9528 Background: Thymidylate synthase (TS), one of the rate-limiting enzymes in the de novo synthesis of DNA, has a unique 28bp tandemly repeated sequence in the promoter region. Patients who are homozygous for the double repeat (2R/2R) of this sequence have been reported to have lower TS expression and better outcome from chemotherapy than patients who are homozygous for the triple repeat (3R/3R). However, loss of heterozygosity (LOH) at the TS locus during tumor development can cause the heterozygous (2R/3R) patients to acquire either the 2R/loss or the loss/3R genotype in their cancer cells, which could affect tumor TS expression as well as clinical outcome. The aim of this study was 1) to verify the relationship between TS mRNA expression and TS genotype in Barrett's associated adenocarcinoma (BA), and 2) to analyze the frequency and timing of LOH at the TS locus in BA and its precursory lesion such as intestinal metaplasia (IM) and high grade dysplasia (HGD). Methods: Sixty-six patients (including 21 with IM, 6 with HGD and 23 with BA) were included in this study. Biopsies were obtained during endoscopy from: (1) normal squamous epithelium at 20cm (HNE); (2) normal squamous epithelium 3cm above the gastroesophageal junction (LNE); (3) IM, HGD or BA. All samples were analyzed for the TS genotype and TS mRNA expression using a quantitative real-time RT-PCR method after laser-capture microdissection. Results: TS mRNA expression from HNE tissue of 3R/3R genotype patients (n=8) was significantly higher than that of 2R/2R genotype patients (1.8-fold; n=14; p=0.0024) and that of 2R/3R genotype patients (1.4-fold; n=44; p=0.0282). Out of 44 patients with heterozygous (2R/3R) genotype in the HNE, no patient lost an allele in the LNE. However, 10 of 18 patients with IM had LOH (55.6%), as had 4/6 patients with HGD (66.7%) and 9/17 patients with BA (52.9%). Conclusions: Our results demonstrate 1) an association between TS genotype and TS expression in normal squamous epithelium and 2) that LOH at the TS locus is a frequent and early event in the IM-BA sequence, and thus tumor and normal genotypes will not be the same in these heterozygous patients. No significant financial relationships to disclose.