Genetic and epigenetic changes in P16/CDKN2A and associated expression profiles in Barrett's esophagus and esophageal adenocarcinoma

SJ Darnton, PhD, a, LJ Hardie, PhD, b, YL Wallis, PhD, a, A Chauhan, BSc, a, P Hainaut, PhD, c, CP Wild, PhD, b, AG Casson, FRCSC, d

a Oesophageal Research Laboratory, Heartlands Hospital, Birmingham UK, b Molecular Epidemiology Unit, University of Leeds, UK, c International Agency for Research on Cancer, Lyon, France, d Dalhousie University,Halifax, Nova Scotia Canada

AIM. To study p16/CDKN2A mutation and hypermethylation, mRNA and protein expression in primary esophageal adenocarcinomas and associated premalignant Barrett's epithelium. METHODS Genomic DNA and mRNA were extracted from 54 surgically resected primary esophageal adenocarcinomas, defined according to strict clinicopathologic criteria. Each tumor had matched histologically normal esophageal epithelium (internal control; n=54), and associated Barrett's mucosa (n=24). PCR-based assays (SSCP, methylation-specific and RT-PCR) were used to study molecular alterations of p16/CDKN2A. Immunohistochemistry (monoclonal antibody sc1661, at 1:300 dilution) was used to evaluate cell nuclear protein distribution in formalin-fixed, paraffin-embedded tissue sections, in an indirect immunoperoxidase assay. RESULTS 12% (5/41) of tumors demonstrated an SSCP band shift (indicative of p16/CDKN2A mutation). p16/CDKN2A hypermethylation was highly prevalent, and was detected in 85% (18/21) adenocarcinomas, 77% (14/18) Barrett's and 42% (9/21) normal mucosa. p16/CDKN2A transcript levels were usually raised in tumors relative to, and were correlated (P<0.05) with, matched normal and Barrett's mucosa, for individual cases only. Heterogenous p16/MTS1 protein distribution was seen in all tumors, but was localized to proliferative regions in Barrett's and squamous esophageal epithelia. CONCLUSIONS We conclude that 1) p16/CDKN2A gene mutations, and homozygous deletion, are uncommon in human esophageal adenocarcinoma, and 2) inactivation of p16/CDKN2A mRNA expression, by hypermethylation or in conjunction with loss of heterozygosity, appears to affect a defined subset of tumor or epithelial cells during the progression of Barrett's epithelium to invasive esophageal adenocarcinoma.

KEY WORDS: Barrett's esophagus, adenocarcinoma, p16/CDKN2A.

For more information, contact

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Gene Expression, Part 2.