Inhibitor development according to concentrate in severe hemophilia: reporting on 1392 Previously Untreated Patients from Europe and Canada.

Background: Clotting factor concentrates have been the mainstay of severe hemophilia treatment over the last 50 years. Differences in risk of neutralizing antibody (inhibitor) formation according to concentrate used remain clinically relevant. Objectives: To assess inhibitor development according to type of clotting factor concentrate in previously untreated patients (PUPs) with severe hemophilia A and B. Methods: The European Haemophilia Safety Surveillance (EUHASS) and Canadian Bleeding Disorders Registry (CBDR) have been monitoring adverse events overall and according to concentrate for 11 and 8 years, respectively. Inhibitors were reported quarterly, and PUPs completed 50 exposure days without inhibitor development annually. Cumulative inhibitor incidences and 95% confidence intervals (CIs) were compared without adjustment for other risk factors. Results: Fifty-six European and 23 Canadian centers reported inhibitor development in 312 of 1219 (26%; CI, 23%-28%) PUPs with severe hemophilia A and 14 of 173 (8%; CI, 5%-13%) PUPs with severe hemophilia B. Inhibitor development was lower on plasma-derived factor (F)VIII (pdFVIII, 20%; CI, 14%-26%) than on standard half-life recombinant FVIII (SHL-rFVIII, 27%; CI, 24%-30% and odds ratio, 0.67; CI, 0.45%-0.98%; P = .04). Extended half-life recombinant FVIII (EHL-rFVIII, 22%; CI, 12%-36%) showed an intermediate inhibitor rate, while inhibitor rates for Advate (26%; CI, 22%-31%) and Kogenate/Helixate (30%; CI, 24%-36%) overlapped. For other SHL-rFVIII concentrates, inhibitor rates varied from 3% to 43%. Inhibitor development was similar for pdFIX (11%; CI, 3%-25%), SHL-rFIX (8%; CI, 3%-15%), and EHL-rFIX (7%; CI, 1%-22%). Conclusion: While confirming expected rates of inhibitors in PUPs, inhibitor development was lower in pdFVIII than in SHL-rFVIII. Preliminary data suggest variation in inhibitor development among different SHL-rFVIII and EHL-rFVIII concentrates.

Incidence of thrombotic microangiopathies in Quebec: insight from a laboratory centralizing ADAMTS-13 testing.

BACKGROUND: Thrombotic microangiopathies (TMA) are serious medical conditions requiring a prompt diagnosis to adapt treatment. The determination of ADAMTS-13 activity enables discriminating thrombotic thrombocytopenic purpura (TTP) from other forms of TMA. The purpose of this study was to provide an estimate of the incidence of TTP and TMA in the Canadian Quebec province using data collected from a laboratory centralizing ADAMTS-13 testing for the whole province. RESULTS: From 2012 to 2019, 846 patients were evaluated for plasma ADAMTS-13 activity due to a suspicion of TMA. TTP was identified in 147 patients. Of these, 118 patients with a median age of 51.5 years and a male-female ratio of 1:1.4 had their first episode of TTP during the study period. The number of ADAMTS-13 tests performed and the number of patients with suspected TMA increased annually by 19% and 21% respectively. While the incidence of non-TTP TMA increased annually, that for TTP remained unchanged. This averaged 10.2 (95% CI 5.9-14.4) per million persons per year for suspected non-TTP TMA and 1.8 (95% CI 1.3-2.4) for confirmed TTP. The incidence rate of TMA other than TTP was higher in the age group 70-79 years (21.8; 95% CI 5.4-38.1) for females and in the age group 80-89 years (24.4; 95% CI 7.2-41.7) for males compared to other age groups. The incidence rate of TTP was higher in the age group 40-49 years (4.0; 95% CI 2.0-5.9) for women and in the age group 60-69 years (3.4; 95% CI 1.1-5.6) for men compared to other age groups. CONCLUSION: The analysis of centralized data measuring ADAMTS-13 activity allowed us to adequately establish the incidence rate and demographic characteristics of TMA, particularly TTP, in Quebec. TTP incidence remained stable while suspected non-TTP TMA steadily increased from 2012 to 2019.

Intravenous Immunoglobulins Tapering and Withdrawal in Systemic Capillary Leak Syndrome (Clarkson Disease).

BACKGROUND: The systemic capillary leak syndrome (SCLS), also known as Clarkson disease, is a very rare condition characterized by recurrent life-threatening episodes of vascular hyperpermeability in the presence of a monoclonal gammopathy. Extended intravenous immunoglobulin (IVIG) treatment is associated with fewer recurrences and improved survival, but the optimal treatment dosage and duration remain unknown. OBJECTIVE: We aim to evaluate the safety of IVIG tapering and withdrawal in patients with SCLS. METHODS: We conducted a retrospective multicenter study including all adult patients with monoclonal gammopathy-associated SCLS from the EurêClark registry who received at least 1 course of IVIG. The primary end point was overall survival according to IVIG withdrawal. RESULTS: Fifty-nine patients of mean ± SD age 51 ± 13 years were included. Overall cumulative probabilities of 2-, 5-, 10- and 15-year survival were 100%, 85%, 72%, 44%, respectively. The IVIG was withdrawn at least once in 18 patients (31%; W+ group) and never in 41 patients (69%; W- group). Cumulative probabilities of 10-year survival in W+ versus W- groups were 50% and 83% (log rank test, P = .02), respectively. Relapse rate and the median number of relapses in the W+ versus the W- groups were 72% versus 58% (P = 0.3) and 2.5 (0.3-4) versus 1 (0-2) (P = .03), respectively. The IVIG tapering was not statistically associated with increased person-year incidence of attacks using a mixed linear model. CONCLUSIONS: The IVIG withdrawal was associated with increased mortality and higher rate of recurrence in SCLS patients. The IVIG tapering might be cautiously considered in stable SCLS patients.

Picomolar Sensitivity Analysis of Multiple Bradykinin-Related Peptides in the Blood Plasma of Patients With Hereditary Angioedema in Remission: A Pilot Study.

Background: Hereditary angioedema (HAE) is a rare autosomal dominant disease; the most well understood forms concern the haplodeficiency of C1 esterase inhibitor (C1INH) and a gain of function mutation of factor XII (FXII). The acute forms of these conditions are mediated by an excessive bradykinin (BK) formation by plasma kallikrein. Methods: A validated LC-MS/MS platform of picomolar sensitivity developed for the analysis of eleven bradykinin-related peptides was applied to the plasma of HAE-C1INH and HAE-FXII sampled during remission. Results: In HAE-C1INH plasma, the concentrations of the relatively stable BK1-5 fragment (mean ± S.E.M.: 12.0 ± 4.2 pmol/L), of BK2-9 (0.7 ± 0.2 pmol/L) and of the sums of BK and its tested fragments (18.0 ± 6.4 pmol/L) are significantly greater than those recorded in the plasma of healthy volunteers (1.9 ± 0.6, 0.03 ± 0.03 and 4.3 ± 0.8 pmol/L, respectively), consistent with the previous evidence of permanent plasma kallikrein activity in this disease. Kinin levels in the plasma of HAE-FXII patients did not differ from controls, suggesting that triggering factors for contact system activation are not active during remission. Conclusion: BK1-5, BK2-9 and the sum of BK and its fragments determined by the sensitive LC-MS/MS technique are proposed as potential biomarkers of HAE-C1INH in remission while this was not applicable to HAE-FXII patients.

Quantitation of a plasma biomarker profile for the early detection of Gaucher disease type 1 patients.

Aim: Gaucher disease (GD) is caused by a deficiency of the lysosomal enzyme acid ß-glucocerebrosidase. Recent metabolomic studies highlighted several new metabolites increased in the plasma of GD patients. We aimed to develop and validate a UPLC-MS/MS method allowing a relative quantitation of lyso-Gb1 and lyso-Gb1 analogs -28, -12, -2, +14, +16 and +18 Da in addition to sphingosylphosphorylcholine, N-palmitoyl-O-phosphocholine to study potential correlations with clinical manifestations. Methodology & results: Following solid-phase extraction, plasma samples were evaporated and resuspended in 100 µl of resuspension solution. Three microliter is injected into the UPLC-MS/MS for analysis. Conclusion: All biomarkers studied were increased in GD patients. Significant correlations were observed between specific analogs and hematological, and visceral complications, as well as overall disease severity.

Magnetic resonance imaging in boys with severe hemophilia A: Serial and end-of-study findings from the Canadian Hemophilia Primary Prophylaxis Study.

BACKGROUND: This study examined the structural outcomes for joints of boys with severe hemophilia A receiving frequency/dose-escalated primary prophylaxis using magnetic resonance imaging (MRI), and the importance of interval MRI changes. METHODS: Forty-six subjects (27 with interval studies) were evaluated by radiographs (X-rays) and mid- and end-of-study MRIs (using the International Prophylaxis Study Group scale), as part of the Canadian Hemophilia Prophylaxis Study. The primary outcome was the presence of MRI osteochondral findings. RESULTS: The median (range) time on study at the end-of-study MRI examination was 9.6 (4.8-16.0) years, during which 18 of 46 subjects (39%) had osteochondral changes in at least one joint. An interval change in MRI score of at least 1 point was observed in 44% of joints (43 ankles, 21 elbows, 4 knees); at least one joint showed this change in all 27 subjects. Self-reported interval hemarthrosis was associated with a higher likelihood of interval osteochondral change (odds ratio [OR], 1.49; 95% confidence interval [CI] = 1.08-2.06). Presence of synovial hypertrophy or hemosiderin on interval MRIs was associated with an OR of 4.71 (95% CI, 1.92-11.57) and 5.25 (95% CI, 2.05-13.40) of later osteochondral changes on MRI. DISCUSSION: MRI changes were seen in 39% of subjects. Interval index joint bleeding was associated with an increased risk of later MRI changes, and earlier soft-tissue changes were associated with subsequent osteochondral changes.

Predictive significance of anti-FVIII immunoglobulin patterns on bleeding phenotype and outcomes in acquired hemophilia A: Results from the Quebec Reference Center for Inhibitors.

BACKGROUND: Acquired hemophilia A (AHA) is a potentially life-threatening bleeding disorder caused by factor VIII (FVIII) autoantibodies, involving various immunoglobulin (Ig) isotypes and IgG subclasses. OBJECTIVES: We analyzed the profile of Ig against FVIII in patients with AHA to identify Ig patterns predictive of bleeding phenotype and outcomes. PATIENTS/METHODS: Ig detection and titration were determined by enzyme-linked immunosorbent assay (ELISA) at disease presentation in a cohort of 66 subjects from the Quebec Reference Centre for Inhibitors registry. RESULTS: Most of plasma samples analyzed (97%) contained multiple anti-FVIII Ig isotypes and IgG subclasses, IgG(1,2,3,4) (24.2%), [IgG(1,2,3,4),IgA] (16.7%) and IgG(2.4) (13.6%) being the most prevalent combinations of Ig detected. AHA patients who presented with IgA antibodies were more likely to have an associated auto-immune disease (p = .049). The presence of IgG4-was associated with bleeding symptoms at presentation (p = .002). IgG1-positive patients were more likely to require transfusions with red packed cell (p = .014) whereas IgM detection was associated with a higher probability of death linked to AHA (p = .011). CONCLUSION: The Ig pattern of AHA patients at diagnosis is widely heterogeneous and is at least partially associated with some underlying conditions. Our data supports the differential predictive significance for IgG1, IgG4 and IgM on bleeding severity and suggests that the early determination of Ig profile may help to identify AHA patients at higher risk of poorer outcomes.

Glanzmann Thrombasthenia: Perspectives from Clinical Practice on Accurate Diagnosis and Optimal Treatment Strategies.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive disorder of fibrinogen-mediated platelet aggregation due to a quantitative or qualitative deficit of the αIIbß3 integrin at the platelet surface membrane resulting from mutation(s) in ITGA2B and/or ITGB3. Patients tend to present in early childhood with easy bruising and mucocutaneous bleeding. The diagnostic process requires consideration of more common disorders of haemostasis and coagulation prior to confirming the disorder with platelet light transmission aggregation, flow cytometry of CD41 and CD61 expression, and/or exon sequencing of ITGA2B and ITGB3. Antifibrinolytic therapy, recombinant activated factor VII, and platelet transfusions are the mainstay of therapy, although the latter may trigger formation of anti-platelet antibodies in GT patients and inadvertent platelet-refractory disease. The management of these patients therefore remains complex, particularly in the context of trauma, labour and delivery, and perioperative care. Bone marrow transplantation remains the sole curative option, although the venue of gene therapy is being increasingly explored as a future alternative for definitive treatment of GT.

Patterns of joint damage in severe haemophilia A treated with prophylaxis.

OBJECTIVE: The primary objective of this study was to assess whether there are different patterns (classes) of joint health in young boys with severe haemophilia A (SHA) prescribed primary tailored prophylaxis. We also assessed whether age at first index joint bleed, blood group, FVIII gene abnormality variant, factor VIII trough level, first-year bleeding rate and adherence to the prescribed prophylaxis regimen significantly predicted joint damage trajectory, and thus class membership. METHODS: Using data collected prospectively as part of the Canadian Hemophilia Primary Prophylaxis Study (CHPS), we implemented a latent class growth mixture model technique to determine how many joint damage classes existed within the cohort. We used a multinomial logistic regression to predict the odds of class membership based on the above predictors. We fitted a survival model to assess whether there were differences in the rate of dose escalation across the groups. RESULTS: We identified three distinct classes of trajectory: persistently low, moderately increasing and rapidly increasing joint scores. By multinomial regression, we found that only age at first index joint bleed predicted rapidly increasing joint scores. The rapidly increasing joint score class group moved through dose escalation significantly faster than the other two groups. CONCLUSIONS: Using tailored prophylaxis, boys with SHA follow one of three joint health trajectories. By using knowledge of disease trajectories, clinicians may be able to adjust treatment according to a subject's predicted long-term joint health and institute cost-effective programmes of prophylaxis targeted at the individual subject level.

The challenge of genetically unresolved haemophilia A patients: Interest of the combination of whole F8 gene sequencing and functional assays.

BACKGROUND: The causative variant remains unidentified in 2%-5% of haemophilia A (HA) patients despite an exhaustive sequencing of the full F8 coding sequence, splice consensus sequences, 5'/3' untranslated regions and copy number variant (CNV) analysis. Next-generation sequencing (NGS) has provided significant improvements for a complete F8 analysis. AIM: The aim of this study was to identify and characterize pathogenic non-coding variants in F8 of 15 French and Canadian HA patients genetically unresolved, through the use of NGS, mRNA sequencing and functional confirmation of aberrant splicing. METHODS: We sequenced the entire F8 gene using an NGS capture method. We analysed F8 mRNA in order to detect aberrant transcripts. The pathogenic effect of candidate intronic variants was further confirmed using a minigene assay. RESULTS: After bioinformatic analysis, 11 deep intronic variants were identified in 13 patients (8 new variants and 3 previously reported). Three variants were confirmed to be likely pathogenic with the presence of an aberrant transcript during mRNA analysis and minigene assay. We also found a small intronic deletion in 6 patients, recently described as causing mild HA. CONCLUSION: With this comprehensive work combining NGS and functional assays, we report new deep intronic variants that cause HA through splicing alteration mechanism. Functional analyses are critical to confirm the pathogenic effect of these variants and will be invaluable in the future to study the large number of variants of uncertain significance that may affect splicing that will be found in the human genome.