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 clinicopathological characteristics and TS expression or genotypes 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,

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 polymorphism 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 different 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 = 51–75%; and 4 = 76–100%). The by capillary electrophoresis, and then detected using a immunoreactive score (IRS) was then multiplied by the 3730XL DNA detection equipment (Applied Biosystems, staining intensity score and the percentage of positively USA). Polymerase chain reaction (PCR) for TS-3′UTR geno- stained cells. According to IRS, TS immunoreactivity was typing was carried out in a 20 μL solution containing classified as low (IRS: 0–4) or high (IRS: 6–12). 1.0 μLgenomicDNA,9.7μL ultrapure water, 2 μL10× PCR

buffer, 1.2 μL50mmol/LMgCl2,0.5μL10mmol/LdNTPs (10 mM),4μL5× GC buffer, 0.5 μLeachprimer(10 μM), μ - ( μ ) μ - ( μ ) 2.3 Western blot analysis 0.2 LprobeFAM 10 M ,0.2 LprobeVIC 10 M ,and 0.2 μL5U/μL Taq DNA polymerase (Invitrogen).Thecycling parameters were as follows: pre-PCR at 50°C for 1 min; held The tissues were lysed with lysis buffer (Thermofisher at 95°C for 10 min; 40 cycles at 95°C for 15 s; 60°C for 1 min; Scientific, USA), and then the proteins were quantified and post-PCR at 60°C for 1 min. PCR was performed using by the BCA method. Then protein extracts were separated 7500 FAST qPCR instrument (Applied Biosystems, Foster by 8% SDS-PAGE and transferred onto PVDF membranes. City, CA, USA). PCR products were analyzed using a real- Subsequently, the proteins were probed with primary time fluorescent PCR allele discrimination system for geno- antibodies against TS (1:5,000; Abcam, ab168853) and typing. There are three main polymorphisms in TS gene: 3′ GAPDH (1:10,000; Abcam, GAPDH). After washing, mem- UTR polymorphisms, 5′UTR 28 bp repeat polymorphism, and branes were incubated with goat anti-rabbit IgG H&L (HRP) G-CSNP.3′UTR polymorphism refers to the 6 bp deletion or (1:10,000, Abcam, ab205718) antibody. Band intensity was insertion polymorphism at 1,494 bp of TS gene, which can detected using Image-Pro Plus software. be divided into +6bp/+6bp, +6bp/−6bp,and −6bp/−6bp according to the polymorphism. The 5′UTR 28 bp repeat poly- - morphism refers to multiple repeats of the 28 bp tandem 2.4 Total RNA isolation and qRT PCR sequence upstream of the transcription starting point of TS gene. At present, the genotypes mainly have 2 times and 3 ’ According to the manufacturer s instructions of MagMAX times repeats, so the common genotypes are 3R/3R, 2R/3R, ( fi fi ) Kit Thermo sher Scienti c, USA , total RNA was extracted and 2R/2R. G-C SNP refers to the second 28 bp repeats of 3R from lung cancer tissues and paracancerous tissues. RNA allele, in which the 12th nucleotide has a G-Cmutation,so3R concentration detection and RNA quantity control were has two alleles: 3G and 3C. detected by Lambda 950 UV-visible absorption spectro- meter (Perkin-Elmer Enterprise, China). Subsequently, the total RNAs were reverse transcribed into complementary DNA. Using the ProFlex qRT-PCR system (Thermo Fisher 2.6 Statistical analysis Scientific, USA) and 2× SYBR Green PCR Mastermix Kit (Solarbio, China),5μL of cDNA was prepared for PCR reac- All statistical analyses were performed using SPSS 22.0 2 tion. The PCR reaction conditions were 95℃ for 30 s, 95℃ software (IBM, New York, USA). Fisher exact test, χ test, for 5 s (40 cycles),and60℃ for 30 s. Then, it was annealed and Mann–Whitney U test were used to test whether the at 95℃ for 15 s, extension at 60℃ for 60 s, and 95℃ for 15 s. protein was related to clinicopathological parameters. 2−ΔCt was used for calculating the fold changes in expres- Kaplan–Meier method was used to analyze OS and DFS, sion. All experiments were repeated three times. TS, sense: and log-rank test was used to compare the differences. 5′-CAAATCTGAGGGAGCTGAGT-3′, antisense: 5′-CAGATAA A two-tailed P < 0.05 is considered to be a statistically GTGGCAGTACAGA-3′;GAPDH,sense:5′- CTCTGCTCCTCCT significant relationship. GTTCGAC-3′,antisense:5′-ACCAAATCCGTTGACTCCGA-3′.

2.5 The detection of TS gene polymorphism 3 Results by PCR-RELP 3.1 Clinicopathological data Genomic DNA was extracted from the tissues using Takara Genomic DNA extraction kit (Takara Biotechnology, China). A total of 160 patients with NSCLC were eligible to parti- Polymorphisms were detected by Taqman probes designed cipate in this study. As shown in Table 1, 92 men and 68 42  Jin-Yin Chen et al.

Table 1: The relationship between TS protein expression and clinicopathological factors

Clinical factors No. ++/+++ (n = 94) −/+(n = 66) χ2 P-value

Age (years) ＞60 86 51 (31.875) 35 (21.875) 0.023 0.878 ≤60 74 43 (26.875) 31 (19.375) Gender Male 92 53 (33.125) 39 (24.375) 0.116 0.733 Female 68 41 (25.625) 27 (16.875) Diﬀerentiation Well/moderately 62 32 (20.00) 36 (22.50) 6.670 0.010 Poorly 98 62 (38.75) 30 (18.75) TNM Stage I/II 61 30 (18.75) 32 (20.00) 4.486 0.034 III/IV 99 64 (40.00) 34 (21.25) Lymph node metastases Yes 71 49 (30.625) 22 (13.75) 5.549 0.018 No 89 45 (18.13) 44 (27.50) Pathological types Squamous cell carcinoma 60 35 (21.875) 25 (15.625) 0.699 0.705 Adenocarcinoma 66 37 (23.125) 29 (18.125) Adeno-squamous carcinoma 34 22 (13.75) 12 (7.50) Smoking Yes 96 60 (37.50) 36 (22.50) 1.393 0.238 No 64 34 (21.25) 30 (18.75) Tumor size ＞20 mm 109 66 (41.25) 43 (26.875) 0.457 0.499 ≤20 mm 51 28 (17.5) 23 (14.375)

Note: TS protein expression intensity of the immunohistochemical staining samples was scored from − to +++ (−: negative; +: weak; ++: moderate; and +++: strong). women participated in this study. The average age at and age, gender, pathological type, smoking, and tumor diagnosis was 71.34 ± 9.52 years. 38.75% (62/160) were size (P > 0.05). well/moderately and 38.13% (61/160) were TNM stage I/II. Among NSCLC, squamous cell carcinoma accounts for ( ) 37.50% 60/160 , adenocarcinoma accounts for 41.25% 3.3 Distribution of TS gene polymorphism (66/160), and adenosquamous carcinoma accounts for 21.25% (34/160).Overall,60%(96/160) of patients were Three polymorphic genotypes of TS were successfully smoking and 68.13% (109/160) of the patients had tumors detected in all NSCLC patients. Among 160 patients with larger than 20 mm. NSCLC, the frequencies of +6bp/+6bp, +6bp/−6 bp, and −6bp/−6 bp genotypes were 10% (16/160),43.13%(69/160), and 46.88% (75/160), respectively. The frequencies of 3R/ 3.2 Expression of TS in patients with NSCLC 3R, 3R/2R, and 2R/2R genotypes in 160 patients with NSCLC were 58.75% (94/160), 25.00% (40/160),and16.25%(26/160), Immunohistochemical staining results showed the total respectively.Thefrequenciesof2R/3C,2R/3G,3G/3G,3C/3C, positive expression rate of TS protein was 58.75% (94/ and 3G/3C genotypes were 16.88% (27/160),8.13%(13/160), 160). The positive rates of TS protein in squamous cell 30.63% (49/160),10.63%(17/160),and17.50%(28/160), carcinoma, adenocarcinoma and adenosquamous cell respectively, as shown in Table 2. carcinoma were 21.88, 23.13, and 13.75%, respectively. Figure 1a and b showed that the TS protein level of NSCLC tissue was signiﬁcantly higher than that of paracancerous tissues (P < 0.05). A similar pattern was also observed in the qRT-PCR results, as shown in Figure 1c 3.4 The relationship between TS gene (P < 0.05). polymorphism or TS protein expression The relationship between TS protein positive rate and clinicopathological factors and clinicopathological factors was shown in Table 1. The positive rate of TS protein was positively correlated Three polymorphic genotypes of TS were successfully with diﬀerentiation (P = 0.010), TNM stage (P = 0.034), detected in all NSCLC patients. The overexpression rates and lymph node metastases (P = 0.018). However, there of TS protein in NSCLC patients with +6 bp/+6 bp, +6 bp/ was no correlation between the positive rate of TS protein −6 bp, or −6 bp/−6 bp genotypes were 43.75, 52.17, and Thymidylate synthase in NSCLC  43

Figure 1: The expression of TS in NSCLC patients. (a) The expression of TS protein was detected by Western blot analysis. (b) The expression of TS protein was detected by immunohistochemical staining. (c) The expression of TS gene was detected by qRT-PCR.

53.33%, respectively. There was no statistical difference differentiation (P = 0.001), TNM stage (P = 0.001), and between protein expression and +6bp/+6bp,+6bp/−6bp, lymph node metastases (P = 0.042). However, the TS-5′ and −6bp/−6 bp genotype frequency (P = 0.783).Theover- UTR polymorphism has no correlation with age, gender, expression rates of TS protein in NSCLC patients with 3R/3R, pathological types, smoking, and tumor size (P > 0.05). 3R/2R, and 2R/2R genotypes were 79.79, 65.00, and 57.69%, respectively. Genotype frequency was significantly correlated with protein expression (P = 0.039).Asshownin Table 3, the frequency of different genotypes was signifi- 3.5 Prognostic values of TS protein in OS of cantly correlated with protein expression (P < 0.001). NSCLC patients Table 4 showed the relationship between TS-5′UTR polymorphism and clinicopathological factors. The TS-5′ The results in Figure 2a showed that the median DFS of UTR polymorphism was significantly correlated with the TS protein high expression group was 19 months,

Table 2: The distribution of TS gene polymorphism in NSCLC patients

Genotypes 2R/2R 2R/3R 3R/3R Total

2R/3C 2R/3G 3C/3C 3C/3G 3G/3G

+6 bp/+6bp34141316(10.00) +6 bp/−6 bp 3 13 8 25 7 13 69 (43.13) −6 bp/−6 bp 20 10 4 20 9 12 75 (46.88) Total 26 (16.25) 27 (16.88) 13 (8.13) 49 (30.63) 17 (10.63) 28 (17.50) 40 (25.00) 94 (58.75) 44  Jin-Yin Chen et al.

Table 3: The relationship between TS gene polymorphism and TS protein expression

Genotypes No. ++/+++ (n = 94) −/+(n = 66) χ2 P-value

+6 bp/+6bp 16(10.00) 7 (43.75) 9 (56.25) +6 bp/−6bp 69(43.13) 36 (52.17) 33 (47.83) 0.489 0.783 −6 bp/−6bp 75(46.88) 40 (53.33) 35 (46.67) 2R/2R 26 (16.25) 15 (57.69) 11 (42.31) 2R/3R 40 (25.00) 26 (65.00) 14 (35.00) 0.649 0.039 3R/3R 94 (58.75) 75 (79.79) 19 (20.21) 19.619 <0.001

Note: TS protein expression intensity of the immunohistochemical staining samples was scored from − to +++ (−: negative; +: weak; ++: moderate; and +++: strong). which was significantly lower than the TS protein low characteristics. We found that the expression of TS pro- expression group (33 months, P < 0.01). Furthermore, tein and TS gene in lung cancer tissues was significantly the 3-year DFS rate in the TS protein high expression higher than that in paracancerous tissues. Furthermore, group was 13.83% (13/94), which was also significantly TS protein expression and TS-5′UTR polymorphism were lower than the TS protein low expression group (36.36%, significantly correlated with differentiation, TNM stage, 24/66; P < 0.01). and lymph node metastases. We also studied the TS The results in Figure 2b showed that the median OS gene polymorphism and found that the frequency of of the TS protein high expression group was 21 months, −6 bp/−6 bp genotype in NSCLC patients was signifi- which was significantly lower than the TS protein low cantly higher than that of other patients. In addition, expression group (35 months, P < 0.01).Furthermore,the thepositiverateofTSproteinexpressionin3R/3R 3-year OS rate in the TS protein high expression group was patients was significantly higher than that of the other 20.21% (19/94), which was also significantly lower than the patients. We also found a significant correlation between TS protein low expression group (46.97%, 31/66; P < 0.01). the frequency of different genotypes and protein expression. Prognostic evaluation showed that high levels of TS protein predicted shorter DFS and OS and lower 3-year DFS rate and 3-year OS rate. 4 Discussion With the continuous in-depth research on the patho- genesis, treatment sensitivity, and drug resistance of This study explored the expression and genotypes of TS lung cancer, most scholars believe that the underlying gene in NSCLC patients with different clinicopathological cause of the occurrence, development, prognosis, and

Table 4: The relationship between TS-5′UTR polymorphism and clinicopathological factors

Clinical factors No. 3R/3R (n = 94) 2R/3R + 2R/2R (n = 66) χ2 P-value

Age (years) ＞60 86 48 (30) 36 (22.5) 0.188 0.664 ≤60 74 46 (28.75) 30 (18.75) Gender Male 92 52 (32.5) 40 (25) 0.444 0.505 Female 68 42 (26.25) 26 (16.25) Diﬀerentiation Well/moderately 62 26 (16.25) 36 (22.5) 11.809 0.001 Poorly 98 68 (42.5) 30 (18.75) TNM stage I/II 61 26 (16.25) 35 (21.875) 10.580 0.001 III/IV 99 68 (42.5) 31 (19.375) Lymph node metastases Yes 71 48 (30) 23 (14.375) 4.13 0.042 No 89 46 (28.75) 43 (26.875) Pathological types Squamous cell carcinoma 60 31 (19.375) 29 (18.125) 5.822 0.054 Adenocarcinoma 66 42 (26.25) 24 (15) Adeno-squamous carcinoma 34 21 (13.125) 13 (8.125) Smoking Yes 96 55 (34.375) 41 (25.625) 0.211 0.646 No 64 39 (24.375) 25 (15.625) Tumor size >20 mm 109 63 (39.375) 46 (28.75) 0.128 0.721 ≤20 mm 51 31 (19.375) 20 (12.5) Thymidylate synthase in NSCLC  45

Figure 2: The prognostic value of TS gene in NSCLC. (a) Kaplan–Meier curves of disease-free survival (DFS). (b) Kaplan–Meier curves of overall survival (OS). prognosis of lung cancer may be abnormalities in the genotypes cancer patients was significantly higher than structure and function of certain genes [19,20]. The role other genotypes. The 5′UTR polymorphism of TS gene of TS in DNA synthesis and repair has attracted wide- was significantly related to differentiation, TNM stage, spread attention. TS is a key enzyme in folate metabo- and lymph node metastases. The results of in vitro experi- lism. It methylates deoxyuridine to deoxythymidine. It is ments show that the protein expression efficiency of duck the only way to synthesize thymidine in cells and is plasmids containing 3R promoter was 3 to 4 times that of necessary for nucleic acid biosynthesis [21,22]. Therefore, duck plasmids containing 2R promoter [27]. TS gene poly- the level of TS directly affects the biological function of morphisms would further clarify the etiology and drug cells. This study found that the TS protein and TS gene in resistance of lung cancer. Furthermore, these findings lung cancer tissues were significantly higher than those highlight the usefulness of TS gene polymorphisms as a in paracancerous tissues. Yang et al. [23] observed similar low-cost NSCLC screening method [28]. patterns. These results suggest that the high expression Lung cancer is the most common cancer and the of TS is closely related to the development of lung cancer. leading cause of cancer-related deaths worldwide, causing Subsequently, we evaluated the relationship between more than 1.4 million deaths every year [29].Inaddition, TS expression and clinicopathological characteristics. the expression of TS protein was related to the poor prog- Interestingly, the high expression of TS protein is signifi- nosis of gastric cancer, rectal cancer [30],andothers.Sub- cantly correlated with differentiation, TNM stage, and sequently, the value of TS in the prognosis of NSCLC was lymph node metastases, suggesting that high expression discussed. Prognostic evaluation showed that high levels of TS protein promotes the development of NSCLC and of TS protein predicted shorter DFS and OS and lower indicates a poor prognosis. The high expression of TS 3-year DFS rate and 3-year OS rate. Shitara, et al. [31] protein helps to increase the level of thymidine deoxy- also found that there was a significant correlation between nucleoside. Thymidine deoxynucleoside is the sole source low TS protein expression and disease control (DC) to car- of thymidine and is essential for cell proliferation and dif- boplatin/pemetrexed therapy (P = 0.027),longerprogres- ferentiation [24]. Therefore, the high expression of TS pro- sion-free survival (PFS; P = 0.017),andlongerOS(P = tein promotes the development and metastasis of NSCLC. 0.022) in advanced gastric cancer. Therefore, reducing TS There are three main polymorphisms in TS gene: protein levels will help improve the survival time and sur- 3′UTR polymorphism, 5′-UTR 28 bp repeat polymorphism, vival rate of NSCLC patients. and G-C SNP. The current research on TS mainly focuses In summary, we discussed the expression of TS gene on tumors of the digestive tract and reproductive system in NSCLC patients with different clinicopathological char- [25,26]. However, the distribution of TS polymorphism in acteristics. We found that the expression of TS in NSCLC NSCLC is unclear. In this study, we found that the fre- was significantly increased, and the expression of TS pro- quency of −6 bp/−6 bp genotype in patients with NSCLC, tein and 5′UTR polymorphism of TS gene were signifi- followed by +6 bp/−6 bp and 3C/3C genotype, was signi- cantly correlated with differentiation, TNM stage, and ficantly higher than other patients. In addition, we found lymph node metastases. Furthermore, the frequency of that the positive rate of TS protein expression in 3R/3R −6 bp/−6 bp genotype was significantly higher than other 46  Jin-Yin Chen et al.

genotypes. The high expression of TS protein also indi- [8] Chen X, Yang Y, Katz S. Early detection of thymidylate synthase cates a poor prognosis. TS Examination may help stratify resistance in non-small cell lung cancer with FLT-PET imaging. ( ) – the subgroups of NSCLC and develop individualized ther- Oncotarget. 2017 Jul;8 47 :82705 13. [9] Chen X, Yang Y, Berger I, Khalid U, Patel A, Cai J, et al. Early apeutic regimens. detection of pemetrexed-induced inhibition of thymidylate synthase in non-small cell lung cancer with FLT-PET imaging. Funding information: Authors state no funding involved. Oncotarget. 2017 Apr;8(15):24213–23. [10] Siddiqui A, Vazakidou ME, Schwab A, Napoli F, Fernandez- Conflict of interest: Authors state no conflict of interest. Molina C, Rapa I, et al. Thymidylate synthase is functionally associated with ZEB1 and contributes to the epithelial-to- mesenchymal transition of cancer cells. J Pathol. 2017 Data availability statement: The data that support the Jun;242(2):221–33. findings of this study are available from Zhuji Affiliated [11] Siddiqui A, Gollavilli PN, Schwab A, Vazakidou ME, Ersan PG, Hospital of Wenzhou Medical University, but restric- Ramakrishnan M, et al. Thymidylate synthase maintains the tions apply to the availability of these data, which de-differentiated state of triple negative breast cancers. Cell ff ( ) – were used under license for the current study, and so Death Di er. 2019 Nov;26 11 :2223 36. [12] Li B, Xu WW, Guan XY, Qin YR, Law S, Lee NP, et al. Competitive are not publicly available. Data are, however, available binding between Id1 and E2F1 to Cdc20 regulates E2F1 degra- - from the authors upon reasonable request and with per dation and thymidylate synthase expression to promote eso- mission of Zhuji Affiliated Hospital of Wenzhou Medical phageal cancer chemoresistance. Clin Cancer Res. 2016 University. Mar;22(5):1243–55. [13] Sasako M, Terashima M, Ichikawa W, Ochiai A, Kitada K, Kurahashi I, et al. Impact of the expression of thymidylate synthase and dihydropyrimidine dehydrogenase genes on survival in stage II/III gastric cancer. Gastric Cancer. 2015 Jul;18(3):538–48. References [14] Shen R, Liu H, Wen J, Liu Z, Wang LE, Wang Q, et al. Genetic polymorphisms in the microRNA binding-sites of the thymi- [1] Herbst RS, Morgensztern D, Boshoff C. The biology and man- dylate synthase gene predict risk and survival in gastric agement of non-small cell lung cancer. Nature. 2018 cancer. Mol Carcinog. 2015 Sep;54(9):880–8. Jan;553(7689):446–54. [15] SuX,LiS,ZhangH,XiaoH,ChenC,WangG.Thymidylatesynthase [2] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA gene polymorphism predicts diseasefreesurvivalinstageII–III Cancer J Clin. 2019 Jan;69(1):7–34. rectal adenocarcinoma patients receiving adjuvant 5-FU-based [3] Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. chemotherapy. Chin Clin Oncol. 2019Jun;8(3):28. Global cancer statistics 2018: GLOBOCAN estimates of inci- [16] Wang X, Wang Y, Wang Y, Cheng J, Wang Y, Ha M. Association dence and mortality worldwide for 36 cancers in 185 countries. of thymidylate synthase gene 3′-untranslated region poly- CA Cancer J Clin. 2018 Nov;68(6):394–424. Erratum in: CA morphism with sensitivity of non-small cell lung cancer to Cancer J Clin. 2020 Apr;70(4):313. pemetrexed treatment: TS gene polymorphism and peme- [4] Levy A, Faivre-Finn C, Hasan B, De Maio E, Berghoff AS, trexed sensitivity in NSCLC. J Biomed Sci. 2013 Jan;20(1):5. Girard N, et al. Diversity of brain metastases screening and [17] Hur H, Kang J, Kim NK, Min BS, Lee KY, Shin SJ, et al. management in non-small cell lung cancer in Europe: results Thymidylate synthase gene polymorphism affects the of the European Organisation for Research and Treatment of response to preoperative 5-fluorouracil chemoradiation Cancer Lung Cancer Group survey. Eur J Cancer. 2018 therapy in patients with rectal cancer. Int J Radiat Oncol Biol Apr;93:37–46. Phys. 2011 Nov;81(3):669–76. [5] Iyengar P, Wardak Z, Gerber DE, Tumati V, Ahn C, Hughes RS, [18] Andrade-Santos JL, Carvalho-Silva W, Coelho A, Souto FO, et al. Consolidative radiotherapy for limited metastatic non– Crovella S, Brandão L, et al. IL18 gene polymorphism and its small-cell lung cancer: a phase 2 randomized clinical trial. influence on CD4 + T-cell recovery in HIV-positive patients JAMA Oncol. 2018 Jan;4(1):e173501F. receiving antiretroviral therapy. Infection, genetics and evo- [6] Lee S, Park YH, Kim KH, Cho EY, Ahn YC, Kim K, et al. Thymidine lution: journal of molecular epidemiology and evolutionary synthase, thymidine phosphorylase, and excision repair genetics in infectious diseases. J Clin Pathol. 2019;75:103997. cross-complementation group 1 expression as predictive [19] Rafnar T, Sigurjonsdottir GR, Stacey SN, Halldorsson G, markers of capecitabine plus cisplatin chemotherapy as first- Sulem P, Pardo LM, et al. Association of BRCA2 K3326* with line treatment for patients with advanced oesophageal squa- small cell lung cancer and squamous cell cancer of the skin. mous cell carcinoma. Br J Cancer. 2010 Sep;103(6):845–51. J Natl Cancer Inst. 2018 Sep;110(9):967–74. [7] Kim MJ, Lee JS, Park SE, Yi HJ, Jeong IG, Kang JS, et al. [20] Aisner DL, Sholl LM, Berry LD, Rossi MR, Chen H, Fujimoto J, Combination treatment of renal cell carcinoma with belinostat et al. The impact of smoking and TP53 mutations in lung and 5-fluorouracil: a role for oxidative stress induced DNA adenocarcinoma patients with targetable mutations-the lung damage and HSP90 regulated thymidine synthase. J Urol. 2015 cancer mutation consortium (LCMC2). Clin Cancer Res. 2018 May;193(5):1660–8. Mar;24(5):1038–47. Thymidylate synthase in NSCLC  47

[21] Djaout K, Singh V, Boum Y, Katawera V, Becker HF, Bush NG, [27] Kawakami K, Salonga D, Park JM, Danenberg KD, Uetake H, et al. Predictive modeling targets thymidylate synthase ThyX in Brabender J, et al. Different lengths of a polymorphic repeat Mycobacterium tuberculosis. Sci Rep. 2016 Jun;6(1):27792. sequence in the thymidylate synthase gene affect transla- [22] Islam Z, Gurevic I, Strutzenberg TS, Ghosh AK, Iqbal T, tional efficiency but not its gene expression. Clin Cancer Res. Kohen A. Bacterial versus human thymidylate synthase: kinetics 2001 Dec;7(12):4096–101. and functionality. PLoS One. 2018 May;13(5):e0196506. [28] Cavic M, Spasic J, Krivokuca A, Boljevic I, Kuburovic M, [23] Yang M, Fan WF, Pu XL, Liu FY, Meng LJ, Wang J. Significance of Radosavljevic D, et al. TP53 and DNA-repair gene polymorph- thymidylate synthase expression for resistance to pemetrexed isms genotyping as a low-cost lung adenocarcinoma screening in pulmonary adenocarcinoma. Oncol Lett. 2014 tool. J Clin Pathol. 2019 Jan;72(1):75–80. Jan;7(1):227–32. [29] Yin D, Lu X, Su J, He X, De W, Yang J, et al. Long noncoding [24] Akman HO, Dorado B, López LC, García-CazorlaA,VilàMR, RNA AFAP1-AS1 predicts a poor prognosis and regulates Tanabe LM, et al. Thymidine kinase 2 (H126N) knockin mice show non-small cell lung cancer cell proliferation by epigenetically the essential role of balanced deoxynucleotide pools for mitochon- repressing p21 expression. Mol Cancer. 2018 May;17(1):92. drial DNA maintenance. Hum Mol Genet. 2008 Aug;17(16):2433–40. [30] Páez D, Paré L, Altés A, Sancho-Poch FJ, Petriz L, Garriga J, [25] Finkelstein Y, Blonquist TM, Vijayanathan V, Stevenson KE, et al. Thymidylate synthase germline polymorphisms in rectal Neuberg DS, Silverman LB, et al. A thymidylate synthase cancer patients treated with neoadjuvant chemoradiotherapy polymorphism is associated with increased risk for bone based on 5-fluorouracil. J Cancer Res Clin Oncol. 2010 toxicity among children treated for acute lymphoblastic leu- Nov;136(11):1681–9. kemia. Pediatr Blood Cancer. 2017 Jul;64(7):e26393. [31] Shitara K, Muro K, Ito S, Sawaki A, Tajika M, Kawai H, et al. [26] Wang B, Walsh SJ, Saif MW. Pancytopenia and severe gastro- Folate intake along with genetic polymorphisms in methylene- intestinal toxicities associated with 5-fluorouracil in a patient tetrahydrofolate reductase and thymidylate synthase in with thymidylate synthase (TYMS) polymorphism. Cureus. patients with advanced gastric cancer. Cancer Epidemiol 2016 Sep;8(9):e798. Biomarkers Prev. 2010 May;19(5):1311–9.