Inhibitor Titer Research Articles

FRI549 The See-Saw Theory - Hypothyroidism Due To Hashimoto's Thyroiditis Followed By Hyperthyroidism Due To Graves' Disease

Abstract Disclosure: P. Sharma: None. Introduction: Hashimoto's thyroiditis is an autoimmune disorder characterized by chronic inflammation of the thyroid gland that leads to hypothyroidism due to formation of autoantibodies against thyroid antigens such as thyroid peroxidase (TPO Ab). Graves' disease is caused by stimulating TSH-receptor antibodies (TRAb) that cause hypertrophy of the thyroid gland leading to hyperthyroidism. While the transformation of Graves' disease to Hashimoto's thyroiditis has been reported, flipping from Hashimoto's thyroiditis to Graves' disease is uncommon. Case Report: A 62 year old female with 20 year history of hypothyroidism secondary to Hashimoto's thyroiditis presented to establish care. She had run out of levothyroxine refills one year prior during COVID-19 pandemic restrictions. She had tachycardia, diarrhea, fatigue, tremors, hair loss, dysphagia, and weight gain. Thyroid panel testing showed hyperthyroidism with an undetectable TSH (normal range 0.45-4.5uIU/ml) and elevated free T4 (2.84, normal 0.82-1.77ng/ml). Patient denied recent infections, iodine intake or iodine contrast exposure. Repeat labs confirmed hyperthyroidism with undetectable TSH with elevated free T4 (3.17ng/ml). Antibody testing comfirmed hyperthyroidism secondary to Graves disease (TRAb 20.7, normal <1.75IU/L). Patient has been on methimazole with fluctuating response and is reviewing definitive therapy options. Discussion: We demonstrate the rare phenomenon of thyroid function transformation from long-standing inhibition to stimulation after 20 years. Hashimoto's thyroiditis is characterized by chronic inflammation due to autoantibodies against TPO, thyroglobulin and the TSH receptor with gradual gland destruction and hypothyroidism. Stimulating autoantibodies to the TSH receptor (thyroid stimulating immunoglobulin (TSIg), TRAb) in Graves' disease lead to hyperthyroidism from gland hypertrophy. The flip from underactivity to overactivity can be explained by recovery of the gland from Hashimoto's thyroiditis injury which gradually leads to an overt response to TSIg, causing a hyperfunctioning gland. An imbalance between the inhibitory and stimulating antibodies can also lead to this transformation. Age, pregnancy and immunomodulating drugs such as alemtuzumab are also thought to play a role. The recovery theory is unlikely in our patient with a 20 year history of Hashimoto's thyroiditis. We believe that she had a high inhibitory antibody titre initially and now has higher titre of stimulating antibodies later, leading to Graves' disease. Luckily, she ran out of levothyroxine otherwise the additive effect of levothyroxine and intrinsic gland activity could have led to worse symptoms. This case highlights that autoimmune hypothyroidism and hyperthyroidism are conditions in the same spectrum, rather than two distinct entities as previously thought. Presentation: Friday, June 16, 2023

Stochastic Interventional Vaccine Efficacy and Principal Surrogate Analyses of Antibody Markers as Correlates of Protection against Symptomatic COVID-19 in the COVE mRNA-1273 Trial.

The COVE trial randomized participants to receive two doses of mRNA-1273 vaccine or placebo on Days 1 and 29 (D1, D29). Anti-SARS-CoV-2 Spike IgG binding antibodies (bAbs), anti-receptor binding domain IgG bAbs, 50% inhibitory dilution neutralizing antibody (nAb) titers, and 80% inhibitory dilution nAb titers were measured at D29 and D57. We assessed these markers as correlates of protection (CoPs) against COVID-19 using stochastic interventional vaccine efficacy (SVE) analysis and principal surrogate (PS) analysis, frameworks not used in our previous COVE immune correlates analyses. By SVE analysis, hypothetical shifts of the D57 Spike IgG distribution from a geometric mean concentration (GMC) of 2737 binding antibody units (BAU)/mL (estimated vaccine efficacy (VE): 92.9% (95% CI: 91.7%, 93.9%)) to 274 BAU/mL or to 27,368 BAU/mL resulted in an overall estimated VE of 84.2% (79.0%, 88.1%) and 97.6% (97.4%, 97.7%), respectively. By binary marker PS analysis of Low and High subgroups (cut-point: 2094 BAU/mL), the ignorance interval (IGI) and estimated uncertainty interval (EUI) for VE were [85%, 90%] and (78%, 93%) for Low compared to [95%, 96%] and (92%, 97%) for High. By continuous marker PS analysis, the IGI and 95% EUI for VE at the 2.5th percentile (519.4 BAU/mL) vs. at the 97.5th percentile (9262.9 BAU/mL) of D57 Spike IgG concentration were [92.6%, 93.4%] and (89.2%, 95.7%) vs. [94.3%, 94.6%] and (89.7%, 97.0%). Results were similar for other D29 and D57 markers. Thus, the SVE and PS analyses additionally support all four markers at both time points as CoPs.

Anti-FVIII antibodies in Black and White hemophilia A subjects: do F8 haplotypes play a role?

The most common complication in hemophilia A (HA) treatment, affecting 25% to 30% of patients with severe HA, is the development of alloimmune inhibitors that foreclose the ability of infused factor VIII (FVIII) to participate in coagulation. Inhibitors confer significant pathology on affected individuals and present major complexities in their management. Inhibitors are more common in African American patients, and it has been hypothesized that this is a consequence of haplotype (H)-treatment product mismatch. F8 haplotypes H1 to H5 are defined by nonsynonymous single-nucleotide polymorphisms encoding sequence variations at FVIII residues 1241, 2238, and 484. Haplotypes H2 to H5 are more prevalent in individuals with Black African ancestry, whereas 80% to 90% of the White population has the H1 haplotype. This study used an established multiplex fluorescence immunoassay to determine anti-FVIII antibody titers in plasma from 394 individuals with HA (188 Black, 206 White), measuring their binding to recombinant full-length H1 and H2 and B-domain-deleted (BDD) H1/H2, H3/H5, and H4 FVIII proteins. Inhibitor titers were determined using a chromogenic assay and linear B-cell epitopes characterized using peptide microarrays. FVIII-reactive antibodies were readily detected in most individuals with HA, with higher titers in those with a current inhibitor, as expected. Neither total nor inhibitory antibody titers correlated with F8 haplotype mismatches, andpeptides with D1241E and M2238V polymorphisms did not comprise linear B-cell epitopes. Interestingly, compared with the full-length FVIII products, the BDD-FVIII proteins were markedly more reactive with plasma antibodies. The stronger immunoreactivity of BDD-FVIII suggests that B-domain removal might expose novel B-cell epitopes, perhaps through conformational rearrangements of FVIII domains.

Acquired Hemophilia A after SARS-CoV-2 Infection: A Case Report and an Updated Systematic Review.

The role of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been implicated in the pathogenesis of acquired hemophilia A (AHA). The aim of this study is to report our case and to summarize clinical studies on de novo AHA after SARS-CoV-2 infection. We performed a systematic search on the association of SARS-CoV-2 with AHA in four medical databases up to 28 May 2023. Eligible studies should include de novo AHA patients who had SARS-CoV-2 infection before or concomitant with the diagnosis of AHA. Findings were synthesized narratively. In addition, we report the case of a 62-year-old female patient, who presented to our clinic with left flank pain 2 weeks after SARS-CoV-2 infection. Clinical investigations confirmed AHA and imaging studies revealed retroperitoneal bleeding. Her hemostasis was successfully secured with bypassing agents; however, despite immunosuppressive therapy, high inhibitor titer persisted. In the systematic review, we identified only 12 relevant cases with a questionable cause-effect relationship between SARS-CoV-2 infection and AHA. Based on the qualitative analysis of the relevant publications, current clinical evidence is insufficient to support a cause-effect relationship. The analysis of data from ongoing AHA registries can serve further evidence.

Combining Low-Dose Rituximab and Bortezomib as Immunosuppressive Therapy for Acquired Hemophilia A: 6 Case Series.

Acquired hemophilia A (AHA) is a rare bleeding disorder caused by autoantibodies against factor VIII. Persistently positive inhibitor titers increase the risk of bleeding. Upfront combined regimen with multiple mechanisms has the potential to shorten remission time to lower bleeding risk and reduce immunosuppressor exposure time. We administered lowered dose of rituximab and bortezomib in combination with corticosteroids and cyclophosphamide (rituximab: 100mg weekly × 4 or 500mg once in week 1, bortezomib: 0.65mg/m2 once in week 1, prednisone: 1mg/kg daily, cyclophosphamide: 11.5-2mg/kg daily). We retrospectively analyzed 6 cases (male = 3, female = 3, median age = 51years) treated on this therapeutic regimen. All patients achieved complete remission (range from19 to 66days); adverse event: infection (1/6). This is the first time that this regimen has been reported for treating AHA. It has shown good therapeutic efficacy and the potential to shorten the time to CR. The incidence of adverse events is within a reasonable range. This regimen is feasible and offers a new alternative treatment for AHA.

Early immune tolerance induction is a unique predictor of favorable outcomes in hemophilia A children with intron 22 inversion and high-responding inhibitors

BackgroundThe predictors of immune tolerance induction (ITI) outcomes in hemophilia A (HA) patients with the same F8 genetic background have not yet been evaluated, although the F8 genotype is strongly associated with ITI response. This study aims to explore the predictors of ITI outcomes in the same F8 genetic background by focusing on intron 22 inversion (Inv22) patients with high-responding inhibitors. MethodsHA children with Inv22 and high-responding inhibitors who received low-dose ITI therapy over 24 months were included in this study. ITI outcomes were centrally assessed at the 24th month of treatment. The predictive ability of clinical variables to identify ITI success was determined using the receiver operating characteristic (ROC) curve, and the predictor of ITI outcomes was analyzed on the multivariable Cox model. ResultsAmong the 32 patients investigated, 23 (71.9 %) achieved success. In univariate analysis, interval time from inhibitor diagnosis to ITI start (interval-time) was significantly associated with ITI success (P = 0.001); however, inhibitor titers showed no significance (P > 0.05). The interval-time demonstrated a good predictive value for ITI success with the area under the ROC curve of 0.855 (P = 0.002), and the cutoff value was 25.8 months (sensitivity, 87.0 %; specificity, 88.9 %). In the multivariable Cox model which considered success rate and time to success, interval-time was the only independent predictor (<25.8 months vs. ≥25.8 months, P = 0.002). ConclusionsThe interval-time was first identified as a unique predictor of ITI outcomes in HA patients with high-responding inhibitors under the same F8 genetic background (Inv22). An interval-time of <25.8 months was associated with increased ITI success and reduced time to success.

Low-dose immune tolerance induction therapy in severe hemophilia a children in China: Starting earlier resulted in better inhibitor eradication outcomes

BackgroundShorter interval-time from inhibitor detection to starting immune tolerance induction (ITI) might predict better ITI outcomes for severe Hemophilia A (SHA) patients with high-risk-inhibitors. However, the prediction-impact of interval-time for these patients on low-dose ITI strategy remained unclear. ObjectivesTo explore the relationship between interval-time and low-dose ITI outcomes in Chinese SHA children with high-risk-inhibitors. MethodsThis was a single-center, retrospective study on SHA children with high-risk-inhibitors (each with immediate pre-ITI inhibitor titer>10 Bethesda Units/mL) undergoing low-dose ITI strategy for ≥24 months. ITI outcomes and their predictive factors were evaluated at the 24th month treatment for each patient. The predictive ability of interval-time on ITI success was determined using receiver operating characteristic (ROC) curve. ResultsAmong 47 patients investigated, 34 (72.3 %) achieved success. Independent predictor for ITI-outcome on multivariate analysis included the interval-time (p = 0.007) and peak inhibitor-titer (p = 0.011). Shorter interval-time predicted ITI success [cut-off value = 22.3 months, area under ROC-curve (AUC) = 0.701] and early-ITI success within 12 month (cut-off value = 9.4 months AUC = 0.704). Linear regression analysis suggested each month interval-time delay delayed success by 0.1552 month. Unlike the interval-time, peak inhibitor-titer had no success-predictive value in high-peak inhibitor-titer patients on ITI with immunosuppressants. ConclusionsInterval-time represented a strong predictive value for outcomes in our low-dose ITI strategy for SHA patients with high-risk-inhibitors. Shorter interval-time was associated with higher success rate and earlier success achievement. The respective interval-time cut-off values were 22.3 months for ITI success and 9.4 months for early-success.

Biosynthesis of zinc oxide nanoparticles using saccharomyces boulardii and study their biological activities

Objective: Saccharomyces boulardii, a probiotic, is used in the current study’s manufacture of zinc oxide nanoparticles with the aim of assessing their biological activity. Due to its advantages over the chemical and physical techniques of synthesis in terms of affordability and environmental friendliness, the biological way of creating nanoparticles is becoming increasingly relevant. Methods: Zinc acetate was added at a dose necessary to biosynthesize ZnO NPs from S. boulardii’s cellfree supernatant (1 mM). Results: An evidence that S. bularedii was responsible for the reaction mixture’s color change from light to dark after 150 rpm of incubation was the color change and antibacterial behavior. Atomic force microscopy, scanning electron microscopy, energy dispersive spectroscopy, ultraviolet-visible spectroscopy, and X-ray diffraction (XRD) all contributed to the completion of the characterisation (AFM). The spectra of the ZnO NPs produced in the reaction mixture using UV-visible spectroscopy were (343.75 nm). The XRD showed that ZnO NPs’ crystal size was (13.31 nm). The SEM was provided; the shape was uniform and spherical, and the average size (24.61 nm). EDS was used to analyze the presence of elemental ZnO NPs. ZnO NPs’ three-dimensional structure was seen using AFM, and their average diameter was (62.89 nm). The FTIR spectrum reveals a variety of functional groups that are present at various locations. Gram positive and gram negative bacteria that were isolated from diabetic foot infections were multidrug resistant (MDR), and biosynthesized ZnO NPs demonstrated antibacterial action against these bacteria. (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis). All of the studied bacterial isolates showed the ability to produce biofilms in the form of nanoparticle-treated biofilms on microtiter plates. When pathogenic bacteria were treated with ZnO NPs, this ability was stopped and eliminated. The ZnO NPs (1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, and 0.12 mg/ml) demonstrated their antioxidant ability in vitro by scavenging DPPH free radicals. The combination of DPPH and biogenic ZnONPs at concentration 1 mg/ml showed the highest inhibitory titer (73.79%).

Perioperative therapeutic plasma exchange in a patient with rare Factor XIII inhibitor

IntroductionFactor XIII deficiency is a rare bleeding disorder which could be severe if inherited or less severe if acquired. We report a case of acquired Factor XIII inhibitor in a 75-year-old male with a suspicious left renal mass treated perioperatively with therapeutic plasma exchange (TPE). Patient and methodTo perform kidney biopsy and ablation of the renal mass, six daily TPE treatments were performed before and after biopsy to minimize bleeding risk because the patient did not respond to drug therapy. Both thromboelastography (TEG) and laboratory-based coagulation tests were performed to assess coagulation status prior to and after TPE. ResultsThe biopsy indicated oncocytoma which was removed by surgical procedure. Factor XIII activity remained below 15 % throughout TPE treatments, but Factor XIII inhibitor titer reduced from initial positive value of 1:40 to negative following the third TPE and remained negative through the sixth TPE. Unfortunately, the inhibitor titer was positive at 1:20 in the fifth month and 1:5 in the sixth month during follow-up. ConclusionsTPE is useful in removing XIII inhibitory factor, but the effects are only short term.

Recombinant factor VIII Fc fusion protein for first-time immune tolerance induction: final results of the verITI-8 study

AbstractInhibitor development remains a major challenge in factor VIII (FVIII) replacement therapy. verITI-8 is the first prospective study of a recombinant FVIII Fc fusion protein (rFVIIIFc; efmoroctocog alfa) for first-time immune tolerance induction (ITI) in males with severe hemophilia A and high-titer inhibitors (historical peak ≥5 Bethesda units [BU]/mL). In this single-arm, open-label, multicenter study, screening was followed by ITI (rFVIIIFc 200 IU/kg per day until tolerization or maximum of 48 weeks). Those who achieved ITI success entered a tapering period, returning to standard prophylaxis, and then entered follow-up. Primary end point was time to tolerization with rFVIIIFc defined by inhibitor titer <0.6 BU/mL, incremental recovery (IR) ≥66% of expected IR (IR ≥1.32 IU/dL per IU/kg), and half-life (t½) ≥7 hours within 48 weeks. Sixteen patients received ≥1 rFVIIIFc dose. Twelve (75%), 11 (69%), and 10 patients (63%), respectively, achieved negative inhibitor titers, an IR ≥66%, and a t½ ≥7 hours (ie, tolerance) within 48 weeks. Median times in weeks to achieve these markers of success were 7.4 (interquartile range [IQR], 2.2-17.8), 6.8 (IQR, 5.4-22.4), and 11.7 (IQR, 9.8-26.2), respectively. All patients experienced ≥1 treatment-emergent adverse event (TEAE), and 1 reported ≥1 related TEAE (injection site pain). Nine patients experienced ≥1 treatment-emergent serious AE. No thrombotic events, discontinuations because of AEs, or deaths were reported during the study. As the first extended half-life rFVIII with prospective data in ITI, rFVIIIFc offered short time to tolerization with durable responses in almost two-thirds of patients and was well tolerated. This trial was registered at www.clinicaltrials.gov as #NCT03093480.

POSTER ABSTRACTS

POSTER ABSTRACTS

High levels of factor VIII activity in patients with acquired hemophilia A in remission are associated with unusually low coagulation potentials.

Elevated factor VIII activity (FVIII:C) is often observed in patients with acquired hemophilia A (PwAHA) in remission. However, comprehensive coagulation potentials in this patient group remain to be investigated. To evaluate comprehensive coagulation potentials in PwAHA. We investigated coagulation function in eleven PwAHA with high FVIII:C (> 150IU/dL) using thrombin generation assay (TGA) and/or rotational thromboelastometry (ROTEM), and compared findings with results obtained from contrived samples generated by spiking recombinant FVIII. The median FVIII:C and FVIII inhibitor titers during remission in enrolled PwAHA were 206IU/dL and 0.44 BU/mL, respectively. In all patients, lag time and time to peak were either prolonged or normal compared to contrived samples corresponding to their FVIII:C. However, higher values of peak thrombin and endogenous thrombin potentials compared to contrived samples were observed in two patients. ROTEM parameters were within normal ranges in all cases. One patient (FVIII:C 171IU/dL) developed venous thrombosis and pulmonary embolism, but TGA parameters showed low or normal coagulation potential compared to contrived samples corresponding to his FVIII:C. PwAHA with high FVIII:C could exhibit lower coagulation potentials than those corresponding to their FVIII:C.

The diagnosis and Management of HIV Associated Acquired Hemophilia A: A Case Series From Durban, South Africa.

In acquired hemophilia A (AHA), the body produces auto-antibodies against Factor VIII. Although AHA is rare, with an incidence of 1.5 patients/1 million population/year, there is a strong association with human immunodeficiency virus (HIV) infection. The accurate interpretation of screening coagulation tests is critical to identify patients with AHA, as the mortality rate secondary to bleeding is high. This was a retrospective case series which included all newly diagnosed AHA patients that were referred to Hemophilia care unit at King Edward VIII Hospital, Durban, South Africa from January 2011 to December 2021. The clinical presentation and laboratory results were documented. Five patients were included in this case series. All patients were females aged between 28 and 64 years of age and they were HIV seropositive. They presented with spontaneous cutaneous and intramuscular bleeding. Four patients were virologically suppressed on anti-retroviral therapy, and no patient had a family history of congenital bleeding diathesis. Laboratory investigations confirmed AHA with high Factor VIII inhibitor titers, which ranged from 41 to 900 Bethesda Units (BU). All patients were managed with bypassing agents and oral corticosteroids. The monitoring of patients after the initiation of treatment was difficult as they all defaulted treatment. In view of the prevalence of HIV in sub-Saharan Africa, there is a possibility that AHA is under-diagnosed in our setting. The clinician and the laboratory have a combined critical role in identifying patients with AHA as the investigation of a prolonged APTT is mandatory. There are challenges in managing AHA patients in a resource-constrained setting.

Determinants of successful immune tolerance induction in hemophilia A: systematic review and meta-analysis

BackgroundImmune tolerance induction (ITI) aims to eradicate anti-factor VIII (FVIII) antibodies (inhibitors) in persons with hemophilia A. However, this burdensome treatment fails in 10% to 40%. To estimate the chance of ITI success in clinical decision making, it is important to identify the predictors of ITI success. ObjectivesWe performed a systematic review and meta-analysis to summarize the current evidence on determinants of ITI outcome in persons with hemophilia A. MethodsA literature search was conducted to identify randomized controlled trials, cohort, or case-control studies reporting on the predictors for ITI outcome in persons with hemophilia A. The main outcome was ITI success. Methodological quality was assessed using an adapted Joanna Briggs Institute checklist, rating as high if ≥11 of 13 criteria were met. Pooled odds ratios (ORs) for ITI success were calculated for each determinant. ITI success was defined as negative inhibitor titer (<0.6 BU/mL), FVIII recovery ≥66% of expected, and FVIII half-life ≥6 hours in 16 (59.3%) studies. ResultsWe included 27 studies, involving 1,734 participants. Methodological quality of 6 (22.2%) studies (418 participants) was rated as high. Twenty different determinants were assessed. Historical peak titer ≤100 BU/mL (compared with >100 BU/mL, OR, 1.7; 95% CI, 1.4-2.1), pre-ITI titer ≤10 BU/mL (compared with >10 BU/mL, OR, 1.8; 95% CI, 1.4-2.3), and peak titer during ITI ≤100 BU/mL (compared with >100 BU/mL, OR, 2.7; 95% CI, 1.9-3.8) were associated with a higher chance of ITI success. ConclusionOur results suggest that determinants related to the inhibitor titer are associated with ITI success.

FVIII inhibitor surveillance in children with hemophilia A in Indonesia: a report from the Indonesian Pediatric Hematology-Oncology Working Group.

Factor VIII (FVIII) inhibitor diagnosis and surveillance in Indonesia are challenging owing to geographic conditions and the lack of laboratory facilities nationwide for inhibitor assays. This study aimed to determine the prevalence of FVIII inhibitors in children diagnosed with hemophilia A (HA) in Indonesia. A cross-sectional study was conducted in 12 hospitals in eight provinces of Indonesia between 2020 and 2021. Factor VIII inhibitor screening was performed in a central hemostasis laboratory for all children with HA (≤18 yr) who had received a minimum of 10 exposure days to clotting factor concentrates. The FVIII inhibitor titer was determined using the Bethesda assay. Children (388) were enrolled in this study, including 219 (56.4%), 131 (33.8%), and 38 (9.4%) with severe, moderate, and mild HA, respectively. The prevalence of children who developed FVIII inhibitors was 37 out of 388 (9.6%). Factor VIII inhibitors were found in 25/219 (11.4%) severe, 11/131 (8.3%) moderate, and 1/38 (2.6%) children with mild HA. Thirteen children had low-titer inhibitors and 24 had high-titer inhibitors, with a median of 9.44 (1.48‒412.0) Bethesda Units. Among 13 children with low-titer inhibitors, eight underwent a confirmation test, of which five tested negative and were classified as transient. A significant difference in annual joint bleeding rate was found between patients with low and high inhibitor titers and those without inhibitors (P＜0.001). Factor VIII inhibitor prevalence in Indonesia was relatively low. However, the risk factors that may contribute to FVIII inhibitor development among Indonesian patients require further study.

Postpartum Acquired Hemophilia (PAH) – A Rare Entity

Objective: Acquired Hemophilia A (AHA) is a rare autoimmune disease that can result in life threatening bleeding. Formation of auto-antibodies (inhibitors) against coagulation factor VIII develops the hemorrhagic syndrome that may appear at any age; however, prevalence of AHA is chiefly observed in post-partum and elderly persons. Idiopathic acquired hemophilia is commonest type of acquired hemophilia whereas it is also associated with autoimmune disease, malignant neoplasm, medication and vaccinations. Post-partum acquired hemophilia (PAH) may develop due to development of antibodies against fetal FVIII. In this case report, 36-year-old Asian woman presented with complain of bruises from 2 months. There was history of cesarean section four months ago. Family history, past medical and drug history were not significant. As there was no active bleeding, patient was started an oral prednisolone along with azathioprine and was counseled regarding disease and precautions. Patient presented after 2 weeks with severe PV bleed, anemia secondary to self-induced medicine abortion. For this she receives pack cells and cryoprecipitate. Steroids and azathioprine were continued and patient was discharged in stable condition. Her ultrasound pelvis however showed small fibroids. Patient went back to her village where she again develops severe PV bleed and for that she underwent hysterectomy. Patient presented in our hospital with severe operative site bleeding, pain and anemia. She was managed with Packed Red Blood (PRBCs), Fresh Frozen Plasma (FFP), Recombinant Activated Clotting Factor VII (rFVIIa), prednisolone IV and azathioprine PO. Her Activated Partial Thromboplastin, Hemoglobin were continuously monitored. After a week bleeding was stopped but Activated Partial Thromboplastin Time (APTT) was still prolonged so we tapered rFVIIa dose and advice two sessions of plasmapheresis. APTT, hemoglobin levels, inhibitor titer, High-Sensitivity C – reactive protein (hs-CRP) levels were improved. Patient was discharged in a stable condition and was advised to continue medication at home with supportive care and wound dressing.

Follow-up and Evaluation of the Efficacy of the Hemostatic Treatment in Congenital FVII Deficiency during Development of Inhibitor

Introduction The development of inhibitors is an extremely rare complication in patients with factor (F)VII deficiency treated with replacement therapies such as recombinant activated FVII (rFVIIa), and represents a major therapeutic challenge in the treatment of this disorder. When inhibitors result in loss of response to high doses of rFVIIa, activated prothrombin complex concentrate (aPCC) is often used as an alternative treatment for these patients. The use of high-dose rFVIIa or aPCC might be accompanied by an increased risk of thromboembolic events, so it is important monitoring properly the effect of the hemostatic treatment Here we describe the therapeutic management and follow-up of a patient with elevated inhibitor titres, resistant to rFVIIa treatment who switched to prophylaxis with aPCC, using several global coagulation tests. Methods The coagulation status of the patient and the reponse to in vitro spiking of different doses of rFVIIa or aPCC were assessed with several global tests: Prothrombine time (PT) was tested using HemosIL®RecombiPlasTin 2G. Clot formation was evaluated with Rotational Thromboelastometry (ROTEM®) using blood with CTI (to inhibit contact activation) and activated with a low amount of Tissue Factor (TF) as trigger (dilution 1:50,000 of EXTEM reagent). The thrombin generation (TG) was tested with the Calibrated Automated Thrombogram (CAT) system using plasma collected with CTI and activated with 1 pM TF plus phospholipids (PPP-Low®, Stago). Thrombus formation under flow conditions was assessed with the Total Thrombus-formation Analysis System (T-TAS®) in AR-chips coated with collagen and thromboplastin. The Area under the flow pressure curve (AUC) was calculated over 30 min after starting. Results Three year-old girl affected by severe congenital FVII deficiency (FVII 9.1%) with significant severe bleeding history (spontaneous intracranial hemorrhage at 1st month of life). Since then she is in prophylaxis treatment with rFVIIa. At 6 months of life she developed an inhibitor (9.8 BU). Despite prophylaxis she presents frequent spontaneous mucocutaneous bleedings. When she was 2 years old she needed to be admitted at hospital because of port-A-Cath infection. Due to the administration of intensive treatment with rFVIIa during admission she developed thrombosis of peripheral venous access, so prophylaxis treatment was temporary suspended. After 5 days without prophylaxis treatment, and increasing inhibitor titres, she developed a new intracranial haemorrhage, which needed intensive treatment with continuous rFVIIa infusion (90 µg/Kg/12h), immunoglobulines and rituximab for lowering inhibitor titre. Hemostatic parameters improved during the administration of these treatments and inhibitor level was reduced. After restarting prophylaxis treatment with rFVIIa (50 µg/Kg/48h), inhibitors and a hypocoagulable state were observed with all global coagulation test showing extended PT, clotting time (CT), TG Lag-time (LT) and anomalous thrombus formation (AUC) but presented very good response to low dosis (2.5 IU/Kg) of aPCC in vitro (Table-1). At the age of three years she required admission at hospital because of port-A-Cath infection again, needing its replacement by a new one after administration of high doses of rFVIIa without bleeding complications. After this, inhibitor titre raised again (> 50 BU), presenting frequent bleeding episodes (spontaneous muscle bleedings and tonsillar bleeding), and due to the clinical worsening, treatment was modified to prophylaxis with aPCC (30 UI/Kg/48h). Despite the treatment change she continued presenting bleeding episodes, global tests continued showing an hypocoagulable state, and required 10 times higher doses in vitro of aPCC (25 IU/Kg) to restore all the monitored hemostatic parameters (Table-1), probably related to the important increase in inhibitor titre. This situation led to employing higher doses of aPCC prophylaxis (60 UI/Kg/48 h) to try to avoid bleeding episodes without increasing the risk of suffering thrombotic events. Conclusions Global coagulation tests used in this study were sufficiently sensitive methods to show an abnormal hemostatic profile in FVII-deficient patients with clinical hemorrhagic symptoms with inhibitor development, supporting their use to manage the risk of bleeding or thrombosis in this life-threatening condition. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Protocol and Primary Analysis of Lome: A Randomized Trial of Low- Versus Intermediate-Dose Immune Tolerance Induction with Recombinant Factor VIII (Omfiloctocog alfa, SCT800) in China

Background: Although the cohort and registry data support low-dose (LD; 50 IU/kg once every other day) and intermediate-dose (MD; 100 IU/kg daily) immune tolerance induction (ITI) are almost equally effective in inducing tolerance in people with severe hemophilia A, with inhibitors (PwHI)1-3, there has been no prospective randomized trials, especially in China. Here, we present the protocol and results of the primary analysis in LOME (ChiCTR2200056603). Methods LOME is a multicenter, prospective, randomized comparison of LD and MD ITI, in severe hemophilia A (HA) patients (FVIII level < 1%) with peak historical inhibitor ≥ 5 and < 200 Bethesda Unite (BU/ml). In the first year of recruitment (Part A), patients with age ≤ 8 years at time of randomized will be included. In the second year of recruitment (Part B), patients with age ≤ 12 years at time of randomized will be included. Patients have received ITI therapy before randomization will be excluded. Subjects were randomized 1:1 to LD or MD ITI with the first domestic recombinant factor VIII (rFVIII) (Omfiloctocog alfa, SCT800) approved in China. Factor III (FVIII) inhibitor screening, ITI monitoring and dose tapering will be based on the consensus from United Kingdom Hemophilia Center Doctors' Organization (UKHCDO)4. If (a) there is an upward trend in titer over 3-6 months period; or (b) fall in chromogenic Bethesda titer of less than 20% in 6 months period after initial peak inhibitor titer; or (c) titer ≥ 200BU/ml during ITI therapy; or (d) spontaneous bleeding > 1 monthly over 3-6 months period, modify the regimen as below: In the LD arm, increase to 100IU/kg daily;In the MD arm, rituximab 375 mg/m2 weekly × 4 weeks (maximum 600 mg) and prednisone 2 mg/kg daily × 1 month (maximum 60 mg, tapering over 3 months) would be added. Once the patient had achieved Success, taper dose as below: In the LD arm, the FVIII dose would be reduced slowly to 25IU/kg three times a week for continuing prophylaxis;In the MD arm, the FVIII dose would be reduced slowly to LD ITI, then same as LD arm. The primary endpoints are ITI outcomes (Success: negative inhibitor titer and FVIII recovery ≥ 66% of expected) and the time taken to ITI Success, in PwHI with LD ITI compared to those with MD ITI. Key secondary endpoints include annualized bleeding rate (ABR), annualized joint bleeding rate (AJBR) for any bleeds and treated bleeds; joint and bone health assessed by Hemophilia Joint Health Score 2.0 (HJHS 2.0) and ultrasound; quality of life measured by the Canadian Hemophilia Outcomes-Kids Life Assessment Tool 2.0 (CHO-KLAT 2.0) and numbers of days out of school; average consumption cost (per kg body weight) was calculated based on the intermediate number of treatment doses consumed to achieve Success. This included the cost of rFVIII and rituximab for ITI, prothrombin complex concentrate (PCC) or recombinant activated factor VII (rFVIIa) for treatment of breakthrough bleed, and intravenous immunoglobulin (IVIG) for infection prevention in patients receiving immunosuppressants. Safety was also a secondary endpoint. Results: Ten subjects were randomized into the study with median follow-up time of 1.64 [range (minimum, maximum), 0.23, 3.91] months in LD arm, and 2.66 (0.26,3.91) months in MD arm. Baseline includes age at time of randomized, age of inhibitor detection, exposure days before inhibitor detection, initial inhibitor titer, inhibitor titer at start of ITI and so on, between two arms are summarized in Table 1. Twenty percent (1/5) in MD arm achieved Success after 7 weeks of treatment and still maintained until the latest follow-up. No one achieved Success in LD arm. The bleeds between the treatment arms are summarized in Table 2. Nearly 2-fold difference in mean ABR and AJBR for treated bleeds, between treatment arms, and 3- to 4-fold in ABR and AJBR for any bleeds. Conclusions: It is planned to enroll 40 patients in Part A and 60 patients in Part B, who will be followed for up to 2 years. The study is ongoing and, currently, 10/40 (25%, in Part A) PwHI from 4 HTCs have been included. LOME study is the first, multicenter, prospective, randomized trial of LD vs MD ITI with domestic rFVIII (Omfiloctocog alfa, SCT800) in Chinese PwHI. This study is likely to contribute with the choice of optimal ITI regimen. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Prophylactic Treatment of Severe Haemophilia a Patients with Inhibitors to FVIII with Peglip-FVIII

Background: FVIII replacement therapy is ineffective for severe haemophilia A (HA) patients who develop inhibitors to FVIII. Patients with intractable inhibitors currently require FVIII mimetics and/or bypassing agents to prevent bleeding. A previously presented interim analysis[1] demonstrated that severe haemophilia A patients with a clinical history of developing inhibitors to FVIII could be prophylactically treated with recombinant FVIII co-injected with PEGylated liposomes (PEGLip), termed PEGLip-FVIII and may present an option for the prophylactic treatment of inhibitor patients. Aims: To establish safety and efficacy of PEGLip-FVIII and demonstrate (a) that PEGLip-FVIII administered intravenously (IV) to severe HA patients with history of inhibitors to FVIII enhances their clotting activity and (b) that PEGLip-FVIII administered to severe HA patients with active inhibitors to FVIII enhances their clotting activity. Methods: Stage A: Thirteen patients that were clinically defined as inhibitor patients were given single IV injections of PEGLip-FVIII (simoctocog alfa) at a dose of 22mg/kg PEGLip + 35 IU/kg FVIII and assessed for clotting activity at 0 hours (pre-injection) and at 20min, 1, 2, 4, 8, 24 hours, and daily thereafter up to 7 days using Rotational Thromboelastometry. Stage B: Patients received IV injections of PEGLip-FVIII for 6-weeks at a frequency determined by the investigator based on results obtained during Stage A. Eight of the patients had a history of developing inhibitors to FVIII but did not have active inhibitors at screening; the remaining five patients had active low-titre inhibitors at screening. Inhibitor titres were monitored throughout. Results: Results are shown in the table. Conclusion: Treatment with PEGLip-FVIII was well tolerated with no clinically significant changes in inhibitor titres in inhibitor patients in both groups. No adverse drug reactions were reported. The mean frequency of administration of PEGLip-FVIII was every 5.5±1.3 days. The mean number of bleeding episodes reported during Stage B was significantly reduced by 74% to 0.3±0.6 per month compared with 1.0±0.4 per month recorded during the 24 weeks prior to enrollment. Conclusion: PEGLip-FVIII in inhibitor patients demonstrated efficacy in preventing spontaneous bleeds without increasing inhibitor titres, indicating a novel FVIII-based treatment for this cohort. The dosing interval of 5.5days at 35IU/kg represents a significant reduction in FVIII infusion frequency. Acknowledgements: The Authors acknowledge the co-operation of the following clinicians in undertaking the study: Nadezhda Zozulia1, Margarita Timofeeva2, Tatiana Pospelov32, Marina Kosinova4, Igor Kurtov5,Heghine Khachatryan6. 1National Medical Research Centre of Hematology, Moscow.2Scientific Research Institute of Hematology and Blood Transfusion, Kirov, Russian Federation, 3State Medical University, City Hematology Centre, Novosibirsk, Russian Federation, 4District Clinical Hospital, Kemerovo, Russian Federation, 5State Medical University, Samara, Russian Federation, 6Haematology Center after professor R. Yeolyan, Yerevan, Armenia. [1] Timofeeva M et al. Prophylactic treatment of severe haemophilia A patients with inhibitors to FVIII with PEGLip-FVIII. Blood 2021; 138 (Suppl 1): 1040 (abstract) Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Inhibiting Activated Protein C Cleavage of Factor VIII for Hemophilia a Gene Therapy

Background Despite progress in adeno-associated virus (AAV) mediated factor VIII (FVIII) gene transfer for hemophilia A (HA), durable and sustained FVIII expression sufficient to ameliorate bleeding in all participants is an unrealized goal. Following the success of using the gain of function FIX-Padua variant in hemophilia B gene therapy, we hypothesized that use of an enhanced hemostatic function FVIII would similarly improve HA gene therapy by: 1) permitting use of lower vector doses, thereby overcoming dose-dependent safety and efficacy limitations and 2) achieving efficacy with lower FVIII antigen. We previously published a FVIII variant resistant to activated protein C (APC) cleavage (FVIII-R336Q/R562Q or FVIII-QQ) with normal specific activity and 4-5 fold improved hemostatic function in recombinant protein experiments (Wilhelm et al. Blood 2021). Aims We evaluated the possible clinical translation of FVIII-QQ for HA AAV-mediated gene therapy by determining the hemostatic benefit and potential prothrombotic and immunological risks in mice. Additionally, FVIII-QQ steady-state expression was 2.5 to 5-fold higher than FVIII-WT. We hypothesized this was due to disrupted cleavage by APC or other serine proteases at R336 and/or R562. Results Survival studies in HA/CD4KO and WT/CD4KO mice treated with AAV vectors to express (h)uman FVIII-WT and hFVIII-QQ in the range of mild HA, normal, and supratherapeutic FVIII levels thus far demonstrate no significant differences in survival at 7-11 month follow up or D-dimer levels after 8 months. Additionally, FVIII-QQ mice were generated using CRISPR/Cas9 to introduce R562Q (WT mice are Q336) for endogenous (m)ouse FVIII-QQ expression. Endogenous mFVIII-QQ mice were viable, fertile, normal weight, and D-dimers did not significantly differ from WT mice, while FVIII activity was ~2-fold higher than WT mice. HA immune competent mice treated with either 6 weekly doses of hFVIII-WT or hFVIII-QQ protein (0.2 μg) or AAV vector to express hFVIII-QQ or hFVIII-WT had no significant differences in inhibitor titers. To evaluate hemostasis, HA/CD4KO mice were treated with AAV vector only differing by 2 amino acids (R336, R362) at 3 different doses (n=6-12 mice/cohort) to express hFVIII-QQ or hFVIII-WT and underwent tail clip challenge. Estimated EC50 and EC80 of vector doses required to normalize blood loss to WT mice were 5 and 10-fold lower, respectively, for hFVIII-QQ versus hFVIII-WT supporting a hemostatic benefit to using the hFVIII-QQ transgene. Interestingly, across 3 different vector constructs differing only by F8 codon optimization, we observed hFVIII-QQ antigen values 2.5 to 5-fold higher than in mice expressing hFVIII-WT. Analysis of liver tissue from HA/CD4KO mice treated with the same vector construct and dose to express hFVIII-WT or hFVIII-QQ demonstrated approximately the same mRNA values, but plasma hFVIII-QQ antigen was 4-fold higher than hFVIII-WT and intracellular hFVIII-QQ antigen was the same or lower than hFVIII-WT antigen. Consistent with our in vivo AAV experiments, preliminary studies of intracellular versus secreted hFVIII antigen in BHK stable cell lines expressing hFVIII-WT or hFVIII-QQ demonstrated similar mRNA values with higher relative secreted hFVIII-QQ protein. To further probe the mechanism of higher hFVIII-QQ steady-state expression, we evaluated a panel of serine proteases (APC, FIXa, FXa, plasmin, PACE/furin and thrombin) for cleavage of FVIII-QQ relative to FVIII-WT protein. As expected, hFVIII-QQ was resistant to APC cleavage. Surprisingly, all other serine proteases tested demonstrated cleavage of hFVIII-WT, but not hFVIII-QQ, at R336 and/or R562 suggesting protease attack at these cleavage sites may occur more than previously appreciated, though the physiologic significance remains to be determined. Conclusion Our studies outline the use of hFVIII-QQ transgene expression thus far does not demonstrate safety concerns and confers hemostatic benefit over hFVIII-WT via: 1) improved hemostatic function and 2) higher steady-state expression. Enhanced steady state hFVIII-QQ expression relative to hFVIII-WT was observed across multiple model systems. Preliminary data demonstrate multiple serine proteases can cleave at the described FVIII APC cleavage sites (R336, R562) suggesting hFVIII-QQ may generally be more proteolytically stable thereby permitting improved steady-state expression.