Loss of p16 protein expression associated with methylation of the p16(INK4A) gene is a frequent finding in Hodgkin's disease

p16 protein binds and inactivates cyclin D-CDK4/6 complexes, stopping the c ell cycle at the G1/S boundary. Loss of p16 expression is found frequently in human cancer tissues, often resulting from allelic loss or promoter regi on hypermethylation in non-Hodgkin's lymphomas. Hodgkin's disease has been shown to be a monoclonal neoplasm of B-cells in which a majority of cells a re cycling. In the attempt to identify hypothetical CDK inhibitor inactivat ion that could explain the accumulation of proliferating cells, we decided to focus on the p16(INK4A) gene. To determine whether inactivation of this gene is implicated in the development of Hodgkin's disease, we immunostaine d 40 cases with a monoclonal antibody for the p16 protein. At the same time , we used a methylation-specific PCR technique to determine the methylation status of exon 1 of the p16(INK4A) gene in 23 cases in this series. Loss o f p16 expression was found in 30 of 37 cases (absence of expression in most Hodgkin's/Reed-Sternberg cells, with a normal scattered pattern of p16 exp ression in the reactive background). Only seven samples showed nuclear p16 expression in a significant proportion of large tumoral cells. In agreement with this finding, hypermethylation of p16(INK4A) gene was found in 14 of 23 cases by PCR. All the p16 cases found positive by immunohistochemistry a lso showed unmethylated DNA. These results show that loss of p16 protein ex pression is usually observed in Hodgkin's/Reed-Sternberg cells in Hodgkin's disease, frequently associated with p16I(NK4A) gene hypermethylation. The high frequency of abnormal methylation found in this study suggests that th is genetic event may play an important role in the pathogenesis of the dise ase.