Gain of chromosome 17 is an early genetic abnormality in neuroblastoma with PPM1D emerging as a strong candidate oncogene driving tumor progression

Gain of chromosome 17 is an early genetic abnormality in neuroblastoma with PPM1D emerging as a strong candidate oncogene driving tumor progression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Gain of chromosome 17 is an early genetic abnormality in neuroblastoma with PPM1D emerging as a strong candidate oncogene driving tumor progression Jelena Milosevic, Susanne Fransson, Johanna Svensson, Jörg Otte, and 21 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5961130/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Segmental gain of chromosome 17q is the most common genetic aberration in high-risk neuroblastoma, but its role in disease progression is poorly understood. This study aims to address the contribution of 17q gain to neuroblastoma malignancy. Patients and methods: We analyzed the genetic and transcriptional landscape of 417 neuroblastoma patients across various risk groups and clinical stages using multi-omic approaches. Single-cell RNA/DNA sequencing and SNP arrays were combined to characterize genomic aberrations, while evolutionary trajectories were mapped to explore the accumulation of genetic changes in patients with neuroblastoma. Additionally, DNA and RNA sequencing were used to assess mutational burden and gene expression patterns. Results Our findings suggest that chromosome 17 gain is an early genetic event acquired during neuroblastoma development, correlating with the accumulation of additional chromosomal aberrations and poor prognosis. Increased segmental gains of chromosome 17q were observed during clonal evolution, relapse disease and metastasis. We identified PPM1D , a p53-inducible Ser/Thr phosphatase located on chr17q22.3, as a key player activated by segmental 17q-gain, gene-fusion, or gain-of-function somatic and germline mutations, further promoting neuroblastoma development/progression. Conclusion Gain of chromosome 17 is an early driver of genetic instability in neuroblastoma, with PPM1D emerging as a potential candidate gene implicated in high-risk disease progression. Neuroblastoma chromosome 17q p53 WIP1 PPM1D Figures Figure 1 Figure 2 Figure 3 Figure 4 KEY POINTS Gain of chromosome 17 is an initial genetic event in neuroblastoma development Numbers of segmental chromosome 17q copies is increased during clonal evolution in high-risk disease PPM1D gene fusions and gain-of-function somatic and germline mutations are present in high-risk neuroblastoma IMPORTANCE OF THE STUDY Chromosomal instability resulting in structural and numerical chromosomal aberrations are frequently observed in cancers. Neuroblastoma, a childhood tumor of the sympathetic neural system, is characterized by a heterogenic clinical behavior which in high-risk patients is manifested by segmental chromosomal aberrations. The most frequent chromosomal abnormality and the strongest indicator of adverse outcome. is segmental gain of chromosome 17q. We used multi-omic approaches to investigate the genetic development of neuroblastoma and show that gain of chromosome 17 is an initial genetic event during neuroblastoma development with additional copies of chromosome 17q acquired through clonal evolution in aggressive and metastatic disease. Furthermore, gene fusions, gain-of-function somatic and germ-line mutations and elevated expression of PPM1D, a regulator of p53 activity, located on chromosome 17q22.3 were detected, suggesting a pivotal role of PPM1D in neuroblastoma disease progression. INTRODUCTION Chromosomal instability (CIN), caused by errors in DNA replication and chromosomal segregation during mitosis, is a hallmark of most human cancers 1 . CIN frequently results in structural and numerical chromosomal abnormalities, leading to intra- and inter-tumor heterogeneity, which disrupts key cancer pathways that give rise to phenotypic variations, posing challenges for therapeutic interventions 2 . Neuroblastoma, a childhood tumor of the sympathetic neural system, typically exhibits, like most childhood tumors, a low burden of somatic mutations 3 . Instead, copy number alterations play a dominant role, where focal amplification of the MYCN oncogene, re-arrangements within the TERT locus on chromosome 5, segmental gains of chromosome 17q, and loss of chromosomes 1p36 and 11q all associated with high-risk disease and poor survival 4 . The most common cytogenetic abnormality in neuroblastoma is segmental or whole chromosomal gain of chromosome 17, detected in over 80% of tumors 5 . Segmental gain of 17q21 extending to 17qter is observed in more than 60% of tumors and is the most frequent genetic aberration linked to high-risk disease and poor prognosis of neuroblastoma patients 6 . Recent studies have added to the genomic complexity by revealing significant spatial genetic heterogeneity within neuroblastomas 7 , 8 . Furthermore, these tumors display distinct cell-type states, which may coexist within the same tumor 9 . While no genetic differences between these cell-type states have been reported, they are distinguished by variations in their transcriptional and epigenetic profiles 10 . Characterization of the genetic events during neuroblastoma evolution has shown that chromosomal gains crucial to disease pathogenesis typically occur early, within the first trimester of pregnancy 11 . This suggests that the intra-tumor heterogeneity may arise from the stepwise accumulation of genetic aberrations during embryonic development, with cells at different developmental maturation stages contributing to the heterogeneity observed in patient samples. Inactivating mutations of the tumor suppressor gene TP53 is a hallmark of adult cancers and lead to impaired DNA repair, cell cycle checkpoint arrest and apoptosis, ultimately resulting in accumulation of DNA mutations and CIN´s 12 . Pediatric cancers, on the other hand, including neuroblastoma, rarely exhibit TP53 mutations 13 . Nevertheless, p53 activity is frequently compromised in neuroblastoma, particularly in relapsed tumors, where the incidence of TP53 mutations increases 14 . This suggests that inactivation of p53 remains critical for tumorigenesis, with alternative mechanisms likely contributing to its attenuation during early neuroblastoma development. One such mechanism may involve PPM1D , a gene located on chr17q22.3, which is frequently gained in high-risk neuroblastomas 5 . PPM1D encodes the nuclear serine/threonine phosphatase WIP1 (wild-type p53 induced phosphatase 1), a key regulator of DNA damage response and cell cycle progression, controlling the activity of p53, ATM, CHK1/2 and other key molecules involved in DNA repair, cell cycle progression and apoptosis 15 – 19 . In concordance, PPM1D mutations, amplifications and WIP1 overexpression have been identified in various cancers 20 – 28 , and we recently demonstrated that overexpression of WIP1 in mice can drive tumor development, including neuroblastoma, by repressing p53 activity, promoting apoptotic escape, inducing genomic instability, and impairing growth arrest 29 , 30 . This suggests that PPM1D might acts as an oncogene, facilitating malignant tumor development. In this study, we use multi-omics approaches to investigate the genetic development of neuroblastoma, focusing on the most common aberration, gain of chromosome 17q, and the oncogenic role of PPM1D and the p53 regulating phosphatase WIP1. We demonstrate that gain of chromosome 17 is an early event in CIN during neuroblastoma development, with additional copies acquired through clonal evolution in aggressive and metastatic disease. Moreover, PPM1D gene fusions resulting in high expression levels of PPM1D as well as activating germline and somatic mutations were detected, suggesting a pivotal role of PPM1D in neuroblastoma disease progression. MATERIALS AND METHODS Patient-derived tumor material and clinical data The collection of neuroblastoma tissue samples from Swedish patients was performed after either written or verbal consent obtained from parents/guardians according to ethical permits approved by the local Ethics Committee (Karolinska Institutet and Karolinska University Hospital, registration number 03-736, 2009/1369-31/1 and 2022-07254-1. Fresh tumor tissue was collected at surgery and snap-frozen and stored at -80 o C until analysis. All patients were diagnosed, managed and treated according to national and international guidelines and protocols. Neuroblastoma tissue genomics and transcriptomics Patient material for genetic analysis DNA was extracted from frozen tumors or blood using DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol and evaluated through absorbance measurements, fluorometric quantitation and DNA integrity assessment on Agilent Tapestation (Agilent, Santa Clara, CA). SNP-microarray analysis Microarray analysis was performed on a consecutive series of samples from an unselected cohort of 417 neuroblastoma patients using Affymetrix CytoScan HD SNP microarrays (Thermo Fisher Scientific, Waltham, MA, USA). These analyses include the majority of cases from Sweden. All tumors were staged according to the International Neuroblastoma Staging System (INSS) and INRG criteria. Handling of the microarrays has been described previously 6 , 31 . Primary data analysis was performed using GeneChip Command Console Software version 5.0.0 (Thermo Fisher Scientific) prior copy number analysis using Chromosome Analysis Suite (ChAS v.3.3.0; Thermo Fisher Scientific). All cases of chromosomal gain, loss, or amplification or allelic imbalances were scored, both segmental and numerical aberrations, including detailed information about the breakpoint positions when applicable. For practical reasons, all notations of gain and loss were considered in relation to a nominal tumor karyotype and a chromosomal break in a tumor was defined as a clear change in gene dose level in the genomic profile. All genomic position annotations are stated based on the hg19 build ( http://genome.ucsc.edu/ ) of the human genome. Analysis of 17q clonal evolution Single nucleotide polymorphism (SNP) arrays including the Oncoscan (formalin fixed paraffins embedded samples) and Cytoscan HD (fresh frozen samples) platforms (Affymetrix Inc.) were retrieved from and subjected to comparisons of 17q copy number. Evolutionary trajectories were formalized as phylogenetic ideograms along the lines described in the original publication 7 . In all, 100 multiregional samples collected from 23 patients with neuroblastoma were analyzed. Each patient was assigned to one of three simplified clinical-genetic risk groups, including children < 18 months of age with only numerical aberrations found in their tumor’s phyologenetic stem by SNP array (low-risk patients), tumors with MYCN amplification in the stem (high-risk patients), and tumors with structural rearrangements in the stem (high-risk patients). DNTR sequencing, processing of RNA and DNA and data analysis were done as described previously 32 . Genome sequencing In total, tumor material from 73 Swedish neuroblastoma patients was subjected to either whole genome sequencing (WGS) or exome sequencing with procedures for handling as described previously 33 , 34 . Exome sequencing was performed through paired-end sequencing on Illumina platforms (Illumina, San Diego, CA) after enrichment with Agilent SureSelect All Exome enrichment kit (Agilent technologies, Santa Clara, CA) on DNA for 18 tumor/normal pairs and additional 20 tumors lacking corresponding constitutional DNA. Alignment against human reference genome hg19 was performed using BWA with GATK local realignment followed by SNV calling using SNPeff. For tumors sequenced without constitutional DNA, filtering was performed by retention of only non-synonymous SNVs and removal of common variants, e.g. showing an allele frequency above 0.01 in the Genome Aggregation Database (gnomAD v2.1.1). WGS was performed on tumor DNA and corresponding constitutional DNA (n = 50) also using paired-end sequencing on Illumina instrumentation (Illumina) at Clinical Genomics, SciLife Laboratories, Gothenburg/Stockholm, Sweden, while mapping to hg19 and variant calling were done using the Sentieon suite of bioinformatics tools (Sentieon Inc., Mountain View, CA, USA) (Sentieon version v.201808.03). Only high-quality called SNVs with a minimum of 10% variant allele frequency and a total read coverage of ten were considered. All synonymous variants or variants in non-coding regions (except those affecting canonical splice sites) were discarded. Remaining SNVs were kept for further analysis and evaluation including manual assessment through the Integrative Genomics Viewer (IGV) 35 for removal of calls due to mapping artifacts and paralogs. FusionCatcher was applied to discover PPM1D -containing fusion transcripts in the National Cancer Institute TARGET dataset comprising paired-end RNA sequenced neuroblastoma patient samples (dbGap Study Accession:phs000218.v16.p6). Immunohistochemistry Twenty-seven neuroblastoma samples derived from children of different ages and all clinical stages, including different biological subsets, were analyzed. Formalin-fixed and paraffin-embedded human tissue sections were deparaffinized in xylene and graded alcohols, hydrated and washed in phosphate-buffered saline (PBS). After antigen retrieval in sodium citrate buffer (pH 6) in a microwave oven, the endogenous peroxidase was blocked by 0.3% H2O2 for 15 min. Sections from human neuroblastoma were incubated overnight at 4°C with a primary antibody against PPM1D (ab31270, Abcam). As a secondary antibody, the anti-rabbit-horseradish peroxidase (HRP) SignalStain Boost IHC detection kit was used (Cell Signal Technology; #8114). A matched isotype control was used as a control for nonspecific background staining (not shown). Case description, germline PPM1D mutation detection (see supplementary methods) Gene expression analysis Patient cohort and methodology of RNA-seq data analysis pipeline are extensively described in 36 . We used gene expression data of 498 neuroblastoma patients from seven countries. According to the Neuroblastoma Risk Group (INRG) classification system 37 we classified patients with MYCN amplifications and patients with a metastatic disease and older than 18 months at diagnosis as high-risk patients (n=176). Events were defined according to a revised version of the International Neuroblastoma Response Criteria 38 . RNA-seq gene expression analyses have been done using the MAGIC-AceView pipeline as previously described 36 . Copy number data for PPM1D was derived from array comparative hybridization (aCGH); 39 , whole exome sequencing and whole genome sequencing analyses 40 . Statistical analyses Gene expression of PPM1D was investigated displayed on the gene level ( PPM1D ) or transcript level for two selected transcript variants (AceView annotation, as a result of the analysis, i.e. PPM1D.aAug10 and PPM1D.bAug10 , corresponding to transcripts ENST00000305921 and ENST00000392995 respectively). PPM1D expression in defined NB subgroups (according to INRG high-risk status, INSS stage, amplification status of MYCN, age, CN status of PPM1D on chromosome 17) was displayed in Tukey box-and-whisker plots (box range: inter-quartile range, whiskers: 1.5x IQR). Where appropriate, either Mann–Whitney–Wilcoxon or Kruskal-Wallis tests were performed to investigate differences in expression of two or more analyzed groups, respectively. Pearson correlation analyses have been applied to investigate correlation between PPM1D expression and copy number. To investigate the prognostic value of PPM1D expression, optimal cut-point expression values separating the cohort into PPM1D -high and PPM1D -low groups that correlate best with outcome in terms of event-free survival (EFS; recurrence, progression and death from disease were considered as events), and overall-survival (OS) were calculated by means of maximally selected rank statistics (maxstat package in R v3.4.3) 41,42 in training sets. Differences in survival of PPM1D high and low-expressing subgroups according to dichotomization with the established cutpoints was then tested by Mantel-Cox (log-rank) tests in corresponding validation sets. For this procedure, equally sized training and validation subsets (n=296) were randomly sampled from the entire cohort (n=498). Resampling was done 1000 times and the mode of those established cutpoint values which passed p < 0.05 in maxstat 41,42 as well as in the log-rank test was eventually selected to define PPM1D -high and PPM1D -low expressing subgroups (this procedure was independently done for EFS and OS). Subsequently, Kaplan-Meier survival estimates for the entire cohort were calculated and displayed from the time of diagnosis and clinical outcome of respective subgroups was compared using the log-rank test. Principal Components Analysis (PCA) on expression data Raw data expression: CEL-files (HU133A chips from Affymetrix) from 30 primary neuroblastoma cases from two published microarray studies 43–45 were pre-processed using gcRMA normalization in Bioconducter for R 2.9.2 (library BioC 2.4). For each gene, the mean expression level from probe-sets was calculated, resulting in 8105 genes/variables 43 . Principal Components Analysis (PCA) was performed on the pre-processed McArdle/Wilzén expression data set using Omics Explorer 3.2 from Qlucore (www.qlucore.se). Genes with the lowest variance were filtered out (variance cut-off =0.4), displaying a PCA plot based on 716 genes/variables and 30 samples/cases according to the method previously described 43 . Samples/cases were joined to their nearest neighbor using Euclidean distances, and they subdivided into four separate groups. The differential expression of PPM1D between molecular subgroups was calculated by fold change and change and significance by Student’s t-test (2-sided comparison, unequal variance). RESULTS Chromosome 17 gain is the most common genetic aberration in neuroblastoma To investigate the genetic aberrations in neuroblastoma, we performed array-based comparative genomic hybridization (CGH) on tumor samples from 417 Swedish neuroblastoma patients collected between 1986 and 2020. The most common genetic anomaly identified was gain of chromosome arm 17q, detected in 76.5% of the tumors, either as segmental gains of 17q (n = 192) or as whole chromosome 17 gains (n = 127) (Fig. 1 A). Segmental 17q gains were predominately observed in unfavorable neuroblastomas, whereas favorable neuroblastomas typically exhibited whole chromosome 17 gains, which includes TP53 on 17p13.1. Importantly, neuroblastoma patients with segmental 17q gain had significantly worse clinical outcomes compared to those without this aberration, with 5-year overall survival probability of 42.9% versus 88.2%, respectively ( P = 4.6x10 − 14 . Figure 1 B). Children with neuroblastoma lacking both segmental 17q-gain and other high-risk features such as MYCN amplification and/or 11q-deletion, showed favorable long-term survival (94.2%) 8–20 years from diagnosis, in most cases without requiring any treatment. In contrast, children with 17q-gain, despite receiving active multimodal therapy, had poorer survival (50.2%) and an increased risk of late sequelae ( Figure S1 ), further confirming 17q gain as a robust prognostic marker in neuroblastoma 4 – 6 . Given the close propinquity to the proximal breakpoints commonly associated with 17q gain and their presence in all tumors with segmental 17q gain, we identified three prominent gene candidates with tumorigenic capacity within this region: RAD51C , PPM1D and BRIP1 (Fig. 1 A). Among these, only PPM1D has previously been proposed as an oncogene in neuroblastoma, highlighting its potential role in driving tumorigenesis in cases with 17q gain 29 , 30 (http//: www.cancer.sanger.ac.uk/cosmic/mutation/ ). Copy number gain of chromosome 17 is an early genetic event in neuroblastoma and additional gene copies are acquired through clonal evolution To further delineate the chromosomal aberrations involved in neuroblastoma development and progression, we employed evolutionary trajectory analysis 7 . We analyzed SNP array data from 100 multiregional tumor samples collected from 23 neuroblastoma patients 7 , with a focus on chromosome 17. Most tumors (57%, 13/23) were found to harbor gain of chromosome 17q in over 90% of tumor cells in all samples, indicating that this aberration was present early in tumor evolution, persisting as a stable feature throughout disease progression. In these cases, no additional copy number changes were observed (Figs. 2 A, I and Table S1 ). However, 30% (7/23) of tumors showed a progressive accumulation of additional 17q copies as tumors evolved, either regionally or during the transition from primary tumor to metastasis or relapse. In four of these cases, gain of 17q occurred early in the phylogenetic stem, followed by further gains of additional copies of these genes as regional clones evolved (Fig. 2 A, II ). In three cases, clones with 17q gain emerged in subset of samples and expanded through selective sweeps, eventually encompassing all tumor cells in the samples (Fig. 2 A, III ). The remaining 13% of patients (3/23) showed regional variation in copy number, making conclusive evolutionary analysis difficult. Among the 20 patients with informative evolutionary data, the successive accumulation of 17q copies was observed in more than half of high-risk cases (7/13; Table S1 ). In contrast, additional changes in 17q copy number were not found in any of the low-risk patients, i.e. children under 18 months with only numerical changes in the stem (0/7; P = 0.0445; two-sided Fisher’s exact test). In additional neuroblastoma cases with multiple tumor samples available for analysis, we confirmed that 17q gain is an early event in neuroblastoma development (Fig. 2 B, S2 ), observed in both a child without known predisposition and in a child with a germline PHOX2B mutation 46 . Our recent single-cell RNA-sequencing data from neuroblastoma patient samples revealed the presence of pre-malignant Schwann cell precursor (SCP)-like cells in addition to the previously described adrenergic phenotype tumor cells 32 . This suggests that SCP-like cells might be the cell-of-origin in some neuroblastoma cases. To dissect the genetic landscape of neuroblastoma at the single-cell level, we used the Numbat algorithm 47 to identify copy number variations and DNTR-Seq (Direct Nuclear Tagmentation and RNA sequencing) 48 to investigate the subclonal structure of tumor cells at the single-cell level. SCP-like cells were identified using the receptor tyrosine kinase ERBB3 as a specific marker, whereas adrenergic cells were identified by CD24 32 . An example of striking intra-tumoral heterogeneity with early chromosome 17 gain was detected with DNTR-seq (single-cell whole-genome sequencing) which revealed five major tumor cell clones (Fig. 2 C). In one cluster of cells with a SCP-like signature (Clone A), gain of chromosome 17 was the only detected genomic aberrations. The remaining malignant clones (clone 1–4), which all had adrenergic transcriptional profiles, displayed multiple whole-chromosome gains which included chromosome 17. Clone 2 which showed further evolution, had acquired segmental chromosomal aberrations that included loss of chromosome 1p and gain of chromosome 2p. Clones 3 and 4 shared the same genomic aberration as clone 2, except that Clone 3 had acquired one additional copy of chromosome 17, while Clone 4 had retained a normal diploid chromosome 5 (Fig. 2 C). Importantly, the gain of chromosome 17 was the only genomic event, shared by all aberrant cell populations (Fig. 2 C). It is also worthy of note that this was gained independently in SCP-like and adrenergic cell populations. PPM1D is activated by gene fusions and, somatic- and germ-line mutations in neuroblastoma Whole exome sequencing (WES) or whole genome sequencing (WGS) of 73 neuroblastoma patient samples, revealed a somatic pathogenic truncating mutation in in exon 6 of PPM1D (c.1344_1345insT; p.L450fs) in a MYCN -amplified tumor from an infant girl (Figure S3A) . Her neuroblastoma rapidly progressed from localized INSS stage 1 to metastatic INSS stage 4, ultimately leading to a fatal clinical outcome despite aggressive treatment (Figs. 3 A and S2 A). Additionally, we identified a de novo germline mutation located in exon 6 of PPM1D l (c.1528C > T; p.Q510*), resulting in a premature truncation of the protein (Fig. 3 A). This mutation was found in a 26-month-old boy diagnosed with metastatic stage 4 (INSS/stage M, INRGSS) neuroblastoma originating in the left adrenal gland with bone metastases. The patient exhibited clinical features resembling previously described cases of children with intellectual disabilities and dysmorphic features associated with PPM1D germline mutations ( Figure S3B ). The tumor lacked MYCN amplification, and the patient had a poor clinical outcome after relapse and disease progression, despite multimodal clinical therapy. A detailed clinical description of this patient can be found in the Supplementary Methods section. Hence, the neuroblastoma-associated PPM1D mutations both resulted in C-terminal truncated variants of WIP1, similar to those gain-of-function variants identified in other cancers (Fig. 3 A) (http//: www.cancer.sanger.ac.uk/cosmic/mutation/ ). In addition, by FusionCatcher analysis of RNA-sequencing data from neuroblastoma tumors we identified a gene fusion between PPM1D and the breast carcinoma amplified sequence 3 (BCAS3) gene located on chromosome 17q23.2 in a tumor from a patient enrolled in the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) neuroblastoma dataset. This fusion was associated with high expression of PPM1D and a predicted WIP1 isoform with a truncated C-terminal (Fig. 3 B). These findings, together with the evidence that chromosome 17q gain is an early event in neuroblastoma tumorigenesis, suggest that genes within this chromosomal region, particularly PPM1D , play a key role in neuroblastoma progression. Given PPM1D ’s known functions in DNA repair, cell cycle regulation, and apoptosis, it is a strong candidate for driving further genomic instability and tumor evolution. Overexpression of PPM1D in neuroblastoma correlates with unfavorable clinical and biological features Our data demonstrating that the PPM1D gene is altered in copy number and/or structure in the majority of 17q gained high-risk neuroblastomas, incited us to investigate the clinical prognostic and biological features of PPM1D gene expression in neuroblastoma. Our results demonstrate that higher expression levels of PPM1D was associated with several neuroblastoma risk factors, including metastatic stage 4 disease, older age at diagnosis (> 18 months), MYCN -gene amplification and classification into the INRG high-risk (HR) group as defined by combined clinical and biological features associated with unfavorable clinical outcome 37 (Fig. 4 A). Notably, the highest PPM1D expression was observed in non- MYCN amplified HR-neuroblastoma tumors (nMN HR; Fig. 4 A), which predominantly correspond to cases with 11q-deletions, a group frequently harboring segmental 17q gains. Next, we correlated PPM1D expression with copy number (CN) alterations, using data from CGH arrays, WES and WGS. CN gains were separated into numerical gains that included whole chromosome 17 and segmental gains that included the sub-region containing PPM1D . Correlation analysis revealed a significant gene dosage effect, with PPM1D expression increasing proportionally with the number of 17q copies (Figs. 4 A, B). Moreover, high PPM1D expression was strongly linked to poor clinical outcome, with significantly lower overall survival (OS; 59% vs. 83% at five years, p < 0.001) and event-free survival (EFS; 47% vs. 68%, p < 0.001) in patients with PPM1D high-expressing tumors (Fig. 4 C). We also compared gene expression patterns across different neuroblastoma subtypes 49 . Using principal component analysis (PCA) on gene expression profiles from 30 primary neuroblastoma samples from two published microarray studies 44 , 45 we observed that high PPM1D expression was associated with the more aggressive subtypes Type 2A (17q gain with 14q- and/or 11q-deletions) and Type 2B (1p-deletion and MYCN -amplification) neuroblastomas. In contrast, low PPM1D expression was characteristic of low-risk, near-triploid tumors with high TrkA expression (Type 1) ( Figure S4A ). Additionally, immunohistochemical analysis confirmed consistent WIP1 expression in all 17q-gained neuroblastoma samples ( Figure S4B ). These findings underscore the role of PPM1D in neuroblastoma aggressiveness and suggest that high WIP1-expressing tumors correlate with worse patient outcomes, further reinforcing the significance of 17q gain as a prognostic marker (Figs. 1 A and S1 ) and highlighting the strong association between PPM1D expression and 17q gain (Figs. 1 B, 2 A, 4 A, B and S4 A). DISCUSSION Neuroblastoma, a childhood tumor originating from the developing peripheral nervous system, remains challenging to treat despite intensified multimodal therapy, and survival rates are still comparatively poor compared to pediatric cancers in general. Advancing our molecular understanding of neuroblastoma is therefore crucial to improve the outcome for these children/patients. We therefore investigated common genetic aberrations in neuroblastoma, identifying chromosomal gain of 17q as an early genetic event in tumorigenesis. Segmental gain of chromosome17q is the most common chromosomal aberration and predictor of poor prognosis in neuroblastoma 5 , 50 . Also, gain of 17q is frequently found in tumors of epithelial, neural and hematopoietic origin, suggesting that 17q harbors key genes involved in oncogenesis 51 – 56 . Our findings indicate that chromosome 17 gain may occur independently in pre-malignant Schwann cell precursor-like cell populations and in adrenergic tumor clones, and that subsequent evolving malignant cell clones may acquire additional copies of chromosome 17. Evolutionary trajectory analysis revealed that 17q gain typically appears at the phylogenetic stem of neuroblastoma tumors, with subsequent gains accumulating as the tumor progresses, supporting its role as an initial driver of malignancy. Together, these observations align with a recent study using molecular clock analysis and population genetic modelling to time the genetic event of neuroblastoma evolution, suggesting that neuroblastoma pathogenesis may be initiated in the first trimester of pregnancy, when rapidly proliferating neuroblasts are susceptible to CIN and aneuploidy 11 . The frequent gain of 17q, as opposed to losses on the 17p arm where TP53 resides, may further favor the accumulation of genomic aberrations and mutations, driving tumorigenesis 57 . Several cancer-associated genes have been identified on chromosome 17q including PPM1D, EME1, BRCA1, ERBB2, NF1, RAD51C, BRIP1, IGF2BP1, NME1 and BIRC5 . Our analysis of 417 Swedish neuroblastoma samples shows, that only RAD51C, PPM1D and BRIP1 are included in the shortest region of overlap of 17q gains and are similar to observations made in breast cancer 25 , 58 , 59 . Notably, PPM1D copy number gains were more frequent in high-risk, metastatic, and relapsed neuroblastomas than low-risk tumors, indicating its potential role in clonal expansion and disease progression. In our patient cohort, we identified both a somatic PPM1D mutation (p.L450fs) and one de novo germline mutation (p.Gln510*) in exon 6, both resulting in truncated, gain-of-function WIP1 proteins that exhibit enhanced stability and lead to increased suppression of the p53 pathway 25 , 58 – 61 , previously reported in numerous other malignancies 62 . PPM1D de novo germline mutations, predominantly in the 5th and 6th exons, have previously been reported in children with the neurodevelopmental disorder Jansen-de Vries syndrome and have not yet been described in the context of cancer. However, an identical c.1528C > T (p.Gln510*) somatic mutation has been reported previously in a malignant melanoma 63 . We also detected a gene fusion of PPM1D and BCAS3 expressed at high levels in a neuroblastoma patient. The fusion is predicted to generate a C-terminal truncation and cause augmented WIP1 protein levels. Similarly, a fusion between PPM1D and C1QTNF1 in an Ewing sarcoma patient has been reported to drive high WIP1 expression ( https://pecan.stjude.cloud ). Additional PPM1D fusions, such as an intragenic region of RPSK6B1 and a PPM1D-ZNS655 fusion, have been described in patients with diffuse cerebellar glioma and acute myeloid leukemia (AML), although without further investigation on the effect on WIP1 protein stability and abundance 64 ( https://pecan.stjude.cloud ). Given that PPM1D gene amplifications and C-terminal mutations are frequently detected in a wide spectrum of cancers 62 suggests that PPM1D is a pivotal gene on 17q contributing to neuroblastoma pathogenesis by promoting CIN and allowing tumor cells to evade p53-mediated apoptosis. In primary neuroblastoma, mutations of genes in the p53 pathway are rare, occurring in only 1–2% of primary cases 40 , but they become more common in relapsed disease (15%) 40 , where p53 inactivation appears to be linked to therapy resistance 65 , 66 . Mechanisms that inactivate p53 in neuroblastoma include MDM2 gene amplification or overexpression mediated by MYCN amplification, hypermethylation or deletion of CDKN2A , miR-380-5p-mediated repression of p53, or its inactivation by the methyltransferase SETD8 14,67–70 . Under normal conditions, WIP1 attenuates p53 activation by inhibiting p53-mediated transactivation of promoters by dephosphorylating p53 at Ser15 71 . Additionally, WIP1 indirectly inhibits p53 activity by dephosphorylating Mdm2 and upstream p53 activating kinases 15 – 19 . Amplification and gain-of-function mutations of PPM1D are proposed to only partially inhibit p53 activity, given that p53 undergoes multiple post-translational modifications such as phosphorylation, acetylation and ubiquitination that mediate p53-dependent beyond WIP1’s regulatory influence 72 . Our recent mouse studies support this, showing that transgenic mice overexpressing PPM1D generate tumors phenotypically similar to Trp53 loss-of-function derived tumors 29 rather than the spectra of tumors observed in Trp53 knock-out mice. The effects of this partial inhibition of p53 activity together with the WIP1-mediated delay in DNA repair processes 62 suggest that PPM1D activating mutations and/or overexpression disrupt the fidelity of DNA repair and DNA replication, allowing for accumulation of mutations and chromosomal aberrations that eventually result in malignant transformation. Our study demonstrates that gain of chromosome 17 is an early event in neuroblastoma and that additional copies of 17q, harboring PPM1D locus, are associated with high-risk, metastatic and relapsed disease. High WIP1 expression in neuroblastoma appears to allow for the accumulation of cells with SNV and CIN in neural crest cells during development, ultimately resulting in the emergence of neuroblastoma with increased tumor aggressiveness and resistance to treatment. Therefore, inhibiting WIP1 could be a valuable addition to neuroblastoma treatment, with potential benefits extending to other pediatric and adult cancers exhibiting WIP1 dysregulation. In summary, our data position 17q gain as an early and possibly the initial event in neuroblastoma development, with PPM1D emerging as a strong candidate oncogene driving tumor progression through p53 pathway modulation and CIN promotion. Targeting WIP1 in neuroblastoma in combinations with current treatment modalities may address the challenge of genetic instability and therapy resistance, representing an avenue for improving outcomes in high-risk and relapsed pediatric neuroblastoma as well as other PPM1D -driven cancers. Declarations Ackowledgements Clinical Genomics, SciLifeLab, Gothenburg, Sweden and the Bioinformatics Core Facility platforms at the Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden for assistance with the bioinformatical analysis of sequencing data. We thank Catarina Träger, Inger Bodin and Susanne Ahlberg for their help and contribution to this work. Funding This work was supported with grants from the Swedish Childhood Cancer Foundation, the Swedish Research Council, the Swedish Cancer Foundation, the Swedish Foundation for Strategic Research (www.nnbcr.se), Karolinska Institutet, Märta and Gunnar V Philipson Foundation, and The Cancer Research Foundations of Radiumhemmet. The study sponsors had no role in the design of the study; the collection, analysis, and interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication. Author contributions Conceptualization, J.M., J.I.J., and P.K.; Investigation and validation, J.M., S.F., B.S., J.Y.H., A.M., G.T.; Computational investigation and analysis, J.M., S.F., J.S., J.O., B.S., T.K.O., F.H., C.B., S.R., A.D., F.A., N.J., Y.S., J.H., D.G., M.J., T.M., J.I.J and P.K.; Writing – Original draft, J.M, J.I.J. and P.K.; Writing – Review & Editing, J.M., S.F., J.J.M., M.F., N.B., J.I.J. and P.K. Resources, M.F., J.J.M., G.T., M.J., D.G., T.M. J.I.J and P.K.; Final editing and manuscript approval, All authors; Funding Acquisition & Supervision, J.I.J. and P.K. Declaration of Interests. The authors have no conflicts of interest to declare. 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23:08:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5961130/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5961130/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":75708204,"identity":"c974140a-1d6b-40ca-bafb-338c821de4eb","added_by":"auto","created_at":"2025-02-07 10:39:05","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1605166,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSegmental chromosome 17q gain in neuroblastoma. A. \u003c/strong\u003eChromosome 17 gain with indicated break-points. Summary of segmental gains of chromosome 17 in the extended Swedish neuroblastoma cohort (n=417). 192 samples displayed segmental gain of chromosome 17 as indicated by red horizontal bars with regions displaying additional level of gain (more copies) indicated by dark red color. An additional 127 cases showed whole chromosome gain of chromosome 17 (not shown in the figure). \u003cstrong\u003eB. \u003c/strong\u003eGain of 17q correlates with poor survival in neuroblastoma. Neuroblastoma survival probability according to Kaplan-Meier analysis in a Swedish population-based patient material in relation to chromosome 17 status in the tumor tissue shows worse overall survival (OS) for children with 17q segmental gain (n=124; green line, 48.0% 5 year OS) vs children with no segmental 17q gain (n=141; blue line, 82.8% 5 year OS).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5961130/v1/10a2a3fe34e05f7a933d122d.png"},{"id":75708189,"identity":"77f08500-d649-426d-80eb-a27eacee9ce6","added_by":"auto","created_at":"2025-02-07 10:39:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1607703,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromosome 17 gain is an early event in neuroblastoma development. A\u003c/strong\u003e. Evolutionary trajectories of chromosomal aberrations in neuroblastoma. Multiregional tumor sampling discloses chromosome 17 copy number accumulation in high-risk neuroblastoma. \u003cstrong\u003e(I) \u003c/strong\u003eGenome array analysis of pre-treatment biopsies (B1-B3), post-chemotherapy resection specimens (P1-P2) and concurrent bone marrow metastasis (M) from a female (F) 6-month-old (mo.) patient with a low-risk neuroblastoma (NB), having a whole genome profile with only numerical (NUM) changes (I, left). Evolutionary reconstruction (I, center) shows 15 aberrations in the phylogenetic stem, i.e. present in \u0026gt;90% of tumor cells in all samples, followed by several additional whole chromosome changes at subclonal levels (arrows with numbers in blue type), distributed over a subset of samples. The stem aberrations include four copies (CN=4) of chromosome 17, as shown by a higher log2 ratio (I, right) than for the trisomic chromosomes (chr. 16 and 18). No further chromosome 17 aberrations were detected. \u003cstrong\u003e(II)\u003c/strong\u003e Multiregional array analysis of a \u003cem\u003eMYCN \u003c/em\u003eamplified (MYCNA) high-risk NB in a 2-year-old (y.) with a metastatic relapse in the bone marrow indicates a stem dominated by structural (STR) rearrangements including segmental gain (CN=3) of chromosome 17q. Additional 17q copies (CN=4) are gained in samples B, M1, M2 and P4 either clonally (\u0026gt;90% of tumor cells, red line) or subclonally (red arrow). \u003cstrong\u003e(III)\u003c/strong\u003e Another high-risk \u003cem\u003eMYCN\u003c/em\u003e amplified neuroblastoma demonstrates parallel evolution of chromosome 17q gain in different tumor regions, as evidenced by different genomic breakpoints (III, right) in different sample sets, including two temporally distinct relapses (R1 and R2). \u003cstrong\u003eB. \u003c/strong\u003eGain of chromosome 17q is an early genetic aberration in a high-risk metastatic neuroblastoma. Whole genome sequencing of different metastatic sites at diagnosis and relapse revealed an unbalanced translocation t(11;17) as the only common aberration whereas multiple\u0026nbsp; but different genetic aberrations unique to respective sample were present in the two metastatic clones sequenced. \u003cstrong\u003eC. \u003c/strong\u003eMultiple cellular subclones harboring different CNV´s are detected in neuroblastoma samples. Reconstructing of tumor phylogeny based on CNV profiles obtained by DNTR-sequencing of a tumor sample from a 21-month-old patient with low-risk disease\u003csup\u003e32\u003c/sup\u003e. Upper row: \u003cem\u003eClone A;\u003c/em\u003e cells with one additional copy of Chr 17. Bottom row: \u003cem\u003eClone 1;\u003c/em\u003e cells with whole chromosome gains of chromosomes 1, 5, 7, 13, 17.\u0026nbsp;\u0026nbsp; \u003cem\u003eClone 2;\u003c/em\u003e Consists of cells with similar CNV´s as Clone C but with segmental loss of chromosome 1p and additional gain of chromosome 2p. This clone reflects the dominating malignant clone in this tumor as reflected by the bulk DNA CGH analysis of the sample. \u003cem\u003eClone 3\u003c/em\u003e cells are identical to clone 2 but have gained an extra copy of chromosome 17, \u003cem\u003eClone 4;\u003c/em\u003e Cells with similar CNV´s as Clone 2, but have lost the extra copy of chromosome 5. Haplotype analysis based on common germline SNPs revealed that Chr17 haplotypes of Clone A and Clones 1-4 are distinct and these clones are not directly related.\u0026nbsp;\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5961130/v1/7cac93f051d32416f6d4badc.png"},{"id":75708190,"identity":"edb7ebfc-a3eb-4521-bdd5-caa4bb72b613","added_by":"auto","created_at":"2025-02-07 10:39:05","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":310366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePPM1D \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egene located on chromosome 17q is altered in neuroblastoma. A. \u003c/strong\u003eSchematic representation of coding and protein sequence of \u003cem\u003ePPM1D\u003c/em\u003e/WIP1 with mutations shown according location in protein and predicted amino acid sequences resulting from the\u003cem\u003e PPM1D\u003c/em\u003evariants detected in neuroblastoma. Amino acids translated from exon 6 depicted in red with mutated amino acids depicted in blue. \u003cstrong\u003eB.\u003c/strong\u003e A neuroblastoma tumor bearing transcript of the \u003cem\u003ePPM1D\u003c/em\u003e–\u003cem\u003eBCAS3 \u003c/em\u003efusion expected to cause loss of the c-terminal terminal region show high \u003cem\u003ePPM1D\u003c/em\u003e expression levels (sample in red)\u003cem\u003e, \u003c/em\u003eshown by FPKM (Fragments Per Kilobase per Million mapped reads).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5961130/v1/2432382ca03ca174b37e6eb4.png"},{"id":75708210,"identity":"984e51b7-57e1-4926-8edc-d922834e5502","added_by":"auto","created_at":"2025-02-07 10:39:06","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":554164,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePPM1D\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e expression correlates with unfavorable prognosis markers, decreased\u003c/strong\u003e \u003cstrong\u003esurvival of neuroblastoma patients, and is gene-dosage dependent\u003c/strong\u003e. \u003cstrong\u003eA.\u003c/strong\u003e Expression of \u003cem\u003ePPM1D\u003c/em\u003e is associated with unfavorable prognostic markers. \u003cem\u003ePPM1D \u003c/em\u003eexpression is shown in box-and whisker plots for neuroblastoma tumor stages according to the INSS classification, age at diagnosis, amplification status of \u003cem\u003eMYCN\u003c/em\u003e, INRG risk group classification (LR=low risk, IMR= intermediate risk, NMN HR= non-\u003cem\u003eMYCN\u003c/em\u003e amplified high risk, MNA=\u003cem\u003eMYCN\u003c/em\u003e amplified), and chromosome 17 copy number (CN) gains. \u003cstrong\u003eB.\u003c/strong\u003e Correlation analysis showing a gene-dosage dependent expression pattern of \u003cem\u003ePPM1D\u003c/em\u003e. Quantitative copy number information for \u003cem\u003ePPM1D\u003c/em\u003e based on CGH, whole exome (WES) and whole genome sequencing (WGS) data (x-axis) is shown against normalized log2 expression values from paired RNA-Seq data (y-axis). CN gains are highlighted and separated into numerical gains copying the whole chromosome 17 (blue) and segmental gains affecting only a sub-region involving \u003cem\u003ePPM1D\u003c/em\u003e (red, min/max/mean size 2.4/70.8/35.8 Mb). Correlation and P-values were obtained by Pearson's correlation coefficient.\u003cstrong\u003e C.\u003c/strong\u003e High expression of \u003cem\u003ePPM1D\u003c/em\u003e is associated with adverse patient outcome. Kaplan–Meier survival estimates are shown for overall survival (OS, left) and event-free survival (EFS, right) in the whole cohort (n=498). P values were obtained by log-rank test. The cohort was dichotomized according to the optimal cut-off expression for \u003cem\u003ePPM1D\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5961130/v1/fe2b018f88ce723e1901e238.png"},{"id":76287013,"identity":"dbbb4fc6-7a34-4830-adec-5584af95928e","added_by":"auto","created_at":"2025-02-14 11:24:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5677014,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5961130/v1/613c0156-3975-4bcf-8d33-31401ca41e58.pdf"},{"id":75708176,"identity":"39cb389c-d3ee-401d-a0a8-395c62325e13","added_by":"auto","created_at":"2025-02-07 10:39:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4351175,"visible":true,"origin":"","legend":"","description":"","filename":"SUPPLEMENTALFIGUREandTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-5961130/v1/2b097d9b64c3532ad2a62e5c.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Gain of chromosome 17 is an early genetic abnormality in neuroblastoma with PPM1D emerging as a strong candidate oncogene driving tumor progression","fulltext":[{"header":"KEY POINTS","content":"\u003cul\u003e\n \u003cli\u003eGain of chromosome 17 is an initial genetic event in neuroblastoma development\u003c/li\u003e\n \u003cli\u003eNumbers of segmental chromosome 17q copies is increased during clonal evolution in high-risk disease\u0026nbsp;\u003c/li\u003e\n \u003cli\u003ePPM1D gene fusions and gain-of-function somatic and germline mutations are present in high-risk neuroblastoma\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"IMPORTANCE OF THE STUDY","content":"\u003cp\u003eChromosomal instability resulting in structural and numerical chromosomal aberrations are frequently observed in cancers. Neuroblastoma, a childhood tumor of the sympathetic neural system, is characterized by a heterogenic clinical behavior which in high-risk patients is manifested by segmental chromosomal aberrations. The most frequent chromosomal abnormality and the strongest indicator of adverse outcome. is segmental gain of chromosome 17q. We used multi-omic approaches to investigate the genetic development of neuroblastoma and show that gain of chromosome 17 is an initial genetic event during neuroblastoma development with additional copies of chromosome 17q acquired through clonal evolution in aggressive and metastatic disease. Furthermore, gene fusions, gain-of-function somatic and germ-line mutations and elevated expression of PPM1D, a regulator of p53 activity, located on chromosome 17q22.3 were detected, suggesting a pivotal role of PPM1D in neuroblastoma disease progression.\u003c/p\u003e\n"},{"header":"INTRODUCTION","content":"\u003cp\u003eChromosomal instability (CIN), caused by errors in DNA replication and chromosomal segregation during mitosis, is a hallmark of most human cancers\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. CIN frequently results in structural and numerical chromosomal abnormalities, leading to intra- and inter-tumor heterogeneity, which disrupts key cancer pathways that give rise to phenotypic variations, posing challenges for therapeutic interventions\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Neuroblastoma, a childhood tumor of the sympathetic neural system, typically exhibits, like most childhood tumors, a low burden of somatic mutations\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Instead, copy number alterations play a dominant role, where focal amplification of the \u003cem\u003eMYCN\u003c/em\u003e oncogene, re-arrangements within the \u003cem\u003eTERT\u003c/em\u003e locus on chromosome 5, segmental gains of chromosome 17q, and loss of chromosomes 1p36 and 11q all associated with high-risk disease and poor survival\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. The most common cytogenetic abnormality in neuroblastoma is segmental or whole chromosomal gain of chromosome 17, detected in over 80% of tumors\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Segmental gain of 17q21 extending to 17qter is observed in more than 60% of tumors and is the most frequent genetic aberration linked to high-risk disease and poor prognosis of neuroblastoma patients\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent studies have added to the genomic complexity by revealing significant spatial genetic heterogeneity within neuroblastomas\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Furthermore, these tumors display distinct cell-type states, which may coexist within the same tumor\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. While no genetic differences between these cell-type states have been reported, they are distinguished by variations in their transcriptional and epigenetic profiles\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Characterization of the genetic events during neuroblastoma evolution has shown that chromosomal gains crucial to disease pathogenesis typically occur early, within the first trimester of pregnancy\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This suggests that the intra-tumor heterogeneity may arise from the stepwise accumulation of genetic aberrations during embryonic development, with cells at different developmental maturation stages contributing to the heterogeneity observed in patient samples.\u003c/p\u003e \u003cp\u003eInactivating mutations of the tumor suppressor gene \u003cem\u003eTP53\u003c/em\u003e is a hallmark of adult cancers and lead to impaired DNA repair, cell cycle checkpoint arrest and apoptosis, ultimately resulting in accumulation of DNA mutations and CIN\u0026acute;s\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Pediatric cancers, on the other hand, including neuroblastoma, rarely exhibit \u003cem\u003eTP53\u003c/em\u003e mutations\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. Nevertheless, p53 activity is frequently compromised in neuroblastoma, particularly in relapsed tumors, where the incidence of \u003cem\u003eTP53\u003c/em\u003e mutations increases\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. This suggests that inactivation of p53 remains critical for tumorigenesis, with alternative mechanisms likely contributing to its attenuation during early neuroblastoma development.\u003c/p\u003e \u003cp\u003eOne such mechanism may involve \u003cem\u003ePPM1D\u003c/em\u003e, a gene located on chr17q22.3, which is frequently gained in high-risk neuroblastomas\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. \u003cem\u003ePPM1D\u003c/em\u003e encodes the nuclear serine/threonine phosphatase WIP1 (wild-type p53 induced phosphatase 1), a key regulator of DNA damage response and cell cycle progression, controlling the activity of p53, ATM, CHK1/2 and other key molecules involved in DNA repair, cell cycle progression and apoptosis\u003csup\u003e\u003cem\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. In concordance, \u003cem\u003ePPM1D\u003c/em\u003e mutations, amplifications and WIP1 overexpression have been identified in various cancers\u003csup\u003e\u003cem\u003e\u003cspan additionalcitationids=\"CR21 CR22 CR23 CR24 CR25 CR26 CR27\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e, and we recently demonstrated that overexpression of WIP1 in mice can drive tumor development, including neuroblastoma, by repressing p53 activity, promoting apoptotic escape, inducing genomic instability, and impairing growth arrest\u003csup\u003e\u003cem\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/em\u003e\u003c/sup\u003e. This suggests that \u003cem\u003ePPM1D\u003c/em\u003e might acts as an oncogene, facilitating malignant tumor development.\u003c/p\u003e \u003cp\u003eIn this study, we use multi-omics approaches to investigate the genetic development of neuroblastoma, focusing on the most common aberration, gain of chromosome 17q, and the oncogenic role of \u003cem\u003ePPM1D\u003c/em\u003e and the p53 regulating phosphatase WIP1. We demonstrate that gain of chromosome 17 is an early event in CIN during neuroblastoma development, with additional copies acquired through clonal evolution in aggressive and metastatic disease. Moreover, \u003cem\u003ePPM1D\u003c/em\u003e gene fusions resulting in high expression levels of \u003cem\u003ePPM1D\u003c/em\u003e as well as activating germline and somatic mutations were detected, suggesting a pivotal role of \u003cem\u003ePPM1D\u003c/em\u003e in neuroblastoma disease progression.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePatient-derived tumor material and clinical data\u003c/h2\u003e \u003cp\u003eThe collection of neuroblastoma tissue samples from Swedish patients was performed after either written or verbal consent obtained from parents/guardians according to ethical permits approved by the local Ethics Committee (Karolinska Institutet and Karolinska University Hospital, registration number 03-736, 2009/1369-31/1 and 2022-07254-1. Fresh tumor tissue was collected at surgery and snap-frozen and stored at -80\u003csup\u003eo\u003c/sup\u003eC until analysis. All patients were diagnosed, managed and treated according to national and international guidelines and protocols.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eNeuroblastoma tissue genomics and transcriptomics\u003c/h3\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePatient material for genetic analysis\u003c/h2\u003e \u003cp\u003eDNA was extracted from frozen tumors or blood using DNeasy blood and tissue kit (Qiagen, Hilden, Germany) according to manufacturer\u0026rsquo;s protocol and evaluated through absorbance measurements, fluorometric quantitation and DNA integrity assessment on Agilent Tapestation (Agilent, Santa Clara, CA).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSNP-microarray analysis\u003c/h3\u003e\n\u003cp\u003eMicroarray analysis was performed on a consecutive series of samples from an unselected cohort of 417 neuroblastoma patients using Affymetrix CytoScan HD SNP microarrays (Thermo Fisher Scientific, Waltham, MA, USA). These analyses include the majority of cases from Sweden. All tumors were staged according to the International Neuroblastoma Staging System (INSS) and INRG criteria. Handling of the microarrays has been described previously\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrimary data analysis was performed using GeneChip Command Console Software version 5.0.0 (Thermo Fisher Scientific) prior copy number analysis using Chromosome Analysis Suite (ChAS v.3.3.0; Thermo Fisher Scientific). All cases of chromosomal gain, loss, or amplification or allelic imbalances were scored, both segmental and numerical aberrations, including detailed information about the breakpoint positions when applicable. For practical reasons, all notations of gain and loss were considered in relation to a nominal tumor karyotype and a chromosomal break in a tumor was defined as a clear change in gene dose level in the genomic profile. All genomic position annotations are stated based on the hg19 build (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genome.ucsc.edu/\u003c/span\u003e\u003cspan address=\"http://genome.ucsc.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) of the human genome.\u003c/p\u003e\n\u003ch3\u003eAnalysis of 17q clonal evolution\u003c/h3\u003e\n\u003cp\u003eSingle nucleotide polymorphism (SNP) arrays including the Oncoscan (formalin fixed paraffins embedded samples) and Cytoscan HD (fresh frozen samples) platforms (Affymetrix Inc.) were retrieved from and subjected to comparisons of 17q copy number. Evolutionary trajectories were formalized as phylogenetic ideograms along the lines described in the original publication\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. In all, 100 multiregional samples collected from 23 patients with neuroblastoma were analyzed. Each patient was assigned to one of three simplified clinical-genetic risk groups, including children\u0026thinsp;\u0026lt;\u0026thinsp;18 months of age with only numerical aberrations found in their tumor\u0026rsquo;s phyologenetic stem by SNP array (low-risk patients), tumors with \u003cem\u003eMYCN\u003c/em\u003e amplification in the stem (high-risk patients), and tumors with structural rearrangements in the stem (high-risk patients). DNTR sequencing, processing of RNA and DNA and data analysis were done as described previously\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eGenome sequencing\u003c/h2\u003e \u003cp\u003eIn total, tumor material from 73 Swedish neuroblastoma patients was subjected to either whole genome sequencing (WGS) or exome sequencing with procedures for handling as described previously\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Exome sequencing was performed through paired-end sequencing on Illumina platforms (Illumina, San Diego, CA) after enrichment with Agilent SureSelect All Exome enrichment kit (Agilent technologies, Santa Clara, CA) on DNA for 18 tumor/normal pairs and additional 20 tumors lacking corresponding constitutional DNA. Alignment against human reference genome hg19 was performed using BWA with GATK local realignment followed by SNV calling using SNPeff. For tumors sequenced without constitutional DNA, filtering was performed by retention of only non-synonymous SNVs and removal of common variants, e.g. showing an allele frequency above 0.01 in the Genome Aggregation Database (gnomAD v2.1.1).\u003c/p\u003e \u003cp\u003eWGS was performed on tumor DNA and corresponding constitutional DNA (n\u0026thinsp;=\u0026thinsp;50) also using paired-end sequencing on Illumina instrumentation (Illumina) at Clinical Genomics, SciLife Laboratories, Gothenburg/Stockholm, Sweden, while mapping to hg19 and variant calling were done using the Sentieon suite of bioinformatics tools (Sentieon Inc., Mountain View, CA, USA) (Sentieon version v.201808.03). Only high-quality called SNVs with a minimum of 10% variant allele frequency and a total read coverage of ten were considered. All synonymous variants or variants in non-coding regions (except those affecting canonical splice sites) were discarded. Remaining SNVs were kept for further analysis and evaluation including manual assessment through the Integrative Genomics Viewer (IGV)\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e for removal of calls due to mapping artifacts and paralogs.\u003c/p\u003e \u003cp\u003eFusionCatcher was applied to discover \u003cem\u003ePPM1D\u003c/em\u003e-containing fusion transcripts in the National Cancer Institute TARGET dataset comprising paired-end RNA sequenced neuroblastoma patient samples (dbGap Study Accession:phs000218.v16.p6).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eImmunohistochemistry\u003c/h3\u003e\n\u003cp\u003eTwenty-seven neuroblastoma samples derived from children of different ages and all clinical stages, including different biological subsets, were analyzed. Formalin-fixed and paraffin-embedded human tissue sections were deparaffinized in xylene and graded alcohols, hydrated and washed in phosphate-buffered saline (PBS). After antigen retrieval in sodium citrate buffer (pH 6) in a microwave oven, the endogenous peroxidase was blocked by 0.3% H2O2 for 15 min. Sections from human neuroblastoma were incubated overnight at 4\u0026deg;C with a primary antibody against PPM1D (ab31270, Abcam). As a secondary antibody, the anti-rabbit-horseradish peroxidase (HRP) SignalStain Boost IHC detection kit was used (Cell Signal Technology; #8114). A matched isotype control was used as a control for nonspecific background staining (not shown).\u003c/p\u003e\u003cp\u003e\u003cem\u003eCase description, germline PPM1D mutation detection (see supplementary methods)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cem\u003eGene expression analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePatient cohort and methodology of RNA-seq data analysis pipeline are extensively described in\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e36\u003c/span\u003e\u003c/sup\u003e. We used gene expression data of 498 neuroblastoma patients from seven countries. According to the Neuroblastoma Risk Group (INRG) classification system\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e37\u003c/span\u003e\u003c/sup\u003e we classified patients with \u003cem\u003eMYCN\u003c/em\u003e amplifications and patients with a metastatic disease and older than 18 months at diagnosis as high-risk patients (n=176). Events were defined according to a revised version of the International Neuroblastoma Response Criteria\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRNA-seq gene expression analyses have been done using the MAGIC-AceView pipeline as previously described\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Copy number data for \u003cem\u003ePPM1D\u003c/em\u003e was derived from array comparative hybridization (aCGH);\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e39\u003c/span\u003e\u003c/sup\u003e, whole exome sequencing and whole genome sequencing analyses\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eStatistical analyses\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGene expression of \u003cem\u003ePPM1D\u003c/em\u003e was investigated displayed on the gene level (\u003cem\u003ePPM1D\u003c/em\u003e) or transcript level for two selected transcript variants (AceView annotation, as a result of the analysis, i.e. \u003cem\u003ePPM1D.aAug10\u003c/em\u003e and \u003cem\u003ePPM1D.bAug10\u003c/em\u003e, corresponding to transcripts ENST00000305921 and ENST00000392995 respectively).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePPM1D\u003c/em\u003e expression in defined NB subgroups (according to INRG high-risk status, INSS stage, amplification status of MYCN, age, CN status of \u003cem\u003ePPM1D\u003c/em\u003e on chromosome 17) was displayed in Tukey box-and-whisker plots (box range: inter-quartile range, whiskers: 1.5x IQR). Where appropriate, either Mann\u0026ndash;Whitney\u0026ndash;Wilcoxon or Kruskal-Wallis tests were performed to investigate differences in expression of two or more analyzed groups, respectively. Pearson correlation analyses have been applied to investigate correlation between \u003cem\u003ePPM1D\u003c/em\u003e expression and copy number.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;To investigate the prognostic value of \u003cem\u003ePPM1D\u003c/em\u003e expression, optimal cut-point expression values separating the cohort into \u003cem\u003ePPM1D\u003c/em\u003e-high and \u003cem\u003ePPM1D\u003c/em\u003e-low groups that correlate best with outcome in terms of event-free survival (EFS; recurrence, progression and death from disease were considered as events), and overall-survival (OS) were calculated by means of maximally selected rank statistics (maxstat package in R v3.4.3)\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e41,42\u003c/span\u003e\u003c/sup\u003e in training sets. Differences in survival of \u003cem\u003ePPM1D\u003c/em\u003e high and low-expressing subgroups according to dichotomization with the established cutpoints was then tested by Mantel-Cox (log-rank) tests in corresponding validation sets. For this procedure, equally sized training and validation subsets (n=296) were randomly sampled from the entire cohort (n=498). Resampling was done 1000 times and the mode of those established cutpoint values which passed p \u0026lt; 0.05 in maxstat\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e41,42\u003c/span\u003e\u003c/sup\u003e as well as in the log-rank test was eventually selected to define \u003cem\u003ePPM1D\u003c/em\u003e-high and \u003cem\u003ePPM1D\u003c/em\u003e-low expressing subgroups (this procedure was independently done for EFS and OS). Subsequently, Kaplan-Meier survival estimates for the entire cohort were calculated and displayed from the time of diagnosis and clinical outcome of respective subgroups was compared using the log-rank test.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003ePrincipal Components Analysis (PCA) on expression data\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRaw data expression: CEL-files (HU133A chips from Affymetrix) from 30 primary neuroblastoma cases from two published microarray studies\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e43\u0026ndash;45\u003c/span\u003e\u003c/sup\u003e were pre-processed using gcRMA normalization in Bioconducter for R 2.9.2 (library BioC 2.4). For each gene, the mean expression level from probe-sets was calculated, resulting in 8105 genes/variables\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Principal Components Analysis (PCA) was performed on the pre-processed McArdle/Wilz\u0026eacute;n expression data set using Omics Explorer 3.2 from Qlucore (www.qlucore.se). Genes with the lowest variance were filtered out (variance cut-off =0.4), displaying a PCA plot based on 716 genes/variables and 30 samples/cases according to the method previously described\u003csup\u003e\u003cspan lang=\"EN-US\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Samples/cases were joined to their nearest neighbor using Euclidean distances, and they subdivided into four separate groups. The differential expression of \u003cem\u003ePPM1D\u003c/em\u003e between molecular subgroups was calculated by fold change and change and significance by Student\u0026rsquo;s t-test (2-sided comparison, unequal variance).\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003eChromosome 17 gain is the most common genetic aberration in neuroblastoma\u003c/h2\u003e\n \u003cp\u003eTo investigate the genetic aberrations in neuroblastoma, we performed array-based comparative genomic hybridization (CGH) on tumor samples from 417 Swedish neuroblastoma patients collected between 1986 and 2020. The most common genetic anomaly identified was gain of chromosome arm 17q, detected in 76.5% of the tumors, either as segmental gains of 17q (n\u0026thinsp;=\u0026thinsp;192) or as whole chromosome 17 gains (n\u0026thinsp;=\u0026thinsp;127) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Segmental 17q gains were predominately observed in unfavorable neuroblastomas, whereas favorable neuroblastomas typically exhibited whole chromosome 17 gains, which includes \u003cem\u003eTP53\u003c/em\u003e on 17p13.1. Importantly, neuroblastoma patients with segmental 17q gain had significantly worse clinical outcomes compared to those without this aberration, with 5-year overall survival probability of 42.9% versus 88.2%, respectively (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.6x10\u003csup\u003e\u0026minus;\u0026thinsp;14\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e\n \u003cp\u003eChildren with neuroblastoma lacking both segmental 17q-gain and other high-risk features such as \u003cem\u003eMYCN\u003c/em\u003e amplification and/or 11q-deletion, showed favorable long-term survival (94.2%) 8\u0026ndash;20 years from diagnosis, in most cases without requiring any treatment. In contrast, children with 17q-gain, despite receiving active multimodal therapy, had poorer survival (50.2%) and an increased risk of late sequelae (\u003cstrong\u003eFigure S1\u003c/strong\u003e), further confirming 17q gain as a robust prognostic marker in neuroblastoma\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e\n \u003cp\u003eGiven the close propinquity to the proximal breakpoints commonly associated with 17q gain and their presence in all tumors with segmental 17q gain, we identified three prominent gene candidates with tumorigenic capacity within this region: \u003cem\u003eRAD51C\u003c/em\u003e, \u003cem\u003ePPM1D\u003c/em\u003e and \u003cem\u003eBRIP1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Among these, only \u003cem\u003ePPM1D\u003c/em\u003e has previously been proposed as an oncogene in neuroblastoma, highlighting its potential role in driving tumorigenesis in cases with 17q gain \u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e (http//:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.cancer.sanger.ac.uk/cosmic/mutation/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eCopy number gain of chromosome 17 is an early genetic event in neuroblastoma and additional gene copies are acquired through clonal evolution\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo further delineate the chromosomal aberrations involved in neuroblastoma development and progression, we employed evolutionary trajectory analysis\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. We analyzed SNP array data from 100 multiregional tumor samples collected from 23 neuroblastoma patients\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, with a focus on chromosome 17. Most tumors (57%, 13/23) were found to harbor gain of chromosome 17q in over 90% of tumor cells in all samples, indicating that this aberration was present early in tumor evolution, persisting as a stable feature throughout disease progression. In these cases, no additional copy number changes were observed (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, I \u003cstrong\u003eand Table S1\u003c/strong\u003e). However, 30% (7/23) of tumors showed a progressive accumulation of additional 17q copies as tumors evolved, either regionally or during the transition from primary tumor to metastasis or relapse. In four of these cases, gain of 17q occurred early in the phylogenetic stem, followed by further gains of additional copies of these genes as regional clones evolved (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cstrong\u003eII\u003c/strong\u003e). In three cases, clones with 17q gain emerged in subset of samples and expanded through selective sweeps, eventually encompassing all tumor cells in the samples (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cstrong\u003eIII\u003c/strong\u003e). The remaining 13% of patients (3/23) showed regional variation in copy number, making conclusive evolutionary analysis difficult. Among the 20 patients with informative evolutionary data, the successive accumulation of 17q copies was observed in more than half of high-risk cases (7/13; \u003cstrong\u003eTable S1\u003c/strong\u003e). In contrast, additional changes in 17q copy number were not found in any of the low-risk patients, i.e. children under 18 months with only numerical changes in the stem (0/7; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0445; two-sided Fisher\u0026rsquo;s exact test).\u003c/p\u003e\n \u003cp\u003eIn additional neuroblastoma cases with multiple tumor samples available for analysis, we confirmed that 17q gain is an early event in neuroblastoma development (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e), observed in both a child without known predisposition and in a child with a germline \u003cem\u003ePHOX2B\u003c/em\u003e mutation\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eOur recent single-cell RNA-sequencing data from neuroblastoma patient samples revealed the presence of pre-malignant Schwann cell precursor (SCP)-like cells in addition to the previously described adrenergic phenotype tumor cells\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. This suggests that SCP-like cells might be the cell-of-origin in some neuroblastoma cases. To dissect the genetic landscape of neuroblastoma at the single-cell level, we used the Numbat algorithm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e to identify copy number variations and DNTR-Seq (Direct Nuclear Tagmentation and RNA sequencing)\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e to investigate the subclonal structure of tumor cells at the single-cell level. SCP-like cells were identified using the receptor tyrosine kinase ERBB3 as a specific marker, whereas adrenergic cells were identified by CD24\u003csup\u003e32\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003eAn example of striking intra-tumoral heterogeneity with early chromosome 17 gain was detected with DNTR-seq (single-cell whole-genome sequencing) which revealed five major tumor cell clones (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). In one cluster of cells with a SCP-like signature (Clone A), gain of chromosome 17 was the only detected genomic aberrations. The remaining malignant clones (clone 1\u0026ndash;4), which all had adrenergic transcriptional profiles, displayed multiple whole-chromosome gains which included chromosome 17. Clone 2 which showed further evolution, had acquired segmental chromosomal aberrations that included loss of chromosome 1p and gain of chromosome 2p. Clones 3 and 4 shared the same genomic aberration as clone 2, except that Clone 3 had acquired one additional copy of chromosome 17, while Clone 4 had retained a normal diploid chromosome 5 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). Importantly, the gain of chromosome 17 was the only genomic event, shared by all aberrant cell populations (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). It is also worthy of note that this was gained independently in SCP-like and adrenergic cell populations.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003ePPM1D\u003c/strong\u003e \u003cstrong\u003eis activated by gene fusions and, somatic- and germ-line mutations in neuroblastoma\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eWhole exome sequencing (WES) or whole genome sequencing (WGS) of 73 neuroblastoma patient samples, revealed a somatic pathogenic truncating mutation in in exon 6 of \u003cem\u003ePPM1D\u003c/em\u003e (c.1344_1345insT; p.L450fs) in a \u003cem\u003eMYCN\u003c/em\u003e-amplified tumor from an infant girl \u003cstrong\u003e(Figure S3A)\u003c/strong\u003e. Her neuroblastoma rapidly progressed from localized INSS stage 1 to metastatic INSS stage 4, ultimately leading to a fatal clinical outcome despite aggressive treatment (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003eA).\u003c/p\u003e\n \u003cp\u003eAdditionally, we identified a \u003cem\u003ede novo\u003c/em\u003e germline mutation located in exon 6 of \u003cem\u003ePPM1D\u003c/em\u003e l (c.1528C\u0026thinsp;\u0026gt;\u0026thinsp;T; p.Q510*), resulting in a premature truncation of the protein (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). This mutation was found in a 26-month-old boy diagnosed with metastatic stage 4 (INSS/stage M, INRGSS) neuroblastoma originating in the left adrenal gland with bone metastases. The patient exhibited clinical features resembling previously described cases of children with intellectual disabilities and dysmorphic features associated with \u003cem\u003ePPM1D\u003c/em\u003e germline mutations (\u003cstrong\u003eFigure S3B\u003c/strong\u003e). The tumor lacked \u003cem\u003eMYCN\u003c/em\u003e amplification, and the patient had a poor clinical outcome after relapse and disease progression, despite multimodal clinical therapy. A detailed clinical description of this patient can be found in the Supplementary Methods section. Hence, the neuroblastoma-associated \u003cem\u003ePPM1D\u003c/em\u003e mutations both resulted in C-terminal truncated variants of WIP1, similar to those gain-of-function variants identified in other cancers (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA) (http//:\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.cancer.sanger.ac.uk/cosmic/mutation/\u003c/span\u003e\u003c/span\u003e). In addition, by FusionCatcher analysis of RNA-sequencing data from neuroblastoma tumors we identified a gene fusion between \u003cem\u003ePPM1D\u003c/em\u003e and the breast carcinoma amplified sequence 3 \u003cem\u003e(BCAS3)\u003c/em\u003e gene located on chromosome 17q23.2 in a tumor from a patient enrolled in the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) neuroblastoma dataset. This fusion was associated with high expression of \u003cem\u003ePPM1D\u003c/em\u003e and a predicted WIP1 isoform with a truncated C-terminal (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). These findings, together with the evidence that chromosome 17q gain is an early event in neuroblastoma tumorigenesis, suggest that genes within this chromosomal region, particularly \u003cem\u003ePPM1D\u003c/em\u003e, play a key role in neuroblastoma progression. Given \u003cem\u003ePPM1D\u003c/em\u003e\u0026rsquo;s known functions in DNA repair, cell cycle regulation, and apoptosis, it is a strong candidate for driving further genomic instability and tumor evolution.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eOverexpression of\u003c/strong\u003e \u003cstrong\u003ePPM1D\u003c/strong\u003e \u003cstrong\u003ein neuroblastoma correlates with unfavorable clinical and biological features\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eOur data demonstrating that the \u003cem\u003ePPM1D\u003c/em\u003e gene is altered in copy number and/or structure in the majority of 17q gained high-risk neuroblastomas, incited us to investigate the clinical prognostic and biological features of \u003cem\u003ePPM1D\u003c/em\u003e gene expression in neuroblastoma. Our results demonstrate that higher expression levels of \u003cem\u003ePPM1D\u003c/em\u003e was associated with several neuroblastoma risk factors, including metastatic stage 4 disease, older age at diagnosis (\u0026gt;\u0026thinsp;18 months), \u003cem\u003eMYCN\u003c/em\u003e-gene amplification and classification into the INRG high-risk (HR) group as defined by combined clinical and biological features associated with unfavorable clinical outcome\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Notably, the highest \u003cem\u003ePPM1D\u003c/em\u003e expression was observed in non-\u003cem\u003eMYCN\u003c/em\u003e amplified HR-neuroblastoma tumors (nMN HR; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), which predominantly correspond to cases with 11q-deletions, a group frequently harboring segmental 17q gains. Next, we correlated \u003cem\u003ePPM1D\u003c/em\u003e expression with copy number (CN) alterations, using data from CGH arrays, WES and WGS. CN gains were separated into numerical gains that included whole chromosome 17 and segmental gains that included the sub-region containing \u003cem\u003ePPM1D\u003c/em\u003e. Correlation analysis revealed a significant gene dosage effect, with \u003cem\u003ePPM1D\u003c/em\u003e expression increasing proportionally with the number of 17q copies (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). Moreover, high \u003cem\u003ePPM1D\u003c/em\u003e expression was strongly linked to poor clinical outcome, with significantly lower overall survival (OS; 59% vs. 83% at five years, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and event-free survival (EFS; 47% vs. 68%, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in patients with \u003cem\u003ePPM1D\u003c/em\u003e high-expressing tumors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC). We also compared gene expression patterns across different neuroblastoma subtypes\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Using principal component analysis (PCA) on gene expression profiles from 30 primary neuroblastoma samples from two published microarray studies\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e we observed that high \u003cem\u003ePPM1D\u003c/em\u003e expression was associated with the more aggressive subtypes Type 2A (17q gain with 14q- and/or 11q-deletions) and Type 2B (1p-deletion and \u003cem\u003eMYCN\u003c/em\u003e-amplification) neuroblastomas. In contrast, low \u003cem\u003ePPM1D\u003c/em\u003e expression was characteristic of low-risk, near-triploid tumors with high TrkA expression (Type 1) (\u003cstrong\u003eFigure S4A\u003c/strong\u003e). Additionally, immunohistochemical analysis confirmed consistent WIP1 expression in all 17q-gained neuroblastoma samples (\u003cstrong\u003eFigure S4B\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eThese findings underscore the role of \u003cem\u003ePPM1D\u003c/em\u003e in neuroblastoma aggressiveness and suggest that high WIP1-expressing tumors correlate with worse patient outcomes, further reinforcing the significance of 17q gain as a prognostic marker (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e) and highlighting the strong association between \u003cem\u003ePPM1D\u003c/em\u003e expression and 17q gain (Figs.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA, B and \u003cspan class=\"InternalRef\"\u003eS4\u003c/span\u003eA).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eNeuroblastoma, a childhood tumor originating from the developing peripheral nervous system, remains challenging to treat despite intensified multimodal therapy, and survival rates are still comparatively poor compared to pediatric cancers in general. Advancing our molecular understanding of neuroblastoma is therefore crucial to improve the outcome for these children/patients. We therefore investigated common genetic aberrations in neuroblastoma, identifying chromosomal gain of 17q as an early genetic event in tumorigenesis.\u003c/p\u003e \u003cp\u003eSegmental gain of chromosome17q is the most common chromosomal aberration and predictor of poor prognosis in neuroblastoma\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Also, gain of 17q is frequently found in tumors of epithelial, neural and hematopoietic origin, suggesting that 17q harbors key genes involved in oncogenesis\u003csup\u003e\u003cspan additionalcitationids=\"CR52 CR53 CR54 CR55\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. Our findings indicate that chromosome 17 gain may occur independently in pre-malignant Schwann cell precursor-like cell populations and in adrenergic tumor clones, and that subsequent evolving malignant cell clones may acquire additional copies of chromosome 17. Evolutionary trajectory analysis revealed that 17q gain typically appears at the phylogenetic stem of neuroblastoma tumors, with subsequent gains accumulating as the tumor progresses, supporting its role as an initial driver of malignancy. Together, these observations align with a recent study using molecular clock analysis and population genetic modelling to time the genetic event of neuroblastoma evolution, suggesting that neuroblastoma pathogenesis may be initiated in the first trimester of pregnancy, when rapidly proliferating neuroblasts are susceptible to CIN and aneuploidy\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. The frequent gain of 17q, as opposed to losses on the 17p arm where \u003cem\u003eTP53\u003c/em\u003e resides, may further favor the accumulation of genomic aberrations and mutations, driving tumorigenesis\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSeveral cancer-associated genes have been identified on chromosome 17q including \u003cem\u003ePPM1D, EME1, BRCA1, ERBB2, NF1, RAD51C, BRIP1, IGF2BP1, NME1\u003c/em\u003e and \u003cem\u003eBIRC5\u003c/em\u003e. Our analysis of 417 Swedish neuroblastoma samples shows, that only \u003cem\u003eRAD51C, PPM1D\u003c/em\u003e and \u003cem\u003eBRIP1\u003c/em\u003e are included in the shortest region of overlap of 17q gains and are similar to observations made in breast cancer\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNotably, \u003cem\u003ePPM1D\u003c/em\u003e copy number gains were more frequent in high-risk, metastatic, and relapsed neuroblastomas than low-risk tumors, indicating its potential role in clonal expansion and disease progression.\u003c/p\u003e \u003cp\u003eIn our patient cohort, we identified both a somatic \u003cem\u003ePPM1D\u003c/em\u003e mutation (p.L450fs) and one \u003cem\u003ede novo\u003c/em\u003e germline mutation (p.Gln510*) in exon 6, both resulting in truncated, gain-of-function WIP1 proteins that exhibit enhanced stability and lead to increased suppression of the p53 pathway \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan additionalcitationids=\"CR59 CR60\" citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e, previously reported in numerous other malignancies\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003ePPM1D de novo\u003c/em\u003e germline mutations, predominantly in the 5th and 6th exons, have previously been reported in children with the neurodevelopmental disorder Jansen-de Vries syndrome and have not yet been described in the context of cancer. However, an identical c.1528C\u0026thinsp;\u0026gt;\u0026thinsp;T (p.Gln510*) somatic mutation has been reported previously in a malignant melanoma\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. We also detected a gene fusion of \u003cem\u003ePPM1D\u003c/em\u003e and \u003cem\u003eBCAS3\u003c/em\u003e expressed at high levels in a neuroblastoma patient. The fusion is predicted to generate a C-terminal truncation and cause augmented WIP1 protein levels. Similarly, a fusion between \u003cem\u003ePPM1D\u003c/em\u003e and \u003cem\u003eC1QTNF1\u003c/em\u003e in an Ewing sarcoma patient has been reported to drive high WIP1 expression (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pecan.stjude.cloud\u003c/span\u003e\u003cspan address=\"https://pecan.stjude.cloud\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cem\u003e).\u003c/em\u003e Additional \u003cem\u003ePPM1D\u003c/em\u003e fusions, such as an intragenic region of \u003cem\u003eRPSK6B1\u003c/em\u003e and a \u003cem\u003ePPM1D-ZNS655\u003c/em\u003e fusion, have been described in patients with diffuse cerebellar glioma and acute myeloid leukemia (AML), although without further investigation on the effect on WIP1 protein stability and abundance\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pecan.stjude.cloud\u003c/span\u003e\u003cspan address=\"https://pecan.stjude.cloud\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cem\u003e).\u003c/em\u003e Given that \u003cem\u003ePPM1D\u003c/em\u003e gene amplifications and C-terminal mutations are frequently detected in a wide spectrum of cancers\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e suggests that \u003cem\u003ePPM1D\u003c/em\u003e is a pivotal gene on 17q contributing to neuroblastoma pathogenesis by promoting CIN and allowing tumor cells to evade p53-mediated apoptosis.\u003c/p\u003e \u003cp\u003eIn primary neuroblastoma, mutations of genes in the p53 pathway are rare, occurring in only 1\u0026ndash;2% of primary cases\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, but they become more common in relapsed disease (15%) \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, where p53 inactivation appears to be linked to therapy resistance\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e,\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Mechanisms that inactivate p53 in neuroblastoma include \u003cem\u003eMDM2\u003c/em\u003e gene amplification or overexpression mediated by \u003cem\u003eMYCN\u003c/em\u003e amplification, hypermethylation or deletion of \u003cem\u003eCDKN2A\u003c/em\u003e, miR-380-5p-mediated repression of p53, or its inactivation by the methyltransferase SETD8\u003csup\u003e14,67\u0026ndash;70\u003c/sup\u003e. Under normal conditions, WIP1 attenuates p53 activation by inhibiting p53-mediated transactivation of promoters by dephosphorylating p53 at Ser15\u003csup\u003e71\u003c/sup\u003e. Additionally, WIP1 indirectly inhibits p53 activity by dephosphorylating Mdm2 and upstream p53 activating kinases\u003csup\u003e\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Amplification and gain-of-function mutations of \u003cem\u003ePPM1D\u003c/em\u003e are proposed to only partially inhibit p53 activity, given that p53 undergoes multiple post-translational modifications such as phosphorylation, acetylation and ubiquitination that mediate p53-dependent beyond WIP1\u0026rsquo;s regulatory influence\u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Our recent mouse studies support this, showing that transgenic mice overexpressing \u003cem\u003ePPM1D\u003c/em\u003e generate tumors phenotypically similar to \u003cem\u003eTrp53\u003c/em\u003e loss-of-function derived tumors\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e rather than the spectra of tumors observed in \u003cem\u003eTrp53\u003c/em\u003e knock-out mice. The effects of this partial inhibition of p53 activity together with the WIP1-mediated delay in DNA repair processes\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e suggest that \u003cem\u003ePPM1D\u003c/em\u003e activating mutations and/or overexpression disrupt the fidelity of DNA repair and DNA replication, allowing for accumulation of mutations and chromosomal aberrations that eventually result in malignant transformation. Our study demonstrates that gain of chromosome 17 is an early event in neuroblastoma and that additional copies of 17q, harboring \u003cem\u003ePPM1D\u003c/em\u003e locus, are associated with high-risk, metastatic and relapsed disease. High WIP1 expression in neuroblastoma appears to allow for the accumulation of cells with SNV and CIN in neural crest cells during development, ultimately resulting in the emergence of neuroblastoma with increased tumor aggressiveness and resistance to treatment. Therefore, inhibiting WIP1 could be a valuable addition to neuroblastoma treatment, with potential benefits extending to other pediatric and adult cancers exhibiting WIP1 dysregulation.\u003c/p\u003e \u003cp\u003eIn summary, our data position 17q gain as an early and possibly the initial event in neuroblastoma development, with \u003cem\u003ePPM1D\u003c/em\u003e emerging as a strong candidate oncogene driving tumor progression through p53 pathway modulation and CIN promotion. Targeting WIP1 in neuroblastoma in combinations with current treatment modalities may address the challenge of genetic instability and therapy resistance, representing an avenue for improving outcomes in high-risk and relapsed pediatric neuroblastoma as well as other \u003cem\u003ePPM1D\u003c/em\u003e-driven cancers.\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAckowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical Genomics, SciLifeLab, Gothenburg, Sweden and the Bioinformatics Core Facility platforms at the Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden for assistance with the bioinformatical analysis of sequencing data. We thank Catarina Tr\u0026auml;ger, Inger Bodin and Susanne Ahlberg for their help and contribution to this work.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported with grants from the Swedish Childhood Cancer Foundation, the Swedish Research Council, the Swedish Cancer Foundation, the Swedish Foundation for Strategic Research (www.nnbcr.se), Karolinska Institutet, M\u0026auml;rta and Gunnar V Philipson Foundation, and The Cancer Research Foundations of Radiumhemmet.\u003c/p\u003e\n\u003cp\u003eThe study sponsors had no role in the design of the study; the collection, analysis, and interpretation of the data; the writing of the manuscript; or the decision to submit the manuscript for publication.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization, J.M., J.I.J., and P.K.; Investigation and validation, \u0026nbsp;J.M., S.F., B.S., J.Y.H., A.M., G.T.; Computational investigation and analysis, J.M., S.F., J.S., J.O., B.S., T.K.O., F.H., C.B., S.R., A.D., F.A., N.J., Y.S., J.H., D.G., M.J., T.M., J.I.J and P.K.; Writing \u0026ndash; Original draft, J.M, J.I.J. and P.K.; Writing \u0026ndash; Review \u0026amp; Editing, J.M., S.F., J.J.M., M.F., N.B., J.I.J. and P.K. Resources, M.F., \u0026nbsp;J.J.M., G.T., M.J., D.G., T.M. J.I.J and P.K.; Final editing and manuscript approval, All authors; Funding Acquisition \u0026amp; Supervision, J.I.J. and P.K.\u003c/p\u003e\n\u003cp\u003eDeclaration of Interests. The authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe deidentified data that support the findings of this study are available from the authors upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBakhoum SF, Cantley LC. The Multifaceted Role of Chromosomal Instability in Cancer and Its Microenvironment. \u003cem\u003eCell\u003c/em\u003e. 2018;174(6):1347\u0026ndash;1360.\u003c/li\u003e\n\u003cli\u003eBurrell RA, McGranahan N, Bartek J, Swanton C. 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Cell Biol.\u003c/em\u003e 2019;11(7):564\u0026ndash;577.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Neuroblastoma, chromosome 17q, p53, WIP1, PPM1D","lastPublishedDoi":"10.21203/rs.3.rs-5961130/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5961130/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eSegmental gain of chromosome 17q is the most common genetic aberration in high-risk neuroblastoma, but its role in disease progression is poorly understood. This study aims to address the contribution of 17q gain to neuroblastoma malignancy.\u003c/p\u003e\u003ch2\u003ePatients and methods:\u003c/h2\u003e \u003cp\u003eWe analyzed the genetic and transcriptional landscape of 417 neuroblastoma patients across various risk groups and clinical stages using multi-omic approaches. Single-cell RNA/DNA sequencing and SNP arrays were combined to characterize genomic aberrations, while evolutionary trajectories were mapped to explore the accumulation of genetic changes in patients with neuroblastoma. Additionally, DNA and RNA sequencing were used to assess mutational burden and gene expression patterns.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eOur findings suggest that chromosome 17 gain is an early genetic event acquired during neuroblastoma development, correlating with the accumulation of additional chromosomal aberrations and poor prognosis. Increased segmental gains of chromosome 17q were observed during clonal evolution, relapse disease and metastasis. We identified \u003cem\u003ePPM1D\u003c/em\u003e, a p53-inducible Ser/Thr phosphatase located on chr17q22.3, as a key player activated by segmental 17q-gain, gene-fusion, or gain-of-function somatic and germline mutations, further promoting neuroblastoma development/progression.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eGain of chromosome 17 is an early driver of genetic instability in neuroblastoma, with \u003cem\u003ePPM1D\u003c/em\u003e emerging as a potential candidate gene implicated in high-risk disease progression.\u003c/p\u003e","manuscriptTitle":"Gain of chromosome 17 is an early genetic abnormality in neuroblastoma with PPM1D emerging as a strong candidate oncogene driving tumor progression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-07 10:38:56","doi":"10.21203/rs.3.rs-5961130/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4f720d09-7b10-4ec3-8b4f-369cb41994f7","owner":[],"postedDate":"February 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-14T11:23:35+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-07 10:38:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5961130","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5961130","identity":"rs-5961130","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}