08 (95% CI: 1.11-23.31) respectively. ID according to FVIII concentrate used was: Advate (®) 18/50 (36%), Kogenate FS(®) or Helixate FS(®) 15/36 (42%), Wilate(®) 0/11 and Xyntha(®) 1/2. In multivariate analysis, Aboriginal ethnicity (OR=11.69; 95% CI: 1.11-122.86) and haemarthrosis (OR=4.49; 95% CI: 1.08-18.61) were statistically significant. The cumulative incidence of ID in severe haemophilia A PUPs was 34% and varied according to ethnicity, type of bleeding at first ED, type of FVIII product and dose intensity at first exposure.

A cross-sectional survey of coagulation factor VIII inhibitor in children with hemophilia A

To study the current situation of coagulation factor VIII (FVIII) inhibitor development in children with hemophilia A (HA) through a cross-sectional survey, and to explore the risk factors of inhibitor development in order to provide evidence for further prevention and management strategies. The clinical data of outpatients with hemophilia A in Beijing Children's Hospital seen from November 2012 to May 2013 were collected, FVIII inhibitor was screened and analyzed its risk factors. A total of 102 HA children were enrolled, 5 were mild cases, 32 were moderate, and 65 were severe cases; the median age on enrollment was 55.5 (3.0-200.0) months:19(18.6%) of patients had inhibitors and 9 (8.8%) had low-titer inhibitors, 10 (9.8%) had high-titer inhibitors. Receiving FVIII treatment for life-threatening bleeding (P = 0.03) ,OR 4.10 (95%CI:1.17-14.32) was a risk factor for inhibitor generation and patients within 20 exposure days have more chances of inhibitor development (P = 0.04) ,OR 3.32 (95%CI:1.02-10.86) . High and intense FVIII exposure within short term was the risk factor for high titer inhibitor development (P = 0.01) ,OR 5.25 (95%CI:1.45-21.92) . Intense FVIII exposure for severe hemorrhage was the risk factor of inhibitors development especially of high titer inhibitors.

Predictive immunogenicity of Refacto®AF

The administration of therapeutic factor VIII (FVIII) to treat or prevent haemorrhages in haemophilia A patients results, in up to 30% of the cases, in the development of inhibitory anti-FVIII antibodies. Much debate has taken place on the relevance of the nature of the FVIII product as a risk factor for inhibitor development. Thus, the plasma-derived vs. recombinant origin, the second vs. third generation of the product, or the presence of the B domain have been controversially evoked. A few years ago, Refacto AF, a third-generation recombinant B domain-deleted FVIII was marketed. The aim of this study was to compare the immunogenicity of Refacto AF to that of two recombinant full-length FVIII products: Helixate and Advate. For the three recombinant FVIII products, we compared the binding to the mannose-sensitive endocytic receptor CD206, the dose-dependent endocytosis by immature monocyte-derived dendritic cells (DCs), the activation by FVIII-loaded DCs of a FVIII-specific HLA-DRB1*0101-restricted mouse T-cell hybridoma and the induction of inhibitory anti-FVIII IgG in FVIII-deficient mice. At elevated FVIII concentrations, Refacto AF was less endocytosed than full-length recombinant products. At lower concentrations, however, Refacto AF was endocytosed by DCs and activated T cells as well as Helixate and Advate. The levels of inhibitory anti-FVIII IgG induced by Refacto AF in FVIII-deficient mice were lower or equal to that induced by Helixate and Advate respectively. The predicted immunogenicity of Refacto AF is identical to or lower than that of the two recombinant full-length FVIII products available on the French market.

Key issues in inhibitor management in patients with haemophilia.

Significant advances in the management of haemophilia have been achieved in the past several decades. These include the development of safe and efficacious plasma-derived and recombinant clotting factor products, use of prophylaxis as standard of care in bleeding prevention and appropriate surgical management of haemophilic arthropathy. Despite these advances, the development of high-titre anti-factor antibodies (inhibitors) remains an unresolved challenge in the management of people with haemophilia. Inhibitors develop in 25–30% of patients with severe haemophilia A and in 1–5% of those with severe haemophilia B1–3. They were first described by Lawrence and Johnson in 19414, and in the seven decades that followed our knowledge of their pathophysiology and risk factors remains incomplete and continues to evolve. This is partly a result of the small inhibitor population with significant intra- and inter-individual variability making the conduct of studies and interpretation of results difficult. The main reason for our incomplete knowledge in this area is that inhibitor development is a multi-factorial event resulting from the variable interplay between several genetic (non-modifiable) and non-genetic (modifiable to some extent) risk factors. Despite these limitations, a number of clinical evaluations analysing treatment of inhibitor patients have been performed including randomised controlled prospective studies5,6. These studies provide, in part, the evidence basis for our current inhibitor management principles and practice. Inhibitors remain a popular subject of haemostasis scientific meetings and many issues related to their pathophysiology and management are discussed and debated in the published literature. The main issues include: (i) the identification of clinically relevant risk factors for inhibitor development, (ii) the definition of the bleeding phenotype and clinical management of these patients, (iii) the usefulness and feasibility of prophylaxis with bypassing agents in the treatment of patients with inhibitors, (iv) the identification of predictive factors for inhibitor eradication, and (v) novel therapeutic approaches and molecules for the treatment and/or eradication of inhibitors. The aim of this review is to give an update on each of these issues with a focus on the current state-of-the-art knowledge and practice from the authors’ personal perspective. This review focuses mainly on factor VIII inhibitors as these occur much more frequently in clinical practice.

Identifying Nongenetic Risk Factors for Inhibitor Development in Severe Hemophilia A

The development of inhibitory antibodies against infused factor VIII (FVIII) is a major challenge in hemophilia treatment. As the antibodies swiftly inactivate administered therapeutic FVIII concentrates, FVIII is no longer effective in controlling bleeding. To achieve adequate hemostasis, bypassing therapies are required, with accompanying clinical challenges and financial expense. Extensive research has aimed to elucidate the multifactorial etiology of inhibitor development. Both genetic and nongenetic causes have been identified. Identification of treatment-related risk factors would offer the possibility to modify treatment strategies in high-risk patients, thereby reducing the risk of inhibitor development. Have we presently gained sufficient knowledge to make a prediction of the inhibitor risk possible and justify changes in treatment regimens for specific patient groups? This review summarizes and evaluates the research findings on nongenetic risk factors of inhibitor development, with special focus on treatment-related factors.

Inhibitor development in previously treated hemophilia A patients: a systematic review, meta‐analysis, and meta‐regression

The development of neutralizing alloantibodies (inhibitors) is the most serious complication of factor VIII (FVIII) replacement therapy in patients with hemophilia A. Unlike previously untreated patients, no definite risk factors for inhibitor development are known for previously treated patients (PTPs). The investigation of the development of inhibitors in PTPs is hindered by several methodological limitations in the available literature. We conducted a systematic review to account for these limitations. We considered the studies reporting on PTPs that were included in the Wight and Paisley meta-analysis and a systematic search of MEDLINE, EMBASE, and The Cochrane Library was conducted to identify studies published after 2003. Studies that investigated the development of inhibitors in hemophilia A PTPs who were treated with any type of FVIII concentrate and that included at least 25 patients with follow-up were included in the analysis. Thirty-three independent cohorts of PTPs with 4323 subjects and 43 incident de novo inhibitors were found and analyzed. The pooled incidence rate of inhibitor development for the 25 studies providing data on follow-up was 3 (95% confidence interval 1-4) per 1000 person-years. A significant association was not found between putative risk factors and inhibitor development in PTPs at meta-regression analysis and subgroup analysis, but the model was sensitive enough to the inclusion of the reports on the Belgian-Dutch experience with a highly immunogenic factor VIII. We confirmed a low overall rate of de novo inhibitors in PTPs, without any significant effect of putative predictors, including the type of factor VIII concentrate.

Cancer detection and management in patients with haemophilia: a retrospective European multicentre study

Lymphomas or hepatocarcinomas related to blood-borne transmitted diseases are well-known malignancies in persons with haemophilia (PWH). However, rising life expectancy has increased the number of PWH suffering from other malignancies. This study aimed to collect cancer occurrence data in PWH followed in five European haemophilia treatment centres (Brussels, Geneva, Marseille, Montpellier and Paris-Bicêtre) over the last 10 years and to analyse some particular features of cancer occurring in PWH. In total, 45 malignancies were diagnosed in 1067 PWH. The most common malignancies were hepatocellular carcinoma (12/45) and urogenital tract tumours (9/45). Bleeding at presentation or changes in bleeding pattern was indicative of cancer in four patients. Three patients with mild haemophilia developed anti-factor VIII inhibitors after intensive substitution therapy prior to surgery or invasive procedures. There was no bleeding associated with chemotherapy or radiotherapy. A few bleeding complications occurred following invasive (3/39) or surgical procedures (2/27) as a result of insufficient hemostatic coverage or in spite of adequate substitution. No bleeding was noted after liver or prostate biopsies. Following cancer diagnosis, five patients were switched from on-demand to prolonged prophylaxis substitution. In the majority of cases, the standard cancer treatment protocol was not modified on account of concomitant haemophilia. Thus, oncological treatments are not contraindicated and should not be withheld in PWH assuming that adequate haemostasis correction is undertaken. As shown by our study results, a change in bleeding pattern in adult PWH should raise suspicion of a malignancy. Intensive substitution must be considered a risk factor for inhibitor development.