Association of thymidylate synthase gene 3'-untranslated region polymorphism with sensitivity of non-small cell lung cancer to pemetrexed treatment: TS gene polymorphism and pemetrexed sensitivity in NSCLC

Abstract

Background: Thymidylate synthase (TS) is a key enzyme responsible for DNA synthesis and repair. Altered expression of TS protein or TS gene polymorphisms has been associated with cancer progression and treatment response. This study investigated the expressions of TS and its gene SNPs in non-small cell lung cancer (NSCLC), and then its association with sensitivity to pemetrexed treatment. Immunohistochemistry and qRT-PCR were performed on 160 resected NSCLC specimens and corresponding normal tissues to assess the expressions of TS protein and TS mRNA, and for associations with clinicopathological data. Blood samples of 106 lung adenocarcinoma patients were examined for polymorphisms of the TS gene 3'-UTR 1494del 6 bp, which was then investigated for associations with responses of the patients to pemetrexed treatment and survival.

Results: Expression of both TS protein and its mRNA was elevated in NSCLC tissues compared with matched normal tissues, and significantly higher in lung squamous cell carcinoma than in lung adenocarcinoma. TS expression was associated with poor tumor differentiation. Furthermore, the genotyping data showed that 56% of lung adenocarcinoma patients had the TS gene 3'-UTR 1494 bp (-6 bp/-6 bp) genotype and the rest had TS gene 3'-UTR 1494 bp (-6 bp/+6 bp). There was no TS 3'-UTR 1494 bp (+6 bp/+6 bp) genotype in any patients. Statistical analysis revealed that gender, tumor stage, and TS 3'-UTR 1494del 6 bp polymorphism were significant prognostic factors after short-term pemetrexed treatment. Log-rank analysis revealed that patients with the (-6 bp/-6 bp) genotype had significantly better progression-free and overall survival than patients with (-6 bp/+6 bp).

Conclusions: This study showed that TS protein is highly expressed in NSCLC and that polymorphisms of TS 3'-UTR 1494del 6 bp are associated with sensitivity of lung adenocarcinoma patients to pemetrexed treatment. This suggests that TS gene polymorphisms should be further evaluated as prognostic markers for personalized therapy in lung adenocarcinoma.

Figures

Figure 1

Detection of TS gene 3’-UTR 6 bp polymorphism at 1494 bp. Genomic DNA was extracted from blood from patients and subjected to qPCR analysis of the TS gene polymorphism and then to DNA sequencing analysis. A and C, qPCR amplification curves show TS 3’-UTR 1494 bp (−6 bp/-6 bp) and (−6 bp/+6 bp) polymorphisms. B and D, DNA sequencing data on TS 3’-UTR 1494 bp (−6 bp/-6 bp) and (−6 bp/+6 bp).

Figure 2

Elevated expression of TS mRNA in NSCLC tissue specimens compared to normal tissues. Surgical specimens were taken from patients with lung adenocarcinoma (A) or squamous cell carcinoma (S) and the corresponding normal mucosae (N) and subjected to semi-quantitative RT-PCR analysis of TS expression. The data are summarized as mean ± standard deviation relative to β-actin. The size of TS and β-actin PCR products was 337 bp and 500 bp, respectively. S (0.712 ± 0.183) compared with A (0.712 ± 0.183), P < 0.05.

Figure 3

Enhanced expression of TS protein in NSCLC compared with the corresponding normal tissues. Paraffin sections from patients with NSCLC were prepared and stained with anti-TS antibody and then reviewed and scored under a microscope. A-C, Representative low, medium, and high levels of TS expression, respectively. D, Negative staining of normal tissues. E, strong expression of TS protein in lung squamous cell carcinoma. F, low level of TS expression in lung adenocarcinoma.

Figure 4

Kaplan-Meier analysis of survival in lung adenocarcinoma and TS 3’-UTR 6 bp 1494del polymorphisms. Kaplan-Meier plot of progression-free survival (PFS, A) and overall survival (OS, B) of the patients by genotypes (−6 bp/-6 bp) (blue line, n = 59) or (−6 bp/+6 bp) (green line, n = 47).