The role of overexpression and gene amplification of cyclin D1 in intrahepatic cholangiocarcinoma

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Abstract

Background/Aims: Intrahepatic cholangiocarcinoma (ICC) is a primary liver malignant tumor with an extremely poor prognosis, but less attention has been directed to factors related to molecular carcinogenesis, including cell cycle proteins. We examined the expression and gene amplification of cyclin D1, the cell cycle regulating protein. Our objective was to evaluate correlations with clinicopathological factors in ICC.

Methods: Cyclin D1 overexpression and cellular proliferative activity (Ki-67 labeling index) were investigated immunohistochemically, and 20 cases were further investigated for cyclin D1 gene amplification, using differential PCR. We examined the correlation between the expression and gene amplification of cyclin D1 and clinicopathological factors, including overall survival in patients with ICC.

Results: Immunohistochemical analysis revealed an overexpression of cyclin D1 protein in 28 of 66 subjects with ICCs (42%). The cyclin D1 overexpression was associated with poor histological differentiation (P=0.04), high cellular proliferative activity (P<0.01), and a poor prognosis (P=0.02) by univariate analysis, although it is not an independent prognostic factor by multivariate analysis. Cyclin D1 gene amplification was confirmed in five of the 20 patients. Of those five cases of ICC, all had poor histological differentiation, and four of the five ICCs (80%) showed evidence of cyclin D1 immunoreactivity.

Conclusions: Overexpression and gene amplification of cyclin D1 are frequent and contribute to dedifferentiation and cellular proliferative activity of ICCs, and overexpression also indicates a poor prognosis for patients with ICC.

Introduction

Intrahepatic cholangiocarcinomas (ICC) occur in approximately 5% of the primary liver cancers in Japan, usually followed by occurrences of hepatocellular carcinoma (HCC) [1]. Despite advances in diagnostic and therapeutic procedures for liver cancers, the prognosis of ICC remains poor compared with that of HCC [1], [2], [3], [4]. In ICC, tumor biological characteristics such as molecular carcinogenesis, including cell cycle proteins, the mechanism of proliferation, differentiation or metastasis and prognosis are not well understood, compared with the case of HCC [5]. A better understanding of the molecular mechanisms in ICC should contribute not only to knowledge of tumor characteristics, but may pave the way towards more effective therapy.

Cyclin D1, an essential G1 cyclin is involved in regulating the G1–S transition, the most important checkpoint in the mammalian cell cycle [6], [7]. Overexpression of cyclin D1 in cultured cells promotes rapid progression from G1 to the S phase and increases cell proliferation [8]. Cyclin D1 is encoded by the CCND1 gene on chromosome 11q13, originally identified as an oncogene related to the parathyroid tumor [9], [10]. The cyclin D1 gene is amplified in breast cancer and the amplicon contains FGF4 and FGF3 (formerly termed the Hst-1 and Int-2) regions [11]. The cyclin D1 protein was found to be overexpressed as a result of amplification and rearrangement of the cyclin D1/CCND1 gene in various human cancers [12], [13], [14], [15], [16]. Studies revealed that overexpression and/or gene amplification of cyclin D1 were associated with histological differentiation, lymph node metastases, and/or poor prognosis in breast cancer, non-small cell lung cancer, esophageal cancer, and head/neck squamous cell carcinoma [17], [18], [19], [20].

Several genetic alterations in ICC, such as the p53 tumor suppressor gene and ras oncogene, were reported to be involved in the development and progression of the tumor [21], [22], [23], but much less is known of cell cycle regulatory molecules in ICC. We examined the protein expression and gene amplification of cyclin D1 to search for possible correlations with clinicopathological findings, cellular proliferative activity and the prognosis for patients with ICC.

Section snippets

Patients

Data on 70 Japanese patients who underwent curative hepatectomy for ICC were collected in the Department of Anatomic Pathology, Graduate School of Medical Sciences of Kyushu University, Fukuoka, Japan from July 1987 to June 1999. Among them, four recurrent cases were excluded, so the remaining 66 cases were included in this retrospective study. All tumors were defined as primary ICCs arising from an intrahepatic bile duct, and tumors arising from extrahepatic bile ducts were excluded from this

Comparison between cyclin D1 expression and clinicopathological variables

The relationship between cyclin D1 positivity and clinicopathological features is summarized in Table 1. Cyclin D1 overexpression correlated with the histological type of the tumor. Fourteen of 23 with the poorly differentiated type (61%) and eleven of 25 with the moderately differentiated type (44%) showed positive immunoreactivity for cyclin D1. Only one of seven with the papillary type (14%) and two of 11 with the well differentiated type (22%) showed cyclin D1-positive immunoreactivity,

Discussion

As the restriction point in the late G1 phase may be deregulated, allowing cells to become insensitive to external signals and to replicate unrepaired mutations, the accumulating genetic changes could contribute to oncogenesis. This can be consequent to aberrant expression of positive regulators, such as the cyclins, or the loss of negative regulators, such as the cyclin-dependent kinase (CDK) inhibitors. For both types, changes have been noted in various human malignant tumors [6], [7], [30].

Acknowledgements

The authors would like to thank the staff in the Department of Surgery and Science, Graduate school of Medical Science, Kyushu University, and affiliated hospitals (Fukuoka City Hospital, Iizuka Hospital, National Oita Hospital, Munakata Ishikai Hospital, Fukuoka Saiseikai Hospital and Kyushu Central Hospital) for providing materials, M. Ohara for helpful comments on the manuscript, and Y. Nozuka and M. Hirata for excellent technical assistance.

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