Elsevier

Journal of Hepatology

Volume 58, Issue 2, February 2013, Pages 350-357
Journal of Hepatology

Research Article
Molecular characterization of hepatocellular adenomas developed in patients with glycogen storage disease type I

https://doi.org/10.1016/j.jhep.2012.09.030Get rights and content

Background & Aims

Hepatocellular adenomas (HCA) are benign liver tumors mainly related to oral contraception and classified into 4 molecular subgroups: inflammatory (IHCA), HNF1A-inactivated (H-HCA), β-catenin-activated (bHCA) or unclassified (UHCA). Glycogen storage disease type I (GSD) is a rare hereditary metabolic disease that predisposes to HCA development. The aim of our study was to characterize the molecular profile of GSD-associated HCA.

Methods

We characterized a series of 25 HCAs developed in 15 patients with GSD by gene expression and DNA sequence of HNF1A, CTNNB1, IL6ST, GNAS, and STAT3 genes. Moreover, we searched for glycolysis, gluconeogenesis, and fatty acid synthesis alterations in GSD non-tumor livers and compared our results to those observed in a series of sporadic H-HCA and various non-GSD liver samples.

Results

GSD adenomas were classified as IHCA (52%) mutated for IL6ST or GNAS, bHCA (28%) or UHCA (20%). In contrast, no HNF1A inactivation was observed, showing a different molecular subtype distribution in GSD-associated HCA from that observed in sporadic HCA (p = 0.0008). In non-tumor GSD liver samples, we identified glycolysis and fatty acid synthesis activation with gluconeogenesis repression. Interestingly, this gene expression profile was similar to that observed in sporadic H-HCA.

Conclusions

Our study showed a particular molecular profile in GSD-related HCA characterized by a lack of HNF1A inactivation. This exclusion could be explained by similar metabolic defects observed with HNF1A inactivation and glucose-6-phosphatase deficiency. Inversely, the high frequency of β-catenin mutations could be related to the increased frequency of malignant transformation in hepatocellular carcinoma.

Introduction

Glycogen storage disease type 1 (GSD) is an autosomal recessive disorder characterized by mutations of glucose-6-phosphatase-α (G6PC, type Ia) or glucose-6-phosphate transporter (G6PT, type Ib), which play a central role in glucose homeostasis [1], [2]. These mutations, resulting in impaired gluconeogenesis and glycogenolysis, lead to excessive glycogen accumulation in various organs, such as liver, kidney or intestinal mucosa [1], [2], [3]. Among the wide range of complications associated with GSD I, development of hepatocellular adenomas (HCA) is considered as a major cause of mortality and morbidity, as malignant transformation into hepatocellular carcinoma (HCC) and intratumor hemorrhage may be life-threatening complications [2]. In a European cohort study, HCAs were observed in 80% of the patients with GSD above the age of 30 years and they were multiple in two thirds of the cases [2].

Following a large study of molecular alterations in sporadic HCA and their correlation with microscopic features, a classification which comprises four HCA subgroups has recently been proposed [4], [5], [6], [7], [8]:

  • Inflammatory HCA (IHCA) account for 20–50% of the cases [5], [9], [10]. They are characterized by marked activation of inflammatory signaling pathways with high expression of CRP (C-reactive protein) and SAA2 (serum amyloid A2). Somatic gain-of-function mutations in the IL6ST gene, which encodes the signaling interleukin 6 (IL6) transducer of signal gp130, have been reported in up to 60% of inflammatory HCA [8]. Other mutated genes (GNAS and STAT3) involved in inflammatory pathways have been recently identified [11], [12].

  • HNF1A (Hepatocytic Nuclear Factor 1 alpha) mutated HCAs (H-HCAs) represent 20–50% of the HCA cases [5], [9], [10]. They harbor bi-allelic inactivating mutations of HNF1A [7]. As transcription of both FABP1 (Fatty Acid Binding Protein 1) and UGT2B7 (UDP-Glucuronosyltransferase-2B7) is positively regulated by HNF1A, H-HCAs demonstrate drastically low FABP1 and UGT2B7 mRNA level and protein expression. H-HCA show several hepatic metabolic pathways alterations, such as gluconeogenesis repression, and glycolysis and fatty acid synthesis activation [13].

  • CTNNB1 mutated HCAs (bHCA) are characterized by activating mutations of the gene encoding β-catenin and account for 7–14% of HCA [5], [9], [10]. These mutations lead to Wnt/β-catenin signaling pathway activation [4], which plays a key role in hepatocyte differentiation and proliferation [14], [15], [16]. In most cases, bHCA show high mRNA levels of 2 main β-catenin transcriptional targets, LGR5 (leucine-rich repeat containing G protein-coupled receptor 5) and GLUL (glutamate-ammonia ligase) [6]. Of note is that about 50% of bHCA also harbor IL6ST activating mutations and that bHCA have been reported to be at greater risk of malignant transformation than other subtypes [4], [8], [15], [17], [18].

  • Currently, less than 10% of HCAs remain unclassified (UHCA) [4].

Relevance of this classification in GSD-associated HCA (GSD HCA) remains to be investigated, as the largest studies of GSD HCA published to date include comparative genomic hybridization [19] or immunohistochemical characterization [20], without systematic gene mutation screening and gene expression analysis.

Our objective was to characterize the molecular profile of a series of GSD HCA. Metabolic pathway alterations were also investigated in non-tumor GSD liver samples and results were compared to those observed in sporadic H-HCA, steatotic and non-steatotic non-tumor liver samples.

Section snippets

Patients and samples

A series of 25 tumor and 7 non-tumor frozen samples from 15 patients with GSD Ia (n = 14) and GSD Ib (n = 1) were collected from 7 university hepatobiliary surgical centers. Liver samples were frozen immediately after surgery and stored at −80 °C. All cases had typical histological features of HCA, except for one case that demonstrated areas of malignant transformation (No 11b). Patient’s consent was obtained if appropriate.

For analysis of metabolic pathways, steatotic (steatosis involving between

Patients and pathological features

GSD-related HCA are rare, and represent less than 5% of overall HCA surgically treated in university hospitals in France [5], [9]. From 2004 to 2012, we collected 25 frozen tumors from 15 GSD patients (see Table 1 for clinical features). When compared with sporadic HCA [5], [9], GSD patients with HCA were more frequently male (sex ratio 0.66 vs. 0.12; p = 0.009), and mean age at diagnosis was also significantly younger (29 vs. 39 years, p = 0.0005). Interestingly, in 5 cases, patients ranging from

Discussion

In the present work, we provided a molecular characterization to classify 25 GSD-related HCA according to all molecular subtypes currently described [4], [5], [6], [8], [9]. With dietary treatment, metabolic control has improved and GSD patients are now living into adulthood [2], [22], [23]. With aging, more complications, including those related to HCA, develop and lead to increased morbidity and mortality [2]. Our molecular classification of sporadic HCA is now widely recognized [4], [5], [6]

Financial support

Julien Calderaro was supported by a fellowship from Assistance Publique Hôpitaux de Paris (Concours de la Medaille d’Or). This study was supported by the Association pour la recherche Contre le Cancer (ARC, Grant N° 3194), Association Nationale de la Recherche (Grant ANR-07-MRAR-011-01), Inserm (Réseaux de recherche clinique et réseaux de recherche en santé des populations), and the BioIntelligence collaborative program.

Conflict of interest

The Authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Acknowledgments

We warmly thank Gabrielle Couchy for her participation in this work. We also thank Jean Saric, Christophe Laurent, Brigitte Le Bail, Anne Rullier, Antonio Sa Cunha (CHU Bordeaux), the Plateforme des Ressouces Biologiques of the Henri Mondor Hospital and the Réseau des CRB Foie for contributing to the tissue collection and all the participants to GENTHEP network (Génétique des tumeurs hépatiques).

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