Detection of p16 {9p} LOH in Barrett's Esophagus using LATE·PCR

Abstract

Background: Early detection of cancer biomarkers is a major strategy for decreasing rates of cancer mortality and morbidity. The type of cancer studied here, Esophageal Adenocarcinoma, is associated with a precancerous condition termed Barrett's Esophagus. Present techniques to predict progression from Barrett’s Esophagus to Esophageal Adenocarcinoma require repetitive biopsies and interpretation of their results are subjective. Loss of Heterozygosity (LOH) of the p16 tumor suppressor gene is the earliest event in the progression from Barrett's Esophagus (BE) to Esophageal Adenocarcinoma (EA) and is studied in the work described below. , this thesis discusses a proof-of-principle panel using Linear-After-The-Exponential PCR to analyze for p16 LOH as an objective, clinically-compatible approach to early cancer detection. Methods: The Pubmed Database was used to identify SNP sites that had high heterozygosity indices and were located near the p16 gene on chromosome 9p. Twelve such sites were chosen for design and construction of a panel of LATE-PCR assays. Visual OMP software was used to design the Limiting Primer and the Excess Primer for each assay, as well as a probe that could hybridize to two allele variants of the target sequence. One variant hybridized to the probe at 55 °C, while both variants hybridized to the probe at 45 °C. By using this probe and normalizing fluorescence values at three temperatures, it was possible to discriminate between alleles and determine the genotype of cells taken from the esophageal mucosa. Furthermore, it was possible to test for the presence of LOH in epithelial cells of BE patient biopsies prepared by flow sorting. Touch ~on, and EDTA. Results: 12 Probes were designed to hybridize to variants at the Tms listed in the methods section, As a result, there was accurate discrimination between homozygous and heterozygous alleles with 99.7% accuracy. Of 12 Single Nucleotide Polymorphism used in this 9pLOH panel, genotyping experiments indicated that 10 of the 12 were heterozygous for esophageal normal cells. Of the 10 that were informative, all 10 were used to perform 9pLOH detection on known Barrett's Esophagus patient tissues, prepared by cell sorting, Touch Preparation, or EDTA solution. All patients tested bad 9pLOH, and the preparation method did not affect the outcome of these results. Conclusions: LATE-PCR assays with mis-match tolerant probes can be used for convenient and accurate detection of 9pLOH. These assays are robust and can be effectively applied to a clinical setting. The benefit of this panel is that for patients that have Barrett's Esophagus, objective management and early treatment protocols can be implemented to prevent morbidity and mortality rates associated with Esophageal Adenocarcinoma. Ultimately, this panel will be utilized for other cancers as well that have the same kind of deletion event.