Elsevier

Journal of Hepatology

Volume 40, Issue 4, April 2004, Pages 660-668
Journal of Hepatology

Multiple chromosomal abnormalities in human liver (pre)neoplasia

https://doi.org/10.1016/j.jhep.2003.12.020Get rights and content

Abstract

Background/Aims

In human hepatocarcinogenesis the tumor precursor lesions and the sequence of genetic aberrations are not known. We therefore compared genetic alterations of different types of benign liver lesions to those of hepatocellular carcinoma.

Methods

By comparative genomic hybridisation (CGH) 40 cases, including cirrhotic liver (CL), focal nodular hyperplasia (FNHs), hepatocellular adenoma (HCAs), dysplastic nodules (DNs), primary hepatocellular carcinoma (HCCs), and hepatocellular metastases to the lung were studied.

Results

FNHs and HCAs exhibited few chromosomal abnormalities. Frequency and pattern of genetic alterations in DNs highly resembled those in HCCs: gains of DNA clustered in chromosome arms 1p/q, 7q, 15q, 16p, 17q, and 20q and losses were often found at 3p, 4q, 9p, and 11q. Aberrations on 1p, 6q, 8p/q, and 13q occurred almost exclusively in HCCs; the gain at 8q encompassed amplification of c-myc, as verified by fluorescence in situ hybridisation.

Conclusions

The pattern of genetic alterations in HCCs resembled more the alterations found in DNs than in FNHs and HCAs, suggesting that DNs may be the actual tumor precursors. Furthermore, alterations at 4q, 9p, 11q, 16p, and 17q appear as early genetic events being crucial for hepatocarcinogenesis.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide with increasing incidence in Europe and the United States [1]. Etiological factors of HCC include infection with hepatitis B- or C-virus, chronic ethanol abuse, or dietary exposure to aflatoxin-B1 [2]. The mechanisms of liver carcinogenesis in humans are poorly understood. Most of the available knowledge stems from rodent models of experimental hepatocarcinogenesis. Herein the development of HCC has been described as a multistage process starting in single cells that may develop to liver (pre)neoplasia [3], [4]. Preneoplastic lesions are genetically and phenotypically fairly well characterized in a few human malignancies, notably colon, but not in the liver [5]. Hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH) are the two most common benign neoplasms of the liver occurring predominantly in women; a few cases of malignant transformation have been reported, especially for HCA [6]. Further putative preneoplastic lesions in human liver are dysplastic nodules (DNs). Their frequent association with HCCs suggests a preneoplastic nature of this lesion [7]. Opinions on the severity attributed to such lesions vary widely among pathologists due to the lack of genetic and other unequivocal markers [2].

Most studies have focused on genetic alterations in HCCs, that generally show considerable loss of heterozygosity (LOH) and low levels of microsatellite instability. LOH frequently occurs on chromosomal loci that contain genes known to function as tumor suppressors, such as LOH of p53 in about 50% of the HCCs [8], [9]. Point mutations have been reported for the tumor-suppressor genes β-catenin and axin in about 25 and 10% of HCCs, respectively [9]. A great portion of HCCs and DNs showed LOH for the mannose 6-phosphate/insulin-like growth factor II receptor combined with deletions or mutations in the remaining allele [10]. Rb-1, E-cadherin, p73, PTEN, and Smad 2 and 4 genes are less frequently altered [2]. On the other side, genes, that may act as oncogenes, are often amplified in HCCs, as reported for c-myc or cyclin D1 [11], [12]. The huge number of genomic alterations in HCC complicates the identification of initial genetic events being crucial for hepatocarcinogenesis. This highlights the necessity to study precancerous lesions to clarify the sequence of genomic alterations underlying liver cancer development.

Until now only a small number of benign liver lesions, such as DNs, FNHs, and HCAs has been investigated by a genome-wide screening [2], [13], [14], [15], [16]. Identification is difficult and opinions on the premalignant nature of these lesions vary. A further complicating factor is the extraction of DNA of sufficient amount and quality from small and often formalin-/paraffin-processed liver samples. Recently, techniques have been developed that allow amplification of total genomic DNA. While degenerate oligonucleotide-primed PCR (DOP-PCR) uses degenerate PCR primers, linker-adapter-PCR (LA-PCR) is based on restriction enzyme digest, ligation of one primer and PCR amplification [17], [18], [19], [20], [21]. In the present study, we used both, DOP-PCR and LA-PCR, to amplify total DNA from DNs, FNHs, HCAs, and HCCs for subsequent CGH-analyses. We found that the frequency and pattern of genetic alterations in HCCs highly resembled the alterations found in the DNs. This substantiates that DNs are the actual precancerous lesion in human liver and points towards a sequence of possible genetic events in human hepatocarcinogenesis.

Section snippets

Human liver samples

Four patients suffering from cirrhosis were subjected to liver transplantation; two further cases were resected. Chemotherapy had not been applied before surgery. Informed consent was obtained from all patients. Classification of liver lesions and stage of disease followed published guidelines [22], [23], [24] (Table 1). Samples were obtained from conventional formalin-fixed and paraffin-embedded pathology specimens; additional samples were snap-frozen and stored in liquid nitrogen.

Paraffin-embedded material

Three to

LA-PCR- and DOP-PCR-based CGHs of frozen and paraffin-embedded material

The amplification efficiency of DOP-PCR and LA-PCR was generally higher for frozen than for formalin/paraffin pre-treated tissues, since the quality of the extracted DNA suffers from pre-treatment. In case of severely degraded DNA or no amplification by DOP-PCR we applied LA-PCR and often yielded sufficient amounts of DNA (Table 1). Direct comparison of the CGH results based on the two different PCR methods showed good agreement, as described in detail by Pirker et al. (submitted).

Any gross

Discussion

In the present CGH-analyses, a subset of HCC-associated genetic changes was disclosed in DNs. The HCAs and FNHs studied exhibited genetic aberrations mostly differing to that of DNs and HCCs. Recent studies on few DNs agree with our data, e.g. the pattern and frequency of the genetic alterations in DNs resembled the alterations seen in HCCs [13], [14]. This is strong evidence that DNs are the actual precursors for HCCs, which offers the chance to study the sequence of genetic events in the

Acknowledgements

This study was supported by the Austrian ‘Gen-Au-Program’ and the Jubiläumsfond der Österreichischen Nationalbank, Project-No. 8817.

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