REVIEWS IN BASIC AND CLINICAL GASTROENTEROLOGY AND HEPATOLOGY

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1 GASTROENTEROLOGY 2013;144: IN BASIC CLINICAL GASTROENTEROLOGY HEPATOLOGY Robert F. Schwabe and John W. Wiley, Section Editors Hepatocellular Benign Tumors From Molecular Classification to Personalized Clinical Care JEAN CHARLES NAULT, 1,2 PAULETTE BIOULAC SAGE, 3,4 and JESSICA ZUCMAN ROSSI 1,2,5 1 INSERM, UMR-674, Génomique Fonctionnelle des Tumeurs Solides, IUH, Paris; 2 Université Paris Descartes, Labex Immuno-oncology, Sorbonne Paris Cité, Faculté de Médecine, Paris; 3 INSERM, UMR-1053, Université Victor Segalen Bordeaux 2, Bordeaux; 4 CHU de Bordeaux, Department of Pathology, Pellegrin Hospital, Bordeaux; and 5 Assistance Publique-Hôpitaux de Paris, Hopital Européen Georges Pompidou, Paris, France Podcast interview: Also available on itunes. Focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA) are benign hepatocellular tumors that develop most frequently in women without cirrhosis. Genomic approaches have identified signaling pathways related to these benign hepatocyte proliferations. FNH, a polyclonal lesion, is characterized by local vascular abnormalities and heterogeneous activation of Wnt/ -catenin and transforming growth factor signaling. Four major subgroups of HCAs have been identified based on mutations in specific oncogenes and tumor suppressor genes. Each molecular subtype of HCA has been associated with specific pathways, providing new information about benign tumorigenesis. Key features include metabolic alterations (induced by defects in HNF1A), oncogene-induced inflammation (activation of JAK-STAT signaling in inflammatory adenomas), and an association between activation of Wnt/ -catenin signaling and progression of HCAs in hepatocellular carcinomas. Benign hepatocellular tumors can be classified using immunohistochemical analyses. Studies of genotypes and phenotypes of FNH and HCAs have led to the identification of risk factors and improved invasive and noninvasive diagnostic techniques, evaluation of prognosis, and treatment. We review the molecular pathways involved in benign hepatocyte proliferation and discuss how this basic knowledge has been progressively translated into personalized clinical care. Keywords: CTNNB1; gp130; STAT3; GNAS. Hepatocellular benign tumors are defined by benign proliferation of hepatocytes with a mass-forming presentation and are mainly categorized as focal nodular hyperplasia (FNH) and hepatocellular adenoma (HCA). Benign liver tumors derived from cholangiocytes, mesenchymal cells, or endothelial cells (cystadenoma, angioma, angiomyolipoma, or polycystic liver disease) are not included in this definition and will not be discussed in this review. 1,2 HCA and FNH share common features. First, a large female predominance is observed in both cases, with a female/male ratio of 8:1 for FNH and 9:1 for HCA. 3 Moreover, FNH and HCA derive from the proliferation of mature hepatocytes and arise on otherwise normal liver. Consequently, these tumors represent a model to study liver tumorigenesis outside the classic background of cirrhosis. However, FNH and HCA also exhibit striking differences in terms of incidence or risk factors. The reported prevalence of FNH and HCA is a rough estimation, but FNH is certainly the leading benign hepatocellular tumor with an estimated prevalence of approximately 0.4 to 3 per 100 in 2 series of 95 and 2500 autopsies more than 20 years ago. 4,5 It should be noted that the prevalence of hemangiomas is 10-fold more than that of FNH. 2,6 Even if debated, oral contraception might play a limited role in the development of FNH. 7,8 In contrast, for HCA, a fold lower prevalence was reported in the 1970s at approximately 3 per 100,000 in patients exposed to high-dose oral contraceptives. 9 However, the prevalence of both FNH and HCA should be reevaluated in the general occidental and Asian population, taking into account the recent evolution of HCA risk factors. Despite the lack of precise and recent epidemiologic data, the incidence of HCA is higher in Western than in Eastern countries. 10,11 This could be partly explained by the lower use of estrogen contraceptives in the Asian population compared with the Western population. 12 Worldwide, the epidemi- Abbreviations used in this paper: bhca, -catenin mutated hepatocellular adenoma; FNH, focal nodular hyperplasia; HCA, hepatocellular adenoma; HCC, hepatocellular carcinoma; H-HCA, HNF1A-mutated hepatocellular adenoma; IHCA, inflammatory hepatocellular adenoma; IL, interleukin; LFABP, liver fatty acid binding protein; MODY3, maturityonset diabetes type 3; MRI, magnetic resonance imaging; SAA, serum amyloid A by the AGA Institute /$

2 May 2013 HEPATOCELLULAR BENIGN TUMORS 889 ologic landscape of HCA could be deeply modified by the emergence of new risk factors such as obesity and alcohol consumption, which are associated with inflammatory HCA (IHCA), but also by the recent development of the third generation of oral contraceptives with a low dose of estrogen. 10,13 16 Recently, molecular analyses of FNH and HCA have highlighted new mechanisms activated in the course of benign liver tumorigenesis and have revealed the genetic heterogeneity of these tumors The aim of this work was to review recent advances in molecular mechanisms and classification and to evaluate how this new knowledge can improve the management of patients with a benign hepatocellular tumor. 15,21,22 FNH Physiopathology of FNH: The Vascular Hypothesis FNH is defined by normal-like hepatocytes arranged in 1- to 2-cell-thick plates with a plurinodular pattern separated by fibrous bands and surrounding a central fibrous scar with a dystrophic arterial vessel. 23 The lesion is also characterized by the absence of a portal vein and presence of a ductular reaction at the interface of fibrous bands/parenchyma. The most accepted theory about the origin of FNH was proposed by Wanless et al 24 : FNH could originate from a hyperplastic lesion composed of reactive polyclonal proliferating hepatocytes. This proliferation of abnormal hepatocytes could be caused by an increased blood flow due to local vascular malformation. 25 Accordingly, clonality studies, mainly using HUMARA assays, have revealed the polyclonal origin of FNH in 50% to 100% of cases. 3,13,26 29 It is noteworthy that most of the latter studies were performed more than 10 years ago, including the so-called telangiectatic FNH entity that was recently reclassified as inflammatory adenoma. 13,20 This bias could partly explain the overestimated number of FNH reported with a monoclonal origin in various studies. No somatic mutations in genes involved in benign and malignant liver tumorigenesis have been described thus far in FNH, thereby confirming the polyclonal origin of these tumors. 19,27 Despite its polyclonal origin, FNH is usually considered as a benign hepatocellular tumor due to its mass-forming presentation. However, FNH appears to better correspond to a hyperplastic lesion than to a neoplastic lesion. 24,27,30 Gene expression studies have revealed molecular features supporting the theory of vascular abnormalities as a driving event in the formation of FNH. Indeed, FNH presents abnormal expression of genes involved in angiogenesis with an increase in expression of the ANGPT1/ ANGPT2 ratio. 19,31 ANGPT1 is classically responsible for vessel formation, and ANGPT2 acts as an antagonist of ANGPT1; thus, increased expression of ANGPT1/ ANGPT2 is expected to promote angiogenesis. Moreover, genome expression using microarray in FNH identified an overexpression of several genes of the extracellular matrix, particularly at the central fibrous scar, related to activation of the transforming growth factor pathway. 19,30 Another major finding was the overexpression of Wnt/ catenin target genes, including GLUL coding for glutamine synthase. In the normal liver lobule, -catenin is activated in 2 to 3 thick of hepatocytes around the central vein and repressed in hepatocytes in the portal area. 32 In contrast, in FNH, overexpression of glutamine synthase is enlarged and shows a specific location with a map-like pattern predominantly in the periphery of the nodules (Figure 1). 19,33 The heterogeneous distribution of glutamine synthase is caused by -catenin activation without -catenin activating mutations in accordance with FNH polyclonal origin. Again, this could result from an abnormal arterial blood flow. 19 Because this map-like pattern of glutamine synthase expression is characteristic, easy to recognize, and specifically observed in FNH, it is now used as a routine diagnostic tool (see the following text). Diagnosis of FNH: Pivotal Role of Radiology With Additional Help From Immunohistochemistry FNH is usually an asymptomatic benign tumor with normal liver enzyme levels and tumor markers (AFP, CA19-9, ACE) 34 that are mostly found incidentally using imaging. FNH is frequently associated with liver hemangioma (20% of cases 35 ), with other FNH (multiple FNH in 20% 30% of cases 23,35,36 ), and more rarely with HCA. 35,37 Some rare diseases such as Rendu Osler disease, Budd Chiari syndrome, and portal vein agenesis are also associated with FNH Interestingly, these diseases are characterized by vascular abnormalities in the liver and constitute a strong link with the vascular hypothesis, explaining the origin of FNH. However, the vast majority of patients with FNH do not have any of those known risk factors. FNH is not associated with significant complications and should usually be managed with conservative treatment. 34 Consequently, the major clinical problem remains the differential diagnosis of FNH from other benign (HCA) or malignant (hepatocellular carcinoma [HCC]) hepatocellular tumors. Radiology constitutes the bona fide approach for the diagnosis of FNH, and contrastenhanced magnetic resonance imaging (MRI) is considered as the gold standard (Figure 1). 41 Using contrastenhanced MRI, FNH is defined by an isosignal or a faint hyposignal in T1, homogeneity of the nodule except for the central scar, diffuse enhancement of the FNH at the arterial phase, a lesion in the isosignal during the portal and late phase, absence of a capsule, a central scar in a hypersignal in T2, and enhancement of the central scar during the late phase for nodules 3 cm. 42 In practice, tumors fulfilling these criteria are considered FNH without histologic analysis. However, some FNH do not present these typical features and require biopsy for a firm diagnosis. 43 Interpretation of the biopsy specimen could also be challenging due to the small size of the tumor or the absence of a fibrous scar. 23 The immunohistochemical staining of glutamine synthase is useful to reveal 5 to 10

3 890 NAULT ET AL GASTROENTEROLOGY Vol. 144, No. 5 Figure 1. Composite algorithm for the diagnosis and treatment of FNH and HCAs. Clinical, radiologic, pathologic, and molecular data are combined in a step-by-step diagnostic and treatment algorithm stratified by the risk of complications (hemorrhage and malignant transformation for HCA, absence of complications for FNH). Diagnosis of FNH is usually at MRI. In atypical cases, a biopsy specimen showing glutamine synthase in a map-like pattern staining is very useful for diagnosis of FNH. CT, computed tomographic; CRP, C-reactive protein; OC, oral contraception; GS, glutamine synthase; SAA,serum amyloid A. * Surgery should be considered in the absence of adenoma regression after oral contraception or androgen withdrawal. thick plates of hepatocytes stained around the veins without staining around the fibrous scar ( map-like pattern; Figure 1). 33 Hence, we propose the introduction of immunohistological analyses in a diagnostic algorithm to better diagnose difficult cases (Figure 1). Treatment The lack of complications justifies abstention of treatment as a general rule in the clinical care of patients with FNH. 34,44 The natural history of FNH shows that the size of the tumor remains generally stable. Consequently, patients with FNH do not require clinical and radiologic follow-up if the initial diagnosis is certain. Because estrogen exposition does not influence the clinical course of FNH, oral contraception can be pursued and pregnancy is possible and safe. 7 Exceptionally, huge FNH nodules can cause abdominal pain and biliary or vessel compression, and surgical resection can be discussed after strict elimination of a differential diagnosis. 45

4 May 2013 HEPATOCELLULAR BENIGN TUMORS 891 Figure 2. Molecular classification of HCAs. Each molecular subgroup of HCA is related to specific pathologic and clinical features (genotype/phenotype relationship). They are identified using immunohistochemistry with 5 antibodies (FABP, SAA, C-reactive protein [CRP], -catenin, and glutamine synthase) and/or specific features at MRI. HCA: More Than a Uniform Benign Tumor HCAs are defined as the monoclonal proliferation of well-differentiated hepatocytes arranged in sheets and cords. They are also characterized by the absence of a portal triad and interlobular bile ducts. 46 A specific clinical entity, liver adenomatosis, was defined by Flejou et al as the occurrence of more than 10 HCAs. 47 Interestingly, liver adenomatosis is not clearly related to oral contraception. 47,48 For more than 40 years, HCA was considered as a well-defined homogeneous entity. However, the recent molecular dissection of HCA in several homogeneous subclasses revealed the heterogeneity of the disease and the genotype/phenotype classification enabled reevaluation of the predisposition and initiation of liver tumors (Figure 2 and Table 1). 3,15,17,18 Those analyses also refined our understanding of the oncogenic mechanisms activated in benign liver tumorigenesis and that are specific to the different molecular subtypes. Finally, this molecular classification has been used in pathological routine practice. Indeed, immunohistochemical staining performed with a panel of antibodies has led to the validation of the genotype/phenotype correlation by several groups worldwide (Table 1) Molecular Classification of HCAs HCAs are classified into 4 major molecular subgroups according to their genetic and phenotype characteristics. HNF1A-mutated adenomas. The first group of HCAs is defined by HNF1A (coding for hepatocyte nuclear factor 1 ) mutations in 30% to 40% of all adenomas (Figure 2 and Table 1). 56 HNF1A mutations were identified in 2002 using genomic approaches that identified recurrent losses of heterozygosity at chromosome 12q with subsequent analyses that revealed biallelic somatic mutations in the HNF1A gene. 56 Most of the time, both inactivating HNF1A mutations are somatic and thus found only in tumor cells. However, some patients carry a germline inherited HNF1A mutation in one allele. This condition is associated with maturity-onset diabetes type 3 (MODY3), a monogenic non insulin-dependent diabetes. 57 Adenomas that develop in patients with MODY3 present another somatic mutation inactivating the second allele of HNF1A in the tumor hepatocytes according to the 2-hits model described for inactivation of tumor suppressor genes. 58 Moreover, patients with germline mutations of HNF1A are predisposed to develop liver adenomatosis A mouse model harboring a germline HNF1A inactivation was developed by Pontoglio et al. 63 hnf1a / mice exhibit hepatomegaly with marked steatosis leading to liver dysfunction. These mice did not develop any liver tumors but were dying precociously from severe metabolic disorders. A second mouse model with HNF1A germline inactivation was developed by Lee et al 64 and confirmed hepatomegaly without nodular lesions in hnf1a / mice. However, these mice showed an increased hepatocyte proliferation with liver dysplasia. In a recent study, Tward et al induced HCAs in mice by overexpressing MET and a dominant negative form of HNF1A using the hydrodynamic injection in a mouse tail approach. 65 In contrast, mice injected with MET or with a dominant negative form of HNF1A alone did not develop any tumor. These results

5 Table 1. Genotype/Phenotype Classification of HCAs Worldwide Reference Country/model Type of analysis No. of analyzed HCAs HCA molecular subgroup (%) H-HCA IHCA IHCA and bhca bhca Unclassified adenomas Percentage of malignant transformation (risk factors) France/multicentric Molecular analysis of surgical Zucman-Rossi et al NS (bhca) specimen Bioulac-Sage et al, France/monocentric IHC of surgical specimen (bhca, male, size 5 cm) Van der Borght et al, Belgium/monocentric IHC of surgical specimen 33 NS NS NS 9 NS 18 (bhca) Bioulac-Sage et al, France/monocentric Molecular analysis plus IHC of (bhca) surgical specimen Dokmak et al, France/monocentric IHC of surgical specimen a a 2 a 16 8 (bhca, male, size 5 cm) ( -catenin alone) ( -catenin alone) Farges et al, France/monocentric IHC of surgical specimen 25 malignant transformation among 218 patients (bhca, male, size 5 cm) Ronot et al, France/monocentric IHC of biopsy specimen NS 2 a 3 NS Van Aalten et al, The Netherlands/monocentric IHC of surgical specimen Sasaki et al, Japan/monocentric IHC of surgical specimen NS (unclassified) Bellamy et al, United Kingdom/monocentric IHC of surgical specimen (bhca) Evason et al, United States/monocentric Molecular analysis plus IHC of 70 NS NS 6 (bhca) surgical and biopsy specimens Bioulac-Sage et al, France/multicentric IHC of biopsy specimen NS Sakellariou et al, United Kingdom/monocentric Molecular analysis plus IHC of 33 (glycogenosis) surgical specimen Calderaro et al, European Union/multicentric Molecular analysis plus IHC of 25 (glycogenosis) (bhca) surgical specimen Fonesca et al, Belgium/monocentric IHC of surgical specimen (bhca, male) NS, not specified; IHC, immunohistochemistry. a Only a subset of HCA has been classified using immunohistochemistry. bhca was defined by -catenin immunostaining alone without assessment with glutamine synthase. 892 NAULT ET AL GASTROENTEROLOGY Vol. 144, No. 5

6 May 2013 HEPATOCELLULAR BENIGN TUMORS 893 obtained in mouse models argue for a necessary cooperation between HNF1A inactivation and additional genetic alterations to promote liver tumorigenesis. However, until now in humans, no additional genetic alterations have thus far been identified in HNF1A-mutated adenomas (H-HCAs) and biallelic inactivating mutations of HNF1A are exclusive of CTNNB1, IL6ST, GNAS, and STAT3 mutations classically found in other HCA subtypes (see the following text). HNF1A belongs to the hepatocyte nuclear factor family and is a key transcription factor involved in development of the liver. In addition, HNF1A controls hepatocyte differentiation as well as glucose and lipid metabolism Transcriptomic analysis of H-HCAs using microarrays revealed the dysregulation of genes involved in glucose and lipid metabolism (Figure 3). The main features observed included repression of gluconeogenesis, activation of glycolysis, and stimulation of aberrant fatty acid synthesis. 69 Similar metabolic alterations were also identified in the liver of hnf1a / mice. 66,67 Moreover, in human H-HCA, several oncogenic factors lead to mtor activation, angiogenesis, cell cycle activation (CCND1), and cell proliferation (PDGFRA and B, ERRB2) (Figure 3). 70 Interestingly, these observations were also observed on HNF1A silencing in different hepatocellular cell lines, thereby showing the specific role of HNF1A inactivation in inducing the metabolic and pro-proliferative disorders observed in H- HCA Strikingly, genotype/phenotype correlations have also corroborated these observations. Indeed, pathological analysis has revealed that inactivating biallelic mutations of HNF1A define a very homogeneous subgroup of adenomas characterized by a marked steatosis. 15,18 However, steatosis alone is insufficient to diagnose H-HCA because it could also be observed in more than 35% of inflammatory and unclassified HCAs. 15 Expression of liver fatty acid binding protein (LFABP), one of the most abundant proteins expressed in normal hepatocytes involved in the cytoplasmic trafficking of fatty acid, is specifically down-regulated in H-HCAs. LFABP staining belongs now to the panel of immunohistological markers used to classify H-HCAs (specificity, 100%; sensitivity, 100%), 18 and finally HNF1A stands at the crossroads between metabolism and tumorigenesis in the liver. -catenin activated adenomas. -catenin activated mutations were described for the first time by Chen et al in 3 of 10 HCAs that developed in Taiwanese patients. 73 Mutations of -catenin are localized at hot spots in exon 3, 15,73 and -catenin mutated HCAs constitute approximately 10% to 15% of all HCAs (Figure 2 and Table 1). Strikingly, -catenin activated mutations are exclusive of HNF1A mutations but can be combined with gp130 or GNAS mutations, and half of the -catenin activated adenomas (bh- CAs) are also inflammatory (bihca) (see the following text). Under physiological conditions, in the absence of activating ligand, the Wnt/ -catenin pathway is inactivated and -catenin is phosphorylated by the GSKB/APC/ AXIN complex. 74 Phosphorylation of -catenin leads to its ubiquitination and subsequent degradation by the proteasome. In tumors, mutations of -catenin impair phosphorylation by the GSK3B/APC/AXIN complex and decrease its degradation; as a consequence, -catenin accumulates and translocates to the nucleus to act as a cotranscription factor via the association with the T-cell factor/lymphoid-enhancing complex. 75 The Wnt/ catenin pathway plays a key role in amino acid metabolism, cell-cell interactions, liver regeneration, and zonation. 75,76 It is also a well-known oncogenic pathway involved in several types of cancer and particularly HCC. 77,78 However, a genetically engineered mouse model harboring liver-specific expression of -catenin activating mutations developed neither HCA nor HCC. 79,80 In contrast, liver-specific deletion of APC or coexpression of HRAS with CTNNB1 mutant leads to formation of HCC. 77,81 These results show that activation of -catenin is not sufficient alone to induce liver carcinogenesis but can cooperate with other pathways to promote tumor development. Genotype/phenotype correlation has revealed striking hallmarks of -catenin mutated HCAs. First, few male subjects develop HCAs; however, when it occurs, it is usually a bhca. At the pathological level, these adenomas exhibit cholestasis and cell dysplasia. 15,82 Importantly, -catenin mutations are associated with a high risk of malignant transformation (Table 1). 15,54,55,83 -catenin mutated adenomas show strong overexpression of GLUL (coding for glutamine synthase) and LGR5 (coding for leucine-rich repeat-containing G-protein coupled receptor 5), 2 -catenin target genes as revealed by quantitative reverse-transcription polymerase chain reaction analysis. 18 Using immunohistochemistry, diagnosis of a -catenin mutated adenoma is performed using -catenin and glutamine synthase immunostaining. bhcas are characterized by a nuclear localization of -catenin and a strong homogeneous cytoplasmic expression of glutamine synthase. 18 This pattern is homogeneous and diffuse in bhcas and differs from the map-like pattern observed in FNH. Because nuclear -catenin is frequently found only in a few nuclei, it is therefore difficult to interpret. Consequently, the analysis of -catenin expression has to be combined with glutamine synthase staining to increase detection sensitivity for -catenin activation. However, although these 2 markers have very good specificity (approximately 100%), they show insufficient sensitivity (75% 85%) for the diagnosis of -catenin mutations. 15,18 Because of the high risk of malignant transformation, HCAs without overexpression of -catenin target genes and not mutated for HNF1A (exclusive with -catenin mutations) should be screened for -catenin mutations. Inflammatory adenomas. The third group of HCAs is composed of inflammatory adenomas and accounts for 40% to 50% of all adenomas (Figure 2 and Table 1). 18 The cardinal feature of IHCAs is the activation of the JAK/STAT pathway (Figure 4). 84 At the histologic level, IHCAs are characterized by polymorphic inflammatory infiltrates, dystrophic vessels, and sinusoidal dilatations. 13,15 They also exhibit overexpression of serum am-

7 894 NAULT ET AL GASTROENTEROLOGY Vol. 144, No. 5 Figure 3. Dysregulation of the metabolic and proliferative pathway in HNF1A-inactivated adenomas. Genes are down-regulated (green) and up-regulated (red) in HNF1A-inactivated HCAs. Biallelic inactivating mutations of HNF1A in hepatocytes leads to metabolic defects: activation of glycolysis (solid arrows), repression of gluconeogenesis (dashed arrows), and activation of fatty acid synthesis. In addition, this subtype of HCA harbors activation of the mtor pathway and dysregulation of cell cycle genes. Abnormal estrogen metabolism with possible accumulation of 17 estradiol could explain the role of oral contraception in the development of HCA.

8 May 2013 HEPATOCELLULAR BENIGN TUMORS 895 Figure 4. Hepatocytes as a pivotal inflammatory cell involved in benign liver tumorigenesis. The driving oncogenes found recurrently mutated and responsible of the inflammatory phenotype are in red. The role of chronic inflammatory response in the genesis of dystrophic artery and sinusoidal dilatation remains unexplored. yloid A (SAA) and C-reactive protein, 2 proteins of the acute phase of inflammation. 18 Identification of IHCAs reveals obesity and high alcohol intake as 2 new risk factors for occurrence of inflammatory adenomas but not for the other subtypes. 14,18,22 Importantly, a subset of IHCAs also harbors an activating mutation of -catenin and, consequently, shows an increased risk of malignant transformation. 84,85 Recently, 3 different molecular drivers, namely IL6ST (coding for gp130), 84 STAT3, 86 and GNAS, 87 were discovered as oncogenes activated by mutations in IHCA (Figure 4). In tumors, each mutation is exclusive of the 2 others and the 3 genes (IL6ST, STAT3, and GNAS) can explain more than 75% of the overall IHCAs. In the canonical IL-6/JAK/STAT pathway, interleukin (IL)-6 binds to gp80, the IL-6 receptor (IL6R), that dimerizes with gp130, the coreceptor signal transducer (IL6ST); then, 2 IL-6/gp130/ gp80 complexes associate and form an hexameric complex at the cell membrane 88 (Figure 4). This leads to the recruitment of JAK1 and JAK2, which phosphorylate in turn STAT3 and STAT1. Activated phospho-stat3 and phospho-stat1 homodimerize and/or heterodimerize and translocate to the nucleus to act as transcription factors. 88 Somatic heterozygous mutations activating gp130 are found in 65% of inflammatory adenomas. 84 Mutations of gp130 are mainly small in-frame deletions targeting the IL-6 binding site. 84 Gp130 mutant leads to gp130 dimerization and constitutive STAT3 activation in the absence of IL-6. 84,89 Rare cases of HCC also harbor gp130- activating mutations. 84,85 Interestingly, these HCCs develop on normal livers and also show -catenin activating mutations, arguing for malignant transformation of HCAs. The second oncogene mutated in IHCA, STAT3, ismu- tated in 5% of IHCAs. 86 These mutations lead to amino acid substitutions mainly localized in the SH2 domain involved in STAT3 dimerization. STAT3 mutants are phosphorylated and activated in the absence of IL-6 and lead to an uncontrolled constitutive activation of the inflammatory pathway. The third gene, GNAS, is also found to be mutated in 5% of IHCAs. GNAS encodes the subunit of the Gs protein. 87 The Gs protein complex is composed of the and / subunits. In their inactive forms, and / subunits are associated. 90 When a stimulus leads to G protein coupled receptor activation, guanosine triphosphate replaces guanosine diphosphate on the subunit. Then, the latter dissociates and activates the adenylate cyclase, thereby increasing levels of intracellular cyclic adenosine monophosphate, which acts as an intracellular second messenger. 90 Thus, the subunit is inactivated due to an intrinsic guanosine triphosphatase activity that converts guanosine triphosphate into guanosine diphosphate. Activating mutations of GNAS localize at hot spots in the guanosine triphosphatase domain. 90 Guanosine triphosphatase activity of the subunit is consequently impaired and leads to aberrant activation of adenylate cyclase and accumulation of cyclic adenosine monophosphate. GNAS is considered an oncogene recurrently mutated in benign lesions such as pituitary and thyroid adenomas, myelodysplastic syndrome, and intraductal papillary mucinous neoplasm but also in cancers such as cholangiocarcinoma. 90,91 We identified typical GNAS-activating mutations in sporadic HCAs and in adenomas arising in patients with McCune Albright syndrome. 92 McCune Albright syndrome is a rare disease defined by café au lait skin macula, fibrous dysplasia of bone, and high risk of pituitary and thyroid adenomas. 93 McCune Albright syndrome is caused by somatic mosaic-activating mutations of GNAS genes and is a condition that promotes occurrence of HCAs. We showed that GNAS-activating mutations lead to mild STAT3 activation in hepatocellular cell lines, which underlines a cross talk between the cyclic adenosine monophosphate and JAK/STAT pathways. 87 Finally, in approximately 25% of IHCAs, we did not identify any gene defect that could explain the phenotype. This led us to postulate that other driver genes leading to JAK/STAT3 activation remain to be identified. Mutations in IL6ST/gp130, STAT3, orgnas act by a similar mechanism called oncogene-induced inflammation that leads to constitutive STAT3 activation (Figure 4). Uncontrolled activation of the JAK/STAT pathway in

9 896 NAULT ET AL GASTROENTEROLOGY Vol. 144, No. 5 Figure 5. Clinical and genetic determinants of the occurrence of HCAs. Patients with germline mutations of HNF1A, McCune Albright syndrome, or glycogenosis 1a should avoid the use of estrogen and should be screened for HCA. Moreover, patients with HNF1A germline mutations should be screened for MODY3 and genetic counseling should be proposed to the family. tumor hepatocytes explains the inflammatory phenotype observed at both the histologic and clinical levels. Cytokine and chemokine induced by STAT3 and secreted by hepatocytes act as chemoattractants that promote tumor infiltration by lymphocytes. 84 The origin of dystrophic artery and sinusoidal dilatation observed in the tumor is unknown, but a direct or indirect role of the release of inflammatory proteins by the mutated hepatocytes could be hypothesized. Inflammatory anemia and systemic inflammatory syndrome are often associated with IHCAs. It could be considered a paraneoplastic syndrome that regresses after resection of the tumor and could be caused by chronic inflammation and the hepcidin produced by IHCAs. 14,94,95 All of these data highlight that hepatocytes are major inflammatory cells and that inflammation is a central mechanism of liver tumorigenesis. The role of STAT3 activation by gp130 in tumorigenesis has been recapitulated in a mouse model with a knock-in activating mutant of gp130. These mice developed spontaneous gastric tumors. 96 However, despite recent advances in our knowledge of the genetic drivers of human IHCA, liver-specific mouse models are still lacking. These animal models will be very useful to dissect the roles of the inflammatory pathway in benign tumorigenesis, to understand the cooperation between the IL-6/JAK/ STAT and -catenin pathways in malignant transformation, and to test for new biotherapies. Unclassified adenomas. The last group of adenomas represents approximately 10% of all HCAs. It is composed of tumors that lack the previously described mutations or the inflammatory phenotype (Figure 2 and Table 1). 15 Translation in Clinical Care Defining the soil where adenomas grow. Different genetic and environmental factors can predispose to development of HCAs (Figure 5). First, several rare genetic syndromes are strongly associated with the occurrence of adenomas. Some patients with MODY3 (monoallelic germline mutations of HNF1A) develop liver adenomatosis with biallelic H-HCA. 48,59,60 Following this mechanism, HNF1A meets the criteria of a classic tumor suppressor gene with a transmitted predisposition to develop tumors as defined by Knudson. 58 To our knowledge, currently all familial transmission of liver adenomatosis identified worldwide is associated with germline HNF1A mutations ,97 In these patients, familial genetic counseling is required to propose screening for diabetes and HCA. However, liver adenomatosis could also be related to other molecular subtypes. 22,51,94,98 Other genetic disorders associated with the occurrence of adenomas are glycogen storage diseases and particularly type 1a glycogenosis. 99,100 Glycogenosis 1a is a rare recessive metabolic disease characterized by germline mutations inactivating glucose-6-phosphatase. 101,102 More than 50% of these patients develop HCAs during follow-up. 99,103 As a mirror of the human disease, mice with a liver-specific deletion of glucose-6-phosphatase harbored the same metabolic disorder and developed HCA. 102 A recent study showed that gene therapy using adenovirus could correct the metabolic disorder and prevent development of HCA in this mouse model. 104 Two studies have recently shown that all HCAs observed in patients with glycogenosis were bhcas, IHCAs, or unclassified, whereas no HNF1A mutations were identified. 100,105 Furthermore, metabolic disorders induced by HNF1A and G6PC inactivation appear to be very similar, at least partly explaining the exclusive occurrence of these genetic defects in patients (Figure 5). 105 Finally, McCune Albright syndrome, an orphan disease characterized by mosaic somatic GNAS-activating mutations, predisposes patients to the development of IHCA. 87 Strikingly, these genetic diseases predispose to the development of specific molecular subtypes: H-HCA in patients with MODY3, IHCA in patients with McCune Albright syndrome, and absence of H-HCA in glycogenosis (Figure 5). However, whereas genetic diseases constitute strong risk factors, penetrance of the HCA phenotype is incom-

10 May 2013 HEPATOCELLULAR BENIGN TUMORS 897 plete and particularly rare in patients with MODY3. Thus, additional modifier genes or environmental factors remain to be identified to fully understand which patients will develop HCA. Most cases of HCA are not associated with rare genetic diseases, but a clinical study conducted at a hospital in Bordeaux showed that 39% of HCAs are multifocal macroscopically. 22 In addition to the macroscopic tumors, pathological studies have revealed the presence of multiple microscopic foci of IHCA easily identified using SAA immunostaining. 106 Microscopic adenomas inactivated for HNF1A and negative for LFABP are also observed in patients with adenomatosis developed on a germline mutation of HNF1A background or in multiple H-HCAs without MODY3. 107,108 We can hypothesize that a modifier genetic background associated with environmental factors could favor multiple occurrences of HCA. 22 Oral contraception is also strongly associated with occurrence of HCAs. Indeed, a dose-dependent association has been described and estrogen exposure could act as a promoting HNF1A gene mutations. 9, Spontaneous regression of HCAs after withdrawal of estrogen has also been described, thereby suggesting that development of HCAs could be in particular cases dependent on the hormonal context. 112,113 Androgen consumption is a well-known risk factor for HCAs, particularly bhcas HCAs have been reported in patients with Fanconi anemia treated with an androgen, in cases of anabolic androgen steroid abuse in athletes, or due to an endogenous high level of androgen such as in Klinefelter s syndrome or polycystic ovary syndrome. 115,117,118 Dramatic regression of HCAs after withdrawal of androgen has been recurrently described. 112,119 Several associations between HCA and antiepileptic drugs have been reported, but a causative effect remains to be shown. 120,121 However, only a small number of patients using oral contraceptives develop HCA (about 3 per 100,000 in exposed patients). 9 A role for an additional unknown minor allele associated with environmental factors is of concern. Supporting this hypothesis, it was observed that some patients with H- HCAs also harbor germline heterozygous mutations of CYP1B These mutations lead to decrease enzymatic activity and could play a role in estrogen metabolism. Moreover, patients with adenomatosis in a large French family harbored 2 monoallelic germline mutations inactivating HNF1A and CYP1B These results argue for the cooperation between genetic and environmental factors to modulate the risk of HCA occurrence. The last risk factors significantly associated with occurrence of IHCA are alcohol consumption and obesity. 14,15,22,123 The mechanisms linking these 2 metabolic disorders with the occurrence of HCA showing an autonomous IL-6/JAK/ STAT3 activation remain to be investigated, taking into account both environmental factors and genetic backgrounds. 124 The direct toxic role of alcohol or a role of cytokine production observed during long-term alcohol consumption and in obese patients cannot be excluded. In summary, these data sustain a model in which the liver could be predisposed to the development of multiple HCAs in association or not with a major genetic syndrome. This model challenges the current dogma according to which the nontumor liver surrounding adenomas is really normal. 125 Because a carcinogenic field effect is proposed in cirrhosis to explain development of HCC, we propose that appearance of multiple adenomas could be caused by a benign tumorigenic field effect. This predisposition could be the result of a conjunction between genetic and genotoxic factors leading to proliferation of benign hepatocytes. Diagnosis of HCAs. At the present time, HCA lesions are mainly discovered following abdominal pain and incidentally on imaging-based exploration. Sometimes, complications such as hemorrhage or malignant transformation can also reveal HCAs. 22,34,52 Liver enzyme levels are often normal, although anicteric cholestasis and inflammatory syndrome could be observed in association with IHCAs. 13 All tumor markers routinely tested are normal. The first contribution of the HCA molecular classification in diagnosis was achieved by the redefinition of telangiectatic focal nodular hyperplasia as IHCA (sometimes called telangiectatic adenomas). 13,15,20 In contrast to classic FNH, telangiectatic focal nodular hyperplasia is monoclonal and harbors activation of the JAK/STAT pathway driven by oncogenic mutations. 84,86 This observation led to the reclassification of telangiectatic focal nodular hyperplasia into the IHCA subgroup. This is clinically relevant because of the risk of hemorrhage and malignant transformation of these tumors, particularly when -catenin is also mutated in addition to JAK/STAT activation (Table 1). 22,83,84 Moreover, the dissection of HCA in molecular subgroups with related immunohistochemical markers is of help to diagnose HCA. 18,51 Histologic analysis is the backbone for the diagnosis of HCA, but diagnosis between HCA and FNH or HCA and well-differentiated HCC can still be challenging, even for an expert pathologist. Histologic analysis of resected or biopsied hepatocellular tumors developed in an otherwise normal liver is refined by the use of a panel of markers qualified through the HCA molecular classification (glutamine synthase, -catenin, LFABP, C-reactive protein, and SAA). 18,126 MRI characteristics can also be relevant to identify H-HCAs and IH- CAs. 50, As such, contrast-enhanced MRI could be used to diagnose an H-HCA in a well-defined clinical situation (noninvasive diagnosis) (Figures 1 and 2). Recently, several studies have tested MRI with a liver-specific contrast agent such as gadobenate dimeglumine (Gd- BOPTA; MultiHance, Bracco Diagnostics Inc, Milan, Italy) or gadoxetate (Gd-EOB-DTPA; Primovist, Bayer, Leverkusen, Germany) to distinguish HCA from FNH. 130,131 Preliminary results are promising but require validation in larger series of tumors with molecular and immunohistochemical classification. 132

11 898 NAULT ET AL GASTROENTEROLOGY Vol. 144, No. 5 Clinical and molecular determinants of prognosis and consequences for treatment. The 2 main complications of HCA are hemorrhage and malignant transformation in HCC Even if rare, these complications are difficult to predict and are particularly frightening in young patients. The risk of hemorrhage is directly correlated with the size of the tumor, and HCAs greater than 5 cm have a high risk of hemorrhage (Figure 1). 52,133 The magnitude of the risk of malignant transformation varies between 4% and 8% in the largest series in the literature. 83,134 However, most of the series that report malignant transformation focused on HCA treated by resection. 134 This could lead to an overestimation of the risk of malignant transformation. 136 Sex (male) and -catenin activating mutations are the main risk factors associated with malignant transformation into HCC, and these 2 features are significantly associated. 15,54,133 In all cases, oral contraception or intake of androgen must be discontinued at diagnosis of HCA because regression of HCA has been described particularly after withdrawal of hormones. In an HCA that does not regress after the withdrawal of oral contraception or androgen, 112,113 surgical treatment has been the gold standard for a long time. 137 However, widely accepted guidelines for the management of patients with HCAs are still lacking. In the past 10 years, a more conservative approach has been proposed by several centers to selectively adjust the surgical approach to patients stratified by the risk of complications. 21,22,44,52,138 First, the diagnostic doubt with a well-differentiated HCA still constitutes an indication for surgical resection. Additionally, surgical indication could be modulated by clinical risk of hemorrhage (tumor size 5 cm) or malignant transformation (b-hca, male sex, and tumor size 5 cm) together with the molecular subgroup of HCAs assessed using MRI and/or biopsy (Figure 1). 15,52,134 To summarize, all large HCAs ( 5 cm) or HCAs that develop in male patients should be resected. For small HCAs ( 5 cm) that develop in women, surgical indication should be driven by the presence of -catenin mutation. Because H-HCAs show a lower risk of malignant transformation than the other subtypes, typical features of small H-HCAs at MRI in women could avoid biopsy and lead to radiologic follow-up, particularly for liver adenomatosis. 50, In the absence of typical features of H-HCA at MRI, including IHCA, 10,74 a tumor biopsy should be proposed to search for -catenin activation/mutation and to better assess the risk of malignant transformation. 15,50,126 Herein we propose a diagnostic and therapeutic algorithm that associates clinical, radiologic, histologic, immunohistochemical, and molecular features to optimize the management of these patients (Figure 1). Apart from the classic surgical resection, which remains the gold standard procedure, 21 several studies have supported the development of alternative treatments such as laparoscopic resection in an expert center 139,140 and radiofrequency 141 to reduce postoperative complications. Liver transplantation should be avoided except for patients with glycogenosis 1a associated adenomatosis because liver transplantation treats both the HCA and the underlying metabolic disorder. 101,142 For adenomatosis, even despite the lack of robust clinical studies and of consensus, the same rules as for sporadic HCAs could be proposed: resection for an HCA 5 cm and/or with -catenin activating mutations and follow-up for other lesions. 48,52 Even if some cases of bleeding HCAs were observed during pregnancy, larger series have shown that pregnancy in patients with residual HCAs is usually safe. 52,143,144 Thus, pregnancy should not be contraindicated but close follow-up by imaging should be proposed. If possible, treatment of an HCA 5 cm and/or mutated for -catenin should be performed before pregnancy. In case of bleeding with hemodynamic instability, urgent arterial embolization could be proposed as first-line treatment to stop the hemorrhage. 145 This aims to avoid an urgent hepatectomy and the related significant surgical risk. Reduction in tumor size is frequently observed after arterial embolization, and this could also limit the extent of the resection. In a second step, surgical resection should be performed a few months after embolization. 146,147 Future targeted treatments and additional insights for other types of tumors. Genetic alterations that modulate benign liver tumorigenesis are affecting few driver genes. Deciphering the effect of these mutations helps to understand the role of the select signaling pathway in liver tumorigenesis. Three signaling pathways have emerged from these molecular analyses: (1) inactivation of HNF1A, 56 (2) activation of the Wnt/ -catenin pathway, 15,73 and (3) activation of the IL-6/JAK/STAT3 pathway. 84 We can predict that targeted inhibition of these driver pathways could have a strong and sustainable effect on HCAs. Genetically engineered mouse models that recapitulate benign tumorigenesis in the liver are still lacking. Their generation will consequently be useful to test new target drugs to support a clinical trial in humans. Several drugs affecting the IL-6/JAK/STAT3 pathway will be ideally tested in patients with unresectable IHCA or adenomatosis, a frightening condition in a young patient. As an example, src inhibitors were shown to shut down the JAK/STAT activation induced by STAT3 mutants. 86 Moreover, the dissection of benign tumorigenesis could be translated into new treatment for malignant diseases. Indeed, STAT3-activating mutations were initially identified in inflammatory adenoma, 86 but recently similar mutations were also described in 40% of T-cell large granular lymphocytic leukemia 148 ; Src inhibitors could therefore also be considered to treat these patients. Unmet needs. There are still unmet needs in the field of hepatocellular benign tumors, particularly in the pathogenesis and clinical care of HCAs. New epidemiologic data are warranted to understand the worldwide difference in the incidence of HCAs through the scope of the genotype/phenotype classification. In addition, only a few patients exposed to estrogen developed HCAs, which

12 May 2013 HEPATOCELLULAR BENIGN TUMORS 899 suggests that additional genetic and/or environmental factors remain to be discovered. Also, driver genetic alterations in the group of nonmutated inflammatory and unclassified HCAs are still unknown. One of the most controversial topics is malignant transformation of HCAs. Both basic and clinical studies are warranted to accurately evaluate the risk of malignant transformation and understand the underlying molecular events. Finally, management of patients with liver adenomatosis or pregnancy in patients with HCAs is still debated and will require multicentric observational studies to better evaluate the benefit and risk associated with the observational versus interventional treatments. Conclusion In the past 10 years, there has been a huge effort to characterize and understand the pathophysiology of hepatocellular benign tumors. 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