Molecular and immunohistochemical study of the inactivation of the p16 gene in primary hepatocellular carcinoma

Objective To determine whether p16 gene is involved in the genesis of prima ry hepatocellular carcinoma (HCC). Methods Twenty-five HCC tumor samples with corresponding non-tumor liver ti ssue specimens were examined for p16 gene alterations. The identification o f deletion of exon 1 and exon 2 in p16 gene was performed using comparative multiplex polymerase chain reaction (PCR) analysis. The point mutation of exon 2 in p16 gene was investigated by single strand conformational polymor phism (SSCP) analysis, and the status of p16 gene methylation was screened using a PCR based methylation analysis. 35 parafin-embedded specimens of HC C with corresponding nontumor liver tissues, including the 25 cases describ ed above for screening p16 gene alterations, were investigated for p16 prot ein expression immunohistochemical analysis. Results Among 25 cases, 2 homozygous deletions and 1 hemizygous deletion we re found in HCC samples. No point mutation was identified in the remaining 22 tumor samples without p16 gene deletions. Hypermethylation was detected in 24% (6/25) of tumor samples. However, the corresponding non-tumor liver tissue specimens were always unmethylated at the p16 locus. Loss of p16 pro tein expression occurred in 16 of 35 (45.7%) tumor samples, and all the non -tumor liver tissue specimens showed positive p16 staining. For the 25 case s examined for p16 gene alterations, the loss of p16 protein expression was observed in all tumors with p16 gene alterations and also in 3 tumors with out p16 gene alterations. Conclusion Inactivation of the p16 gene may play an important role in the g enesis of primary hepatocellular carcinoma.