Thymidylate synthase locus LOH in combination with genotype has prognostic and predictive significance in colorectal cancer

Abstract

The aim of the current study was to investigate the prognostic and predictive significance of polymorphisms in the thymidylate synthase (TS) gene, alongside the loss of heterozygocity (LOH) at this gene locus in patients with colorectal cancer. Genotyping was carried out for a variable number tandem repeat (VNTR) polymorphism in the TS 5'-untranslated region, a G/C single nucleotide polymorphism (SNP) located within this VNTR, and for TS LOH status in 246 colorectal cancer and paired normal DNA samples. The results were analyzed in relation to clinicopathological features, including the prognostic and predictive significance of TS genotype in patients who underwent curative surgery. Complete VNTR, SNP and LOH information for TS was obtained in 226 cases. No significant associations were observed between normal tissue TS genotype status and clinicopathological features. LOH of TS was observed in 58% of tumor samples and was associated with poor prognosis independently of clinical stage. Cases exhibiting TS LOH were classified into the three groups of 2R/loss, 3G/loss and 3C/loss. Patients with 3C/loss genotype status had poor outcomes when treated by surgery alone, but their survival was similar to patients with other genotypes following Fluorouracil (5-FU)-based adjuvant chemotherapy. The results suggested that LOH of the TS locus may be a significant prognostic factor in colorectal cancer, with the genotype of the residual allele also demonstrating an influence on prognosis. In conclusion, LOH status should be considered when TS genotype is explored as a potential prognostic and predictive marker for 5-FU-based adjuvant chemotherapy in colorectal cancer.

Keywords: 5-fluorouracil; colorectal cancer; genetic polymorphism; loss of heterozygocity; thymidylate synthase.

Figure 1

TS VNTR and SNP analysis. (A) VNTR analysis using PCR amplification and separation of products on a 3% agarose gel. (B) SNP analysis in the 2R allele using PERFLP followed by separation on a 3% agarose gel and scanning with a fluoroimager. (C) SNP analysis in the 3R allele by RFLP and separation on a 3% agarose gel. The DNA fragments stained using ethidium bromide are displayed as white pixels on a black background, whereas those labeled by fluoresein are displayed as black pixels on a white background. The numbers on these panels indicate the same samples being analyzed. The genotypes are as follows: 1, 2G/2C; 2, 2C/2C; 3, 2G/3C; 4, 2C/3G; 5, 2C/3C; 6, 3G/3G; 7, 3G/3C; and 8, 3C/3C. M indicates the size marker (50, 100, 200 and 300 bp) and NC indicates that there was no template control. TS, thymidylate synthase gene; VNTR, variable number tandem repeat; SNP, single nucleotide polymorphisml RFLP, restriction fragment length polymorphism.

Figure 2

Flow chart of LOH analysis and the frequency of TS genotypes. The LOH status of tumors was analyzed according to the TS genotype in normal sample. The TS genotype frequency in relation to LOH status is shown in the box. LOH, loss of heterozygocity; TS, thymidylate synthase gene; RFLP, restriction fragment length polymorphism.

Figure 3

Representative cases of LOH analysis in 3G/3C genotype samples. The PCR-primer extention restriction fragment length polymorphism method detailed in the materials and methods was used to avoid interference by the heteroduplex product. The image of DNA fragments labeled by fluoresein are displayed as black pixels on a white background. Thymidylate synthase genotype and LOH status is indicated on the top of matched T and N lanes. LOH, loss of heterozygocity; T, tumor; N, normal.

Figure 4

Overall survival of patients with curatively resected colorectal cancer according to their TS LOH status. TS, thymidylate synthase gene; LOH, loss of heterozygocity.

Figure 5

LOH status of the TS locus and of 18q in 41 randomly selected tumor samples. White squares indicate that the LOH status could not be determined either due to microsatellite instability (I) or homozygosity (H). Light grey squares indicate no LOH and dark grey squares indicate LOH. LOH, loss of heterozygocity; TS, thymidylate synthase gene.

Figure 6

Survival analysis of patients with colorectal cancer exhibiting TS LOH. The survival rates of patients with 2R/loss (broken line), 3G/loss (dotted line) and 3C/loss (continuous line) genotype were compared using the Kaplan-Meier method in (A) all patients with TS LOH, (B) patients treated with surgery alone and (C) patients who received adjuvant chemotherapy. (D) Survival of patients with the 3C/loss genotype who did (broken line) or did not (continous line) receive 5-FU-based adjuvant chemotherapy. TS, thymidylate synthase gene; LOH, loss of heterozygocity; 5-FU, Fluorouracil.