Original Paper
Chromosomal abnormality in hepatocellular carcinoma by comparative genomic hybridisation in Taiwan

https://doi.org/10.1016/S0959-8049(98)00430-4Get rights and content

Abstract

The elucidation of the genetic changes of hepatocellular carcinoma (HCC) is very important for understanding the molecular mechanism of liver carcinogenesis. In order to identify the gains or losses in DNA sequence copy number in HCC, we used comparative genomic hybridisation to study 40 cases (44 tumours) of HCC. Tumour DNA and DNA from non-neoplastic liver tissue were labelled with different fluorochromes and then simultaneously hybridised to normal metaphase spread chromosomes. An image acquisition system was used to quantitate signal intensities contributed by tumour and reference DNA along the entire length of each chromosome. Regions of amplification and deletion were demonstrated as quantitative alterations. Losses were prevalent on chromosome regions 16q (43%), 17p (20%), 13q (20%), 4q (15%) and 8p (15%). Gains frequently occurred on 8q (30%), 1q (20%), 6p (20%) and 17q (18%). Hepatitis B virus carriers had a significantly higher frequency of losses on chromosome 16q. Furthermore, the minimal region of losses was narrowed down to 16q11-q22. This study confirms the presence of previously known chromosomal aberrations in HCC and highlights a new significant correlation between losses on chromosome 16q and hepatitis B virus carriers.

Introduction

Hepatocellular carcinoma (HCC) is one of the leading malignancies in the world[1]. In Taiwan, it ranks first in terms of cancer mortality2, 3. Different aetiological factors such as hepatitis viral infection, dietary aflatoxins, or chemical carcinogens are associated with the development of liver cancer2, 3, 4, 5. Nevertheless, the molecular mechanism remains to be clarified.

The genesis of human cancers is a multistep process reflecting cumulative genetic alterations that include the activation of oncogenes or the inactivation of tumour suppressor genes6, 7. Traditionally, cytogenetics have been used to detect the genetic changes of cancer[8]. Chromosomal aberrations have been reported in leukaemia for many years7, 8. For solid tumours, the identification of chromosomal aberrations is in its infancy because of technical difficulties in obtaining sufficient numbers of dividing cells9, 10, 11. Molecular genetic studies of isolated tumour DNA have been more successful and have been used to detect common regions of allelic loss, mutation, or amplification12, 13. In HCC, by using restriction fragment length polymorphism (RFLP), loss of heterozygosity (LOH) on chromosome 1p, 4q, 5q, 8p, 11p, 13q, 16q and 17p has been described14, 15, 16, 17, 18. Microsatellite analysis provides another simple way to study systematically genetic changes in tumour tissue19, 20, 21, 22. However, genome-wide surveys of LOH are tedious and time consuming[23].

Recently, a new molecular cytogenetic method has been developed[11]. This method, comparative genomic hybridisation (CGH), is capable of detecting and mapping relative DNA sequence copy number between genomes. Tumour DNA and DNA from non-neoplastic tissue are labelled with different fluorochromes and then simultaneously hybridised into normal metaphase spread chromosomes. An image acquisition system is used to quantitate signal intensities contributed by tumour and reference DNA along the entire length of each chromosome. Regions of amplification and deletion are demonstrated as quantitative alterations.

CGH adapts molecular biological techniques for simultaneous analysis of the entire genome24, 25, 26, 27, 28. As no specific probes or previous knowledge of aberrations are required, CGH is especially suitable for the identification and mapping of previously unknown DNA copy number changes that may highlight the locations of important genes in neoplasia. Furthermore, the possibility that more than one locus is involved in tumour initiation and progression can be assessed26, 29, 30. Genomic DNA from tumour specimens is used so that genetic alterations identified with CGH are not artifactually altered by propagation in cell culture24, 27. In this study, we used CGH to identify the gains or losses in DNA sequence copy number in 40 HCC patients.

Section snippets

Patients

Primary HCC tissues and non-neoplastic liver tissues were obtained from 40 HCC patients who underwent surgical resection in National Taiwan University Hospital. The diagnosis of HCC was confirmed by histology. A total of 40 HCC patients were included in this study (Table 1). One patient (case 29) had two operations for primary and recurrent HCCs and there were two tumours in each operation. Another patient (case 30) had two small tumours. Therefore, a total of 44 tumours were studied. The

Gains and losses of DNA sequence in HCC detected by CGH

Of the 40 HCC patients analysed, 30 HCC patients (75%, 30/40) clearly showed a number of chromosomal alterations, whilst the remaining 10 HCCs had no detectable changes (Table 1 and Table 2). Among the 30 HCC patients who had chromosomal aberrations, 21 HCCs had both gains and losses, seven HCCs had gains only, whilst two HCCs had losses only (Table 1). After analysing the 24 chromosomes, a total of 15 chromosomes had abnormalities (Fig. 1). Among the 15 chromosomes which had aberrations, eight

Discussion

In the 44 HCCs studied by CGH, 77% of the tumours showed losses and gains of DNA sequences in at least one chromosome arm. Furthermore, most of the changes involved many different chromosomal regions. Losses were prevalent on chromosome regions 16q (43%), 17p (20%), 13q (20%), 4q (15%) and 8p (15%). Gains frequently occurred on 8q (30%), 1q (20%), 6p (20%) and 17q (18%). These results suggest that the genetic changes of HCC are highly complex. Similar complexity has been reported in a variety

Acknowledgements

This study was supported by grants from the Department of Health and National Science Council, Executive Yuan and Liver Disease Prevention and Treatment Research Foundation, Taiwan, Republic of China.

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