In the esophagus, both in vitro and in vivo studies indicate exposure to gastroduodenal reflux results in measurable oxidative damage to DNA. As well, p16 alterations have been observed in animal models in response to oxidative stress, and it has been proposed that oxidative damage may be responsible for the loss of heterozygosity frequently observed at multiple chromosomal loci in BE. Oxidative stress can also induce p16 mediated senescence; thus, the oxidative damage induced by gastroduodenal reflux provides both a mechanism for generating genetic alterations as well as a selective pressure for loss of p16 function. To our knowledge, this is the largest study of p16 mutation spectrum yet reported in a premalignant condition. The study was based on a cohort design and all biopsies were obtained prospectively using standardized protocols. The characteristics of our cohort, comparable to studies from specialty centers. Our study was performed in a tertiary Danthron referral center and our research cohort therefore has a higher percentage of patients with a diagnosis of dysplasia than the general BE population; however, this is Gambogic-acid unlikely to have affected the p16 mutation spectrum reported here because we also detected p16 mutations in patients without high-grade dysplasia, indicating p16 mutations can occur very early during neoplastic progression. The observation that frequency of mutation increased with histologic grade suggests these patients may have experienced a longer or more intense exposure to oxidative damage induced by reflux. All of the data obtained in this study are consistent with Knudsen��s two-hit model for inactivating tumor suppressor genes. LOH is the predominant mechanism of inactivation for p16, occurring in almost 60% of patients with BE, compared to 14.5% with mutation. This may represent the fact that the genetic mechanisms that result in LOH are more common than the development of point mutations. Alternatively, LOH events may involve additional genes, leading to a greater selective advantage over clones containing mutations.