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Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma, but resistance to ALK tyrosine kinase inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing neuroblastoma; inhibition of miR-1304-5p decreases, while mimics of this miRNA increase the sensitivity of neuroblastoma cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in neuroblastoma, inducing apoptosis in vitro. In particular, on combined treatment of neuroblastoma patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth is observed although tumours rapidly regrow on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk neuroblastoma although prolonged therapy is likely required to prevent relapse. Targeting oncogenic ALK activity in neuroblastoma is an attractive therapeutic strategy but success has been limited by resistance to ALK inhibitors. Here, the authors identify loss of miR-1304-5p as a driver of ALK inhibitor resistance via regulation of NRAS, and therapeutically target this axis with the addition of a farnesyltransferase inhibitor in preclinical models of neuroblastoma. [ABSTRACT FROM AUTHOR]

Neutrophils are evolutionarily conserved innate immune cells playing pivotal roles in host defense. Zebrafish models have contributed substantially to our understanding of neutrophil functions but similarities to human neutrophil maturation have not been systematically characterized, which limits their applicability to studying human disease. Here we show, by generating and analysing transgenic zebrafish strains representing distinct neutrophil differentiation stages, a high-resolution transcriptional profile of neutrophil maturation. We link gene expression at each stage to characteristic transcription factors, including C/ebp-β, which is important for late neutrophil maturation. Cross-species comparison of zebrafish, mouse, and human samples confirms high molecular similarity of immature stages and discriminates zebrafish-specific from pan-species gene signatures. Applying the pan-species neutrophil maturation signature to RNA-sequencing data from human neuroblastoma patients reveals association between metastatic tumor cell infiltration in the bone marrow and an overall increase in mature neutrophils. Our detailed neutrophil maturation atlas thus provides a valuable resource for studying neutrophil function at different stages across species in health and disease. Maturation of innate immune cells is a graded stereotypic process which is often conserved across species. Here authors label distinct neutrophil leukocyte developmental stages via generating combinations of transgenic zebrafish reporter strains, followed by transcriptome analysis of different neutrophil maturation stages and comparison to the gene expression profile of developing neutrophils from humans and mice. [ABSTRACT FROM AUTHOR]

Purpose: Neuroblastoma (NB) is the most frequent extracranial tumor in children. The detection of bone marrow (BM) involvement is crucial for correct staging and risk-adapted treatment. We compared three methods regarding the detection of NB involvement in BM. Methods: Eighty-one patients with NB were included in this retrospective study. BM samples were obtained at designated time points at study entry and during treatment or follow-up. The diagnostic tools for BM analysis included cytomorphology (CM), flow cytometry (FCM) and automatic immunofluorescence plus fluorescence in situ hybridization (AIPF). Results: We analyzed 369 aspirates in 81 patients in whom AIPF, CM, and FCM were simultaneously available. During the observation period, NB cells were detected in 86/369 (23.3%) cases, by CM in 32/369 (8.7%), by FCM in 52 (14.1%), and by AIPF in 72 (19.5%) samples. AIPF and/or FCM confirmed all positive results obtained in CM and detected 11 additional positive BM aspirates in 294 CM negative samples (p < 0,001). Survival of patients with BM involvement at study entry identified solely by FCM/AIPF was 17.4% versus 0% for patients in whom BM involvement was already identified by CM. Conclusion: The combination of AIPF/FCM yielded the highest detection rate of NB cells in BM. AIPF was the single, most sensitive method in detecting these cells. Although CM did not provide any additional positive results, it is still a useful, readily available and cost-effective tool. The prognostic significance of FCM and AIPF should be confirmed in a prospective study with a larger number of patients. [ABSTRACT FROM AUTHOR]

In neuroblastoma, MYCN amplification and 11q-deletion are important, although incomplete, markers of high-risk disease. It is therefore relevant to characterize additional alterations that can function as prognostic and/or predictive markers. Using SNP-microarrays, a group of neuroblastoma patients showing amplification of one or multiple 12q loci was identified. Two loci containing CDK4 and MDM2 were commonly co-amplified, although amplification of either locus in the absence of the other was observed. Pharmacological inhibition of CDK4/6 with ribociclib or abemaciclib decreased proliferation in a broad set of neuroblastoma cell lines, including CDK4/MDM2-amplified, whereas MDM2 inhibition by Nutlin-3a was only effective in p53wild-type cells. Combined CDK4/MDM2 targeting had an additive effect in p53wild-type cell lines, while no or negative additive effect was observed in p53mutated cells. Most 12q-amplified primary tumors were of abdominal origin, including those of intrarenal origin initially suspected of being Wilms' tumor. An atypical metastatic pattern was also observed with low degree of bone marrow involvement, favoring other sites such as the lungs. Here we present detailed biological data of an aggressive neuroblastoma subgroup hallmarked by 12q amplification and atypical clinical presentation for which our in vitro studies indicate that CDK4 and/or MDM2 inhibition also could be beneficial. [ABSTRACT FROM AUTHOR]

Sequencing of cell-free DNA in the blood of cancer patients (liquid biopsy) provides attractive opportunities for early diagnosis, assessment of treatment response, and minimally invasive disease monitoring. To unlock liquid biopsy analysis for pediatric tumors with few genetic aberrations, we introduce an integrated genetic/epigenetic analysis method and demonstrate its utility on 241 deep whole-genome sequencing profiles of 95 patients with Ewing sarcoma and 31 patients with other pediatric sarcomas. Our method achieves sensitive detection and classification of circulating tumor DNA in peripheral blood independent of any genetic alterations. Moreover, we benchmark different metrics for cell-free DNA fragmentation analysis, and we introduce the LIQUORICE algorithm for detecting circulating tumor DNA based on cancer-specific chromatin signatures. Finally, we combine several fragmentation-based metrics into an integrated machine learning classifier for liquid biopsy analysis that exploits widespread epigenetic deregulation and is tailored to cancers with low mutation rates. Clinical associations highlight the potential value of cfDNA fragmentation patterns as prognostic biomarkers in Ewing sarcoma. In summary, our study provides a comprehensive analysis of circulating tumor DNA beyond recurrent genetic aberrations, and it renders the benefits of liquid biopsy more readily accessible for childhood cancers. Liquid biopsies enable minimally invasive applications for diagnosis and treatment monitoring. Here the authors analyse fragmentation patterns of circulating tumour DNA on multiple levels and develop a bioinformatic tool, LIQUORICE, to accurately detect and classify paediatric cancers with low mutational burden. [ABSTRACT FROM AUTHOR]

Adult Schwann cells (SCs) possess an inherent plastic potential. This plasticity allows SCs to acquire repair-specific functions essential for peripheral nerve regeneration. Here, we investigate whether stromal SCs in benign-behaving peripheral neuroblastic tumors adopt a similar cellular state. We profile ganglioneuromas and neuroblastomas, rich and poor in SC stroma, respectively, and peripheral nerves after injury, rich in repair SCs. Indeed, stromal SCs in ganglioneuromas and repair SCs share the expression of nerve repair-associated genes. Neuroblastoma cells, derived from aggressive tumors, respond to primary repair-related SCs and their secretome with increased neuronal differentiation and reduced proliferation. Within the pool of secreted stromal and repair SC factors, we identify EGFL8, a matricellular protein with so far undescribed function, to act as neuritogen and to rewire cellular signaling by activating kinases involved in neurogenesis. In summary, we report that human SCs undergo a similar adaptive response in two patho-physiologically distinct situations, peripheral nerve injury and tumor development. Schwann cells (SCs) can acquire a repair phenotype following nerve injury. Here, the authors show that stromal SCs in ganglioneuromas express nerve-repair genes. Importantly, neuroblastoma cells respond to repair-related SCs increasing neuronal differentiation and reducing proliferation via EGFL8. [ABSTRACT FROM AUTHOR]

Neuroblastoma (NB) is a pediatric cancer of the sympathetic nervous system and one of the most common solid tumors in infancy. Amplification of MYCN, copy number alterations, numerical and segmental chromosomal aberrations, mutations, and rearrangements on a handful of genes, such as ALK, ATRX, TP53, RAS/MAPK pathway genes, and TERT, are attributed as underlying causes that give rise to NB. However, the heterogeneous nature of the disease—along with the relative paucity of recurrent somatic mutations—reinforces the need to understand the interplay of genetic factors and epigenetic alterations in the context of NB. Epigenetic mechanisms tightly control gene expression, embryogenesis, imprinting, chromosomal stability, and tumorigenesis, thereby playing a pivotal role in physio- and pathological settings. The main epigenetic alterations include aberrant DNA methylation, disrupted patterns of posttranslational histone modifications, alterations in chromatin composition and/or architecture, and aberrant expression of non-coding RNAs. DNA methylation and demethylation are mediated by DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) proteins, respectively, while histone modifications are coordinated by histone acetyltransferases and deacetylases (HATs, HDACs), and histone methyltransferases and demethylases (HMTs, HDMs). This article focuses predominately on the crosstalk between the epigenome and NB, and the implications it has on disease diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Fully-automated nuclear image segmentation is the prerequisite to ensure statistically significant, quantitative analyses of tissue preparations,applied in digital pathology or quantitative microscopy. The design of segmentation methods that work independently of the tissue type or preparation is complex, due to variations in nuclear morphology, staining intensity, cell density and nuclei aggregations. Machine learning-based segmentation methods can overcome these challenges, however high quality expert-annotated images are required for training. Currently, the limited number of annotated fluorescence image datasets publicly available do not cover a broad range of tissues and preparations. We present a comprehensive, annotated dataset including tightly aggregated nuclei of multiple tissues for the training of machine learning-based nuclear segmentation algorithms. The proposed dataset covers sample preparation methods frequently used in quantitative immunofluorescence microscopy. We demonstrate the heterogeneity of the dataset with respect to multiple parameters such as magnification, modality, signal-to-noise ratio and diagnosis. Based on a suggested split into training and test sets and additional single-nuclei expert annotations, machine learning-based image segmentation methods can be trained and evaluated. Measurement(s) nucleus • Annotation • Frozen Section • Neuroblastoma • Touch Prep Slide • Centrifuged Smear Slide • cells grown on slide • Ganglioneuroblastoma • Wilms Tumor • HaCaT cell Technology Type(s) Fluorescence Imaging • machine learning Factor Type(s) nucleus segmentation Sample Characteristic - Organism Homo sapiens Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12570854 [ABSTRACT FROM AUTHOR]