Recently, we reported that p16 protein expression was nondetectable in 49.5% of 107 resected nonsmall cell lung cancers (NSCLCs), suggesting that the p16INK4a gene is frequently inactivated in primary NSCLC. To identify the molecular basis for this p16 immunohistochemical negativity further, we performed a genetic and epigenetic study of p16INK4a status in a series of 115 NSCLC samples parallel to the clinicopathologic and prognostic analyses. Microdissected tumor DNA samples were screened for homozygous deletion using comparative multiplex-polymerase chain reaction (PCR), for intragenic mutation using direct sequencing and for loss of heterozygosity (LOH) using an intragenic microsatellite marker, D9S942. Of these samples, 67 were further analyzed by Smal-based PCR methylation assay to evaluate aberrant methylation at the gene. To examine the correlation of aberrant methylation in tumor and sputum samples, sputum samples from 12 matched patients were assessed for this change. We found that methylation of the p16INK4a gene was present in 38 of the 67 (56.7%) tumors and was significantly associated with negative p 16 protein expression (p = 0.029). A 92% (11/12) concordance of sputum samples with matched resected tumors was found. The survival rates among adenocarcinoma patients with p16INK4a methylation were lower, but at a level of borderline significance compared with those patients without methylation (p = 0.071). In addition, 29.4% of the informative cases were found to harbor LOH at D9S942. None of the 115 microdissected tumors exhibited homozygous deletion in the p16INK4a gene. Only 1 patient exhibited a complex mutation at the fourth ankyrin repeat consensus sequence and concordantly demonstrated p16 immunohistochemical negativity. Overall, 69% (79/115) of NSCLC tumors had at least 1 type of p16INK4a alteration. Our data provide compelling evidence that p16INK4a alterations are involved in NSCLC tumorigenesis and that promoter methylation is the predominant mechanism in p16INK4a deregulation.
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