Promoter methylation of CDKN2A and lack of p16 expression characterize patients with hepatocellular carcinoma

Abstract

Background: The product of CDKN2A, p16 is an essential regulator of the cell cycle controlling the entry into the S-phase. Herein, we evaluated CDKN2A promoter methylation and p16 protein expression for the differentiation of hepatocellular carcinoma (HCC) from other liver tumors.

Methods: Tumor and corresponding non-tumor liver tissue samples were obtained from 85 patients with liver tumors. CDKN2A promoter methylation was studied using MethyLight technique and methylation-specific PCR (MSP). In the MethyLight analysis, samples with > or = 4% of PMR (percentage of methylated reference) were regarded as hypermethylated. p16 expression was evaluated by immunohistochemistry in tissue sections (n = 148) obtained from 81 patients using an immunoreactivity score (IRS) ranging from 0 (no expression) to 6 (strong expression).

Results: Hypermethylation of the CDKN2A promoter was found in 23 HCCs (69.7%; mean PMR = 42.34 +/- 27.8%), six (20.7%; mean PMR = 31.85 +/- 18%) liver metastases and in the extralesional tissue of only one patient. Using MSP, 32% of the non-tumor (n = 85), 70% of the HCCs, 40% of the CCCs and 24% of the liver metastases were hypermethylated. Correspondingly, nuclear p16 expression was found immunohistochemically in five (10.9%, mean IRS = 0.5) HCCs, 23 (92%; mean IRS = 4.9) metastases and only occasionally in hepatocytes of non-lesional liver tissues (mean IRS = 1.2). The difference of CDKN2A-methylation and p16 protein expression between HCCs and liver metastases was statistically significant (p < 0.01, respectively).

Conclusion: Promoter methylation of CDKN2A gene and lack of p16 expression characterize patients with HCC.

Figure 1

Results of Methylight analysis. Frequency of promoter methylation of CDKN2A gene is shown in patients with liver tumors detected by Methylight assay.

Figure 2

Results of MSP analysis. Frequency of promoter methylation of CDKN2A gene is shown in patients with liver tumors detected by MSP.

Figure 3

Quantitative differences of p16 protein expression. In liver tumors and non-neoplastic tissue samples the quantitative differences of p16 expression evaluated by immunohistochemical detection of p16 using an immunoreactivity score (IRS) are shown.

Figure 4

Immunohistochemical expression of p16 in non-neoplastic liver tissue and hepatocellular carcinoma. A moderately differentiated HCC is completely immunonegative for p16 (left). The non-neoplastic liver tissue shows immunostaining for p16 in scattered ductular reactions and mild cytoplasmic immunostaining in few periportal and periseptal hepatocytes (right). Anti-p16-antibody. Hematoxylin-counterstain.

Figure 5

Immunohistochemical expression of p16 in non-tumor liver sample. The non-neoplastic liver tissue shows strong immunostaining for p16 in the ductular reactions and mild cytoplasmic immunostaining in few periportal hepatocytes. Anti-p16-antibody. Hematoxylin-counterstain.

Figure 6

Immunohistochemical detection of p16 in a liver metastasis. Liver metastasis of a moderately differentiated colorectal cancer with strong cytoplasmic and also nuclear expression of p16 in tumor cells. Non-neoplastic liver tissue adjacent to colorectal cancer metastasis is completely devoid of p16 expression. Anti-p16-antibody. Hematoxylin-counterstain.