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Pediatric liver cancers (PLC) comprise diverse diseases affecting infants, children, and adolescents. Despite overall good prognosis, PLCs display heterogeneous response to chemotherapy. Integrated genomic analysis of 126 pediatric liver tumors showed a continuum of driver mechanisms associated with patient age, including new targetable oncogenes. In 10% of patients with hepatoblastoma, all before three years old, we identified a mosaic premalignant clonal expansion of cells altered at the 11p15.5 locus. Analysis of spatial and longitudinal heterogeneity revealed an important plasticity between “hepatocytic,” “liver progenitor,” and “mesenchymal” molecular subgroups of hepatoblastoma. We showed that during chemotherapy, “liver progenitor” cells accumulated massive loads of cisplatin-induced mutations with a specific mutational signature, leading to the development of heavily mutated relapses and metastases. Drug screening in PLC cell lines identified promising targets for cisplatin-resistant progenitor cells, validated in mouse xenograft experiments. These data provide new insights into cisplatin resistance mechanisms in PLC and suggest alternative therapies.Significance:PLCs are deadly when they resist chemotherapy, with limited alternative treatment options. Using a multiomics approach, we identified PLC driver genes and the cellular phenotype at the origin of cisplatin resistance. We validated new treatments targeting these molecular features in cell lines and xenografts.This article is highlighted in the In This Issue feature, p. 2355

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Purpose:Macrotrabecular-massive hepatocellular carcinoma (MTM-HCC) is a novel morphological subtype of HCC associated with early relapse after resection or percutaneous ablation, independently of classical clinical and radiological prognostic factors. The aim of the present study was to identify immunohistochemical markers of MTM-HCC, to ease its diagnosis and implementation into clinical practice.Experimental Design:To identify potential biomarkers of MTM-HCC, we first analyzed gene expression profiling data from The Cancer Genome Atlas study and further selected two candidate biomarkers. Performance of both biomarkers for diagnosis of MTM-HCC was further tested by immunohistochemistry in two independent series of 67 and 132 HCC biopsy samples.Results:Analysis of RNA sequencing data showed that MTM-HCC was characterized by a high expression of neoangiogenesis-related genes. Two candidate biomarkers, Endothelial-Specific Molecule 1 (ESM1) and Carbonic Anhydrase IX (CAIX), were selected. In the discovery series, sensitivity and specificity of ESM1 expression by stromal endothelial cells for the detection of MTM-HCC were 97% (28/29), and 92% (35/38), respectively. Sensitivity and specificity of CAIX were 48% (14/29) and 89% (34/38). In the validation set, sensitivity and specificity of ESM1 for the identification of MTM-HCC were 93% (14/15) and 91% (107/117), respectively. Interobserver agreement for ESM1 assessment was good in both series (Cohen Kappa 0.77 and 0.76).Conclusions:Using a molecular-driven selection of biomarkers, we identified ESM1 as a reliable microenvironment immunohistochemical marker of MTM-HCC. The results represent a step toward the implementation of HCC morpho-molecular subtyping into clinical practice.

Pediatric liver cancers (PLCs) comprise diverse diseases affecting infants, children and adolescents. Despite overall good prognosis, PLCs display heterogeneous response to chemotherapy. Integrated genomic analysis of 126 pediatric liver tumors showed a continuum of driver mechanisms associated with patient age, including new targetable oncogenes. In 10% of hepatoblastoma patients, all before 3 years old, we identified a mosaic premalignant clonal expansion of cells altered at the 11p15.5 locus. Analysis of spatial and longitudinal heterogeneity revealed an important plasticity between 'Hepatocytic', 'Liver Progenitor' and 'Mesenchymal' molecular subgroups of hepatoblastoma. We showed that during chemotherapy, 'Liver Progenitor' cells accumulated massive loads of cisplatin-induced mutations with a specific mutational signature, leading to the development of heavily mutated relapses and metastases. Drug screening in PLC cell lines identified promising targets for cisplatin-resistant progenitor cells, validated in mouse xenograft experiments. These data provide new insights into cisplatin resistance mechanisms in PLC and suggest alternative therapies., info:eu-repo/semantics/published

Purpose: Macrotrabecular-massive hepatocellular carcinoma (MTM-HCC) is a novel morphological subtype of HCC associated with early relapse after resection or percutaneous ablation, independently of classical clinical and radiological prognostic factors. The aim of the present study was to identify immunohistochemical markers of MTM-HCC, to ease its diagnosis and implementation into clinical practice. Experimental Design: To identify potential biomarkers of MTM-HCC, we first analyzed gene expression profiling data from The Cancer Genome Atlas study and further selected two candidate biomarkers. Performance of both biomarkers for diagnosis of MTM-HCC was further tested by immunohistochemistry in two independent series of 67 and 132 HCC biopsy samples. Results: Analysis of RNA sequencing data showed that MTM-HCC was characterized by a high expression of neoangiogenesis-related genes. Two candidate biomarkers, Endothelial-Specific Molecule 1 (ESM1) and Carbonic Anhydrase IX (CAIX), were selected. In the discovery series, sensitivity and specificity of ESM1 expression by stromal endothelial cells for the detection of MTM-HCC were 97% (28/29), and 92% (35/38), respectively. Sensitivity and specificity of CAIX were 48% (14/29) and 89% (34/38). In the validation set, sensitivity and specificity of ESM1 for the identification of MTM-HCC were 93% (14/15) and 91% (107/117), respectively. Interobserver agreement for ESM1 assessment was good in both series (Cohen Kappa 0.77 and 0.76). Conclusions: Using a molecular-driven selection of biomarkers, we identified ESM1 as a reliable microenvironment immunohistochemical marker of MTM-HCC. The results represent a step toward the implementation of HCC morpho-molecular subtyping into clinical practice.

International audience; BACKGROUND AND AIMS: Hepatocellular carcinomas (HCCs) are heterogeneous aggressive tumors with low rates of response to treatment at advanced stages. We screened a large panel of liver cancer cell lines (LCCLs) to identify agents that might be effective against HCC and markers of therapeutic response.METHODS: We performed whole-exome RNA and microRNA sequencing and quantification of 126 proteins in 34 LCCLs. We screened 31 anticancer agents for their ability to decrease cell viability. We compared genetic, RNA, and protein profiles of LCCLs with those of primary HCC samples and searched for markers of response.RESULTS: The protein, RNA and mutational signatures of the LCCLs were similar to those of the proliferation class of HCC, which is the most aggressive tumor type. Cell lines with alterations in genes encoding members of the Ras-MAPK signaling pathway and that required fibroblast growth factor (FGF)19 signaling via FGF receptor 4 for survival were more sensitive to trametinib than to FGF receptor 4 inhibitors. Amplification of FGF19 resulted in increased activity of FGF19 only in tumor cells that kept a gene expression pattern of hepatocyte differentiation. We identified single agents and combinations of agents that reduced viability of cells with features of the progenitor subclass of HCC. LCCLs with inactivating mutations in TSC1 and TSC2 were sensitive to the mammalian target of rapamycin inhibitor rapamycin, and cells with inactivating mutations in TP53 were sensitive to the Aurora kinase A inhibitor alisertib. Amplification of MET was associated with hypersensitivity to cabozantinib and the combination of sorafenib and inhibitors of MAP kinase 1 and MAP kinase2 had a synergistic antiproliferative effect.CONCLUSION: LCCLs can be screened for drugs and agents that might be effective for treatment of HCC. We identified genetic alterations and gene expression patterns associated with response to these agents. This information might be used to select patients for clinical trials.

International audience; DNA methylation patterns are highly rearranged in hepatocellular carcinomas (HCCs). However, diverse sources of variation are intermingled in cancer methylomes, precluding the precise characterization of underlying molecular mechanisms. We developed a computational framework (methylation signature analysis with independent component analysis [MethICA]), leveraging independent component analysis (ICA) to disentangle the diverse processes contributing to DNA methylation changes in tumors. Applied to a collection of 738 HCCs, MethICA unraveled 13 stable methylation components (MCs) preferentially active in specific chromatin states, sequence contexts, and replication timings. These included signatures of general processes associated with gender and age but also new signatures related to specific driver events and molecular subgroups. Catenin beta 1 (CTNNB1) mutations were major modulators of methylation patterns in HCC, characterized by a targeted hypomethylation of transcription factor 7 (TCF7)‐bound enhancers in the vicinity of Wnt target genes as well as a widespread hypomethylation of late‐replicated partially methylated domains (PMDs). By contrast, demethylation of early‐replicated highly methylated domains (HMDs) was a signature of replication stress, leading to an extensive hypomethylator phenotype in cyclin (CCN)‐activated HCC. Inactivating mutations of the chromatin remodeler AT‐rich interactive domain‐containing protein 1A (ARID1A) were associated with epigenetic silencing of differentiation‐promoting transcriptional networks, also detectable in cirrhotic liver. Finally, a hypermethylation signature targeting Polycomb‐repressed chromatin domains was identified in the G1 molecular subgroup with progenitor features. Conclusion: This study elucidates the diversity of processes remodeling HCC methylomes and reveals the epigenetic and transcriptional impact of driver alterations.

Genetic alterations define different molecular subclasses of hepatocellular adenoma (HCA) linked with risk factors, histology and clinical behavior. Recently, Argininosuccinate Synthase 1 (ASS1), a major periportal protein, was proposed as a marker of HCA with a high risk of hemorrhage. We aimed to assess the significance of ASS1 expression through the scope of the HCA molecular classification. ASS1 expression was evaluated using RNAseq, quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and Immunohistochemistry. ASS1 and glioma-associated oncogene 1 (GLI1) expression were analyzed in vitro after modulation of GLI1 expression. Using RNAseq in 27 HCA and five nontumor liver samples, ASS1 expression was highly correlated with GLI1 expression (P

DNAJB1-PRKACA fusion is a specific driver event in fibrolamellar carcinoma (FLC), a rare subtype of hepatocellular carcinoma (HCC) that occurs in adolescents and young adults. In older patients, molecular determinants of HCC with mixed histological features of HCC and FLC (mixed-FLC/HCC) remain to be discovered.A series of 151 liver tumors including 126 HCC, 15 FLC, and 10 mixed-FLC/HCC were analyzed by RNAseq and whole-genome- or whole-exome sequencing. Western blots were performed to validate genomic discoveries. Results were validated using the TCGA database.Most of the mixed-FLC/HCC RNAseq clustered in a robust subgroup of 17 tumors, which all had mutations or translocations inactivating BAP1, the gene encoding BRCA1-associated protein-1. Like FLC, BAP1-HCC were significantly enriched in females, patients with a lack of chronic liver disease, and fibrotic tumors compared to non-BAP1 HCC. However, patients were older and had a poorer prognosis than those with FLC. BAP1 tumors were immune hot, showed progenitor features and did not show DNAJB1-PRKACA fusion, while almost none of these tumors had mutations in CTNNB1, TP53 and TERT promoter. In contrast, 80% of the BAP1 tumors showed a chromosome gain of PRKACA at 19p13, combined with a loss of PRKAR2A (coding for the inhibitory regulatory subunit of PKA) at 3p21, leading to a high PRKACA/PRKAR2A ratio at the mRNA and protein levels.We have characterized a subgroup of BAP1-driven HCC with fibrolamellar-like features and a dysregulation of the PKA pathway, which could be at the root of the clinical and histological similarities between BAP1 tumors and DNAJB1-PRKACA FLCs.Herein, we have defined a homogeneous subgroup of hepatocellular carcinomas in which the BAP1 gene is inactivated. This leads to the development of cancers with features similar to those of fibrolamellar carcinoma. These tumors more frequently develop in females without chronic liver disease or cirrhosis. The presence of PKA activation and T cell infiltrates suggest that these tumors could be treated with PKA inhibitors or immunomodulators.

Introduction: Malignant pleural mesothelioma (MPM) is a heterogeneous cancer characterized by a diffuse locoregional growth within the thoracic cavity. Better knowledge of molecular and cellular intra-tumor heterogeneity throughout the thoracic cavity is required to develop efficient therapies. Methods: Intra-tumor heterogeneity was investigated in 16 patients biopsied at distinct anatomic sites (apex, side wall, costo-diaphragmatic and highly metabolic site detected by PET scan when present). Paired biopsies and five derived primary cell lines were screened by targeted sequencing. Part of the cohort was also deeply characterized by whole exome sequencing, RNA-seq and DNA methylation profiling. Molecular classification and recently defined histo-molecular gradients were assessed, as were the cell populations infiltration of the tumor microenvironment. Results: Sequencing analysis showed that most of the protein-altering somatic variants were common between paired samples from a given patient. However, we identified heterogeneous variants notably in NF2, the well-known mesothelioma driver tumor suppressor gene. Furthermore, we highlighted subclonal tumor populations shared among paired biopsies, suggesting a polyclonal dissemination of the tumor. Chromosomal abnormalities profiles were similar between different regions within the same tumor suggesting that the main chromosomal alterations occurring in MPM are early events of mesothelial carcinogenesis. Transcriptomic analysis showed major differential gene expression between anatomic sites, leading to the dysregulation of specific pathways notably linked to cell adhesion and extracellular matrix. Accordingly, histo-molecular gradient determining the proportions of epithelioid-like and sarcomatoid-like cellular entities varied between these samples. These changes were linked to epigenetic mechanisms in two patients. Finally, we found consequent spatial intra-tumor heterogeneity of the immune microenvironment with differential expression of immune mediators and immune checkpoints, and differential infiltration of immune populations. These changes led to a switch from a hot to a cold immune profile in three patients. Conclusions: This study is the first to report on the multi-omics profiling of a substantial series of multi-site MPM tumor samples. Spatial intra-tumor heterogeneity is complex in MPM and varies among patients. We highlighted multiple types of heterogeneity, i.e. (i) genetic, (ii) transcriptomic, (iii) epigenetic and (iv) in the immune landscape of the tumor microenvironment. These results support the need for multi-sampling for the implementation of molecular-based precision medicine in MPM. Citation Format: Clément Meiller, François Montagne, Théo Z. Hirsch, Stefano Caruso, Julien de Wolf, Quentin Bayard, Jean-Baptiste Assié, Léa Meunier, Yuna Blum, Lisa Quetel, Laure Gibault, Ecaterina Pintilie, Cécile Badoual, Marie-Christine Copin, Eric Letouzé, Arnaud Scherpereel, Jessica Zucman-Rossi, Françoise Le Pimpec-Barthes, Marie-Claude Jaurand, Didier Jean. Spatial intra-tumor molecular heterogeneity in malignant pleural mesothelioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3128.

SummaryLiver cancer is a highly heterogeneous disease in terms of etiology, tissue and cellular morphology, tumor molecular characteristics, microenvironment composition and prognosis. Several studies, based on tumor gene-expression profiling (GEP) data, have dissected the molecular heterogeneity of liver cancer. They resulted in various tools, either delineating homogeneous tumor subtypes or calculating molecular scores of prognostic or biological functions. Here, we present MS.liverK, an easy-to-use R package providing a comprehensive implementation of these tools, for research use.Availability and implementationThe MS.liverK R package is available from GitHub (https://github.com/cit-bioinfo/MS.liverK).