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Purpose: Wilms' tumor (WT), the most common pediatric renal malignancy, is associated with mutations in several well-characterized genes, most notably WT1, CTNNB1, WTX, and TP53. However, the majority of cases do not harbor mutations in these genes. We hypothesized that additional drivers of tumor behavior would be contained within areas of consistent genomic copy number change, especially those associated with the WT risk groups defined by the International Society of Paediatric Oncology (SIOP).Experimental Design: We analyzed high-resolution (Affymetrix 250K single nucleotide polymorphism array) genomic copy number profiles of over 100 tumors from selected risk groups treated under the SIOP protocols, further characterizing genes of interest by sequencing, Multiplex Ligation–dependent Probe Amplification, or fluorescence in situ hybridization.Results: We identified FBXW7, an E3 ubiquitin ligase component, as a novel Wilms' tumor gene, mutated or deleted in ∼4% of tumors examined. Strikingly, 3 of 14 (21%) of tumors with epithelial type histology after neoadjuvant chemotherapy had FBXW7 aberrations, whereas a fourth WT patient had germline mutations in both FBXW7 and WT1. We also showed that MYCN copy number gain, detected in 9 of 104 (8.7%) of cases, is relatively common in WT and significantly more so in tumors of the high risk diffuse anaplastic subtype (6 of 19, 32%).Conclusions: Because MYCN is itself a target of FBXW7-mediated ubiquitination and degradation, these results suggest that a common pathway is dysregulated by different mechanisms in various WT subtypes. Emerging therapies that target MYCN, which is amplified in several other pediatric cancers, may therefore be of value in high risk Wilms' tumor. Clin Cancer Res; 16(7); 2036–45. ©2010 AACR.

Supplementary Data from Subtype-Specific FBXW7 Mutation and MYCN Copy Number Gain in Wilms' Tumor

The tumor suppressor TP53 promotes nerve growth factor receptor (NTRK1) -Y674/Y675 phosphorylation (NTRK1-pY674/pY675) via repression of the NTRK1 phosphatase PTPN6 in a ligand-independent manner, resulting in suppression of breast cancer cell proliferation. Moreover, NTRK1-pY674/pY675 together with low levels of PTPN6 and TP53 expression is associated with favorable disease-free survival of breast cancer patients. We determined whether in neuroblastoma this protein expression pattern impacts relapse-free survival (RFS). NTRK1-pY674/pY675, PTPN6, and TP53 expression was assessed in 98 neuroblastoma samples by immunohistochemistry. Association between expression levels and RFS was investigated by multivariate and Kaplan-Meier analysis. Mutant or wild-type TP53 was identified by sequencing tumor DNA. Tumors expressing NTRK1-pY674/pY675 and low or undetectable levels of PTPN6 and TP53 were significantly associated with 5-year RFS (P = .014) when the dataset was stratified by MYCN amplification, segmental chromosomal abnormalities and histology. Similar results were observed with tumors expressing wild-type TP53, NTRK1-pY674/pY675 and low or undetectable levels of PTPN6. Kaplan-Meier analysis demonstrated a significant correlation (P = .004), with a 50% probability of RFS (median survival 4.73 years) when present compared with 19.51% (median survival 11.63 months) when absent. Similar results were seen with non-amplified MYCN or unfavorable/undifferentiating samples and tumors from patients aged 18 months or less. Importantly, NTRK1-pY674/pY675 is an independent predictor of improved RFS. These results strongly suggest that NTRK1-pY674/pY675 together with wild-type TP53 and undetectable or low levels of PTPN6 expression is a potential biomarker of improved RFS of neuroblastoma patients. The predictive value of NTRK1-pY674/pY675 together with wild-type TP53 and low PTPN6 expression could contribute to neuroblastoma patient prognosis.

Background and Purpose: Wilms tumor (WT) is the commonest pediatric renal malignancy. A significant minority of cases have mutations in known genes or recurrent copy number aberrations, but few of these have prognostic value. We have previously observed MYCN copy number gain in WT, an aberration associated with poor outcome in several other childhood cancers. We therefore set out to assess the prognostic significance of MYCN gain in a large WT series, and to explore other mechanisms by which the function of this gene may be dysregulated. Methods: MYCN copy number was measured in 293 tumours by Multiplex ligation-dependent probe amplification. Whole exome sequencing (Agilent SureSelect V4+UTR/Illumina HiSeq) was carried out on 51 WT tumour/germline pairs. Sanger sequencing was used to confirm variants of interest and to assess an independent panel of 168 WTs. 65 WTs were profiled on Affymetrix SNP arrays (250K Nsp or SNP 6.0), Illumina HumanMethylation450 arrays, and Illumina HumanHT-12 expression arrays, and additional selected tumors on Illumina HumanCytoSNP-12 arrays. Results: MYCN gain is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. A somatic p.P44L (c.131C>t) mutation was detected in 3/45 exomes that gave informative data at this position, and in 5/168 additional tumors screened for this variant, an overall frequency of 3.8%. MYCN overexpression in high risk tumors was associated with hypomethylation at specific loci. We observed parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumors of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolved over time. In a second bilateral case, we detected MYCN gain as a germline aberration. Conclusions: MYCN gain is associated with poor outcome and anaplastic histology in WT. MYCN also appears to be disrupted by other mechanisms in WT, including DNA hypomethylation and a recurrent potential gain of function mutation, P44L. The discovery of a germline aberration suggests that MYCN dysregulation may contribute to tumor predisposition. Citation Format: Richard D. Williams, Tasnim Chagtai, Marisa Alcaide-German, John Apps, Jenny Wegert, Sergey Popov, Gordan Vujanic, Harm van Tinteren, Marry M. van den Heuvel-Eibrink, Marcel Kool, David Gisselsson, Norbert Graf, Manfred Gessler, Kathy Pritchard-Jones. Multiple mechanisms of MYCN dysregulation in Wilms tumor. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-59.

Background and Purpose: Treatment of Wilms tumor (WT) patients under International Society of Paediatric Oncology (SIOP) protocols is currently stratified by staging and histopathology at nephrectomy after neoadjuvant chemotherapy. However, most relapses occur in cases without specific histopathological risk factors, and there is a clinical need for better prognostic biomarkers. Combined loss of heterozygosity (LOH) of 1p and 16q has recently been introduced in the US as an adverse prognostic indicator, while previous work in our and other laboratories suggests that 1q gain may have a similar association with poor outcome. To examine the clinical significance of 1q gain and assess its potential as a WT biomarker, we developed a simple, effective assay that measures its genomic copy number together with that of several other loci of interest, and applied it to a large tumor series. Methods: 686 frozen tumor samples from the SIOP WT 2001 trial (from a total of 7 countries) were assayed using a rapid multiplex ligation-dependent probe amplification (MLPA) assay that was developed and optimized in association with MRC-Holland b.v. to assess the copy number status of 1p, 1q, 16q, WT1, WTX, TP53, MYCN and FBXW7. Analyses were conducted in 3 laboratories, with exchange of a blinded quality assurance sample set. Results: 1q gain was present in 28% (190/686) of the cases. The 5- year Event Free Survival (EFS) rate was 72.6% (95% Confidence Interval (CI), 66.3%-85%) for those with 1q gain and 86.4% (95% CI, 83.4%-89.6%) for those who lacked 1q gain (p= Conclusion: Gain of 1q is a potential adverse biomarker for WT. Its association with high risk histological features after pre-operative chemotherapy and independent impact on survival require assessment in a larger number of patients before consideration for clinical use. Citation Format: Tasnim Chagtai, Christina Zill, Linda Dainese, Jenny Wegert, Mariana Maschietto, Gordan Vujanic, Neil Sebire, Ivo Leuschner, Peter Ambros, Maureen O'Sullivan, Christophe Bergeron, David Gisselsson, Marcel Kool, Marry van den Heuvel-Eibrink, Norbert Graf, Harm van Tinteren, Aurore Coulomb, Manfred Gessler, Richard Williams, Kathy Pritchard-Jones. Prognostic significance of copy number aberrations in Wilms tumor. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-67.

SummaryBlastemal histology in chemotherapy-treated pediatric Wilms tumors (nephroblastoma) is associated with adverse prognosis. To uncover the underlying tumor biology and find therapeutic leads for this subgroup, we analyzed 58 blastemal type Wilms tumors by exome and transcriptome sequencing and validated our findings in a large replication cohort. Recurrent mutations included a hotspot mutation (Q177R) in the homeo-domain of SIX1 and SIX2 in tumors with high proliferative potential (18.1% of blastemal cases); mutations in the DROSHA/DGCR8 microprocessor genes (18.2% of blastemal cases); mutations in DICER1 and DIS3L2; and alterations in IGF2, MYCN, and TP53, the latter being strongly associated with dismal outcome. DROSHA and DGCR8 mutations strongly altered miRNA expression patterns in tumors, which was functionally validated in cell lines expressing mutant DROSHA.

Purpose Wilms tumor (WT) is the most common pediatric renal tumor. Treatment planning under International Society of Paediatric Oncology (SIOP) protocols is based on staging and histologic assessment of response to preoperative chemotherapy. Despite high overall survival (OS), many relapses occur in patients without specific risk factors, and many successfully treated patients are exposed to treatments with significant risks of late effects. To investigate whether molecular biomarkers could improve risk stratification, we assessed 1q status and other potential copy number biomarkers in a large WT series. Materials and Methods WT nephrectomy samples from 586 SIOP WT 2001 patients were analyzed using a multiplex ligation-dependent probe amplification (MLPA) assay that measured the copy number of 1q and other regions of interest. Results One hundred sixty-seven (28%) of 586 WTs had 1q gain. Five-year event-free survival (EFS) was 75.0% in patients with 1q gain (95% CI, 68.5% to 82.0%) and 88.2% in patients without gain (95% CI, 85.0% to 91.4%). OS was 88.4% with gain (95% CI, 83.5% to 93.6%) and 94.4% without gain (95% CI, 92.1% to 96.7%). In univariable analysis, 1q gain was associated with poorer EFS (P

Background Wilms tumours (WTs) are characterised by several hallmarks that suggest epimutations such as aberrant DNA methylation are involved in tumour progression: loss of imprinting at 11p15, lack of recurrent mutations and formation of nephrogenic rests (NRs), which are lesions of retained undifferentiated embryonic tissue that can give rise to WTs. Methods To identify such epimutations, we performed a comprehensive methylome analysis on 20 matched trios of micro-dissected WTs, NRs and surrounding normal kidneys (NKs) using Illumina Infinium HumanMethylation450 Bead Chips and functionally validated findings using RNA sequencing. Results Comparison of NRs with NK revealed prominent tissue biomarkers: 629 differentially methylated regions, of which 55% were hypermethylated and enriched for domains that are bivalent in embryonic stem cells and for genes expressed during development (P = 2.49 × 10-5). Comparison of WTs with NRs revealed two WT subgroups; group-2 WTs and NRs were epigenetically indistinguishable whereas group-1 WTs showed an increase in methylation variability, hypomethylation of renal development genes, hypermethylation and relative loss of expression of cell adhesion genes and known and potential new WT tumour suppressor genes (CASP8, H19, MIR195, RB1 and TSPAN32) and was strongly associated with bilateral disease (P = 0.032). Comparison of WTs and NRs to embryonic kidney highlighted the significance of polycomb target methylation in Wilms tumourigenesis. Conclusions Methylation levels vary during cancer evolution. We have described biomarkers related to WT evolution from its precursor NRs which may be useful to differentiate between these tissues for patients with bilateral disease. Electronic supplementary material The online version of this article (doi:10.1186/s13073-015-0136-4) contains supplementary material, which is available to authorized users.

Purpose The presence of diffuse anaplasia in Wilms tumours (DAWT) is associated with TP53 mutations and poor outcome. As patients receive intensified treatment, we sought to identify whether TP53 mutational status confers additional prognostic information. Patients and Methods We studied 40 patients with DAWT with anaplasia in the tissue from which DNA was extracted and analysed for TP53 mutations and 17p loss. The majority of cases were profiled by copy number (n = 32) and gene expression (n = 36) arrays. TP53 mutational status was correlated with patient event-free and overall survival, genomic copy number instability and gene expression profiling. Results From the 40 cases, 22 (55%) had TP53 mutations (2 detected only after deep-sequencing), 20 of which also had 17p loss (91%); 18 (45%) cases had no detectable mutation but three had 17p loss. Tumours with TP53 mutations and/or 17p loss (n = 25) had an increased risk of recurrence as a first event (p = 0.03, hazard ratio (HR), 3.89; 95% confidence interval (CI), 1.26–16.0) and death (p = 0.04, HR, 4.95; 95% CI, 1.36–31.7) compared to tumours lacking TP53 abnormalities. DAWT carrying TP53 mutations showed increased copy number alterations compared to those with wild-type, suggesting a more unstable genome (p = 0.03). These tumours showed deregulation of genes associated with cell cycle and DNA repair biological processes. Conclusion This study provides evidence that TP53 mutational analysis improves risk stratification in DAWT. This requires validation in an independent cohort before clinical use as a biomarker.

We identified constitutional truncating mutations of the BRCA1-interacting helicase BRIP1 in 9/1,212 individuals with breast cancer from BRCA1/BRCA2 mutation-negative families but in only 2/2,081 controls (P = 0.0030), and we estimate that BRIP1 mutations confer a relative risk of breast cancer of 2.0 (95% confidence interval = 1.2-3.2, P = 0.012). Biallelic BRIP1 mutations were recently shown to cause Fanconi anemia complementation group J. Thus, inactivating truncating mutations of BRIP1, similar to those in BRCA2, cause Fanconi anemia in biallelic carriers and confer susceptibility to breast cancer in monoallelic carriers.

We screened individuals from 443 familial breast cancer pedigrees and 521 controls for ATM sequence variants and identified 12 mutations in affected individuals and two in controls (P = 0.0047). The results demonstrate that ATM mutations that cause ataxia-telangiectasia in biallelic carriers are breast cancer susceptibility alleles in monoallelic carriers, with an estimated relative risk of 2.37 (95% confidence interval (c.i.) = 1.51-3.78, P = 0.0003). There was no evidence that other classes of ATM variant confer a risk of breast cancer.

Wilms tumor (WT) with diffuse anaplasia has an unfavorable prognosis and is often (>70%) associated with mutations in the TP53 gene. Although most WTs are unilateral, 5–10% are bilateral, and they are almost always present with nephrogenic rests. The latter are considered a precursor of WT. Two cases of bilateral WTs with nephroblastomatosis, in which anaplastic changes were detected over a period of time, were analyzed using clinical, radiological, histopathological, and molecular-genetic data. TP53 was analyzed by direct sequencing of its full coding sequence and intron-exon boundaries in 11 fragments. DNA was extracted from paraffin-embedded or frozen specimens. High-resolution genomic copy number profiling was carried out by UCL Genomics on the Affymetrix Human Mapping 250K Nsp or Genome-Wide Human SNP Array 6.0 platform. Both cases demonstrated a strong association between the appearance of anaplastic clones and TP53 mutations. Synchronous ganglioneuroma was diagnosed in one case. Our cases are unique as they represent a long disease history and demonstrate the difficulties in managing rare cases of bilateral WT with anaplasia. These cases also emphasize the practical importance of modern molecular-genetic techniques and their clinical application. Moreover, they highlight the issue of the adequate sampling needed in order to gather comprehensive, efficient, and sufficient information about genetic events in a single tumor.

Anaplasia in Wilms tumor, a distinctive histology characterized by abnormal mitoses, is associated with poor patient outcome. While anaplastic tumors frequently harbour TP53 mutations, little is otherwise known about their molecular biology. We have used array comparative genomic hybridization (aCGH) and cDNA microarray expression profiling to compare anaplastic and favorable histology Wilms tumors to determine their common and differentiating features. In addition to changes on 17p, consistent with TP53 deletion, recurrent anaplasia-specific genomic loss and under-expression were noted in several other regions, most strikingly 4q and 14q. Further aberrations, including gain of 1q and loss of 16q were common to both histologies. Focal gain of MYCN, initially detected by high resolution aCGH profiling in 6/61 anaplastic samples, was confirmed in a significant proportion of both tumor types by a genomic quantitative PCR survey of over 400 tumors. Overall, these results are consistent with a model where anaplasia, rather than forming an entirely distinct molecular entity, arises from the general continuum of Wilms tumor by the acquisition of additional genomic changes at multiple loci.

Purpose: Wilms' tumor (WT), the most common pediatric renal malignancy, is associated with mutations in several well-characterized genes, most notably WT1, CTNNB1, WTX, and TP53. However, the majority of cases do not harbor mutations in these genes. We hypothesized that additional drivers of tumor behavior would be contained within areas of consistent genomic copy number change, especially those associated with the WT risk groups defined by the International Society of Paediatric Oncology (SIOP). Experimental Design: We analyzed high-resolution (Affymetrix 250K single nucleotide polymorphism array) genomic copy number profiles of over 100 tumors from selected risk groups treated under the SIOP protocols, further characterizing genes of interest by sequencing, Multiplex Ligation–dependent Probe Amplification, or fluorescence in situ hybridization. Results: We identified FBXW7, an E3 ubiquitin ligase component, as a novel Wilms' tumor gene, mutated or deleted in ∼4% of tumors examined. Strikingly, 3 of 14 (21%) of tumors with epithelial type histology after neoadjuvant chemotherapy had FBXW7 aberrations, whereas a fourth WT patient had germline mutations in both FBXW7 and WT1. We also showed that MYCN copy number gain, detected in 9 of 104 (8.7%) of cases, is relatively common in WT and significantly more so in tumors of the high risk diffuse anaplastic subtype (6 of 19, 32%). Conclusions: Because MYCN is itself a target of FBXW7-mediated ubiquitination and degradation, these results suggest that a common pathway is dysregulated by different mechanisms in various WT subtypes. Emerging therapies that target MYCN, which is amplified in several other pediatric cancers, may therefore be of value in high risk Wilms' tumor. Clin Cancer Res; 16(7); 2036–45. ©2010 AACR.

PALB2 interacts with BRCA2, and biallelic mutations in PALB2 (also known as FANCN), similar to biallelic BRCA2 mutations, cause Fanconi anemia. We identified monoallelic truncating PALB2 mutations in 10/923 individuals with familial breast cancer compared with 0/1,084 controls (P = 0.0004) and show that such mutations confer a 2.3-fold higher risk of breast cancer (95% confidence interval (c.i.) = 1.4–3.9, P = 0.0025). The results show that PALB2 is a breast cancer susceptibility gene and further demonstrate the close relationship of the Fanconi anemia–DNA repair pathway and breast cancer predisposition.

Introduction: Wilms tumour (WT) is the most common paediatric renal tumour, thought to arise from disruption in normal kidney development. WT typically presents triphasic histology with blastemal, stromal and epithelial components. The International Society of Paediatric Oncology (SIOP) categorises Wilms tumours based on their in vivo response to neoadjuvant chemotherapy. Tumours where a viable blastemal component predominates at nephrectomy (high risk ‘blastemal type’, BT) are associated with poorer outcome than tumours with primarily stromal, epithelial or mixed histology (intermediate risk, IR). Although a number of WT-associated genes have been defined, most tumours do not harbour mutations in any of these genes. Global hypo-methylation as well as hyper-methylation and silencing of tumour suppressor genes have been frequently reported in cancer. In WT, several epigenetic lesions have previously been identified, particularly loss of imprinting at 11p13 and 11p15. However, to date, no genome wide comprehensive DNA methylation study in WT has been reported. Aim: To perform genome wide DNA methylation profiling in WT and identify molecular signatures associated with BT WT. Methods: Genomic DNA from 69 frozen clinical WT samples was extracted and bisulphite- converted with the Zymo EZ DNA Methylation Kit. The methylation levels were assessed on the Infinium HumanMethylation 450 BeadChip array, which interrogates over 480,000 CpG sites. For 56 of the samples, HumanHT-12 v3 Expression BeadChip data was available. Bioinformatic and statistical analyses were carried out using the Bioconductor minfi and limma packages. Preliminary analysis was performed integrating expression and methylation data. Results: 930 genes showed differential methylation between BT and IR. Over 70% of the samples showed aberrant methylation at the 11p15 locus, as previously reported. 75 of these differentially methylated genes were also differentially expressed. Of interest, the well characterised WT gene, WT1, was significantly down regulated in IR, and correlated with hypermethylation in this region. Gene Ontology pathway analysis indicated that almost 50% of these 75 genes were involved in developmental processes, supporting the current model of WT. Conclusions: This study highlighted several key genes and pathways dysregulated in BT compared to IR. Moreover, global hypomethylation was found in the BT cohort, which could be associated with genome instability or the dysregulation of DNA methyl-transferase maintenance enzymes. Further studies are required to validate these molecular routes as potential biomarkers for high risk WT. Citation Format: Marisa Alcaide German, Richard Williams, Jocelyn Charlton, Tasnim Chagtai, Tiffany Morris, Kerra Pearce, Kathy Pritchard-Jones. Genome Wide DNA methylation profiling in Wilms tumor. Integration with expression and identification of molecular signatures associated with high risk blastemal type histology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 660. doi:10.1158/1538-7445.AM2013-660

Wilms tumor (WT) is a paediatric renal tumor with a relapse rate of 10 to 15%. Tumor stage and the histological presence of diffuse anaplasia (DA) are the most important adverse prognostic factors in WT. Previous studies have described TP53 mutations at a frequency of up to 75% in DA WT cases, however only small cohorts were analysed. The aim of this study was to evaluate the frequency of TP53 mutations in a large group of DA WT and correlate these data with tumor and patient characteristics. DA WTs from 61 patients were analysed for TP53 mutations using Sanger sequencing. A paediatric pathologist confirmed the presence of anaplastic cells in the specimen used for DNA extraction. Sequence alterations were classified as damaging or neutral mutations with reference to databases of known mutations and functional prediction algorithms (SIFT, PolyPhen2). Loss of 17p, where TP53 is located, was evaluated using CGH or SNP arrays and gene expression profiling was carried out using cDNA microarrays. Kaplan-Meier Survival analysis and Breslow's test were used to compare TP53 alterations with clinico-pathological characteristics. Of the 61 cases, 29 (47.5%) had at least one damaging TP53 mutation and 26 of these also had TP53 loss. Most of the cases (20 out of 29, 68.9%) presented TP53 mutations located within exons 5 to 8. These exons comprise the DNA-binding domain where the hotspots are located. The other 32 DA WT cases did not harbour mutations but six had 17p loss. Patients with DA WT that carry TP53 mutations and/or 17p loss had a worse response to treatment than those patients that had wild type TP53 (p The CGH profiles showed an increase in the number of gains and losses seen in DA WT with TP53 mutations, compared to those without, suggesting a more unstable genome. Gene expression profiling revealed 71 differentially expressed genes between DA WT with and without mutations. These genes may be associated with the anaplastic phenotype, but their role in the pathogenesis of WT remains to be confirmed. Evaluating a large series of anaplastic WT, we found that the frequency of TP53 mutations in DA WT was lower than previously reported. However, samples with apparent wild type TP53 will be further evaluated by deep-sequencing to determine if mutations are present in a minority cell population. The relatively high frequency of wild type TP53 in DA WT suggests that TP53 mutations are neither necessary nor sufficient to generate anaplasia. Grants: Cancer Research UK; EU FP7 P-medicine, Sophie Barringer Trust and GOSHCC. Citation Format: Mariana Maschietto, Tasnim Chagtai, Sergey D. Popov, Neil Sebire, Gordan Vujanic, Sandra Hing, Paul Grundy, Jeffrey S. Dome, Kathy Pritchard-Jones, Richard D. Williams. TP53 mutation status defines two distinct classes of diffuse anaplastic Wilms tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3829. doi:10.1158/1538-7445.AM2013-3829

Introduction Biomarkers are increasingly used to support risk stratification and clinical decisions in Paediatric Oncology. Implicit in the clonal evolutionary theory of cancer development is the evolution of cells derived from an initiating clone through natural selection acting upon intra-tumoural genetic heterogeneity. Wilms tumours frequently present as large macroscopically heterogeneous masses, and can be multifocal in origin. Several studies have indicated the presence of intra-tumoural genetic heterogeneity in a range of tumours. This study aimed to characterise intra-tumoural genetic heterogeneity within Wilms tumours. Methods Multiple snap frozen samples were taken from consecutive post chemotherapy Wilms tumour nephrectomy specimens at Great Ormond Street Hospital, May-October 2011. DNA was extracted from frozen specimens and analysed using the Illumina CytoSNP array for copy number variation. Results 23 samples were analysed from 10 separate tumours. 2 cases showed clear heterogeneity between samples. In one case gains of chromosome 8 and 13 were conserved across two tumour samples however gains of chromosomes 2 and 12 were present in one sample and of chromosome 7 in the other. In a separate case partial gain of 22q was present in both samples and 1q and partial 1p gain in only one sample. A third case showed localised heterogeneity with loss of 7p and gain of 7q present in two samples whilst gain of chromosome 8 was more apparent in only one sample. In three other cases, the changes observed were conserved between two or more samples (gain of 12, 13 and 20 in one case, gain of 12 in the second, and partial 9q21-22 deletions in the third). 4 tumours had no apparent copy number changes. Discussion 6 of 10 tumours showed copy number variation. In all of these cases certain copy number variations were conserved between multiple samples of an individual tumour, consistent with a clonal origin. In three cases there was intra-tumoural genetic heterogeneity. Further understanding such heterogeneity may aid in deciphering the genetic changes in the evolution of Wilms tumours and help identify driver mutations. In addition, as the use of biomarkers, such as 1q gain, enters clinical practice understanding such heterogeneity will be important for establishing appropriate sampling strategies.

Genomic gain of the proto-oncogene transcription factor gene MYCN is associated with poor prognosis in several childhood cancers. Here we present a comprehensive copy number analysis of MYCN in Wilms tumour (WT), demonstrating that gain of this gene is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. Using whole exome and gene-specific sequencing, together with methylation and expression profiling, we show that MYCN is targeted by other mechanisms, including a recurrent somatic mutation, P44L, and specific DNA hypomethylation events associated with MYCN overexpression in tumours with high risk histologies. We describe parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumours of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolve over time. We report a second bilateral case in which MYCN gain is a germline aberration. Our results suggest a significant role for MYCN dysregulation in the molecular biology of Wilms tumour. We conclude that MYCN gain is prognostically significant, and suggest that the novel P44L somatic variant is likely to be an activating mutation.