Loss of heterozygosity at thymidylate synthase locus in Barrett's metaplasia, dysplasia, and carcinoma sequences

Hidekazu Kuramochi,Kazumi Uchida,Kathleen D Danenberg,Peter V Danenberg,Daniel Vallbohmer,Daisuke Shimizu,Sylke Schneider,Jeffery H Peters

doi:10.1186/1471-2407-9-157

Abstract

BackgroundThymidylate synthase (TS) is known to have a unique 28 bp tandemly repeated sequence in the promoter region, and the majorities of subjects have a heterozygous double repeat/triple repeat genotype in their non-cancerous tissue. Loss of heterozygosity (LOH) at the TS locus is known to occur in cancer patients, but there is no evidence that it is present in precancerous tissue. The aim of this study was to analyze the frequency and timing of LOH at the TS locus in Barrett-associated adenocarcinoma (BA) and its precursory lesions, such as intestinal metaplasia (IM) and dysplasia.MethodsOne hundred twenty-three samples (including 37 with gastroesophageal reflux disease (GERD), 29 with IM, 13 with dysplasia, and 44 with BA) were obtained from 100 patients. Biopsies were obtained from the lower esophageal mucosa/IM/dysplasia/BA, when available. Normal squamous tissue from the upper esophagus was taken as a control. All tissues were analyzed for the TS genotype and TS mRNA expression using the real-time reverse-transcription polymerase chain reaction (RT-PCR) method after laser-capture microdissection.ResultsAmong the patients with informative heterozygous genotype in their control samples, no sample with LOH at the TS locus was observed in the lower esophageal mucosa in GERD patients (0/22 samples). However, 6 out of 21 samples (28.6%) had LOH in IM, 2 of 7 (28.6%) in dysplasia, and 10 of 25 (40.0%) in BA. No significant difference in TS mRNA expression levels was observed between TS genotypes.ConclusionOur results demonstrate that LOH is a relatively frequent and early event in the IM-BA sequence.

Highlights

Thymidylate synthase (TS) is known to have a unique 28 bp tandemly repeated sequence in the promoter region, and the majorities of subjects have a heterozygous double repeat/triple repeat genotype in their non-cancerous tissue
The 3RC allele can abolish the increased transcriptional activity of the 3R variant in vitro by altering a transcription factor-binding site [10]. Another polymorphism, a 6 bp insertion/deletion at bp 1494 in the 3'-untranslated region (3'-UTR) of TS gene, was identified [11] and is thought to influence the intratumoral TS mRNA level in vivo [12]. Using this 5'-untranslated region (5'-UTR) polymorphism as a heterozygous marker, we have reported that there is a high incidence of loss of heterozygosity (LOH) at the TS locus in colorectal cancer tissue, and have demonstrated that the TS genotype in cancer tissue modulated by Loss of heterozygosity (LOH) to give either a 2R/loss or 3R/loss situation influences the chemosensitivity to 5FU-related drugs and the prognosis of colorectal cancer patients [13]
Frequency of LOH at TS 5'-UTR locus in intestinal metaplasia (IM)-Barrett-associated adenocarcinoma (BA) sequence The patients with informative heterozygous genotype in the 5'-UTR polymorphism in their normal squamous tissues were evaluated the presence of LOH in their IM, dysplasia, and BA tissues

Summary

Introduction

Thymidylate synthase (TS) is known to have a unique 28 bp tandemly repeated sequence in the promoter region, and the majorities of subjects have a heterozygous double repeat/triple repeat genotype in their non-cancerous tissue. 5-fluorouracil (5-FU) inhibits TS by forming a stable ternary complex among 5,10-methylenetetrahydrofolate, TS, and fluoro-dUMP, the metabolite of 5-FU Based on this mechanism, the TS expression level is regarded as a predictor of response to 5-FU-based chemotherapy, and patients with low TS reported having better prognoses than those with high TS [2,3]. The 3RC allele can abolish the increased transcriptional activity of the 3R variant in vitro by altering a transcription factor-binding site [10] Another polymorphism, a 6 bp insertion/deletion at bp 1494 in the 3'-untranslated region (3'-UTR) of TS gene, was identified [11] and is thought to influence the intratumoral TS mRNA level in vivo [12]

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