Mechanisms of p16INK4A inactivation in non small-cell lung cancers

The cyclin-dependent kinase inhibitor p16 (p16(INK4A)/CDKN2/MTS1) is a potent inhibitor of the cyclin D-dependent phosphorylation of the retinoblastoma gene (Rb) product, the inactivation of which induces loss of Rb-dependent G1 arrest through inappropriate phosphorylation of the Rb protein. To analyse the role of po16(INK4A) as a tumor suppressor in the genesis of non small cell lung cancers (NSCLC) and correlate loss of p16(INK4A) protein expression to genetic or epigenetic mechanisms, we have performed a comprehensive study of p16 status in a series of 43 NSCLC. To this end, we have investigated p16(INK4A) protein expression with immunohistochemistry, deletions of the gene by FISH, and determined the methylation status of exon 1 alpha using a PCR-based methylation assay. Finally, possible mutations were studied by SSCP and subsequent sequencing, Twenty one of the 43 (49%) NSCLC studied exhibited an absence of p16(INK4A) nuclear staining. Of these, three (14%) had frameshift or missense mutations, seven (33%) displayed methylation of exon1 alpha and 10 (48%) displayed homozygous deletions, In total, 95% of the tumors with p16(INK4A) negative staining carried one of these three alternative genetic or epigenetic alterations. Furthermore, a high degree of chromosome 9 polysomy was found (58%) in those tumors with p16(INK4A) inactivation. Taken together these results suggest that deregulation of the p16 gene locus is a frequently occurring event in NSCLC through distinct mechanisms including rare point mutations, promotor methylation and frequent homozygous deletions. Furthermore, our data show that immunohistochemistry is a rapid and an accurate technique for screening of p16(INK4A) gene inactivation events that result in loss of protein expression.