| Objective: Esophageal cancer and gastric cardiac cancerare prevalent tumours in China,especially in high incidenceareas. Both tumor types are characterized by a particularly poorprognosis because most patients are currently diagnosed atadvanced stages of the disease. Detection of esophageal cancerand gastric cardiac cancer at earlier stages proably gives the bestchance to improve prognosis. Determination of individuals withhigh risk toward esophageal or cardiac cancer provides apromising approach to achieve this goal. The development ofboth tumor types was found to be associated with environmentalfactors including the presence of nitrosocompounds in theenvironment and the consumed foods, physical damage causedby ingesting hard foods or hot liquids, deficiency of nutrients,and the consumption of alcohol and tobacco. However, only asubset of individuals exposed to the above listed exogenous riskfactors will develop esophageal or cardiac cancer, suggesting arole of endogenous host susceptibility factors in the cancerdevelopment.Thymidylate synthase (TS) is a key enzyme in DNAsynthesis. It catalyzes the conversion of deoxyuridinemonophosphate (dUMP) to deoxythymidine monophosphate(dTMP), playing an important role in DNA synthesis and repair.TS is also a target enzyme for major chemotherapeutic drugs.Polymorphisms in the untranslated regions (UTRs) of thethymidylate synthase (TS) gene, which may modulate TStranscription and expression, have been described. It isimportant to know whether TS polymorphisms resulting inalteration of TS expression may affect risk of the occurrence andprogression of ESCC and GCAThe aim of this study is to explore the influence of thethree TS polymorphisms, the 28-bp tandem repeatpolymorphism and the G/C single nucleotide polymorphism(SNP) in the 5' UTR, the 6bp deletion polymorphism in the 3'UTR, on the development and lymphatic metastases of ESCCand GCA.Methods: This hospital-based case-control study included465 cancer patients (232 with ESCC and 233 with GCA) and348 healthy controls. Genomic DNA was extracted by usingproteinase K digestion followed by a salting out procedure. TSpolymorphisms were analyzed by polymerase chain reaction(PCR)-fragment length analysis and PCR-restriction fragmentlength polymorphism analysis (RFLP) , respectively. DNAsequencing analysis was used to confirm the results of TSgenotyping at the 5'UTR tandem repeat locus in a subset of 12representative samples.Tumor cells and corresponding normalepithelium cells from 66 tumor tissues were separated bymicrodissection under the light microscope and DNAs wereprepared from dissected tissues by proteinase K digestionmethod for genotype stability analysis. TS expression wasdetected by immunohistochemistry in 51 tissues of esophagealsquamous cell carcinoma.Statistical analysis was performed using SPSS12.0 softwarepackage.P<0.05 was considered significant for all statisticalanalyses. Hardy-Weinberg analysis was performed to comparethe observed and expected genotype frequencies usingChi-square test. Comparison of the TS genotype, allelotype andhaplotype distribution in the study groups was performed bymeans of two-sided contingency tables using Chi-square test.The TS haplotype frequencies and linkage disequilibriumcoefficient were estimated by using EH linkage software and2LD software. The odds ratio (OR) and 95% confidence Interval(CI) were calculated using an unconditional logistic regressionmodel and adjusted by age and sex accordingly.Results:1 The frequency of positive family history of upper gastrointestinal cancer (UGIC) in ESCC (31.0%) and GCA (34.3%) patients was significantly higher than that in healthy controls (4.2%) (χ2=39.01 and 46.00, P<0.0001). The proportion of smokers in ESCC and GCA patients (56.9% and 56.7% respectively) was not significantly different from that in healthy controls(48.6%) (χ2=3.53 and 3.33, P=0.06 and 0.07 , respectively).2 The distribution of the three TS variants did not significantly deviate from those expected from Hardy-Weinberg equilibrium in ESCC,GCA patients and healthy controls. The frequency of the 5'UTR 3G/3G,3G/3C,3C/3C,2R/3G,2R/3C,2R/2R and other genotypes in healthy controls was 17.5%, 17.3%, 29.3%,12.9%,17.8%,3.7% and 1.5%,in ESCC patients was 16.0%,16.0%,29.3%,13.8%, 17.6%,4.3% and 3.0%, in GCA patients was 18.5%, 18.9%,26.2%,12.4%,20.2%,3.4% and 0.4%. The frequency of the 3G,3C,2R and other alleles in healthy controls was 32.8%,47.0%,19.5% and 0.7%,in ESCC patients was 31.2%,46.8%,20.5% and 1.5%, in GCA patients was 34.1%,45.9%,19.8% and 0.2%。The overall genotype and allelotype distributions of the TS 5'UTR and 3'UTR polymorphisms in ESCC and GCA patients were not significantly different from those in healthy controls (P > 0.05).3 The linkage analysis performed with the EH program suggested that the TS 5'UTR and 3'UTR polymorphisms were imperfectly in linkage disequilibrium in Chinese population (D'= 0.26, χ~2 = 37.35, P < 0.0001). The 6bp- allele tends to be linked to the 3R allele (3G or 3C).4 Using the 6bp-/3G haplotype as the reference, the frequency of the 6bp-/2R haplotype in GCA patients was significantly less than that in healthy controls (4.9% versus 9.2%; OR=0.48, 95%CI=0.28-0.81). A marginal difference in the frequency of the 6bp-/2R haplotype between ESCC patients (6.0%) and healthy controls was also observed (OR=0.61, 95%CI=0.37-1.00). In addition, the 6bp+/3G haplotype was significantly less common in ESCC patients than in healthy controls (1.8% versus 5.4%, OR=0.30, 95%CI=0.14-0.67).5 The frequency of the 2R/3G genotype in ESCC patients with lymphatic metastasis was significantly higher than that in lymph node negative ones (27.1% versus 4.9%; χ2 = 13.94, P < 0.001). In contrast, the genotype distribution of the TS 3'UTR polymorphism was not significantly different between subgroups of ESCC patients with and without lymphatic metastasis (P > 0.05). Unlike ESCC, neither the TS 5'UTR nor the 3'UTR polymorphism showed significant association with the risk of lymphatic metastasis of GCA. Moreover, the effect of the TS haplotypes on the risk of lymphatic metastasis was not observed in both ESCC and GCA.6 Among 32 pairs of samples showing TS homozygous genotypes (28 cases with 3R/3R and 4 with 2R/2R) in genomic DNA, the genotypes obtained from tumor and normal epithelium cell DNAs were exactly the same asthose found with blood DNA. However, among 28 pairs of informative samples, LOH was found in two ESCC tissues (7.1%) and both lost their 3C allele in tumor cells.7 TS protein expresson was not related to gender ,age , pathological stage and lymphatic metastasis in ESCC patients (P>0.05). The protein expression of the 2R/3R genotype was significantly higher than that of the 3R/3R genotype in ESCC patients (P=0.03).Conclusions:1 Family history of UGIC significantly increases the risk of developing ESCC and GCA.2 The genotype distribution in healthy controls is not correlated to age, gender and smoking status. TS 5'UTR and 3'UTR polymorphisms is imperfectly in linkage disequilibrium in Chinese. The 6bp-allele tends to be linked to the 3R allele (3G or 3C).3 Neither the combined polymorphisms in the TS 5'UTR nor the 6bp deletion polymorphism in the TS 3'UTR is independently associated with susceptibility to ESCC and GCA.4 The combined effects of the TS polymorphisms on risk of the development of ESCC and GCA was observed. The 6bp-/2R haplotype significantly reduce the risk of developing GCA and ESCC. Additionally, individuals... |
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