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

Abstract

Background: 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. 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.

Methods: One 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.

Results: Among 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.

Conclusion: Our results demonstrate that LOH is a relatively frequent and early event in the IM-BA sequence.

Figure 1

A (left side): Detection of TS 5'-UTR polymorphism. Thymidylate synthase (TS) 5'-UTR genotype analysis in matched normal (N) and tumor (T) DNA. The upper and lower bands represent PCR products from amplification of the TS segment containing 3R and 2R, respectively. Each patient has a heterozygous 2R/3R genotype in normal tissue, as indicated by the presence of both bands. Case 1: Loss of heterozygosity (LOH) gives rise to a tumor with a 2R/loss genotype. Case 2: LOH does not occur. Case 3: LOH gives rise to a tumor with a 3R/loss genotype. 1B (right side): Detection of TS 3'-UTR polymorphism. Thymidylate synthase (TS) 3'-UTR genotype analysis in matched normal(N) and tumor (T) DNA. The middle lane shows the double bands in 109 bp and 103 bp, representing the heterozygous 6 bp insertion/deletion. The right lane shows the single band in 109 bp, representing the loss of the 103 bp band.

Figure 2

Comparison of intratumoral TS mRNA levels with the 5'-UTR genotype. A: Grouped by the number of repeats. No difference was observed between 2R/2R, 2R/3R, and 3R/3R groups. B: Grouped by the number of repeats and G/C SNPs. The patients with 2R/3RG, 3RC/3RG, and 3RG/3RG genotypes were classified as the 3RG group, while those with 2R/2R, 2R/3RC, and 3RC/3RC were classified as the non-3RG group. There was no difference in TS mRNA levels between these two groups. Boxes indicate the first and third quartiles (median inside); bars represent the range of values falling within 1.5-fold the interquartile range.

Figure 3

Comparison of intratumoral TS mRNA levels with TS 3'-UTR genotype. No significant difference in TS mRNA levels was observed between the patients with ins/ins, ins/del, and del/del. Ins, 6 bp inserted allele; del, 6 bp deleted allele.