Frequency of p16INK4A alterations and k-ras mutations in intrahepatic cholangiocarcinoma of the liver

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Abstract
BACKGROUND Inactivation of the tumour suppressor gene p16 (CDKN2/MTS-1/INK4A) and K-ras mutations are among the most frequent genetic alterations in human malignancies. AIMS To investigate the tumour suppressor gene p16 and its possible association with K-ras mutations in intrahepatic cholangiocarcinomas of the liver. METHODS The status of p16 was evaluated in 41 cholangiocarcinomas by methylation specific polymerase chain reaction, microsatellite analysis, DNA sequencing, and immunohistochemical staining. K-rasmutations were determined by direct DNA sequencing analyses after microdissection. The results obtained were correlated with histopathological variables and patient survival. RESULTS Hypermethylation of the 5′ CpG island of the p16 gene was found in 34 of 41 (83%) carcinomas. Homozygous deletion at the p16 region was present in two (5%), and loss of heterozygosity (LOH) in eight cases (20%). We failed to detect p16 gene missense mutations. K-rasmutations were found in 22 of 41 (54%) cholangiocarcinomas and in two cases of tumour surrounding non-neoplastic liver tissue. All 22 cancers with K-ras mutations also exhibited methylated p16. We failed to observe a correlation between K-ras or p16 status and histopathological factors or prognosis of patients. CONCLUSION These data suggest that inactivation of the p16 gene is a frequent event in cholangiocarcinoma. The most common somatic alteration is promotor methylation of the p16 gene which is closely associated with K-ras mutations. We failed to establish p16 or K-ras status as independent prognostic factors in these tumours.