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Transfusion-dependent β-thalassaemia (TDT) is a severe disease, resulting in lifelong blood transfusions, iron overload, and associated complications. Betibeglogene autotemcel (beti-cel) gene therapy uses autologous haematopoietic stem and progenitor cells (HSPCs) transduced with BB305 lentiviral vector to enable transfusion independence. HGB-212 was a non-randomised, multicentre, single-arm, open-label, phase 3 study of beti-cel in patients with TDT conducted at eight centres in France, Germany, Greece, Italy, the UK, and the USA. Patients with β0/β0, β0/β+IVS-I-110, or β+IVS-I-110/β+IVS-I-110 genotypes, clinically stable TDT, and a transfusion history of at least 100 mL/kg per year of packed red blood cells (pRBCs) or at least eight transfusions of pRBCs per year in the 2 years before enrolment were eligible for participation. After undergoing HSPC mobilisation and busulfan-based, pharmacokinetic-adjusted myeloablative conditioning, patients were infused with beti-cel and followed up for 24 months. The primary efficacy outcome was transfusion independence, defined as weighted average haemoglobin level of 9 g/dL or above without pRBC transfusions for 12 or more months. The primary outcome was measured in all patients who received an infusion of beti-cel (transplant population); safety was evaluated in all patients who initiated study treatment (intention-to-treat population). Patients were eligible to enrol in the ongoing 13-year long-term follow-up study (for a total of 15 years), LTF-303 (registered at ClinicalTrials.gov , NCT02633943). This trial, HGB-212, was registered at ClinicalTrials.gov (NCT03207009), and is complete. From June 8, 2017, to March 12, 2020, 20 patients were screened for eligibility. One patient was ineligible and one withdrew consent before HSPC mobilisation and myeloablative conditioning. Of the 18 patients who received beti-cel, ten (56%) were male and eight (44%) were female; 13 (72%) were younger than 18 years at the time of informed consent, and five (28%) were older than 18 years. 12 (67%) patients had β0/β0 genotypes, three (17%) had β0/ β+IVS-I-110, and three (17%) had β+IVS-I-110/β+IVS-I-110. As of Jan 30, 2023, all patients enrolled in the long-term follow-up study and the median follow-up was 47·9 months (range 23·8–59·0). All 18 patients were evaluable for transfusion independence, with 16 (89%) of 18 reaching and maintaining transfusion independence to last follow=up (estimated effect size 89·9% [95% CI 65·3–98·6]). All patients had at least one adverse event after beti-cel infusion. There were no serious adverse events considered to be related to beti-cel, and no deaths. These data demonstrate that beti-cel can allow patients with genotypes that cause severe β-thalassaemia (β0/β0, β0/β+IVS-I-110, or β+IVS-I-110/β+IVS-I-110) to reach transfusion independence. Beti-cel offers the potential to attain near-normal haemoglobin levels for those with severe forms of TDT, and a potentially curative option without the risks and limitations of allogeneic HSPC transplantation. Patients are being followed up for a total of 15 years to assess the durability of transfusion independence and long-term safety profile of beti-cel. Bluebird Bio, Somerville, MA, USA. [ABSTRACT FROM AUTHOR]

Examines the genetic mechanism called nonsense-mediated mRNA decay (NMD). Biological and medical importance of NMD; RNA metabolism; Nuclear aspects of mammalian NMD; Cytoplasmic aspects of mammalian NMD.

Patients with sickle cell disease (SCD) in Germany exhibit a substantial genetic diversity in the β‐globin genotype. Data collected by the national German SCD registry reflect this diversity and allowed us to analyze the phenotypes associated with different SCD genotypes. Our study focused on 90 patients with HbS/β‐thalassaemia (HbS/β‐thal) and compared these to patients with HbSS and HbSC. Patients with HbS/β‐thal were classified into three groups: HbS/β0‐thal (no HbA), HbS/β+‐thal (HbA < 14%), and HbS/β++‐thal (HbA≥14%). In comparison to HbSS, patients with HbS/β++‐thal had higher Hb‐levels, lower hemolytic activity and rarely required red blood cell transfusions. HbS/β0‐thal and HbS/β+‐thal closely resembled each other and are jointly referred to as HbS/β0/+‐thal. Compared to HbSS, patients with HbS/β0/+‐thal experienced a similar frequency of vasoocclusive crises and degree of hemolysis. However, the frequency of red blood cell transfusions (0.6 vs. 0.39/year, p =.0049) and splenic sequestration crises (42.4 vs. 15.5% of patients, p = 3.799e‐05) was higher in HbS/β0/+‐thal than in HbSS, but close to zero in HbS/β++‐thal. In conclusion, the level of HbA expression determines the phenotype of HbS/β+‐thal. HbS/β‐thal expressing no or little HbA is hematologically similar to HbSS, but causes a previously unknown high risk of splenic sequestration. [ABSTRACT FROM AUTHOR]

In this issue of Molecular Cell (), regulation of the key nonsense-mediated RNA decay (NMD) proteins UPF1 and MNL51 (BTZ) by miRNA 128 is discovered to fine-tune gene expression in brain differentiation, thus documenting a novel cooperation of two posttranscriptional mechanisms of gene regulation in developmental decision making. [ABSTRACT FROM AUTHOR]

Background: Third-generation chimeric antigen receptor (CAR)-engineered T cells (CARTs) might improve clinical outcome of patients with B cell malignancies. This is the first report on a third-generation CART dose-escalating, phase-1/2 investigator-initiated trial treating adult patients with refractory and/or relapsed (r/r) acute lymphoblastic leukemia (ALL). Methods: Thirteen patients were treated with escalating doses of CD19-directed CARTs between 1 × 106 and 50 × 106 CARTs/m2. Leukapheresis, manufacturing and administration of CARTs were performed in-house. Results: For all patients, CART manufacturing was feasible. None of the patients developed any grade of Immune effector cell-associated neurotoxicity syndrome (ICANS) or a higher-grade (≥ grade III) catokine release syndrome (CRS). CART expansion and long-term CART persistence were evident in the peripheral blood (PB) of evaluable patients. At end of study on day 90 after CARTs, ten patients were evaluable for response: Eight patients (80%) achieved a complete remission (CR), including five patients (50%) with minimal residual disease (MRD)-negative CR. Response and outcome were associated with the administered CART dose. At 1-year follow-up, median overall survival was not reached and progression-free survival (PFS) was 38%. Median PFS was reached on day 120. Lack of CD39-expression on memory-like T cells was more frequent in CART products of responders when compared to CART products of non-responders. After CART administration, higher CD8 + and γδ-T cell frequencies, a physiological pattern of immune cells and lower monocyte counts in the PB were associated with response. Conclusion: In conclusion, third-generation CARTs were associated with promising clinical efficacy and remarkably low procedure-specific toxicity, thereby opening new therapeutic perspectives for patients with r/r ALL. Trial registration This trial was registered at www.clinicaltrials.gov as NCT03676504. [ABSTRACT FROM AUTHOR]

Somatic structural variants (SVs) are widespread in cancer, but their impact on disease evolution is understudied due to a lack of methods to directly characterize their functional consequences. We present a computational method, scNOVA, which uses Strand-seq to perform haplotype-aware integration of SV discovery and molecular phenotyping in single cells by using nucleosome occupancy to infer gene expression as a readout. Application to leukemias and cell lines identifies local effects of copy-balanced rearrangements on gene deregulation, and consequences of SVs on aberrant signaling pathways in subclones. We discovered distinct SV subclones with dysregulated Wnt signaling in a chronic lymphocytic leukemia patient. We further uncovered the consequences of subclonal chromothripsis in T cell acute lymphoblastic leukemia, which revealed c-Myb activation, enrichment of a primitive cell state and informed successful targeting of the subclone in cell culture, using a Notch inhibitor. By directly linking SVs to their functional effects, scNOVA enables systematic single-cell multiomic studies of structural variation in heterogeneous cell populations. Linking structural variants to gene expression information identifies aberrant signaling pathways. [ABSTRACT FROM AUTHOR]

Children with sickle cell disease (SCD) benefit from newborn screening, because life-threatening complications can be prevented by pre-symptomatic diagnosis. In Germany, the immigration of people from endemic countries is steadily growing. Comprehensive data about the epidemiology and prevalence of SCD in Germany are however lacking, and SCD is not included in the national newborn screening program. We provide data on the prevalence of SCD in a population from both urban and rural areas in Southwest Germany. Anonymized dried blood spots from 37,838 unselected newborns were analyzed by allele-specific PCR for the HbS mutation. Samples tested positive were subjected to Sanger sequencing of the entire β-globin coding sequence firstly to validate the screening and secondly to identify compound heterozygous SCD patients with other mutations of the β-globin gene. We identified 83 carriers of the sickle cell trait, three compound heterozygous SCD patients (two with sickle cell-β-thalassemia, one with sickle cell-Hb Tianshui) but no homozygous SCD patients. The novel molecular method and strategy for newborn screening for SCD presented here compares favorably in terms of sensitivity (1.0 for homozygous HbS, 0.996 for heterozygous HbS), specificity (0.996), practicability, and costs with conventional biochemical screening. Our results demonstrate a significant prevalence of SCD of approximately 1:12,000 in an unselected urban and rural population in Southwest Germany. Together with previously published even higher results from exclusively urban populations in Berlin and Hamburg, our data provide the basis for the decision on a newborn screening program for SCD in Germany. [ABSTRACT FROM AUTHOR]

Background: Characterization of clinical phenotypes in context with tumor and host genomic information can aid in the development of more effective and less toxic risk-adapted and targeted treatment strategies. To analyze the impact of therapy-related hyperbilirubinemia on treatment outcome and to identify contributing genetic risk factors of this well-recognized adverse effect we evaluated serum bilirubin levels in 1547 pediatric patients with acute lymphoblastic leukemia (ALL) and conducted a genome-wide association study (GWAS). Patients and methods: Patients were treated in multicenter trial AIEOP-BFM ALL 2000 for pediatric ALL. Bilirubin toxicity was graded 0 to 4 according to the Common Toxicity Criteria (CTC) of the National Cancer Institute. In the GWAS discovery cohort, including 650 of the 1547 individuals, genotype frequencies of 745,895 single nucleotide variants were compared between 435 patients with hyperbilirubinemia (CTC grades 1-4) during induction/consolidation treatment and 215 patients without it (grade 0). Replication analyses included 224 patients from the same trial. Results: Compared to patients with no (grade 0) or moderate hyperbilirubinemia (grades 1-2) during induction/consolidation, patients with grades 3-4 had a poorer 5-year event free survival (76.6 ± 3% versus 87.7 ± 1% for grades 1-2, P = 0.003; 85.2 ± 2% for grade 0, P < 0.001) and a higher cumulative incidence of relapse (15.6 ± 3% versus 9.0 ± 1% for grades 1-2, P = 0.08; 11.1 ± 1% for grade 0, P = 0.007). GWAS identified a strong association of the rs6744284 variant T allele in the UGT1A gene cluster with risk of hyperbilirubinemia (allelic odds ratio (OR) = 2.1, P = 7 × 10− 8). TT-homozygotes had a 6.5-fold increased risk of hyperbilirubinemia (grades 1-4; 95% confidence interval (CI) = 2.9-14.6, P = 7 × 10− 6) and a 16.4-fold higher risk of grade 3-4 hyperbilirubinemia (95% CI 6.1-43.8, P = 2 × 10− 8). Replication analyses confirmed these associations with joint analysis yielding genome-wide significance (allelic OR = 2.1, P = 6 × 10− 11; 95% CI 1.7-2.7). Moreover, rs6744284 genotypes were strongly linked to the Gilbert's syndrome-associated UGT1A1*28/*37 allele (r2 = 0.70), providing functional support for study findings. Of clinical importance, the rs6744284 TT genotype counterbalanced the adverse prognostic impact of high hyperbilirubinemia on therapy outcome. Conclusions: Chemotherapy-related hyperbilirubinemia is a prognostic factor for treatment outcome in pediatric ALL and genetic variation in UGT1A aids in predicting the clinical impact of hyperbilirubinemia. Trial registration: http://www.clinicaltrials.gov; #NCT00430118. [ABSTRACT FROM AUTHOR]

Objective: To report the diagnostic challenges of newborn screening for abnormal haemoglobins. Setting: Cord blood samples from 13 hospitals in southwest Jamaica taken in 2008–2019. Methods: Blood spots, collected from the umbilical cord, were analysed by high pressure liquid chromatography (HPLC) to reveal phenotypes for HbSS and HbCC, but genotype confirmation may require parental studies or gene sequencing. Such cases that were successfully traced were analysed in this follow-up study. Results: HPLC screening of 121,306 samples detected HbAS in 11,846 (9.8%), HbAC in 4508 (3.7%) and other electrophoretic abnormalities in 1090 babies. Among 101 previously unconfirmed cases, 34/90 (38%) with HPLC evidence of a HbSS phenotype had other genotypes, and 7/11 (64%) with a HbCC phenotype had other genotypes. Syndromes from the interaction of β thalassaemia occurred in 112 babies (85 with HbS, 27 with HbC) and of genes for hereditary persistence of fetal haemoglobin (HPFH) in 18 (12 with HbS, 6 with HbC). Variants other than HbS and HbC occurred in 270 babies, 16 in combination with either HbS or HbC, and 254 as traits. Most variants are benign even when inherited with HbS, although HbO Arab, HbD Punjab, or Hb Lepore Washington, which occurred in 6 cases, may cause sickle cell disease. Conclusions: Genes for β thalassaemia and HPFH are common in western Jamaica and when associated with HbS may present diagnostic challenges in newborns, as HbF and HbA2 have not reached diagnostic levels. Family and DNA studies may be necessary for genotype confirmation. [ABSTRACT FROM AUTHOR]

• Cystic fibrosis (CF) newborn screening with pancreatitis associated protein (PAP) as second-tier test might be an option in multiethnic populations. • PAP-based protocols may have advantages in detectig fewer newborns designated as CFSPID (Cystic fibrosis screening positive, inconclusive diagnosis) • PAP-based CF screening protocols achieve adequate sensitivity when PAP is evaluated as a second-tier test together with the first-tier test IRT, but this comes at the expense of PPV. 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. 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. 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. 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. [ABSTRACT FROM AUTHOR]

Thrombin is the protease involved in blood coagulation. Its deregulation can lead to hemostatic abnormalities, which range from subtle subclinical to serious life-threatening coagulopathies, i.e., during septicemia. Additionally, thrombin plays important roles in many (patho)physiological conditions that reach far beyond its well-established role in stemming blood loss and thrombosis, including embryonic development and angiogenesis but also extending to inflammatory processes, complement activation, and even tumor biology. In this review, we will address thrombin's broad roles in diverse (patho)physiological processes in an integrative way. We will also discuss thrombin as an emerging major target for novel therapies. [ABSTRACT FROM AUTHOR]

Summary: Exon junction complexes (EJCs) are deposited onto mRNAs during splicing, serve as positional landmarks for the intron exon structure of genes, and direct posttranscriptional processes in the cytoplasm. EJC removal and recycling by translation are ill understood and have been attributed to ribosomal passage. This work identifies the ribosome-associated protein PYM as an EJC disassembly factor and defines its mechanism of function. Whereas EJC assembly intermediates are resistant to PYM, fully assembled EJCs are dissociated from spliced mRNAs by PYM. This disassembly involves PYM binding to the EJC proteins MAGOH-Y14. PYM overexpression in cells disrupts EJC association with spliced mRNA and inhibits nonsense-mediated mRNA decay. In cells depleted of PYM, EJCs accumulate on spliced mRNAs and EJC protein recycling is impaired. Hence, PYM is an EJC disassembly factor that acts both in vitro and in living cells, and that antagonizes important EJC functions. [Copyright &y& Elsevier]

Abstract: Treatment of high-risk neuroblastoma (NB) is difficult. Novel therapeutics improving survival rates are urgently required. We have previously shown that the histone deacetylase inhibitor (HDACI) Helminthosporium carbonum (HC)-toxin induces differentiation of neuroblastoma (NB) cells. Here, we show that HC-toxin inhibits the growth of both established NB cell lines and primary cultures with and without amplified MYCN stronger than retinoids (RAs) and other HDACIs (MS-275, n-butyric acid, suberoylanilide hydroxamic acid, trichostatin A, valproic acid). Nanomolar dosages suppress E2F-1, N-myc, Skp2, Mad2 and survivin proteins, found at high levels in high-risk NBs, more efficiently than both RAs and other HDACIs. The level of hypophosphorylated active retinoblastoma (RB) tumor suppressor protein is increased most effectively. HC-toxin’s epoxy group is essential for inhibiting HDACs and promoting anti-NB activity. Without this functional group, those cellular effects are not observed. In conclusion, the anti-NB activity of HC-toxin is superior to that of RAs and that of all other HDACIs tested. [Copyright &y& Elsevier]

Recent advances in the understanding of the molecular mechanism of mRNA 3′ end processing have uncovered a previously unanticipated integrated network of transcriptional and RNA-processing mechanisms. A variety of human diseases impressively reflect the importance of the precision of the complex 3′ end-processing machinery and gene specific deregulation of 3′ end processing can result from mutations of RNA sequence elements that bind key specific processing factors. Interestingly, more general deregulation of 3′ end processing can be caused either by mutations of these processing factors or by the disturbance of the well-coordinated equilibrium between these factors. From a medical perspective, both loss of function and gain of function can be functionally relevant, and an increasing number of different disease entities exemplifies that inappropriate 3′ end formation of human mRNAs can have a tremendous impact on health and disease. Here, we review the mechanistic hallmarks of mRNA 3′ end processing, highlight the medical relevance of deregulation of this important step of mRNA maturation and illustrate the implications for diagnostic and therapeutic strategies. [ABSTRACT FROM AUTHOR]

Background: The present study evaluates the outcome of 19 children with rhabdomyosarcoma of the head-and-neck region treated with Intensity Modulated Radiotherapy (IMRT) or Fractionated Stereotactic Radiotherapy (FSRT) between August 1995 and November 2005. Methods: We treated 19 children with head-and-neck rhabdomyosarcoma with FSRT (n = 14) or IMRT (n = 5) as a part of multimodal therapy. Median age at the time of radiation therapy was 5 years (range 2-15 years). All children received systemic chemotherapy according to the German Soft Tissue Sarcoma Study protocols. Median size of treatment volume for RT was 93,4 ml. We applied a median total dose of 45 Gy (range 32 Gy -- 54 Gy) using a median fractionation of 5 x 1,8 Gy/week (range 1,6 Gy -- 1,8 Gy). The median time interval between primary diagnosis and radiation therapy was 5 months (range 3-9 months). Results: After RT, the 3- and 5-year survival rate was 94%. The 3- and 5-year actuarial local control rate after RT was 89%. The actuarial freedom of distant metastases rate at 3- and 5-years was 89% for all patients. Radiotherapy was well tolerated in all children and could be completed without interruptions > 4 days. No toxicities >CTC grade 2 were observed. The median follow-up time after RT was 17 months. Conclusion: IMRT and FSRT lead to excellent outcome in children with head-and-neck RMS with a low incidence of treatment-related side effects. [ABSTRACT FROM AUTHOR]

• Combined ion-beam radiotherapy for osteosarcoma is a novel local therapeutic approach • Despite unfavorable size/localization toxicity was moderate/local control promising • FDG PET might serve as a surrogate for grading histopathologic regression in future. 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. 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. 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. CIBRT shows a favorable toxicity profile and promising results particularly for patients with inoperable craniofacial osteosarcoma. [ABSTRACT FROM AUTHOR]

We present a case of primary renal chondrosarcoma, its diagnosis and management. Copyright © 2009 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Structural variation (SV), involving deletions, duplications, inversions and translocations of DNA segments, is a major source of genetic variability in somatic cells and can dysregulate cancer-related pathways. However, discovering somatic SVs in single cells has been challenging, with copy-number-neutral and complex variants typically escaping detection. Here we describe single-cell tri-channel processing (scTRIP), a computational framework that integrates read depth, template strand and haplotype phase to comprehensively discover SVs in individual cells. We surveyed SV landscapes of 565 single cells, including transformed epithelial cells and patient-derived leukemic samples, to discover abundant SV classes, including inversions, translocations and complex DNA rearrangements. Analysis of the leukemic samples revealed four times more somatic SVs than cytogenetic karyotyping, submicroscopic copy-number alterations, oncogenic copy-neutral rearrangements and a subclonal chromothripsis event. Advancing current methods, single-cell tri-channel processing can directly measure SV mutational processes in individual cells, such as breakage–fusion–bridge cycles, facilitating studies of clonal evolution, genetic mosaicism and SV formation mechanisms, which could improve disease classification for precision medicine. Complex structural variations in single cells are detected by integrating read depth, template strand and haplotype phase information. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: 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‐Münster (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. [ABSTRACT FROM AUTHOR]

Background: Deletions of 6q15-16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which has a crucial role in innate immune signaling and was observed to be functionally and prognostically relevant in different cancer entities. Therefore, we correlated the presence of MAP3K7 deletions with clinical parameters in a cohort of 327 pediatric T-ALL patients and investigated the function of MAP3K7 in the T-ALL cell lines CCRF-CEM, Jurkat and MOLT-4.Methods: MAP3K7 deletions were detected by multiplex ligation-dependent probe amplification (MLPA). T-ALL cell lines were transduced with adeno-associated virus (AAV) vectors expressing anti-MAP3K7 shRNA or a non-silencing shRNA together with a GFP reporter. Transduction efficiency was measured by flow cytometry and depletion efficiency by RT-PCR and Western blots. Induction of apoptosis was measured by flow cytometry after staining with PE-conjugated Annexin V. In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR.Results: MAP3K7 is deleted in approximately 10% and point-mutated in approximately 1% of children with T-ALL. In 32 of 33 leukemias the deletion of MAP3K7 also included the adjacent CASP8AP2 gene. MAP3K7 deletions were associated with the occurrence of SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and outcome. Depletion of MAP3K7 expression in T-ALL cell lines by shRNAs slowed down proliferation and induced apoptosis, but neither changed protein levels of components of NF-κB signaling nor NF-κB target gene expression after stimulation with TNF-α.Conclusions: This study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts. [ABSTRACT FROM AUTHOR]

The article presents a study for examining the protocol II vs. protocol III of reinduction therapy for children with acute lymphoblastic leukemia (ALL). Topics include dosage of dexamethasone, thyoguanine, and arabinoside were given to children during therapy; usefulness of therapy in reducing disease relapse rate; and study shows improved outcome of patients with prednisone-poor response.

Nonsense-mediated mRNA decay (NMD) is a cellular surveillance pathway that recognizes and degrades mRNAs with premature termination codons (PTCs). The mechanisms underlying translation termination are key to the understanding of RNA surveillance mechanisms such as NMD and crucial for the development of therapeutic strategies for NMD-related diseases. Here, we have used a fully reconstituted in vitro translation system to probe the NMD proteins for interaction with the termination apparatus. We discovered that UPF3B (i) interacts with the release factors, (ii) delays translation termination and (iii) dissociates post-termination ribosomal complexes that are devoid of the nascent peptide. Furthermore, we identified UPF1 and ribosomes as new interaction partners of UPF3B. These previously unknown functions of UPF3B during the early and late phases of translation termination suggest that UPF3B is involved in the crosstalk between the NMD machinery and the PTC-bound ribosome, a central mechanistic step of RNA surveillance. [ABSTRACT FROM AUTHOR]

Wereport 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 allshowedpoor 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Histone 3.3 (H3.3) hotspot mutations in bone tumors occur in the vast majority of giant cell tumors of bone (GCTBs; 96%), chondroblastomas (95%) and in a few cases of osteosarcomas. However, clinical presentation, histopathological features, and additional molecular characteristics of H3.3 mutant osteosarcomas are largely unknown. Methods: In this multicentre, retrospective study, a total of 106 conventional high-grade osteosarcomas, across all age groups were re-examined for hotspot mutations in the H3.3 coding genes H3F3A and H3F3B. H3.3 mutant osteosarcomas were re-evaluated in a multidisciplinary manner and analyzed for genome-wide DNA-methylation patterns and DNA copy number aberrations alongside H3.3 wild-type osteosarcomas and H3F3A G34W/L mutant GCTBs. Results: Six osteosarcomas (6/106) carried H3F3A hotspot mutations. No mutations were found in H3F3B. All patients with H3F3A mutant osteosarcoma were older than 30 years with a median age of 65 years. Copy number aberrations that are commonly encountered in high-grade osteosarcomas also occurred in H3F3A mutant osteosarcomas. Unlike a single osteosarcoma with a H3F3A K27M mutation, the DNA methylation profiles of H3F3A G34W/R mutant osteosarcomas were clearly different from H3.3 wild-type osteosarcomas, but more closely related to GCTBs. The most differentially methylated promoters between H3F3A G34W/R mutant and H3.3 wild-type osteosarcomas were in KLLN/PTEN (p < 0.00005) and HIST1H2BB (p < 0.0005). Conclusions: H3.3 mutations in osteosarcomas may occur in H3F3A at mutational hotspots. They are overall rare, but become more frequent in osteosarcoma patients older than 30 years. Osteosarcomas carrying H3F3A G34W/R mutations are associated with epigenetic dysregulation of KLLN/PTEN and HIST1H2BB. [ABSTRACT FROM AUTHOR]

The prognosis of children with severe combined immunodeficiency (SCID) depends on a presymptomatic diagnosis and early treatment before complications occur. We established and tested a simplified, practical and economic newborn screening method based on the quantification of T-cell receptor excision circles (TRECs) on dried blood spots (DBSs) through qPCR. Our method was validated by the analysis of 11 positive controls, which all showed an absence of TRECs, thus yielding a sensitivity of 100%. Further, we analyzed 6034 anonymized newborns of whom 6031 (99,95%) showed a normal TREC qPCR with a median of 600 estimated TREC copies/1.6 mm punch. The test showed a recall-rate of 0.05%. We present a highly sensitive, specific and time- and cost-effective method of TREC quantification, which is suitable for SCID newborn screening. In comparison to established methods, our test requires only 25% of the input material, doesn't require DNA purification and significantly reduces time and cost requirement. [ABSTRACT FROM AUTHOR]

The blue rubber bleb nevus syndrome (BRBNS, syn. bean syndrome) is a rare dise ase characterized by multiple cutaneous and gastrointestinal venous malformations associated with severe bleeding. However, the underlying molecular mechanisms are unknown and no targeted therapeutic approach exists to date. Here we report the case of a 19-year-old male patient with severe BRBNS in whom we analyzed the expression of tyrosine kinases frequently involved in tumor development by immunohistochemistry (vascular endothelial growth factor receptor-2, stem cell growth factor receptor (c-kit), platelet-derived growth factor receptor-β, and stem cell tyrosine kinase-1). A prominent expression of c-kit was detectable in smaller blood vessels, which also showed a moderate expression of the proliferation marker MIB1. Surprisingly, other growth factor receptors stained negatively. We therefore conclude that pharmacological inhibition of the c-kit signaling pathway in cavernous hemangiomas by selective kinase inhibitors may offer options in the treatment of BRBNS patients. [ABSTRACT FROM AUTHOR]

The blue rubber bleb nevus syndrome (BRBNS, syn. bean syndrome) is a rare disease characterized by multiple cutaneous and gastrointestinal venous malformations associated with severe bleeding. However, the underlying molecular mechanisms are unknown and no targeted therapeutic approach exists to date. Here we report the case of a 19-year-old male patient with severe BRBNS in whom we analyzed the expression of tyrosine kinases frequently involved in tumor development by immunohistochemistry (vascular endothelial growth factor receptor-2, stem cell growth factor receptor (c-kit), platelet-derived growth factor receptor-β, and stem cell tyrosine kinase-1). A prominent expression of c-kit was detectable in smaller blood vessels, which also showed a moderate expression of the proliferation marker MIB1. Surprisingly, other growth factor receptors stained negatively. We therefore conclude that pharmacological inhibition of the c-kit signaling pathway in cavernous hemangiomas by selective kinase inhibitors may offer options in the treatment of BRBNS patients. [ABSTRACT FROM AUTHOR]

Cancer- and treatment-related side effects in patients with childhood cancer may cause limitations in motor performance affecting activities of daily living (ADLs). Data focusing on long-term effects are available, but little is known with regard to the short-term perspective. Therefore, the purpose of this study was to assess muscle strength performance and quality of life (QoL) in children and adolescents with cancer at the beginning of primary treatment. Forty children and adolescents aged 5–18 years (mean: 11.39 ± 4.08 years) with different types of childhood cancer were enrolled. On average 36 ± 20.5 days after diagnosis, strength performance in 7 muscle groups was assessed by handheld dynamometry. KINDL questionnaires were completed to evaluate QoL (children's self-report and parents' report). All parameters were compared with age- and gender-matched reference values. Patients with childhood cancer showed significantly lower strength values in all muscle groups (P< .01) compared with age- and gender-matched controls. Most affected were the lower extremities, with a −57.1% ± 10.4%, median: −59.2%, minimum: −75.4%, maximum: −41.4% percentage deviation in knee flexion from healthy peers. Children themselves and parents assessed total QoL significantly below age- and gender-matched reference values (P< .01). Correlation between elbow flexion and self-reported QoL was detected. Broader correlations were found for the parents' report. Muscle weakness and decreased QoL in children and adolescents seem to persist already at the beginning of anticancer treatment. This underlines the need of counteracting measures, such as exercise intervention programs, starting as early as possible during the treatment process. Efforts on this topic are currently being carried out by our group. [ABSTRACT FROM PUBLISHER]

The 'pediatric targeted therapy' (PTT) program aims to identify the presence and activity of druggable targets and evaluate the clinical benefit of a personalized treatment approach in relapsed or progressive tumors on an individual basis. 10 markers (HDAC2, HR23B, p-AKT, p-ERK, p-S6, p-EGFR, PDGFR-alpha/beta, p53 and BRAFV600E) were analyzed by immunohistochemistry. Pediatric patients with tumors independent of the histological diagnosis, with relapse or progression after treatment according to standard protocols were included. N = 61/145 (42%) cases were eligible for analysis between 2009 and 2013, the most common entities being brain tumors. Immunohistochemical stainings were evaluated by the H-Score (0-300). In 93% of the cases potentially actionable targets were identified. The expressed or activated pathways were histone deacetylase (HDACs; 83.0% of cases positive), EGFR (87.2%), PDGFR (75.9%), p53 (50.0%), MAPK/ERK (43.3%) and PI3K/mTOR (36.1%). Follow-up revealed partial or full implementation of PTT results in treatment decision-making in 41% of the cases. Prolonged disease stabilization responses in single cases were noticed, however, response rates did not differ from cases treated with other modalities. Further studies evaluating the feasibility and clinical benefit of personalized diagnostic approaches using paraffin material are warranted. [ABSTRACT FROM AUTHOR]

Induction therapy for childhood acute lymphoblastic leukemia (ALL) traditionally includes prednisone; yet, dexamethasone may have higher antileukemic potency, leading to fewer relapses and improved survival. After a 7-day prednisone prephase, 3720 patients enrolled on trial Associazione Italiana di Ematologia e Oncologia Pediatrica and Berlin-Frankfurt-Münster (AIEOP-BFM) ALL 2000 were randomly selected to receive either dexamethasone (10 mg/m2 per day) or prednisone (60 mg/m2 per day) for 3 weeks plus tapering in induction. The 5-year cumulative incidence of relapse (± standard error) was 10.8 ± 0.7% in the dexamethasone and 15.6 ± 0.8% in the prednisone group (P < .0001), showing the largest effect on extramedullary relapses. The benefit of dexamethasone was partially counterbalanced by a significantly higher induction-related death rate (2.5% vs 0.9%, P = .00013), resulting in 5-year event-free survival rates of 83.9 ± 0.9% for dexamethasone and 80.8 ± 0.9% for prednisone (P = .024). No difference was seen in 5-year overall survival (OS) in the total cohort (dexamethasone, 90.3 ± 0.7%; prednisone, 90.5 ± 0.7%). Retrospective analyses of predefined subgroups revealed a significant survival benefit from dexamethasone only for patients with T-cell ALL and good response to the prednisone prephase (prednisone good-response [PGR]) (dexamethasone, 91.4 ± 2.4%; prednisone, 82.6 ± 3.2%; P = .036). In patients with precursor B-cell ALL and PGR, survival after relapse was found to be significantly worse if patients were previously assigned to the dexamethasone arm. We conclude that, for patients with PGR in the large subgroup of precursor B-cell ALL, dexamethasone especially reduced the incidence of better salvageable relapses, resulting in inferior survival after relapse. This explains the lack of benefit from dexamethasone in overall survival that we observed in the total cohort except in the subset of T-cell ALL patients with PGR. This trial was registered at www.clinicaltrials.gov (BFM: NCT00430118, AIEOP: NCT00613457). [ABSTRACT FROM AUTHOR]

Low-dose azacitidine is efficient and safe in the therapy of malignant myeloid disorders in adults but data in children are lacking. We present a retrospective analysis of 24 children and young adults with myelodysplastic syndrome ( MDS) who received azacitidine at the time of first diagnosis or relapse after allotransplant (2 children were treated with azacitidine both initially and for relapse). Diagnoses were refractory cytopenia of childhood ( N = 4), advanced primary MDS ( N = 9) and secondary MDS ( N = 11). The median duration of treatment was four cycles. Azacitidine was well tolerated, but cytopenias led to dose reduction in five cases. Treatment was discontinued in one child because of impaired renal function. Sixteen MDS patients were treated with azacitidine at first diagnosis. One complete clinical remission was observed and one child showed complete marrow remission; six children experienced stable disease with haematological improvement. Ten children received azacitidine for relapsed MDS after transplant: of these, seven experienced stable disease for 2-30 cycles (median 3), including one patient with haematological improvement for seven cycles. In summary, azacitidine is effective in some children with MDS and appears to be a non-toxic option in palliative situations to prolong survival. [ABSTRACT FROM AUTHOR]

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.20A>T)]- or Hb C (HBB: c.19G>A)-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, β0-thal genes accounted for 10.0-27.0% of these groups and most common was IVS-II-849 (A>G) (HBB: c.316-2A>G). Of the β+ mutations, seven subjects had severe genes with low levels of β chain synthesis but the majority were benign mutations in the promoter region. The -29 (A>G) (HBB: c.-79A>G) mutation dominated in the newborn study in Kingston, similar to experiences in Guadeloupe and African Americans but the -88 (C>T) (HBB: c.-138C>T) mutation was more common among school students in central Jamaica. Caribbean populations are genetically heterogeneous but variations within different parts of Jamaica is of potential importance for prenatal diagnosis and genetic counseling. This information may also be useful among the large Jamaican diaspora. [ABSTRACT FROM AUTHOR]

Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking. [ABSTRACT FROM AUTHOR]

Curative treatment of pediatric cancer not only focuses on long-term survival, but also on reducing treatment-related side effects. Advantages of particle therapy are mainly due to their physical ability of significantly reducing integral dose. Methods. Between January 2009 and December 2012, we treated 83 pediatric patients (aged 21 and younger) at the Heidelberg Ion Therapy Center at University Hospital of Heidelberg (HIT). In total 56 patients (67%) received proton irradiation, while 25 (30%) patients were treated with carbon ions (C12). Two patients received both treatments (3%). Treatment toxicity was analyzed retrospectively and documented according to the CTCAE/RTOG classification. In a second step, treatment toxicity from ion therapy was analyzed in comparison to treatment toxicity during photon irradiation of a comparable historical group of 19 pediatric patients. Results. In all patients, particle therapy was tolerated well (median follow-up time 3.7 months), children (20 patients) with at least two follow-up visits showed a median follow-up time of 10.2 months. During the first two months patients mainly suffered from radiogenic skin reaction (63%), mucositis (30%), headache and dizziness (35%) as well as nausea and vomiting (13%). Severe toxicity reaction (grade II-IV) was only seen in patients who had intensive simultaneous chemotherapy or who had undergone several operations in the irradiated area before radiotherapy (18%). Treatment toxicity during ion therapy was comparable to treatment toxicity from photon irradiation of a historical group. Conclusions. In comparison to conventional therapy, patients with particle therapy do not suffer from increased acute treatment-related toxicity during the first months. More experience with particle therapy will be needed during the next years to help to thoroughly evaluate the high potential of ion therapy. [ABSTRACT FROM AUTHOR]

Background: Minimal improvements in treatment or survival of patients with osteosarcoma have been achieved during the last three decades. Especially in the case of incomplete tumor resection, prognosis remains poor. Heavy ion radiotherapy (HIT) and modern anticancer drugs like histone deacetylase inhibitors (HDACi) have shown promising effects in osteosarcoma in vitro. In this study, we tested the effect of HIT and the combination of HIT and the HDACi suberoylanilide hydroxamic acid (SAHA) in a xenograft mouse model. Methods: Osteosarcoma xenografts were established by subcutaneous injection of KHOS-24OS cells and treated with either vehicle (DMSO), SAHA, HIT or HIT and SAHA. Tumor growth was determined and tumor necrosis, proliferation rate, apoptotic rate as well as vessel density were evaluated. Results: Here, we show that the combination of HIT and SAHA induced a significant delay of tumor growth through increased rate of apoptosis, increased expression of p53 and p21Waf1/Cip1, inhibition of proliferation and angiogenesis compared to tumors treated with HIT only. Conclusion: HIT and in particular the combination of HIT and histone deacetylase inhibition is a promising treatment strategy in OS and may be tested in clinical trials. [ABSTRACT FROM AUTHOR]

Background: Minimal improvements in treatment or survival of patients with osteosarcoma have been achieved during the last three decades. Especially in the case of incomplete tumor resection, prognosis remains poor. Heavy ion radiotherapy (HIT) and modern anticancer drugs like histone deacetylase inhibitors (HDACi) have shown promising effects in osteosarcoma in vitro. In this study, we tested the effect of HIT and the combination of HIT and the HDACi suberoylanilide hydroxamic acid (SAHA) in a xenograft mouse model. Methods: Osteosarcoma xenografts were established by subcutaneous injection of KHOS-24OS cells and treated with either vehicle (DMSO), SAHA, HIT or HIT and SAHA. Tumor growth was determined and tumor necrosis, proliferation rate, apoptotic rate as well as vessel density were evaluated. Results: Here, we show that the combination of HIT and SAHA induced a significant delay of tumor growth through increased rate of apoptosis, increased expression of p53 and p21Waf1/Cip1, inhibition of proliferation and angiogenesis compared to tumors treated with HIT only. Conclusion: HIT and in particular the combination of HIT and histone deacetylase inhibition is a promising treatment strategy in OS and may be tested in clinical trials. [ABSTRACT FROM AUTHOR]

Pediatric glioblastoma (pedGBM) is amongst the most common malignant brain tumors of childhood and carries a dismal prognosis. In contrast to adult GBM, few molecular prognostic markers for the pediatric counterpart have been established. We, therefore, investigated the prognostic significance of genomic and epigenetic alterations through molecular analysis of 202 pedGBM (1-18 years) with comprehensive clinical annotation. Routinely prepared formalin-fixed paraffin-embedded tumor samples were assessed for genome-wide DNA methylation profiles, with known candidate genes screened for alterations via direct sequencing or FISH. Unexpectedly, a subset of histologically diagnosed GBM ( n = 40, 20 %) displayed methylation profiles similar to those of either low-grade gliomas or pleomorphic xanthoastrocytomas (PXA). These tumors showed a markedly better prognosis, with molecularly PXA-like tumors frequently harboring BRAF V600E mutations and 9p21 ( CDKN2A) homozygous deletion. The remaining 162 tumors with pedGBM molecular signatures comprised four subgroups: H3.3 G34-mutant (15 %), H3.3/H3.1 K27-mutant (43 %), IDH1-mutant (6 %), and H3/IDH wild-type (wt) GBM (36 %). These subgroups were associated with specific cytogenetic aberrations, MGMT methylation patterns and clinical outcomes. Analysis of follow-up data identified a set of biomarkers feasible for use in risk stratification: pedGBM with any oncogene amplification and/or K27M mutation ( n = 124) represents a particularly unfavorable group, with 3-year overall survival (OS) of 5 %, whereas tumors without these markers ( n = 38) define a more favorable group (3-year OS ~70 %).Combined with the lower grade-like lesions, almost 40 % of pedGBM cases had distinct molecular features associated with a more favorable outcome. This refined prognostication method for pedGBM using a molecular risk algorithm may allow for improved therapeutic choices and better planning of clinical trial stratification for this otherwise devastating disease. [ABSTRACT FROM AUTHOR]

Background: Osteosarcoma (OS) is the most common pediatric primary malignant bone tumor. As the prognosis for patients following standard treatment did not improve for almost three decades, functional preclinical models that closely reflect important clinical cancer characteristics are urgently needed to develop and evaluate new treatment strategies. The objective of this study was to establish an orthotopic xenotransplanted mouse model using patient-derived tumor tissue. Methods: Fresh tumor tissue from an adolescent female patient with osteosarcoma after relapse was surgically xenografted into the right tibia of 6 immunodeficient BALB/c Nu/Nu mice as well as cultured into medium. Tumor growth was serially assessed by palpation and with magnetic resonance imaging (MRI). In parallel, a primary cell line of the same tumor was established. Histology and high-resolution array-based comparative genomic hybridization (aCGH) were used to investigate both phenotypic and genotypic characteristics of different passages of human xenografts and the cell line compared to the tissue of origin. Results: A primary OS cell line and a primary patient-derived orthotopic xenotranplanted mouse model were established. MRI analyses and histopathology demonstrated an identical architecture in the primary tumor and in the xenografts. Array-CGH analyses of the cell line and all xenografts showed highly comparable patterns of genomic progression. So far, three further primary patient-derived orthotopic xenotranplanted mouse models could be established. Conclusion: We report the first orthotopic OS mouse model generated by transplantation of tumor fragments directly harvested from the patient. This model represents the morphologic and genomic identity of the primary tumor and provides a preclinical platform to evaluate new treatment strategies in OS. [ABSTRACT FROM AUTHOR]

The interaction between thrombopoietin (THPO) and its receptor c-Mpi regulates downstream cytokine signaling and platelet homeostasis. Hereditary mutations of c-Mpl can either result in loss-of-function and thrombocytopenia or in gain-of-function and throm-bocythemia (HT), and are important models to analyze the mechanism of c-Mpl activity. We have analyzed the effect of the c-Mpi P103L gain-of-function and the nearby loss-of-function R102P and F104S mutations, which cause HT or thrombocytopenia, respectively, on posttranslational processing, intracellular trafficking, cell surface expression, and cell proliferation. In contrast to R102P and F1043, the P106L mutant confers cytokine-independent growth and stimulates downstream signaling after THPO treatment in Ba/F3 cells. Despite their opposite function, R102P and P106L, both lead to abnormal subcellular receptor distribution, lack of membrane localization, impaired glycosylation, and elevated THPO serum levels in effected patients. These findings indicate that the activation of downstream signaling by c-Mpl P106L does not require correct processing, trafficking, and cell surface expression of c-Mpi, whereas the negative feedback loop controlling THPO serum levels requires cell surface expression of the receptor. Thus, we propose that the P106L mutation functionally separates the activity of c-Mpi in downstream signaling from that in maintaining platelet homeostasis. [ABSTRACT FROM AUTHOR]

Gain-of-function somatic mutations introducing cysteines to either the extracellular or to the transmembrane domain (TMD) in interleukin-7 receptor α (IL7R) or cytokine receptorlike factor 2 (CRLF2) have been described in acute lymphoblastic leukemias. Here we report noncysteine in-frame mutations in IL7R and CRLF2 located in a region of the TMD closer to the cytosolic domain. Biochemical and functional assays showed that these are activating mutations conferring cytokine-independent growth of progenitor lymphoid cells in vitro and are transforming in vivo. Protein fragment complementation assays suggest that despite the absence of cysteines, the mechanism of activation is through ligand-independent dimerization. Mutagenesis experiments and ConSurf calculations suggest that the mutations stabilize the homodimeric conformation, positioning the cytosolic kinases in predefined orientation to each other, thereby inducing spontaneous receptor activation independently of external signals. Hence, type I cytokine receptors may be activated in leukemia through 2 types of transmembrane somatic dimerizing mutations. [ABSTRACT FROM AUTHOR]

Neuroblastoma is an embryonic solid tumor of neural crest origin and accounts for 11% of all cancer-related deaths in children. Novel therapeutic strategies are therefore urgently required. MYCN oncogene amplification, which occurs in 20% of neuroblastomas, is a hallmark of high risk. Here, we aimed to exploit molecular mechanisms that can be pharmacologically addressed with epigenetically modifying drugs, such as histone deacetylase (HDAC) inhibitors. Grainyhead-like 1 (GRHL1), a gene critical for Drosophila neural development, belonged to the genes most strongly responding to HDAC inhibitor treatment of neuroblastoma cells in a genome-wide screen. An increase in the histone H4 pan-acetylation associated with its promoter preceded transcriptional activation. Physically adjacent, HDAC3 and MYCN colocalized to the GRHL1 promoter and repressed its transcription. High-level GRHL1 expression in primary neuroblastomas correlated on transcriptional and translational levels with favorable patient survival and established clinical and molecular markers for favorable tumor biology, including lack of MYCN amplification. Enforced GRHL1 expression in MYCN-amplified neuroblastoma cells with low endogenous GRHL1 levels abrogated anchorage-independent colony formation, inhibited proliferation, and retarded xenograft growth in mice. GRHL1 knockdown in MYCN single-copy cells with high endogenous GRHL1 levels promoted colony formation. GRHL1 regulated 170 genes genome-wide, and most were involved in pathways regulated during neuroblastomagenesis, including nervous system development, proliferation, cell-cell adhesion, cell spreading, and cellular differentiation. In summary, the data presented here indicate a significant role of HDAC3 in the MYCN-mediated repression of GRHL1 and suggest drugs that block HDAC3 activity and suppress MYCN expression as promising candidates for novel treatment strategies of high-risk neuroblastoma. [ABSTRACT FROM AUTHOR]

Abstract: Background: In recent years different IRT/PAP protocols have been evaluated, but the individual performance remains unclear. To optimize the IRT/PAP strategy we compared protocols from three regional CF newborn screening centers (Heidelberg, Dresden, and Prague). Methods: We evaluated the effect of elevating the IRT-cut-off from 50 to 65μg/l (~97.5th to ~99.0th percentile), the need of a failsafe protocol (FS, IRT≥99.9th percentile) and the relative performance using either two IRT-dependent PAP-cut-offs or one PAP-cut-off. Findings: Elevation of the IRT cut-off to 65μg/l (~99.0th percentile) increased the PPV significantly (Dresden: 0.065 vs. 0.080, p<0.0001, Prague: 0.052 vs. 0.074, p<0.0001) without reducing sensitivity. All three IRT/PAP protocols showed a trend towards a higher sensitivity with FS than without and when using one PAP-cut-off instead of two IRT-dependent PAP-cut-offs. Conclusions: For best performance we suggest an IRT/PAP protocol with an IRT-cut-off close to the 99.0th percentile, FS, and a single PAP-cut-off. [Copyright &y& Elsevier]

Children with Down syndrome ( DS) have a greater risk for developing both acute lymphoblastic leukaemia ( ALL) and significant adverse effects of chemotherapy. We investigated their outcome with, and tolerance of, treatment protocols for relapsed ALL optimized in the paediatric population without DS. Probability of survival and causes of treatment failure were determined for 49 children with DS and a matched cohort of 98 children without DS among 2160 children treated for relapsed ALL in clinical trials conducted by the Berlin- Frankfurt- Münster ALL Relapse Study Group between 1983 and 2012. Despite more favourable ALL relapse characteristics, children with DS experienced lower event-free ( EFS) and overall survival ( OS) than the control group without DS ( EFS 17 ± 08% vs. non- DS 41 ± 06%, P = 0·006; OS 17 ± 09% vs. non- DS 51 ± 06%, P < 0·001). Children with DS developed more frequently fatal complications of treatment (34 ± 07% vs. non- DS 10 ± 04%, P < 0·001). During the last decade, EFS and OS were no longer significantly different in children with and without DS ( EFS 31 ± 09% vs. 36 ± 09%, P = 0·399; OS 31 ± 12% vs. 53 ± 09%, P = 0·151). DS proved an independent prognostic factor of outcome after ALL relapse. Induction deaths and treatment-related mortality but not subsequent relapse were the main barrier to successful outcomes of relapse therapy in children with DS. [ABSTRACT FROM AUTHOR]

Synapsins are a family of neuronal phosphoproteins associated with the cytosolic surface of synaptic vesicles. Experimental evidence suggests a role for synapsins in synaptic vesicle clustering and recycling at the presynaptic terminal, as well as in neuronal development and synaptogenesis. Synapsin knock-out (Syn1−/−) mice display an epileptic phenotype and mutations in the SYN1 gene have been identified in individuals affected by epilepsy and/or autism spectrum disorder. We investigated the impact of the c.1067G>A nonsense transition, the first mutation described in a family affected by X-linked syndromic epilepsy, on the expression and functional properties of the synapsin I protein. We found that the presence of a premature termination codon in the human SYN1 transcript renders it susceptible to nonsense-mediated mRNA decay (NMD). Given that the NMD efficiency is highly variable among individuals and cell types, we investigated also the effects of expression of the mutant protein and found that it is expressed at lower levels compared to wild-type synapsin I, forms perinuclear aggregates and is unable to reach presynaptic terminals in mature hippocampal neurons grown in culture. Taken together, these data indicate that in patients carrying the W356× mutation the function of synapsin I is markedly impaired, due to both the strongly decreased translation and the altered function of the NMD-escaped protein, and support the value of Syn1−/− mice as an experimental model mimicking the human pathology. [ABSTRACT FROM AUTHOR]

Ependymomas are primary brain tumors found throughout the central nervous system (CNS) in children and adults. Currently, many treatment protocols stratify grade I and II ependymomas as low-risk tumors, whereas grade III anaplastic ependymomas are considered high-risk tumors. The prognostic significance of World Health Organization (WHO) grade II or III, however, remains debated, and it is furthermore increasingly recognized that the pathologic differentiation between grades II and III is arbitrary in daily practice, thus resulting in imprecise risk stratification. Therefore, prognostic markers enabling more precise stratification to guide treatment decisions are urgently needed. An analysis of n = 379 tumor samples revealed that protein expression of nestin, a marker for neural stem and progenitor cells established as a routine staining in most neuropathology centers, is associated with poor outcome in intracranial ependymomas. Most importantly, nestin-positive grade II ependymomas have the same prognosis as grade III ependymomas. Multivariable analysis demonstrates that nestin positivity is an independent marker for poor progression-free survival (PFS) and overall survival (OS). Gene expression analysis for transcriptionally co-regulated genes revealed a strong association of developmental and epigenetic processes with nestin. In summary, our data implicate nestin as a useful novel marker for intracranial ependymoma risk stratification easily implementable in routine diagnostics. [ABSTRACT FROM AUTHOR]

Hereditary thrombocythemia (HT) has been described as a rare benign disorder caused by mutations in the thrombopoietin (THPO) or the c-Mpl receptor genes. Here we report two families with HT resulting from a THPO c.13+1 G>C mutation in the splice donor of intron 3. In one family there were coexisting distal limb defects, whereas in the other one member developed early-onset multiple myeloma. These observations, together with previously reported patients, suggest that THPO gain of function may dysregulate the hemangioblast and disturb vasculogenesis and hematopoietic development. Overstimulation of the THPO pathway might therefore predispose to clonal hematopoietic disease and to congenital abnormalities. [ABSTRACT FROM AUTHOR]

Summary Transient myeloproliferative disorder ( TMD) of the newborn and acute megakaryoblastic leukaemia ( AMKL) in children with Down syndrome ( DS) represent paradigmatic models of leukaemogenesis. Chromosome 21 gene dosage effects and truncating mutations of the X-chromosomal transcription factor GATA1 synergize to trigger TMD and AMKL in most patients. Here, we report the occurrence of TMD, which spontaneously remitted and later progressed to AMKL in a patient without DS but with a distinct dysmorphic syndrome. Genetic analysis of the leukaemic clone revealed somatic trisomy 21 and a truncating GATA1 mutation. The analysis of the patient's normal blood cell DNA on a genomic single nucleotide polymorphism ( SNP) array revealed a de novo germ line 2·58 Mb 15q24 microdeletion including 41 known genes encompassing the tumour suppressor PML. Genomic context analysis of proteins encoded by genes that are included in the microdeletion, chromosome 21-encoded proteins and GATA1 suggests that the microdeletion may trigger leukaemogenesis by disturbing the balance of a hypothetical regulatory network of normal megakaryopoiesis involving PML, SUMO3 and GATA1. The 15q24 microdeletion may thus represent the first genetic hit to initiate leukaemogenesis and implicates PML and SUMO3 as novel components of the leukaemogenic network in TMD/ AMKL. [ABSTRACT FROM AUTHOR]