Original articles
Investigation of Chromosomal Aberrations in Hepatocellular Carcinoma by Fluorescence In Situ Hybridization

https://doi.org/10.1016/S0165-4608(98)00215-5Get rights and content

Abstract

Molecular cytogenetic approaches have been applied only rarely in the characterization of hepatocellular carcinoma (HCC). The aim in this study was to evaluate aberrations, particularly deletions, of specific chromosomal regions in HCC. Dual-color fluorescence in situ hybridization (FISH) was performed on intact nuclei from touch preparations of 17 HCCs and 1 hepatic adenoma. Each touch preparation was hybridized with a digoxigenin-labeled centromere probe and a biotin-labeled unique sequence probe from the same chromosome. This approach permitted the simultaneous evaluation of ploidy changes and chromosome arm deletions. Eight noncentromeric chromosome regions, 3p14, 4q21, 6q14, 6q21, 8p12, 8p22, 9p21, and 9p24 were selected for study on the basis of their having been implicated as tumor suppressor regions in HCC or other common types of carcinoma. Together with the 5 corresponding centromeric probes on chromosomes 3, 4, 6, 8, and 9, a total of 13 chromosome loci were evaluated. All cases of hepatocellular carcinoma showed at least one deletion or aneuploidy. The hepatic adenoma was all diploid. Chromosome 4q21 showed the highest rate of deletion (76.5%) and aneusomy (88%). The second and the third were chromosome 8p22 and 6q14, which showed 59% and 47% of deletion, respectively. A 4q21 deletion is also the most frequent single chromosome aberration. Prominent tumor heterogeneity and variable deletion patterns were noted. Interphase FISH was an efficient means for evaluating numerical and structural chromosome aberrations in HCCs. Most HCCs contained deletions of known tumor suppressor regions (4q and 8p), and a novel deletion hotspot was demonstrated on chromosome band 6q14.

Introduction

Hepatocellular carcinoma (HCC) is one of the most common cancers in humans and is the leading cause of cancer death in many countries in Asia and Africa. Although extensive study has been done on this important tumor to search for the underlying pathogenesis, including the strong association with hepatitis B and C viruses and afla-toxin, the mechanism of hepatocarcinogenesis remains nearly unknown. Study of the chromosome aberration patterns is a helpful way to understand tumorigenesis by the specific chromosomal association. To date, chromosome changes in primary hepatocellular carcinoma have been reported in only 10 cases 1, 2, 3, 4. The paucity of HCC cytogenetic data is largely attributable to the need for short-term tissue culture in conventional karyotyping. Primary HCCs are difficult to establish in tissue culture, and the HCC metaphase cells isolated from tissue culture contain complex chromosomal aberrations that can be impossible to classify by using cytogenetic banding methods. Chromosome 1p is the region most commonly noted to have aberrations, but the aberration patterns are quite variable 1, 3. Microsatellite polymorphism analyses for allelic loss also were evaluated for HCC and showed various proportions of loss of heterozygosity at several chromosome arms, including 1p 5, 6, 7, 4q 6, 8, 9, 5q [10], 6q 11, 12, 8p 13, 14, 10q [10], 11p 10, 15, 16, 13q 15, 16, 17, 16q 6, 8, 9, 17p 16, 18, and 22q [19]. Among them, a few chromosome regions were specified for higher frequency of allelic loss, such as 1p35–36, 4q12–23, 6q26–27, 8p12–22, 13q12–13, and 16q22–23, suggesting the presence of candidate tumor suppressor genes in these loci. Many of these same chromosome regions were also found to be aberrant in a HCC comparative genomic hybridization study [20]. Boige et al. [21] and Nagai et al. [22] both performed comprehensive and high-resolution allelotyping of HCC recently, which revealed results similar to the aforedescribed results; that is, frequent loss in 1p, 4q, 6q, 8p, 13q, and 16q. But the data for each chromosome locus from the aforementioned studies are sometimes quite different. Chromosome 4q, 6q, and 8p are three regions worth further investigation. Chromosome 4q is rarely known to have been deleted in major cancers cytogenetically in the past, so it might be quite unique for HCC. Chromosome 8p12–22 is also noted to be frequently deleted in colon, lung, and prostate cancers and appeared to be an early event in prostate tumorigenesis 13, 23. Chromosome 6q is a very common deletion site in major human cancers, such as lung and breast cancers, melanoma, and lymphoma 24, 25, 26. But study of the 6q deletion in HCC is still very limited. We are also interested in chromosome 9p, because 9p21 is already known to be the locus of the CDKN2A (p16) and CDKN2B (p15) tumor suppressor genes.

Fluorescence in situ hybridization (FISH) is a relatively new technique that can be used to detect genetic alterations in either metaphase or interphase nuclei by appropriate probes. Interphase FISH is especially suitable for the analysis of tumor samples that are difficult to culture or that contain significant normal background cells, because it requires only intact nuclei and is evaluated on a single-cell level. So far, only six reports of interphase FISH study on HCC have been found in the English literature 27, 28, 29, 30, 31, 32, all of which used α-satellite probes to evaluate chromosome copy numbers, and none of them had selected chromosomes 4, 6, and 9 as their target regions. These centromere probes are very useful for aneuploid study, but they are of limited use in the study of allelic loss. Cosmid probes have been used for the study of tumor by interphase FISH [33]. However, the cosmid size is too small to achieve good and stable efficiency when dealing with solid tumor. The CEPH collection of yeast artificial chromosome (YAC) libraries contains megabase-size human DNA inserts. After labeling, these YAC probes can produce relatively intense signals and much improved hybridization efficiency [34]. Another advantage of the mega-YACs is that their chromosomal localization and sequence-tag-site (STS) content are usually well known through World Wide Web Genome Data Base search, and it is readily available commercially (Research Genetics).

In this study, we wished to determine whether FISH with unique sequence probes might be an effective means for demonstrating chromosomal losses in HCC. FISH enable the evaluation of chromosome aberrations on a cell-by-cell basis: we hypothesized that this approach might enable unambiguous detection of chromosomal deletions and gains, even in genetically heterogeneous populations.

Five chromosomes arms were selected. Chromosome 4q21, 8p12, and 8p22 were selected on the basis of previous microsatellite studies as frequent deletion sites in HCC or other major cancers. Common deletion sites on chromosome 3p (p14), 6q (q14, q21), and 9p (p21, p24) in major human cancers by cytogenetic study 24, 25, 26, 35, 36, 37, but rarely studied in HCC, also were selected to compare their deletion frequency in HCC.

Section snippets

Materials and methods

The initial study population was composed of 19 HCCs and 1 hepatic adenoma resected at National Taiwan University Hospital in 1989 and 1990. All cases were frozen at −130°C immediately after hepatectomy. Nonneoplastic liver tissue also was frozen from each hepatectomy specimen. Prior to the FISH evaluations, one hematoxylin- and eosin-stained section was prepared from each frozen specimen to confirm the original histologic diagnosis and to establish that at least 50% of the cells in the

Results

Control hybridization values were established, by using nontumor liver as target, for each of the 13 centromeric and YAC probes. The cutoff criteria used in categorizing results as normal versus aberrant were derived from the third standard deviations of FISH distributions obtained from the nonneoplastic hepatocyte hybridizations. For all probes, the third standard deviations for monosomies were ≤25%. The third standard deviations for trisomies and tetrasomies were ≤7% and ≤12%, respectively.

Discussion

This is the first study to concomitantly demonstrate ploidy aberrations and specific locus deletions in a series of HCCs. This concomitant demonstration can be achieved only by interphase FISH with the use of α-satellite and unique-sequence FISH probes, inasmuch as traditional cytogenetic study of HCC has been very difficult. In this study, we demonstrated frequent deletions in HCC, even in the cases of polysomy with only partial loss of alleles. In many cases, more than two alleles are still

Acknowledgements

The authors thank Dr. Pei-Jer Chen and Ms. Shiou-Hwei Yeh, for DNA clones, Drs. Sheng Xiao and Yung-Ming Cheng, for technical assistance, and Dr. Ting-Chang Chang for biostatistical advice.

Supported by grant no. NSC85-2331-B002-065 from the National Science Council, Taiwan.

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