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Association of Expression and Genotypes of Thymidylate Synthase in Non-Small Cell Lung Cancer Patients with Different Clinicopathological Characteristics

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Association of Expression and Genotypes of Thymidylate Synthase in Non-Small Cell Lung Cancer Patients with Different Clinicopathological Characteristics Pteridines 2021; 32: 39–47 Research Article Jin-Yin Chen,He-Jian Chen, Pei-Feng Chen* Association of expression and genotypes of thymidylate synthase in non-small cell lung cancer patients with different clinicopathological characteristics https://doi.org/10.1515/pteridines-2020-0013 addition, the rate of TS protein overexpression in NSCLC received April 29, 2020; accepted November 13, 2020 patients with 3R/3R was 79.79%, which was higher than Abstract others. Interestingly, high expression of TS protein predicted - - Objective ‒ To explore the expression and genotypes of shorter DFS and OS and lower 3 year DFS rate and 3 year thymidylate synthase (TS) in patients of non-small cell OS rate. Conclusions ‒ lung cancer (NSCLC) with different clinicopathological The expression levels of TS in NSCLC were fi characteristics. signi cantly increased and may help to predict the prognosis ′ Methods ‒ The expression profiles of TS were examined of NSCLC, and high expression of TS protein and 5 UTR fi ff - by immunohistochemical staining and quantitative polymorphism of TS gene were signi cantly related to di er real-time reverse transcription polymerase chain reaction entiation, TNM stage, and lymph node metastases. (qRT-PCR) in 160 patients with NSCLC. Polymerase chain Keywords: thymidylate synthase, TS, non-small cell lung reaction-restriction fragment length polymorphism (PCR- cancer, NSCLC, clinicopathological characteristics, gene RFLP) was used to detect TS-5′UTR tandem repeats, G/C polymorphism, prognosis nucleotide polymorphisms, and 3′UTR 6 bp deletion/ insertion polymorphisms. The relationships between clin- icopathological characteristics and TS expression or geno- types were investigated through χ2 test. Kaplan–Meier 1 Introduction survival analysis was used to analyze the association fi between TS expression and overall survival (OS) and dis- Lung cancer currently ranks rst in the occurrence of ease-free survival (DFS) of NSCLC patients. tumors in China, and it is a serious threat to lives and Results ‒ The expression levels of TS protein and TS health. Clinical studies have shown that 80% of lung cancer - ( ) gene in NSCLC tissues were significantly higher than patients have non smallcelllungcancer NSCLC ,andits [ – ] that in paracancerous tissues (P < 0.05). Furthermore, morbidity and mortality are still on the rise 1 3 .Recent high expression of TS protein and 5′UTR polymorphism studieshaveshownthattheoccurrenceanddevelopmentof of TS gene showed significant correlation with differen- NSCLC are regulated by multiple genes and related to cell [ ] fi tiation, TNM stage, and lymph node metastases. The fre- proliferation and apoptosis 4,5 .Thespeci c mechanism of quency of −6bp/−6 bp genotypes in patients with NSCLC this complex process is currently not very clear. ( ) - was 43.13% (69/160), which was higher than others. In Thymidylate synthase TS is the rate limiting enzyme in the synthesis of dTMP. Mechanisms research has shown that TS catalyzes the methylation of deoxyuridine nucleo- - * Corresponding author: Pei-Feng Chen, Department of Respiratory tide to deoxythymidine nucleotides and regulates the bal Medicine, Zhuji Affiliated Hospital of Wenzhou Medical University ance between the nucleotides required for cell DNA replica- (Zhuji People’s Hospital of Zhejiang Province), 9 Jianmin Street, tion and repair [6,7]. In recent years, the biological functions Taozhu Sub-district, Zhuji, Zhejiang 311800, China, of TS have been studied, and it is found that the expression - + - - - e mail: [email protected], tel: 86 575 8178 2135 intensity of TS is closely related to the malignant biological - - Jin Yin Chen, He Jian Chen: Department of Respiratory Medicine, [ ] [ ] Zhuji Affiliated Hospital of Wenzhou Medical University (Zhuji behavior of tumors 8,9 .Siddiquietal. 10 reported that TS People’s Hospital of Zhejiang Province), 9 Jianmin Street, Taozhu is functionally related to ZEB1 and contributes to the epithelial- Sub-district, Zhuji, Zhejiang 311800, China mesenchymal transition of cancer cells. Furthermore, Open Access. © 2021 Jin-Yin Chen et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. 40 Jin-Yin Chen et al. Siddiqui et al. [11] also reported that TS maintained the history of liver, heart, kidney disease, or diabetes mellitus; dedifferentiated state of triple-negative breast cancers. (2) a double primary malignant tumor; (3) the patient had Several studies have found that cancer patients with high a history of acute or chronic inflammatory diseases and TS expression have significantly higher tumor-associated infectious diseases; (4) loss of follow-up after treatment; antigen levels, vascular invasion, and distant metastasis (5) the patient had a history of lung cancer. These rates than patients with low TS expression [10,12].Importantly, patients did not receive any preoperative chemotherapy the high expression of TS enhances 5-fluorouracil-related chemo- or radiotherapy. The TNM staging and lymph node therapy resistance and is significantly associated with metastasis were evaluated according to the American poor overall survival (OS) and disease-free survival (DFS) Joint Committee on Cancer/TNM staging system of lung [13]. Therefore, understanding the expression profile of TS is cancer (seventh edition). Before chemotherapy, patients helpful for the treatment and prognosis prediction of NSCLC. with NSCLC were treated with radical or palliative resection, TS gene polymorphism has attracted widespread and lung cancer tissues and adjacent tissues were collected. attention. TS gene polymorphism including TS-5′UTR It was then immediately frozen in liquid nitrogen and stored tandem repeats, G/C mononuclear protein polymor- at −80℃ until immunohistochemistry. The follow-up ended phisms, and 3′UTR-6 bp deletion or insertion polymor- on May 31, 2019. The date of death and recurrence is verified phisms may lead to changes in enzyme activity or function, through hospital records or telephone contact with the thereby changing the susceptibility to cancer, changing patient or his/her relatives. Determine the DFS and OS time the sensitivity of cancer patients to chemotherapeutic according to the time after treatment. All patients have reg- drugs, and even affecting the prognosis [14,15]. Wang ular outpatient and telephone follow-ups every 1 month. et al. [16] found that TS 3′-UTR 1494del 6 bp poly- morphism is related to the sensitivity of patients with Ethical approval: The research related to human use has lung adenocarcinoma to pemetrexed treatment, and the been complied with all the relevant national regulations, OS and DFS of patients with −6 bp/−6 bp genotype were institutional policies and in accordance with the tenets of significantly higher than those of patients with −6bp/+6bp the Helsinki Declaration, and has been approved by the genotype. Hur et al. [17] showed that the single nucleo- Ethics Committee of Zhuji Affiliated Hospital of Wenzhou tide polymorphisms (SNPs) in the TS enhancer region Medical University. affected the tumor response of preoperative 5-fluorouracil chemotherapy for rectal cancer. Therefore, under- Informed consent: Informed consent has been obtained standing TS gene polymorphism is helpful to the treat- from all individuals included in this study. ment and prognosis predication of NSCLC. However, the expression of TS and TS gene polymorphisms in NSCLC patients with different clinical-pathological characteristics remains unclear. This study aims to investigate the expres- 2.2 Immunohistochemical staining sion and genotype of TS gene in NSCLC patients with dif- ferent clinicopathological characteristics, find new targets, All tissue specimens were formalin-fixed and paraffin- and try to improve the prognosis. embedded. Before deparaffinization and dehydration, 4 μm sections were taken for immunohistochemistry. With 0.01 M citrate buffer (pH 6.0) in a microwave oven for antigen ( ) 2 Materials and methods retrieval, they were treated with H2O2 3% to quench the endogenous peroxidase. Add the normal serum of the host animal to block nonspecific binding. Then anti-TS antibody 2.1 Patients and clinical samples (Santa Cruz Biotechnology, Santa Cruz, CA) was added and incubated at 37℃ for 1 h. DAB color reagent kit (Shanghai The clinical and pathological data of patients with NSCLC Medici Biomedical Co., Ltd., Shanghai, China) was applied from January 2014 to August 2015 in Zhuji Affiliated for 30 min to visualize the positive signal. The negative con- Hospital of Wenzhou Medical University were retrospec- trol stains without the primary antibody to determine tively analyzed. NSCLC is diagnosed by pathological the specificity of the antibody reaction. Mayer hematoxylin examination, ultrasound, X-ray, radiological imaging, was used for light counterstaining. or cytological examination. The demographic data as The immunohistochemical results were evaluated by well as laboratory data, therapy protocol, imaging data, three independent pathologists. The protein expression and follow-up records of patients were collected. The intensity of the immunohistochemical staining samples exclusion criteria were as follows: (1) the patient had a was scored from − to +++ (−: negative; +: weak; ++: Thymidylate synthase in NSCLC 41 moderate; and +++: strong). The percentage of positively and synthesized by Invitrogen. PCR analysis was performed stained cells was based on a 0 to 4 scoring system (1 = as previously reported [18]. The PCR products were confirmed 0–25%; 2 = 26–50%; 3
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