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 ...

A patient with b E /b 0 -thalassemia major was converted to transfusion-independence 4.5 years ago by lentiviral gene transfer in hematopoietic stem cells while showing a myeloid-biased cell clone. Induced pluripotent stem cells (iPSCs) are a potential alternative source of hematopoietic stem cells. If fetal to adult globin class, switching does not occur in vivo in iPSC-derived erythroid cells, b-globin gene transfer would be unnecessary. To investigate both vector integration skewing and the potential use of iPSCs for the treatment of thalassemia, we derived iPSCs from the thalassemia gene therapy patient and compared iPSCderived hematopoietic cells to their natural isogenic somatic counterparts. In NSG immunodeficient mice, embryonic to fetal and a partial fetal to adult globin class switching were observed, indicating that the gene transfer is likely necessary for iPSC-based therapy of the bhemoglobinopathies. Lentivector integration occurred in regions of low and high genotoxicity. Surprisingly, common integration sites (CIS) were identified across those iPSCs and cells retrieved from isogenic and nonisogenic gene therapy patients with b-thalassemia and adrenoleukodystrophy, respectively. This suggests that CIS observed in the absence of overt tumorigenesis result from nonrandom lentiviral integration rather than oncogenic in vivo selection. These findings bring the use of iPSCs closer to practicality and further clarify our interpretation of genome-wide lentivector integration.

Lentiviral vectors hold great promise for the genetic correction of various inherited diseases. However, lentiviral vector biology is still not completely understood and warrants the precise decoding of molecular mechanisms underlying integration and post-translational modification. This study investigated a series of self-inactivating (SIN) and full long terminal repeat (LTR) lentiviral vectors that contained different types of promoters with or without a transgene to gain deeper insights in lentiviral target site selection and potential perturbation of cellular gene expression. Using an optimized nonrestrictive linear amplification-mediated polymerase chain reaction (nrLAM-PCR) protocol, vector structure-dependent integration site profiles were observed upon transduction of mouse lin hematopoietic progenitors in vitro. Initial target site selection mainly depended on the presence of the promoter while being independent of its nature. Despite the increased propensity for read-through transcription of SIN lentiviral vectors, the incidence of viral-cellular fusion transcript formation involving the canonical viral splice donor or cryptic splice sites was reduced in both unselected primary lin cells and transformed 32D cells. Moreover, the strength of the internal promoter in vectors with SIN LTRs is decisive for in vitro selection and for the abundance of chimeric transcripts, which are decreased by moderately active promoters. These results will help to better understand vector biology and to optimize therapeutic vectors for future gene therapy applications.

Next generation sequencing approaches are identifying a plethora of mutations in a variety of human cancers. However, the clinical implications of these new findings are still limited, since the identification of cancer driving mutations is hampered by the co-occurrence of several bystander and progression related events. Here we developed a forward genetics approach based on a new lentiviral vector-based insertional mutagen aimed at identifying cancer initiating genes that are relevant in human hepatocarcinogenesis. We generated a replication-defective lentiviral vector (LV) engineered with long terminal repeats carrying hepatospecific enhancers able to deregulate genes upon integration. Differently from retroviruses and transposons, our replication-defective LV provides a single round of integration shortly after the administration, thus tagging mainly early events in carcinogenesis. A single administration of LV in newborn mice was able to induce hepatocellular carcinoma (HCC) in 3 clinically relevant models of hepatocarcinogenesis: in 30% of Cdkn2a deficient mice (P=0.005 Vs untreated), in 27% of liver-specific Pten deficient mice (P=0.04) and in 75% of wild type mice coupled to CCl4 administration (P=0.002). From 30 LV-induced HCCs we retrieved 172 LV integrations that allowed the identification of Braf, Fign, Sos1 and Rtl1 as candidate cancer loci. The causative role of these 4 genes in HCC was experimentally validated in vivo by forced expression in the mouse liver. Whole transcriptome gene expression analysis allowed unveiling the molecular pathways deregulated by the newly identified cancer genes. HCC induced by integration at the paternally expressed gene Rtl1 (mapping within the imprinted Dlk1-Dio3 region) displayed the peculiar upregulation of oxidative phosphorylation genes. Conversely, LV-mediated upregulation of Braf and Fign caused the overexpression of the maternally expressed microRNAs encoded within the Dlk1-Dio3 region in HCCs which display the downregulation of oxidative phosphorylation genes. These findings highlighted a relevant role of Dlk1-Dio3 region in HCCs and in the regulation of metabolism. Additionally, we showed that WNT pathway may represent a target of the new enigmatic oncogene Fign. We then analyzed different human HCCs collections induced by HBV or HCV (N tot of HCC=221). We found that all the newly identified cancer genes were significantly upregulated and amplified or deleted in human HCCs. Moreover, we showed that the expression level of the new liver cancer genes or the specific gene expression signature caused by their upregulation can efficiently distinguish HCC patients characterized by poor survival. Overall, we developed a new insertional mutagen by which we identified new clinically relevant liver cancer genes that may provide novel prognostic markers and therapeutic targets for the diagnosis and treatment of human HCCs. Citation Format: Marco Ranzani, Daniela Cesana, Cynthia C. Bartholomä, Francesca Sanvito, Michela Riba, Mauro Pala, Fabrizio Benedicenti, Pierangela Gallina, Stefano Annunziato, Lucia Sergi Sergi, Stefania Merella, Alessandro Bulfone, Claudio Doglioni, Christof von Kalle, Yoon Jun Kim, Manfred Schmidt, Elia Stupka, Giovanni Tonon, Luigi Naldini, Eugenio Montini. Lentiviral vector-based insertional mutagenesis identifies new clinically relevant liver cancer genes. [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 3169. doi:10.1158/1538-7445.AM2013-3169

Gene TarGeTinG and Gene CorreCTion ii transcription start site, coding region start and end sites etc. Our tool is highly appropriate for in-depth quantitative analysis of biosafety and transduction efficiency of viral vectors.

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