Electronic diaries in the management of haemophilia gene therapy: Perspective of an expert group from the German, Austrian and Swiss Society on Thrombosis and Haemostasis (GTH).

INTRODUCTION: Gene therapy (GT) is becoming a realistic treatment option for patients with haemophilia. Outside clinical trials, the complexity and potential complications of GT will pose unprecedented challenges to haemophilia care centres. AIM: To explore the potential use of electronic tools to improve the delivery of GT under real-world conditions. METHODS: Considering the hub-and-spoke model, the GTH working group on GT considered the entire patient pathway and reached consensus on requirements for an integrative software tool to secure documenting and sharing information between treaters, pharmacies and patients. RESULTS: Six steps of the gene therapy process were identified, each requiring completion of the previous step as a prerequisite for entry. The responsibilities of GT dosing and follow-up treatment centres, read/write access rules, and the minimum data set were outlined. Data contributed by patients through mobile devices was also considered. CONCLUSION: Important information needs to be shared between patients and treatment centres in a real-world GT hub-and-spoke model. Collecting and sharing this information in well-organised electronic applications will not only improve patient care but also enable national and international data collection in clinical registries.

Bleeding signs due to acquired von Willebrand syndrome at diagnosis of chronic myeloid leukaemia in children.

A considerable proportion of patients with chronic myeloid leukaemia (CML) may present at diagnosis with high platelet counts. This may result in thrombosis or bleeding complications due to binding of von Willebrand factor (VWF) multimers to platelets. Paediatric CML is very rare and no systematic investigation on clinical complications of elevated platelets has been reported. Data on platelet count and associated haemostaseological complications were retrospectively analysed in a cohort of 156 children with CML. Fifty-one percent (81/156) patients presented with thrombocytosis (platelet count> 500 × 109 /l), and were extreme (>1 000 × 109 /l) in 23/156 (16%). There were no cases of thrombosis but mild bleeding signs were present in 12% (n = 9) children with thrombocytosis. Bleeding occurred without correlation to elevated platelet counts and was associated with reduced large VWF multimers, indicating a diagnosis of acquired von Willebrand syndrome (AVWS), which resolved after initiation of CML treatment. Patients with paediatric CML frequently exhibit high platelet counts not resulting in thrombosis. In patients with thrombocytosis mild bleeding signs due to a low percentage of large VWF multimers can be demonstrated. AVWS may be underdiagnosed in paediatric CML (Clinical-Trials.gov NCT00445822, 9 March 2007).

Platelet dysfunction caused by a novel thromboxane A2 receptor mutation and congenital thrombocytopenia in a case of mild bleeding.

Chronic hemorrhagic diathesis in patients showing normal levels of plasmatic clotting factors strongly suggests for congenital platelet disorders. We report on a pediatric patient (male, 3 years, D1) with mild bleeding. A sibling (D2), his mother (D3) and father (D4) were included for laboratory investigation. Platelet counts in D1, D2 and D4 indicated mild thrombocytopenia (100 Gpt/L). D1 and D3 platelets showed significantly diminished aggregation response on arachidonic acid and U46619 stimulation. Immunostaining for platelet proteins on blood smears of D1 and D2 indicated defects in ß1-tubulin. Exon sequencing of TBXA2R and TUBB1 revealed heterozygosity for the novel TBXA2R*c.908T>C (p.L303P) mutation in D1 and D3. TUBB1 was either wild type (D2, D3) or heterozygous (D1, D4) for the common polymorphism TUBB1*c.920G>A (rs6070697; p.R307H). In conclusion, the bleeding phenotype of the index patient can be explained by a diminished platelet function caused by the TBXA2R*c.908T>C mutation inherited from the mother and a mild thrombocytopenia with unknown molecular basis that is inherited from the father.

Duodenocolonic Fistula As A Rare Complication of Intestinal Burkitt Lymphoma in a Three-Year-Old Boy.

BACKGROUND: Burkitt lymphoma (BL) in children often presents with abdominal localization. Intestinal perforations have been described mainly during treatment. We report on a three-year-old patient with abdominal BL who was diagnosed with a duodenocolonic fistula. CASE REPORT: A three-year-old boy presented with diarrhea, crampy abdominal pain, and a four-week history of loss of appetite and weight. Ultrasound and MRI detected a colonic tumor forming a duodenocolonic fistula which was verified by gastroduodenoscopy. A surgical biopsy revealed BL. The stage III BL with low LDH was treated with four courses of BFM-type short-pulse chemotherapy. After two courses of chemotherapy the patient developed a mechanic ileus. A segmental resection of a short segment of the colon at the right flexure carrying the residual tumor mass with cicatricial stenosis and fistula followed by colonic end to end anastomosis and covering of the fistula by omentum major were carried out without complication. 15 days after surgery, two additional courses of chemotherapy could be administrated and the boy is in ongoing remission and free of any symptoms with a follow-up interval of 18 months. CONCLUSIONS: Duodeonocolonic fistula at presentation in a child with abdominal BL is extremely rare. Delayed surgery after size of the tumor bulk has been reduced by chemotherapy might represent a risk adapted approach. However, due to limited experience with duodenocolonic fistulas even in larger pediatric lymphoma trials any decision has to be based on the problems to be faced in individual cases.

Childhood primary large vessel CNS vasculitis: single-centre experience and review of the literature.

Ischaemic brain injuries are rare conditions in the paediatric age group. Main causes include non-arteriosclerotic arteriopathies, which in childhood usually result from primary vasculitis of large or small vessels and lead to impaired perfusion and subsequent ischaemic brain lesions. In accordance with the nomenclature of systemic forms, CNS vasculitis is subdivided into groups, based on the size of affected vessels: angiography-positive primary angiitis of medium-sized and large vessels (pPACNS), and angiography-negative angiitis of small vessels (svPACNS). We report the clinical presentation, diagnostic approach, and therapy of four children with progressive pPACNS. Patients were treated with high-dose corticosteroids and anticoagulation with unfractionated heparin in the acute phase, followed by immune modulatory treatment with mycophenolate mofetil (MMF) and dual antiplatelet therapy with acetylsalicylic acid and clopidogrel. In this manuscript, we illustrate the experience gained in our hospital, resulting in significantly faster diagnosis and treatment initiation, and discuss the applied immune modulating treatment regimen in the context of the literature. Based on our observations, we conclude that immune modulating therapy with initial high-dose corticosteroids, followed by steroid-sparing maintenance treatment with MMF, may be safe and effective in childhood progressive pPACNS.

Clinical and laboratory characteristics of children with venous thromboembolism and protein C-deficiency: an observational Israeli-German cohort study.

Venous thromboembolism [TE] is a multifactorial disease and protein C deficiency [PCD] constitutes a major risk factor. In the present study the prevalence of PCD and the clinical presentation at TE onset, including neonatal purpura fulminans, in a cohort of children are reported. In 367 unselected children (0·1-19 years) recruited between July 1996 and December 2013, a comprehensive thrombophilia screening was performed along with recording of anamnestic data. Twenty-five of 338 children (7·4%) had PCD. Mean age at first TE onset was 10 years (range 0·1-18). Leading thromboembolic manifestations were neonatal purpura fulminans (n = 5), TE of cerebral veins (n = 3), stroke (n = 2) deep veinthrombosis (DVT) of the leg (n = 10), DVT & pulmonary embolism (n = 2) and DVT & pelvic veins (n = 3). Concomitant risk factors for TE were identified in 12 patients, whereas 13 children spontaneously developed TE. A positive family history of DVT was found in 10 children. In this unselected cohort of paediatric patients with symptomatic TE the overall prevalence of PCD was 7·4%; 1·5% presented with neonatal purpura fulminans. Given its clinical implication for patients and family members, thrombophilia testing should be performed and the benefit of medical or educational interventions should be evaluated in this high-risk population.

Treatment of Inherited Platelet Disorders: Current Status and Future Options.

Inherited platelet disorders (IPDs) comprise a heterogeneous group of entities that manifest with variable bleeding tendencies. For successful treatment, the underlying platelet disorder, bleeding severity and location, age, and sex must be considered in the broader clinical context. Previous information from the AWMF S2K guideline #086-004 (www.awmf.org) is evaluated for validity and supplemented by information of new available and future treatment options and clinical scenarios that need specific measures. Special attention is given to the treatment of menorrhagia and risk management during pregnancy in women with IPDs. Established treatment options of IPDs include local hemostatic treatment, tranexamic acid, desmopressin, platelet concentrates, and recombinant activated factor VII. Hematopoietic stem cell therapy is a curative approach for selected patients. We also provide an outlook on promising new therapies. These include autologous hematopoietic stem cell gene therapy, artificial platelets and nanoparticles, and various other procoagulant treatments that are currently tested in clinical trials in the context of hemophilia.

Impact of Tyrosine Kinase Inhibitors Applied for First-Line Chronic Myeloid Leukemia Treatment on Platelet Function in Whole Blood of Healthy Volunteers In Vitro.

The tyrosine kinase inhibitors (TKIs) imatinib, dasatinib, bosutinib, and nilotinib are established for first-line treatment of chronic myeloid leukemia (CML) but may cause side effects such as bleeding and thrombotic complications. We investigated the impact of TKIs on platelet function ex vivo in anticoagulated whole blood (WB) samples from healthy adults by lumiaggregometry and PFA-100 test. Samples (n = 15 per TKI) were incubated for 30 minutes with TKI at therapeutically relevant final concentrations. Aggregation and ATP release were induced by collagen (1 µg/mL), arachidonic acid (0.5 mmol/L), and thrombin (0.5 U/mL). Imatinib, bosutinib, and nilotinib significantly increased collagen-induced aggregation compared with controls. In addition, for bosutinib and nilotinib, a significant increase in aggregation after induction with arachidonic acid was detected. ATP-release and PFA-100 closure times were not influenced significantly by these three TKI. In contrast, dasatinib demonstrated a concentration-dependent inhibition of collagen-induced aggregation and ATP release and a significant prolongation of the PFA-100 closure time with the collagen/epinephrine cartridge. Aggregation and ATP release by other agonists as well as closure time with the collagen/ADP cartridge were not influenced significantly. In conclusion, we clearly show a concentration-dependent inhibition of collagen-induced platelet function in WB by dasatinib confirming prior results obtained in platelet-rich plasma. Bosutinib and nilotinib exerted no impairment of platelet activation. On the contrary, both TKI showed signs of platelet activation. When comparing our results with existing data, imatinib in therapeutic relevant concentrations does not impair platelet function.

[Gene therapy of Hemophilia: Recommendations from the German, Austrian, and Swiss Society for Thrombosis and Haemostasis Research (GTH)]. / Gentherapie der Hämophilie: Empfehlung der Gesellschaft für Thrombose- und Hämostaseforschung (GTH).

Gene therapy has recently become a realistic treatment perspective for patients with hemophilia. Reviewing the literature and our personal experience from clinical trials, we discuss key aspects of hemophilia A and B gene therapy with vectors derived from adeno-associated virus, including predictable results, risks, adverse events, and patient-reported outcomes. Patient selection, informed consent, administration, and monitoring of gene therapy as well as data collection are explained. We also discuss the need for interdisciplinary cooperation with hepatology and other specialties. We emphasize structural and organizational requirements for treatment centers according to the hub-and-spoke model and recommend the use of electronic diaries to ensure safe and timely collection and exchange of data. Electronic diaries will play a key role as a primary source of data for pharmacovigilance, postmarketing clinical studies, national and international registries, as well as health technology and benefit assessment. Reimbursement aspects and the future of gene therapy in adolescents and children are also considered. In a rapidly evolving scientific environment, these recommendations aim to support treatment providers and payers to prepare for the implementation of gene therapy following marketing authorization.

Influencing Factors and Differences in Born Aggregometry in Specialized Hemostaseological Centers: Results of a Multicenter Laboratory Comparison.

Introduction Light transmission aggregometry (LTA) is regarded as the gold standard in platelet function diagnostics. However, there is a relevant degree of interlaboratory variability in practical applications. Objective The aim of the present study was to develop a practicable laboratory comparison on LTA and to analyze differences and influencing factors in regard to standardization in five specialized hemostaseological centers. Methods The study was performed on 30 patients in total. Each center performed LTA on blood samples from six healthy volunteers (three men and three women) using the inductors collagen (Col), adenosine diphosphate (ADP), arachidonic acid (ARA), and ristocetin. The LTA was performed three times using different methods as follows: (1) International Society on Thrombosis and Haemostasis recommendations with identical reagents, (2) in-house protocols and the identical reagents; and (3) in-house protocols and in-house reagents. Results A total of 396 measurements of 30 probands were performed. Even after standardization of the protocol and using identical reagents, there were significant differences between the centers regarding the final and maximum aggregation ( p = 0.002 and <0.001) and further significant differences in the maximum and final aggregation according to the wavelength of the device used to measure the LTA (PAP-8: 430 nm, APACT 4004: 740 nm [ p < 0.001 each]). Using identical reagents but individual inductor concentrations and laboratory protocols also resulted in different maximum and final aggregation. The largest differences were seen with Col and ristocetin; there were significant influences from the reagents' manufacturers in the results of aggregometry for the inductor Col ( p < 0.01) but not for ADP, ARA, and ristocetin. Conclusion In this study, we proved that there are significant influences from the used aggregometers, inductors concentrations, and manufacturers. These results illustrate the challenges and importance of standardization of LTA.

Correction of a Factor VIII genomic inversion with designer-recombinases.

Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions.

A rare PALB2 germline variant causing G2/M cell cycle arrest is associated with isolated myelosarcoma in infancy.

BACKGROUND: Isolated myelosarcoma of infancy is a rare presentation of acute myelogenous leukaemia (AML). Because of its rarity and early onset in infancy underlying genetic predisposition is potentially relevant in disease initiation. METHODS AND RESULTS: We report an oncologic emergency in an infant with thoracic and intraspinal aleukaemic myeloid sarcoma causing acute myelon compression and lower leg palsy. Whole-exome sequencing of the patient's germline DNA identified a rare PALB2 (OMIM 610355) variant (p.A1079S), which is located in a domain critical for the gene's proper function within the homology-directed repair pathway. In line with potential DNA damage repair defects mediated by the PALB2 deregulation, the patient's fibroblasts showed increased sensitivity towards radiation and DNA intercalating agents. CONCLUSION: Therefore, we suggest PALB2 p.A1079S as a pathogenic variant potentially contributing to the here observed patient phenotype.

One Gene, Many Facets: Multiple Immune Pathway Dysregulation in SOCS1 Haploinsufficiency.

Background: Inborn errors of immunity (IEI) present with a large phenotypic spectrum of disease, which can pose diagnostic and therapeutic challenges. Suppressor of cytokine signaling 1 (SOCS1) is a key negative regulator of cytokine signaling, and has recently been associated with a novel IEI. Of patients described to date, it is apparent that SOCS1 haploinsufficiency has a pleiotropic effect in humans. Objective: We sought to investigate whether dysregulation of immune pathways, in addition to STAT1, play a role in the broad clinical manifestations of SOCS1 haploinsufficiency. Methods: We assessed impacts of reduced SOCS1 expression across multiple immune cell pathways utilizing patient cells and CRISPR/Cas9 edited primary human T cells. Results: SOCS1 haploinsufficiency phenotypes straddled across the International Union of Immunological Societies classifications of IEI. We found that reduced SOCS1 expression led to dysregulation of multiple intracellular pathways in immune cells. STAT1 phosphorylation is enhanced, comparably with STAT1 gain-of-function mutations, and STAT3 phosphorylation is similarly reduced with concurrent reduction of Th17 cells. Furthermore, reduced SOCS1 E3 ligase function was associated with increased FAK1 in immune cells, and increased AKT and p70 ribosomal protein S6 kinase phosphorylation. We also found Toll-like receptor responses are increased in SOCS1 haploinsufficiency patients. Conclusions: SOCS1 haploinsufficiency is a pleiotropic monogenic IEI. Dysregulation of multiple immune cell pathways may explain the variable clinical phenotype associated with this new condition. Knowledge of these additional dysregulated immune pathways is important when considering the optimum management for SOCS1 haploinsufficient patients.

Strategies in Clinical and Laboratory Diagnosis of Inherited Platelet Function Disorders in Children.

Inherited disorders of platelet function are a rare and heterogeneous group of diseases usually characterised by a mild to moderate bleeding tendency. Typical bleeding symptoms are easy bruising, epistaxis, menorrhagia as well as mucocutaneous and perioperative bleeding. The performance of platelet function diagnostics in children is hampered by age-dependent restriction of blood sample size, poor venous access, and the lack of reproducible test reference ranges for children of different age groups. Platelet function testing is limited to specialised centres, because platelet function test procedures are complicated and time-consuming, which most likely results in a relevant number of undiagnosed and incorrectly classified children with clinically relevant platelet function defects. Evaluation of bleeding history and bleeding symptoms is essential for a rational step-by-step approach to diagnosis. Platelet function diagnostics should be preceded by the exclusion of thrombocytopenia, von Willebrand disease, and secondary haemostasis defects. Light transmission aggregometry is still considered the standard for the assessment of platelet function. Every effort should be made to classify the specific platelet function defect in the patient, because this is essential for accurate treatment and counselling.

Investigations of Platelet Function in Whole Blood with BAPA as Anticoagulant.

BACKGROUND: Citrate anticoagulation of blood results in non-physiologically low calcium concentration and rapid deterioration of platelet viability. Benzylsulfonyl-D-Arg-Pro-4-amidinobenzylamide (BAPA) as anticoagulant maintains the physiological calcium level and seems to retain platelet function (PF) over a time period of at least 24 h. We evaluated PF in BAPA-anticoagulated peripheral whole blood (WB) between 0.5 and 30 h after blood collection. METHODS: In WB from 21 healthy volunteers (15 women, 6 men, age range 19-57 years) platelet aggregation (PA) was determined by impedance method and ATP release by luminometry. Platelet response was tested by ADP (10 and 20 µmol/l) and collagen (1 and 2 µg/ml) between 0.5 and 30 h after blood collection. RESULTS: Parameters of ADP-induced PA showed stable values until 6.5 h after blood collection followed by a significant decline. PA in response to collagen was stable up to 25 h of storage. ATP release induced by collagen displayed a continuous, significant decrease over time. CONCLUSION: Preservation of platelet response in BAPA-anticoagulated blood depends on the applied agonist showing that collagen-induced PA is more stable compared to ADP known as a weak agonist in WB.

Real-Time Live Confocal Fluorescence Microscopy as a New Tool for Assessing Platelet Vitality.

BACKGROUND: Assessment of platelet vitality is important for patients presenting with inherited or acquired disorders of platelet function and for quality assessment of platelet concentrates. METHODS: Herein we combined live stains with intra-vital confocal fluorescence microscopy in order to obtain an imaging method that allows fast and accurate assessment of platelet vitality. Three fluorescent dyes, FITC-coupled wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and acetoxymethylester (Rhod-2), were used to assess platelet morphology, mitochondrial activity and intra-platelet calcium levels. Microscopy was performed with a microlens-enhanced Nipkow spinning disk-based system allowing live confocal imaging. RESULTS: Comparison of ten samples of donor platelets collected before apheresis and platelets collected on days 5 and 7 of storage showed an increase in the percentage of Rhod-2-positive platelets from 3.6 to 47 and finally to 71%. Mitochondrial potential was demonstrated in 95.4% of donor platelets and in 92.5% of platelets stored for 7 days. CONCLUSION: Such fast and accurate visualization of known key parameters of platelet function could be of relevance for studies addressing the quality of platelets after storage and additional manipulation, such as pathogen inactivation, as well as for the analysis of inherited platelet function disorders.

Novel Mutation in Bernard-Soulier Syndrome.

BACKGROUND: Bernard-Soulier syndrome (BSS) is a severe congenital bleeding disorder characterized by thrombocytopenia, thrombocytopathy and decreased platelet adhesion. BSS results from genetic alterations of the glycoprotein (GP) Ib/IX/V complex. METHODS: We report on a patient demonstrating typical BSS phenotype (thrombocytopenia with giant platelets, bleeding symptoms). However, BSS was not diagnosed until he reached the age of 39 years. RESULTS: Flow cytometry of the patient's platelets revealed absence of GPIb/IX/V receptor surface expression. In addition, immunofluorescence analysis of patient's platelets demonstrated very faint staining of GPIX. A novel homozygous deletion comprising 11 nucleotides starting at position 1644 of the GPIX gene was identified using molecular genetic analysis. CONCLUSIONS: The novel 11-nucleotide deletion (g.1644_1654del11) was identified as causing the bleeding disorder in the BSS patient. This homozygous deletion includes the last 4 nucleotides of the Kozak sequence as well as the start codon and the following 4 nucleotides of the coding sequence. The Kozak sequence is a region indispensable for the initiation of the protein translation process, thus preventing synthesis of functional GPIX protein in the case of deletion.

Perinatal Management of Haemophilia.

The aim of this review is to provide practical guidance for the treatment of carriers of haemophilia and newborns presenting with haemophilia. Both mother and newborn have an increased risk for clinically relevant bleeding. An experienced team should manage genetic counselling, prenatal diagnosis, pregnancy, delivery and the newborn presenting with haemophilia. Published and regularly updated guidelines must guide this team. Vaginal and caesarean deliveries before labour entail a comparable bleeding risk. Haemophilia carriers should receive factor concentrate (FC) at the time of delivery if their factor level is below normal. Evidence remains insufficient to recommend systemic desmopressin and tranexamic acid for the prevention of peripartum haemorrhage. Primary prophylaxis with FC for all newborns with severe haemophilia is not justified. The pattern of bleeding seen in the affected newborns is essentially different from that seen in older children. Estimated frequency of intracranial haemorrhage (ICH) is 2 to 3%. Cranial ultrasound is a good screening method for ICH in newborns. Many neonatal bleeds are iatrogenic in origin. The most prominent concerns regarding neonatal factor replacement are the risk for inhibitor development, followed by local bleeding and issues related to poor vascular access. The preference for plasma-derived FC and recombinant FC differs widely between centres and countries. Replacement therapy should be monitored since newborns may require higher doses of FC. Emicizumab, licensed for all age groups since 2019, should not be used in newborns with severe haemophilia A and acute bleeding, although "non-factor" agents are expected to revolutionise haemophilia therapy.