p16 inactivation by methylation of the CDKN2A promoter occurs early during neoplastic progression in Barrett's esophagus.
OBJECTIVE
The potential role of p16 inactivation by CDKN2A/p16 promoter hypermethylation and/or loss of heterozygosity (LOH) of the CDKN2A gene was investigated in neoplastic progression of Barrett's esophagus.


METHODS
CDKN2A promoter hypermethylation was studied by methylation sensitive single-strand conformation analysis and sequencing using bisulfite modified DNA in Barrett's esophageal adenocarcinomas, premalignant lesions, and normal squamous esophageal epithelium. All of the lesions of interest were sampled by microdissection from paraffin-embedded fixed tissue sections.


RESULTS
No methylation of the CDKN2A promoter was found in normal esophageal squamous cell epithelia, whereas methylation was detected in 18 of 22 (82%) adenocarcinomas and 10 of 33 (30%) premalignant lesions, including 4 of 12 (33%) samples with intestinal metaplasia only. LOH at the CDKN2A gene locus was found in 68% of adenocarcinomas and in 55% of premalignant lesions. Of 28 samples without p16 immunoreactivity, 25 (89%) showed CDKN2A promoter hypermethylation with or without LOH of CDKN2A. Only 2 (8%) samples expressing p16 protein were found to be methylated; these showed a mixture of completely methylated and unmethylated CDKN2A promoters. In 7 of 19 (37%) informative samples without LOH of CDKN2A, the CDKN2A promoter was found to be methylated at both alleles. Loss of p16 protein expression was strongly associated with CDKN2A promoter hypermethylation (P < 0.00001), but not with LOH (P = 0.33).


CONCLUSIONS
Our results indicate that methylation of the CDKN2A promoter is the predominant mechanism for p16 inactivation. This hypermethylation is a very common event in esophageal adenocarcinoma and occurs as early as metaplasia.
Bian
Yan-Song
YS
Institute of Pathology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland.
Osterheld
Maria-Chiara
MC
Fontolliet
Charlotte
C
Bosman
Fred T
FT
Benhattar
Jean
J
D000230:Adenocarcinoma / Q000235:genetics / Q000503:physiopathology*; D001471:Barrett Esophagus / Q000235:genetics / Q000503:physiopathology*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics* / Q000378:metabolism*; D019175:DNA Methylation; D018450:Disease Progression; D004938:Esophageal Neoplasms / Q000235:genetics / Q000503:physiopathology*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D019656:Loss of Heterozygosity; D011230:Precancerous Conditions / Q000235:genetics / Q000503:physiopathology; D011401:Promoter Regions, Genetic / Q000502:physiology; D014407:Tumor Cells, Cultured
D019941:Cyclin-Dependent Kinase Inhibitor p16