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LOH and copy number changes on chromosomes 11, 3, 4, 1, 17, and 7. Global view of common areas of LOH (left) and copy number change (right) in 22 primary neuroblastomas. Each sample is depicted as a series of vertical bars in both the LOH and copy number panels. Blue areas represent regions of LOH, while yellow denotes retention of heterozygosity. Copy number is marked by shades of red, with ≤1 copy in light red and ≥3 copies in dark red (see scale at the bottom of the panel).
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Neuroblastomas are characterized by chromosomal alterations with biological and clinical significance. We analyzed paired blood and primary tumor samples from 22 children with high-risk neuroblastoma for loss of heterozygosity (LOH) and DNA copy number change using the Affymetrix 10K single nucleotide polymorphism (SNP) array.
Multiple areas of LOH...
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Citations
... ITH of both MNA (hetMNA) and typical deletion at 1p36 of the NB have been detected by fluorescence in situ hybridisation (FISH) in different studies [5,25], one of which reports hetMNA as the apparent culprit of aggressive local growth and development of metastases in patients ≥18 months of age at diagnosis [5]. Moreover, ITH has also been detected in other large SCAs, as well as in single nucleotide variations (SNVs), such as those of the ALK gene [21,22,26,27]. ITH is commonly associated with progressive disease and treatment resistance, likely because of selection of treatment-resistant clones [2]. ...
Spatial ITH is defined by genomic and biological variations within a tumour acquired by tumour cell evolution under diverse microenvironments, and its role in NB patient prognosis is understudied. In this work, we applied pangenomic techniques to detect chromosomal aberrations in at least two different areas of each tumour and/or in simultaneously obtained solid and liquid biopsies, detecting ITH in the genomic profile of almost 40% of HR-NB. ITH was better detected when comparing one or more tumour pieces and liquid biopsy (50%) than between different tumour pieces (21%). Interestingly, we found that patients with ITH analysed by pangenomic techniques had a significantly better survival rate that those with non-heterogeneous tumours, especially in cases without MYCN amplification. Moreover, all patients in the studied cohort with high ITH (defined as 50% or more genomic aberration differences between areas of a tumour or simultaneously obtained samples) survived after 48 months. These results clearly support analysing at least two solid tumour areas (separately or mixed) and liquid samples to provide more accurate genomic diagnosis, prognosis and therapy options in HR-NB.
... . The use of CRISPR/Cas9 allows to directly target the endogenous MAPT and generate complete knock-out lines or induce mutations on a single base level that are thought to be involved in TAU (mis)sorting, e.g. by using the improved CRISPR/Cas9 prime editing method 90 .However, one has to consider the genetic predispositions of SH-SY5Y cells, as large-scale chromosomal abnormalities and imbalances are reported for neuroblastoma cell lines in general[91][92][93][94][95] . Accordingly, SH-SY5Y cells show trisomy of chromosome 7 (chr7), a duplication of the qarm of chr1, and further complex rearrangements on the majority of chromosomes leading to both copy number gains and losses96 . ...
1. Abstract The human-derived SH-SY5Y neuroblastoma cell line is widely used for studying TAU physiology and TAU-related pathology in Alzheimer's disease (AD) and related tauopathies. SH-SY5Y cells can be differentiated into neuron-like cells (SH-SY5Y-derived neurons), which resemble noradrenergic, dopaminergic or cholinergic neurons, by using various substances. This review evaluates whether SH-SY5Y-derived neurons are a suitable model for investigating intracellular TAU sorting mechanisms in general, and with respect to neuron subtype-specific TAU vulnerability. SH-SY5Y-derived neurons show pronounced axodendritic polarity resembling neuronal cell polarity, high TAU protein levels, axonal TAU localization, expression of the six major human brain isoforms, and TAU phosphorylation similar to AD. This enables studying the isoform-and phosphorylation-dependent impact on TAU subcellular distribution and axodendritic trafficking of TAU. As SH-SY5Y cells are accessible for genetic engineering, stable transgene integration and leading-edge genome editing are valuable and often-used tools for TAU-related research in these cells. Furthermore, SH-SY5Y-derived neurons resemble cells of distinct subcortical nuclei, i.e. the Locus coeruleus (LC), Nucleus basalis (NB) and Substantia nigra (SN), depending on the used differentiation procedure. This allows to study neuron-specific TAU isoform expression and intracellular localization in the context of vulnerability to TAU pathology. Limitations are e.g. the lack of mimicking age-related tauopathy risk factors and the difficulty to define the exact neuronal subtype of SH-SY5Y-derived neurons. In brief, this review discusses the suitability of SH-SY5Y-derived neurons for investigating TAU sorting mechanisms and neuron-specific TAU vulnerability in human-like conditions.
... . The use of CRISPR/Cas9 allows to directly target the endogenous MAPT and generate complete knock-out lines or induce mutations on a single base level that are thought to be involved in TAU (mis)sorting, e.g. by using the improved CRISPR/Cas9 prime editing method 90 .However, one has to consider the genetic predispositions of SH-SY5Y cells, as large-scale chromosomal abnormalities and imbalances are reported for neuroblastoma cell lines in general[91][92][93][94][95] . Accordingly, SH-SY5Y cells show trisomy of chromosome 7 (chr7), a duplication of the qarm of chr1, and further complex rearrangements on the majority of chromosomes leading to both copy number gains and losses96 . ...
The human-derived SH-SY5Y neuroblastoma cell line is widely used for studying TAU physiology and TAU-related pathology in Alzheimer’s disease (AD) and related tauopathies. SH-SY5Y cells can be differentiated into neuron-like cells (SH-SY5Y-derived neurons), which resemble noradrenergic, dopaminergic or cholinergic neurons, by using various substances. This review evaluates whether SH-SY5Y-derived neurons are a suitable model for investigating intracellular TAU sorting mechanisms in general, and with respect to neuron subtype-specific TAU vulnerability. SH-SY5Y-derived neurons show pronounced axodendritic polarity resembling neuronal cell polarity, high TAU protein levels, axonal TAU localization, expression of the six major human brain isoforms, and TAU phosphorylation similar to AD. This enables studying the isoform- and phosphorylation-dependent impact on TAU subcellular distribution and axodendritic trafficking of TAU. As SH-SY5Y cells are accessible for genetic engineering, stable transgene integration and leading-edge genome editing are valuable and often-used tools for TAU-related research in these cells. Furthermore, SH-SY5Y-derived neurons resemble cells of distinct subcortical nuclei, i.e. the Locus coeruleus (LC), Nucleus basalis (NB) and Substantia nigra (SN), depending on the used differentiation procedure. This allows to study neuron-specific TAU isoform expression and intracellular localization in the context of vulnerability to TAU pathology. Limitations are e.g. the lack of mimicking age-related tauopathy risk factors and the difficulty to define the exact neuronal subtype of SH-SY5Y-derived neurons. In brief, this review discusses the suitability of SH-SY5Y-derived neurons for investigating TAU sorting mechanisms and neuron-specific TAU vulnerability in human-like conditions.
... We used VCF2CNA to perform cghMCR analysis with CNA profiles and observed a genome pattern similar to that reported for SNP array platforms (Fig. 6A) 32 . In addition to loss of large regions on chr1p, 3p, and 11q and a broad gain of chr17q, VCF2CNA found frequent focal amplifications of MYCN in NBL tumors and www.nature.com/scientificreports ...
VCF2CNA is a tool (Linux commandline or web-interface) for copy-number alteration (CNA) analysis and tumor purity estimation of paired tumor-normal VCF variant file formats. It operates on whole genome and whole exome datasets. To benchmark its performance, we applied it to 46 adult glioblastoma and 146 pediatric neuroblastoma samples sequenced by Illumina and Complete Genomics (CGI) platforms respectively. VCF2CNA was highly consistent with a state-of-the-art algorithm using raw sequencing data (mean F1-score = 0.994) in high-quality whole genome glioblastoma samples and was robust to uneven coverage introduced by library artifacts. In the whole genome neuroblastoma set, VCF2CNA identified MYCN high-level amplifications in 31 of 32 clinically validated samples compared to 15 found by CGI’s HMM-based CNA model. Moreover, VCF2CNA achieved highly consistent CNA profiles between WGS and WXS platforms (mean F1 score 0.97 on a set of 15 rhabdomyosarcoma samples). In addition, VCF2CNA provides accurate tumor purity estimates for samples with sufficient CNAs. These results suggest that VCF2CNA is an accurate, efficient and platform-independent tool for CNA and tumor purity analyses without accessing raw sequence data.
... Gain of 17q is the most frequent genetic alteration in NB and is associated with 1p deletion [54]. Loss of 11q is almost mutually exclusive of MNA but is also a feature of high-risk disease and portends an unfavourable prognosis [14,55]. Interestingly, tumours with 11q loss tend to display numerous SCAs, suggestive of a chromosomal instability phenotype [56,57]. ...
Molecular analysis of nucleic acid and protein biomarkers is becoming increasingly common in paediatric oncology for diagnosis, risk stratification and molecularly targeted therapeutics. However, many current and emerging biomarkers are based on analysis of tumour tissue, which is obtained through invasive surgical procedures and in some cases may not be accessible. Over the past decade, there has been growing interest in the utility of circulating biomarkers such as cell-free nucleic acids, circulating tumour cells and extracellular vesicles as a so-called liquid biopsy of cancer. Here, we review the potential of emerging circulating biomarkers in the management of neuroblastoma and highlight challenges to their implementation in the clinic.
... A few years later a variant of this technique, array CGH (aCGH), was developed, in which the tumor and reference DNA are hybridized on oligonucleotide probes fixed on a glass slide 67,72,[102][103][104][105][106][107] . In addition, genome- wide SNP arrays (SNPa) allowed to determine allelic imbalances on top of copy number status 108,109 . This approach has the advantage of informing on copy-neutral loss of heterozygosity (LOH), however, the precise prognostic impact of the rarely occurring copy- neutral LOH in neuroblastoma remains to be determined. ...
Cancer is one of the four major non-communicable diseases. It is estimated that, overall, it affects one in three individuals. Cancer develops by the accumulation of genomic alterations, which activate oncogenes and inactivate tumor suppressor genes. DNA copy number imbalances are a form of genomic alterations frequently observed in cancer cells. Copy number gains and losses typically target oncogenes and tumor suppressor genes, respectively, and can be associated with patient survival. Neuroblastoma, a pediatric tumor of the peripheral nervous system, is characterized by remarkable clinical heterogeneity and abundant copy number alterations. Despite intensive treatment, high-risk patients still have low survival probabilities. Therefore, we searched for copy number alterations that are associated with patient outcome in high-risk patients. Through an international collaboration, we collected copy number profiles of 556 high-risk neuroblastoma patients. In this large dataset I identified two rare types of copy number alterations, namely distal 6q losses and genomic amplifications, that were associated with poor survival in high-risk patients. To facilitate reuse of this unique dataset, I provide detailed information on data access in the form of a data descriptor paper. Moreover, in this manuscript I offer multiple options for data analysis suited for researchers with different experience levels in bioinformatics. In the second part of the project, I evaluated the presence of genomic amplifications in a pan-cancer context using the TCGA dataset, comprising data of around 11,000 cancer patients of 33 different cancer types. Applying an optimized selection method, we identified amplifications in 31 cancer types, including the known hotspot regions like EGFR, CCND1 and ERBB2. In this large dataset I identified several very rare amplifications that are only recurrent across different cancer types. This can lead to the discovery of new oncogenic amplifications, but also select patients that might benefit from targeted therapy, especially when the presence of amplifications is associated with poor survival. I will provide an overview of the observed amplifications in the form of an online application that allows genome-wide browsing and zooming in on specific regions. In conclusion, by using large datasets I succeeded to identify rare DNA copy number events that are associated with poor patient survival and can possibly serve as new entry points for targeted therapy.
... Although the mutation rate of the targeted genes was relatively low, considerable variations in copy number changes were observed in most neuroblastoma individuals (frequency, 81.6%). Recapitulating previous reports [6,[20][21][22], neuroblastoma genomes were characterized by common 17q gains with or without 1p, 3p, 4p, 11q, 19p, and 19q LOH, and 1q, 2p, 7q, and 12q gains with varying combinations ( Figure 2A). As shown in Figure 2A, the CNVs found in more than 5% of the samples were 1p LOH (30.2%), 1q gain (13.2%), 2p gain (35.8%), 3p LOH (15.8%), 4p LOH (8.8%), 5p gain (7.6%), 6q gain (7.0%), 7q gain (36.2%), 8p LOH (5.8%), 11q LOH (28.2%), 12q gain (29.4%), 17q gain (73.4%), 19p LOH (6.6%), 19q LOH (7.4%), and 22q LOH (6.2%). ...
To provide better insight into the genetic signatures of neuroblastomas, we analyzed 500 neuroblastomas (included specimens from JNBSG) using targeted-deep sequencing for 10 neuroblastoma-related genes and SNP arrays analysis. ALK expression was evaluated using immunohistochemical analysis in 259 samples. Based on genetic alterations, the following 6 subgroups were identified: groups A (ALK abnormalities), B (other gene mutations), C (MYCN amplification), D (11q loss of heterozygosity [LOH]), E (at least 1 copy number variants), and F (no genetic changes). Groups A to D showed advanced disease and poor prognosis, whereas groups E and F showed excellent prognosis. Intriguingly, in group A, MYCN amplification was not a significant prognostic marker, while high ALK expression was a relevant indicator for prognosis (P = 0.033). Notably, the co-existence of MYCN amplification and 1p LOH, and the co-deletion of 3p and 11q were significant predictors of relapse (P = 0.043 and P = 0.040). Additionally, 6q/8p LOH and 17q gain were promising indicators of survival in patients older than 5 years, and 1p, 4p, and 11q LOH potentially contributed to outcome prediction in the intermediate-risk group. Our genetic overview clarifies the clinical impact of genetic signatures and aids in the better understanding of genetic basis of neuroblastoma.
... Chromosome 17q gain, partial loss of chromosome 1p or 11q and v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) amplification are frequently observed genetic aberrations in neuroblastoma tumours [29]. EZH2 is located on chromosome 7q35, and frequent gains of whole chromosome 7 have been observed in neuroblastoma [30,31]. A functional role for EZH2 in neuroblastoma was reported, whereas EZH2 caused histone hypermethylation in the promoter regions of known tumour suppressor genes CASZ1, CLU, RUNX3 and NGFR resulting in the silencing and downregulation of these genes [32]. ...
To understand the genetic mechanism of disease, finding biomarkers and drug targets are a major focus of academic and pharmaceutical research. However, it has become clear that many important genes driving diseases like cancer, are deregulated in a relatively small group of outliers, e.g. amplified and fusion genes. Cancer outlier profile analysis (COPA), introduced by Tomlins et al, has raised interest in detecting genes with outlier expression within a group, particularly in cancer patients. However, the magnitudes of the COPA scores are not directly comparable across different studies or with different input parameters. Also, integrating COPA scores across multiple studies in a meta-analysis is problematic. Current solutions to performing meta-analyses, including rank-based methods and binomial test, require large study numbers without adjustment for the differences in study size. Also, within a single study COPA scores alone do not provide a P-value, and it is impossible to know which outlier genes are statistically significant. Here we developed three novel methods to address these problems by computing a significance for COPA scores, including Bootstrap resampling, Bootstrap + generalized Pareto distribution (GPD) and Binomial method for statistical significance of COPA (ssCOPA), to enable the comparison of COPA score across studies or parameters. We first evaluated our methods using two breast cancer studies. The P-values generated by Binomial method match closely to Bootstrap resampling using 10,000 randomizations, better than Bootstrap + GPD. In addition, the Binomial method requires significantly less computing time than the other methods. We further validated our methods with a meta-analysis of five breast cancer studies and compared the results with the top-ranking genes of a large-scale meta-analysis of 31 breast cancer studies by ONCOMINE. We show that our P-value-based approaches (P = 10⁻¹⁷~10⁻²⁴) achieves significantly better concordance than the Binomial test (P = 10⁻¹⁴). Data and methods are available for use on the Illumina BaseSpace® CohortAnalyzer platform (formerly known as NextBio Clinical).
Citation Format: Hong Gao, Sam Ng, Hinco J. Gierman. ssCOPA: a novel method with improved sensitivity to assess statistical significance for cancer outlier profile analysis of gene expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4552. doi:10.1158/1538-7445.AM2017-4552
... While whole (or numerical) chromosome changes without segmental alterations (NCA) [8] are frequently observed in low risk NBs with good outcome, SCA defined by gains or losses of partial chromosome material are associated with poor prognosis in most cases [7,8,28,29]. With losses of 1p, 3p, 4p and gains of 1q, 2p and 17q; 11q loss is one of the most frequent SCA observed, reported in 13 to 68% of the samples depending of the cohort analysed [5,7,8,28,30,31]. As previously reported, 11q loss was more frequently found in NBs with high-risk (HR) features (47 to 68% of the HR vs. 47 to 50% for MNA) [30,31], and regularly found associated with poor prognosis [5,7,8,29,32]. ...
... With losses of 1p, 3p, 4p and gains of 1q, 2p and 17q; 11q loss is one of the most frequent SCA observed, reported in 13 to 68% of the samples depending of the cohort analysed [5,7,8,28,30,31]. As previously reported, 11q loss was more frequently found in NBs with high-risk (HR) features (47 to 68% of the HR vs. 47 to 50% for MNA) [30,31], and regularly found associated with poor prognosis [5,7,8,29,32]. Schleiermacher et al. reported that in 147 NBs without MNA, a SCA profile was the strongest independent prognosis factor. ...
... Similar results concerning the age were recently reported in the Swedish cohort of unfavourable NBs where the median age at diagnosis was 58.5 months in NBs with 11q deletion vs 18 months in the MNA group [30]. While 11q alteration is detected mostly in older patients, over 18 months of age at diagnosis [31], recent analysis of the INRG database showed that in the youngest patients (< 18 months) with stage 3 NB, 11q deletion is the only factor found independently associated with poor EFS and OS [32]. ...
Deletion of the long arm of chromosome 11 (11q deletion) is one of the most frequent events that occur during the development of aggressive neuroblastoma. Clinically, 11q deletion is associated with higher disease stage and decreased survival probability. During the last 25 years, extensive efforts have been invested to identify the precise frequency of 11q aberrations in neuroblastoma, the recurrently involved genes, and to understand the molecular mechanisms of 11q deletion, but definitive answers are still unclear. In this review, it is our intent to compile and review the evidence acquired to date on 11q deletion in neuroblastoma.
... Chromosome 17q gain, partial loss of chromosome 1p or 11q and v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN) amplification are frequently observed genetic aberrations in neuroblastoma tumours [29]. EZH2 is located on chromosome 7q35, and frequent gains of whole chromosome 7 have been observed in neuroblastoma [30,31]. A functional role for EZH2 in neuroblastoma was reported, whereas EZH2 caused histone hypermethylation in the promoter regions of known tumour suppressor genes CASZ1, CLU, RUNX3 and NGFR resulting in the silencing and downregulation of these genes [32]. ...
Neuroblastoma is predominantly characterised by chromosomal rearrangements. Next to V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma Derived Homolog (MYCN) amplification, chromosome 7 and 17q gains are frequently observed. We identified a neuroblastoma patient with a regional 7q36 gain, encompassing the enhancer of zeste homologue 2 (EZH2) gene. EZH2 is the histone methyltransferase of lysine 27 of histone H3 (H3K27me3) that forms the catalytic subunit of the polycomb repressive complex 2. H3K27me3 is commonly associated with the silencing of genes involved in cellular processes such as cell cycle regulation, cellular differentiation and cancer. High EZH2 expression correlated with poor prognosis and overall survival independent of MYCN amplification status. Unexpectedly, treatment of 3 EZH2-high expressing neuroblastoma cell lines (IMR32, CHP134 and NMB), with EZH2-specific inhibitors (GSK126 and EPZ6438) resulted in only a slight G1 arrest, despite maximum histone methyltransferase activity inhibition. Furthermore, colony formation in cell lines treated with the inhibitors was reduced only at concentrations much higher than necessary for complete inhibition of EZH2 histone methyltransferase activity. Knockdown of the complete protein with three independent shRNAs resulted in a strong apoptotic response and decreased cyclin D1 levels. This apoptotic response could be rescued by overexpressing EZH2ΔSET, a truncated form of wild-type EZH2 lacking the SET transactivation domain necessary for histone methyltransferase activity. Our findings suggest that high EZH2 expression, at least in neuroblastoma, has a survival function independent of its methyltransferase activity. This important finding highlights the need for studies on EZH2 beyond its methyltransferase function and the requirement for compounds that will target EZH2 as a complete protein.
