Factor VIII Products and Inhibitor Development in Severe Hemophilia A

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1 original article Factor VIII Products and Inhibitor Development in Severe Hemophilia A Samantha C. Gouw, M.D., Ph.D., Johanna G. van der Bom, M.D., Ph.D., Rolf Ljung, M.D., Ph.D., Carmen Escuriola, M.D., Ana R. Cid, M.D., Ségolène Claeyssens-Donadel, M.D., Christel van Geet, M.D., Ph.D., Gili Kenet, M.D., Anne Mäkipernaa, M.D., Ph.D., Angelo Claudio Molinari, M.D., Wolfgang Muntean, M.D., Rainer Kobelt, M.D., George Rivard, M.D., Elena Santagostino, M.D., Ph.D., Angela Thomas, M.D., Ph.D., and H. Marijke van den Berg, M.D., Ph.D., for the PedNet and RODIN Study Group* A bs tr ac t Background For previously untreated children with severe hemophilia A, it is unclear whether the type of factor VIII product administered and switching among products are associated with the development of clinically relevant inhibitory antibodies (inhibitor development). Methods We evaluated 574 consecutive patients with severe hemophilia A (factor VIII activity, <0.01 IU per milliliter) who were born between 2000 and 2010 and collected data on all clotting-factor administration for up to 75 exposure days. The primary outcome was inhibitor development, which was defined as at least two positive inhibitor tests with decreased in vivo recovery of factor VIII levels. Results Inhibitory antibodies developed in 177 of the 574 children (cumulative incidence, 32.4%); 116 patients had a high-titer inhibitory antibody, defined as a peak titer of at least 5 Bethesda units per milliliter (cumulative incidence, 22.4%). Plasma-derived products conferred a risk of inhibitor development that was similar to the risk with recombinant products (adjusted hazard ratio as compared with recombinant products, 0.96; 95% confidence interval [CI], 0.62 to 1.49). As compared with thirdgeneration full-length recombinant products (derived from the full-length complementary DNA sequence of human factor VIII), second-generation full-length products were associated with an increased risk of inhibitor development (adjusted hazard ratio, 1.60; 95% CI, 1.08 to 2.37). The content of von Willebrand factor in the products and switching among products were not associated with the risk of inhibitor development. Conclusions Recombinant and plasma-derived factor VIII products conferred similar risks of inhibitor development, and the content of von Willebrand factor in the products and switching among products were not associated with the risk of inhibitor development. Second-generation full-length recombinant products were associated with an increased risk, as compared with third-generation products. (Funded by Bayer Healthcare and Baxter BioScience.) The authors affiliations are listed in the Appendix. Address reprint requests to Dr. van den Berg at the University Medical Center Utrecht, Julius Center for Health Sciences and Primary Care, Rm. No. Stratenum 5.125, P.O. Box 85500, 3508 GA Utrecht, the Netherlands, or at h.m.vandeberg@umcutrecht.nl. * Investigators in the European Pediatric Network for Hemophilia Management (PedNet) and the Research of Determinants of Inhibitor Development (RODIN) study group are listed in the Supplementary Appendix, available at NEJM.org. N Engl J Med 2013;368: DOI: /NEJMoa Copyright 2013 Massachusetts Medical Society. n engl j med 368;3 nejm.org january 17,

2 Patients with severe hemophilia A have a deficiency of functional clotting factor VIII (<0.01 IU per milliliter) and have bleeding in the joints and muscles. To prevent joint destruction, the current standard of care for children with severe hemophilia A is primary prophylaxis. This includes regular infusions of factor VIII, which are initiated at the time of the first episode of bleeding in a joint or earlier, aiming at the prevention of joint damage. 1 However, in about 30% of children, inhibitory antibodies to infused factor VIII products develop, making usual treatment with factor VIII and prophylaxis impossible. There are multiple risk factors for the development of inhibitory antibodies (inhibitor development) It has been suggested that recombinant factor VIII products are more immunogenic than plasmaderived products. However, the outcomes of numerous studies and systematic reviews have been contradictory The studies have been limited by the enrollment of small, heterogeneous study populations and the use of several factor VIII products, and comparisons among studies have been difficult because of different study designs. 22,24 The inclusion of minimally treated patients and patients who were still at risk for subsequent development of inhibitory antibodies has led to an underestimation of the incidence of inhibitor development. 25 In addition, prospective postmarketing studies could not include high-risk children who started bleeding at an early age, which meant that the risk of inhibitor development was underestimated. Furthermore, small studies with extreme results are more likely to be published than are those with less extreme findings. 22 For these reasons, three systematic reviews of the immunogenicity of factor VIII products resulted in different conclusions Randomized trials comparing the immunogenicity of factor VIII products have not yet been completed. 26 A finding that recombinant and plasmaderived products had a differential risk with respect to inhibitor development would influence both the decision about which type of product to administer in individual patients and the availability of the preferred product. Therefore, knowledge of the risk of inhibitor development associated with recombinant and plasma-derived products is important for both the individual patient with hemophilia and the hemophilia population as a whole. We assessed whether the type of factor VIII product and switching among products were associated with inhibitor development in previously untreated children with severe hemophilia A. Me thods Patients We enrolled consecutive, previously untreated patients with severe hemophilia A (factor VIII activity, <0.01 IU per milliliter) that had been diagnosed in 1 of the 29 participating hemophilia treatment centers. All the children in the study were born between January 1, 2000, and January 1, Children who had been referred to the centers because of the presence of inhibitory antibodies were excluded from the study. Approval was obtained from the institutional review board at each study center. Parents or guardians of all children provided written informed consent. Data Collection We uniformly collected detailed data on all infusions of factor VIII for up to 75 exposure days or until the development of inhibitory antibodies, including dates of infusion, doses and brands of factor VIII products, reasons for treatment, types of bleeding, extravasation of products, and surgery. Patients were followed until the development of a clinically relevant inhibitory antibody or a cumulative number of 75 exposure days. (After 75 exposure days, inhibitor development becomes rare [approximately 2 to 5 cases per 1000 patientyears]). 27 Outcomes The primary outcome was the development of clinically relevant inhibitory antibodies, which was defined as at least two positive inhibitor titers combined with decreased in vivo recovery of factor VIII levels up to the 75th exposure day. The secondary outcome was the development of a high-titer inhibitor, which was defined as a peak titer of at least 5 Bethesda units per milliliter up to the 75th exposure day. 28 A positive inhibitor titer was defined according to the cutoff level of the inhibitor assay used in the laboratory at each center. Factor VIII recovery was described as decreased if the level of factor VIII activity was less than 66% of the expected level 15 minutes after the infusion of factor VIII. The expected level of factor VIII activity was calculated according to the criteria of Lee et al n engl j med 368;3 nejm.org january 17, 2013

3 Development of Factor VIII Inhibitors in Hemophilia A In the majority of centers (92%), patients were routinely screened for inhibitor development after every 1 to 5 exposure days during the first 20 exposure days and at least every 3 months thereafter. At all centers, patients were closely monitored for signs of inhibitor development, and investigators performed inhibitor and recovery testing if there was any suspicion that inhibitory antibodies had developed. Types of Factor VIII Products We assessed the incidence of inhibitor development according to the type of product used at subsequent exposure days (time-varying determinant). We categorized factor VIII products in several ways. First, we compared the inhibitor risk between plasma-derived factor VIII products and recombinant products. Second, to investigate whether the content of von Willebrand factor was associated with the risk of inhibitor development, we categorized factor VIII products into products containing no von Willebrand factor (all recombinant products), products containing less than 0.01 IU of von Willebrand factor antigen per international unit of factor VIII antigen (monoclonal antibody purified plasma-derived products), and products containing 0.01 IU or more of von Willebrand factor per international unit of factor VIII antigen (other plasma-derived products). 30 Third, we compared inhibitor incidence among the following categories of factor VIII products: plasmaderived products, first-generation full-length recombinant product (derived from the full-length complementary DNA sequence of human factor VIII) (Recombinate, Baxter BioScience), secondgeneration B-domain deleted recombinant product, and second- and third-generation full-length recombinant products. We did not evaluate Kogenate (Bayer Healthcare), a first-generation full-length recombinant product, or Refacto AF (Pfizer), a third-generation B-domain deleted product, because of the small numbers of patients who received these products (10 patients [7 as first-use product] and 3 patients [3 as first-use product], respectively). The product type that was used most frequently was selected as the reference category. Switching among Products We evaluated the risk of inhibitor development in children who were receiving a plasma-derived product who were then switched to a recombinant product, as compared with those who were still receiving a plasma-derived product. We similarly assessed the association between switching among various types of factor VIII products and the development of inhibitory antibodies. Study Conduct The study was supported by unrestricted research grants from Bayer Healthcare and Baxter Bio- Science. The companies did not have a role in the study design, data collection, data analysis, or writing of the manuscript. Representatives of the companies reviewed the manuscript before it was submitted for publication. No one who is not an author contributed to the writing of the manuscript. Three of the authors (including the first author) designed the study, performed statistical analyses, interpreted the data, and vouch for the integrity of the data, the fidelity of the study to the protocol, and the accuracy of the data analyses. The first author wrote the first draft of the manuscript. All the other authors collected the data, critically reviewed the manuscript, and made the decision to submit the manuscript for publication. Statistical Analysis The absolute risk of inhibitor development varies according to the cumulative number of exposure days. To account for this varying risk, we used pooled logistic regression with the cumulative number of exposure days as the time variable instead of calendar time. We pooled observations over all exposure days for all patients into a single sample and then used a logistic-regression model with stratification according to number of exposure days to relate the risk factors to inhibitor development. This method accounts for varying risks according to the cumulative number of exposure days and is equivalent to Cox regression with exposure days as time-variable and timedependent covariates. 31 Relative hazard rates were interpreted as relative risks. We calculated both unadjusted and adjusted hazard ratios, with the latter adjusted for race or ethnic group; age at first exposure to factor VIII; reason for first treatment; interval between exposure days; dose of factor VIII; F8 genotype; and status with respect to family history of hemophilia and inhibitors, history of switching among product brands, peak treatment episodes (defined as treatment with factor VIII for bleeding or for surgery on either 3 consecutive days or 5 consecutive days), a history of major surgery, and regular prophylaxis. Coding details are n engl j med 368;3 nejm.org january 17,

4 648 Patients were eligible 606 Were included 584 Remained in study 574 Remained in study 25 Were excluded 5 Did not provide informed consent 10 Did not have available data 8 Were lost to follow-up 1 Died from intracranial hemorrhage 1 Had unknown reason 17 Had pending informed consent 22 Had baseline-only data 6 Were not yet treated 3 Were lost to follow-up 8 Had data of insufficient quality 5 Had unspecified reasons 7 Had no inhibitor development 15 Had unknown inhibitor status 8 Had insufficient data on exposure days (reached study end point) 2 Had inhibitor development 6 Had no inhibitor development 2 Received unknown factor VIII product at first exposure efficacy) for previously untreated patients. An independent statistician who was unaware of product types repeated all results by means of Cox proportional-hazards regression models. R esult s Patients A total of 648 patients were eligible for the study. Of these, 17 patients were excluded because of pending informed consent, and 25 patients were excluded by the investigators for various other reasons (Fig. 1). Baseline data were available for the remaining 606 patients; the analysis included 574 of these patients (94.7%), for whom detailed exposure data were available. Their characteristics according to the type of factor VIII product that was first used are presented in Table 1 (see also Table 1S in the Supplementary Appendix). Primary Outcome Clinically relevant inhibitory antibodies developed in 177 patients (cumulative incidence, 32.4%; 95% confidence interval [CI], 28.5 to 36.3). Of these patients, 116 had high-titer inhibitors (cumulative incidence, 22.4%; 95% CI, 18.8 to 26.0). Inhibitory antibodies developed after a median of 15 exposure days (interquartile range, 10 to 20) at a median age of 15.5 months (interquartile range, 10.7 to 19.6). 516 Reached study end point 58 Did not reach study end point 47 Had treatment data updated until Jan. 9, Did not have treatment data updated 3 Moved to another center 2 Died from intracranial hemorrhage 1 Was lost to follow-up 5 Had unspecified reasons Figure 1. Enrollment and Outcomes. provided in the Supplementary Appendix, available with the full text of this article at NEJM.org. To assess whether our findings were robust, we also compared the incidence of inhibitor development according to the product brands used at the first exposure to factor VIII (a time-fixed determinant). We repeated the analyses in the subgroup of patients who were not included in registration trials (primary studies of safety and Plasma-Derived versus Recombinant Products Plasma-derived products were used on 4018 exposure days, and recombinant products were used on 25,661 exposure days. Plasma-derived products carried a risk of inhibitor development that was similar to the risk with recombinant products (adjusted hazard ratio as compared with recombinant products, 0.96; 95% CI, 0.62 to 1.49) (Table 2 and Fig. 2). Content of von Willebrand Factor Seven patients received products with a low von Willebrand factor content on 1 to 11 exposure days (total, 26 exposure days). Inhibitory antibodies developed in two patients after receiving a product with a low von Willebrand factor content. The risk of inhibitor development with products containing a high amount of von Willebrand factor was similar to the risk with products containing no von Willebrand factor (adjusted hazard ratio, 0.90; 95% CI, 0.57 to 1.41). 234 n engl j med 368;3 nejm.org january 17, 2013

5 Development of Factor VIII Inhibitors in Hemophilia A Table 1. Characteristics of the Patients and the Type of Factor VIII Product Administered during the First Treatment. Characteristic Recombinant Product* Third-Generation Full-Length (N = 157) Second-Generation Full-Length (N = 183) First-Generation Full-Length (N = 59) Second-Generation B-Domain Deleted (N = 77) Plasma-Derived Product (N = 88) All Product Types (N = 574) Patients who switched product brands no. (%) 4 (2.5) 16 (8.7) 25 (42.4) 15 (19.5) 28 (31.8) 94 (16.4) Patients who switched from plasma-derived to recombinant products no. (%) 0 2 (1.1) 0 1 (1.3) 17 (19.3) 20 (3.5) Median age (interquartile range) yr 4.6 ( ) 6.1 ( ) 9.3 ( ) 9.1 ( ) 6.4 ( ) 6.4 ( ) White race no. (%) 137 (87.3) 167 (91.3) 54 (91.5) 73 (94.8) 81 (92.0) 521 (90.8) Family history of hemophilia no. (%) No 71 (45.2) 117 (63.9) 27 (45.8) 42 (54.5) 44 (50.0) 304 (53.0) Yes Negative for inhibitors 64 (40.8) 50 (27.3) 21 (35.6) 21 (27.3) 24 (27.3) 187 (32.6) Positive for inhibitors 22 (14.0) 16 (8.7) 11 (18.6) 14 (18.2) 20 (22.7) 83 (14.5) F8 genotype no. (%) High-risk 95 (60.5) 100 (54.6) 35 (59.3) 37 (48.1) 56 (63.6) 331 (57.7) Low-risk 45 (28.7) 60 (32.8) 18 (30.5) 20 (26.0) 28 (31.8) 172 (30.0) Median age at first exposure to factor VIII (interquartile range) mo Initiation of regular prophylaxis within first 50 exposure days no. (%) Median cumulative exposure days at start of prophylaxis (interquartile range) no. History of peak treatment episode on first exposure day no. (%) 9.9 ( ) 10.2 ( ) 9.7 ( ) 8.8 ( ) 7.9 ( ) 9.8 ( ) 110 (70.1) 125 (68.3) 40 (67.8) 62 (80.5) 67 (76.1) 411 (71.6) 17.0 ( ) 12.0 ( ) 18.0 ( ) 18.5 ( ) 12 (5 21) 15 (7 25) 3 days 40 (25.5) 41 (22.4) 15 (25.4) 20 (26.0) 30 (34.1) 149 (26.0) 5 days 28 (17.8) 21 (11.5) 9 (15.3) 14 (18.2) 24 (27.3) 98 (17.1) History of surgical procedure no. (%) 46 (29.3) 33 (18.0) 18 (30.5) 28 (36.4) 16 (18.2) 144 (25.1) Inhibitor development no. (%) Clinically relevant 41 (28.2) 64 (37.7) 17 (29.0) 23 (30.3) 29 (33.1) 177 (32.4) High-titer 25 (17.9) 40 (25.2) 14 (24.6) 13 (18.0) 21 (25.7) 116 (22.4) * Among the recombinant products, data are not included for seven patients who were first treated with Kogenate (Bayer Healthcare), a first-generation full-length product, and three patients who were first treated with Refacto AF (Wyeth), a third-generation B-domain deleted product. However, these patients are included in the total number of patients who received any product. Race was self-reported. Low-risk genotypes included those with small deletions and insertions, missense mutations, and splice-site mutations. High-risk genotypes included those with large deletions, nonsense mutations, and intron 1 and 22 inversions. The start of regular prophylaxis was defined as the moment at which at least three consecutive prophylactic infusions of factor VIII had been given within a period of at least 15 days. A peak treatment episode was defined as treatment with factor VIII for bleeding or for surgery on either at least 3 consecutive days or at least 5 consecutive days. The values for inhibitor development are cumulative incidences, which were calculated by means of the Kaplan Meier method. n engl j med 368;3 nejm.org january 17,

6 Specific Product Types Table 2. Risk of Inhibitor Development, According to the Type of Factor VIII Product.* Product Any Inhibitor Development High-Titer Inhibitor Development Adjusted Hazard Ratio P Value Unadjusted Hazard Ratio P Value No. of Exposure Days Adjusted Hazard Ratio P Value Unadjusted Hazard Ratio P Value No. of Exposure Days All recombinant vs. all plasma-derived products Recombinant 25, NA 1.00 NA 25, NA 1.00 NA Plasma-derived 4, ( ) ( ) , ( ) ( ) 0.85 Specific products Recombinant Third-generation full-length 9, NA 1.00 NA 9, NA 1.00 NA Second-generation full-length 9, ( ) ( ) , ( ) ( ) 0.02 First-generation full-length 2, ( ) ( ) , ( ) ( ) 0.53 Second-generation B-domain deleted 4, ( ) ( ) , ( ) ( ) 0.92 Plasma-derived 4, ( ) ( ) , ( ) ( ) 0.51 * NA denotes not applicable. Hazard ratios were adjusted for race or ethnic group; age at first exposure to factor VIII; reason for first treatment; interval between exposure days; dose of factor VIII; F8 genotype; and status with respect to family history of hemophilia and inhibitors, history of switching among product brands, peak treatment episodes of either at least 3 consecutive days or at least 5 consecutive days, history of major surgery, and regular prophylaxis. A first-generation full-length recombinant product, Kogenate (Bayer Healthcare), and a third-generation B-domain deleted recombinant product, Refacto AF (Wyeth), were used only in 10 and 3 patients on 103 and 163 exposure days, respectively, so the effect of these products on inhibitor development was not studied. The only first-generation full-length product in this category was Recombinate (Baxter BioScience). The risk of inhibitor development was similar among plasma-derived products, first-generation full-length recombinant products, second-generation B-domain deleted recombinant products, and third-generation recombinant products. Firstgeneration recombinant products were associated with an unadjusted hazard ratio of 1.44 (95% CI, 0.71 to 2.90) for high-titer inhibitor development; however, after adjustment, the hazard ratio was lower. Second-generation full-length recombinant products were associated with a significantly higher risk of inhibitor development than were third-generation products (adjusted hazard ratio, 1.60; 95% CI, 1.08 to 2.37; P = 0.02); for high-titer inhibitor development, the adjusted hazard ratio was 1.79 (95% CI, 1.09 to 2.94; P = 0.02) (Table 2). Switching among Products Details about the analyses of switching among brands of factor VIII are provided in the Supplementary Appendix. Switching among products was not associated with the risk of inhibitor development (adjusted hazard ratio as compared with no switching, 0.99; 95% CI, 0.63 to 1.56) (Table 3S in the Supplementary Appendix). Sensitivity Analyses The results of sensitivity analyses regarding the use of factor VIII products (plasma-derived vs. recombinant products and specific product types) were similar to those of the primary analysis. Details regarding the sensitivity analyses are provided in the Supplementary Appendix. Discussion In this cohort study involving 574 consecutive, previously untreated children with severe hemophilia A who were born between 2000 and 2010, recombinant factor VIII products conferred a risk of inhibitor development that was similar to the risk conferred by plasma-derived products. The von Willebrand factor content in factor VIII products was not associated with inhibitor development. Second-generation full-length recombinant products were associated with a higher risk of inhibitor development than were third-generation full-length products. Switching from a plasmaderived product to a recombinant product or switching among brands of factor VIII products 236 n engl j med 368;3 nejm.org january 17, 2013

7 Development of Factor VIII Inhibitors in Hemophilia A did not result in an increased risk of inhibitor development. We directly compared the use of recombinant products and plasma-derived products in one study cohort. We avoided selection bias by including all consecutive patients who were born between January 1, 2000, and January 1, We excluded all patients who were referred from nonparticipating hemophilia centers because of inhibitor development. We used survival analysis because at the moment of data analysis a number of patients had not yet reached the study end point and were still at risk for inhibitor development. This enabled us to include all patients up to the last exposure day and to calculate cumulative incidences. Collection of detailed information on all 75 exposure days allowed us to adjust the associations for potential confounding factors. These findings were robust in sensitivity analyses. Even though we adjusted for potentially confounding factors, we cannot rule out residual confounding. The observed associations may have been affected by information bias, if frequencies and methods of inhibitor screening among centers had differed according to the particular factor VIII product. However, we would not expect that the cumulative incidence of high-titer inhibitor development would be influenced by variations in inhibitor screening because of a lack of central laboratory testing, since these inhibitory antibodies will always be detected clinically. Since the associations were similar with respect to both all clinically relevant inhibitor development and high-titer inhibitor development, information bias would therefore not have played a major role. Because a relatively small number of patients were treated with plasma-derived products and because of the variety of plasmaderived products, we may not have been able to detect potential differences in the risk of inhibitor development among various plasma-derived products. Several reports have suggested that plasmaderived factor VIII products, especially those containing considerable amounts of von Willebrand factor, are less immunogenic than recombinant products. 19,32,33 However, several systematic reviews have yielded inconclusive results Our results are in agreement with the findings of a similarly designed study, the Concerted Action on Neutralizing Antibodies in Severe Hemophilia A Adjusted Relative Risk (95% CI) All Recombinant All Plasma-Derived 3rd Generation Full-Length 1st Generation Full-Length 2nd Generation Full-Length 2nd Generation B-Domain Deleted (CANAL) study, in which the risk of inhibitor development was not clearly lower with plasmaderived products than with recombinant products (relative risk, 0.79; 95% CI, 0.49 to 1.28). 20 Unexpectedly, the risk of inhibitor development was 60% higher among children receiving a second-generation full-length recombinant product than among those receiving a thirdgeneration full-length product. This association may be a biased finding (through confounding, selection bias, or information bias), a chance finding, or a causal effect. We accounted for bias from confounding by adjusting the association for multiple potential confounding factors. We summarized potentially confounding factors according to the product type used at the first treatment (Table 1). Children at increased risk for inhibitor development did not receive second-generation full-length factor VIII products more often than they did thirdgeneration products. Therefore, confounding does not seem to explain this association. We avoided selection bias by including all con- Plasma-Derived Figure 2. Adjusted Relative Risk of Inhibitor Development, According to the Type of Factor VIII Product. Data are for 574 previously untreated children with severe hemophilia A. In the comparison between all recombinant products and all plasma-derived products (at left), the reference group was the recombinant products. In the comparison of specific products (at right), the reference group was third-generation full-length recombinant products. The only first-generation full-length recombinant product that was evaluated was Recombinate (Baxter BioScience). The I bars indicate 95% confidence intervals. n engl j med 368;3 nejm.org january 17,

8 secutive patients and by excluding all patients who were referred to the participating center because of a known increased risk for inhibitors. In addition, to further confirm the absence of selection bias, we performed a sensitivity analysis among patients who were not in a safety and efficacy trial. Patients who were included in such a trial might have been at reduced risk for inhibitor development, since they did not have early bleeding. The observed increase in the risk of inhibitor development with second-generation full-length recombinant products as compared with thirdgeneration full-length products is not likely to be affected by information bias, since it is unlikely that patients who were treated with a secondgeneration full-length product were more often screened for inhibitors than those treated with a third-generation product. Furthermore, we observed a similar association in high-titer development. Thus, selection bias and information bias do not explain the observed increase in risk in second-generation full-length recombinant products, as compared with third-generation fulllength products. This difference in risk between recombinant products may be due to chance, which seems unlikely, given the precision of our estimate of effect. But as long as the observation in our study is not confirmed in other studies, we cannot exclude the possibility. However, since bias is unlikely and the probability of a chance finding is low, the observed increase in the risk of inhibitor development in patients receiving second-generation full-length factor VIII products may be real. Other studies including a systematic review 23 and reports of the Kogenate Bayer Study Group 25,34 have not shown significant differences in the risk of inhibitor development among various recombinant factor VIII products. In the registration studies, the incidence of inhibitor development may have been underestimated because of the inclusion of patients who had already been treated with factor VIII on several exposure days and a short follow-up period for the subgroup of patients who were still at risk for inhibitor development. There is no straightforward biologic explanation for a difference in immunogenicity among recombinant factor VIII products. Further studies are needed to verify these observations and to identify biologic explanations. In conclusion, the use of recombinant factor VIII products in children with severe hemophilia A did not have a significant effect on the risk of inhibitor development, as compared with the use of plasma-derived products, nor was the von Willebrand factor content of the products or switching among them associated with the risk of inhibitor development. An unexpected finding was that second-generation full-length recombinant products were associated with an increased risk of inhibitor development, as compared with third-generation products. Supported by unrestricted research grants from Bayer Healthcare and Baxter BioScience. Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. We thank the study coordinator, Ella Smink; Emma Smid and Mojtaba Hashemi for their support in data cleaning; Yves Guillaume, Kate Khair, Karin Lindvall, Monique Spoor, and Bep Verkerk for their assistance in the study; and J. Michael Soucie (Division of Blood Disorders, National Center on Birth Defects and Developmental Disabilities, Centers for Disease Control and Prevention) for repeating the analyses. Appendix The authors affiliations are as follows: the Department of Pediatrics, Wilhelmina Children s Hospital (S.C.G.), and the Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht (S.C.G., H.M.B.), Utrecht, and the Department of Clinical Epidemiology, Leiden University Medical Center, and the Center for Clinical Transfusion Research, Sanquin Foundation, Leiden (J.G.B.) all in the Netherlands; Lund University, Department of Pediatrics and Malmö Center for Thrombosis and Hemostasis, Skånes Universitetssjukhus, Malmö, Sweden (R.L.); Department of Pediatrics, J.W. Goethe University Hospital, Frankfurt, Germany (C.E.); Unidad de Hemostasia y Trombosis, Hospital Universitario y Politécnico La Fe, Valencia, Spain (A.R.C.); Centre Regional d Hemophilie, Centre Hospitalier Universitaire, Toulouse, France (S.C.-D.); the Department of Pediatrics, University Hospitals Leuven, and the Department of Cardiovascular Sciences, KU Leuven both in Leuven, Belgium (C.G.); National Hemophilia Center, Ministry of Health, Sheba Medical Center, Tel Hashomer, Israel (G.K.); Hospital for Children and Adolescents, University of Helsinki, Helsinki (A.M.); Dipartimento di Ematologia ed Oncologia, Unità Trombosi ed Emostasi, Ospedale Pediatrico Giannina Gaslini, Genoa (A.C.M.), and Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione IRCCS Ca Granda, Ospedale Maggiore Policlinico, Milan (E.S.) both in Italy; Universitätsklinik für Kinder- und Jugendheilkunde, Graz, Austria (W.M.); Hämophiliezentrum, Wabern and Children s Hospital of the University of Bern, Bern, Switzerland (R.K.); Division of Hematology/Oncology, Hôpital St. Justine, Montreal (G.R.); and Royal Hospital for Sick Children, Edinburgh (A.T.). 238 n engl j med 368;3 nejm.org january 17, 2013

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