Frequency of p16(INK4A) alterations and K-ras mutations in intrahepatic cholangiocarcinoma of the liver

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-ras mutations 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-ras mutations 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.

Figure 1

Methylation analysis of p16 in cholangiocarcinoma. Methylation specific PCR (MSP) results are expressed as unmethylated p16 specific bands (U) or methylated p16 specific bands (M). (A) Bisulphite converted DNA from normal liver tissue (N) served as a negative control as indicated by the presence of the U but not the M band. (B) MSP results of case No 28. The tumour surrounding non-neoplastic liver tissue (NT) with unmethylated p16. (C) Representative cases of cholangiocarcinoma. The numbers of the cholangiocarcinoma cases are shown above the corresponding lanes.

Figure 2

Immunostaining of p16 protein in cholangiocarcinoma. (A) Patient No 28 (same patient as in figure 1B) with a methylated p16 gene. Complete loss of p16 expression. (B) Immunohistochemical staining of p16 in a moderately differentiated cholangiocarcinoma without p16 methylation. Strong nuclear positivity of the tumour cells (brown reaction product). (Original magnification ×40.)

Figure 3

Immunostaining of the ras protein with perinuclear staining (red reaction product) of tumour cells. (Original magnification ×60.)