| BackgroundEsophageal squamous cell carcinoma(ESCC)is one of the most common carcinomas in the world.China has the highest incidence and mortality of ESCC in the world.There are about 250 000 newly diagnosed patients with ESCC every year. Linxian in Henan province and Ci county in Hebei province have an incidence of 100/100 000 and over,which are the areas with the highest incidence in China. Feicheng was found to be a high-incidence area of ESCC in the first malignant tumor death survey in China.Its ESCC mortality rate was 63.19/100 000 in the periods of 1970-1974,and was only inferior to Linzhou.It is harmful to local residents because of its high ESCC risk.Esophageal squamous cell carcinoma is a disease with contributions of multi-factors and multi-genes and its development process can divide into several stages.In China,an immigration epidemiology found that after 100 years of the residents of Linzhou in Henan province moving to Changzhi in Shanxi province,the detection rate of ESCC of the immigrations is similar with that of the residents in Lizhou.It indicated that changes in environment and time can't affect the incidence rate of ESCC,and the genetic factor plays an important role in it.Several studies indicated that the risk factors for ESCC are different among different areas and races.In summary,the influencing factors of ESCC include deficiency of nutrition elements,diet,life habits(such as smoking and drinking), family history of esophageal cancer(EC)etc.Among the people who expose to identical living environment and have similar life behavior,only a few develop ESCC. Most researchers think that this phenomenon is caused by different hereditary susceptibilities to genes related to tumor.Furthermore,with the development of proteomics and gene expression profile,it is found that the function of protease is caused by the expression of gene,and the mutation in gene certain site cause changing of enzyme activity.The mutation in gene certain site may be inherited or caused by carcinogen outside.So it is a hot issue to study the susceptibilities of genes in carcinoma epidemiology including metabolic gene,DNA repair gene,oncogene and anti-oncogene.As to gene susceptibility,it has been reported that gene polymorphisms of MTHFR, CYP1A1,CYP2A6,CYP2E1,GSTM1,GSTT1,hOGG1,XRCC1,XPD and p53 are correlated with EC,but the results are inconsistent.However,there are few studies about the gene susceptibility of precancerous lesions.As to serum protein marker,it has been reported that CEA,CA19-9 and SCCA are associated with prognosis of EC, but there are few studies about the association with precancerous lesions.As to expression of tissue protein at EC site and side of cancer,it has been reported that p53, CK19 and CK20 are associated with carcinogenesis and development of EC,however, there are not any studies about precancerous lesions.As to the association of gene mRNA in peripheral blood and EC,the studies are restricted to EC,however,there are not any studies about precancerous lesions.In conclusion,there are many studies about the biomarker of EC from different aspects,but there are few studies about precancerous lesions.Pathological and epidemiological studies since 1970s suggest that the malignant transformation of human esophageal mucosa is a progressive process which starts from normal epithelium to basal cell hyperplasia,dysplasia or carcinoma in situ,and finally to invasive ESCC.The dysplasia is considered as an important precancerous lesion of EC.Follow-up studies in the sites with high incidence of EC show that mild dysplasia and moderate dysplasia of the esophagus are an active and unstable stage.Most of the lesions are reversible,and only about 3%to 5%of the lesions develop to severe dysplasia.Severe dysplasia is a relative stable stage.The possibility of reverse is rare and it will develop to cancer.Therefore,it means a lot to the prevention of EC that mild dysplasia and moderate dysplasia are discovered early and interfere in these patients and urge them to reverse.Endoscopic staining examination with 1.2%iodine solution method is an effective method to discover esophageal diseases early. However,the accept rate is rare for its trauma and high cost.As a result,the study about the biomarkers of ESCC and dysplasia in peripheral blood is helpful to the discovery of non-invasive screening method and monitoring the pathological changes. Because of the sensitivity of the expression of gene mRNA in peripheral blood is higher than the relative protein in serum,it has become the hot issue to detect the expression of gene mRNA in peripheral blood.Previously,the studies on EC-related factors were carried out using case-control study.Because cancer patients were recruited as case in hospital,these studies unavoidably have bias resulted from representative difference of a population and design difference.The incidence of EC is variant among different areas,so there are discrepant conclusions.Most of the esophagus precancerous lesions are insidious and the cost of endoscopic detection is high,which make it inevitable that few studies use precancerous lesion as indicator cases.If we can pick out cancerous and precancerous patients as subjects from a high-risk population and carry out the study in traditional epidemiology and molecular epidemiology,it would be valuable and practical for exploring the risk factors and biomarkers of EC and precancerous lesions.Although Feicheng is an area with a high incidence of ESCC,no intervention study has been given.We carried out a program of endoscopic staining examination with 1.2%iodine solution method in screening for esophageal lesions in Feicheng,China between January 2004 and December 2006.We obtain the data and biology specimens from subjects.It brings advantageous conditions for exploring the risk factors and biomarkers of esophageal cancer and precancerous lesions.ObjectivesThe purpose of the present study was to prove the risk factors and predisposing genes in the different stages of esophageal squamous cell carcinogenesis,and it can provide theoretical bases to definite the high-risk population and find biomarkers in monitoring the transformation from precancerous lesions of esophagus to malignant lesions.The trend and specificity of ESCC mortality during from 1970 to 2004 in Feicheng were analyzed.The associations of MTHFR,CYP2E1 and hOGG1 with ESCC and other esophageal diseases,and the expression of SCCA2,hTERT and EYA4 in peripheral blood within the different stages of esophageal carcinogenesis were studied by the methods of epidemiology and molecular biology.Methods 1 Study on the trend and specificity of ESCC mortalityThe death data of ESCC including the periods of 1970-1974,1985-1989 and 1990-1992 were collected in the survey of total death cause by health department and the office of treatment and prevention of tumor in Shandong province.The death data of ESCC in the periods of 1997-1999 and 2000-2004 came from the registration of death in Feicheng by Shandong Academy of Medical Science.2 Study on the risk factors for the different stages of esophageal squamous cell carcinogenesis2.1 SubjectsThe subjects in this study were from 40-years old country dwellers and consisted of 260 patients with basal cell hyperplasia,563 patients with esophageal squamous cell dysplasia,84 patients with ESCC,and 7789 controls with normal esophageal squamous epithelium cells.All subjects took part in the screening program,which employed an endoscopic staining examination with 1.2%iodine solution in Feicheng, China between January 2004 and December 2006.Moreover,biopsies were taken from a non-staining area of the mucosa,and the subjects then underwent separate pathologic evaluations carried out by two pathologists.2.2 Questionnaire investigationInvestigation contents were as follows:questionnaire investigation included basic information,such as sex,age,education,profession,family average income per year; family history of ESCC;life habits(such as smoking and drinking habits);and diet, such as staple food,nonstaple food,vegetables and fruits.3 Study on gene polymorphisms in the different stages of esophageal squamous cell carcinogenesis3.1 SubjectsThe subjects and questionnaire investigations were the same as the above.From each subject,4 ml of peripheral vein blood was drawn into degerming cry vials,which contained 0.5 ml anticoagulation reagent.The samples were centrifuged for 10 min at 3000 r/min to separate plasma and obtain blood cells,then nucleated cell was suspended in the buffer of TNE and were stored at -80℃for assay.3.2 Test methodsThe genotypes of MTHFR and CYP2E1 were detected by the polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)assay.The genotypes of hOGG1 were detected by the polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP)assay.4 The expression of SCCA2,hTERT and EYA4 mRNA in peripheral blood within the different stages of esophageal squamous cell carcinogenesis4.1 SubjectsThe subjects and questionnaire investigations were the same as the above.The subjects consisted of 50 patients with basal cell hyperplasia,50 patients with dysplasia,50 patients with ESCC,and 50 controls.From each subject,4 ml of peripheral vein blood was drawn into degerming cry vials,which contained 0.5 ml anticoagulation reagent and was soaked in 0.1%DEPC.Then the samples were stored at -80℃for assay.4.2 Test methodsThe expression of SCCA2,hTERT and EYA4 mRNA in peripheral blood were detected by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR).5 Statistical analysisThe mortality of ESCC in Feicheng was analyzed by negative binomial regression model,and the observation indexes were relative risks(RRs)and 95%confidence intervals(95%CIs).Pearson'sχ~2 test was used to examine the differences in the distribution of sociodemographic characteristics,alcohol use,tobacco use,and family history of esophageal cancer among the cancer,dysplasia,basal cell hyperplasia,and control groups.Multinomial logistic regression was used to analyze the risk factors for the different esophageal lesions,the association between the genes and the different stages of esophageal squamous cell carcinogenesis,the interaction between gene and enviroment factors,and the association between the expression of SCCA2, EYA4,HTERT mRNA and the different esophageal lesions.The observation indexes were odds ratios(ORs)and 95%confidence intervals(95%CIs).In addition,both sensitivity and specificity of SCCA2 were also calculated.Finally,all statistical analyses were performed using version 11.5 of the SPSS software and version 8.0 of the Stata software.Results1 Study on the trend and specificity of ESCC mortalityThe ESCC mortality in male was higher than that in female in the period of 1970-2004 and the ratio of male to female in the different stages was 2.16-2.67 to 1. The higher ratio than Linzhou's showed that the cause of ESCC had specificity.The results of analysis by negative binomial regression model showed that the risk of EC mortality was associated with age and gender.The risk of esophageal cancer mortality increased by 1.67(95%CI:1.56-1.78)times from 40- years old group to every other five years old groups.The risk is 2.38(95%CI:1.78-3.18)times higher in male than that in female.There was no significant association between the risk of esophageal cancer mortality with periods or birth cohorts.2 Study on the risk factors of the different stages of esophageal squamous cell carcinogenesisAfter adjustment of sex,age,education,and family average income per year,the results by multinomial logistic regression showed as follow:(1)The ORs(95%CIs)of alcohol drinking index≥130 were 1.78(1.22-2.59),1.46(1.13-1.90)and 1.44(0.79-2.63)in basal cell hyperplasia group,dysplasia group and ESCC group respectively,which did not reach the significant level in ESCC group.(2)The ORs(95%CIs)of smoking index≥600 were 2.45(1.61-3.73),2.52(1.14-4.44)and 1.16(0.87-1.55)in basal cell hyperplasia group,ESCC group and dysplasia group respectively,which did not reach the significant level in dysplasia group.(3)The ORs(95%CIs)of family history of ESCC in first degree relatives were 1.19(0.81-1.75), 2.11(1.68-2.51)and 2.26(1.29-3.96)in basal cell hyperplasia group,dysplasia group and ESCC group respectively,which did not reach the significant level in basal cell hyperplasia group.(4)With the increase of dietary cellulose intake,the risk of ESCC tended to decrease and reached the significant level.(5)With the increase of vitamin C intake,the risk of ESCC tended to decrease and reached the significant level.(6) With the increase of protein intake,the ORs in three groups tended to decrease,but did not reach the significant level.The risk factors for ESCC reported in 1970s in Linzhou were deficiency of nutritionélements,and neither smoking nor drinking was associated with ESCC.With the improvement of economy,it's reported that drinking becomes a new risk factor for ESCC in Linzhou in the recent years.3 Study on gene polymorphisms in the different stages of esophageal squamous cell carcinogenesis3.1 Association of MTHFR C677T genotypes with the defferent stages of esophageal squamous cell carcinogenesisThe subjects in this assay consisted of 184 patients with basal cell hyperplasia,106 patients with esophageal squamous cell dysplasia,79 patients with ESCC,and 114 controls with normal esophageal squamous epithelium cells.The distributions of the genotypes of MTHFR C677T were statistically different among four groups (χ~2=23.527,P=0.001).When MTHFR C/C genotype was used as the baseline(OR=1.0),C/T and T/T genotypes combined was significantly associated with basal cell hyperplasia and dysplasia,with ORs(95%CIs)being 0.32(0.16-0.63)and 0.25(0.12-0.51)respectively after adjustment for sex,age,culture,and family average income,and OR(95%CI)being 0.82(0.33-2.05)in ESCC group.To analyze the combined effect of genotype with alcohol drinking or smoking,smoking + 'C/T and T/T' was significantly associated with dysplasia(OR=0.20,95%CI:0.07-0.57)and alcohol drinking + 'C/T and T/T' was significantly associated with dysplasia (OR=0.21,95%CI:0.07-0.60).3.2 Association of CYP2E1 genotypes with the defferent stages of esophageal squamous cell carcinogenesisThe subjects in this assay consisted of 131 patients with basal cell hyperplasia,61 patients with esophageal squamous cell dysplasia,27 patients with ESCC,and 44 controls with normal esophageal squamous epithelium cells.The distributions of the genotypes of CYP2E1 were statistically different among four groups(χ~2=4.654, P=0.589).When CYP2E1 C1/C1 genotype was used as the baseline(OR=1.0),C1/C2 and C2/C2 genotype combined was not significantly associated with basal cell hyperplasia,dysplasia or ESCC with ORs(95%CIs)being 1.07(0.50-2.32), 0.79(0.32-1.96)and 0.87(0.29-2.58)respectively.To analyze the combined effect of genotype with alcohol drinking or smoking,there were no interactions in three groups.3.3 Association of hOGG1 genotypes with the defferent stages of esophageal squamous cell carcinogenesisThe subjects in this assay consisted of 138 patients with basal cell hyperplasia,69 patients with esophageal squamous cell dysplasia,34 patients with ESCC,and 48 controls with normal esophageal squamous epithelium cells.The distributions of the genotypes of hOGG1 were not statistically different among four groups(χ~2=6.592, P=0.360).When hOGG1 C/C genotype was used as the baseline(OR=1.0),C/G and G/G genotyne combined was not significantly associated with basal cell hyperplasia, dysplasia or ESCC with ORs(95%CIs)being 1.05(0.53-2.07),1.61(0.72-3.58)and 1.07(0.43-2.68)respectively.To analyze the combined effect of genotype with alcohol drinking or smoking,there were no interactions in three groups.4 The expression of SCCA2,hTERT and EYA4 mRNA in peripheral blood within the different stages of esophageal squamous cell carcinogenesis4.1 Association of the expression of SCCA2 mRNA in peripheral blood within the defferent stages of esophageal squamous cell carcinogenesisBy using the band intensity ratios of SCCA2 toβ-actin,with a positive cut-off value of≥0.4,the positive rates of the SCCA2 mRNA expression in peripheral blood were found to be 82%(41/50),60%(30/50),48%(24/50),and 36%(18/50)in the cancer,dysplasia,basal cell hyperplasia,and control groups,respectively.The positive rate of the cancer group was significantly different from the three other groups(P＜0.05),and there was also a significant difference of the SCCA2 mRNA expression between the dysplasia group and the control group(χ~2=5.769,P=0.016). In the multinomial logistic regression analysis,the OR(95%CI)were 1.71(0.73-3.99) in the basal cell hyperplasia group,2.77(1.14-6.71)in the dysplasia group,and 7.87 (2.88-21.55)in the cancer group after adjustment for age,gender,smoking index, alcohol drinking index,and family history of esophageal cancer.The SCCA2 mRNA expression in peripheral blood was then divided into different grades according to the band intensity ratios of SCCA2 toβ-actin.By using a positive cut-off value of≥0.4, the testing sensitivities in the basal cell hyperplasia,dysplasia,and cancer groups were found to be 48%,60%,and 82%,respectively,with the same testing specificity being 64%.4.2 Association of the expression of hTERT mRNA in peripheral blood within the defferent stages of esophageal squamous cell carcinogenesisBy using the band intensity ratios of hTERT toβ-actin,with a positive cut-off value of≥0.4,the positive rates of the hTERT mRNA expression in peripheral blood were found to be 90%(45/50),84%(42/50),80%(40/50)and 80%(40/50)in the cancer, dysplasia,basal cell hyperplasia,and control groups,respectively.There were no significant differences of the positive rates of the hTERT mRNA expression among the four groups(χ~2=2.432,P=0.488).In the multinomial logistic regression analysis, the OR(95%CI)were 1.36(0.47-3.96)in the basal cell hyperplasia group,1.41 (0.45-4.44)in the dysplasia group,and 2.87(0.78-10.60)in the cancer group after adjustment for age,gender,smoking index,alcohol drinking index,and family history of esophageal cancer.4.3 Association of the expression of EYA4 mRNA in peripheral blood within the defferent stages of esophageal squamous cell carcinogenesisBy using the band intensity ratios of EYA4 toβ-actin,with a positive cut-off value of≥0.4,the positive rates of the EYA4 mRNA expression in peripheral blood were found to be 10%(5/50),4%(2/50),4%(2/50)and 4%(2/50)in the cancer,dysplasia, basal cell hyperplasia,and control groups,respectively.There were no significant differences of the positive rates of the EYA4 mRNA expression among the four groups(χ~2=2.597,P=0.458).In the multinomial logistic regression analysis,the OR (95%CI)were 1.35(0.17-10.80)in the basal cell hyperplasia group,1.29(0.15-11.10) in the dysplasia group,and 4.22(0.63-28.33)in the cancer group after adjustment for age,gender,smoking index,alcohol drinking index,and family history of esophageal cancer.Conclusions1 The mortality rate of ESCC showed consistently increased in early days and a little decreased in the near future in recent thirty years in Feicheng,which probably associated with the stabile life style and(or)genetic factors.The mortality rate for male was higher than that for female and the ratio for male to female was much higher than that of Linzhou,Henan province,thus it showed autospecific in ESCC occurrence in Feicheng.2 The important risk factors for ESCC in Feicheng were first degree relatives having a family history of ESCC,smoking,the deficiency of Vitamin C and dietary cellulose intake;the risk factors for dysplasia were first degree relatives having a family history of ESCC and alcohol drinking;the risk factors for basal cell hyperplasia were smoking and drinking.3 Genotypes of MTHFR 677 CT and 677TT were not associated with ESCC whereas they negatively associated with dysplasia and basal cell hyperplasia.Both genotypes(C/T,T/T)could decrease the risk of dysplasia in those with smoking and alcohol drinking habit.The genotypes of CYP2E1 and hOGG1 had no association with the different stages of esophageal carcinogenesis.4 SCCA2 mRNA expression in peripheral blood was linked with the different stages of esophageal carcinogenesis,and hTERT and EYA4 mRNA expressions were not associated with the different stages of esophageal carcinogenesis.This indicated that SCCA2 may be useful in monitoring the processes of changes that occur in esophageal premalignant lesions among subjects who live in a high-incidence area, but can not provide the evidence for therapy of moderate or severe dysplasia.Innovation and meaning1 In this study,epidemiologic data and biological samples were collected from endoscopic staining examination with 1.2%iodine solution method,combined with pathologic evaluation in screening for esophageal lesions in Feicheng,an area with a high incidence of esophageal cancer,China.People with normal esophageal mucous and with the different stages esophageal squamous cell carcinogenesis lesions were included as subjects.The case-control study was operated including three-case groups and one control group.Multinomial Logistic regression was used to analyze the risk factors,predisposing genes and the expression of mRNA in peripheral blood in the different stages esophageal squamous cell carcinogenesis.This study is more scientific than those case-control studies based on hospital.2 In this study,the specificity of risk factors for ESCC in Feicheng was found by analyzing death surveillance data of ESCC and comparing with the data in Linzhou.It can provide the evidence for prevention of ESCC.3 In this study,several predisposing genes and the expression of gene mRNA in different stages of esophageal carcinogenesis were investigated.This makes it possible to study the biomarkers associated with ESCC and dysplasia further and monitor the transformation of precancerous lesions to malignant direction.Shortcomings and improvementsShortcomings1 We only detect one polymorphism for each gene,making it impossible to analyze the association of haplotype map with disease.2 The samples for genes polymorphisms detection were relatively small and their interactions were not analyzed.Improvements1 Analyze the genes polymorphisms with a relative large sample size and research the association of haplotype map with the different stages of esophageal carcinogenesis.2 Use the biomarkers which were found significant in this study to monitor the status of changes that occur in esophageal precancerous lesions,and go on exploring other biomarkers,thus provide the evidence for therapy of precancerous lesions. |
PDF Full Text Request