Homozygous deletions of the CDKN2 gene and loss of heterozygosity of 9p in primary hepatocellular carcinoma

doi:10.1016/S0304-3835(97)00403-5

Cancer Letters

Volume 122, Issues 1–2, 9 January 1998, Pages 201-207

https://doi.org/10.1016/S0304-3835(97)00403-5 Get rights and content

Abstract

To elucidate the alterations of CDKN2 in hepatocarcinogenesis, we performed a loss of heterozygosity (LOH) study using eight polymorphic markers surrounding the CDKN2 gene and analyzed the homozygous deletions and mutations of the CDKN2 gene in 41 primary hepatocellular carcinomas (HCCs). Frequent LOH (27.8–44%) was found in the eight loci on chromosome 9p, however, no intragenic mutations of CDKN2 were observed by PCR-SSCP analysis. Homozygous deletions were detected in 25 of 41 HCCs (61%) by a comparative multiplex PCR. No expression of the CDKN2 protein was noted in five out of nine available HCCs by Western blot analysis. These results suggest that inactivation of the CDKN2 gene in HCC is a frequent event in which homozygous deletions are the most common mechanism of CDKN2 inactivation.

Introduction

Hepatocellular carcinoma (HCC) is one of the most frequent malignant neoplasms in the world, particularly in Asia and Africa [1]. Hepatitis B or C virus infection, subsequent chronic inflammation and hepatic regeneration seem to be important risk factors for HCC 2, 3, 4. Molecular genetic studies have revealed that oncogene activation is rarely detected in HCC, whereas inactivation of the tumor suppressor gene is thought to be critical for hepatocarcinogenesis and involves both alleles of the genes 5, 6. The recently identified tumor suppressor gene, CDKN2, is known to be localized on chromosome 9p21 7, 8. The product of the CDKN2 gene, p16, inhibits the cyclin D/CDK4 and cyclin D/CDK6 kinase complexes [9]and negatively regulates cell-cycle progression. Homozygous deletions of the CDKN2 gene have been reported in many types of tumor cell lines and primary tumors 7, 8, 9, 10, 11, 12. In HCCs, loss of heterozygosity (LOH) of chromosome 9p and alteration of CDKN2 exon 2 have been reported previously 13, 14, 15, 16. Although some abnormalities of the CDKN2 gene are known to exist in HCCs, the patterns and incidences of CDKN2 abnormalities in hepatocarcinogenesis have not been studied in detail. Therefore, we investigated the LOH of 9p, homozygous deletions and mutation of the CDKN2 gene in 41 primary HCCs. We also analyzed the expression of CDKN2 in nine available HCCs.

Section snippets

HCC tissue preparation and DNA extraction

Forty-one HCCs and corresponding non-tumorous tissues were obtained from surgical resections performed at the Yonsei University College of Medicine, Seoul, South Korea, from January 1995 to September 1996. Patient information (tumor size and serum viral markers) was obtained prospectively without any knowledge of genetic alterations. Small HCC was defined as ≤3 cm and advanced HCC as >3 cm in diameter [17]. The HCC differentiation was classified into two groups, i.e. well-differentiated HCC

LOH on chromosome 9p

We evaluated LOH on chromosome 9p using eight polymorphic microsatellite markers located between D9S162 and D9S171; the distance between these two loci was about 9 cm 26, 27. The most frequent LOH was found at D9S265 (11 of 24 informative cases, 44%). When the results of eight markers were combined, 51.2% (21/41) of LOH was demonstrated on this 9 cm region (Fig. 1). Representative LOH results are shown in Fig. 2.

Mutation analysis of the CDKN2 gene

For the reason that 99% of the CDKN2 coding sequence is contained in exons 1 and 2

Discussion

Accumulated evidence has shown that CDKN2 is a major inhibitor of cellular proliferation [29]. The frequent inactivation of the CDKN2 gene has been reported in many cancer types including carcinomas of the esophagus, pancreas, breast and urinary bladder 8, 10, 26, 30, 31. The principal mechanism for inactivation of the CDKN2 gene is homozygous deletions or point mutation [32]. Several previous studies on HCCs have shown that no homozygous deletions 13, 14, 15or infrequent homozygous deletions

Acknowledgements

This study was supported by a basic research medical fund from the Ministry of Education, South Korea for 1996 and 1997.

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Primary hepatocellular carcinoma (HCC) is a significant cause of cancer morbidity and mortality on the global scale. Although epidemiologic studies have identified major risk factors for HCC, the sequence of oncogenic events at the molecular level remains poorly understood. While genetic allele loss appears to be a common event, the significance of the loss is not clear. In order to determine whether allele loss appears to be a random event among HCCs or whether patterns of loss cluster in groups of tumors, a phylogenetic approach was used to examine 32 tumors for genome-wide loss of heterozygosity employing 391 markers. Clusters identified by the phylogenetic analysis were then contrasted to compare candidate locus variation among individuals and to determine whether certain clusters exhibited higher loss rates than other clusters. The analysis found that 3 major and 1 minor cluster of loss could be identified and, further, these clusters were distinguished by variable rates of loss (cluster 1, 29%; cluster 2, 21%; cluster 3, 16%). The analyses also indicated that the allele loss rates in HCC were not insignificant and that the patterns of allele loss were complex. In addition, the results indicated that an individual's constitutional genotype at the EPHX1 locus may be a critical factor in determining the path of tumor evolution. In conclusion, it appears that in HCC, allele loss is not random, but clusters into definable groups that are characterized by distinctive rates of loss. (HEPATOLOGY 2002;36:1341-1348.)
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We examined the chromosomal changes of 22 hepatocellular carcinomas (HCCs) by comparative genomic hybridization (CGH) analysis and compared the results with that of allelotype by polymerase chain reaction based loss of heterozygosity (PCR-LOH) analysis. By CGH analysis, frequent chromosomal losses were noted in the chromosomal region of 4q (59%), 8p (77%), and 16q (50%), whereas gains were noted in 1q (86%) and 8q (77%). All of these chromosomal arms were revealed to have frequent allelic imbalances by PCR-LOH analysis, however, 9% of chromosomal aberrations were detected only by CGH analysis and 2% were detected only by PCR-LOH analysis. Our results suggest that CGH analysis gives more precise results for the screening of chromosomal aberrations in HCCs than that of PCR-LOH analysis with randomly selected microsatellite markers.
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Homozygous deletions of the CDKN2 gene and loss of heterozygosity of 9p in primary hepatocellular carcinoma

Abstract

Introduction

Section snippets

HCC tissue preparation and DNA extraction

LOH on chromosome 9p

Mutation analysis of the CDKN2 gene

Discussion

Acknowledgements

Lancet

Hepatology

Biochem. Biophys. Res. Commun.

Cancer Lett.

Hepatocellular carcinogenesis: recent advances and speculations

Cancer Cells

Increasing incidence of hepatocellular carcinoma possibly associated with non-A non-B hepatitis in Japan, disclosed by hepatitis B virus DNA analysis of surgically resected cases

Cancer Res.

Hepatitis C virus infection is associated with the development of hepatocellular carcinoma

Proc. Natl. Acad. Sci. USA

Low incidence of point mutation of c-Ki-ras and N-ras oncogenes in human hepatocellular carcinoma

Jpn. J. Cancer Res.

Analysis of ras gene mutation in human hepatic malignant tumors by polymerase chain reaction and direct sequencing

Cancer Res.

A cell cycle regulator potentially involved in genesis of many tumor types

Science

Deletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers

Nature

A new regulatory motif in cell-cycle control causing specific inhibition of cyclin D/CDK4

Nature

Deletion of the p16 and p15 gene in human bladder tumors

J. Natl. Cancer Inst.

CDKN2/p16 or RB alterations occur in the majority of glioblastomas and are inversely correlated

Cancer Res.

p16 gene in uncultured tumors

Nature

Frequency of mutation and deletion of the tumor suppressor gene CDKN2A (MTS1/p16) in hepatocellular carcinoma from an Australian population

Hepatology

Infrequent alterations of the p16INK4 gene in liver cancer

Int. J. Cancer

Alterations of CDKN2 (p16/MTS1) exon 2 in human hepatocellular carcinoma

Oncol. Rep.

Loss of heterozygosity and analysis of mutation of p53 in hepatocellular carcinoma

J. Gastroenterol. Hepatol.

Primary carcinoma of the liver. A study of 100 cases among 48 900 necropsies

Cancer

Infrequent alterations of the p16^INK4 gene in liver cancer