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Background & Aims: Hepatoblastoma (HB) is a rare disease. Nevertheless, it is the predominant pediatric liver cancer, with limited therapeutic options for patients with aggressive tumors. Herein, we aimed to uncover the mechanisms of HB pathobiology and to identify new biomarkers and therapeutic targets in a move towards precision medicine for patients with advanced HB. Methods: We performed a comprehensive genomic, transcriptomic and epigenomic characterization of 159 clinically annotated samples from 113 patients with HB, using high-throughput technologies. Results: We discovered a widespread epigenetic footprint of HB that includes hyperediting of the tumor suppressor BLCAP concomitant with a genome-wide dysregulation of RNA editing and the overexpression of mainly non-coding genes of the oncogenic 14q32 DLK1-DIO3 locus. By unsupervised analysis, we identified 2 epigenomic clusters (Epi-CA, Epi-CB) with distinct degrees of DNA hypomethylation and CpG island hypermethylation that are associated with the C1/C2/C2B transcriptomic subtypes. Based on these findings, we defined the first molecular risk stratification of HB (MRS-HB), which encompasses 3 main prognostic categories and improves the current clinical risk stratification approach. The MRS-3 category (28%), defined by strong 14q32 locus expression and Epi-CB methylation features, was characterized by CTNNB1 and NFE2L2 mutations, a progenitor-like phenotype and clinical aggressiveness. Finally, we identified choline kinase alpha as a promising therapeutic target for intermediate and high-risk HBs, as its inhibition in HB cell lines and patient-derived xenografts strongly abrogated tumor growth. Conclusions: These findings provide a detailed insight into the molecular features of HB and could be used to improve current clinical stratification approaches and to develop treatments for patients with HB. Lay summary: Hepatoblastoma is a rare childhood liver cancer that has been understudied. We have used cutting-edge technologies to expand our molecular knowledge of this cancer. Our biological findings can be used to improve clinical management and pave the way for the development of novel therapies for this cancer. (c) 2020 European Association for the Study of the Liver. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)., This article was possible thanks to the inputs from the Instituto de Salud Carlos III, ISCIII (PI09/00751, PI10/02082, PI13/02340). The project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 668596 (ChiLTERN) and grant agreement No 826121 (iPC). JCR is supported by the Catalan Agency for Management of University and Research Grants (AGAUR, 2019 FI_B01024). LT is supported by an Accelerator Award (CRUCK, AECC, AIRC) (HUNTER, C9380/A26813). DS is supported by the Gilead Research Scholar in Liver Disease. JML is supported by the European Union's Horizon 2020 research and innovation programme (HEPCAR, 667273-2), Institucio Catalana de Recerca i Estudis Avancats (ICREA), U.S. Department of Defense (CA150272P3), an Accelerator Award (CRUCK, AECC, AIRC) (HUNTER, C9380/A26813), National Cancer Institute, Tisch Cancer Institute (P30-CA196521), Samuel Waxman Cancer Research Foundation, Spanish National Health Institute (SAF2016-76390) and AGAUR (SGR-1358). CA and MRS were supported by Ramon y Cajal (RYC-2010-07249) and Miguel Servet (CPII14/00021) programs of the Ministry of Science and Innovation of Spain and ISCIII, respectively. CA, MRS and MS received funding from CIBERehd (CB06/04/0033) and AGAUR (2017-SGR-490). IGTP is a member of the CERCA network of institutes. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Identification of new treatments for relapsing pediatric cancer is an unmet clinical need and a societal challenge. Liver cancer occurrence in infancy, 1.5 for million children per year, falls far below the threshold of interest for dedicated drug development programs, and this disease is so rare that it is very difficult to gather enough children into a phase II clinical trial. Here, we present the establishment of an unprecedented preclinical platform of 24 pediatric liver cancer patient-derived xenografts (PLC-PDXs) from 20 hepatoblastomas (HBs), 1 transitional liver cell tumor (TCLT), 1 hepatocellular carcinoma, and 2 malignant rhabdoid tumors. Cytogenetic array and mutational analysis of the parental tumors and the corresponding PLC-PDXs show high conservation of the molecular features of the parental tumors. The histology of PLC-PDXs is strikingly similar to that observed in primary tumors and recapitulates the heterogeneity of recurrent disease observed in the clinic. Tumor growth in the mouse is strongly associated with elevated circulating alpha-fetoprotein (AFP), low rate of necrosis/fibrosis after treatment, and gain of chromosome 20, all indicators of resistance to chemotherapy and poor outcome. Accordingly, the ability of a tumor to generate PLC-PDX is predictive of poor prognosis. Exposure of PLC-PDXs to standards of care or therapeutic options already in use for other pediatric malignancies revealed unique response profiles in these models. Among these, the irinotecan/temozolomide combination induced strong tumor regression in the TCLT and in a model derived from an AFP-negative relapsing HB. Conclusion: These results provide evidence that PLC-PDX preclinical platform can strongly contribute to accelerate the identification and diversification of anticancer treatment for aggressive subtypes of pediatric liver cancer. (Hepatology 2016;64:1121-1135)

Introduction Advances in chemotherapy and surgery have greatly improved overall survival rates of children with hepatoblastoma (HB). However, prognosis still remains quite poor for HB patients with high risk characteristics such as portal/hepatic venous macrovascular involvement, extrahepatic growth, high extent of the disease, and metastasis, as shown by studies of the International Childhood Liver Tumours Strategy Group (SIOPEL). The aim of our trinational study was to validate recently identified genetic and transcriptional characteristics of HB as prognostic biomarkers to aid development of a new risk stratification system based on clinical and biological factors. Experimental procedures Tumor tissue of a cohort of 171 hepatoblastoma patients from Germany, France and Spain was analyzed for mutations in CTNNB1, NFE2L2 and TERT (Eichenmüller et al., J Hepatol 2014; 61:1312-1320) by Sanger sequencing and the 16-gene signature (Cairo et al., Cancer Cell 2008; 14:471-84) by real-time PCR. Kaplan-Meier estimates of specific survival time in the various groups were compared using the log-rank Mantel-Cox test. Results Mutations of CTNNB1, NFE2L2, and TERT were found in 133 (78%), 10 (6%), and 10 (6%) patients, respectively. The adverse C2 subtype of the 16-gene signature was detected in 60 (35%) patients. Kaplan-Meier analyses depicted a significant association of the 16-gene signature (P Conclusions We have validated the 16-gene signature and NFE2L2 mutations as highly prognostic biomarkers in HB and propose a new stratification system that is based on the combination of clinical and biological factors, which might facilitate more tailored and risk-adapted therapies and thus better outcome of high risk patients in the future. Citation Format: Stefano Cairo, Carolina Armengol, Beate Häberle, Marina Simon-Coma, Catherine Guettier, Ivo Leuschner, Marie-Annick Buendia, Dietrich von Schweinitz, Roland Kappler. Combined clinical and biological risk stratification in pediatric hepatoblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4724. doi:10.1158/1538-7445.AM2017-4724