IMMUNOHISTOCHEMICAL ANALYSIS OF THE P16(INK4) CYCLIN-DEPENDENT KINASEINHIBITOR IN MALIGNANT MESOTHELIOMA

Background: The identification in 1994 of the CDKN2 gene as a target f or mutations in a wide range of human cancers, including malignant mes othelioma, has been controversial because subsequent studies have dete cted a lower frequency of CDKN2 gene mutations in primary tumors than in cultured cell lines, These reports raised the hypothesis that anoth er gene, distinct from CDKN2, might be the target of the chromosome 9p 21 deletions frequently observed in these tumors, Purpose: To address whether inactivation of CDKN2 function is an essential event in the et iology of malignant mesothelioma, we examined p16(INK4) protein expres sion in primary thoracic mesotheliomas, in nonmalignant pleural tissue s, and in independent mesothelioma cell lines, We also studied the gro wth rate of tumor cell lines following stable transfection of CDKN2 ge ne, Methods: Retinoblastoma (Rb) and p16(INK4) protein expression was determined by immunohistochemical analysis from archival paraffin spec imens of 12 primary thoracic mesotheliomas and a nonmalignant pleural biopsy specimen, In addition, protein immunoblot analysis for Rb and p 16(INK4) expression was conducted on 15 independent mesothelioma cell lines, and the ability of a transfected CDKN2 gene to suppress the gro wth of the mesothelioma cell lines H2373 and H2461 in vitro was examin ed, Results: We demonstrated abnormal p16(INK4) expression in 12 of 12 primary mesothelioma specimens and in 15 of 15 mesothelioma cell line s, All tumor specimens and the tumor cell lines showed expression of w ild-type Rb protein, In addition, we have confirmed the ability of a t ransfected CDKN2 gene to suppress growth of two independent mesothelio ma cell lines, Conclusions: Immunohistochemical analysis of the p16(IN K4) gene product is feasible in archival biopsy samples, With this ana lysis, CDKN2 gene inactivation can be determined in tumors that are co ntaminated with nonmalignant cells, Furthermore, since loss of p16(INK 4) protein expression can result from both genetic (gene mutations) an d epigenetic (abnormal DNA hypermethylation) mechanisms, as we and oth ers have shown recently, examination of protein expression is a highly sensitive method for analyzing the CDKN2 status in large numbers of t umor samples, Implications: This study suggests that inactivation of t he CDKN2 gene is an essential step in the etiology of malignant mesoth eliomas, Defining the role of the p16(INK4):Rb tumor suppressor pathwa y and its immediate downstream substrates will be an important goal in designing future therapeutic strategies.