The landscape of genomic alterations across childhood cancers a list of authors and affiliations appears at the end of the paper. OPENPan-cancer analyses that examine commonalities and differences among various cancer types have emerged as a powerful way to obtain novel insights into cancer biology. Here we present a comprehensive analysis of genetic alterations in a pan-cancer cohort including 961 tumours from children, adolescents, and young adults, comprising 24 distinct molecular types of cancer. Using a standardized workflow, we identified marked differences in terms of mutation frequency and significantly mutated genes in comparison to previously analysed adult cancers. Genetic alterations in 149 putative cancer driver genes separate the tumours into two classes: small mutation and structural/copy-number variant (correlating with germline variants). Structural variants, hyperdiploidy, and chromothripsis are linked to TP53 mutation status and mutational signatures. Our data suggest that 7-8% of the children in this cohort carry an unambiguous predisposing germline variant and that nearly 50% of paediatric neoplasms harbour a potentially druggable event, which is highly relevant for the design of future clinical trials.Cure rates for childhood cancers have increased to about 80% in recent decades, but cancer is still the leading cause of death by disease in the developed world among children over one year of age 1,2 . Furthermore, many children who survive cancer suffer from long-term sequelae of surgery, cytotoxic chemotherapy, and radiotherapy, including mental disabilities, organ toxicities, and secondary cancers 3 . A crucial step in developing more specific and less damaging therapies is the unravelling of the complete genetic repertoire of paediatric malignancies, which differ from adult malignancies in terms of their histopathological entities and molecular subtypes 4 . Over the past few years, many entityspecific sequencing efforts have been launched, but the few paediatric pan-cancer studies thus far have focused only on mutation frequencies, germline predisposition, and alterations in epigenetic regulators [4][5][6] .We have carried out a broad exploration of cancers in children, adolescents, and young adults, by incorporating small mutations and copy-number or structural variants on somatic and germline levels, and by identifying putative cancer genes and comparing them to those previously reported in adult cancers by The Cancer Genome Atlas (TCGA) 7 . We have also examined mutational signatures and potential drug targets. The compendium of genetic alterations presented here is available to the scientific community at http://www.pedpancan.com.This integrative analysis includes 24 types of cancer and covers all major childhood cancer entities, many of which occur exclusively in children 8 (Fig. 1, Supplementary Table 1). Ninety-five per cent of the patients in this study were diagnosed during childhood or adolescence (aged 18 years or younger) and 5% as young adults (up to 25 years) (Extended Data ...

Ewing tumors (ET) are highly malignant, localized in bone or soft tissue, and are molecularly defined by ews/ets translocations. DNA microarray analysis revealed a relationship of ET to both endothelium and fetal neural crest. We identified expression of histone methyltransferase enhancer of Zeste, Drosophila, Homolog 2 (EZH2) to be increased in ET. Suppressive activity of EZH2 maintains stemness in normal and malignant cells. Here, we found EWS/FLI1 bound to the EZH2 promoter in vivo, and induced EZH2 expression in ET and mesenchymal stem cells. Down-regulation of EZH2 by RNA interference in ET suppressed oncogenic transformation by inhibiting clonogenicity in vitro. Similarly, tumor development and metastasis was suppressed in immunodeficient Rag2 ؊/؊ ␥C ؊/؊ mice. EZH2-mediated gene silencing was shown to be dependent on histone deacetylase (HDAC) activity. Subsequent microarray analysis of EZH2 knock down, HDAC-inhibitor treatment and confirmation in independent assays revealed an undifferentiated phenotype maintained by EZH2 in ET. EZH2 regulated stemness genes such as nerve growth factor receptor (NGFR), as well as genes involved in neuroectodermal and endothelial differentiation (EMP1, EPHB2, GFAP, and GAP43). These data suggest that EZH2 might have a central role in ET pathology by shaping the oncogenicity and stem cell phenotype of this tumor.epigenetic regulation ͉ Ewing tumor ͉ stemness E wing tumors (ET) are highly malignant tumors with an approximate incidence of 3.3/10 6 in children under the age of 15. ET are characterized by early metastases, and metastatic spread is commonly hematogeneous. ET were originally described by Ewing in 1921 as endothelioma of the bone (1), and we confirmed this endothelial signature by microarray analysis (2). ET are molecularly defined by ews/ets translocations. Translocation-derived chimeric transcription factors yield transactivation, transformation, and the highly malignant phenotype. In mice, EWS/FLI1 transforms bone marrow derived or mesenchymal progenitor cells, and generates tumors (3, 4), which have features of ET. Also, inhibition of EWS/FLI1 expression may allow ET cells to recover the phenotype of their presumed mesenchymal stem cell (MSC) progenitor (5). Multipotent MSCs represent a leading candidate for primary transformation in ET. We revealed a relationship of ET to both endothelial and fetal neural crest-derived cells (2), after having demonstrated neuroectodermal histogenesis of ET in 1985 (6). Based on our recent study, we postulated in 2004 that the ET stem cell is arrested at early mesenchyme development from the neuroectodermal germ layer, and, thus, the ET stem cell is a neuronal crest-derived stem cell at transition to mesenchymal endothelial development, residing in the bone marrow. However, ectopic EWS/FLI1 expression resulted in a neural phenotype, raising the possibility that transdifferentiation or lineage promiscuity may be an alternative to the MSC histogenetic origin hypothesis of ET (7).We used high density DNA microarrays for the ident...

Cytotoxic T lymphocyte (CTL) mediated tumor immunity against major histocompatibility antigen (MHC) class I-positive but class II-negative tumors often requires help from CD4+ T cells. These CD4 cells are activated by MHC class II-positive cells that present tumor derived antigens. Considering that different antigen presenting cells, such as B cells, macrophages and dendritic cells compete for antigen and influence the outcome of an immune response, we analyzed tumor immunity in B cell-deficient mice. These mice appear normal with regard to T cell immunity and tolerance to some pure foreign antigens. We show here that the low immunogenicity of tumors is caused by B cells whose presence in the priming phase results in disabled CD4+ T cell help for CTL mediated tumor immunity. Instead, in the presence of B cells, a non-protective humoral immune response is induced. Our results may explain the enigmatic observation that tumor-reactive antibodies occur frequently in cancer patients.

Round cell sarcomas harboring CIC-DUX4 fusions have recently been described as highly aggressive soft tissue tumors of children and young adults. Due to partial morphologic and immunohistochemical overlap with Ewing sarcoma (ES), CIC-DUX4-positive tumors have generally been classified as Ewing sarcoma-like and managed similarly, however, a systematic comparison at the molecular and immunohistochemical levels between these two groups has not yet been conducted. Based on an initial observation that CIC-DUX4-positive tumors show nuclear immunoreactivity for WT1 and ETS transcription factors, FLI1 and ERG, we performed a detailed immunohistochemical and molecular analysis including these markers, to further investigate the relationship between CIC-DUX4 tumors and ES. The study group included 21 CIC-DUX4-positive sarcomas and 20 EWSR1-rearranged ES. Immunohistochemically, CIC-DUX4 sarcomas showed membranous CD99 positivity in 18 (86%) cases, but only 5 (24%) with a diffuse pattern, while WT1 and FLI1 were strongly positive in all cases. ERG was positive in 18% of cases. All ES expressed CD99 and FLI1, while ERG positivity was only seen in EWSR1-ERG fusion positive ES. WT1 was negative in all ES. Expression profiling validated by q-PCR revealed a distinct gene signature associated with CIC-DUX4 fusion, with upregulation of ETS transcription factors (ETV4, ETV1 and ETV5) and WT1, among top overexpressed genes compared to ES, other sarcomas and normal tissue. In conclusion, the distinct gene signature and immunoprofile of CIC-DUX4 sarcomas suggest a distinct pathogenesis from ES. The consistent WT1 expression may provide a useful clue in the diagnosis in the context of round cell sarcomas negative for EWSR1-rearrangement.