BACKGROUND:The neuroinflammatory cerebral form of X-linked adrenoleukodystrophy (CALD) is among the most severe neurological diseases affecting children. Early intervention by hematopoietic stem cell transplantation (HSCT) or gene therapy halts CALD, justifying its inclusion in newborn screening programmes. Currently, eligibility for treatment is assessed using MRI-based Loes and neurological scoring; however, grading poorly differentiates advanced stages or atypical lesion patterns. We recently identified blood neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP), indicative of axonal and glial damage, as valid biomarkers reflecting CALD activity. METHODS:Here, we investigated how pre-treatment biomarker levels relate to outcome of HSCT or gene therapy in a retrospective cohort of 14 paediatric CALD patients aged 5-13 years. Patients were monitored longitudinally before and for up to 5.5 years following HSCT or gene therapy. Disease progression was assessed using MRI-based Loes scoring and neurological evaluations. Plasma NfL and GFAP were quantified using single molecule array (Simoa) technology. RESULTS:Here we show that blood NfL and GFAP transiently increase due to myeloablative conditioning and gradually normalize post-treatment. Pre-treatment NfL ≤ 113 pg/ml, but not GFAP, correlate with CALD stabilization or, for Loes >9, only minor progression. In contrast, NfL >243 pg/ml associates with major progression irrespective of baseline Loes score. In three boys with atypical lesions or advanced disease, NfL outperforms Loes-scoring in predicting outcome. CONCLUSIONS:Blood NfL can complement clinical decision-making, particularly in patients with advanced CALD or atypical lesions. These findings are especially relevant for clinical management in countries that have not yet implemented X-linked adrenoleukodystrophy into newborn screening.

Deficiency of adenosine deaminase 2 (DADA2) is an autoinflammatory disease with diverse phenotypes. We describe the genetics, phenotypes, and treatment of n = 48 DADA2 patients from Germany, Austria, and Switzerland. We report a high incidence of hematological (83%) and immunological features (85%), a comparatively low anti-tumor necrosis factor full-response rate (58%), and a high decision probability (21%) for hematopoietic cell transplantation (HCT). We establish a correlation between genetic variant ADA2 activity and patient ADA2 activity. Remarkably, lower patient ADA2 activity is predictive of neutropenia and shows a trend toward HCT decision, whereas higher patient ADA2 activity is predictive of vasculitis symptoms. Genetic variants with low residual ADA2 activity are significantly more common among patients receiving HCT. Our study corroborates previous observations connecting ADA2 activity and clinical phenotype, which up to now have been mainly based on in vitro data.

Supplementary Figure 1 from The Coamplification Pattern of the MYCN Amplicon Is an Invariable Attribute of Most MYCN-Amplified Human Neuroblastomas

Background: Genetic defects in components of inflammasomes can cause autoinflammation. Biallelic loss-of-function mutations in dipeptidyl peptidase 9 (DPP9), a negative regulator of the NLRP1 and CARD8 inflammasomes, have recently been shown to cause an inborn error of immunity characterized by pancytopenia, skin manifestations, and increased susceptibility to infections.Objective: We sought to study the molecular basis of autoinflammation in a patient with severe infancy-onset hyperinflammation associated with signs of fulminant hemophagocytic lymphohistiocytosis.Methods: Using heterologous cell models as well as patient cells, we performed genetic, immunologic, and molecular investigations to identify the genetic cause and to assess the impact of the identified mutation on inflammasome activation.Results: The patient exhibited pancytopenia with decreased neutrophils and T, B, and natural killer cells, and markedly elevated levels of lactate dehydrogenase, ferritin, soluble IL-2 receptor, and triglycerides. In addition, serum levels of IL-1 beta and IL-18 were massively increased, consistent with inflammasome activation. Genetic analysis revealed a previously undescribed de novo mutation in DPP9 (c.755G>C, p.Arg252Pro) affecting a highly conserved amino acid residue. The mutation led to destabilization of the DPP9 protein as shown in transiently transfected HEK293T cells and in patient-derived induced pluripotent stem cells. Using functional inflammasome assays in HEK293T cells, we demonstrated that mutant DPP9 failed to restrain the NLRP1 and CARD8 inflammasomes, resulting in constitutive inflammasome activation. These findings suggest that the Arg252Pro DPP9 mutation acts in a dominant-negative manner.Conclusions: A de novo mutation in DPP9 leads to severe infancy-onset autoinflammation because of unleashed inflammasome activation.

Deficiency of adenosine deaminase 2 (DADA2) is a rare Mendelian, autoinflammatory multiorgan disease. We report the case of a 3.8-year-old female patient who was admitted with an acute brainstem stroke and was diagnosed with DADA2 by early initiation of exome sequencing. We recommend that DADA2 and a genetic workup should be taken into account, when evaluating strokes in children even if no other than neurological symptoms are evident.

Heme oxygenase (HO)−1 catalyzes the degradation of cytotoxic heme into biliverdin and blocks antitumor immune responses, thus protecting cancer against host defense. Whether this scenario also applies to neuroblastoma (NB), the most common extracranial solid childhood tumor, is not known. Here, we demonstrate for the first time a prognostic relevance of HO‐1 expression in samples from NB patients and show that targeting of HO‐1 prevents both cancer resistance against cellular stress and immune escape in the syngeneic NXS2 A/J mouse model of NB. High HO‐1 RNA expression in NB tissues emerged as unfavorable prognostic marker, in particular for patients older than 18 months as indicated by univariate as well as multivariate survival probability analyses including disease stage and MYCN status. On the basis of this observation we aimed to target HO‐1 by systemic as well as tumor‐specific zinc protoporphyrin‐mediated HO‐1 suppression in a syngeneic immunocompetent NB mouse model. This resulted in 50% reduction of primary tumor growth and a suppression of spontaneous liver metastases. Importantly, HO‐1 inhibition abrogated immune cell paralysis affecting CD4 and CD8 T‐effector cells. This in turn reverted HO‐1‐dependent immune escape mechanisms in NB by increasing NB apoptosis and improved DC maturation. In summary, HO‐1 emerges as a novel immune regulator in NB and emerges as a promising target for the development of therapeutic approaches.

Evasion from the immune system has emerged as a novel hallmark in the genesis of neuroblastoma as well as in its progression. A profound knowledge of underlying immune regulatory mechanisms in neuroblastoma may be of vital importance to improve current and future immunotherapeutic approaches against this challenging childhood malignancy. This chapter highlights the basic immune escape mechanisms in NB with a particular focus on immune regulatory cells and regulatory molecules that drive immunity to tolerance. (C) 2015 S. Karger AG, Basel

Background: Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is caused by germ line mutations in the PTEN gene. Symptoms include cancer predisposition, immune deviations, and lipomas/lipomatosis. No causal standard therapy is available. We describe a therapeutic attempt with the mammalian target of rapamycin (mTOR) inhibitor sirolimus for a PHTS patient suffering from thymus hyperplasia and lipomatosis. We furthermore assessed the in vitro effects of sirolimus and other inhibitors on lipoma cells of the patient. Methods: The patient underwent clinical and blood examinations and whole-body magnetic resonance imaging to assess tumor sizes. Lipoma cells of the patient were incubated with inhibitors of the phosphoinositide-3-kinase (PI3K)/AKT/mTOR signaling pathway to analyze the effects on proliferation, adipocyte differentiation, and survival in vitro . Results: Sirolimus treatment improved somatic growth and reduced thymus volume. These effects diminished over the treatment period of 19 mo. Sirolimus decreased lipoma cell proliferation and adipocyte differentiation in vitro but did not cause apoptosis. PI3K and AKT inhibitors induced apoptosis significantly. Conclusion: Sirolimus treatment led to an improvement of the patient’s clinical status and a transient reduction of the thymus. Our in vitro findings point to PI3K and AKT inhibitors as potential treatment options for patients with severe forms of PHTS.

The clinical course of a female patient affected by a progeroid syndrome with Restrictive Dermopathy (RD)-like features was followed up. Besides missing hairiness, stagnating weight and growth, RD-like features including progressive skin swelling and solidification, acrocontractures, osteolysis and muscular hypotension were observed until the patient died at the age of 11 months. A homozygous LMNA mutation c.1303C>T (p.R435C) was found by Sanger sequencing. Haplotyping revealed a partial uniparental disomy of chromosome 1 (1q21.3 to 1q23.1) including the LMNA gene. In contrast to reported RD patients with LMNA mutations, LMNA p.R435C is not located at the cleavage site necessary for processing of prelamin A by ZMPSTE24 and leads to a distinct phenotype combining clinical features of Restrictive Dermopathy, Mandibuloacral Dysplasia and Hutchinson-Gilford Progeria. Functionally, LMNA p.R435C is associated with increasing DNA double strand breaks and decreased recruitment of P53 binding protein 1 (53BP1) to DNA-damage sites indicating delayed DNA repair. The follow-up of the complete clinical course in the patient combined with functional studies showed for the first time that a progressive loss of lamin A rather than abnormal accumulation of prelamin A species could be a pathophysiological mechanism in progeroid laminopathies, which leads to DNA repair deficiency accompanied by advancing tissue degeneration.

Background. Cancer in childhood is rare, but nevertheless one of the most frequent causes of disease related death. Initial symptoms are often unspecific, frequently leading to a delay of cancer diagnosis. As a timely diagnosis can be crucial for the clinical outcome, our aim is to point out when unspecific symptoms should be considered suspect of being associated with specific cancer entities. Data sources. A systematic literature research in PubMed and current biliographies, as well as an evaluation of published epidemiologic data was performed. Results. This article reviews the typical presenting features and epidemiologic characteristics of the more common childhood malignancies, elucidates when specific and virtually unspecific symptoms require further evaluation, and gives advice how to start a rational diagnostic workup. Furthermore, genetic syndromes requiring increased watchfulness for cancer in childhood are demonstrated. Conclusion. Patients showing suspect symptoms should early be referred to specialized centres to assure optimal diagnostic and therapeutic capabilities.

Reliable diagnostic strategies for individuals with cancer demand practical methods for highly sensitive and specific detection of tumor cells. Amplification of genomic regions that include putative oncogenes is common in tumor cells of various types. Genomic array platforms offer the opportunity to identify and precisely map amplified genomic regions (ampGRs). The stable existence of these tumor cell–specific genomic aberrations during and after therapy, in theory, make ampGRs optimal targets for cancer diagnostics. In this study, we mapped ampGRs around the proto-oncogene MYCN of human neuroblastomas using a high-resolution tiling array (HR-TA). Based on the HR-TA data, we were able to precisely describe the telomeric and centromeric borders of the ampGRs and deduce virtual fusion sites of the joined ampGRs (amplicon fusion sites [AFSs]). These AFSs served as blueprints for the subsequent design of AFS bridging PCR assays (AFS-PCRs). Strikingly, these assays were absolutely tumor cell specific and capable of detecting 1 tumor cell in 1 × 10(6) to 8 × 10(6) control cells. We successfully proved the in vivo practicability of AFS-PCR by detecting and quantifying the specific AFS DNA of human MYCN-amplified neuroblastomas in the patients’ corresponding peripheral blood and bone marrow samples. Thus, we believe AFS-PCR could become a powerful and nevertheless feasible personalized diagnostic tool applicable to a large number of cancer patients, including children with MYCN-amplified neuroblastomas.

A very rare case of a congenital orbital fat herniation is demonstrated. Clinically, the child presented a progressive, small yellowish and mobile mass on the temporal orbital wall of her right eye. Magnetic resonance imaging showed a fat isointensive structure in T1-weighing without contrast enhancement. The possible aetiology of the herniation could be a congenital weakness of the Tenon's capsule or the conjunctival fornix.

In response to stimuli that activate p53, cells can undergo either apoptosis or cell cycle arrest, depending on the precise pattern of p53 target genes that is activated. We show here that Zbtb4, a transcriptional repressor protein, associates with the Sin3/histone deacetylase co-repressor and represses expression of P21CIP1 as part of a heterodimeric complex with Miz1. In vivo, expression of ZBTB4 is downregulated in advanced stages of multiple human tumours. In cell culture, depletion of ZBTB4 promotes cell cycle arrest in response to activation of p53 and suppresses apoptosis through regulation of P21CIP1, thereby promoting long-term cell survival. Our data suggest that Zbtb4 is a critical determinant of the cellular response to p53 activation and reinforce the notion that p21Cip1 can provide an essential survival signal in cells with activated p53.

Abstract Purpose: Fifteen percent to 20% of human neuroblastomas show amplification of the MYCN oncogene physiologically located at chromosome 2p24-25, indicating an aggressive subtype of human neuroblastoma with a poor clinical outcome. Recent findings revealed that the structure of the amplicon differs interindividually and that coamplification of genes in telomeric proximity to MYCN might play a relevant role in neuroblastoma development and response to treatment, respectively. We now asked if the amplicon structure is an invariable attribute of an individual tumor or if the coamplification pattern could change during progress or in case of recurrent disease. Experimental Design: We used a previously described multiplex PCR approach to analyze the coamplification status of MYCN-amplified human neuroblastomas (n = 33) in tumor tissue at the time of initial diagnosis and in consecutive tissue specimens at later time points after initial treatment or from relapsing disease. The MYCN copy number per haploid genome (Mcn/hg) in these specimens was determined in a separate duplex PCR. Results: In 32 of the 33 investigated tumors, the amplicon structure showed no changes after initial chemotherapy and in recurrent disease. Mcn/hg showed a decrease after initial treatment (n = 23), whereas we found a significant increase in recurrent disease (n = 10). Conclusion: Our data indicate that the initial determined structure of the 2p24-25 amplicon is a consistent attribute in the great majority of the individual MYCN-amplified neuroblastomas and shows no plasticity during or after chemotherapy. Observed changes in the Mcn/hg over the course of disease are in line with preexisting cell culture findings.