p16INK4A hypermethylation is associated with hepatitis virus infection, age, and gender in hepatocellular carcinoma.
OBJECTIVE
The tumor suppressor gene p16INK4A is mainly inactivated by an epigenetic change involving promoter hypermethylation in hepatocarcinogenesis. The possible clinical impact of p16INK4A methylation and the potential risk factors for this epigenetic alteration have not been thoroughly investigated.


METHODS
We studied the methylation status and mRNA and protein expression of p16INK4A in 50 hepatocellular carcinomas and corresponding nonneoplastic liver lesions using methylation-specific PCR, reverse transcription-PCR, and immunohistochemical techniques.


RESULTS
p16INK4A hypermethylation was observed in 58% (29 of 50) of the hepatocellular carcinomas and 16% (6 of 38) of the corresponding chronic hepatitis and cirrhosis tissue samples. p16INK4A methylation was significantly associated with mRNA and protein expression (P <0.001 and P=0.003, respectively). All of the p16INK4A-methylated tumors were positive for hepatitis B virus or hepatitis C virus markers, but none of the virus-negative tumors exhibited p16INK4A methylation (P=0.006). The frequency of p16INK4A hypermethylation tended to be higher in hepatitis C virus-related tumors (23 of 32, 72%) than in hepatitis B virus-related tumors (6 of 13, 46%; P=0.1). Aberrant methylation of p16INK4A was also related significantly to increasing age, female gender, and normal levels of serum PIVKA-II (P=0.02, 0.04, and 0.04, respectively). No statistically significant difference in survival was observed between patients with p16INK4A hypermethylation and those without.


CONCLUSIONS
Our observations suggest that p16INK4A hypermethylation may contribute to hepatocarcinogenesis from an early stage and that multiple risk factors, such as viral infections, age, and gender, may be associated with p16INK4A hypermethylation in hepatocarcinogenesis.
Li
Xin
X
Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, the University of Tokyo, Tokyo, Japan.
Hui
Ai-Min
AM
Sun
Lin
L
Hasegawa
Kiyoshi
K
Torzilli
Guido
G
Minagawa
Masami
M
Takayama
Tadatoshi
T
Makuuchi
Masatoshi
M
D000328:Adult; D000367:Age Factors; D000368:Aged; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000821:virology*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D019175:DNA Methylation; D018572:Disease-Free Survival; D005260:Female; D005355:Fibrosis / Q000378:metabolism; D006518:Hepatitis Viruses / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics* / Q000821:virology*; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012307:Risk Factors; D012737:Sex Factors; D013997:Time Factors
D019941:Cyclin-Dependent Kinase Inhibitor p16; D012333:RNA, Messenger