[Transition in sickle cell disease - recommendations of the transition initiative sickle cell disease]. / Transition bei der Sichelzellkrankheit ­ Empfehlungen der Transitionsinitiative Sichelzellkrankheit.

Sickle cell disease is a rare, but complex multi-systemic disorder with high need of interdisciplinary and specialized care and new structural requirements. Besides care of those chronically sick patients, transition process is a vulnerable phase which highly influences further treatment. To make matters worse, patients often have migration background with subsequent communication problems. A national guidance for a standardized transition process is lacking in Germany. In context of a structured consensus process, the "transition initiative sickle cell disease" developed specific recommendations for a structured transition of sickle cell patients on the basis of the S3 transition guideline of the DGfTM. These recommendations should improve this vulnerable process in this complex disease to ensure adequate further treatment and to avoid acute and chronic complications but also mental, social or job-related issues. Besides improvement of quality of life, medical treatment and survival, health economic aspects arise. Documents were developed to support and facilitate the transition process and are available under www.sichelzellkrankheit.info/transition/.

Hydroxyurea is associated with later onset of acute splenic sequestration crisis in sickle cell disease: Lessons from the European Sickle Cell Disease Cohort-Hydroxyurea (ESCORT-HU) study.

Acute splenic sequestration crisis (ASSC) is a potentially life-threatening complication of sickle cell disease (SCD), typically occurring in young patients under 5 years of age, with a median age at first episode of less than 2 years. Because a beneficial effect of hydroxyurea (HU) on spleen perfusion and splenic function has been suspected, we hypothesized that HU treatment might be associated with later onset of ASSC in patients with SCD. To investigate this hypothesis, we analyzed data from the ESCORT-HU study on a large cohort of patients with SCD receiving HU, enrolled between January 2009 and June 2017 with a follow-up of 7309 patient-years of observation. The median age at ASSC of the 14 patients who experienced a first episode of ASSC during the study period was 8.0 [IQR: 5.0-24.1] years. The median age at HU initiation was significantly lower in these 14 patients (4.8 [IQR: 3.3-18.7] years) compared to the 1664 patients without ASSC (19.9 [8.8-33.4] years, p = .0008). These findings suggest that ASSC may occur at an unusually late age in patients receiving HU, possibly reflecting longer preservation of spleen perfusion and function secondary to early initiation of HU. Further studies are needed to better characterize the effects of HU on spleen perfusion/function and on the occurrence of ASSC in patients with SCD (ClinicalTrials.gov identifier: NCT02516579; European registry ENCEPP/SDPP/10565).

Single-cell multi-omics of mitochondrial DNA disorders reveals dynamics of purifying selection across human immune cells.

Pathogenic mutations in mitochondrial DNA (mtDNA) compromise cellular metabolism, contributing to cellular heterogeneity and disease. Diverse mutations are associated with diverse clinical phenotypes, suggesting distinct organ- and cell-type-specific metabolic vulnerabilities. Here we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from six patients across the phenotypic spectrum of single large-scale mtDNA deletions (SLSMDs). By profiling 206,663 cells, we reveal the dynamics of pathogenic mtDNA deletion heteroplasmy consistent with purifying selection and distinct metabolic vulnerabilities across T-cell states in vivo and validate these observations in vitro. By extending analyses to hematopoietic and erythroid progenitors, we reveal mtDNA dynamics and cell-type-specific gene regulatory adaptations, demonstrating the context-dependence of perturbing mitochondrial genomic integrity. Collectively, we report pathogenic mtDNA heteroplasmy dynamics of individual blood and immune cells across lineages, demonstrating the power of single-cell multi-omics for revealing fundamental properties of mitochondrial genetics.

Current German Guidelines on Diagnosis and Treatment of Secondary Hemochromatosis in Patients with Congenital Anemias.

Adequate diagnosis and treatment of secondary hemochromatosis in patients with congenital anemias is important for reducing long-term mortality and morbidity as for improving patients' quality of life. Due to the strong migration movements during recent years, the number of patients in Germany suffering from hemoglobinopathies as some of the most relevant disorders in the context of iron overload (IOL) has increased enormously. Many of these patients had received inadequate medical care in their countries of origin prior to migration, including diagnosis and treatment of IOL. In parallel, various medical developments and achievements took place, including the expansion of stem cell transplant as curative therapeutic option and the introduction of gene therapy for patients with hemoglobinopathies. Diagnostic tools to assess both liver and heart IOL became available at more sites in Germany. Overall experience with iron elimination therapy either as monotherapy or as combination of different chelators increased. All these aspects were considered during revision of the consensus-based guideline on the diagnosis and treatment of secondary IOL in patients with congenital anemias (AWMF Reg. No. 025/029). Here, we briefly summarize the procedure for the revision of the guideline, provide a brief overview of innovations as compared to the previous version dated from 2015, and finally present the consensus recommendations adopted in the current version.

Transition in Sickle Cell Disease (SCD): A German Consensus Recommendation.

Sickle cell disease (SCD) is considered a rare disease in Germany. Due to the increasing prevalence, the acute and chronic morbidities associated with the disease and the sharp increase in the mortality rate of young adults, a need-based transition structure for patients with SCD in Germany is explicitly required. This is the first multicenter German consensus statement addressing the importance of implementing a standardized transition guideline that allows adolescents and young adults to safely transition from pediatric to adult care. Early identification of medical needs and intervention remains important in the context of chronic diseases. Effective measures can improve health care in general, as they lead to a reduction in disease and the consequential economic burden. It is noteworthy that improving structural barriers remains a key challenge even in highly developed countries such as Germany. Inclusion of these transition services for patients with SCD into the regular care of chronically ill adolescents and young adults should be ensured, as well as the coverage of costs associated with a structured transition process.

Targeted Analysis of Cell-free Circulating Tumor DNA is Suitable for Early Relapse and Actionable Target Detection in Patients with Neuroblastoma.

PURPOSE: Treating refractory or relapsed neuroblastoma remains challenging. Monitoring body fluids for tumor-derived molecular information indicating minimal residual disease supports more frequent diagnostic surveillance and may have the power to detect resistant subclones before they give rise to relapses. If actionable targets are identified from liquid biopsies, targeted treatment options can be considered earlier. EXPERIMENTAL DESIGN: Droplet digital PCR assays assessing MYCN and ALK copy numbers and allelic frequencies of ALK p.F1174L and ALK p.R1275Q mutations were applied to longitudinally collected liquid biopsies and matched tumor tissue samples from 31 patients with high-risk neuroblastoma. Total cell-free DNA (cfDNA) levels and marker detection were compared with data from routine clinical diagnostics. RESULTS: Total cfDNA concentrations in blood plasma from patients with high-risk neuroblastoma were higher than in healthy controls and consistently correlated with neuron-specific enolase levels and lactate dehydrogenase activity but not with 123I-meta-iodobenzylguanidine scores at relapse diagnosis. Targeted cfDNA diagnostics proved superior for early relapse detection to all current diagnostics in 2 patients. Marker analysis in cfDNA indicated intratumor heterogeneity for cell clones harboring MYCN amplifications and druggable ALK alterations that were not detectable in matched tumor tissue samples in 17 patients from our cohort. Proof of concept is provided for molecular target detection in cerebrospinal fluid from patients with isolated central nervous system relapses. CONCLUSIONS: Tumor-specific alterations can be identified and monitored during disease course in liquid biopsies from pediatric patients with high-risk neuroblastoma. This approach to cfDNA surveillance warrants further prospective validation and exploitation for diagnostic purposes and to guide therapeutic decisions.

Genetic modifiers of fetal hemoglobin affect the course of sickle cell disease in patients treated with hydroxyurea.

The course of sickle cell disease (SCD) is modified by polymorphisms boosting fetal hemoglobin (HbF) synthesis. However, it has remained an open question how these polymorphisms affect patients who are treated with the HbF-inducing drug hydroxyurea/ hydroxycarbamide. The German SCD registry offers the opportunity to answer this question, because >90% of patients are treated according to national guidelines recommending the use of hydroxyurea in all patients above 2 years of age. We analyzed the modifying effect of HbF-related genetic polymorphisms in 417 patients with homozygous SCD >2 years old who received hydroxyurea. HbF levels were correlated with higher total hemoglobin levels, lower rates of hemolysis, a lower frequency of painful crises and of red blood cell transfusions. The minor alleles of the polymorphisms in the γ-globin promoter (rs7482144), BCL11A (rs1427407) and HMIP (rs66650371) were strongly associated with increased HbF levels. However, these associations did not translate into lower frequencies of vaso-occlusive events which did not differ between patients either carrying or not carrying the HMIP and BCL11A polymorphisms. Patients on hydroxyurea carrying the γ-globin promoter polymorphism demonstrated substantially higher hemoglobin levels (P<10-4) but also higher frequencies of painful crises and hospitalizations (P<0.01) when compared to patients without this polymorphism. Taken together, these data indicate that the γ-globin, HMIP and BCL11A polymorphisms correlate with increased HbF in SCD patients on hydroxyurea. While HbF is negatively correlated with the frequency of painful crises and hospitalizations, this was not observed for the presence of known HbF-boosting alleles.

Benefits of a Disease Management Program for Sickle Cell Disease in Germany 2011-2019: The Increased Use of Hydroxyurea Correlates with a Reduced Frequency of Acute Chest Syndrome.

Sickle Cell Disease (SCD) is the most common monogenic disorder globally but qualifies as a rare disease in Germany. In 2012, the German Society for Paediatric Oncology and Haematology (GPOH) mandated a consortium of five university hospitals to develop a disease management program for patients with SCD. Besides other activities, this consortium issued treatment guidelines for SCD that strongly favour the use of hydroxyurea and propagated these guidelines in physician and patient education events. In order to quantify the effect of these recommendations, we made use of claims data that were collected by the research institute (WIdO) of the major German insurance company, the Allgemeine Ortskrankenkasse (AOK), and of publicly accessible data collected by the Federal Statistical Office (Statistisches Bundesamt, Destatis). While the number of patients with SCD in Germany increased from approximately 2200 in 2011 to approximately 3200 in 2019, important components of the recently issued treatment guidelines have been largely implemented. Specifically, the use of hydroxyurea has more than doubled, resulting in a proportion of approximately 44% of all patients with SCD being treated with hydroxyurea in 2019. In strong negative correlation with the use of hydroxyurea, the frequency of acute chest syndromes decreased. Similarly, the proportion of patients who required analgesics and hospitals admissions declined. In sum, these data demonstrate an association between the dissemination of treatment guidelines and changes in clinical practice. The close temporal relationship between the increased use of hydroxyurea and the reduction in the incidence of acute chest syndrome in a representative population-based analysis implies that these changes in clinical practice contributed to an improvement in key measures of disease activity.

Real-Life experience with hydroxyurea in patients with sickle cell disease: Results from the prospective ESCORT-HU cohort study.

Several controlled studies have evidenced good efficacy and short-term and mid-term safety profiles for hydroxyurea (HU), which has become the cornerstone for prevention of sickle-cell disease (SCD)-related vaso-occlusive crises. However, there are few large-scale reports on its long-term use and certain caregivers and patients have concerns about its safety. Following the licensing of HU in Europe for children and adults with severe forms of SCD, ESCORT-HU was designed as a Phase IV observational cohort study. It included 1906 participants, of whom 55% were adults. The most common hemoglobin (Hb) genotypes were HbSS (84.7%) and HbSß+ (7.0%). The median duration of follow-up was 45 months, for a total of 7309 patient-years of observation. The dose of HU after 1 year was 20.6 mg/kg/d for children and 16.3 mg/kg/d for adults. There was a statistically significant decrease in the number of vaso-occlusive episodes lasting >48 h, acute chest syndrome episodes, hospitalizations, and the percentage of patients requiring blood transfusions within the first 12 months relative to the year before enrolment. Neutropenia and thrombocytopenia were the most commonly reported adverse effects. No new HU toxicity was identified. Overall, 125 pregnancies were reported in 101 women and no malformations were observed in the neonates. There were 12 pregnancies for partners of male patients treated with HU. One case of fatal myelodysplastic syndrome was reported, for which a causal association with HU could not be excluded. This cohort study of patients with SCD highlights the positive benefit-to-risk ratio of HU in children and adults.

Hematopoietic Stem Cell Transplantation Cures Therapy-refractory Aspergillosis in Chronic Granulomatous Disease.

BACKGROUND: Pulmonary invasive aspergillosis is a frequent and life-threatening complication for patients with chronic granulomatous disease (CGD). Despite combined treatment with several groups of antifungal agents, conservative treatment of invasive aspergillosis often remains refractory. Pulmonary invasive aspergillosis is often treated by surgical resection of consolidated lobes or segments, donor granulocyte transfusions and allogeneic hematopoietic stem cell transplantation (HSCT). These options are not mutually exclusive and often combined. METHODS AND RESULTS: We here describe the treatment of 3 patients with CGD who received HSCT upon active pulmonary invasive aspergillosis: Two of them received HSCT as salvage therapy for refractory aspergillosis, and 1 patient received elective HSCT in infancy but developed pulmonary aspergillosis during secondary graft failure. Based on our experience and available literature, we discuss indication as well as timing of HSCT, granulocyte transfusions and surgery in patients with CGD and pulmonary invasive aspergillosis. CONCLUSIONS: Upon diagnosis with invasive aspergillosis in CGD, we propose to start antifungal treatment and preparation for HSCT at the same time. Remission of pulmonary invasive aspergillosis before HSCT remains preferable but is not mandatory. When pulmonary aspergillosis in patients with CGD remains refractory for longer than 3 months on conservative treatment, HSCT without prior surgery or accompanying granulocyte transfusions is a feasible option.

Introduction of Universal Newborn Screening for Sickle Cell Disease in Germany-A Brief Narrative Review.

Sickle cell disease (SCD) is a severe non-malignant disorder of hemoglobin and is inherited in an autosomal-recessive manner [...].

Allogeneic hematopoietic stem cell transplantation from sibling and unrelated donors in pediatric patients with sickle cell disease-A single center experience.

HSCT is curative in SCD. Patients with HLA-identical sibling donor have an excellent outcome ranging from 90%-100% overall and event-free survival. However, due to the lack of matched sibling donors this option is out of reach for 70% of patients with SCD. The pool of potential donors needs to be extended. Transplantations from HLA-matched unrelated donors were reported to be less successful with shorter event-free survival and higher incidences of complications including graft-vs-host disease, especially in patients with advanced stage SCD. Here we report transplantation outcomes for 25 children with SCD transplanted using HLA-matched grafts from related or unrelated donors. Overall survival was 100% with no severe (grade III-IV) graft-vs-host disease and a 12% rejection rate. Mixed donor chimerisms only occurred in transplantations from siblings, while transplantations from unrelated donors resulted in either complete donor chimerism or rejection. Despite the small patient number, overall and disease-free survival for unrelated donor transplantations is excellent in this cohort. The advanced disease state, higher alloreactive effect and stronger immunosuppression in unrelated donor transplantations raises patient risk, for which possible solutions could be found in optimization of transplant preparation, graft manipulation or haploidentical transplantation using T cell receptor α/ß-depleted grafts.

Tumor-Derived Extracellular Vesicles Impair CD171-Specific CD4+ CAR T Cell Efficacy.

Chimeric antigen receptor (CAR) T cell efficacy against solid tumors is currently limited by several immune escape mechanisms, which may include tumor-derived extracellular vesicles. Advanced neuroblastoma is an aggressive childhood tumor without curative treatment options for most relapsed patients today. We here evaluated the role of tumor-derived extracellular vesicles on the efficacy of CAR T cells targeting the neuroblastoma-specific antigen, CD171. For this purpose, CAR T cell activation, cytokine production, exhaustion, and tumor cell-directed cytotoxicity upon co-culture was evaluated. Tumor-derived extracellular vesicles isolated from SH-SY5Y neuroblastoma cells neither affected CAR T cell activation nor expression of inhibitory markers. Importantly, exposure of CD4+ CD171-specific CAR T cells to tumor-derived extracellular vesicles significantly impaired tumor cytotoxicity of CAR T cells. This effect was independent of neurotrophic receptor tyrosine kinases 1 or 2 (NTRK1, NTRK2) expression, which is known to impact immune responses against neuroblastoma. Our results demonstrate for the first time the impact of tumor-derived extracellular vesicles and non-cell-mediated tumor-suppressive effects on CD4+ CAR T cell efficacy in a preclinical setting. We conclude that these factors should be considered for any CAR T cell-based therapy to make CAR T cell therapy successful against solid tumors.

Donor selection in a pediatric stem cell transplantation cohort using PIRCHE and HLA-DPB1 typing.

BACKGROUND: New strategies to optimize donor selection for hematopoietic stem cell transplantation (HSCT) have mainly been evaluated in adults, but the disease spectrum requiring HSCT differs significantly in children and has consequences for the risk of complications, such as graft-versus-host disease (GvHD). PROCEDURES: Here we evaluated whether HLA-DPB1 and Predicted Indirectly ReCognizable HLA-Epitope (PIRCHE) matching can improve donor selection and minimize risks specific for a pediatric cohort undergoing HSCT in Berlin between 2014 and 2016. RESULTS: The percentage of HLA-DPB1-mismatched HSCT in the pediatric cohort was in line with the general distribution among matched unrelated donor HSCT. Nonpermissive HLA-DPB1 mismatches were not associated with a higher incidence of GvHD, but the incidence of relapse was higher in patients undergoing HSCT from HLA-DPB1-matched transplantations. High PIRCHE-I scores were associated with a significantly higher risk for developing GvHD in patients undergoing HSCT from nine of ten matched unrelated donors. This finding persisted after including HLA-DPB1 into the PIRCHE analysis. CONCLUSIONS: Implementing PIRCHE typing in the donor selection process for HSCT in children could particularly benefit children with nonmalignant diseases and support further validation of PIRCHE-based donor selection in a larger number of children treated at different sites.

Sickle cell disease in Germany: Results from a national registry.

BACKGROUND: Limited data on the prevalence and medical care of sickle cell disease (SCD) in Germany are available. Here, we make use of a patient registry to characterize the burden of disease and the treatment modalities for patients with SCD in Germany. PROCEDURE: A nationwide German registry for patients with SCD documents basic data on diagnosis and patient history retrospectively at the time of registration. A prospective annual documentation provides more details on complications and treatment of SCD. For the current analyses, data of 439 patients were available. RESULTS: Most patients had homozygous SCD (HbSS 75.1%, HbS/ß-thalassemia 13.2%, and HbSC 11.3%). The median age at diagnosis was 1.9 years (interquartile range, 0.6-4.4 years), most patients were diagnosed when characteristic symptoms occurred. Sepsis and stroke had affected 3.2% and 4.2% of patients, respectively. During the first year of observation, 48.3% of patients were admitted to a hospital and 10.1% required intensive care. Prophylactic penicillin was prescribed to 95.6% of patients with homozygous SCD or HbS/ß thalassemia below the age of six and hydroxycarbamide to 90.4% of patients above the age of two years. At least one annual transcranial Doppler ultrasound was documented for 74.8% of patients between 2 and 18 years. CONCLUSION: With an estimated number of at least 2000, the prevalence of SCD in Germany remains low. Prospectively, we expect that the quality of care for children with SCD will be further improved by an earlier diagnosis after the anticipated introduction of a newborn screening program for SCD.

HLA-haploidentical hematopoietic stem cell transplantation in pediatric patients with hemoglobinopathies: current practice and new approaches.

We review current approaches in HLA-haploidentical hematopoietic stem cell transplantation (haplo-HSCT) for pediatric patients with hemoglobinopathies with a focus on recent developments using TCRα/ß+/CD19+ depleted grafts in patients with ß-thalassemia major (TM) or sickle cell disease (SCD) in two European transplant units. Eleven TM and three SCD patients (Roma cohort) received a preparative regimen consisting of busulfan/thiotepa/cyclophosphamide/ATG preceded by fludarabine/hydroxyurea/azathioprine. The preparative regimen for 5 SCD patients included treosulfan/thiotepa/fludarabine/ATG (Berlin pilot cohort). All grafts were PBSC engineered by TCR-α/ß+/CD19+ depletion. In both cohorts, rates for graft failure, treatment related mortality (TRM) and GvHD were encouraging. Overall survival (OS) and disease-free survival (DFS) in the Roma cohort were 84 and 69%, respectively, while OS and DFS are 100% in the Berlin cohort. Immune reconstitution was satisfactory. Although asymptomatic viral reactivation was common, no severe viral infection occured. These data confirm that TCR-α/ß+/CD19+ depletion is a well-suited haplo-HSCT strategy for children with hemoglobinopathies. We discuss the results in the context of additional optimization strategies and introduce our concepts for multicenter trial protocols in Germany.

Presence of centromeric but absence of telomeric group B KIR haplotypes in stem cell donors improve leukaemia control after HSCT for childhood ALL.

Although allogeneic hematopoietic stem-cell transplantation (HSCT) provides high cure rates for children with high-risk acute lymphoblastic leukaemia (ALL), relapses remain the main cause of treatment failure. Whereas donor killer cell immunoglobulin-like receptor (KIR) genotype was shown to impact on relapse incidence in adult myeloid leukaemia similar studies in paediatric ALL are largely missing. Effect of donor KIR genotype on transplant outcome was evaluated in 317 children receiving a first myeloablative HSCT from an HLA-matched unrelated donor or sibling within the prospective ALL-SCT-BFM-2003 trial. Analysis of donor KIR gene polymorphism revealed that centromeric presence and telomeric absence of KIR B haplotypes was associated with reduced relapse risk. A centromeric/telomeric KIR score (ct-KIR score) integrating these observations correlated with relapse risk (hazard ratio (HR) 0.58; P = 0.002) while it had no impact on graft-versus-host disease or non-relapse mortality. In multivariable analyses ct-KIR score was associated with reduced relapse risk (HR 0.58; P = 0.003) and a trend towards improved event-free survival (HR 0.76; P = 0.059). This effect proved independent of MRD level prior to HSCT. Our data suggest that in children with ALL undergoing HSCT after myeloablative conditioning, donor selection based on KIR genotyping holds promise to improve clinical outcome by decreasing relapse risk and prolonged event-free survival.