Sandra Rebouissou

MD Jean-Charles Nault
email: naultjc@gmail.com

Advances in molecular classification and precision oncology in hepatocellular carcinoma

Keywords: liver cancer, mutation, hepatitis B, targeted therapy, hepatocellular adenoma, malignant transformation

Hepatocellular carcinoma (HCC) arises from hepatocytes through the sequential accumulation of multiple genomic and epigenomic alterations resulting from a Darwinian selection. Genes from various signaling pathways such as telomere maintenance, Wnt/ß-catenin, P53/cell cycle regulation, oxidative stress, epigenetic modifiers, AKT/mTOR and MAP kinase are frequently mutated in HCC. Several subclasses of HCC have been identified based on transcriptomic dysregulation and genetic alterations that are closely related to the risk factors, pathological features and prognosis. Undoubtedly, integration of data obtained from both preclinical models and human studies can be helpful in accelerating the identification of robust predictive biomarkers of response to targeted biotherapy and immunotherapy. The aim of this review was to describe the main advances in HCC in term of molecular biology and how this knowledge could be used in clinical practice in the future.

Key points:

1) Single nucleotide polymorphisms of PNPLA3 (rs738409), TM6SF2 (rs58542926) and HSD17B13 (rs72613567) modulate the risk of chronic liver disease and HCC development, mainly in patients with alcoholic and nonalcoholic fatty liver disease 2) Telomerase reactivation is one of the key events in malignant transformation of cirrhotic hepatocytes. The main mechanisms of telomerase reactivation were: hot spot mutations in the promoter of TERT, TERT amplification, TERT translocation and, in HBV related HCC, viral insertion in the promoter of TERT 3) Each HCC is a unique combination of somatic genetic alterations. The median rate of protein altering mutations per tumor was 50 to 70 including mutations in 2 to 6 driver genes. 4) Several types of tumor heterogeneity have been described in HCC developed on cirrhosis: intra-tumor heterogeneity and inter-tumor heterogeneity due to multifocal independent carcinogenesis and/or intra-hepatic metastasis 5) Genetic features of HCC are strongly linked with risk factors, histological and clinical features. Several genomic alterations are candidate predictive biomarkers of response to targeted therapy and immunotherapy.

Introduction

Despite several advances in the management of hepatocellular carcinoma (HCC), the prognosis of patients with HCC remains poor mainly due to the high rate of diagnosis at advanced stages [START_REF] Forner | Hepatocellular carcinoma[END_REF]. Several tyrosine kinase inhibitors (sorafenib, lenvatinib, cabozantinib and regorafenib) or monoclonal antibody directed against VEGFR2 (ramucirumab) have shown to improve survival when used as first line (sorafenib and lenvatinib) or second line (regorafenib, cabozantinib and ramucirumab) in patients with advanced HCC [START_REF] Llovet | Sorafenib in advanced hepatocellular carcinoma[END_REF][START_REF] Bruix | Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF][START_REF] Kudo | Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial[END_REF][START_REF] Abou-Alfa | Cabozantinib in Patients with Advanced and Progressing Hepatocellular Carcinoma[END_REF][START_REF] Zhu | Ramucirumab after sorafenib in patients with advanced hepatocellular carcinoma and increased αfetoprotein concentrations (REACH-2): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF]. However, these treatments are associated with adverse events and have a limited efficacy with frequent primary or secondary resistance. More data on pathogenesis of HCC are required in order to develop new efficient therapies to increase survival of HCC patients [START_REF] Dhanasekaran | Genomic Medicine and Implications for Hepatocellular Carcinoma Prevention and Therapy[END_REF]. Our knowledge of liver carcinogenesis has increased thanks to the dissection of the mechanisms of development of cirrhosis and the understanding of natural history of risk factors such as chronic viral infection [START_REF] Dhanasekaran | Genomic Medicine and Implications for Hepatocellular Carcinoma Prevention and Therapy[END_REF]. Moreover, the development of mouse model of HCC has helped to recapitulate the molecular pathway of the liver carcinogenesis and the relation with microenvironment and immunity [START_REF] Brown | Mouse models of hepatocellular carcinoma: an overview and highlights for immunotherapy research[END_REF]. Major advancements in our knowledge of molecular profiling and genetic defects have been made possible with the advent of next generation sequencing [START_REF] Guichard | Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma[END_REF][START_REF] Schulze | Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets[END_REF][START_REF] Totoki | Transancestry mutational landscape of hepatocellular carcinoma genomes[END_REF][START_REF] Fujimoto | Wholegenome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators[END_REF][START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF] which has thrown insight into the frequent somatic/genetic alterations in driver genes and the main signaling pathways dysregulated in HCC [START_REF] Nault | The role of molecular enrichment on future therapies in hepatocellular carcinoma[END_REF]. In this review, we aim to describe the major recent advances in genomic studies in the field of HCC and how these studies could be utilized in clinical practice in the future..

Genetic predisposition and mutational processes involved in HCC development

• Genetic predisposition

Single nucleotide polymorphisms (SNPs) are genetic variants present in the general population that could influence the occurrence, severity and the clinical behavior of human diseases. Compared to germline mutations that are rare, constitutional genetic alterations associated with a high odds ratio of diseases development, SNPs are observed usually in more than 5% of the population and, taken alone, only modestly predispose to disease development (Odds ratio most of the time below 5) [START_REF] Nahon | Single nucleotide polymorphisms and risk of hepatocellular carcinoma in cirrhosis[END_REF]. Several approaches have been developed to identify SNP associated with disease development. The first one is a candidate-based approach that test target SNP based on biological hypothesis and analysis of the literature. Several SNPs in genes belonging to inflammatory pathways (TNF alpha, IL1 Beta, IL10, TGF Beta, etc…), iron metabolism (HFE1), oxidative stress pathways (GSTM1, SOD2, MPO, etc…), DNA repair mechanisms (MTHFR, TP53, MDM2, etc…) were found associated with HCC occurrence in the literature [START_REF] Nahon | Single nucleotide polymorphisms and risk of hepatocellular carcinoma in cirrhosis[END_REF][START_REF] Wei | Interleukin-10 gene polymorphisms and hepatocellular carcinoma susceptibility: a meta-analysis[END_REF][START_REF] Yoon | MDM2 and p53 polymorphisms are associated with the development of hepatocellular carcinoma in patients with chronic hepatitis B virus infection[END_REF][START_REF] Nahon | Liver iron, HFE gene mutations, and hepatocellular carcinoma occurrence in patients with cirrhosis[END_REF][START_REF] Nahon | Myeloperoxidase and superoxide dismutase 2 polymorphisms comodulate the risk of hepatocellular carcinoma and death in alcoholic cirrhosis[END_REF]. However, most of these studies used a case/control approach and only few of SNPs have been validated externally using a prospective approach in cirrhotic patients screened by ultrasonography every 6 months in order to bypass the usual bias of a case/control study [START_REF] Nahon | Single nucleotide polymorphisms and risk of hepatocellular carcinoma in cirrhosis[END_REF]. Another way to identify genetic variants is based on an agnostic approach using genome wide association study (GWAS) that tests the association between thousands of SNPs in the genome and the occurrence of a disease (Figure 1). GWAS has identified PNPLA3 rs738409 and TM6SF2 rs58542926 SNPs linked with the development of Alcoholic Liver Disease (ALD) and Non-Alcoholic Fatty Liver Disease (NAFLD) and their progression to cirrhosis [START_REF] Romeo | Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease[END_REF][START_REF] Kozlitina | Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease[END_REF]. PNPLA3 rs738409 is a coding variant (I148M) involved in lipid metabolism and TM6SF2 rs58542926 in fatty acid accumulation in hepatocytes [START_REF] Pirazzi | Patatinlike phospholipase domain-containing 3 (PNPLA3) I148M (rs738409) affects hepatic VLDL secretion in humans and in vitro[END_REF][START_REF] Trépo | PNPLA3 gene in liver diseases[END_REF][START_REF] Luukkonen | Impaired hepatic lipid synthesis from polyunsaturated fatty acids in TM6SF2 E167K variant carriers with NAFLD[END_REF]. Subsequent studies have showed that PNPLA3 rs738409 and TM6SF2 rs58542926 are associated with a higher risk of HCC development according to the etiologies of the underlying liver diseases [START_REF] Trépo | PNPLA3 gene in liver diseases[END_REF][START_REF] Stickel | Genetic variants in PNPLA3 and TM6SF2 predispose to the development of hepatocellular carcinoma in individuals with alcohol-related cirrhosis[END_REF]. If the association with HCC development on ALD or NAFLD seems consistent among studies, the association with HCC risk in hepatitis C is still debatable [START_REF] Trépo | Association between the PNPLA3 (rs738409 C>G) variant and hepatocellular carcinoma: Evidence from a meta-analysis of individual participant data[END_REF][START_REF] Yang | PNPLA3 and TM6SF2 variants as risk factors of hepatocellular carcinoma across various etiologies and severity of underlying liver diseases[END_REF]. Recently, a loss of function variant (rs72613567) of HSD17B13, coding for a hepatic retinol deshydrogenase and protective against the development of ALD and NAFLD, was identified by GWAS [START_REF] Abul-Husn | A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease[END_REF][START_REF] Ma | 17-Beta Hydroxysteroid Dehydrogenase 13 Is a Hepatic Retinol Dehydrogenase Associated With Histological Features of Nonalcoholic Fatty Liver Disease[END_REF]. We recently showed that this variant was also protective in the development of HCC on ALD. However, more data is needed to understand the mechanisms by which HSD17B13 (rs72613567) protects from chronic liver diseases and carcinogenesis [START_REF] Nault | Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma[END_REF]. Currently, there is no data that suggest a potential implementation of SNPs in clinical practice. Overall, the odds ratio of the current SNPs seems to be too low to stratify patients according to the risk of HCC development for intensive screening and the incremental effect of SNPs compared to clinical predictive features is weak [START_REF] Nahon | Single nucleotide polymorphisms and risk of hepatocellular carcinoma in cirrhosis[END_REF]. However, combination of several SNPs (polygenic approach) with clinical features should be adopted in the future in order to refine prediction of risk factors, screening methods and chemopreventive treatment modalities. Currently, liver ultrasonography every 6 months is the standard method for HCC screening in the cirrhotic patients. However, usefulness of HCC screening for low-HCC risk population such as hepatitis B patients or patients with NASH without cirrhosis remains debatable. Recently, in HBV patients, clinical scoring system such as PAGE B has been proposed to identify patients at risk of developing HCC. [START_REF] Papatheodoridis | PAGE-B predicts the risk of developing hepatocellular carcinoma in Caucasians with chronic hepatitis B on 5-year antiviral therapy[END_REF]. Polygenic approach in conjunction with the clinical features could be tested to refine HCC risk score already developed in non-cirrhotic HBV patients or to identify the non-cirrhotic patients with NASH at risk of HCC development.

• Mutational signatures and HCC development

Another way to understand the mechanisms that shape tumor development is to study the mechanisms of accumulation of mutations in DNA during the lifetime of malignant hepatocytes [START_REF] Helleday | Mechanisms underlying mutational signatures in human cancers[END_REF]. Each tumor genome bears the molecular fingerprints of the exogeneous (mainly exposition to carcinogens such as tobacco, ultraviolet etc…) and endogenous processes (such as defect in DNA repair mechanism, age etc…) in DNA [START_REF] Alexandrov | Signatures of mutational processes in human cancer[END_REF]. It helps to understand the combination of processes that induces the malignant transformation of hepatocytes (Figure 1). The type of substitution in the trinucleotide context as well as the small insertion & deletion and chromosomal rearrangement recapitulates the main mutational signatures in carcinogenesis [START_REF] Nik-Zainal | The genome as a record of environmental exposure[END_REF][START_REF] Alexandrov | Mutational signatures associated with tobacco smoking in human cancer[END_REF]. Major mutational processes were observed in more than 50% of HCC and were related to aging (deamination of methylated cytosines for signature 1 and unknown mechanism for signature 5), liver specific signature (signature 16-only observed in primary liver tumors) and exposure to polycyclic aromatic hydrocarbons including but not restricted to tobacco (signature 4 observed in other solid tumors related to tobacco) [START_REF] Schulze | Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets[END_REF][START_REF] Totoki | Transancestry mutational landscape of hepatocellular carcinoma genomes[END_REF][START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF][START_REF] Letouzé | Mutational signatures reveal the dynamic interplay of risk factors and cellular processes during liver tumorigenesis[END_REF]. In a small percentage of tumors, sporadic mutational signatures have been identified such as signature 24 that is pathognomonic of exposure to aflatoxin B1 a mycotoxin that contaminates culture in Africa [START_REF] Letouzé | Mutational signatures reveal the dynamic interplay of risk factors and cellular processes during liver tumorigenesis[END_REF]. There is also rare type of HCC that harbored mutational signature of mismatch repair deficiency (1%, signature 6) [START_REF] Schulze | Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets[END_REF]. Interestingly, a subgroup of HCC, mainly from Asia, harbored A:T to T:A transversions preferentially in a CTG trinucleotide context (signature 22) that is a hallmark of exposure to aristolochic acid. Aristolochic acid is present in plants from the aristolochia species that are widely used in traditional Asian medicine [START_REF] Poon | Genome-wide mutational signatures of aristolochic acid and its application as a screening tool[END_REF][START_REF] Hoang | Mutational signature of aristolochic acid exposure as revealed by whole-exome sequencing[END_REF][START_REF] Rosenquist | Mutational signature of aristolochic acid: Clue to the recognition of a global disease[END_REF]. Interestingly, analysis of mutational signature has helped to identify new risk factors of HCC development such as aristolochic acid in Asia or have provided a biological proof to the epidemiological evidences that suggested tobacco as a risk factor of HCC development. However, the mechanism underlying the development of some sporadic mutational signature (signature 17 and 23) are still unknown and need further research. All these mutational signatures have been described initially in liver tumor. Recent studies have shown the presence of sub-clonal somatic mutations in the non-tumor liver highlighting the occurrence of mutations in known cancer driver genes in non-malignant hepatocytes due to aging, exposure to carcinogens and chronic liver damage [START_REF] Zhu | Somatic Mutations Increase Hepatic Clonal Fitness and Regeneration in Chronic Liver Disease[END_REF][START_REF] Yizhak | RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues[END_REF]. However, the potential usefulness of the detection of these sub-clonal mutations in the cirrhotic liver to predict HCC occurrence is currently unknown.

Apart from being informative on etiology, mutational signatures can be used for precision oncology as biomarkers to guide treatment. This was particularly well documented for cancers with DNA repair deficiency that harbored particular mutational signatures depending on the affected DNA repair pathway. These HCC can benefit from different therapies based on their particular defects using either DNA damaging agents such as PARP inhibitors that induces synthetic lethality) or immunotherapies [START_REF] Ma | The therapeutic significance of mutational signatures from DNA repair deficiency in cancer[END_REF].

In HCC, repair-deficient tumors are exceptional, however, a small subgroup of HCC (7%) harboring aberrant activation of cyclin A2 or E1 was recently associated with a unique signature of structural rearrangements involving hundreds of tandem duplications and templated insertions [START_REF] Bayard | Cyclin A2/E1 activation defines a hepatocellular carcinoma subclass with a rearrangement signature of replication stress[END_REF]. While this subgroup of HCC does not harbor a DNA repair defect, it shares a common signature of genomic instability with BRCA1-altered tumors and thus may be a good candidate for PARP inhibitor based therapies that have demonstrated clinical benefit in BRCA1deficient tumors [START_REF] Lord | PARP inhibitors: Synthetic lethality in the clinic[END_REF].

Mechanisms of malignant transformation

• On cirrhosis

In the setting of chronic liver disease, persistent liver injury due to chronic inflammation and exposure to carcinogens induces repeated cycles of cell necrosis followed by proliferation. This results in telomere shortening due to absence of telomerase activity in the adult liver [START_REF] Farazi | Hepatocellular carcinoma pathogenesis: from genes to environment[END_REF]. Thus, cirrhosis is composed of nodules of senescent hepatocytes with short telomeres. Therefore, mice models with short telomeres are prone to develop cirrhosis when exposed to chemical or genetic liver injury [START_REF] Rudolph | Inhibition of experimental liver cirrhosis in mice by telomerase gene delivery[END_REF]. However, telomerase reactivation in these mice, promoted malignant transformation of hepatocytes by evading telomere shortening and replicative senescence [START_REF] Farazi | Differential impact of telomere dysfunction on initiation and progression of hepatocellular carcinoma[END_REF]. Similar results were observed in human HCC where 95% of the tumors harbored a reexpression of the telomerase reverse transcriptase (TERT), the limiting factor of the telomerase complex [START_REF] Nakayama | Telomerase activation by hTRT in human normal fibroblasts and hepatocellular carcinomas[END_REF][START_REF] Meyerson | hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization[END_REF]. The different mechanisms of telomerase reactivation were: hot spot mutations in the promoter of TERT (40 to 60% of HCC), TERT amplification (5%), TERT translocation (2 to 3%) and, in HBV related HCC, viral insertion in the promoter of TERT (10%) [START_REF] Totoki | Transancestry mutational landscape of hepatocellular carcinoma genomes[END_REF][START_REF] Bayard | Cyclin A2/E1 activation defines a hepatocellular carcinoma subclass with a rearrangement signature of replication stress[END_REF][START_REF] Nault | High frequency of telomerase reverse-transcriptase promoter somatic mutations in hepatocellular carcinoma and preneoplastic lesions[END_REF][START_REF] Paterlini-Bréchot | Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene[END_REF]. The multistep process of cirrhosis could be described as follows: development of premalignant lesions such as low-grade dysplastic nodules and high-grade dysplastic nodules followed by malignant transformation into early HCC. Therefore, telomerase reactivation is required in the early step of carcinogenesis in cirrhosis and TERT promoter mutations are the only recurrent mutations in the driver genes observed in both low grade and high-grade dysplastic nodules (between 5 to 20% of mutations) and up to 60% of early HCC's [START_REF] Nault | Telomerase reverse transcriptase promoter mutation is an early somatic genetic alteration in the transformation of premalignant nodules in hepatocellular carcinoma on cirrhosis[END_REF][START_REF] Torrecilla | Trunk mutational events present minimal intra-and inter-tumoral heterogeneity in hepatocellular carcinoma[END_REF]. These results indicate that TERT is a gatekeeper gene which is mutated very early in liver carcinogenesis to allow reactivation of telomerase in order to bypass senescence of hepatocytes in cirrhosis. Moreover, transcriptomic analysis of premalignant nodules in cirrhosis and early HCC's have highlighted that activation of MYC, early in the course, could be involved in the process of malignant transformation [START_REF] Kaposi-Novak | Central role of c-Myc during malignant conversion in human hepatocarcinogenesis[END_REF] whereas activation of other signaling pathways such as TGFß, WNT or NOTCH occurred later during hepatocarcinogenesis [START_REF] Marquardt | Sequential transcriptome analysis of human liver cancer indicates late stage acquisition of malignant traits[END_REF].

Even an expert pathologist could face difficulties in differentiating premalignant lesions from early HCC on cirrhosis [START_REF] Tommaso | Advanced precancerous lesions in the liver[END_REF]. In this setting, further research is needed to study the potential role of TERT promoter mutations as predictive biomarkers of malignant transformation of premalignant nodules and to better dissect the molecular profile of premalignant lesions and early HCC in order to propose a new nosology of early lesions based on genotype/phenotype correlation.

• On non-cirrhotic liver HCC development in normal liver is a rare event (less than 10% of the cases) but is a pure model that allows the identification of direct mechanisms of liver carcinogenesis outside the background noise of cirrhosis. The first road of carcinogenesis in normal liver is related to viral infections. Hepatitis B virus (HBV) is a DNA virus that could stay into the nucleus of hepatocytes in a circular covalently closed form but could also integrate into human DNA [START_REF] Neuveut | Mechanisms of HBV-related hepatocarcinogenesis[END_REF].

The occurrence of HBV related HCC in normal liver (without fibrosis) is due to the action of viral oncoprotein such as HbX and to a mechanism called insertional mutagenesis [START_REF] Neuveut | Mechanisms of HBV-related hepatocarcinogenesis[END_REF]. Viral insertion near a cancer gene could modify the expression or function of the gene and promote malignant transformation in the absence of cirrhosis. The main genes recurrently targeted by HBV insertions are TERT, MLL4, CCNA2 and CCNE1 [START_REF] Paterlini-Bréchot | Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene[END_REF][START_REF] Wang | Hepatitis B virus integration in a cyclin A gene in a hepatocellular carcinoma[END_REF][START_REF] Sung | Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma[END_REF]]. However, it should be noted that viral insertional mutagenesis is not restricted to HBV related HCC developed in normal liver but it could also be seen in HBV related HCC developed on cirrhotic liver.

We recently described HCC developing in normal liver due to recurrent viral insertions of adeno-associated virus type 2 (AAV2) [START_REF] Nault | Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas[END_REF]. AAV2 is a monostrand defective DNA virus which gets inserted in the human DNA. It was previously considered as a non-pathogenic infection observed in more than the half of the population. Recurrent clonal viral insertions of AAV2 in driver genes such as TERT, MLL4, CCNE1, CCNA2 and TNFSF10 were observed in a subset of HCC developed on normal liver without other etiologies [START_REF] Nault | Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas[END_REF]. These results suggest that AAV2 could lead to HCC development in normal liver in rare cases. Interestingly, a subset of HCC developed on normal liver was characterized by activation of cyclin gene (CCNE1, CCNA2) either due to AAV2 or HBV insertion or due to chromosomal rearrangements that induced accumulation of duplications and templated insertions in the genome responsible for the overexpression of TERT [START_REF] Bayard | Cyclin A2/E1 activation defines a hepatocellular carcinoma subclass with a rearrangement signature of replication stress[END_REF].

Finally, the last road known to lead to HCC development in normal liver was the malignant transformation of hepatocellular adenomas (HCA) [START_REF] Nault | Molecular classification of hepatocellular adenoma in clinical practice[END_REF]. HCA developed mostly in normal liver in young women taking oral contraception and could rarely be complicated by transformation into HCC (5% of surgical series). Several genetic subgroups of HCA have been described and were linked with patho-radiological features and the risk of complications: HCA with inactivating mutations of HNF1A, HCA with mutations of CTNNB1 in exon 3 or in exon 7 or 8, inflammatory HCA and sonic hedgehog HCA [START_REF] Nault | Molecular Classification of Hepatocellular Adenoma Associates With Risk Factors, Bleeding, and Malignant Transformation[END_REF]. 10 to 15% of HCA harbored an activating mutation of CTNNB1 (coding for B-catenin) in exon 3 and has a high risk of malignant transformation [START_REF] Zucman-Rossi | Genotype-phenotype correlation in hepatocellular adenoma: new classification and relationship with HCC[END_REF]. Interestingly, reactivation of telomerase due to mutations of the TERT promoter is required in a second step to foster a full malignant transformation of HCA [START_REF] Pilati | Genomic profiling of hepatocellular adenomas reveals recurrent FRK-activating mutations and the mechanisms of malignant transformation[END_REF].

Molecular classification of Hepatocellular carcinoma

• Mutational, transcriptomic and epigenetic landscape HCC is a complex disease arising from hepatocytes through the sequential accumulation of multiple genomic and epigenomic alterations that have undergone Darwinian selection. Next generation sequencing has allowed to draw the precise genetic landscape of HCC and identified a median rate of approximately 50 to 70 protein altering mutations per tumor [START_REF] Schulze | Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets[END_REF][START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF][START_REF] Ahn | Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification[END_REF], with similar mutational burden across the different stages from BCLC 0/A to advanced HCCs (BCLC B/C) [START_REF] Nault | Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma[END_REF]. The vast majority of these mutations are passengers, they accumulate randomly and are not involved in carcinogenesis while only a few of them, about 2 to 6, are considered as functional "driver" mutations and alter key signaling pathways leading to selective advantage. Advanced computational methods for distinguishing between driver and passenger mutations [START_REF] Lawrence | Mutational heterogeneity in cancer and the search for new cancer-associated genes[END_REF] have allowed to identify the main mutated genes causally implicated in HCCs that can be classified in six major biological pathways including telomere maintenance, Wnt/ß-catenin, P53/cell cycle regulation, AKT/mTOR, MAP kinase, epigenetic modifiers and oxidative stress (Figure 1) [START_REF] Guichard | Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma[END_REF][START_REF] Schulze | Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets[END_REF][START_REF] Totoki | Transancestry mutational landscape of hepatocellular carcinoma genomes[END_REF][START_REF] Fujimoto | Wholegenome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators[END_REF][START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF][START_REF] Nault | High frequency of telomerase reverse-transcriptase promoter somatic mutations in hepatocellular carcinoma and preneoplastic lesions[END_REF][START_REF] Ahn | Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification[END_REF][START_REF] Chiang | Focal gains of VEGFA and molecular classification of hepatocellular carcinoma[END_REF]. The most prevalently mutated genes are TERT (promoter), TP53, CTNNB1, AXIN1, ARID1A and ARID2 while the others are mutated in less than 10% of the cases (Figure 1). The interplay between mutated driver genes, also defined as epistatic interactions, is a major determinant of the carcinogenesis process. Indeed, in cancer, some mutations tend to co-occur suggesting that they cooperate together in tumor development while mutual exclusivity is usually explained either by the functional redundancy of these mutations or by their deleterious effect when arising simultaneously. In HCC, three main clusters of positive epistatic interactions have been described, the first one between CTNNB1 mutations and TERT promoter, ARID2 and NFE2L2 mutations, the second one between AXIN1 mutations and RPS6KA3 and ARID1A mutations and the third one between TP53 mutations and KEAP1, TSC2 mutations and CCND1/FGF19 amplification[10] (Figure 2). By contrast, a negative interaction was identified between CTNNB1 mutations and AXIN1 and TP53 mutations [START_REF] Schulze | Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets[END_REF] (Figure 2).

Epigenetic dysregulation also plays a crucial role in hepatocarcinogenesis by altering gene expression through various mechanisms including modifications in DNA methylation, histones, chromatin remodeling and changes in levels of small (microRNAs) and long (lncRNAs) noncoding RNAs. This is further highlighted by the frequent genetic alterations occurring in epigenetic modifiers accounting for around 20-50% of HCC cases [START_REF] Guichard | Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma[END_REF][START_REF] Schulze | Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets[END_REF][START_REF] Totoki | Transancestry mutational landscape of hepatocellular carcinoma genomes[END_REF][START_REF] Fujimoto | Wholegenome sequencing of liver cancers identifies etiological influences on mutation patterns and recurrent mutations in chromatin regulators[END_REF][START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF][START_REF] Ahn | Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification[END_REF]. DNA methylation and microRNA expression are so far, the most well investigated epigenetic alterations in HCC. Methylome profiling has reported aberrant promoter methylation in multiple genes some of which have been directly involved in liver tumorigenesis such as, among others, CDKN2A that is preferentially inactivated by promoter hypermethylation in 50% of the cases or IGF2 that is activated through hypomethylation of its fetal promoter [START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF][START_REF] Villanueva | DNA methylation-based prognosis and epidrivers in hepatocellular carcinoma[END_REF][START_REF] Martinez-Quetglas | IGF2 Is Up-regulated by Epigenetic Mechanisms in Hepatocellular Carcinomas and Is an Actionable Oncogene Product in Experimental Models[END_REF]. Some risk factors such as HBV and HCV can directly modulate DNA methylation in tumor cells and participate in carcinogenesis [START_REF] Dhanasekaran | Genomic Medicine and Implications for Hepatocellular Carcinoma Prevention and Therapy[END_REF]. There are also interactions between genetic and epigenetic alterations, for example, an extensive CpG hypermethylation of multiple gene promoters was associated with CTNNB1 mutations [START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF][START_REF] Nishida | Extensive methylation is associated with beta-catenin mutations in hepatocellular carcinoma: evidence for two distinct pathways of human hepatocarcinogenesis[END_REF], likewise, CDKN2A promoter methylation was linked to TERT promoter mutations [START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF] and genome-wide hypomethylation was correlated with genomic instability [START_REF] Calvisi | Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma[END_REF]. Aberrant expression of microRNAs has been also largely implicated in the development and progression of HCC through deregulation of key cellular processes such as proliferation, apoptosis, angiogenesis, invasion, metastasis and autophagy [START_REF] Xu | The Role of MicroRNAs in Hepatocellular Carcinoma[END_REF]. Both genetic and epigenetic alterations participate in reprogramming gene expression in tumor cells. In HCC, multiple genome-wide gene-expression studies have allowed to identify the main regulatory networks transcriptionally altered. Pathways that are commonly aberrantly activated include the canonical Wnt/ß-catenin, TGFß, cell cycle, interferon, MYC, PI3K/AKT and MET [START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF][START_REF] Chiang | Focal gains of VEGFA and molecular classification of hepatocellular carcinoma[END_REF][START_REF] Lee | Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling[END_REF][START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Hoshida | Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma[END_REF][START_REF] Kaposi-Novak | Met-regulated expression signature defines a subset of human hepatocellular carcinomas with poor prognosis and aggressive phenotype[END_REF]. Transcriptomic profiling also revealed different differentiation patterns among HCCs, some of them are well differentiated and retain a hepatocyte-like phenotype while others are less differentiated and are characterized by expression of progenitor markers such as IGF2, EPCAM, AFP or KRT19 [START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Hoshida | Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma[END_REF][START_REF] Calderaro | Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification[END_REF]. More recently, deconvolution methods applied to transcriptomic data have enabled to identify an "immune class" of HCC representing 30% of the cases and characterized by an immune gene expression signature including two clusters of HCCs with either active or exhausted immune responses [START_REF] Sia | Identification of an Immune-specific Class of Hepatocellular Carcinoma, Based on Molecular Features[END_REF]. Integrated transcriptional and genomic analysis has also allowed deciphering the similarities and differences between HCC, intra-hepatic cholangiocarcinoma (ICC) and the combined/mixed form of HCC-ICC, which were considered as clinically different entities. In particular, Chaisaingmongkol et al. have identified common molecular subtypes between HCC and ICC with similar prognosis that are specifically found in Asian patients [START_REF] Chaisaingmongkol | Common Molecular Subtypes Among Asian Hepatocellular Carcinoma and Cholangiocarcinoma[END_REF]. Another recent study showed that mixed HCC-ICC are more similar to HCC while combined HCC-ICC share more common molecular features with ICC. Interestingly, nestin expression was shown as a reliable biomarker for identifying these two subtypes of tumors and may represent a potential new therapeutic target for both combined and mixed HCC-ICC [START_REF] Xue | Genomic and Transcriptomic Profiling of Combined Hepatocellular and Intrahepatic Cholangiocarcinoma Reveals Distinct Molecular Subtypes[END_REF].

• Integrative analysis and correlation with clinical and pathological features

Over the past decade, worldwide efforts in HCC molecular subtyping combined with analysis of the associated clinical, etiological and histopathological features have significantly refined the nosological classification of HCC leading to the definition of several subgroups. HCC are first classified in two major classes: a "proliferation class" and a "non-proliferation class", each of which integrated several subclasses previously defined across multiple studies [START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF][START_REF] Chiang | Focal gains of VEGFA and molecular classification of hepatocellular carcinoma[END_REF][START_REF] Lee | Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling[END_REF][START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Hoshida | Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma[END_REF] (Figure 3). The "proliferation class" is associated with HBV-related etiology and includes clinically aggressive tumors, poorly differentiated with frequent vascular invasion and is characterized by an enrichment in TP53 inactivating mutations, amplification of FGF19 and CCND1, as well as frequent activation of pro-survival signaling pathways including cell cycle, mTOR (hyperphosphorylation of RPS6), RAS-MAPK and MET [START_REF] Chiang | Focal gains of VEGFA and molecular classification of hepatocellular carcinoma[END_REF][START_REF] Lee | Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling[END_REF][START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Hoshida | Molecular classification and novel targets in hepatocellular carcinoma: recent advancements[END_REF]. This class is also characterized by a chromosomal instability and a global DNA hypomethylation profile [START_REF] Calvisi | Mechanistic and prognostic significance of aberrant methylation in the molecular pathogenesis of human hepatocellular carcinoma[END_REF][START_REF] Laurent-Puig | Genetic alterations associated with hepatocellular carcinomas define distinct pathways of hepatocarcinogenesis[END_REF]. The proliferation class is further subdivided in two subclasses: a "Wnt-TGFß subclass" characterized by an activation of both TGFß and Wnt pathways and an exhausted immune response, and a "progenitor subclass" defined by the transcriptional and protein overexpression of hepatic progenitor markers, inactivating mutations in RPS6KA3 and AXIN1 and hyperphosphorylation of ERK [START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Hoshida | Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma[END_REF][START_REF] Calderaro | Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification[END_REF][START_REF] Sia | Identification of an Immune-specific Class of Hepatocellular Carcinoma, Based on Molecular Features[END_REF]. The main signaling pathways specifically activated in the progenitor subclass are IGF1R and AKT [START_REF] Chiang | Focal gains of VEGFA and molecular classification of hepatocellular carcinoma[END_REF][START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF]. The "Wnt-TGFß subclass" also includes a more aggressive subgroup of HCC "G3" distinguished by a specific histological phenotype designed as "macrotrabecular massive" [START_REF] Calderaro | Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification[END_REF][START_REF] Ziol | Macrotrabecularmassive hepatocellular carcinoma: A distinctive histological subtype with clinical relevance[END_REF], the occurrence of TSC1 and TSC2 mutations, a more pronounced enrichment in FGF19/CCND1 amplification as well as overexpression of genes related to the cell cycle and nucleus pore [START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Calderaro | Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification[END_REF].

The "non-proliferation class" of HCC is more heterogeneous and includes less aggressive tumors, chromosomally stable, related to HCV or alcohol. They are more differentiated and tend to retain hepatocyte-like features [START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF][START_REF] Lee | Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling[END_REF][START_REF] Hoshida | Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma[END_REF][START_REF] Hoshida | Molecular classification and novel targets in hepatocellular carcinoma: recent advancements[END_REF]. This class contains at least two subclasses with, as the best defined, a subset of HCC harboring CTNNB1 mutations leading to an activation of the Wnt/ß-catenin pathway that is higher in the "G6" subclass compared to "G5" [START_REF] Chiang | Focal gains of VEGFA and molecular classification of hepatocellular carcinoma[END_REF][START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF].

TERT promoter mutations are also more frequent in this subclass as well as hypermethylation of CDKN2A and CDH1 promoters [START_REF]Electronic address: wheeler@bcm.edu, Cancer Genome Atlas Research Network. Comprehensive and Integrative Genomic Characterization of Hepatocellular Carcinoma[END_REF][START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF]. In addition, CTNNB1-mutated HCC are immunologically cold and are characterized by cholestasis, nuclear accumulation of ß-catenin and positive immunohistochemical staining of glutamine-synthetase (GS) [START_REF] Calderaro | Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification[END_REF][START_REF] Sia | Identification of an Immune-specific Class of Hepatocellular Carcinoma, Based on Molecular Features[END_REF]. The second subclass belonging to the non-proliferation class named "G4" is less well defined and a subgroup of HCC inside the G4 class harbored a frequent steatohepatitic phenotype and an activation of the IL6/JAK-STAT pathway defined by a positive immunohistochemical expression of the C-reactive-protein (CRP) [START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Calderaro | Histological subtypes of hepatocellular carcinoma are related to gene mutations and molecular tumour classification[END_REF]. The G4 subclass encompasses two others subclasses named "Polysomy 7" (Poly7) and "Interferon" (IFN) that include respectively a small group of HCC with polysomy of chromosome 7 and a group of HCC overexpressing interferon-stimulated genes with an active immune response [START_REF] Chiang | Focal gains of VEGFA and molecular classification of hepatocellular carcinoma[END_REF][START_REF] Sia | Identification of an Immune-specific Class of Hepatocellular Carcinoma, Based on Molecular Features[END_REF].

Tumor progression and tumor heterogeneity • Tumor aggressiveness and mechanisms of progression

Several molecular features have been linked with tumor aggressiveness and the risk of relapse [START_REF] Dhanasekaran | Genomic Medicine and Implications for Hepatocellular Carcinoma Prevention and Therapy[END_REF]. First, several transcriptomic molecular signatures have been identified as predictive of tumor recurrence: hepatoblast like signature, stem cell signature, proliferative subclass, G3 subclasses, 5 gene score, etc… [START_REF] Lee | Classification and prediction of survival in hepatocellular carcinoma by gene expression profiling[END_REF][START_REF] Boyault | Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets[END_REF][START_REF] Nault | A hepatocellular carcinoma 5-gene score associated with survival of patients after liver resection[END_REF][START_REF] Villanueva | Combining clinical, pathology, and gene expression data to predict recurrence of hepatocellular carcinoma[END_REF][START_REF] Lee | A novel prognostic subtype of human hepatocellular carcinoma derived from hepatic progenitor cells[END_REF] Molecular signatures derived from the non-tumor liver also have been developed to predict late relapse related to de novo carcinogenesis on cirrhosis [START_REF] Hoshida | Gene expression in fixed tissues and outcome in hepatocellular carcinoma[END_REF]. However, these molecular signatures have not been validated in prospective studies and are currently not used in clinical practice. Other markers of poor prognosis in patients treated by liver resection have been also identified such as mutations of TP53, RB1, CDKN2A or amplification of FGF19 [START_REF] Schulze | Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets[END_REF][START_REF] Ahn | Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification[END_REF][START_REF] Woo | Association of TP53 mutations with stem cell-like gene expression and survival of patients with hepatocellular carcinoma[END_REF]. Data available on genomic of advanced HCC is sparse mainly due to mainly due to the absence of tumor biopsy performed in advanced stages in clinical practice. A recent study has showed an enrichment in TP53, RB1 and SF3B1 mutations in advanced HCC suggesting a role of these genes in tumor progression [START_REF] Nault | Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma[END_REF]. More data are needed with longitudinal samples in order to assess the mechanisms of tumor progression across the different stages of the disease.

• Tumor heterogeneity in HCC

Another issue in the field of molecular biology of HCC is related to tumor heterogeneity.

Several studies have shown the presence of intratumor heterogeneity with trunk mutations present in all the cells of the tumor and private mutations present in only one part of the tumor (Figure 4) [START_REF] Zhai | The spatial organization of intra-tumour heterogeneity and evolutionary trajectories of metastases in hepatocellular carcinoma[END_REF][START_REF] Friemel | Intratumor heterogeneity in hepatocellular carcinoma[END_REF]. Preliminary data in HCC have suggested that the main driver genes such as CTNNB1, TP53 and TERT are clonal trunk mutations and are consequently ideal therapeutic target [START_REF] Torrecilla | Trunk mutational events present minimal intra-and inter-tumoral heterogeneity in hepatocellular carcinoma[END_REF][START_REF] Friemel | Intratumor heterogeneity in hepatocellular carcinoma[END_REF][START_REF] Martins-Filho | A phenotypical map of disseminated hepatocellular carcinoma suggests clonal constraints in metastatic sites[END_REF][START_REF] Huang | Circumventing intratumoral heterogeneity to identify potential therapeutic targets in hepatocellular carcinoma[END_REF]. However, more data are required to clarify this using ultradeep sequencing and combination with methylation and transcriptomic analysis in order to capture the complexity of intratumor heterogeneity in large cohorts of HCC. Another type of genomic heterogeneity observed in HCC is inter tumor heterogeneity that could be related to de novo independent carcinogenesis on cirrhosis and/or intrahepatic metastasis (Figure 4). According to the study and the method used to assess tumor clonality, intra hepatic metastasis are observed in 20 to 40% of the patients whereas the rest of the tumors are related to de novo carcinogenesis on cirrhosis [START_REF] Ahn | Genomic portrait of resectable hepatocellular carcinomas: implications of RB1 and FGF19 aberrations for patient stratification[END_REF][START_REF] Furuta | Whole genome sequencing discriminates hepatocellular carcinoma with intrahepatic metastasis from multi-centric tumors[END_REF][START_REF] Chen | Chromosomal changes and clonality relationship between primary and recurrent hepatocellular carcinoma[END_REF][START_REF] An | Tumor heterogeneity in small hepatocellular carcinoma: analysis of tumor cell proliferation, expression and mutation of p53 AND beta-catenin[END_REF]. However, we have to underline that most of these studies have been performed on surgical samples of resection or transplantation and the results may be different in more advanced stages. Obviously, a better understanding of intra tumor and inter tumor heterogeneity are mandatory to understand their role in primary and secondary resistance to systemic treatments.

Precision oncology in HCC • Biomarkers of response to tyrosine kinase inhibitors

Targeted therapies including Tyrosine kinase inhibitors (TKIs such as sorafenib, lenvatinib, cabozantinib and regorafenib) and anti VEGFR2 antibody (ramucirumab) have substantially improved the systemic treatment of advanced HCC. Since the approval of sorafenib as the frontline standard of care [START_REF] Llovet | Sorafenib in advanced hepatocellular carcinoma[END_REF], four additional targeted therapy have recently demonstrated clinical benefits in phase 3 trials including lenvatinib in first-line and regorafenib, cabozantinib and ramucirumab in second-line [START_REF] Bruix | Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF][START_REF] Kudo | Lenvatinib versus sorafenib in first-line treatment of patients with unresectable hepatocellular carcinoma: a randomised phase 3 non-inferiority trial[END_REF][START_REF] Abou-Alfa | Cabozantinib in Patients with Advanced and Progressing Hepatocellular Carcinoma[END_REF][START_REF] Zhu | Ramucirumab after sorafenib in patients with advanced hepatocellular carcinoma and increased αfetoprotein concentrations (REACH-2): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF]. However, all these drugs confer only a modest extension in median survival (2-3 months) and low response rates, which underscores the need of predictive biomarkers to improve clinical management. Several potential markers have been associated with sorafenib response, such as FGF3/FGF4 and VEGFA genomic amplification, overexpression of active VEGF receptors or elevated Mapk14-Atf2 [START_REF] Arao | FGF3/FGF4 amplification and multiple lung metastases in responders to sorafenib in hepatocellular carcinoma[END_REF][START_REF] Horwitz | Human and mouse VEGFA-amplified hepatocellular carcinomas are highly sensitive to sorafenib treatment[END_REF][START_REF] Peng | Autocrine vascular endothelial growth factor signaling promotes cell proliferation and modulates sorafenib treatment efficacy in hepatocellular carcinoma[END_REF][START_REF] Rudalska | In vivo RNAi screening identifies a mechanism of sorafenib resistance in liver cancer[END_REF] and more recently, a clinical study has linked sorafenib primary resistance to the occurrence of activating mutations in the PI3K/AKT/mTOR pathway in HCC [START_REF] Harding | Prospective Genotyping of Hepatocellular Carcinoma: Clinical Implications of Next-Generation Sequencing for Matching Patients to Targeted and Immune Therapies[END_REF]. However, currently, there are still no validated predictive factors of sorafenib response that can be used in routine patient care.

Similarly, an exploratory retrospective analysis of the RESORCE trial has reported that candidate plasma biomarkers including 5 proteins and 9 miRNAs are able to predict regorafenib response, while these findings also need further confirmation [START_REF] Teufel | Biomarkers Associated With Response to Regorafenib in Patients With Hepatocellular Carcinoma[END_REF]. Interestingly, the phase 3 REACH-2 study confirmed the predictive value of elevated serum alpha-fetoprotein (>=400 ng/ml) for ramucirumab sensitivity which represents so far, the first drug in HCC with a consistent predictive biomarker applicability in daily clinical practice [START_REF] Zhu | Ramucirumab after sorafenib in patients with advanced hepatocellular carcinoma and increased αfetoprotein concentrations (REACH-2): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF]. Other biomarkers of TKI response have also been proposed in HCC such as high levels of MET detected by immunohistochemistry as a predictor of tivantinib sensitivity [START_REF] Santoro | Tivantinib for second-line treatment of advanced hepatocellular carcinoma: a randomised, placebo-controlled phase 2 study[END_REF] but this finding could not be subsequently validated in phase 3 trial [START_REF] Rimassa | Tivantinib for second-line treatment of MET-high, advanced hepatocellular carcinoma (METIV-HCC): a final analysis of a phase 3, randomised, placebo-controlled study[END_REF]. This failure may be explained firstly by an inappropriate selection criterion as MET overexpression may not be a surrogate marker of oncogenic addiction and secondly by an inadequate treatment since tivantinib does not act as a MET inhibitor, as reported by multiple studies [START_REF] Basilico | Tivantinib (ARQ197) displays cytotoxic activity that is independent of its ability to bind MET[END_REF][START_REF] Katayama | Cytotoxic activity of tivantinib (ARQ 197) is not due solely to c-MET inhibition[END_REF][START_REF] Rebouissou | Proliferation Markers Are Associated with MET Expression in Hepatocellular Carcinoma and Predict Tivantinib Sensitivity In Vitro[END_REF]. Alternatively, true MET inhibitors may be effective in the small subset of patients with MET amplified tumors as we have recently reported in liver cancer cell lines and in one patient who achieved a complete tumor response after treatment by teponinib, a specific Met inhibitor [START_REF] Nault | Clinical impact of genomic diversity from early to advanced hepatocellular carcinoma[END_REF][START_REF] Rebouissou | Proliferation Markers Are Associated with MET Expression in Hepatocellular Carcinoma and Predict Tivantinib Sensitivity In Vitro[END_REF][START_REF] Caruso | Analysis of Liver Cancer Cell Lines Identifies Agents With Likely Efficacy Against Hepatocellular Carcinoma and Markers of Response[END_REF].

• Biomarkers of response to immunotherapy

In recent years, immunotherapy and especially, immune checkpoint inhibitors have revolutionized the management of broad spectrum of malignancies. HCC is a typical inflammation-associated cancer and is known to arise in a context of immunosuppression making immunotherapy a potential attractive therapeutic option. In this line, two phase 2 studies have reported objective responses to nivolumab and pembrolizumab, two immune checkpoint inhibitors targeting PD-1, in 20% and 17% of advanced HCCs, respectively [START_REF] El-Khoueiry | Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, noncomparative, phase 1/2 dose escalation and expansion trial[END_REF][START_REF] Zhu | Pembrolizumab in patients with advanced hepatocellular carcinoma previously treated with sorafenib (KEYNOTE-224): a non-randomised, open-label phase 2 trial[END_REF]. These results have led to an accelerated approval of these two checkpoint inhibitors by the FDA for the treatment of advanced HCC in second-line. However, recent press release reported that the phase 3 randomized controlled trial comparing nivolumab versus sorafenib in first line as well as the phase 3 randomized controlled trial comparing pembrolizumab versus placebo in first line did not reach their prespecified endpoints. It suggests that additional data are required to identify patients who will benefit from immunotherapy. In both the phase 2 studies, PD-L1 expression status on tumor cells was not predictive of the response rates, thus, alternative predictive biomarkers remain to be identified. Apart from PD-L1 expression, in other cancer types, several biomarkers have been proposed such as high levels of tumor-infiltrating lymphocytes, especially effector cells, an increased secretion of IFN-γ and other cytokines, as well as, a high somatic mutational burden and neoantigen load [START_REF] Darvin | Immune checkpoint inhibitors: recent progress and potential biomarkers[END_REF]. Interestingly, the newly identified "immune class" of HCC harbors features that closely resemble those of cancers vulnerable to checkpoint inhibitors including high infiltration of immune cells, active interferon gamma signaling and expression of PD-1 and PD-L1, making it a promising candidate for immunotherapy [START_REF] Sia | Identification of an Immune-specific Class of Hepatocellular Carcinoma, Based on Molecular Features[END_REF]. However, future clinical investigations will be required to confirm this hypothesis. By contrast, CTNNB1 mutated HCCs are characterized by an immune exclusion, accordingly, a recent clinical study has shown that this subgroup was refractory to immune checkpoint blockers [START_REF] Harding | Prospective Genotyping of Hepatocellular Carcinoma: Clinical Implications of Next-Generation Sequencing for Matching Patients to Targeted and Immune Therapies[END_REF]. A recent study confirmed in a mouse model that β-catenin activation blocked T cell activity, promoted immune escape and were responsible of resistance to immunotherapy [START_REF] Ruiz De Galarreta | β-catenin activation promotes immune escape and resistance to anti-PD-1 therapy in hepatocellular carcinoma[END_REF]. In light of these recent findings it would be interesting to reanalyze the negative phase 3 nivolumab and pembrolizumab trials by excluding CTNNB1 mutated HCC.

Hence, development of efficient immune checkpoint-inhibitor-based therapy in HCC is still challenged by the identification of accurate predictors of response. An additional way to increase the efficacy of anti PD1/PDL1 antibodies is their combinations with either tyrosine kinase inhibitor such as lenvatinib, an anti VEGF antibody such as bevacizumab or an anti CTLA4 antibody such as tremelimumab. The first results of phase 2 released at the ASCO and ESMO meetings seem encouraging in term of tumor response and phase 3 clinical trials are currently on-going.

• How to modelize response to target therapy?

Despite the significant success of targeted therapies in the management of many cancers, HCC is one of the malignancies that have so far benefited the least from these advances. This lack of progress relies in part on the scarcity of HCC tissues, especially for advanced stages, which represents a major barrier to understand the relationship between tumor biology and the therapeutic response. In this context, the development of relevant preclinical models is particularly needed. In cancer, the most commonly used models include tumor-derived cell lines and patient-derived tumor xenografts (PDTXs), as they have been shown to retain most of the common molecular features found in primary tumors [START_REF] Barretina | The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity[END_REF][START_REF] Garnett | Systematic identification of genomic markers of drug sensitivity in cancer cells[END_REF][START_REF] Kopetz | The promise of patient-derived xenografts: the best laid plans of mice and men[END_REF][START_REF] Lee | Pharmacogenomic landscape of patient-derived tumor cells informs precision oncology therapy[END_REF][START_REF] Iorio | A Landscape of Pharmacogenomic Interactions in Cancer[END_REF][START_REF] Gao | High-throughput screening using patient-derived tumor xenografts to predict clinical trial drug response[END_REF]. Over the past decade, several pan-cancer large-scale pharmacogenomics screenings have been conducted on monolayer-cultured cancer cells [START_REF] Barretina | The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity[END_REF][START_REF] Garnett | Systematic identification of genomic markers of drug sensitivity in cancer cells[END_REF][START_REF] Iorio | A Landscape of Pharmacogenomic Interactions in Cancer[END_REF]. These studies on large set of cell lines are representative of tumor diversity and combined with deep molecular characterizations have proven to be powerful tools for molecular prediction of drug response and biomarker discovery [START_REF] Barretina | The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity[END_REF][START_REF] Garnett | Systematic identification of genomic markers of drug sensitivity in cancer cells[END_REF][START_REF] Iorio | A Landscape of Pharmacogenomic Interactions in Cancer[END_REF]. In HCC, until now the vast majority of the in vitro pharmacological studies have been scattered in the literature and restricted to an insufficient number of cell lines to capture HCC diversity. To fill this gap, we recently performed an extensive multi-omic characterization of the most widely used liver cancer cell lines (LCCL) including 34 models, combined with a pharmacological screen of 31 anti-cancer agents [START_REF] Caruso | Analysis of Liver Cancer Cell Lines Identifies Agents With Likely Efficacy Against Hepatocellular Carcinoma and Markers of Response[END_REF]. This study showed that the current available LCCL faithfully recapitulate protein, mutational and transcriptional signatures of the proliferation class of HCC and may serve as platform to identify molecular contexts associated with drug vulnerabilities that may help to guide clinical trials [START_REF] Caruso | Analysis of Liver Cancer Cell Lines Identifies Agents With Likely Efficacy Against Hepatocellular Carcinoma and Markers of Response[END_REF]. This dataset is publicly available through an online portal with data visualization (http://lccl.zucmanlab.com) and provides a rich resource that may help to accelerate research in liver cancer (Figure 5). However, cell line models for the non-proliferation class are still lacking, probably due to the poor success rate of establishment [START_REF] Qiu | Hepatocellular carcinoma cell lines retain the genomic and transcriptomic landscapes of primary human cancers[END_REF][START_REF] Park | Characterization of cell lines established from human hepatocellular carcinoma[END_REF]. Large-scale functional screenings using shRNA or CRISPR/CAS9 have also emerged as promising methods to draw a comprehensive mapping of cancer genes and identify potential new therapeutic targets, but for now, very few or no HCC cell lines were included in these studies [START_REF] Behan | Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens[END_REF][START_REF] Mcdonald | Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening[END_REF].

Although tumor-derived cell lines have the advantage of being easy to grow, inexpensive and suitable for high throughput studies, two-dimensional culture condition does not fully recapitulate the native 3D environment of tumor cells and may influence drug response. PDTXs mimic primary tumors more closely than 2D culture, to date, a total of 116 PDTX generated from HCC have been reported in the literature and all the available clinical annotations, molecular and drug response data have been regrouped in the PDXliver database (http://www.picb.ac.cn/PDXliver/) [START_REF] He | PDXliver: a database of liver cancer patient derived xenograft mouse models[END_REF]. However, this resource is limited by the small number of models with drug response data (only 26) and by an incomplete molecular characterization (Figure 5) [START_REF] He | PDXliver: a database of liver cancer patient derived xenograft mouse models[END_REF]. Moreover, the relatively low engraftment efficiency and long engraftment periods (several months) of HCC is still limiting for large-scale drug screenings [START_REF] Huynh | Xenografts of human hepatocellular carcinoma: a useful model for testing drugs[END_REF][START_REF] Xin | Establishment and characterization of 7 novel hepatocellular carcinoma cell lines from patient-derived tumor xenografts[END_REF][START_REF] Gu | Genomic characterization of a large panel of patient-derived hepatocellular carcinoma xenograft tumor models for preclinical development[END_REF].

Alternatively, liver cancer organoid cultures generated from fresh tumor tissues also emerge as valuable models that mimic more closely parental tumors than 2D culture and can be used in large drug screenings (Figure 5) [START_REF] Broutier | Human primary liver cancer-derived organoid cultures for disease modeling and drug screening[END_REF][START_REF] Nuciforo | Organoid Models of Human Liver Cancers Derived from Tumor Needle Biopsies[END_REF]. The major drawback of both organoids and 2D culture cell lines is that they lack immune system and stromal components, consequently, they are not suitable for evaluating therapies targeted against tumor microenvironment such as antiangiogenic, the most effective agents to date in the systemic treatment of HCC, or immunotherapies which cannot be explored either in PTDX models (Figure 5B). Dozens of HCC models in immunocompetent mice have also been developed including spontaneous, chemically-induced and genetically engineered models (Mouse Tumor Biology database http://tumor.informatics.jax.org) (Figure 5) [START_REF] Gengenbacher | Preclinical mouse solid tumour models: status quo, challenges and perspectives[END_REF][START_REF] Krupke | The Mouse Tumor Biology Database: A Comprehensive Resource for Mouse Models of Human Cancer[END_REF][START_REF] Maronpot | Mutations in the ras protooncogene: clues to etiology and molecular pathogenesis of mouse liver tumors[END_REF]. While they are useful for studying basic tumor biology, their use as relevant preclinical platform to evaluate the therapeutic response in HCC is still limited by the lack of systematic cross-species comparative molecular analyses [START_REF] Dow | Integrative genomic analysis of mouse and human hepatocellular carcinoma[END_REF][START_REF] Lee | Application of comparative functional genomics to identify best-fit mouse models to study human cancer[END_REF][START_REF] Connor | Mutational landscape of a chemically-induced mouse model of liver cancer[END_REF].

• New biomarkers

While, tumor tissue is still the gold standard source for identifying cancer specific biomarkers, liquid biopsy has become a promising alternative non-invasive procedure allowing the isolation and detection of cancer-derived subcellular components that are released in biological fluids such as blood [START_REF] Shigeyasu | Emerging Role of MicroRNAs as Liquid Biopsy Biomarkers in Gastrointestinal Cancers[END_REF]. Thus, it enables to overcome the difficulty in obtaining tissue biopsies, as in HCC. Several circulating biomarkers can be detected in liquid biopsies such as DNA, tumor cells, microRNAs and exosomes that are secretory vesicles containing nucleic acids and proteins [START_REF] Shigeyasu | Emerging Role of MicroRNAs as Liquid Biopsy Biomarkers in Gastrointestinal Cancers[END_REF]. Hence, liquid biopsy has become an appealing source of biomarkers for several applications in cancer such as diagnosis, prognosis and for prediction of response to treatment [START_REF] Shigeyasu | Emerging Role of MicroRNAs as Liquid Biopsy Biomarkers in Gastrointestinal Cancers[END_REF]. Liquid biopsy also has the advantage of allowing longitudinal monitoring of tumors and may capture the spatial and temporal intra-tumor heterogeneity that is not feasible with traditional tissue biopsy. However, although liquid biopsy is likely to revolutionize the clinical management of cancers, this technology is still a research field, which is currently facing multiple technical issues that need to be solved, in particular to improve its sensitivity and specificity before future clinical applications.

Conclusion

Although substantial progress has been made in the treatment of HCC, the implementation of an effective precision medicine is still hampered by several obstacles. Firstly, there is still a lack of reliable biomarkers to predict response to current therapies and newer more effective treatments are needed. Since no clear oncogenic addiction loops have been reported in HCC, a better understanding of the epistatic interactions between mutated driver genes may help to develop more potent combination-based therapies. Also, a more detailed characterization of intra and inter-tumoral heterogeneity using longitudinal samples and single cell approaches may provide new insights into the resistance to treatment. Moreover, much more efforts have to be undertaken in developing newer relevant preclinical models, in particular, taking into account the tumor microenvironment, especially the immune contexture that may be feasible by establishing organoid co-culture systems or PTDX in mice with humanized immune system. Finally, systematic tumor biopsy and blood sampling should be done for patients with HCC and more biomarkers based clinical trials should be implemented in the future. Integration of the data collected in both preclinical models and in humans should enable to accelerate the identification of robust predictive biomarkers and ultimately improve the clinical management of HCC (Figure 5A).

Figure 1: Genetic predisposition and mutational process in HCC

We figured the multistep process of carcinogenesis on cirrhosis as well as the risk factors of HCC development on normal liver. We also underlined the main SNP in genes promoting cirrhosis and HCC development (in red, PNPLA3 and TM6SF2) and protecting from cirrhosis and HCC development (in blue, HSD17B13) as well as genes with SNP potentially involved in HCC development but that deserve further validation (in grey). We also represented the mutational process operative in malignant hepatocytes as well as the main somatic genetic alterations in signaling pathways involved in liver carcinogenesis.

Figure 2: Major epistatic interactions between mutated driver genes identified in HCC

Positive interaction indicates a co-occurrence between the mutated genes, while negative interaction indicates mutual exclusivity between the mutated genes. Multi-omic characterization Ye s Ye s Incomplete [122] NA Few models [131,132] Incomplete [131-133] Incomplete [133] Human relevance ++ +++ +++ ++ Variable, model dependant [131] Poor (DEN model) [131,133] Not well characterized [133] Immunocompromised mice Cellular models derived from tumor patients Immunocompetent mice [129]

Figure 3 :

 3 Figure 3: Integrative molecular and pathological classification of HCCThe main molecular subclasses of HCC shown are derived from transcriptomic based classifications previously reported in five studies (Lee, Boyault, Chiang, Hoshida and TCGA) "Unannot": unannotated group of HCC from Chiang's classification without specific features. For each subclass, major associations with pathological, immunohistochemical (IHC), genetic, epigenetic, immunological and clinical features are shown.

Figure 4 :

 4 Figure 4: Tumor heterogeneity in HCCWe represented intra-tumor heterogeneity in the upper panel. As an example of spatial and temporal intratumor heterogeneity, we described a transformation of HCA in HCC with the HCC part who relapse 2 years after. In the lower panel, we figured description of inter tumor heterogeneity on cirrhosis including intrahepatic metastasis and multifocal independent de novo carcinogenesis. Intrahepatic metastasis and multifocal independent carcinogenesis could coexist in the same liver.

Figure 5 :

 5 Figure 5: Modeling of the therapeutic response in HCC A) Strategy to improve precision medicine in HCC based on integration of preclinical models and analyses from HCC patients. B) Main features of HCC preclinical models.

This work was supported by Institut National du Cancer (INCa) with the International Cancer Genome Consortium (ICGC LICA-FR project) and NoFLIC projects (PAIR HCC, INCa and ARC), INSERM with the « Cancer et Environnement » (plan Cancer), MUTHEC projects (INCa) and the HECAM project (BPI). The group is supported by the Ligue Nationale contre le Cancer (Equipe Labellisée), Labex OncoImmunology (investissement d'avenir), grant IREB, Coup d'Elan de la Fondation Bettencourt-Shueller, the SIRIC CARPEM and Fondation Mérieux.

Author contributions:

Drafting of the manuscript, revision of the manuscript and approval of the final

3D tumor organoids [START_REF] Broutier | Human primary liver cancer-derived organoid cultures for disease modeling and drug screening[END_REF][START_REF] Nuciforo | Organoid Models of Human Liver Cancers Derived from Tumor Needle Biopsies[END_REF] Patient-derived xenografts (PDTX) [START_REF] He | PDXliver: a database of liver cancer patient derived xenograft mouse models[END_REF] Cancer Establishment success rate 14-29% [118,119] 18-26% [126,127] 26-35% [124,125] NA / / / Ease of propagation High High Moderate to difficult Moderate to difficult / / / Suitable for high-throughput screening