Jessica Zucman-Rossi

Signatures of mutational processes in human cancer

All cancers are caused by somatic mutations; however, understanding of the biological processes generating these mutations is limited. The catalogue of somatic mutations from a cancer genome bears the signatures of the mutational processes that have been operative. Here we analysed 4,938,362 mutations from 7,042 cancers and extracted more than 20 distinct mutational signatures. Some are present in many cancer types, notably a signature attributed to the APOBEC family of cytidine deaminases, whereas others are confined to a single cancer class. Certain signatures are associated with age of the patient at cancer diagnosis, known mutagenic exposures or defects in DNA maintenance, but many are of cryptic origin. In addition to these genome-wide mutational signatures, hypermutation localized to small genomic regions, ‘kataegis’, is found in many cancer types. The results reveal the diversity of mutational processes underlying the development of cancer, with potential implications for understanding of cancer aetiology, prevention and therapy.

Integrated analysis of somatic mutations and focal copy-number changes identifies key genes and pathways in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy. Here, we performed high-resolution copy-number analysis on 125 HCC tumors and whole-exome sequencing on 24 of these tumors. We identified 135 homozygous deletions and 994 somatic mutations of genes with predicted functional consequences. We found new recurrent alterations in four genes (ARID1A, RPS6KA3, NFE2L2 and IRF2) not previously described in HCC. Functional analyses showed tumor suppressor properties for IRF2, whose inactivation, exclusively found in hepatitis B virus (HBV)-related tumors, led to impaired TP53 function. In contrast, inactivation of chromatin remodelers was frequent and predominant in alcohol-related tumors. Moreover, association of mutations in specific genes (RPS6KA3-AXIN1 and NFE2L2-CTNNB1) suggested that Wnt/β-catenin signaling might cooperate in liver carcinogenesis with both oxidative stress metabolism and Ras/mitogen-activated protein kinase (MAPK) pathways. This study provides insight into the somatic mutational landscape in HCC and identifies interactions between mutations in oncogene and tumor suppressor gene mutations related to specific risk factors.

Transcriptome classification of HCC is related to gene alterations and to new therapeutic targets

Hepatocellular carcinomas (HCCs) are a heterogeneous group of tumors that differ in risk factors and genetic alterations. We further investigated transcriptome‐genotype‐phenotype correlations in HCC. Global transcriptome analyses were performed on 57 HCCs and 3 hepatocellular adenomas and validated by quantitative RT‐PCR using 63 additional HCCs. We determined loss of heterozygosity, gene mutations, promoter methylation of CDH1 and CDKN2A, and HBV DNA copy number for each tumor. Unsupervised transcriptome analysis identified 6 robust subgroups of HCC (G1‐G6) associated with clinical and genetic characteristics. G1 tumors were associated with low copy number of HBV and overexpression of genes expressed in fetal liver and controlled by parental imprinting. G2 included HCCs infected with a high copy number of HBV and mutations in PIK3CA and TP53. In these first groups, we detected specific activation of the AKT pathway. G3 tumors were typified by mutation of TP53 and overexpression of genes controlling the cell cycle. G4 was a heterogeneous subgroup of tumors including TCF1‐mutated hepatocellular adenomas and carcinomas. G5 and G6 were strongly related to β‐catenin mutations that lead to Wnt pathway activation; in particular, G6 tumors were characterized by satellite nodules, higher activation of the Wnt pathway, and E‐cadherin underexpression. Conclusion: These results have furthered our understanding of the genetic diversity of human HCC and have provided specific identifiers for classifying tumors. In addition, our classification has potential therapeutic implications because 50% of the tumors were related to WNT or AKT pathway activation, which potentially could be targeted by specific inhibiting therapies. (HEPATOLOGY 2007;45:42–52.rpar;

MicroRNA Profiling in Hepatocellular Tumors Is Associated with Clinical Features and Oncogene/Tumor Suppressor Gene Mutations

Molecular classifications defining new tumor subtypes have been recently refined with genetic and transcriptomic analyses of benign and malignant hepatocellular tumors. Here, we performed microRNA (miRNA) profiling in two series of fully annotated liver tumors to uncover associations between oncogene/tumor suppressor mutations and clinical and pathological features. Expression levels of 250 miRNAs in 46 benign and malignant hepatocellular tumors were compared to those of 4 normal liver samples with quantitative reverse‐transcriptase polymerase chain reaction. miRNAs associated with genetic and clinical characteristics were validated in a second series of 43 liver tumor samples and 16 nontumor samples. miRNA profiling unsupervised analysis classified samples in unique clusters characterized by histological features (tumor/nontumor, P < 0.001; benign/malignant tumors, P < 0.01; inflammatory adenoma and focal nodular hyperplasia, P < 0.01), clinical characteristics [hepatitis B virus (HBV) infection, P < 0.001; alcohol consumption, P < 0.05], and oncogene/tumor suppressor gene mutations [β‐catenin, P < 0.01; hepatocyte nuclear factor 1α (HNF1α), P < 0.01]. Our study identified and validated miR‐224 overexpression in all tumors and miR‐200c, miR‐200, miR‐21, miR‐224, miR‐10b, and miR‐222 specific deregulation in benign or malignant tumors. Moreover, miR‐96 was overexpressed in HBV tumors, and miR‐126* was down‐regulated in alcohol‐related hepatocellular carcinoma. Down‐regulations of miR‐107 and miR‐375 were specifically associated with HNF1α and β‐catenin gene mutations, respectively. miR‐375 expression was highly correlated to that of β‐catenin–targeted genes as miR‐107 expression was correlated to that of HNF1α in a small interfering RNA cell line model. Thus, this strongly suggests that β‐catenin and HNF1α could regulate miR‐375 and miR‐107 expression levels, respectively. Conclusion: Hepatocellular tumors may have a distinct miRNA expression fingerprint according to malignancy, risk factors, and oncogene/tumor suppressor gene alterations. Dissecting these relationships provides a new hypothesis to understand the functional impact of miRNA deregulation in liver tumorigenesis and the promising use of miRNAs as diagnostic markers. (HEPATOLOGY 2008.)

Genotype–phenotype correlation in hepatocellular adenoma: New classification and relationship with HCC

Hepatocellular adenomas are benign tumors that can be difficult to diagnose. To refine their classification, we performed a comprehensive analysis of their genetic, pathological, and clinical features. A multicentric series of 96 liver tumors with a firm or possible diagnosis of hepatocellular adenoma was reviewed by liver pathologists. In all cases, the genes coding for hepatocyte nuclear factor 1α (HNF1α) and β‐catenin were sequenced. No tumors were mutated in both HNF1α and β‐catenin enabling tumors to be classified into 3 groups, according to genotype. Tumors with HNF1α mutations formed the most important group of adenomas (44 cases). They were phenotypically characterized by marked steatosis (P < 10−4), lack of cytological abnormalities (P < 10−6), and no inflammatory infiltrates (P < 10−4). In contrast, the group of tumors defined by β‐catenin activation included 13 lesions with frequent cytological abnormalities and pseudo‐glandular formation (P < 10−5). The third group of tumors without mutation was divided into two subgroups based on the presence of inflammatory infiltrates. The subgroup of tumors consisting of 17 inflammatory lesions, resembled telangiectatic focal nodular hyperplasias, with frequent cytological abnormalities (P = 10−3), ductular reaction (P < 10−2), and dystrophic vessels (P = .02). In this classification, hepatocellular carcinoma associated with adenoma or borderline lesions between carcinoma and adenoma is found in 46% of the β‐catenin–mutated tumors whereas they are never observed in inflammatory lesions and are rarely found in HNF1α mutated tumors (P = .004). In conclusion, the molecular and pathological classification of hepatocellular adenomas permits the identification of strong genotype–phenotype correlations and suggests that adenomas with β‐catenin activation have a higher risk of malignant transformation. (HEPATOLOGY 2006;43:515–524.)

Hepatocellular Adenoma Subtype Classification Using Molecular Markers and Immunohistochemistry

Hepatocellular adenomas (HCA) with activated β‐catenin present a high risk of malignant transformation. To permit robust routine diagnosis to allow for HCA subtype classification, we searched new useful markers. We analyzed the expression of candidate genes by quantitative reverse transcription polymerase chain reaction QRT‐PCR followed by immunohistochemistry to validate their specificity and sensitivity according to hepatocyte nuclear factor 1 alpha (HNF1α) and β‐catenin mutations as well as inflammatory phenotype. Quantitative RT‐PCR showed that FABP1 (liver fatty acid binding protein) and UGT2B7 were downregulated in HNF1α‐inactivated HCA (P ≤ 0.0002); GLUL (glutamine synthetase) and GPR49 overexpression were associated with β‐catenin–activating mutations (P ≤ 0.0005), and SAA2 (serum amyloid A2) and CRP (C‐reactive protein) were upregulated in inflammatory HCA (P = 0.0001). Immunohistochemistry validation confirmed that the absence of liver‐fatty acid binding protein (L‐FABP) expression rightly indicated HNF1α mutation (100% sensitivity and specificity), the combination of glutamine synthetase overexpression and nuclear β‐catenin staining were excellent predictors of β‐catenin–activating mutation (85% sensitivity, 100% specificity), and SAA hepatocytic staining was ideal to classify inflammatory HCA (91% sensitivity and specificity). Finally, a series of 93 HCA was unambiguously classified using our 4 validated immunohistochemical markers. Importantly, new associations were revealed for inflammatory HCA defined by SAA staining with frequent hemorrhages (P = 0.003), telangiectatic phenotype (P < 0.001), high body mass index, and alcohol intake (P ≤ 0.04). Previously described associations were confirmed and in particular the significant association between β‐catenin–activated HCA and hepatocellular carcinomas (HCC) at diagnosis or during follow‐up (P < 10−5). Conclusion: We refined HCA classification and its phenotypic correlations, providing a routine test to classify hepatocellular adenomas using simple and robust immunohistochemistry. (HEPATOLOGY 2007.)

Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

Genomic analyses promise to improve tumor characterization to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors identified mutational signatures associated with specific risk factors, mainly combined alcohol and tobacco consumption and exposure to aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrently altered pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (hepatitis B virus, HBV) and AXIN1. Analyses according to tumor stage progression identified TERT promoter mutation as an early event, whereas FGF3, FGF4, FGF19 or CCND1 amplification and TP53 and CDKN2A alterations appeared at more advanced stages in aggressive tumors. In 28% of the tumors, we identified genetic alterations potentially targetable by US Food and Drug Administration (FDA)–approved drugs. In conclusion, we identified risk factor–specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC, which will be useful to design clinical trials for targeted therapy.

Frequent in-frame somatic deletions activate gp130 in inflammatory hepatocellular tumours.

Inflammatory hepatocellular adenomas are benign liver tumours defined by the presence of inflammatory infiltrates and by the increased expression of inflammatory proteins in tumour hepatocytes. Here we show a marked activation of the interleukin (IL)-6 signalling pathway in this tumour type; sequencing candidate genes pinpointed this response to somatic gain-of-function mutations in the IL6ST gene, which encodes the signalling co-receptor gp130. Indeed, 60% of inflammatory hepatocellular adenomas harbour small in-frame deletions that target the binding site of gp130 for IL-6, and expression of four different gp130 mutants in hepatocellular cells activates signal transducer and activator of transcription 3 (STAT3) in the absence of ligand. Furthermore, analysis of hepatocellular carcinomas revealed that rare gp130 alterations are always accompanied by β-catenin-activating mutations, suggesting a cooperative effect of these signalling pathways in the malignant conversion of hepatocytes. The recurrent gain-of-function gp130 mutations in these human hepatocellular adenomas fully explains activation of the acute inflammatory phase observed in tumourous hepatocytes, and suggests that similar alterations may occur in other inflammatory epithelial tumours with STAT3 activation.

Hepatocellular adenoma management and phenotypic classification: The Bordeaux experience

We took advantage of the reported genotype/phenotype classification to analyze our surgical series of hepatocellular adenoma (HCA). The series without specific known etiologies included 128 cases (116 women). The number of nodules varies from single, <5, and ≥5 in 78, 38, and 12 cases, respectively. The resection was complete in 95 cases. We identified 46 HNF1α‐inactivated HCAs (44 women), 63 inflammatory HCAs (IHCA, 53 women) of which nine were also β‐catenin–activated, and seven β‐catenin–activated HCAs (all women); six additional cases had no known phenotypic marker and six others could not be phenotypically analyzed. Twenty‐three of 128 HCAs showed bleeding. No differences were observed in solitary or multiple tumors in terms of hemorrhagic manifestations between groups. In contrast, differences were observed between the two main groups. Steatosis (tumor), microadenomas (resected specimen), and additional benign nodules were more frequently observed in HNF1α‐inactivated HCAs (P < 0.01) than in IHCAs. Body mass index > 25, peliosis (tumor), and steatosis in background liver were more frequent in IHCA (P < 0.01). After complete resection, new HCAs in the centimetric range were more frequently found during follow‐up (>1 year) in HNF1α‐inactivated HCA. After incomplete resection (HCA left in nonresected liver), the majority of HCA remained stable in the two main groups and even sometimes regressed. Six patients of 128 developed hepatocellular carcinoma (HCC) (all were β‐catenin–activated, whether inflammatory or not). Conclusion: There were noticeable clinical differences between HNF1α–inactivated HCA and IHCA; there was no increased risk of bleeding or HCC related to the number of HCAs; β‐catenin–activated HCAs are at higher risk of HCC. As a consequence, we believe that management of HCA needs to be adapted to the phenotype of these tumors. (HEPATOLOGY 2009.)

Bi-allelic inactivation of TCF1 in hepatic adenomas

Liver adenomas are benign tumors at risk of malignant transformation. In a genome-wide search for loss of heterozygosity (LOH) associated with liver adenomas, we found a deletion in chromosome 12q in five of ten adenomas. In most cases, LOH at 12q was the only recurrent genetic alteration observed, suggesting the presence of a tumor-suppressor gene in that region. A minimal common region of deletion was defined in 12q24 that included the gene TCF1 (transcription factor 1), encoding hepatocyte nuclear factor 1 (HNF1; refs 1,2). Heterozygous germline mutations of TCF1 have been identified in individuals affected with maturity-onset diabetes of the young type 3 (MODY3; ref. 3). Bi-allelic inactivation of TCF1 was found in 10 of 16 screened adenomas, and heterozygous germline mutation were present in three affected individuals. Furthermore, 2 well-differentiated hepatocellular carcinomas (HCCs) occurring in normal liver contained somatic bi-allelic mutations of 30 screened HCCs. These results indicate that inactivation of TCF1, whether sporadic or associated with MODY3, is an important genetic event in the occurrence of human liver adenoma, and may be an early step in the development of some HCCs.