Alterations of the p16(ink4a) gene in resected nonsmall cell lung tumors and exfoliated cells within sputum

Int J Cancer. 2002 Apr 10;98(5):724-31. doi: 10.1002/ijc.10262.


Recently, we reported that p16 protein expression was nondetectable in 49.5% of 107 resected nonsmall cell lung cancers (NSCLCs), suggesting that the p16(INK4a) 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 p16(INK4a) 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 SmaI-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 p16(INK4a) gene was present in 38 of the 67 (56.7%) tumors and was significantly associated with negative p16 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 p16(INK4a) 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 p16(INK4a) 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 p16(INK4a) alteration. Our data provide compelling evidence that p16(INK4a) alterations are involved in NSCLC tumorigenesis and that promoter methylation is the predominant mechanism in p16(INK4a) deregulation.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Aged, 80 and over
  • Carcinoma, Adenosquamous / genetics
  • Carcinoma, Adenosquamous / pathology
  • Carcinoma, Adenosquamous / surgery
  • Carcinoma, Large Cell / genetics
  • Carcinoma, Large Cell / pathology
  • Carcinoma, Large Cell / surgery
  • Carcinoma, Non-Small-Cell Lung / genetics*
  • Carcinoma, Non-Small-Cell Lung / pathology
  • Carcinoma, Non-Small-Cell Lung / surgery
  • Cyclin-Dependent Kinase Inhibitor p16 / genetics*
  • Cyclin-Dependent Kinase Inhibitor p16 / metabolism
  • DNA Methylation
  • DNA Mutational Analysis
  • DNA, Neoplasm / analysis
  • Female
  • Humans
  • Immunoenzyme Techniques
  • Loss of Heterozygosity
  • Lung Neoplasms / genetics*
  • Lung Neoplasms / pathology
  • Lung Neoplasms / surgery
  • Male
  • Middle Aged
  • Polymerase Chain Reaction
  • Sputum / cytology*


  • Cyclin-Dependent Kinase Inhibitor p16
  • DNA, Neoplasm