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Background/Purpose In Hirschsprung disease (HD) surgery, confirming ganglionic bowel is essential. A faster diagnostic method than the current frozen biopsy is desirable. This study investigated whether aganglionic and ganglionic intestinal wall can be distinguished from each other by ultra high frequency ultrasound (UHF ultrasound). Methods In an HD center during 2019, intestinal walls of recto-sigmoid specimens from HD patients were examined ex vivo with a 70 MHz UHF ultrasound transducer. Data from four sites were described. Histopathologic analysis was compared to the ultrasonography outcome at each site. Each patient's specimen served as its own control. Results 11 resected recto-sigmoid specimens (median 20 cm long [range 6.5–33]) with transition zones of 5 cm (2–11 cm) were taken from children aged 22 days (13–48) weighing 3668 g (3500–5508); 44 key sites were analyzed. There was full concordance for 42/44 (95%) key sites and 10 of 11 (91%) specimens. The specimen with discordance of two key sites contained a segment of aganglionosis (3 cm) and a transition zone (1 cm): the site discordance was limited to the transition zone ends. Conclusions This first report on UHF ultrasound in recto-sigmoid HD shows promising results in identifying aganglionosis, transition zones and ganglionic bowel. Further in vivo studies are required.

Phylogenetic reconstruction of cancer cell populations remains challenging. There is a particular lack of tools that deconvolve clones based on copy number aberration analyses of multiple tumor biopsies separated in time and space from the same patient. This has hampered investigations of tumors rich in aneuploidy but few point mutations, as in many childhood cancers and high-risk adult cancer. Here, we present DEVOLUTION, an algorithm for subclonal deconvolution followed by phylogenetic reconstruction from bulk genotyping data. It integrates copy number and sequencing information across multiple tumor regions throughout the inference process, provided that the mutated clone fraction for each mutation is known. We validate DEVOLUTION on data from 56 pediatric tumors comprising 253 tumor biopsies and show a robust performance on simulations of bulk genotyping data. We also benchmark DEVOLUTION to similar bioinformatic tools using an external dataset. DEVOLUTION holds the potential to facilitate insights into the development, progression, and response to treatment, particularly in tumors with high burden of chromosomal copy number alterations., Natalie Andersson et al. present DEVOLUTION, an algorithm and tool that allows researchers to reconstruct tumor clonal lineages from copy number and/or sequencing data. They validate this method on a diverse cohort of pediatric cancers, demonstrating its potential applications for cancer research.

Background Pancreatic cancer is a devastating disease with a dismal prognosis. Despite profound medical advances in systemic therapies for other types of aggressive tumours during recent years, a diagnosis of pancreatic cancer is still often synonymous with a fatal outcome. The term periampullary cancer includes pancreatic cancer and applies to the group of tumours found in proximity to the ampulla of Vater. Molecular events and immune response in the host during chemotherapy remain largely unexplored in this group of tumours. Therefore, the “Chemotherapy, Host Response and Molecular Dynamics in Periampullary Cancer (CHAMP)” study aims to monitor these processes to gain new insight into this perplexing disease. Methods The CHAMP study is a prospective, single-arm observational study. All patients diagnosed with pancreatic or other periampullary adenocarcinoma undergoing adjuvant or palliative chemotherapy treatment in the Department of Oncology, Skåne University Hospital, are invited to participate. Clinical and pathological data will be compiled at study entry. A single tissue microarray (TMA) block is constructed for each patient with a resected tumour and blood samples are drawn before, during and after chemotherapy in order to sample peripheral blood mononuclear cells (PBMC), cytokines and circulating tumour DNA (ctDNA). Next generation sequencing will be performed on tumour tissue and ctDNA to detect changes in the clonal landscape over space and time. Discussion Despite the recent emergence of some promising biomarkers for periampullary cancer, there has been a lack of success in clinical implementation. Cancer cells continuously adapt and become resistant to treatment during chemotherapy. To be able to keep pace with and hopefully overtake this rapid evolution we must, with the help of new diagnostic tools, be ready to adapt and alter treatment accordingly. It seems to us that the only way forward is to gain a better understanding of the dynamics of the disease during treatment. With insights gained from the CHAMP study we hope to find answers to key questions in this largely unexplored territory. Trial registration This study has been registered 30th October 2018 at clinicaltrials.gov as NCT03724994.

Darwinian evolution of tumor cells remains underexplored in childhood cancer. We here reconstruct the evolutionary histories of 56 pediatric primary tumors, including 24 neuroblastomas, 24 Wilms tumors, and 8 rhabdomyosarcomas. Whole-genome copy-number and whole-exome mutational profiling of multiple regions per tumor were performed, followed by clonal deconvolution to reconstruct a phylogenetic tree for each tumor. Overall, 88% of the tumors exhibited genetic variation among primary tumor regions. This variability typically emerged through collateral phylogenetic branching, leading to spatial variability in the distribution of more than 50% (96/173) of detected diagnostically informative genetic aberrations. Single-cell sequencing of 547 individual cancer cells from eight solid pediatric tumors confirmed branching evolution to be a fundamental underlying principle of genetic variation in all cases. Strikingly, cell-to-cell genetic diversity was almost twice as high in aggressive compared with clinically favorable tumors (median Simpson index of diversity 0.45 vs. 0.88; P = 0.029). Similarly, a comparison of multiregional sampling data from a total of 274 tumor regions showed that new phylogenetic branches emerge at a higher frequency per sample and carry a higher mutational load in high-risk than in low-risk tumors. Timelines based on spatial genetic variation showed that the mutations most influencing relapse risk occur at initiation of clonal expansion in neuroblastoma and rhabdomyosarcoma, whereas in Wilms tumor, they are late events. Thus, from an evolutionary standpoint, some high-risk childhood cancers are born bad, whereas others grow worse over time. Significance: Different pediatric cancers with a high risk of relapse share a common generic pattern of extensively branching evolution of somatic mutations.

The ERBB2 gene encodes a receptor tyrosine kinase also known as HER2. The gene is amplified and overexpressed in one-fifth of breast carcinomas; patients with such tumors benefit from targeted treatment with trastuzumab or other drugs blocking the receptor. In addition, ERBB2 has been shown to be amplified and/or overexpressed in a variety of other malignancies. Notably, both alveolar and embryonal rhabdomyosarcoma (RMS), especially in children, often show increased expression of ERBB2. Although high-level amplification of the gene has not been described in RMS, its frequent expression at the cell surface of RMS cells has been exploited for chimeric antigen receptor T-cell (CAR T)-based treatment strategies. We here describe two cases of pediatric, fusion-negative embryonal RMS with high-level amplification of the ERBB2 gene. One patient is currently treated with conventional chemotherapy for a recently detected standard risk RMS, whereas the other patient died from metastatic disease. Both tumors displayed focal amplicons (210 and 274 Kb, respectively) in chromosome band 17q12, with proximal and distal borders corresponding to those typically seen in breast cancer. In both tumors, the ERBB2 amplicon correlated with high expression at the RNA and protein levels. Thus, breast cancer-like ERBB2 amplification is a very rare, but recurrent feature of pediatric RMS, and should be exploited as an alternative treatment target. Funding Agencies|Governmental Funding of Clinical Research within the National Health Service of Sweden; Swedish Cancer SocietySwedish Cancer Society; Swedish Childhood Cancer Fund

Background: Therapeutic activation of tumor-infiltrating lymphocytes using monoclonal antibodies targeting PD1 or PD-L1 (immune checkpoint inhibitors—ICIs) has revolutionized treatment of specific solid tumors in adult cancer patients, and much hope has been placed on a similar effect in relapsed or refractory solid pediatric tumors. Recent clinical trials have disappointingly shown an almost nonexistent response rate, while case reports have demonstrated that some pediatric patients do achieve durable responses when treated with this type of drug. Aim: To elucidate this paradox, we mapped the landscape of expressed neoantigens as well as the levels of immune cell infiltration in the two most common extracranial solid pediatric tumors: Wilms tumor and neuroblastoma using state-of-the-art in silico analysis of a large cohort of patients with these tumors. Methods: By integration of whole exome sequencing and RNA-sequencing, we mapped the landscape of neoantigens in the TARGET cohorts for these diagnoses and correlated these findings with known genetic prognostic markers. Results: Our analysis shows that these tumors typically have much lower levels of expressed neoantigens than commonly seen in adult cancers, but we also identify subgroups with significantly higher levels of neoantigens. For neuroblastomas, the cases with higher levels of neoantigens were confined to the group without MYCN-amplification and for Wilms tumor restricted to the TP53-mutated cases. Furthermore, we demonstrate that neuroblastomas have an unexpectedly high level of CD8+ tumor-infiltrating lymphocytes, even when compared to adult tumor types where ICI is an approved treatment. Conclusion: These results could be important to consider when designing future clinical trials of ICI treatment in pediatric patients with relapsed or refractory solid tumors. (Less)

Highlights • Analysis of multiple tumor types reveal neuroblastoma sensitivity to rigosertib. • Neuroblastoma 2D and 3D organoid models are sensitive to rigosertib. • Rigosertib causes G2M cell cycle arrest rather than inhibition of Ras binding domains. • Rigosertib prolongs survival of MYCN- amplified patient-derived xenograft tumors. • Combination of rigosertib and vincristine is synergistic against neuroblastoma., High-risk neuroblastoma has a poor prognosis despite intense treatment, demonstrating the need for new therapeutic strategies. Here we evaluated the effects of rigosertib (ON-01910.Na) in preclinical models of high-risk neuroblastoma. Among several hundred cancer cell lines representing 24 tumor types, neuroblastoma was the most sensitive to rigosertib. Treatment of MYCN-amplified neuroblastoma organoids resulted in organoid disintegration, decreased cell viability, and increased apoptotic cell death. Neuroblastoma response to rigosertib involved G2M cell cycle arrest and decreased phosphorylation of AKT (Ser473) and ERK1/2 (Thr202/Tyr204). Rigosertib delayed tumor growth and prolonged survival of mice carrying neuroblastoma MYCN-amplified PDX tumors (median survival: 31 days, treated; 22 days, vehicle) accompanied with increased apoptosis in treated tumors. We further identified vincristine and rigosertib as a potential promising drug combination treatment. Our results show that rigosertib might be a useful therapeutic agent for MYCN-amplified neuroblastomas, especially in combination with existing agents.

A 27-year-old woman (gravida 2, para 1) was transported to the regional university hospital in gestational week (GW) 34 + 4 due to a three-day history of fever, abdominal pain and reduced foetal movements. She had developed a dry cough one day prior to the admission (Figure S1). The woman, was slightly overweight (BMI 27 kg/m2 ) but otherwise healthy. She had normal antenatal check-ups and an obstetric ultrasound at GW 32 + 2 showed a normal foetal weight deviation of +8%1 .

Clonal deconvolution of mutational landscapes is crucial to understand the evolutionary dynamics of cancer. Two limiting factors for clonal deconvolution that have remained unresolved are variation in tumor cell fraction (TCF) and chromosomal copy number across different samples of the same tumor. We developed a semi-supervised algorithm that tracks variant calls through multi-sample spatiotemporal tumor data. While normalizing allele frequencies based on TCF it also adjusts for copy number changes at clonal deconvolution. Absent à priori copy number data, it rendersin silicocopy number estimations from bulk sequences. Using published and simulated tumor sequences, we reliably segregated clonal/subclonal variants even at low sequencing depth (∼50x). Given at least one pure tumor sample (>70% TCF), we could normalize and deconvolve paired samples down to a TCF of 40%. This renders a reliable clonal reconstruction well adapted to multi-regionally sampled solid tumor, which are often aneuploid and contaminated by non-cancer cells.

This study aims to characterize the molecular signatures of sacrococcygeal teratomas (SCTs). Methods: Three SCTs were analyzed with whole genome genotyping. RNA sequencing of 10 SCTs dominated by m...

Neuroblastoma is a childhood malignancy with often dismal prognosis; relapse is common despite intense treatment. Here, we used human tumor organoids representing multiple MYCN-amplified high-risk neuroblastomas to perform a high-throughput drug screen with approved or emerging oncology drugs. Tumor-selective effects were calculated using drug sensitivity scores. Several drugs with previously unreported anti-neuroblastoma effects were identified by stringent selection criteria. ARRY-520, an inhibitor of kinesin spindle protein (KSP), was among those causing reduced viability. High expression of the KSP-encoding gene KIF11 was associated with poor outcome in neuroblastoma. Genome-scale loss-of-function screens in hundreds of human cancer cell lines across 22 tumor types revealed that KIF11 is particularly important for neuroblastoma cell viability. KSP inhibition in neuroblastoma patient-derived xenograft (PDX) cells resulted in the formation of abnormal monoastral spindles, mitotic arrest, up-regulation of mitosis-associated genes, and apoptosis. In vivo, KSP inhibition caused regression of MYCN-amplified neuroblastoma PDX tumors. Furthermore, treatment of mice harboring orthotopic neuroblastoma PDX tumors resulted in increased survival. Our results suggested that KSP inhibition could be a promising treatment strategy in children with high-risk neuroblastoma.

The aim of this study was to examine the prevalence of embryologic remnants in umbilical cords of different gestational ages. Sections from 392 umbilical cords were examined using light microscopy. Of these, 52% contained at least 1 remnant, most commonly of the allantoic duct type. Although there was a significant decrease in vitelline duct remnants over increasing gestational age, from 11% at weeks 11–25 to 1.6% at weeks 36–42 ( P = .009; χ2 test), the allantoic duct remnants remained constant in prevalence irrespective of gestational age.

Wilms tumour is a paediatric malignancy with features of halted kidney development. Here, we demonstrate that the Iroquois homeobox genes IRX3 and IRX5 are essential for mammalian nephrogenesis and govern the differentiation of Wilms tumour. Knock-out Irx3−/Irx5− mice showed a strongly reduced embryonic nephron formation. In human foetal kidney and Wilms tumour, IRX5 expression was already activated in early proliferative blastema, whereas IRX3 protein levels peaked at tubular differentiation. Accordingly, an orthotopic xenograft mouse model of Wilms tumour showed that IRX3−/− cells formed bulky renal tumours dominated by immature mesenchyme and active canonical WNT/β-catenin-signalling. In contrast, IRX5−/− cells displayed activation of Hippo and non-canonical WNT-signalling and generated small tumours with abundant tubulogenesis. Our findings suggest that promotion of IRX3 signalling or inhibition of IRX5 signalling could be a route towards differentiation therapy for Wilms tumour, in which WNT5A is a candidate molecule for enforced tubular maturation. (Less)

Malignant rhabdoid tumour (MRT) is a childhood neoplasm of high malignancy characterised by biallelic mutation and/or loss of the epigenetic master regulator SMARCB1, accompanied by no or few other oncogenic drivers. In spite of their generally low mutational burden, an intratumoural T-cell response has been reported in a subset of MRTs, indicating that immune checkpoint inhibition may be considered a viable therapy option for some patients. We assess here the evolution over time and space of predicted neoantigens and indicators of immune checkpoint status in two MRT patients who progressed under treatment. Both patients showed an accumulation of novel clonal and subclonal mutations, including predicted neoantigens, in metastases compared to their inferred ancestral clones in the primary tumours. The first patient had peritoneal metastases from an MRT of the liver. Clonal deconvolution revealed polyclonal seeding from the primary tumour to a single metastatic site, followed by a local subclonal burst of mutations. The second patient had a renal MRT with multiple pulmonary metastases, each of which could be traced back to a single genetically unique founder cell, with formation of novel subclones in two metastases. Both patients showed a regionally heterogeneous landscape of predicted neoantigens and of tumour-infiltrating lymphocytes expressing CD8 and PD1. In both patients, some tumour regions fulfilled established criteria for PD-L1 positivity ( 1% of tumour cells), while others did not. This suggests that even in a tumour type like MRT, with a single driver mutation, there can be heterogeneity in neoantigen repertoire, immune response, and biomarkers for checkpoint blockade among sampled locations. This must be taken into account when assessing progressed MRT patients for checkpoint inhibition therapy. © 2020 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Pleomorphic myxoid liposarcoma (PML) is an exceptionally rare and poorly studied subtype of liposarcoma, typically occurring in children and adolescents. The few previous genetic studies have shown that PML lacks the gene fusions and amplifications that characterize myxoid liposarcoma, atypical lipomatous tumor, and dedifferentiated liposarcoma. To learn more about its pathogenesis, we performed a comprehensive genetic analysis, including chromosome banding, fluorescence in situ hybridization, single nucleotide polymorphism (SNP) array analysis, deep sequencing of the exome (WES) complemented by targeted sequencing of hotspot regions of selected cancer-associated genes, and transcriptome sequencing (RNA-seq), of a PML in a 10-year-old boy.

The global methylation profile and the mutational status of 633 specific epigenetic regulators were analyzed in the pediatric tumor clear cell sarcoma of the kidney (CCSK). Methylation array analyses of 30 CCSKs revealed CCSK tumor DNA to be globally hypermethylated compared to Wilms tumor, normal fetal kidney, and adult kidney. The aberrant methylation pattern of CCSKs was associated with activation of genes involved in embryonic processes and with silencing of genes linked to normal kidney function. No epigenetic regulator was recurrently mutated in our cohort, but a mutation in the key epigenetic regulator EZH2 was discovered in one case. EZH2 mRNA was significantly higher in CCSK compared to Wilms tumor and normal kidney, and the EZH2 protein was strongly expressed in more than 90 % of CCSK tumor cells in 9/9 tumors analyzed. This was in striking contrast to the lack of EZH2 protein expression in Wilms tumor stromal elements, indicating that EZH2 could be explored further as a diagnostic marker and a potential drug target for CCSK.

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.

Clear cell sarcoma of the kidney (CCSK) is the second most common pediatric renal tumor. Two recurrent genetic aberrations have been described in CCSK. One is a fusion of YWHAE and NUTM2B/E, the other is an internal tandem duplication (ITD) in the BCOR gene. Here it is shown that YWHAE-NUTM2B/E fusion and the BCOR ITD are mutually exclusive events and activated different downstream signaling systems. This has important diagnostic implications and opens up for further mechanistic studies of CCSK pathogenesis.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents. Alveolar (ARMS) and embryonal (ERMS) histologies predominate, but rare cases are classified as spindle cell/sclerosing (SRMS). For treatment stratification, RMS is further subclassified as fusion-positive (FP-RMS) or fusion-negative (FN-RMS), depending on whether a gene fusion involving PAX3 or PAX7 is present or not. We investigated 19 cases of pediatric RMS using high resolution single-nucleotide polymorphism (SNP) array. FP-ARMS displayed, on average, more structural rearrangements than ERMS; the single FN-ARMS had a genomic profile similar to ERMS. Apart from previously known amplification (e.g., MYCN, CDK4, and MIR17HG) and deletion (e.g., NF1, CDKN2A, and CDKN2B) targets, amplification of ERBB2 and homozygous loss of ASCC3 or ODZ3 were seen. Combining SNP array with cytogenetic data revealed that most cases were polyploid, with at least one case having started as a near-haploid tumor. Further bioinformatic analysis of the SNP array data disclosed genetic heterogeneity, in the form of subclonal chromosomal imbalances, in five tumors. The outcome was worse for patients with FP-ARMS than ERMS or FN-ARMS (6/8 vs. 1/9 dead of disease), and the only children with ERMS showing intratumor diversity or with MYOD1 mutation-positive SRMS also died of disease. High resolution SNP array can be useful in evaluating genomic imbalances in pediatric RMS.

Benign fibrous histiocytomas (FH) can be subdivided into several morphological and clinical subgroups. Recently, gene fusions involving either one of two protein kinase C genes (PRKCB and PRKCD) or the ALK gene were described in FH. We here wanted to evaluate the frequency of PRKCB and PRKCD gene fusions in FH. Using interphase fluorescence in situ hybridization on sections from formalin-fixed paraffin-embedded (FFPE) tumors, 36 cases could be analyzed. PRKCB or PRKCD rearrangements were seen in five tumors: 1/7 regular, 0/3 aneurysmal, 0/6 cellular, 2/7 epithelioid, 0/1 atypical, 2/10 deep, and 0/2 metastatic lesions. We also evaluated the status of the ALK gene in selected cases, finding rearrangements in 3/7 epithelioid and 0/1 atypical lesions. To assess the gene fusion status of FH further, deep sequencing of RNA (RNA-Seq) was performed on FFPE tissue from eight cases with unknown gene fusion status, as well as on two FH and six soft tissue sarcomas with known gene fusions; of the latter eight positive controls, the expected fusion transcript was found in all but one, while 2/8 FH with unknown genetic status showed fusion transcripts, including a novel KIRREL/PRKCA chimera. Thus, also a third member of the PRKC family is involved in FH tumorigenesis. We conclude that gene fusions involving PRKC genes occur in several morphological (regular, cellular, aneurysmal, epithelioid) and clinical (cutaneous, deep) subsets of FH, but they seem to account for only a minority of the cases. In epithelioid lesions, however, rearrangements of PRKC or ALK were seen, as mutually exclusive events, in the majority (5/7) of cases. Finally, the study also shows that RNA-Seq is a promising tool for identifying gene fusions in FFPE tissues.

Patient-derived xenografts (PDX) and the Avatar, a single PDX mirroring an individual patient, are emerging tools in preclinical cancer research. However, the consequences of intratumor heterogeneity for PDX modeling of biomarkers, target identification, and treatment decisions remain under-explored. In this study, we undertook serial passaging and comprehensive molecular analysis of neuroblastoma orthotopic PDXs, which revealed strong intrinsic genetic, transcriptional, and phenotypic stability for more than 2 years. The PDXs showed preserved neuroblastoma-associated gene signatures that correlated with poor clinical outcome in a large cohort of patients with neuroblastoma. Furthermore, we captured spatial intratumor heterogeneity using ten PDXs from a single high-risk patient tumor. We observed diverse growth rates, transcriptional, proteomic, and phosphoproteomic profiles. PDX-derived transcriptional profiles were associated with diverse clinical characteristics in patients with high-risk neuroblastoma. These data suggest that high-risk neuroblastoma contains elements of both temporal stability and spatial intratumor heterogeneity, the latter of which complicates clinical translation of personalized PDX-Avatar studies into preclinical cancer research. Significance: These findings underpin the complexity of PDX modeling as a means to advance translational applications against neuroblastoma. (C) 2018 AACR.

Cultured cancer cells serve as important models for preclinical testing of anti-cancer compounds. However, the optimal conditions for retaining original tumor features during in vitro culturing of cancer cells have not been investigated in detail. Here we show that serum-free conditions are critical for maintaining an immature phenotype of neuroblastoma cells isolated from orthotopic patient-derived xenografts (PDXs). PDX cells could be grown either as spheres or adherent on laminin in serum-free conditions with retained patient-specific genomic aberrations as well as tumorigenic and metastatic capabilities. However, addition of serum led to morphological changes, neuronal differentiation and reduced cell proliferation. The epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) were central for PDX cell proliferation and MYCN expression, and also hindered the serum-induced differentiation. Although serum induced a robust expression of neurotrophin receptors, stimulation with their cognate ligands did not induce further sympathetic differentiation, which likely reflects a block in PDX cell differentiation capacity coupled to their tumor genotype. Finally, PDX cells cultured as spheres or adherent on laminin responded similarly to various cytotoxic drugs, suggesting that both conditions are suitable in vitro screening models for neuroblastoma-targeting compounds.

A major challenge to personalized oncology is that driver mutations vary among cancer cells inhabiting the same tumor. Whether this reflects principally disparate patterns of Darwinian evolution in different tumor regions has remained unexplored1–5. We mapped the prevalence of genetically distinct clones over 250 regions in 54 childhood cancers. This showed that primary tumors can simultaneously follow up to four evolutionary trajectories over different anatomic areas. The most common pattern consists of subclones with very few mutations confined to a single tumor region. The second most common is a stable coexistence, over vast areas, of clones characterized by changes in chromosome numbers. This is contrasted by a third, less frequent, pattern where a clone with driver mutations or structural chromosome rearrangements emerges through a clonal sweep to dominate an anatomical region. The fourth and rarest pattern is the local emergence of a myriad of clones with TP53 inactivation. Death from disease was limited to tumors exhibiting the two last, most dynamic patterns.

Background Mitochondrial diseases due to defective respiratory chain complex III (CIII) are relatively uncommon. The assembly of the eleven-subunit CIII is completed by the insertion of the Rieske iron-sulfur protein, a process for which BCS1L protein is indispensable. Mutations in the BCS1L gene constitute the most common diagnosed cause of CIII deficiency, and the phenotypic spectrum arising from mutations in this gene is wide. Results A case of CIII deficiency was investigated in depth to assess respiratory chain function and assembly, and brain, skeletal muscle and liver histology. Exome sequencing was performed to search for the causative mutation(s). The patient’s platelets and muscle mitochondria showed respiration defects and defective assembly of CIII was detected in fibroblast mitochondria. The patient was compound heterozygous for two novel mutations in BCS1L, c.306A > T and c.399delA. In the cerebral cortex a specific pattern of astrogliosis and widespread loss of microglia was observed. Further analysis showed loss of Kupffer cells in the liver. These changes were not found in infants suffering from GRACILE syndrome, the most severe BCS1L-related disorder causing early postnatal mortality, but were partially corroborated in a knock-in mouse model of BCS1L deficiency. Conclusions We describe two novel compound heterozygous mutations in BCS1L causing CIII deficiency. The pathogenicity of one of the mutations was unexpected and points to the importance of combining next generation sequencing with a biochemical approach when investigating these patients. We further show novel manifestations in brain, skeletal muscle and liver, including abnormality in specialized resident macrophages (microglia and Kupffer cells). These novel phenotypes forward our understanding of CIII deficiencies caused by BCS1L mutations. Electronic supplementary material The online version of this article (doi:10.1186/s13023-017-0624-2) contains supplementary material, which is available to authorized users.

Wilms tumor (WT) is a pediatric tumor of the kidney, the treatment of which includes heavy chemotherapy. Affected children would likely benefit from more targeted therapies with limited side effects. Establishment of relevant orthotopic WT xenografts is important to better understand mechanisms of WT growth and for preclinical drug testing.

Whether chromosome abnormalities observed in tumor cells may in some cases reflect low-grade somatic mosaicism for anomalies present already at zygote formation, rather than acquired somatic mutations, has for long remained a speculation. We here report a patient with Wilms tumor, where constitutional somatic mosaicism of trisomy 8 was detected in a previously healthy 2 ½-year-old boy. Single Nucleotide Polymorphism (SNP) array analysis of tumor tissue revealed a complex distribution of allele frequencies for chromosome 8 that could not be explained solely by mitotic events. Combined analysis of allele frequencies, chromosome banding, and fluorescence in situ hybridization revealed that the majority of tumor cells contained four copies of chromosome 8, with three distinct haplotypes at a 2:1:1 ratio. Because the patient had not been subject to organ transplantation, these findings indicated that the tumor karyotype evolved from a cell with trisomy 8 of meiotic origin, with subsequent somatic gain of one additional chromosome copy. Haplotype analysis was consistent with trisomy 8 through nondisjunction at meiosis I. Matched normal renal tissue or peripheral blood did not contain detectable amounts of cells with trisomy 8, consistent with the complete lack of mosaic trisomy 8 syndrome features in the patient. This case provides proof of principle for the hypothesis that tumor genotypes may in rare cases reflect meiotic rather than mitotic events, also in patients lacking syndromic features. © 2014 Wiley Periodicals, Inc.

e21503 Background: Neuroblastoma (NB) is a solid pediatric tumor that causes 15% of childhood cancer deaths. High-risk patients have 50 to 70% mortality after 5 years. Currently, the standard way to treat high risk patients is COJEC induction followed by surgery, chemotherapy with stem cell transplants, irradiation and immunotherapy. This intense treatment has significantly increased overall survival, but mortality is still high, survivors are prone to relapse and treatment resistance is a major clinical problem. COJEC induction consists in the cycled administration of the chemotherapeutics Cisplatin, Vincristine, Etoposide, Cyclophosphamide and Carboplatin. These drugs have been long used in the clinic, but a comprehensive preclinical study combining all of them had not been done. The aim of this project was to establish a PDX model for chemotherapy induction in high-risk NB and investigate tumor characteristics and response upon treatment. Methods: NB cells from previously developed high-risk PDX models were injected subcutaneously in nude mice. When tumors reached an appropriate size, they were treated with either a COJEC-like protocol or single agent induction with high dose cisplatin. After treatment, tumors were analyzed by immunohistochemistry, chromosomal copy number analysis and RNA sequencing. Results: Most mice responded well to COJEC induction and 8/10 tumors were reduced. Within the reduced tumors, 4 were subjected to total resection and relapsed after several weeks, mimicking the clinical situation. Cisplatin treatment alone led to no significant tumor reduction and 6/10 tumors were highly resistant. Thus, as for humans, multidrug induction is more successful in reducing tumor size but presents an uneven response with high relapse rate. The responsive tumors show a higher degree of cellular differentiation when compared to resistant tumors. Cisplatin treated tumors have accumulation of chromosomal aberrations when compared to controls, and subclonal dynamics are enhanced in resistant tumors. Conclusions: We established a PDX model for chemotherapy induction in high-risk NB. The results mimic clinical findings seen in high-risk NB patients. The model is useful to understand the mechanisms of treatment resistance and for the testing of novel therapies against high-risk NB.

Clear cell sarcoma of the kidney (CCSK) is a tumor affecting children with a median age of 3 years at diagnosis. The cell of origin of CCSK is unknown and data on the molecular changes giving rise to CCSK is scarce. This has hindered the identification of positive diagnostic markers and development of molecularly targeted treatment protocols for CCSK. We have characterized a panel of CCSK to gain information regarding its molecular profile and possible origin. High-resolution genomic analysis with single nucleotide polymorphism array of 37 tumors did not reveal any clues to the mechanisms behind tumor development as remarkably few genetic imbalances were found. Gene expression analysis revealed a highly characteristic gene signature, enriched for pathways involved in embryonic development, including kidney formation. The presence of markers for two different developmental lineages in the embryonic kidney was therefore investigated in the tumor cells. FOXD1 which identifies cells giving rise to stromal elements, and CITED1, a marker for cells primed for nephrogenic epithelial differentiation, were both highly expressed in CCSK. In addition, the early embryonic marker OSR1 was expressed at higher levels in CCSK than in Wilms tumor, normal fetal kidney or adult kidney. As this marker discriminates the intermediate mesoderm from other mesodermal structures, our study could suggest that CCSK arises from a mesodermal cell type that retains the capacity to initiate differentiation towards both nephrons and stroma, but remains locked in a primitive state. © 2014 Wiley Periodicals, Inc.

Soft tissue tumors in children under one year of age (infants) are rare. The etiology is usually unknown, with external factors or congenital birth defects and hereditary syndromes being recognized in only a small proportion of the cases. We ascertained the cytogenetic findings in 16 infants from whom tumor tissue had been obtained during a 25-year period. In eight of them, single nucleotide polymorphism (SNP) array analyses could also be performed. No constitutional chromosome aberrations were detected, and assessment of clinical files did not reveal any congenital or later anatomical defects. Three tumors--one infantile fibrosarcoma, one embryonal rhabdomyosarcoma, and one angiomatoid fibrous histiocytoma (AFH)--had abnormal karyotypes. As the AFH had an exchange between chromosome arms 12p and 15q, additional fluorescence in situ hybridization and reverse transcription-polymerase chain reaction analyses were performed, unexpectedly revealing an ETV6/NTRK3 fusion. Three of the eight tumors, including the AFH with an abnormal karyotype, analyzed by SNP array showed aberrations (loss of heterozygosity or imbalances). The present series suggests that the addition of array-based technologies is valuable for detecting underlying pathogenetic mechanisms.

Post-zygotic variation refers to genetic changes that arise in the soma of an individual and that are not usually inherited by the next generation. Although there is a paucity of research on such variation, emerging studies show that it is common: individuals are complex mosaics of genetically distinct cells, to such an extent that no two somatic cells are likely to have the exact same genome. Although most types of mutation can be involved in post-zygotic variation, structural genetic variants are likely to leave the largest genomic footprint. Somatic variation has diverse physiological roles and pathological consequences, particularly when acquired variants influence the clonal trajectories of the affected cells. Post-zygotic variation is an important confounder in medical genetic testing and a promising avenue for research: future studies could involve analyses of sorted and single cells from multiple tissue types to fully explore its potential.

Oncological therapy resembles a military force that eliminates the central power of a country (dominant clone of a cancer) to create a vacuum where insurgents (subclones) thrive and instigate rebellion (relapse). We suggest that military counterinsurgency doctrine can inspire a discussion of cancer that uniquely embraces both cancer cell evolution and tumour microenvironment.

PURPOSE: Whether childhood adrenocortical tumors (ACTs), choroid plexus tumors (CPTs), and rhabdomyosarcoma (RMS) are early manifestation of Li-Fraumeni syndrome (LFS) is uncertain. In this study, we evaluated the frequency of germline TP53 mutations and family history in a population-based series of patients. PATIENTS AND METHODS: We identified children (≤18 years) diagnosed between 1958 and 2008 with ACT (n = 3) or CPT (n = 7), or children ≤5 years with RMS (n = 29). Registry-based pedigree expansion was performed. RESULTS: No patients had a family history of classic LFS but 17 fulfilled Chompret or Eeles criteria. TP53 mutations were found in 1/3 ACT patients and 1/18 RMS patients; both were novel mutations. Of five tested CPT patients none had a detectable mutation. No excess of LFS associated tumors was observed, except for breast cancer in families of CPT patients. An overall increased cancer incidence was observed in families of patients with CPT [standardized incidence ratio (SIR) = 2.0; 95% CI: 1.1-3.5] due to excess of breast and female kidney cancer and in families of patients with RMS (SIR = 1.2; 95% CI: 0.9-1.7), due to excess of early-onset melanoma and male stomach cancer. CONCLUSION: Relatives of patients with childhood ACTs, CPTs, and RMSs showed no increased risk of LFS associated tumors. However, TP53 mutations could be found in these children irrespective of family history. Absence of LFS associated tumors may suggest the presence of other cancer syndromes. Improved knowledge about relatives' cancer risks could be helpful in counseling family members of children with cancer. Pediatr Blood Cancer © 2012 Wiley Periodicals, Inc.

BACKGROUND: Wilms tumor (WT) is the most common renal neoplasm in children. Histologically, most WTs consist of three tissue elements: blastema, epithelium, and stroma. Some cases also show diffuse or focal anaplastic features. Previous studies have shown that a predominance of blastemal cells in post-chemotherapy WT specimens is associated with a poor clinical course. However, there is currently no molecular marker for blastemal cells, and risk stratification for post-nephrectomy treatment is therefore often based on clinico-histological parameters alone. PROCEDURE: In the present study, three public gene expression microarray datasets, including 82 WTs and 8 normal fetal kidneys, were used to establish a consensus gene expression profile of WT. By bioinformatic analyses, 17 genes overexpressed in WT compared to fetal kidney were then selected for evaluation of their protein expression in WT cell lines and in the different histological components in paraffin-embedded WT tissue sections by immunofluorescence. RESULTS: Most of the evaluated proteins were expressed in all three common histological components. A prominent exception was SIX1, being expressed predominantly in blastemal elements in 24/25 pediatric cases containing blastema. Anaplastic elements exhibited highly variable SIX1-positivity. The SIX2 protein, known to be co-expressed with SIX1 during nephrogenesis, only exhibited blastemal-predominant expression in half of the SIX2 evaluated cases. CONCLUSIONS: Genes highly expressed in WT compared to fetal kidney are generally overexpressed in all of the three common WT tissue elements. An exception is the predominant expression of SIX1 in blastemal cells, hereby identifying this protein as a candidate marker for blastema. Pediatr Blood Cancer © 2011 Wiley Periodicals, Inc. (Less)

Neuroblastoma tumors frequently show loss of heterozygosity of chromosome 11q with a shortest region of overlap in the 11q23 region. These deletions are thought to cause inactivation of tumor suppressor genes leading to haploinsufficiency. Alternatively, micro-deletions could lead to gene fusion products that are tumor driving. To identify such events we analyzed a series of neuroblastomas by comparative genomic hybridization and single-nucleotide polymorphism arrays and integrated these data with Affymetrix mRNA profiling data with the bioinformatic tool R2 (http://r2.amc.nl). We identified three neuroblastoma samples with small interstitial deletions at 11q23, upstream of the forkhead-box R1 transcription factor (FOXR1). Genes at the proximal side of the deletion were fused to FOXR1, resulting in fusion transcripts of MLL-FOXR1 and PAFAH1B2-FOXR1. FOXR1 expression has only been detected in early embryogenesis. Affymetrix microarray analysis showed high FOXR1 mRNA expression exclusively in the neuroblastomas with micro-deletions and rare cases of other tumor types, including osteosarcoma cell line HOS. RNAi silencing of FOXR1 strongly inhibited proliferation of HOS cells and triggered apoptosis. Expression profiling of these cells and reporter assays suggested that FOXR1 is a negative regulator of fork-head box factor-mediated transcription. The neural crest stem cell line JoMa1 proliferates in culture conditional to activity of a MYC-ER transgene. Over-expression of the wild-type FOXR1 could functionally replace MYC and drive proliferation of JoMa1. We conclude that FOXR1 is recurrently activated in neuroblastoma by intrachromosomal deletion/fusion events, resulting in overexpression of fusion transcripts. Forkhead-box transcription factors have not been previously implicated in neuroblastoma pathogenesis. Furthermore, this is the first identification of intrachromosomal fusion genes in neuroblastoma. Oncogene (2012) 31, 1571-1581; doi:10.1038/onc.2011.344; published online 22 August 2011

Medulloblastomas are the most common malignant brain tumors in children. They express high levels of COX-2 and produce PGE2, which stimulates tumor cell proliferation. Human cytomegalovirus (HCMV) is prevalent in the human population and encodes proteins that provide immune evasion strategies and promote oncogenic transformation and oncomodulation. In particular, HCMV induces COX-2 expression; STAT3 phosphorylation; production of PGE2, vascular endothelial growth factor, and IL-6; and tumor formation in vivo. Here, we show that a large proportion of primary medulloblastomas and medulloblastoma cell lines are infected with HCMV and that COX-2 expression, along with PGE2 levels, in tumors is directly modulated by the virus. Our analysis indicated that both HCMV immediate-early proteins and late proteins are expressed in the majority of primary medulloblastomas. Remarkably, all of the human medulloblastoma cell lines that we analyzed contained HCMV DNA and RNA and expressed HCMV proteins at various levels in vitro. When engrafted into immunocompromised mice, human medulloblastoma cells induced expression of HCMV proteins. HCMV and COX-2 expression correlated in primary tumors, cell lines, and medulloblastoma xenografts. The antiviral drug valganciclovir and the specific COX-2 inhibitor celecoxib prevented HCMV replication in vitro and inhibited PGE2 production and reduced medulloblastoma tumor cell growth both in vitro and in vivo. Ganciclovir did not affect the growth of HCMV-negative tumor cell lines. These findings imply an important role for HCMV in medulloblastoma and suggest HCMV as a novel therapeutic target for this tumor.

Acute myeloid leukemia (AML) is an aggressive disease associated with poor prognosis, hence, new therapeutic strategies are needed. The AML cells reside in the bone marrow niche and depend on external signals such as cytokines for their growth and survival. In contrast to providing a supporting role, cytokines can act as negative regulators of AML cells. We previously identified that interleukin 4 (IL-4) induces apoptosis of AML cells in a Stat6-dependent manner and demonstrated strong anti-leukemic activity of IL-4 in a murine AML model driven by MLL-AF9 (Peña-Martínez et al., Leukemia 2017). Ectopic expression of IL-4 in AML cells resulted in prolonged survival in C57BL/6 mice, with mice almost devoid of leukemia cells in bone marrow and spleen upon the time of sacrifice. However, the IL-4-induced apoptosis of AML cells did not fully explain the strong antileukemic activity of IL-4 in vivo. To investigate whether the anti-leukemic effect of ectopic IL-4 expression in vivo was microenvironment dependent, we used IL-2 receptor gamma(-/-) immunodeficient mice, which lack a functional IL-4 receptor type I complex, as recipients for transplantation of murine MLL-AF9 AML cells. Notably, there was no antileukemic activity of IL-4 in these mice, suggesting that the in vivo anti-leukemic effect of IL-4 was predominantly microenvironment-dependent. To identify the cell population that was responsible for the antileukemic effect of IL-4 stimulation, we performed flow cytometry analysis of bone marrow and spleen cells in mice exposed to high IL-4 levels. We found that the anti-leukemic activity of IL-4 was associated with a significant expansion of F4/80+ macrophages in both the bone marrow and spleen, which was confirmed by immunohistochemistry. To determine whether macrophages were responsible for the anti-leukemic effect of IL-4, we depleted macrophages with clodronate liposomes in mice receiving IL-4-secreting AML cells. Depletion of macrophages partially rescued the anti-leukemic effect of IL-4, indicating that macrophages stimulated with IL-4 were responsible for the antileukemic activity. Consistent with these findings, in macrophage differentiation assays with either murine or human monocytes, IL-4 enhanced the phagocytosis of leukemia cells. To characterize the IL-4-induced macrophages, we performed RNA sequencing of F4/80+ macrophages from animals exposed to high IL-4 levels. Gene expression analysis revealed IL-4-induced M2 polarization of the macrophages, as key markers of M2 activation such as Arg1, Retnla and Chil3 were upregulated. Moreover, consistent with the in vitro findings, gene set enrichment analysis revealed an enrichment of phagocytosis signatures in the macrophages stimulated with IL-4. Strikingly, we found that exposure of AML cells to IL-4 resulted in strong upregulation of CD47, a 'don't eat me signal', which protects cells from macrophage-mediated phagocytosis. By CRISPR/Cas9-mediated disruption of Stat6 in IL-4 treated AML cells, we found that the CD47 upregulation was Stat6 dependent, recognizing a previously unknown link between IL-4 signalling and CD47 upregulation. Consistent with this finding, combined antibody blockade of CD47 on leukemia cells and IL-4 stimulation of macrophages resulted in enhanced phagocytosis of AML cells. Taken together, this study shows that IL-4 has anti-leukemic activity by activating macrophages, findings that might translate into new immunotherapeutic opportunities in AML and possibly other malignancies. Disclosures No relevant conflicts of interest to declare.

Neuroblastoma is one of the most common paediatric malignancies. Detection of somatic genetic alterations in this tumour is instrumental for its risk stratification and treatment. On the other hand, an absence of detected chromosomal imbalances in neuroblastoma biopsies is difficult to interpret because it is unclear whether this situation truly reflects the tumour genome or if it is due to suboptimal sampling. We here present a neuroblastoma in the left adrenal of a newborn. The tumour was subjected to single-nucleotide polymorphism array analysis of five tumour regions with >80% tumour cells in histological mirror sections. This revealed no aberrations compared with a normal reference sample from the patient. Whole exome sequencing identified two single-nucleotide variants present in most tumour regions, corroborating that the tumour resulted from monoclonal expansion. Our data provide proof-of-principle that rare cases of neuroblastoma can have a normal whole genome copy number and allelic profile.

The 8p11 myeloproliferative syndrome (EMS), also referred to as stem cell leukemia/lymphoma, is a chronic myeloproliferative disorder that rapidly progresses into acute leukemia. Molecularly, EMS is characterized by fusion of various partner genes to the FGFR1 gene, resulting in constitutive activation of the tyrosine kinases in FGFR1. To date, no previous study has addressed the functional consequences of ectopic FGFR1 expression in the potentially most relevant cellular context, that of normal primary human hematopoietic cells. Herein, we report that expression of ZMYM2/FGFR1 (previously known as ZNF198/FGFR1) or BCR/FGFR1 in normal human CD34+ cells from umbilical-cord blood leads to increased cellular proliferation and differentiation toward the erythroid lineage in vitro. In immunodeficient mice, expression of ZMYM2/FGFR1 or BCR/FGFR1 in human cells induces several features of human EMS, including expansion of several myeloid cell lineages and accumulation of blasts in bone marrow. Moreover, bone marrow fibrosis together with increased extramedullary hematopoiesis is observed. This study suggests that FGFR1 fusion oncogenes, by themselves, are capable of initiating an EMS-like disorder, and provides the first humanized model of a myeloproliferative disorder transforming into acute leukemia in mice. The established in vivo EMS model should provide a valuable tool for future studies of this disorder.

Wilms tumors in patients with constitutional WT1 mutations are examples of Knudson's tumor suppressor paradigm, with somatic inactivation of the second allele occurring through 11p loss of heterozygosity. The time point of this second hit has remained unknown. We analyzed seven Wilms tumors from two patients with constitutional WT1 mutations by whole exome sequencing and genomic array. All tumors exhibited wild type WT1 loss through uniparental isodisomy. Each tumor had a unique genomic breakpoint in 11p, typically accompanied by a private activating mutation of CTNNB1. Hence, convergent evolution rather than field carcinogenesis underlies multifocal tumors in WT1 mutation carriers.

High hypoxia-inducible factor-2α (HIF-2α) protein levels predict poor outcome in neuroblastoma, and hypoxia dedifferentiates cultured neuroblastoma cells toward a neural crest-like phenotype. Here, we identify HIF-2α as a marker of normoxic neural crest-like neuroblastoma tumor-initiating/stem cells (TICs) isolated from patient bone marrows. Knockdown of HIF-2α reduced VEGF expression and induced partial sympathetic neuronal differentiation when these TICs were grown in vitro under stem cell-promoting conditions. Xenograft tumors of HIF-2α-silenced cells were widely necrotic, poorly vascularized, and resembled the bulk of tumor cells in clinical neuroblastomas by expressing additional sympathetic neuronal markers, whereas control tumors were immature, well-vascularized, and stroma-rich. Thus, HIF-2α maintains an undifferentiated state of neuroblastoma TICs. Because low differentiation is associated with poor outcome and angiogenesis is crucial for tumor growth, HIF-2α is an attractive target for neuroblastoma therapy.