Immunohistochemical Detection of the Cyclin-dependent Kinase Inhibitor 2/Multiple Tumor Suppressor Gene 1 (CDKN2/MTS1) Product p16INK4A in Archival Human Solid Tumors: Correlation with Retinoblastoma Protein Expression

The retinoblastoma (RB) and cyclin-dependent kinase inhibitor 2/multiple tumor suppressor gene 1 (CDKN2/MTS1) tumor suppressor genes play important roles in the regulation of the cell cycle. The protein products of these two genes, pRB and p16INK4A (“p16”), respectively, inhibit progression from G1 to S phase. Moreover, pl6 has been shown to exert its function through inhibition of CDK4-mediated phosphorylation of pRB. Both genes have been found to be mutated or deleted in a wide range of primary human tumors and tumor cell lines. However, the presence of CDKN2/MTS1 containing nonneoplastic elements in every tumor specimen may contribute to the apparent lower deletion detection rate in resected neoplasms compared to cell lines. We have developed an immunohistochemical assay that allows us to assess pl6 expression in formalin-fixed, paraffin-embedded tissues. As controls, we used paraffin-embedded pellets of cell lines with well-defined pl6 status (four positive and four negative lines), as well as routinely processed nude mouse xenografts of two pl6-positive cell lines. pl6-negative cells were characterized by the absence of nuclear staining, whereas cytoplasmic staining was variable. In neoplastic and normal tissues, the level of pl6 generally appeared to be low. We tested 75 random human malignancies from 4 different anatomic sites for pl6 expression and correlated the findings with the immunohistochemical presence or absence of pRB. Twenty % of tumors selectively lacked pRB, while 37% of neoplasms had undetectable pl6. In 43% of all carcinomas, both pRB and pl6 could be detected. Significant differences existed in the expression of both tumor suppressor genes between carcinomas from different sites. Breast cancers had the highest rate of pl6 negativity (13 of 20). Our data show that: (a) immunohistochemistry may be a suitable modality to screen for RB and CDKN2/MTS1 abnormalities in paraffin-embedded tissues; (b) undetectable levels of pl6 expression occur at a relatively high frequency; (c) pl6 and pRB expression in common human malignancies is not mutually exclusive; (d) loss of function of both tumor suppressor genes appears to be a distinctly uncommon phenomenon; and (e) different types of carcinomas have variable rates of disturbance in the p16/pRB pathway.