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Background Previous studies suggest that PAP-based CF protocols are suitable for newborn screening (NBS) for cystic fibrosis (CF) when newborns designated as CFSPID should not be detected. However, there are still discussions about the performance of IRT/PAP algorithms. We present the final results of a pilot study evaluating a IRT/PAP protocol with an IRT-dependent safety net (SN) conducted from 2008 to 2016 in southwestern Germany on nearly 500,000 newborns. Methods To achieve reliable data, all newborns were screened using both the PAP-based and a DNA-based CFNBS algorithm. PAP quantification and genetic analysis of the four most common CFTR mutations in Germany were performed in all newborns with IRT≥99.0 percentile. NBS was rated positive if either PAP was ≥1.6 µg/l and/or at least one CFTR mutation was detected. In addition, an IRT-dependent SN resulted in positive rating for both protocols if IRT was ≥99.9 percentile. To evaluate the IRT/PAP protocol, its performance was compared to that of the IRT/DNA protocol. Results The IRT/PAP protocol with IRT-based SN used in the study achieved a sensitivity of 94%, if false-negative detected neonates with meconium ileus and those designated as CFSPID were excluded from analysis. CF/CFSPID ratio was 92. However, PPV of the IRT/PAP+SN protocol was with 10.3% very low. Conclusions PAP-based CFNBS protocols can be used, if less detection of CFSPID is desired. The IRT/PAP protocol with IRT-dependent SN evaluated here achieved adequate sensitivity but should probably be used in combination with a third-tier test to also achieve an acceptable PPV.

Cellular stress causes multifaceted reactions to trigger adaptive responses to environmental cues at all levels of the gene expression pathway. RNA-binding proteins (RBP) are key contributors to stress-induced regulation of RNA fate and function. Here, we uncover the plasticity of the RNA interactome in stressed cells, differentiating between responses in the nucleus and in the cytoplasm. We applied enhanced RNA interactome capture (eRIC) analysis preceded by nucleo-cytoplasmic fractionation following arsenite-induced oxidative stress. The data reveal unexpectedly compartmentalized RNA interactomes and their responses to stress, including differential responses of RBPs in the nucleus versus the cytoplasm, which would have been missed by whole cell analyses.

BackgroundAlthough considerable effort has been put into decoding of the osteosarcoma genome, very little is known about germline mutations that underlie this primary malignant tumour of bone.Methods and resultsWe followed here a coincidental finding in a multiple endocrine neoplasia family in which a 32-year-old patient carrying a germline pathogenic RET mutation developed an osteosarcoma 2 years after the resection of a medullary thyroid carcinoma. Sequencing analysis of additional 336 patients with osteosarcoma led to the identification of germline activating mutations in the RET proto-oncogene in three cases and somatic amplifications of the gene locus in five matched tumours (4%, n=5/124 tumours). Functional analysis of the pathogenic variants together with an integrative analysis of osteosarcoma genomes confirmed that the mutant RET proteins couple functional kinase activity to dysfunctional ligand binding. RET mutations further co-operated with alterations in TP53 and RB1, suggesting that osteosarcoma pathogenesis bears reminiscence to the stepwise model of medullary thyroid carcinoma.ConclusionsAfter Li-Fraumeni-predisposing mutations in TP53, RET becomes the second most mutated cancer-predisposing gene in the germline of patients with osteosarcoma. Hence, early identification of RET mutation carriers can help to identify at-risk family members and carry out preventive measures.

INTRODUCTION Granulocyte transfusions have long been used to bridge the time to neutrophil recovery in patients with neutropenia and severe infection. Recent randomized controlled trials did not prove a beneficial effect of granulocyte transfusions, but were likely underpowered and suffered from very heterogeneous study populations. METHODS We retrospectively reviewed data of all patients treated with granulocyte transfusions at our pediatric center from 2004 to 2019. In order to identify parameters that predict the success of granulocyte transfusions, we stratified patients in 3 groups. Patients in group 1 cleared their infection, whereas patients in group 2 succumbed to an infection in neutropenia despite granulocyte transfusions. A third group included all patients who died of causes that were not related to infection. RESULTS We demonstrate that patients without respiratory or cardiocirculatory insufficiency are enriched in group 1 and more likely to benefit from granulocyte transfusions than patients who already require these intensive care measures. The effect of granulocyte transfusions correlates with the cell dose per body weight applied per time. With our standard twice weekly dosing, patients with a body weight below 40 kg are more likely to achieve a sufficient leukocyte increment and clear their infection in comparison to patients with a higher body weight. DISCUSSION/CONCLUSIONS We suggest that future studies on the benefits of granulocyte transfusions stratify patients according to clinical risk factors that include the need for respiratory or cardiocirculatory support and strive for a sufficient dose density of granulocyte transfusions.

Progress in the systemic control of osteosarcoma has been limited over the past decades thus indicating the urgent clinical need for the development of novel treatment strategies. Therefore, we have recently developed new preclinical models to study promising novel agents for the treatment of pediatric osteosarcoma. The checkpoint kinase (chk) inhibitor prexasertib (LY2606368) and its salt form (LSN2940930) have recently been shown to be active in adult and pediatric malignancies, including sarcoma. We have now tested the potency of prexasertib in clonogenic survival assays in two new lines of primary patient‐derived osteosarcoma cells and in two established osteosarcoma cell lines as a single agent and in combination with cisplatin and the poly ADP‐ribose polymerase (PARP) inhibitor talazoparib. Prexasertib alone results in strongly reduced clonogenic survival at low nanomolar concentrations and acts by affecting cell cycle progression, induction of apoptosis and induction of double‐stranded DNA breakage at concentrations that are well below clinically tolerable and safe plasma concentrations. In combination with cisplatin and talazoparib, prexasertib acts in a synergistic fashion. Chk1 inhibition by prexasertib and its combination with the DNA damaging agent cisplatin and the PARP‐inhibitor talazoparib thus emerges as a potential new treatment option for pediatric osteosarcoma which will now have to be tested in preclinical primary patient derived in vivo models and clinical studies., What's new? Treatment options and outcomes for osteosarcoma have changed very little in decades. New treatment strategies are thus urgently needed. In this study, the authors found that the checkpoint inhibitor ‘prexasertib’ increased apoptosis and DNA damage in patient‐derived osteosarcoma cells, at very low concentrations. In addition, when combined with cisplatin and talazoparib, prexasertib acted in a synergistic fashion. This drug thus represents a promising therapeutic candidate for further preclinical and clinical development in osteosarcoma.

Background In the western world, 2-5% of women of child-bearing age suffer from irondeficiency anemia. Iron overload due to chronic treatment with blood transfusions or hereditary hemochromatosis is much rarer. Methods This review is based on pertinent publications retrieved by a selective search on the pathophysiology, clinical features, and diagnostic evaluation of iron deficiency and iron overload. Results The main causes of iron deficiency are malnutrition and blood loss. Its differential diagnosis includes iron-refractory iron deficiency anemia (IRIDA), a rare congenital disease in which the hepcidin level is pathologically elevated, as well as the more common anemia of chronic disease (anemia of chronic inflammation), in which increased amounts of hepcidin are formed under the influence of interleukin-6 and enteric iron uptake is blocked as a result. Iron overload comes about through long-term transfusion treatment or a congenital disturbance of iron metabolism (hemochromatosis). Its diagnostic evaluation is based on clinical and laboratory findings, imaging studies, and specific mutation analyses. Conclusion Our improving understanding of the molecular pathophysiology of iron metabolism aids in the evaluation of iron deficiency and iron overload and may in future enable treatment not just with iron supplementation or iron chelation, but also with targeted pharmacological modulation of the hepcidin regulatory system.

In 1986, a paper in the Lancet was the first to collate hematology, molecular findings, and clinical features of homozygous sickle cell (SS) disease in India. The paper came from the group organized by Professor Bimal Kar in Burla Medical College, Sambalpur University, in western Odisha. Although widely quoted, few readers will be aware of the history of this work that is now attached in an informal summary.

Methemoglobinemia is a rare disorder associated with oxidization of divalent ferro-iron of hemoglobin (Hb) to ferri-iron of methemoglobin (MetHb). Methemoglobinemia can result from either inherited or acquired processes. Acquired forms are the most common, mainly due to the exposure to substances that cause oxidation of the Hb both directly or indirectly. Inherited forms are due either to autosomal recessive variants in the CYB5R3 gene or to autosomal dominant variants in the globin genes, collectively known as HbM disease. Our recommendations are based on a systematic literature search. A series of questions regarding the key signs and symptoms, the methods for diagnosis, the clinical management in neonatal/childhood/adulthood period, and the therapeutic approach of methemoglobinemia were formulated and the relative recommendations were produced. An agreement was obtained using a Delphi-like approach and the experts panel reached a final consensus >75% of agreement for all the questions.

Background Beti-cel ex vivo gene therapy integrates a modified HBB gene into hematopoietic stem cells of patients with TDT, aiming to enable lifelong, stable production of functional adult hemoglobin (Hb). The efficacy and safety of the treatment have been demonstrated in a total of 63 patients treated across 4 clinical trials (HGB-204,-HGB-205, HGB-207, and HGB-212). Here, we present the first patient who received beti-cel outside of the clinical trial setting, a 14-year-old male with a β 0/β + (IVS-1-6) genotype. Methods Following hematopoietic stem cell collection via granulocyte-colony stimulating factor plus plerixafor mobilization and apheresis, CD34+ cells were transduced with the BB305 lentiviral vector encoding HbA T87Q. The patient received hypertransfusion before mobilization and conditioning, maintaining a pre-transfusion Hb level of >11 g/dL. Six days prior to beti-cel infusion, single-agent busulfan myeloablation was initiated (16 single doses at 0.8 mg/kg body weight; 3.2 mg/kg/24 h) with concomitant clonazepam (see Table for treatment timeline). Ursodeoxycholic acid therapy was continued as hepatic veno-occlusive disease (VOD) prophylaxis through inpatient treatment. Results The patient was diagnosed with TDT at the age of 2 years in his home country and has been treated in Germany since the age of 9. Regular transfusion therapy was initiated soon after diagnosis (Table). Aged 9, the patient was started on desferasirox for iron elimination therapy. His annualized red blood cell (RBC) transfusion volume was 174 ml/kg in 2018 and 185 ml/kg in 2019, maintaining his pre-transfusion Hb at or above 9 g/dl. No HLA-related donor was available for allogeneic transplant. At informed consent, the patient was 13 years old and met the eligibility criteria for beti-cel treatment as outlined in the summary of product characteristics (SmPC). The patient was physically fit, with a 90% Lansky score and regular participation in school sports, but reported physical limitations when running extensively. The patient underwent a thorough assessment before admission (Table), which did not reveal any remarkable abnormalities except TDT-related splenomegaly and signs of slight iron overload (liver iron content, 2.0 mg/g dry weight [normal range, 0.17-1.8]). On 11/Feb/2021, the patient was infused with 5.1 × 10 6 CD34+ cells/kg. The patient received 4 RBC and 8 platelet transfusions following infusion until Day 13 and 27, respectively (Table). Neutrophil and platelet engraftment occurred on day 27 post beti-cel infusion. The patient was discharged from inpatient treatment the same day, in excellent general condition, with 90% Lansky score, Hb of 8.2 g/dl, a reticulocyte count of 9.3%, a total white cell count of 1.55/nl, a neutrophil count of 0.75/nl, and a platelet count of 24/nl. At last follow-up (+100 days), the patient felt well and exhibited normal exercise tolerance. He has received neither red blood cell nor platelet transfusions or chelation therapy since discharge. Total Hb was 11.8 g/dl (Table). Granulocytes and lymphocytes had recovered to normal levels. The patient showed continued, albeit slowly improving, thrombocytopenia (platelet count, 31/nl [29/nl at +60 days]), consistent with previous observations after beti-cel therapy. Myeloablation and beti-cel infusion were tolerated well. Adverse events post infusion were febrile neutropenia, elevated C-reactive protein levels, pruritus, gingivitis, mild mucositis, and vertigo, consistent with the SmPC. At +23 and +26 days, the patient experienced transient subjective hearing loss (quickly resolved). No VOD events occurred. Conclusions This is the first real-world patient with TDT treated with beti-cel therapy. The treatment regimen had a tolerability profile consistent with that of mobilization, apheresis, and busulfan myeloablation, matching clinical trial observations. Following treatment, this 14-year-old patient reached a total Hb of 11.8 g/dL at +100 days without requirement of red cell transfusions and continues to exhibit prolonged but slowly improving and asymptomatic thrombocytopenia. Figure 1 Figure 1. Disclosures Schmitt: TolerogenixX Ltd: Current Employment; Therakos/Mallinckrodt: Research Funding; Hexal: Other: Travel grant; Jazz Pharmaceuticals: Other: Travel grant. Schmitt: Bluebird Bio: Other: Travel grants; Novartis: Other: Travel grants, Research Funding; TolerogenixX: Current holder of individual stocks in a privately-held company; Apogenix: Research Funding; MSD: Membership on an entity's Board of Directors or advisory committees; Hexal: Other: Travel grants, Research Funding; Kite Gilead: Other: Travel grants. Kulozik: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria; BioMedX: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Eine nicht fruhzeitig diagnostizierte Sichelzellkrankheit („sickle cell disease“, SCD) ist mit einem erheblichen Morbiditats- und Mortalitatsrisiko im Sauglings- und Kleinkindalter verbunden. An erster Stelle sind lebensbedrohliche Komplikationen durch invasive Infektionen mit bekapselten Bakterien und plotzliche Verschlechterungen der Anamie zu nennen. In zahlreichen Landern ist die SCD daher Zielkrankheit im Neugeborenenscreening (NBS), teilweise schon seit den fruhen 1970er-Jahren. Der Gemeinsame Bundesausschuss (G-BA) hat am 20.11.2020 beschlossen, auch in Deutschland ein NBS auf SCD einzufuhren. Dieser Beitrag gibt einen Uberblick uber die Erkrankung, die Screeninglogistik, -methodik und die Konsequenzen, die sich aus einem positiven Befund ergeben.

Introduction Betibeglogene autotemcel (beti-cel; LentiGlobin for β-thalassemia) gene therapy is being evaluated for the treatment of transfusion-dependent β-thalassemia (TDT). Initial positive results of beti-cel in the phase 3 studies, HGB-207 (NCT02906202; non-β0/β0 genotypes) and HGB-212 (NCT03207009; β0/β0, β0/β+ IVS-I-110 and β+ IVS-I-110/β+ IVS-I-110 genotypes), showed 10/12 adult patients achieved transfusion independence. The studies expanded enrollment to include adolescents and children. We present interim results from pediatric patients Methods After mobilization and apheresis, autologous CD34+ cells were transduced ex vivo with BB305 lentiviral vector, containing a modified human β-globin gene to produce beti-cel drug product (DP). Patients underwent busulfan myeloablation and infusion with beti-cel and were then followed longitudinally. Transfusion independence (TI; weighted average hemoglobin [Hb] ≥9 g/dL without transfusions for ≥12 mo) was the primary endpoint in HGB-207 and a secondary endpoint in HGB-212. Transfusion reduction (≥60% reduction in transfusion volume between Month 12 to 24 versus baseline) is the primary endpoint in HGB-212. Hb levels, TI characteristics, and quality of life were secondary endpoints. Assessments of ineffective erythropoiesis were exploratory. Data presented as median (min-max). Results Twenty-four pediatric patients were treated including 13 patients 3 mo follow-up achieved platelet engraftment and had platelets ≥100 x109/L by Month 12; one 17-yr old patient did not have platelets ≥100 x109/L until Month 15. In HGB-207, 6/7 (86%) patients 3 mo follow-up have stopped transfusions for ≥6 mo. TI was achieved in 3/4 (75%) evaluable patients In HGB-212, 3/5 (60%) patients 3mo follow-up have stopped transfusions for ≥6 mo. TI was achieved in 1/2 evaluable patients Post-infusion non-hematologic grade ≥3 adverse events (AEs) in ≥3 patients aged Summary Interim results in HGB-207 and HGB-212 show that after treatment with beti-cel, pediatric patients Disclosures Thompson: CRISPR/Vertex: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Baxalta: Research Funding; BMS: Consultancy, Research Funding; Biomarin: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding. Kwiatkowski:Novartis: Research Funding; Agios: Consultancy; Sangamo: Research Funding; bluebird bio,Inc.: Consultancy, Research Funding; Apopharma: Research Funding; Imara: Consultancy; BMS: Consultancy; Terumo Co: Research Funding; Celgene: Consultancy. Porter:Vifor Pharmaceuticals: Honoraria; bluebird bio, Inc.: Consultancy, Honoraria; Agios Pharmaceuticals: Consultancy, Honoraria; La Jolla Pharmaceuticals: Honoraria; Silence Therapeutics: Honoraria; Protagonist Therapeutics: Honoraria; BMS: Consultancy, Honoraria. Kulozik:Novartis: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria. Thrasher:Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees. Thuret:Novartis pharma: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials; bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials; Apopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials. Lal:Insight Magnetics: Research Funding; Celgene, BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Terumo Corporation: Research Funding; Agios Pharmaceuticals: Consultancy; Chiesi USA: Consultancy; La Jolla Pharmaceutical Company: Research Funding; bluebird bio, Inc.: Research Funding; Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Guo:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Liu:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Colvin:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Walters:Veevo Biomedicine: Consultancy; AllCells, Inc: Consultancy; Editas: Consultancy. Locatelli:Jazz Pharmaceeutical: Speakers Bureau; Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Introduction We investigated the impact of β-thalassemia genotypes and disease genetic modifiers including HBA and KLF1 genotype and sentinel single-nucleotide polymorphism (SNP) genotypes at 3 major HbF quantitative trait loci (QTL) on clinical outcomes of TDT patients treated with beti-cel gene therapy in two phase 3 studies, HGB-207 (NCT02906202) and HGB-212 (NCT03207009). Methods HBA deletions and triplications were determined by gap-polymerase chain reactions. HBG2 (including rs7482144, Xmn1 site) and HBG1 promoters, HBA2, HBA1, and KLF1 underwent individual nucleotide sequencing. Multiplex amplification refractory mutation system (ARMS) tests were used to identify HbF QTL SNPs (rs10128556 in HBBP1; rs766432, rs1427407, rs10189857 in BCL11A; rs9399137, rs66650371 in HMIP). Thalassemia severity score (TSS) was calculated as defined by Danjou et al, Haematologica, 2015, considering gender, HBB and HBA genotypes, and 4 SNPs in HbF QTL (HBG2, BCL11A, HMIP). Correlative analyses were performed to assess relationships between genotype, presence/absence of non-HBB mutations (HBA2, HBA1, KLF1), presence/absence of HbF QTL SNPs (HBG2, BCL11A, HMIP), and TSS with the achievement of transfusion independence (TI; weighted average hemoglobin [Hb] ≥9 g/dL without red blood cell [RBC] transfusions for ≥12 months). Correlation coefficients used percentage bend correlation. Statistical significance threshold was p ≤ 0.05. Results As of 3 March 2020, 38 patients were treated in HGB-207 and HGB-212 (β0/β0 genotype n=9; non-β0/β0 genotype n=29 [β+/β+ n=8; β0/β+ n=15; βE/β0 n=6]). All patients were heterozygous or homozygous for mutations or SNPs that may modulate disease severity; 20 patients were homozygous for ≥1 mutation or SNP. Patients had the following alleles associated with higher HbF synthesis: HBG2 rs7482144 C>T (Xmn1 site), C/T n=9, T/T n=1; BCL11A rs1427407 G>T, G/T n=8; BCL11A rs10189857 A>G, A/G n=16, G/G n=18; HMIP rs9399137 T>C, T/C n=9, C/C n=2. Three patients were heterozygous for single α-globin gene deletion (-α/αα) and 2 were heterozygous for α-globin gene triplication (αα/ααα). Median TSS was 3.65 (min - max 0.4 - 8.1). TI was achieved by 23/27 (85%) evaluable patients; 4 patients with ≥ 12 months follow-up have been transfusion free for > 10 months but were not yet evaluable for TI (Figure). β-thalassemia genotype did not strongly correlate with TI (two-sided Fisher's Exact Test, p-value = 0.78). Month 12 median (min - max) peripheral blood vector copy number (PB VCN) was 1.5 (0.2 - 5.0) c/dg in TI or transfusion-free patients (n=27) and 0.2 (0.2 - 0.4) c/dg in patients who did not achieve TI (n=4). The transfusion-free patient (β0/β0) with the lowest month 12 PB VCN was homozygous for T/T at rs7482144 (HBG2Xmn1 site) and G/G at rs10189857 (BCL11A), and heterozygous for single α-globin gene deletion. Endogenous Hb (5.9 g/dL HbF + 0.2 g/dL HbA2) and gene therapy-derived HbAT87Q (4.4 g/dL) at month 12 enabled this patient to stop transfusions. Tests of association of SNPs and mutations with TI were not significant; no p-value < 0.31 (chi-squared test). As only 4 patients did not achieve TI, the power to detect an association was limited. Larger sample sizes are needed to determine if individual SNPs and mutations may have an impact on TI. In TI or transfusion-free patients (n=27), TSS correlated strongly with month 12 endogenous unsupported Hb (HbA + HbA2 + HbF + HbE without RBC transfusions for 60 days) (correlation coeff. = -0.76, p < 0.0001), but not with HbAT87Q (correlation coeff. = 0.26, p = 0.19) or unsupported total Hb (correlation coeff. = 0.32, p = 0.10). Beti-cel-related adverse events (AE) in >1 patient were abdominal pain (n=2 non-β0/β0; n=1 β0/β0), thrombocytopenia (n=3 non-b0/b0). Serious AEs in ≥3 patients post-infusion were thrombocytopenia (n=2 non-β0/β0; n=1 β0/β0), pyrexia (n=1 non-b0/b0; n=2 β0/β0), veno-occlusive disease (n=3 non-β0/β0). Summary Genetic characterization of TDT patients treated with beti-cel revealed diverse HBB and non-HBB mutations and polymorphisms that may influence disease severity. Higher PB VCN and HbAT87Q levels were associated with increased likelihood of TI. In instances of lower HbAT87Q, higher endogenous HbF might be a determinant in whether TI is achieved. Despite genetic heterogeneity, beti-cel enabled patients to achieve TI regardless of β-thalassemia genotype, TSS, disease genetic modifiers including HBA, and HbF QTL SNP genotypes. Disclosures Walters: Veevo Biomedicine: Consultancy; AllCells, Inc: Consultancy; Editas: Consultancy. Chui:bluebird bio, Inc.: Other: Payment for lab use for the sequencing analyses done for the studies. Lal:bluebird bio, Inc.: Research Funding; Agios Pharmaceuticals: Consultancy; Celgene, BMS: Membership on an entity's Board of Directors or advisory committees, Research Funding; La Jolla Pharmaceutical Company: Research Funding; Novartis: Research Funding; Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Terumo Corporation: Research Funding; Chiesi USA: Consultancy; Insight Magnetics: Research Funding. Locatelli:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharmaceeutical: Speakers Bureau. Kwiatkowski:bluebird bio, Inc.: Consultancy, Research Funding; Agios: Consultancy; Apopharma: Research Funding; Bristol Myers Squibb: Consultancy; Imara: Consultancy; Celgene: Consultancy; Sangamo: Research Funding; Terumo Corp: Research Funding; Novartis: Research Funding. Porter:bluebird bio, Inc.: Consultancy, Honoraria; Vifor Pharmaceuticals: Honoraria; La Jolla Pharmaceuticals: Honoraria; Protagonist Therapeutics: Honoraria; Agios Pharmaceuticals: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Silence Therapeutics: Honoraria. Thuret:Apopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis pharma: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials; Celgene: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials; bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Investigator in clinical trials. Kulozik:Novartis: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria. Thrasher:4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Equity ownership. Yannaki:bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Speakers Bureau; SANDOZ: Speakers Bureau; Gilead: Speakers Bureau. Yang:bluebird bio,Inc.: Current Employment, Current equity holder in publicly-traded company. Whitney:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Petrusich:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Colvin:bluebird bio, Inc.: Current Employment, Current equity holder in publicly-traded company. Thompson:BMS: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; CRISPR/Vertex: Research Funding; Biomarin: Research Funding; Baxalta: Research Funding.

ZusammenfassungDie β-Thalassämien und die Sichelzellerkrankung sind weltweit die häufigsten Einzelgendefekte, die konventionell nur durch eine lebenslang erforderliche Supportivtherapie oder kurativ mittels allogener Stammzelltransplantation behandelbar sind. Aktuell werden Gentherapiestrategien entwickelt, die nach Transduktion von hämatopoetischen Stammzellen durch lentivirale Vektoren zu einer Expression eines therapeutischen β-Globin-Gens oder zur therapeutischen Reexpression der fetalen Globinsynthese und damit des fetalen Hämoglobins (HbF) führen. Für die β-Thalassämie hat die Genadditionstherapie für eine ausgewählte Gruppe von Patientinnen und Patienten mit transfusionsbedürftiger Non-β0-Thalassaemia major nach konditionaler Zulassung durch die European Medicines Agency (EMA) im Juni 2019 einen ersten Grad klinischer Reife erreicht. In dieser Übersicht diskutieren wir darüber hinaus die Möglichkeiten und Herausforderungen von Genomeditierungsstrategien für die Hämoglobinkrankheiten, insbesondere durch Inaktivierung von BCL11A, des zentralen Inhibitors der postnatalen fetalen Hämoglobinsynthese. Insgesamt verspricht die Entwicklung der Gentherapie für die Hämoglobinkrankheiten eine relevante Chance für diese, auch in Deutschland zahlreicher werdenden Gruppe von Patientinnen und Patienten mit schweren, sowohl Lebensqualität als auch Lebenserwartung erheblich einschränkenden Erkrankungen.

While Wilms tumors (WT) typically present solely with an abdominally palpable mass, rare cases exhibiting vascular tumor growth can also present with circulatory problems. Here, we report the case of a 2.5-year-old girl presenting with upper venous congestion and arterial hypertension as the primary symptoms of intraventricular tumor growth exhibiting remarkable tubular and perfused morphology. Clinical situation stabilized after initiation of neoadjuvant chemotherapy (NAC) with actinomycin D and vincristine, followed by surgical resection via laparotomy and sternotomy supported by cardiopulmonary bypass and deep hypothermia. Our results highlight the previously reported feasibility of this approach, even in primarily unstable patients.

Summary Frameshifted protein sequences elicit tumor-specific T cell-mediated immune responses in microsatellite-unstable (MSI) cancers if presented by HLA class I molecules. However, their expression and presentation are limited by nonsense-mediated RNA decay (NMD). We employed an unbiased immunopeptidomics workflow to analyze MSI HCT-116 cells and identified >10,000 HLA class I-presented peptides including five frameshift-derived InDel neoepitopes. Notably, pharmacological NMD inhibition with 5-azacytidine stabilizes frameshift-bearing transcripts and increases the HLA class I-mediated presentation of InDel neoepitopes. The frameshift mutation underlying one of the identified InDel neoepitopes is highly recurrent in MSI colorectal cancer cell lines and primary patient samples, and immunization with the corresponding neoepitope induces strong CD8+ T cell responses in an HLA-A∗02:01 transgenic mouse model. Our data show directly that pharmacological NMD inhibition augments HLA class I-mediated presentation of immunogenic frameshift-derived InDel neoepitopes thus highlighting the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies., Graphical abstract, Highlights • Immunopeptidomic neoepitope discovery workflow employing public DNA sequencing data • Identification of immunogenic frameshift-derived InDel neoepitopes in an MSI model • NMD inhibition increases HLA class I-mediated presentation of InDel neoepitopes • CKAP2 frameshift mutation is highly recurrent in different types of MSI cancer, Biological Sciences ; Immunology ; Cancer

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

INFORM is a prospective, multinational registry gathering clinical and molecular data of relapsed, progressive, or high-risk pediatric patients with cancer. This report describes long-term follow-up of 519 patients in whom molecular alterations were evaluated according to a predefined seven-scale target prioritization algorithm. Mean turnaround time from sample receipt to report was 25.4 days. The highest target priority level was observed in 42 patients (8.1%). Of these, 20 patients received matched targeted treatment with a median progression-free survival of 204 days [95% confidence interval (CI), 99–not applicable], compared with 117 days (95% CI, 106–143; P = 0.011) in all other patients. The respective molecular targets were shown to be predictive for matched treatment response and not prognostic surrogates for improved outcome. Hereditary cancer predisposition syndromes were identified in 7.5% of patients, half of which were newly identified through the study. Integrated molecular analyses resulted in a change or refinement of diagnoses in 8.2% of cases. Significance: The pediatric precision oncology INFORM registry prospectively tested a target prioritization algorithm in a real-world, multinational setting and identified subgroups of patients benefiting from matched targeted treatment with improved progression-free survival, refinement of diagnosis, and identification of hereditary cancer predisposition syndromes. See related commentary by Eggermont et al., p. 2677 . This article is highlighted in the In This Issue feature, p. 2659

Sarcomas are malignant soft tissue and bone tumours affecting adults, adolescents and children. They represent a morphologically heterogeneous class of tumours and some entities lack defining histopathological features. Therefore, the diagnosis of sarcomas is burdened with a high inter-observer variability and misclassification rate. Here, we demonstrate classification of soft tissue and bone tumours using a machine learning classifier algorithm based on array-generated DNA methylation data. This sarcoma classifier is trained using a dataset of 1077 methylation profiles from comprehensively pre-characterized cases comprising 62 tumour methylation classes constituting a broad range of soft tissue and bone sarcoma subtypes across the entire age spectrum. The performance is validated in a cohort of 428 sarcomatous tumours, of which 322 cases were classified by the sarcoma classifier. Our results demonstrate the potential of the DNA methylation-based sarcoma classification for research and future diagnostic applications., This work was funded by Deutsche Krebshilfe grant 70112499, the NCT Heidelberg and an Illumina Medical Research Grant. Part of this work was funded by the National Institute of Health Research (to S.B. and Z.J.) and to UCLH Biomedical research centre (BRC399/NS/RB/101410). Human tissues were obtained from University College London NHS Foundation Trust as part of the UK Brain Archive Information Network (BRAIN UK, Ref: 18/004) which is funded by the Medical Research Council and Brain Tumour Research UK. The methylation profiling at NYU is supported by a grant from the Friedberg Charitable Foundation (to M.Sn.). M.Mi. would like to thank the Luxembourg National Research Fond (FNR) for the support (FNR PEARL P16/BM/11192868 grant). Open Access funding enabled and organized by Projekt DEAL.

Somatic structural variants (SVs) are widespread in cancer genomes, however, their impact on tumorigenesis and intra-tumour heterogeneity is incompletely understood, since methods to functionally characterize the broad spectrum of SVs arising in cancerous single-cells are lacking. We present a computational method, scNOVA, that couples SV discovery with nucleosome occupancy analysis by haplotype-resolved single-cell sequencing, to systematically uncover SV effects on cis-regulatory elements and gene activity. Application to leukemias and cell lines uncovered SV outcomes at several loci, including dysregulated cancer-related pathways and mono-allelic oncogene expression near SV breakpoints. At the intra-patient level, we identified different yet overlapping subclonal SVs that converge on aberrant Wnt signaling. We also deconvoluted the effects of catastrophic chromosomal rearrangements resulting in oncogenic transcription factor dysregulation. scNOVA directly links SVs to their functional consequences, opening the door for single-cell multiomics of SVs in heterogeneous cell populations.

Secondary thrombocytosis is a frequent secondary finding in childhood infection and inflammation. Primary hereditary thrombocytosis may be caused by germline mutations within the genes encoding key regulators of thrombopoiesis, i.e., thrombopoietin (THPO) and its receptor c-MPL (MPL) or the receptor’s effector kinase Januskinase2 (JAK2). Furthermore, somatic mutations in JAK2, MPL, and in the gene-encoding calreticulin (CALR) have been described to act as driver mutations within the so-called Philadelphia-negative myeloproliferative neoplasms (MPNs), namely essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Increasing knowledge on the molecular mechanisms and on the clinical complications of these diseases is reflected by the WHO diagnostic criteria and European LeukemiaNet (ELN) recommendations on the management of adult MPN. However, data on childhood thrombocytosis are rare, and no consensus guidelines for pediatric thrombocytosis exist. Current literature has highlighted differences in the epidemiology and molecular pathogenesis of childhood thrombocytosis as compared to adults. Furthermore, age-dependent complications and pharmacological specificities suggest that recommendations tailored to the pediatric population are necessary in clinical practice. Here we summarize literature on classification, diagnostics, and clinical management of childhood thrombocytosis.

Microsatellite-unstable (MSI) colorectal cancer is characterized by the accumulation of somatic insertion/deletion (InDel) mutations potentially generating tumor-specific, frameshifted protein sequences. Such mutations typically generate premature translation termination codons targeting the affected mRNAs to degradation by nonsense-mediated RNA decay (NMD), limiting the synthesis and HLA class I-mediated presentation of tumor-specific InDel neoepitopes. We reasoned that the NMD inhibitor 5-azacytidine (5AZA) could serve to increase the expression of NMD-sensitive neoepitopes and analyzed the immunopeptidome of MSI HCT-116 cells using a proteogenomic approach. After immunoprecipitation of HLA:peptide complexes, we identified more than 10,000 HLA class I-presented peptides by LC-MS/MS including five InDel neoepitopes. The InDel neoepitopes were verified on the genomic, transcriptomic, and peptidomic level. Treatment with 5AZA increased the expression of the corresponding frameshift- and premature termination codon-bearing mRNAs and enhanced the presentation of peptides originating from known NMD targets and one of the identified InDel neoepitopes. By analyzing an array of MSI colorectal cancer cell lines and patient samples, we found the underlying frameshift mutation to be highly recurrent and immunization with the corresponding neoepitope induced strong CD8+ T cell responses in an HLA-A*02:01 transgenic mouse model. Our data directly show that peptides originating from frameshifted open reading frames due to InDel mutations in mismatch repair-deficient cells are presented on the cell surface via HLA class I. Moreover, we demonstrate the utility of NMD inhibitor-enhanced HLA class I-mediated presentation of InDel neoepitopes as well as their immunogenicity, uncovering the clinical potential of NMD inhibition in anti-cancer immunotherapy strategies.One Sentence SummaryImmunopeptidomics identified increased HLA class I-mediated presentation of immunogenic, frameshift-derived neoepitopes following NMD inhibition.

We aimed at identifying the developmental stage at which leukemic cells of pediatric T‐ALLs are arrested and at defining leukemogenic mechanisms based on ATAC‐Seq. Chromatin accessibility maps of seven developmental stages of human healthy T cells revealed progressive chromatin condensation during T‐cell maturation. Developmental stages were distinguished by 2,823 signature chromatin regions with 95% accuracy. Open chromatin surrounding SAE1 was identified to best distinguish thymic developmental stages suggesting a potential role of SUMOylation in T‐cell development. Deconvolution using signature regions revealed that T‐ALLs, including those with mature immunophenotypes, resemble the most immature populations, which was confirmed by TF‐binding motif profiles. We integrated ATAC‐Seq and RNA‐Seq and found DAB1, a gene not related to leukemia previously, to be overexpressed, abnormally spliced and hyper‐accessible in T‐ALLs. DAB1‐negative patients formed a distinct subgroup with particularly immature chromatin profiles and hyper‐accessible binding sites for SPI1 (PU.1), a TF crucial for normal T‐cell maturation. In conclusion, our analyses of chromatin accessibility and TF‐binding motifs showed that pediatric T‐ALL cells are most similar to immature thymic precursors, indicating an early developmental arrest., Analysis of chromatin accessibility of human T‐cell precursors revealed progressive chromatin condensation during maturation. Pediatric T‐ALLs resemble the most immature populations indicating that the epigenetic landscape of this type of leukemia is most similar to the earliest thymic precursors.

Background To investigate the role of combined ion-beam radiotherapy (CIBRT) with protons and carbon ions in a multimodal treatment strategy of inoperable osteosarcoma; final analysis of a one-armed, single center phase I/II trial. Methods Between August 2011 until September 2018, 20 patients with primary (N = 18), metastatic (N = 3), or recurrent (N = 2) inoperable pelvic (70%) or craniofacial (30%) osteosarcoma were treated with protons up to 54 Gy (RBE) and a carbon ion boost of 18 Gy (RBE) and followed until May 2019. A Fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) was performed before CIBRT in search for a prognostic factor. The primary endpoint was toxicity. Secondary endpoints included treatment response, global, local and distant progression free survival (PFS, LPFS and DPFS) and overall (OS), among others. Results The median age was 20; all patients finished treatment per protocol. LPFS, DPFS, PFS and OS were 73%, 74%, 60% and 75% after one year and 55%, 65% 65.3%, 45% and 68% after two years, respectively. The median clinical target volume (CTV) was 1042 cc and 415 cc for the primary and boost plan, respectively. Craniofacial localization, lower uptake of FDG in PET/CT and boost plan CTV ≤ median were associated with improved overall survival (p = 0.039, p = 0.016 and p = 0.0043, respectively). No acute toxicities > grade III were observed. We observed one case of secondary acute myeloid leukemia (AML) seven months after CIBRT for recurrent disease and one case of hearing loss. Conclusion CIBRT shows a favorable toxicity profile and promising results particularly for patients with inoperable craniofacial osteosarcoma.

DNA mismatch repair-deficient colorectal cancers (CRCs) accumulate numerous frameshift mutations at repetitive sequences recognized as microsatellite instability (MSI). When coding mononucleotide repeats (cMNRs) are affected, tumors accumulate frameshift mutations and premature termination codons (PTC) potentially leading to truncated proteins. Nonsense-mediated RNA decay (NMD) can degrade PTC-containing transcripts and protect from such faulty proteins. As it also regulates normal transcripts and cellular physiology, we tested whether NMD genes themselves are targets of MSI frameshift mutations. A high frequency of cMNR frameshift mutations in the UPF3A gene was found in MSI CRC cell lines (67.7%), MSI colorectal adenomas (55%) and carcinomas (63%). In normal colonic crypts, UPF3A expression was restricted to single chromogranin A-positive cells. SILAC-based proteomic analysis of KM12 CRC cells revealed UPF3A-dependent down-regulation of several enzymes involved in cholesterol biosynthesis. Furthermore, reconstituted UPF3A expression caused alterations of 85 phosphosites in 52 phosphoproteins. Most of them (38/52, 73%) reside in nuclear phosphoproteins involved in regulation of gene expression and RNA splicing. Since UPF3A mutations can modulate the (phospho)proteomic signature and expression of enzymes involved in cholesterol metabolism in CRC cells, UPF3A may influence other processes than NMD and loss of UPF3A expression might provide a growth advantage to MSI CRC cells.

Sickle cell disease and the ß-thalassemias are caused by mutations of the ß-globin gene and represent the most frequent single gene disorders worldwide. Even in European countries with a previous low frequency of these conditions the prevalence has substantially increased following large scale migration from Africa and the Middle East to Europe. The hemoglobin diseases severely limit both, life expectancy and quality of life and require either life-long supportive therapy if cure cannot be achieved by allogeneic stem cell transplantation. Strategies for ex vivo gene therapy aiming at either re-establishing normal ß-globin chain synthesis or at re-activating fetal γ-globin chain and HbF expression are currently in clinical development. The European Medicine Agency (EMA) conditionally licensed gene addition therapy based on lentiviral transduction of hematopoietic stem cells in 2019 for a selected group of patients with transfusion dependent non-ß° thalassemia major without a suitable stem cell donor. Gene therapy thus offers a relevant chance to this group of patients for whom cure has previously not been on the horizon. In this review, we discuss the potential and the challenges of gene addition and gene editing strategies for the hemoglobin diseases.

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/�����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/�� 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., publishedVersion

PLOS ONE 15(9), e0237792 (2020). doi:10.1371/journal.pone.0237792, Published by PLOS, San Francisco, California, US

This study reports the prognostic impact of the expression of the natural killer cell marker CD56 in a large series of risk-adapted paediatric patients with T cell acute lymphoblastic leukaemia (T-ALL; n = 493) treated within the ALL-Berlin-Frankfurt-Munster (BFM) 2000 protocol. The immunophenotype was analysed centrally at diagnosis using flow cytometry and correlated with clinical parameters and outcome. CD56 expression was detected in 7·1% and early T-cell precursor (ETP) phenotype in 6·7% of all T-ALL patients. The percentage of ETP in the CD56+ T-ALL cohort was 4-fold higher than in the whole cohort. CD56+ T-ALL frequently expressed the progenitor marker CD34 and myeloid antigens CD13 and CD33. The 5-year event-free survival (EFS) rates for the European Group for the Immunological classification of Leukaemias/World Health Organization subgroups and the ETP phenotype were not statistically different. By contrast, patients with CD56 expression had a significantly reduced EFS (60 ± 8%) and overall survival (60 ± 8%) at 5 years, with a hazard ratio of 2·46 (P = 0·002) and 2·99 (P < 0·001), respectively. Moreover, CD56 expression in combination with the minimal residual disease (MRD)-based high risk assignment defined a population with a 'very-high' risk probability of relapse in the ALL-BFM 2000 trial. The CD56 marker has the potential to augment MRD-based risk stratification and may serve as a molecular target for antibody-based treatment strategies in childhood T-ALL.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging - with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Introduction: Transfusion-dependent β-thalassemia (TDT) is a severe genetic disease requiring lifelong, regular packed red blood cell (pRBC) transfusions and iron chelation treatment. Betibeglogene autotemcel (beti-cel) is a one-time ex vivo gene therapy that addresses the underlying cause of TDT to potentially enable lifelong, stable production of modified adult hemoglobin (Hb) sufficient to achieve transfusion independence (TI). Patients (pts) with TDT (N=63) were treated with beti-cel in 2 completed phase 1/2 studies (HGB-204, HGB-205) and 2 ongoing phase 3 studies (HGB-207, HGB-212). After 2 y of follow-up, pts could enroll in a long-term follow-up study (LTF-303 [NCT02633943]) for up to an additional 13 y. We report results from LTF-303 in pts with up to 7 y of follow-up. Methods: Pts underwent single-agent, pharmacokinetic-adjusted busulfan myeloablation and were infused with autologous CD34+ cells transduced with BB305 lentiviral vector. The phase 3 studies used a refined transduction process compared with the phase 1/2 studies. LTF-303 assessments included transfusion status, Hb, iron overload, and safety. Around the time of beti-cel infusion, pts stopped iron chelation; however, chelation could be resumed post-infusion at the physician's discretion. A subanalysis was conducted to assess iron status in pts who restarted then stopped chelation. Pts who achieved TI and had a minimum of 9 mo of follow-up after discontinuation of chelation were included in the subanalysis to assess iron parameters while off chelation. Data are reported as median (min-max) unless otherwise stated. Results: As of March 9, 2021, LTF-303 enrolled 51 pts (phase 1/2: 22 pts; phase 3: 29 pts), of which 55% were female; the median age was 19 (7-35) y. Post-infusion follow-up was 44.2 (22.9-86.5 mo; 1 pt had their Month 24 visit at 22.9 mo). TI (weighted average Hb ≥9 g/dL without pRBC transfusions for ≥12 mo) was achieved in 15/22 (68%) pts treated in the phase 1/2 studies and in 25/29 (86%) pts treated in the phase 3 studies (all during parent study); at last follow-up, all pts had remained TI for 32.2 (13.1-84.1) mo. Median (Q1, Q3) gene therapy-derived Hb (HbA T87Q) in pts treated in the phase 1/2 studies who achieved TI was stable over time: 7.3 (5.4-8.4) g/dL at Month 24 (n=15) and 7.6 (6.6-9.3) g/dL at Month 72 (n=7). Median (Q1, Q3) HbA T87Q in pts treated in the phase 3 studies was also stable: 9.1 (8.5-10.3) g/dL at Month 24 (n=24) and 9.9 (9.8-10.0) g/dL at Month 42 (n=2). HbA T87Q helped to sustain total Hb. Total Hb was a median (Q1, Q3) of 10.0 (9.7-11.8) g/dL at Month 24 and 10.5 (10.3-12.1) g/dL at Month 72 in phase 1/2 studies and 12.1 (10.8-13.1) g/dL at Month 24 and 12.2 (11.7-12.8) g/dL at Month 42 in phase 3 studies. Pts who achieved TI (n=40) experienced reductions in iron burden over time; all pts had normal cardiac T2* levels at last follow-up and liver iron concentration (LIC) and serum ferritin were positively and strongly correlated at Months 24, 36, and 48 (Table). The subanalysis showed iron reduction in response to chelation and stabilization of iron markers after chelation was discontinued (Figure). Of the 15 pts who achieved TI in phase 1/2 studies, 10 (67%) restarted and then stopped iron chelation 25 (12-56) mo after beti-cel infusion; the 5 remaining pts continued on chelation. Of the 25 pts who achieved TI in phase 3 studies, 18 (72%) stopped iron chelation 29 (6-47) mo after beti-cel infusion; 11 (44%) pts never restarted and 7 (28%) restarted and then stopped again. Two pts in the subanalysis treated in a phase 1/2 study also received phlebotomy for approximately 20 and 56 mo post-infusion. Serious AEs occurring after 2 y in LTF-303 were reported in 8 pts (diabetic ketoacidosis, gonadotropic insufficiency, ectopic pregnancy, fetal death, cholelithiasis, gallbladder wall thickening/polyp, bacteremia with neutropenia, pulmonary embolism, and major depression; each n=1). No beti-cel-related AEs were observed beyond 2 y post-infusion. No deaths, replication-competent lentivirus, insertional oncogenesis, or malignancies were reported. Insertion site analysis indicated sustained polyclonal hematopoiesis. Conclusions: Beti-cel is a potentially curative gene therapy for pts with TDT through the ability to achieve TI with near-normal Hb levels. Sustained levels of HbA T87Q and effective restoration of iron homeostasis over time reduced iron management burden in pts treated with beti-cel. Figure 1 Figure 1. Disclosures Thompson: Baxalta: Research Funding; Biomarin: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Celgene/BMS: Consultancy, Research Funding; CRISPR Therapeutics: Research Funding; Vertex: Research Funding; Editas: Research Funding; Graphite Bio: Research Funding; Novartis: Research Funding; Agios: Consultancy; Beam: Consultancy; Global Blood Therapeutics: Current equity holder in publicly-traded company. Locatelli: Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Miltenyi: Speakers Bureau; Medac: Speakers Bureau; Jazz Pharamceutical: Speakers Bureau; Takeda: Speakers Bureau. Yannaki: Novartis: Speakers Bureau; bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; SANDOZ: Speakers Bureau; Gilead: Speakers Bureau. Walters: Vertex pharmaceuticals: Consultancy; Ensoma, Inc.: Consultancy; BioLabs, Inc: Consultancy; AllCells, Inc: Consultancy. Porter: Agios: Consultancy, Honoraria; Protagonism: Honoraria; La Jolla Pharmaceuticals: Honoraria; Celgene (BMS): Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vifor: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kulozik: Sanofi: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BioMedX: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Thrasher: Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees. Thuret: Novartis: Other: investigators for clinical trials, participation on scientific/medical advisory board; Apopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc.: Other: investigators for clinical trials, participation on scientific/medical advisory board; Celgene: Other: investigators for clinical trials, participation on scientific/medical advisory board. Lal: Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Insight Magnetics: Research Funding; Chiesi: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; bluebird bio, Inc.: Research Funding; Agios Pharmaceuticals: Consultancy; Terumo Corporations: Research Funding; Novartis: Research Funding; La Jolla Pharmaceutical Company: Research Funding. Cavazzana: Smart Immune: Other: co-founder. Rasko: Australian Government: Membership on an entity's Board of Directors or advisory committees; Cynata: Honoraria, Speakers Bureau; Genea: Current equity holder in publicly-traded company; Cure the Future Foundation: Membership on an entity's Board of Directors or advisory committees; Imago: Consultancy; Gene Technology Technical Advisory Board: Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Speakers Bureau; NHMRC Mitochondrial Donation Expert Working Committee: Membership on an entity's Board of Directors or advisory committees; Australian Cancer Research: Membership on an entity's Board of Directors or advisory committees; FSHD Global Research Foundation: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer Inc: Honoraria, Speakers Bureau; Glaxo-Smith-Kline: Honoraria, Speakers Bureau; Spark: Honoraria, Speakers Bureau; Takeda: Honoraria, Speakers Bureau; bluebird bio: Honoraria, Speakers Bureau. Du: bluebird bio, Inc.: Current Employment, Other: bluebird bio, Inc.: Employee, Ownership Interest and Salary. Colvin: bluebird bio, Inc.: Current Employment, Other: Employee, Ownership Interest and Salary. Kwiatkowski: Agios: Consultancy; Bioverativ: Research Funding; Imara: Consultancy, Research Funding; bluebird bio,Inc.: Consultancy, Research Funding; Sangamo: Research Funding; Silence Therapeutics: Consultancy; Bristol Myers Squibb: Consultancy; Apopharma: Research Funding; Celgene: Consultancy.

Introduction: Patients with transfusion-dependent β-thalassemia (TDT) have increased morbidity and mortality and reduced health-related quality of life (HRQoL) compared with the general population owing to the mental, physical, and time demands of chronic transfusions, iron chelation, and treatment. Betibeglogene autotemcel (beti-cel) is a one-time ex vivo gene therapy for TDT that enables stable production of functional adult hemoglobin (Hb) sufficient for transfusion independence (TI). In 2 ongoing, fully enrolled, phase 3 studies, HGB-207 (Northstar-2; NCT02906202) and HGB-212 (Northstar-3; NCT03207009), 32/36 (89%) evaluable patients achieved TI (weighted average Hb ≥9 g/dL without packed red blood cell transfusions for ≥12 mo after drug product infusion) with a median (min-max) weighted average Hb of 11.52 (9.3-13.7) g/dL during TI (as of March 9, 2021). In the current analysis, we evaluated the effect of beti-cel on HRQoL in 12 adults and 18 pediatric/adolescent patients with TDT enrolled in phase 3 studies who achieved TI. Methods: CD34+ cells were mobilized using granulocyte-colony stimulating factor and plerixafor, collected via apheresis, transduced with BB305 lentiviral vector, and infused back into patients after single-agent, pharmacokinetic-adjusted, busulfan-based myeloablation. HRQoL over time was assessed using the Pediatric Quality of Life Inventory (PedsQL; range, 0-100) for pediatric and adolescent patients ( Results: Forty-one patients were infused with beti-cel (HGB-207, n=23; HGB-212, n=18). Age at informed consent was 4-34 years; most patients (27/41, 66%) were Conclusions: Beti-cel is potentially curative in patients with TDT through the achievement of TI and near-normal Hb levels. Beti-cel addresses the disease pathophysiology at a genetic level and allows patients to achieve TI. Pediatric and adult patients with TDT who achieved TI after receiving beti-cel in phase 3 studies showed early and sustained improvements in HRQoL, despite relatively high HRQoL with supportive care at baseline. Figure 1 Figure 1. Disclosures Kwiatkowski: bluebird bio,Inc.: Consultancy, Research Funding; Celgene: Consultancy; Sangamo: Research Funding; Apopharma: Research Funding; Bristol Myers Squibb: Consultancy; Imara: Consultancy, Research Funding; Agios: Consultancy; Silence Therapeutics: Consultancy; Bioverativ: Research Funding. Locatelli: Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyl: Honoraria; bluebird bio, Inc.: Consultancy; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Walters: AllCells, Inc: Consultancy; BioLabs, Inc: Consultancy; Vertex pharmaceuticals: Consultancy; Ensoma, Inc.: Consultancy. Porter: Silence Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene (BMS): Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; La Jolla Pharmaceuticals: Honoraria; Protagonism: Honoraria; Vifor: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Honoraria. Yannaki: Gilead: Speakers Bureau; SANDOZ: Speakers Bureau; bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Speakers Bureau. Kulozik: bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BioMedX: Consultancy, Honoraria. Thrasher: Rocket Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; 4Bio Capital: Consultancy, Membership on an entity's Board of Directors or advisory committees; Generation bio: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Thuret: bluebird bio, Inc.: Other: investigators for clinical trials, participation on scientific/medical advisory board; Apopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: investigators for clinical trials, participation on scientific/medical advisory board; Celgene: Other: investigators for clinical trials, participation on scientific/medical advisory board. Lal: Insight Magnetics: Research Funding; Protagonist Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; La Jolla Pharmaceutical Company: Research Funding; Chiesi: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; bluebird bio, Inc.: Research Funding; Agios Pharmaceuticals: Consultancy; Terumo Corporations: Research Funding. Guo: bluebird bio, Inc.: Current Employment, Other: Employee, Ownership Interest and Salary. Colvin: bluebird bio, Inc.: Current Employment, Other: Employee, Ownership Interest and Salary. Gruppioni: bluebird bio, Inc.: Current Employment, Other: Employee, Ownership Interest and Salary. Thompson: Baxalta: Research Funding; bluebird bio, Inc.: Consultancy, Research Funding; Biomarin: Research Funding; Celgene/BMS: Consultancy, Research Funding; CRISPR Therapeutics: Research Funding; Vertex: Research Funding; Editas: Research Funding; Graphite Bio: Research Funding; Novartis: Research Funding; Agios: Consultancy; Beam: Consultancy; Global Blood Therapeutics: Current equity holder in publicly-traded company.

Background Worldwide, Sickle Cell Disease (SCD) is the most common single gene disorder affecting >250,000 newborns annually. In Germany, SCD qualifies as a rare disease and almost exclusively affects immigrants from endemic countries and their descendants. The recent surge of immigration from high-prevalence countries increased the numbers of patients with SCD in Germany and raised awareness for the need of specialized care. In 2012, the German Society for Pediatric Oncology and Hematology (GPOH) mandated a consortium of five university hospitals to develop a disease management program for patients with SCD. This consortium issued treatment guidelines for SCD that strongly favor the use of hydroxyurea, initiated patient and physician education events, prepared for a universal newborn screening program that will start 09/2021, moderated a consensus on the indication of allogeneic stem cell transplantation for patients with SCD, and established a national patient registry. Methods In order to quantify the effect of these activities, 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). ICD10 codes were used to identify patients with SCD and their comorbidities. Pharmacologic treatments were quantified using the German Anatomical Therapeutic Chemical (ATC)-Classification with defined daily doses. Results We estimate that the number of patients with SCD in Germany increased from approximately 2,200 in 2011 to approximately 3,200 in 2019. Analyses of administered treatments illustrate that important components of recently issued national treatment guidelines have been largely implemented. Specifically, the use of hydroxyurea has more than doubled, resulting in a proportion of approximately 45% of all patients with SCD being treated with hydroxyurea in 2019 (Figure 1A). In strong negative correlation with the use of hydroxyurea, the frequency of acute chest syndromes decreased (Figure 1B). While before the widespread use of hydroxyurea (2011-2013) 8.1% of patients with SCD were admitted at least once per year for ACS, this proportion dropped to 6.6% in the period 2017 to 2019. Similarly, the proportion of patients who required analgesics, red blood cell transfusions and hospitals admissions declined from 2011 to 2019, indicating a reduced burden of SCD with the increased use of hydroxyurea. Conclusion In sum, these data demonstrate an association between the dissemination of nationwide treatment guidelines and changes in clinical practice in particular relating to the use of hydroxyurea. These changes translate into a remarkable improvement of key measures of disease activity in a representative population based analysis. Figure 1 Figure 1. Disclosures Lobitz: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AddMedica: Honoraria, Membership on an entity's Board of Directors or advisory committees; Vertex: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kulozik: BioMedX: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Introduction Patients who suffer a relapse of pediatric T-cell acute lymphoblastic leukemia (T-ALL) face a dismal prognosis. Prognostic molecular biomarkers that reliably predict the risk of relapse at the time of first diagnosis are not available. Inactivating mutations in TP53 were previously detected in approximately 10% of relapsed patients (Hof et al. J Clin Oncol. 2011) and are invariably associated with fatal outcome (Richter-Pechanska et al. Blood Cancer J. 2017). Mutations in other genes were identified to be either specific for relapse (NT5C2 and CCDC88A) or to be associated with a poor prognosis in relapse (IL7R, KRAS, NRAS, USP7, CNOT3 and MSH6) (Meyer et al. Nat Genet. 2013; Richter-Pechanska et al. Blood Cancer J. 2017). We hypothesized that subclones bearing such mutations can give rise to relapse and analyzed these 9 genes at initial diagnosis of T-ALL with targeted ultra-deep sequencing. Methods Leukemia samples collected at initial diagnosis of 81 children with T-ALL who later relapsed were analyzed. As a control group, we selected 79 children with T-ALL who remained in first remission for at least three years and were matched with regard to treatment response, treatment, age and sex. Targeted deep sequencing was performed by using the Agilent Haloplex High Sensitivity kit with unique molecular identifiers for reliable detection of mutations with very low allele frequencies (average read depth: 1,012x). Results Overall, we detected 75 mutations among 7 targeted genes in 33 / 81 relapsing and 21 / 79 non-relapsing patients. The average allele frequency (AF) of the identified mutations was 25% (0.8% - 83%; SD ± 18%). More than half of the variants (43/75) showed AFs below 30% and were thus classified as subclonal. Interestingly, 7 pathogenic TP53 mutations (subclonal: n=5, clonal: n=2) with AFs of 4.4% - 49.4% were exclusively discovered in 6 patients who experienced a relapse. While 2 of these patients received an allogeneic stem cell transplantation in first remission because of poor treatment response, the remaining 4 patients were treated by chemotherapy in the high-risk (n=1) or medium-risk (n=3) arm. None of the 79 non-relapsing control patients carried TP53 mutations. Consistent with the hypothesis of clonal evolution as a mechanism of relapse in T-ALL, Sanger Sequencing of the relapse sample of one TP53-positive patient confirmed that the subclone harboring the TP53 mutation A159D at initial diagnosis (AF 5.4%) expanded to a major clone (AF 42%) in relapse. The presence of TP53 mutations in two further TP53-positive patients in at least one available post-remission sample is also compatible with clonal selection. However, in a fourth patient the low allele frequency of the TP53 mutation at relapse indicates that the TP53 subclone persisted but did not expand during the development of relapse. In addition to TP53, we identified pathogenic KRAS mutations to be significantly enriched in relapsing patients (9 / 81) compared to non-relapsing patients (2 / 79) at the time of initial diagnosis (chi-squared test, p= 0.032; Table 1). Conclusion Subclonal and clonal mutations in TP53 and KRAS at initial diagnosis were enriched in T-ALL patients who later relapsed and identified approximately 17% of patients suffering a relapse. We thus propose that (subclonal) mutations of TP53 and KRAS may define a subgroup of high-risk T-ALL patients already at the time of first diagnosis. The identification of such mutations may complement the current risk stratification which depends on treatment response and may determine a new molecularly defined subgroup of T-ALLs that may benefit from intensified treatment strategies. Figure 1 Figure 1. Disclosures Schrappe: SigmaTau: Other: research support; Amgen: Other: research support; Servier: Honoraria; Novartis: Honoraria; JazzPharma: Honoraria; Servier: Honoraria, Other: research support; JazzPharma: Honoraria, Other: research support; SHIRE: Other: research support; Novartis: Honoraria, Other: research support. Cario: Novartis: Other: Lecture Fee. Muckenthaler: Silence Therapeutics: Research Funding. Kulozik: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; BioMedX: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Consultancy, Honoraria.

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.

We report 5 individuals in 3 unrelated families with severe thrombocytopenia progressing to trilineage bone marrow failure (BMF). Four of the children received hematopoietic stem cell transplants and all showed poor graft function with persistent severe cytopenias even after repeated transplants with different donors. Exome and targeted sequencing identified mutations in the gene encoding thrombopoietin (THPO): THPO R99W, homozygous in affected children in 2 families, and THPO R157X, homozygous in the affected child in the third family. Both mutations result in a lack of THPO in the patients' serum. For the 2 surviving patients, improvement in trilineage hematopoiesis was achieved following treatment with a THPO receptor agonist. These studies demonstrate that biallelic loss-of-function mutations in THPO cause BMF, which is unresponsive to transplant due to a hematopoietic cell-extrinsic mechanism. These studies provide further support for the critical role of the MPL-THPO pathway in hematopoiesis and highlight the importance of accurate genetic diagnosis to inform treatment decisions for BMF.

Background Temozolomide (TMZ) is widely used in high-grade glioma (HGG). There is a major concern of treatment-induced secondary haematological malignancies (SHMs). Due to the poor overall survival of HGG patients, the true incidence is yet elusive. Thus, the aim of this study was to determine the risk of SHMs following TMZ in paediatric HGG. Methods We analysed 487 patients from the HIT-HGG database of the German-speaking Society of Pediatric Oncology and Hematology with follow up beyond 1 year. Results The incidence of SHM was 7.7 ± 3.2% at 10 years. No SHM occurred in 194 patients after first-line TMZ therapy, but four out of 131 patients treated with TMZ for relapse following first-line multiagent chemotherapy experienced SHM (20% at 10 years; p = 0.041). SHMs occurred in two out of 162 patients who underwent multiagent chemotherapy without TMZ (4.1% at 10 years). Gender, patient age and acute haematological toxicity during treatment did not affect the incidence of SHMs. Conclusion Data of our cohort do not indicate an increased risk of SHM following TMZ treatment when compared to previous chemotherapy regimen. However, if TMZ is administered as a second-line treatment following conventional chemotherapy regimen, the risk might be disproportionately increasing.

Hintergrund Zur Einfuhrung des Neugeborenenscreening auf Mukoviszidose (CF) in Deutschland hat der Gemeinsame Bundesausschuss (G-BA) ein neues Screeningprotokoll mit der Bestimmung von Immunreaktiven Trypsinogen (IRT) als ersten und Pankreatitis assoziierten Protein (PAP) als zweiten Schritt eingefuhrt. In einem dritten Schritt folgt eine Analytik von 31 CFTR-Mutationen, um den positiven Vorhersagewert (PPV) gegenuber einem rein biochemischen IRT/PAP-Protokoll zu erhohen. Methoden Der Datenpool (n=372 906) einer Studie zur Evaluation eines rein biochemischen IRT/PAP-Protokolls wurde fur den Vergleich des vom G-BA beschlossenen Protokolls mit einem von den Autoren vorgeschlagenen Alternativprotokoll genutzt. Der Unterschied beider Protokolle liegt im weiteren Vorgehen, wenn das IRT>99,9. Perzentile liegt. Im G-BA-Protokoll wird dann sowohl der PAP- als auch der DNA-Schritt, im Alternativprotokoll nur der PAP-Schritt umgangen. Ergebnisse Beide 3-stufigen Protokolle verlieren durch die DNA-Analytik im Vergleich zum 2-stufigen IRT/PAP-Protokoll nicht an Sensitivitat. Unterschiede zeigten sich jedoch im PPV: Das G-BA-Protokoll zeigt mit 351 deutlich mehr falsch-positiv getestete Neugeborene (PPV 20,2%) als das Alternativprotokoll mit 31 (PPV 69,6%). Schlussfolgerung Das G-BA-Protokoll hat im Vergleich zum Alternativprotokoll die schlechtere Performance. Durch die zu erwartende hohere Zahl an falsch-positiv detektierten Neugeborenen im G-BA-Protokoll werden mehr Vorstellungen zur Konfirmationsdiagnostik und fur Schweistests notwendig, mehr Familien verunsichert und hohere Kosten nach dem Screening verursacht.

// Elena Vendramini 1, 2, 3 , Marco Giordan 1 , Emanuela Giarin 1 , Barbara Michielotto 1 , Grazia Fazio 4 , Gianni Cazzaniga 4 , Andrea Biondi 4 , Daniela Silvestri 4 , Maria Grazia Valsecchi 5 , Martina U. Muckenthaler 6 , Andreas E. Kulozik 6 , Valter Gattei 7 , Shai Izraeli 2, 3 , Giuseppe Basso 1 and Geertruy te Kronnie 1 1 Department of Women’s and Children’s Health, University of Padova, Padova, Italy 2 Edmond and Lily Safra Children’s Hospital, Sheba Medical Center, Ramat Gan, Israel 3 Tel Aviv University, Tel Aviv, Israel 4 Centro Ricerca Tettamanti, Clinica Pediatrica, University of Milano-Bicocca, Monza, Italy 5 School of Medicine and Surgery, University of Milano-Bicocca, Milano, Italy 6 Department of Pediatric Oncology Hematology, University of Heidelberg, Heidelberg, Germany 7 Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico, I.R.C.C.S., Aviano (PN), Italy Correspondence to: Elena Vendramini, email: elena.vendramini@gmail.com Keywords: B cell precursor acute lymphoblastic leukemia, ERG aberrations, noncoding RNAs, miR-125, SNORD116 Received: June 29, 2016 Accepted: March 04, 2017 Published: March 21, 2017 ABSTRACT ERG-related leukemia is a B cell precursor acute lymphoblastic leukemia (BCP ALL) subtype characterized by aberrant expression of DUX4 and ERG transcription factors, and highly recurrent ERG intragenic deletions. ERG-related patients have remarkably favorable outcome despite a high incidence of inauspicious IKZF1 aberrations. We describe clinical and genomic features of the ERG-related cases in an unselected cohort of B-other BCP ALL pediatric patients enrolled in the AIEOP ALL 2000 therapeutic protocol. We report a small noncoding RNA signature specific of ERG-related group, with up-regulation of miR-125b-2 cluster on chromosome 21 and several snoRNAs in the Prader-Willi locus at 15q11.2, including the orphan SNORD116 cluster.

Recent developments in sequencing technologies led to the discovery of a novel form of genomic instability, termed chromothripsis. This catastrophic genomic event, involved in tumorigenesis, is characterized by tens to hundreds of simultaneously acquired locally clustered rearrangements on one chromosome. We hypothesized that leukemias developing in individuals with Ataxia Telangiectasia, who are born with two mutated copies of the ATM gene, an essential guardian of genome stability, would show a higher prevalence of chromothripsis due to the associated defect in DNA double-strand break repair. Using whole-genome sequencing, fluorescence in situ hybridization and RNA sequencing, we characterized the genomic landscape of Acute Lymphoblastic Leukemia (ALL) arising in patients with Ataxia Telangiectasia. We detected a high frequency of chromothriptic events in these tumors, specifically on acrocentric chromosomes, as compared with tumors from individuals with other types of DNA repair syndromes (27 cases total, 10 with Ataxia Telangiectasia). Our data suggest that the genomic landscape of Ataxia Telangiectasia ALL is clearly distinct from that of sporadic ALL. Mechanistically, short telomeres and compromised DNA damage response in cells of Ataxia Telangiectasia patients may be linked with frequent chromothripsis. Furthermore, we show that ATM loss is associated with increased chromothripsis prevalence in additional tumor entities.

Despite progress with immunotherapy and targeted agents, treatment of refractory disease remains challenging, in particular for patients with T-ALL. Identification of genomic lesions defining actionable targets had limited impact on patient care so far. The complexity of biological systems highlights the need to develop complementary functional approaches. We and others have established platforms with a library of 120 drugs based on current treatment and (pre-)clinical development, to detect ex-vivo drug response phenotypes on leukemia samples at single cell resolution by high content image analysis. We demonstrated that drug response profiling (DRP) identifies dependencies not predicted by genetic alterations adding a functional information layer for clinicians. Here we report first correlations with clinical outcome using DRP in a non-interventional setting. From 2016 to 2019 we performed DRP in the framework of European ALL first- and second- line protocols upon request by treating centers. Here we analyze retrospectively treatment decisions and outcome for 23 T- and 50 BCP-ALL patients. To evaluate drug responses, we compared dose response curves of individual patients to data recorded for all patients. Sensitivity and resistance were defined based on the IC50 outlier analysis using cut-offs depending on distribution (normal gaussian vs. skewed). From 73 patients tested, clinical outcome data has been available for 36 BCP- and 15 T-ALL patients. NGS data provided by the INFORM registry has been available in 8 BCP- and 2 T-ALL patients. In first line BCP-ALL patients, ex-vivo Dexamethasone response predicted clinical response to prephase prednisone (d8) and minimal residual disease (MRD) reduction measured by flow cytometry at d15 of first line AIEOP BFM 2009 induction (Fig. 1). For refractory and relapsed ALL we observed an association of DRP and response to targeted agents in 14 out of 16 patients (87.5 %; Table 1). Data for the r/r BCP-ALL cohort is limited because most patients underwent CD19 and / or CD22-directed immunotherapy. Sensitivity and resistance to Calicheamicin correlated with clinical response to Inotuzumab, suggesting functional testing to be evaluated in future studies. In contrast, lack of correlation of ex-vivo sensitivity to MEK-inhibitors with presence of RAS-pathway alterations caution the exclusive use of molecular information to predict response to these agents. Most therapeutic decisions based on DRP information were made for patients with r/r T-ALL. Bortezomib ex-vivo sensitivity correlated with clinical responses in 5 T-ALL patients (Fig. 2). Both patients predicted to respond to Bortezomib and treated on Bortezomib + Venetoclax experienced good MRD response providing a bridge to stem cell transplantation (SCT). However, these patients relapsed after SCT emphasizing the need for additional consolidative therapeutic elements for heavily pretreated patients. In line with previous reports, we confirmed a T-ALL with high sensitivity to Dasatinib (IC50 1.9 nM; Fig. 3). Dasatinib monotherapy induced a molecular remission. A 2nd T-ALL showing an ex- vivo Dasatinib IC50 at 80 nM was refractory to treatment with Dasatinib + Daunoxome-FLAG. A 3rd ABL1-fusion positive T-ALL, ex-vivo resistant to Imatinib and Dasatinib, had only short- term response to Imatinib + chemotherapy. Finally, treating a T-ALL patient based on high sensitivity to the XPO1 inhibitor Selinexor as 4th line monotherapy led to significant decrease of PB blasts from d1 25 G/L to 0.7 G/L at d13 of treatment (Fig. 4). The patient experienced improved quality of life, minimizing need of hospitalization with stable disease for 3 months on maintenance with Selinexor. Given the promising preclinical data with this class of agents and current lack of established biomarkers, we propose that DRP should be evaluated for this class of agents. In conclusion, we established first associations between DRP and clinical response for various agents providing a rationale for the evaluation of DRP in prospective clinical trials. Integration of molecular and functional information may improve the selection of more specific treatment options for patients with resistant disease. The international BFM Study Group and ITCC Consortium are planning an international multiarm early clinical trial for treatment of r/r ALL patients that will include DRP for evaluation in order to improve selection of targeted therapy. Disclosures Cario: Jazz Pharmaceuticals: Consultancy, Other: travel support; Novartis: Consultancy, Other: travel support. Hrusak:Amgen: Other: MRD investigations funded by Amgen, Research Funding. Kulozik:Novartis: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria. von Stackelberg:Morphosys: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Jazz: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau; Shire: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees, advisory committees and speakers bureau, Speakers Bureau. Jacoby:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Lonza: Membership on an entity's Board of Directors or advisory committees. Bourquin:Servier: Other: Travel Support.

Antibody application is a promising therapy in hematological malignancies including acute lymphoblastic leukemia (ALL). Unlike for B-cell precursor (BCP-ALL), immunotherapeutic interventions in T-cell ALL (T-ALL) are practically non-existent. Most T-ALL patient samples show substantial surface expression of CD38. Moreover, mice bearing T-ALL patient-derived xenograft (PDX) samples treated with daratumumab (Dara) monotherapy displayed prolonged survival and MRD-negativity in 50% of cases as opposed to animals treated with chemotherapy (Vogiatzi et al., Blood, 2019). Besides CD38, elevated surface expression of CD47 has been described in T-ALL (Chao et al., 2011). CD47 acts as a "don't-eat-me" signal protecting cancer cells from macrophage-dependent phagocytosis. In this study, we explored the efficacy of Dara and a CD47 blocking antibody (Fc-modified version of Hu5F9-G4, termed Hu5F9-IgG2σ) alone or in combination in T-ALL. In vitro phagocytosis assays with M-CSF derived M0 (CD64+, CD80-, CD163+) macrophages from healthy donors were evaluated in T-ALL cell lines. Cells labelled with a pH-dependent dye became fluorescent upon engulfment by macrophages and formation of the acidic phagolysosome, which was automatically measured as an absolute cell count. The highest count was set as 100% in each experiment and all other values expressed as percentages relative to that. Treatment of MOLT-13 cells with Dara or Hu5F9-IgG2σ increased median phagocytosis from 5% in control to 23% and 27%, respectively (p=0.008 for both, Figure 1A). However, combined treatment resulted in a maximal increase of median phagocytosis in MOLT-13 cells (p=0.004 compared to Dara and p=0.008 compared to Hu5F9-IgG2σ, Figure 1A). Similar results were obtained in HSB-2 and P-12 cells (p=0.037/0.004 for HSB-2, p=0.011/0.001 for P-12). In addition, phagocytosis was determined in 12 PDX samples from random T-ALL patients. Hu5F9-IgG2σ alone showed a modest increase in median phagocytosis compared to control (19% vs. 6%, p The effects of the Dara/ Hu5F9-IgG2σ combination were also examined in vivo in phase II-like preclinical trials containing different patients in one experiment. Six random T-ALL-PDX samples were injected into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice and subjected to therapy with Dara and Hu5F9-IgG2σ alone or in combination. In order to mimic minimal residual disease (MRD), antibody therapy was started one day post injection. Mice subjected to Dara displayed prolonged survival in 4/6 (67%) animals compared to control (p=0.007), whereas animals treated with Hu5F9-IgG2σ showed significantly prolonged survival in all cases (p=0.007, Figure 1B). Interestingly, mice not responding to Dara benefitted from Hu5F9-IgG2σ monotherapy and the combination had no further survival advantage in this MRD model. In a second approach, eight r/r T-ALL PDX samples were used, and the same therapy was started upon overt leukemia (1% human blasts in peripheral blood). Compared to control, mice treated with Dara in this overt leukemia and r/r setting showed no survival benefit, while animals subjected to Hu5F9-IgG2σ showed a trend towards a prolonged survival (5/8 cases, 63%, p=0.08). Most importantly, the combination of both agents resulted in a statistically significant survival prolongation in 7/8 cases (88%, p=0.010, Figure 1C). Altogether, we show that combining Dara with CD47 blockade increases phagocytosis in vitro in three T-ALL cell lines and 28 PDX samples. Our in vivo data suggest that this combination is a highly promising therapeutic strategy, especially in the relapsed/refractory setting. Figure Disclosures Cario: Jazz Pharmaceuticals: Consultancy, Other: travel support; Novartis: Consultancy, Other: travel support. Kulozik:bluebird bio, Inc.: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Bourquin:Servier: Other: Travel Support. Schewe:Sobi: Other: was an advisory board member; Bayer: Other: was an advisory board member; Jazz: Other: was an advisory board member; OSE pharmaceuticals: Research Funding.

Introduction A phase 1/2 study of betibeglogene autotemcel (beti-cel; LentiGlobin for β-thalassemia) gene therapy in patients with transfusion-dependent β-thalassemia (TDT) demonstrated stable transgene expression with up to 4.5 years follow-up and enabled some patients to achieve transfusion independence. The interim results of two phase 3 studies, HGB-207 (NCT02906202; non-β0/β0 genotypes) and HGB-212 (NCT03207009; β0/β0, β0/β+ IVS-I-110 or β+ IVS-I-110/β+ IVS-I-110 genotypes), are presented here. Methods After G-CSF and plerixafor mobilization, autologous hematopoietic stem cells were collected via apheresis. Using a refined manufacturing process compared to the phase 1/2 study, CD34+ cells were transduced with BB305 lentiviral vector. Patients were infused with transduced cells following pharmacokinetic-adjusted, single-agent busulfan myeloablation. Target busulfan AUC was 3800–4500 (once daily) or 950–1125 (Q6H) μM*min. Statistics are shown as median (min–max). Results As of 13 Dec 2018 and 12 Apr 2019, 31 patients were treated in HGB-207 and HGB-212 with a follow-up of 8.1 (0.5–22.2) and 4.6 (1.5–15.7) months, respectively. Daily average busulfan AUC was 4450 (3709–9087) μM*min. Neutrophil and platelet engraftment were achieved at 25 (13–38) and 44 (20–84) days, respectively. Hospitalization from conditioning to discharge ranged from 30–92 days. In HGB-207, 10/11 patients followed for ≥6 months have stopped transfusions for ≥5.9 months (Figure 1). HbAT87Q levels at Months 6 (n=11), 12 (n=8) and 18 (n=3) were 9.5, 9.2, and 9.5 g/dL, respectively, which contributed to total hemoglobin (Hb) of 11.9, 12.4, and 11.3 g/dL at these time points, respectively. Marrow myeloid:erythroid ratios in 7/8 patients at Month 12 were 0.63–1.90 versus 0.14–0.48 at baseline, indicating improved erythropoiesis. In HGB-212, 3/4 patients followed for ≥6 months stopped transfusions for ≥6 months. Total Hb at last visit ranged from 10.5–13.6 g/dL and HbAT87Q was 9.5–12.6 g/dL at last assessment. Transfusion independence (weighted average Hb ≥9 g/dL without transfusions for ≥12 months) was achieved in 4/5 evaluable patients in HGB-207 and in the only evaluable patient in HGB-212. Post-infusion non-hematologic grade ≥3 adverse events (AEs) in ≥3 patients in either study were stomatitis (n=17), febrile neutropenia (n=11), pyrexia (n=4), epistaxis (n=3), and veno-occlusive liver disease (VOD; n=3). VOD prophylaxis with ursodiol or ursodiol/defibrotide was used in 24/31 patients. One beti-cel-related serious AE of thrombocytopenia was reported; platelet count was 63 × 109/L at last visit (Month 7). All patients remain alive. Summary Following treatment with beti-cel gene therapy, 10/11 and 3/4 patients with ≥6 months follow-up have stopped transfusions in HGB-207 and HGB-212, respectively. The safety profile of treatment with beti-cel is consistent with busulfan myeloablation.

Introduction Allogeneic hematopoietic stem cell (HSC) transplantation (allo-HSCT) is a treatment option for several monogenic diseases; however, its use is limited by the need for a matched donor and risk of HSCT-related complications. Autologous HSC gene addition does not have some of these limitations and may have similar efficacy with an improved safety profile. Ex vivo gene addition therapy using lentiviral vectors (LVV) is being evaluated in patients with transfusion-dependent β-thalassemia (TDT) using betibeglogene autotemcel (beti-cel, LentiGlobin for TDT) in the HGB-204, -205, -207, and -212 studies, sickle cell disease (SCD) using LentiGlobin for SCD in HGB-205 and -206, and cerebral adrenoleukodystrophy (CALD) using Lenti-D in ALD-102. The safety outcomes following autologous gene modified HSCT in these ongoing studies are summarized. Methods HSCs are collected using disease-appropriate procedures, then CD34+ cells are transduced with LVV encoding disease-specific therapeutic transgenes. After myeloablation with busulfan (SCD, TDT) or busulfan/cyclophosphamide (CALD), patients are infused with LVV-transduced CD34+ HSCs. Patients are followed for two years and offered participation in long term follow-up studies (LTF-303 [NCT02633943] or LTF-304 [NCT02698579]). Results Across all 6 studies, 110 patients have been treated as of most recent analyses (Table 1). Patients have been followed for 2 years follow-up. No patient experienced primary or secondary graft failure. One patient with CALD experienced disease progression and died 22 months after drug product (DP) infusion of disease complications. Two additional patients with CALD withdrew from the study after DP infusion and were referred for allo-HSCT. All other patients with CALD, TDT, and SCD remain alive. Most (107/110) patients had ≥ one grade 3 or 4 adverse event (AE) attributed to conditioning; most common AEs were cytopenia, febrile neutropenia, and stomatitis. Myelodysplastic syndrome was reported in one patient with SCD. After investigation for LVV insertion in malignant cells, the AE was assessed as not related to LentiGlobin insertion or transgene expression. AEs reported as related to the DP are shown in Table 1. There was no GVHD and no clinically relevant clonal dominance or LVV-mediated replication competent lentivirus. Summary Data from 110 patients followed for up to 5 years supports that the safety profile of gene-modified autologous HSCT does not carry the risks of GVHD, graft rejection, and long-term immunosuppression attendant to allo-HSCT. While the safety profile beyond 5-years is still being established, these data suggest that HSC gene therapy may be an acceptable therapy for patients with TDT, SCD, and CALD, particularly in patients at increased risk of complications from allo-HSCT, such as those who lack a suitable donor or are more advanced in age.

Over the last 43 years, surveys of over 200,000 subjects in Jamaica have identified β-thalassemia (β-thal) mutations. In most, these genes were detected at birth in patients with sickle cell-β-thal and so the prevalence and distribution would not be influenced by subsequent clinical course. There were two newborn populations, 100,000 deliveries in the corporate area between 1973-1981 and 84,940 in south and western Jamaica between 2008-2016. A third population, which derived from the Manchester Project in central Jamaica, screened 16,612 secondary school children, aged predominantly 15-19 years, and identified 150 students with the β-thal trait and 11 with sickle cell [Hb S (HBB: c.20AT)]- or Hb C (HBB: c.19GA)-β-thal. The latter patients may have been subject to symptomatic selection, but this should not have affected those with β-thal trait. Of the 24 different molecular mutations, β

TO THE EDITOR: Novel immunotherapies have recently led to a broadened spectrum of therapeutic options in pediatric B-cell precursor acute lymphoblastic leukemia, even in the setting of relapsed/refractory disease. However, antibody therapy for T-cell acute lymphoblastic leukemia (T-ALL) is

Introduction: The German PID-NET registry was founded in 2009, serving as the first national registry of patients with primary immunodeficiencies (PID) in Germany. It is part of the European Society for Immunodeficiencies (ESID) registry. The primary purpose of the registry is to gather data on the epidemiology, diagnostic delay, diagnosis, and treatment of PIDs. Methods: Clinical and laboratory data was collected from 2,453 patients from 36 German PID centres in an online registry. Data was analysed with the software Stata® and Excel. Results: The minimum prevalence of PID in Germany is 2.72 per 100,000 inhabitants. Among patients aged 1-25, there was a clear predominance of males. The median age of living patients ranged between 7 and 40 years, depending on the respective PID. Predominantly antibody disorders were the most prevalent group with 57% of all 2,453 PID patients (including 728 CVID patients). A gene defect was identified in 36% of patients. Familial cases were observed in 21% of patients. The age of onset for presenting symptoms ranged from birth to late adulthood (range 0-88 years). Presenting symptoms comprised infections (74%) and immune dysregulation (22%). Ninety-three patients were diagnosed without prior clinical symptoms. Regarding the general and clinical diagnostic delay, no PID had undergone a slight decrease within the last decade. However, both, SCID and hyper IgE- syndrome showed a substantial improvement in shortening the time between onset of symptoms and genetic diagnosis. Regarding treatment, 49% of all patients received immunoglobulin G (IgG) substitution (70%-subcutaneous; 29%-intravenous; 1%-unknown). Three-hundred patients underwent at least one hematopoietic stem cell transplantation (HSCT). Five patients had gene therapy. Conclusion: The German PID-NET registry is a precious tool for physicians, researchers, the pharmaceutical industry, politicians, and ultimately the patients, for whom the outcomes will eventually lead to a more timely diagnosis and better treatment. peerReviewed