Abstract: The appearance of inhibitors is the most serious complication in haemophilia A (HA) patients. The primary objective is their eradication. Up to date, immune tolerance induction (ITI) was the only therapeutic option to achieve this. Aim To assess the efficacy of moroctocog‐alpha as an ITI regimen in a population of HA patients with high‐titre inhibitors. Methods The REF.IT Registry is a retrospective‐prospective study that collected data on all patients with HA and high‐titre inhibitors treated with moroctocog‐alpha as an ITI regimen at twelve Italian Haemophilia Centres. Results We enrolled 27 patients, 85.2% were children. All patients were high responders, 88.9% had severe HA. We found 69.3% of them had one or more risk factors for poor ITI prognosis, 14.8% were ITI rescue. Overall 59.3% achieved a complete/partial success (complete in 51.9%). ITI failed in 11 patients, 63.6% of them with poor‐prognosis risk factors. Inhibitors appeared after a mean of 27 exposure days. Mean historical peak was 78.8 BU/mL. The primary ITIs started on average 20.2 months after the diagnosis. A partial or complete success after a mean of 15 months of treatment was achieved in 56.6% of the children while the same result was obtained by 75.0% adults after 22 months from ITI onset. Patients who were treated with high‐dose moroctocog‐alpha (200 UI/kg/day) were 63.0%. Conclusion Our Registry showed that the use of moroctocog‐alpha in the setting of ITI was effective and safe also in a population of patients with high‐titre inhibitors, presenting one or more risk factors for poor ITI prognosis.

Abstract: Although primary prophylaxis is a well-established and widely recommended practice for the management of patients with severe hemophilia, there are limited data in the literature documenting the benefit of secondary prophylaxis initiated in adolescence or adulthood. In order to address this issue we have conducted a retrospective study among the 10 Italian Hemophilia Centers on adolescent and adult patients with severe hemophilia switched from on-demand treatment to a long term secondary prophylaxis regimen. On the whole, we collected information on 73 consecutive patients with severe hemophilia (66 hemophilia A and 7 hemophilia B). Their median age started prophylaxis was 12 (range 10–17) for 23 adolescents and 30 (17.4–72) for the 50 adults and continued for a median of years 5.8 (2–14.7) in adolescents and 4 years (1.5–12) in adults. The clinical impact of secondary prophylaxis with respect to the last year on-demand regimen before switching is showed in the following table for each group of patients. Adolescents (n=23) Adults (n=50) On-Demand Prophylaxis On-Demand Prophylaxis Values are reported as mean (standard deviation) Bleedings 32.4(24.8) 2.8 (3.2) 42.7 (24.5) 5.5 (5.2) Joint bleedings 25.6 (22.1) 1.9 (2.3) 36.4 (24.5) 4.2 (4.1) Orthopaedic score 8.4 (7.5) 3.7 (2.4) 21.6 (12.2) 17.7 (12.2) Pettersson score 5.1 (3.4) 5.0 (3.3) 14.7 (13.8) 15.2 (14.3) Work/school days lost 35.1 (34.5) 1.0 (1.5) 33.8 (20.9) 2 (0–17) Concentrate consumption U/Kg/y 2449.8 (1761.9) 3508.3(803.1) 3222.6 (2241.8) 4058.5 (952.3) The decreased joint bleeding, better orthopaedic score, less work/school days lost, delayed progression of arthropathy and improved well-being observed with secondary prophylaxis show the benefit of this therapeutic approach and appear to counterbalance its higher clotting factor consumption. In conclusion large, prospective, long-term randomized studies are needed to definitevely assess the clinical impact of this strategy in adolescent and adult hemophiliacs and to identify those groups of patients who would benefit from this therapeutic approach.

Abstract: Introduction: Immune tolerance therapy (ITT), consists of administration of daily high doses of factor VIII concentrates by intravenous infusions. It can induce tolerance to the exogenous protein and eradicate the antibody response to FVIII, which still represents the main complication of replacement therapy in haemophilia A (HA) severe cases. Methods: We have investigated 71 HA patients with inhibitors (sixty-nine severe, one moderate and one mild cases). We screened the patients for the causative mutations in the F8 gene using Long Range PCR for the intron22 inversion, multiplex-PCR for intron1 inversion or conformation sensitive gel electrophoresis (CSGE) followed by DNA sequencing for other mutation types. Results: Diverse genetic defects were detected in the severe cases, with a predominance of gross mutations: F8 gene inversions, large deletions and nonsense mutations account for 68% of the mutations, whereas in the two non severe patients specific missense mutations were identified. ITT has been attempted in 16 HR patients of this cohort but failed in 5 cases. Conclusion: We confirmed that the presence of inhibitors correlates well with the presence of null mutations, as reported by Schwaab et al (Thromb Haemost 1995). Several predictive factors for ITT outcome have been so far taken into consideration but no correlation has been made between F8 gene defect and ITT response. In our cohort 2 large deletions, 2 intron22 inversions and 1 nonsense mutations failed to respond. For the remaining 2 large deletions no ITT were attempted so no information are available. Large deletions appear to be a high risk genetic factor both for inhibitor development and for long term inhibitor persistence and ITT unresponsiveness. The identification of these patients at very high risk of inhibitor development and ITT unresponsiveness by mutation analysis should therefore be strongly recommended soon after the diagnosis, even because of the high cost of the therapy.

Abstract: Background: A major complication in severe hemophilia A is the formation of persistent neutralizing antibodies against factor(F) VIII, inhibitors, which render subsequent treatment ineffective. The presence of non-neutralizing anti-FVIII IgG antibodies (NNAs) before FVIII treatment initiation has been associated with subsequent development of inhibitors in previously untreated patients (PUPs) with severe hemophilia A in the frame of the SIPPET cohort (Cannavò A et al, Blood 2017). Up to 30% of PUPs develop neutralizing antibodies (inhibitors) within the first 20-30 exposure days (Eds) to FVIII concentrates, of which one third disappear spontaneously over a course of six months due to endogenous immune tolerance, and two thirds progress into persistent inhibitors that require immunotolerance therapy. The role of each anti-FVIII IgG subclasses (e.g., IgG1, IgG2, IgG3 and IgG4) and their possible prediction of persistent anti-FVIII inhibitors is not known yet. Aims: To investigate the predictive value of anti-FVIII IgG subclasses on persistence of the anti-FVIII inhibitor in PUPs with severe hemophilia A within 60 days from the first development of anti-FVIII inhibitor. Methods: From the 76 patients who developed inhibitors in the SIPPET cohort (Peyvandi et al., N Eng J Med 2016), anti-FVIII IgG subclasses were measured by an ELISA assay in 43 patients according to plasma availability (median age 18 months [IQR: 12-29]), median inhibitor titer: 16 Bethesda IU [IQR: 5-135] ). For each IgG subclass, a cutoff of positivity was defined as the mean OD absorbance value + 5 SD, obtained by analyzing the plasma of 150 normal individuals. The association of number of anti-FVIII IgG subclasses and other possible risk factors (age at first treatment, type of FVIII product, number of EDs and type of F8 gene variation) with inhibitor persistence was first estimated by univariate analysis. Predictive associations were assessed by logistic regression, in which inhibitor persistence was the outcome, and number of anti-FVIII IgG subclasses (1= only one [always IgG1], 2 subclasses, 3 subclasses or all 4 subclasses) and age at first treatment with FVIII concentrates were the putative predictors that showed an association with inhibitor persistence. Other risk factors, such as type of FVIII product, number of EDs and type of F8 gene variation, were not associated with inhibitor persistence at univariate analysis. Relative risks (RR) and 95% confidence intervals (95% CI) were recalculated from odds ratios according to Zhang (JAMA, 1998). The predictive capacity was expressed as the area under the receiving operative characteristic (ROC) curve (AUC). Results: Of the 43 patients who developed an inhibitor (31 persistent, 12 transient), 3 had only one IgG subclass (IgG1), 15 two subclasses, 13 three subclasses and 12 were positive for all the four IgG subclasses. The presence of each subclass was associated with an increased risk of inhibitor persistence, both in univariate and multivariate analysis, with relative risks ranging from 1.3 to 1.8. The risk of inhibitor persistence progressively increased with the number of concomitant IgG subclasses. In the model containing also age at first treatment and taking the category with only IgG1 positivity as reference, the RR (95% CI) was 1.7 (0.2 to 2.9) for patients with two IgG subclasses, 2.6 (0.7 to 3.0) for those with three subclasses and 2.8 (1.2 to 3.0) for those with all the four subclasses. The odds of inhibitor persistence increased by 8% for every 1-month increase of age at first treatment (OR 1.08 [0.99 to 1.22]). The AUC of the predictive model was 0.82 (95% CI: 0.68 to 0.96) (Fig 1). Conclusions: The concomitant presence of more than one anti-FVIII IgG subclass within 60 days from the first development of anti-FVIII inhibitor in patients with severe hemophilia A was associated with an increased risk of persistence of the inhibitor. Age at first treatment also predicted inhibitor persistence. In conclusion, this predictive analysis showed a promising discriminative capability for clinicians to select patients with the highest risk of inhibitor persistence who could benefit from immunotolerance therapy. These results need to be confirmed in other cohorts of PUPs with severe hemophilia A. Figure 1. Figure 1. Disclosures Peyvandi: Novo Nordisk: Speakers Bureau; Octapharma US: Honoraria; Novo Nordisk: Speakers Bureau; Shire: Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Roche: Speakers Bureau; Grifols: Speakers Bureau; Sobi: Speakers Bureau; Kedrion: Consultancy; Grifols: Speakers Bureau; Octapharma US: Honoraria; Sobi: Speakers Bureau; Grifols: Speakers Bureau; Grifols: Speakers Bureau; Kedrion: Consultancy; Octapharma US: Honoraria; Octapharma US: Honoraria; Kedrion: Consultancy; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Novo Nordisk: Speakers Bureau; Sobi: Speakers Bureau; Roche: Speakers Bureau; Shire: Speakers Bureau; Sobi: Speakers Bureau; Kedrion: Consultancy; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Shire: Speakers Bureau; Novo Nordisk: Speakers Bureau; Roche: Speakers Bureau; Shire: Speakers Bureau; Grifols: Speakers Bureau; Kedrion: Consultancy; Ablynx: Other: Member of Advisory Board, Speakers Bureau; Shire: Speakers Bureau; Octapharma US: Honoraria; Sobi: Speakers Bureau; Roche: Speakers Bureau; Roche: Speakers Bureau; Novo Nordisk: Speakers Bureau. Palla:Grifols: Other: travel support; Pfizer: Other: travel support. Santagostino:Grifols: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Shire: Membership on an entity's Board of Directors or advisory committees; Kedrion: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees. Young:Novo Nordisk: Consultancy, Honoraria; Bioverativ: Consultancy, Honoraria; Bayer: Consultancy; CSL Behring: Consultancy, Honoraria; Kedrion: Consultancy; Genentech/Roche: Consultancy, Honoraria; Shire: Consultancy, Honoraria. Seth:Shire: Honoraria. Mancuso:Bayer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity's Board of Directors or advisory committees; Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kedrion: Consultancy; Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees; Biotest: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees. Mahlangu:Sanofi: Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; LFB: Consultancy; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; CSL Behring: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy; Catalyst Biosciences: Consultancy, Research Funding; Biomarin: Research Funding, Speakers Bureau; Biogen: Research Funding, Speakers Bureau; Bayer: Research Funding; Amgen: Consultancy; Alnylam: Consultancy, Research Funding, Speakers Bureau; Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Research Funding, Speakers Bureau; Spark: Consultancy, Research Funding. Ewing:Hema Biologics: Honoraria; Novo Nordisk: Honoraria; CSL Behring: Honoraria; Grifols: Honoraria; Bayer: Honoraria; Shire: Honoraria; Genentech: Honoraria; Biogen: Research Funding. Male:Bayer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Octapharma: Speakers Bureau; Novo Nordisk: Speakers Bureau; Biotest: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Speakers Bureau; SOBI: Speakers Bureau; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Majumdar:NIMHD: Research Funding. Manco-Johnson:CSL Behring: Honoraria; Biogentek: Honoraria; Novo Nordisk: Honoraria; Bayer AG: Honoraria, Research Funding; Baxalta, now part of Shire: Honoraria. Mazzucconi:Baxalta-Shire: Consultancy, Speakers Bureau; Bayer: Consultancy, Speakers Bureau; Novartis,: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Novo Nordisk: Consultancy, Speakers Bureau; CSL Behring: Consultancy, Speakers Bureau. Neme:Shire: Consultancy, Honoraria; Novonordisk: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Grifols: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria. Prezotti:Bayer: 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; Roche: 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; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Mannucci:Kedrion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Grifols: Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Baxalta/Shire: Speakers Bureau; Alexion: Speakers Bureau; Novo Nordisk: Speakers Bureau.

Abstract: A case of sporadic hemophilia A in a young child was investigated from a molecular biology point of view. The propositus is a 4-year-old severe hemophiliac who was first seen when he was 2 years old. At that time, easy bruising and hematomas were noted because of accidental falls while toddling. The coagulation study showeda prolonged partial thromboplastin time and a factor VIII level of 1.3% of normal. Molecular biologic analysis showed a large deletion involving intron 13 up to exon 23. In the inversion study, the propositus exhibited only a 10 kb band, and this result suggests that intron 22 was deleted while his mother shows a normal pattern. To further examine the length of the deletion, a long polymerase chain reaction by means of primers amplifying the region from exon 13 to 23. In the index patient, an approximate 13-kb product was obtained, whereas no product was obtained from his mother. The mother investigated by means of polymorphism was shown not to be a carrier.