Gastroenterology

Gastroenterology

Volume 116, Issue 2, February 1999, Pages 394-400
Gastroenterology

Liver, Pancreas, and Biliary Tract
p16INK4 is inactivated by extensive CpG methylation in human hepatocellular carcinoma

https://doi.org/10.1016/S0016-5085(99)70137-XGet rights and content

Abstract

Background & Aims: The molecular status of the p16INK4 tumor-suppressor gene has not been fully elucidated in hepatocellular carcinoma. The aim of this study was to clarify the mechanism that gives rise to inactivation of p16INK4 in hepatocellular carcinoma. Methods: The status of p16INK4 was evaluated in 60 hepatocellular carcinomas by immunohistochemical staining, differential polymerase chain reaction, single-strand conformational polymorphism, methylation-specific polymerase chain reaction, and methylation-sensitive single nucleotide primer extension. Results: Immunohistochemical staining showed that 29 of the 60 tumors exhibited complete loss of p16INK4 expression. High levels of DNA methylation were detected in 24 of 29 cases of hepatocellular carcinoma with negative p16INK4 expression, with methylation of 60%–85% of the CpG islands. In contrast, the level of methylation was <25% in tumors with faint p16INK4 staining, and no methylation was detected in tumors with positive immunostaining. Intragenic alteration of p16INK4 was detected in 4 cases. Conclusions: A strong correlation was found between the extent of methylation and the degree of expression of p16INK4 in tumor tissues, indicating that epigenetic change due to extensive CpG methylation is the main cause of inactivation of p16INK4 in hepatocellular carcinoma.

GASTROENTEROLOGY 1999;116:394-400

Section snippets

Tissue samples

HCC tissue specimens were obtained from 60 individuals (48 men and 12 women), ranging in age between 29 and 75 years (mean age, 58 ± 12 years), undergoing surgical resection or biopsy. Eleven (18%) individuals were positive for hepatitis B surface antigen, 44 (73%) were positive for hepatitis C virus antibody, and 2 were positive for both viral markers. None had hereditary or systemic disorders. The size of the tumors varied from 1.5 to 16 cm (mean size, 5.0 ± 3.8 cm). Tissue samples were fixed

Immunohistochemistry

All normal livers and livers with benign hepatic diseases showed p16-positive nuclear immunostaining (Figure 1A).

. Immunohistochemical patterns for p16. (A) Strong nuclear reactivity for p16 was detected in a normal liver. Cytoplasmic staining in hepatocytes was regarded as nonspecific staining. (B) HCC exhibiting complete loss of expression of p16. Some of the admixed stromal cells in the tumor region, as an internal positive control, show nuclear reactivity (arrowhead). (C) HCC showing

Discussion

We chose to focus on primary HCC tissues because significant differences in the frequency of p16 inactivation have been shown between primary cancers and cultured cells.16, 17 Immunostaining showed that p16 expression was lost completely in approximately half of the HCCs examined, and the proportion of tumors with reduced p16 immunostaining increased according to the histological stage. There was no statistical association between the loss of p16 expression and etiologic factors in individuals

Acknowledgements

The authors thank N. Honda, T. Tsuchida, and H. Koizumi for technical assistance.

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    Address requests for reprints to: Takafumi Ichida, M.D., Department of Internal Medicine III, Niigata University School of Medicine, Asahimachi-dori 1, Niigata City 951-8122, Japan. e-mail: [email protected]; fax: (81) 25-227-0776.

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