| BackgroundThe burden of esophageal cancer(EC)in China is high,with more than 50%of EC cases in the world.The current screening program of EC based on endoscopy initialed in population has obvious prevention effects and is mainly suitable for directly conducting endoscopy screening in the population in high incidence areas.The main problem of current program is the screening efficiency and effectiveness are low,which leading it is difficult to quickly apply it to the general population on a large scale due to the lack of molecular biological indicators for the concentration of high-risk group and the prediction of high-risk individuals.The genetic and epigenetic inactivation of the tumor suppressor gene CNKN2A/P16(P16)is an early event in the development of EC.The frequency of copy deletion of P16 in EC tissues was as high as 60%.At present,whether the variation of P16 genetic and epigenetic inactivation during the occurrence of EC,the consistency of different sampling methods,and its relationship with environmental risk factor exposure can be used for EC screening and precancerous Lesions prediction are lack of study.ObjectivesBased on the resources of the EC screening cohort in high incidence of China,this study aimed to analyze P16 methylation and copy number deletion(CND)in EC tissues and evaluate the effect of intra-tumoral heterogeneity(ITH)in EC on P16 methylation,further to analyze the occurrence of P16 methylation and CND in endoscopic biopsy and exfoliated cells at different stage in the process of EC and its feasibility for EC early screening,and to explore the acceptance of esophageal sponge sampling method and the efficiency of exfoliated cell sampling to provide evidence for endoscopic diagnosis and treatment of EC and optimization of EC screening program in China.Materials and MethodsInpatients were Enrolled in the oncology hospitals at high incidence area of EC,collected the cancer and adjacent tissues of EC to detect P16 methylation and CND.A systematic sampling method was independently designed to collect tumor biopsy,corresponding cut tissue and adjacent biopsy of EC surgical tissue,then pathological diagnosis and P16 methylation were performed on all tissues to evaluate the effect of ITH on pathological diagnosis and P16 methylation quantitatively.Based on the upper gastrointestinal cancer screening population cohort in Linzhou,Henan province,biopsies and exfoliated cells of EC and precancerous lesions of different grades were obtained by endoscopy,then P16 methylation and CND were detected,respectively.Compared the positive rate of P16 methylation of different grades lesion and sample types and analyzed the consistency.Taking pathological diagnosis as the gold standard and combined with the information of environmental exposure risk factors,a predictive model was established to evaluate the efficacy of P16 methylation in the primary screening of EC and its precancerous lesions.Based on the EC screening cohort,esophageal sponge ball was used to collect esophageal exfoliated cells and the acceptance of esophageal sponge examination was investigated subsequently,then endoscopic examination was carried out to objectively and synchronously evaluate the influence of sponge ball sampling on esophageal mucosa.DNA of exfoliated cells collected by sponge balls was extracted.The acceptance and the sampling effectiveness of esophageal sponge ball examination were evaluated.P16 methylation and CND were detected by fluorescence PCR methods,MethyLight and P16Light,respectively.Results Determination:take CoL2A1as the internal reference gene(Ct<29.3),he sample was considered to be positive for P16 methylation,when 2 Ct values of the 3 parallel tests were<40 at least;the P16 relative copy numbers of the target specimen(cancer,precancerous tissue)and the reference specimen(paracancer tissue,white blood cell)were calculated,if the proportion of P16 relative copy number of target specimen is lower than 20%compared to the reference specimen(p<0.05),it is CND positive.DNA concentration and ?-ACT gene expression levels was measured by ultraviolet spectrophotometer and fluorescence quantitative PCR.SPSS 26.0 software was used to collate and analysis data,the significance of a was 0.05.Ct value of P16 methylation in cancer and adjacent tissues from same subject was compared by paired sample T test.Chi-square test was used to compare the difference and determine the trend of positive rate of P16 methylation and CND among subgroups.In the multivariate Logistic regression analysis,1)p<0.05;2)factors with p<0.25,OR>1.7 or OR<0.5 entered the regression model.Sensitivity,specificity,AUC were used to describe the diagnostic accuracy of indicators for esophageal lesions.Results 1.The positive rate of P16 methylation and CND in the cancer tissues collected by conventional sampling method was 63.0%(51/81)and 66.7%(54/81),respectively.P16 methylation might be correlated to CND(p<0.01)with a consistency of 54.3%.The positive rate of the combination of P16 methylation and CND was 88.8%,suggesting that there might be a complementary between the two biomarkers.2.The consistent rate of pathological diagnosis of the paired tumor tissue and tumor biopsy was 65.0%;and the positive rate of P16 methylation was 85.0%,68.9%,with a consistent rate of 55%.The abnormal rate of pathologic diagnosis and the positive rate of P16 methylation in para-cancer tissues were 15.7%and 54.9%,which decreased with the increasing of distance from tumor edge.3.The overall positive rate of P16 methylation and CND in endoscopic biopsies in EC and its precancerous lesions was 20.9%and 37.2%,respectively.P16 methylation was positively correlated to subjects' age and lesion grade,while the positive rate of CND was only significantly correlated to lesion grade.The accuracy of P16 CND in the diagnosis of all grades lesion was higher than P16 methylation,and the accuracy of combined two biomarkers was higher,which reached the maximum accuracy in LGIN and above lesions,with a sensitivity of 58.8%,specificity of 95.2%and AUC of 0.72(0.61-0.83).4.The overall positive rate of P16 methylation of esophageal exfoliated cells was 18.1%(19/105).The positive rate of P16 methylation in normal/esophagitis,LGIN,HGIN and cancer was 4.8%(3/62),25.0%(3/12),37.5%(3/8)and 43.5%(10/23),respectively,gradually increased with the increasing of the lesion grade.P16 methylation detection on esophageal exfoliated cells alone showed the highest accuracy for HGIN and above lesions,with a sensitivity and specificity of 41.9%and 91.9%,respectively,and AUC of 0.67(95%CI:0.55-0.79).The prediction accuracy of environmental exposure multivariate Logistic regression model combined with P16 methylation for esophageal lesions was significantly higher than that of the simple population risk factors model,and yielded a highest accuracy for LGIN and above lesions,the sensitivity and specificity was 86.1%and 87.1%,respectively,and the AUC was 0.93(95%CI:0.88-0.98).5.The positive rate of P16 methylation of exfoliated cells obtained by endoscopy in LGIN and above lesions was 37.2%(16/43),significantly higher than that in endoscopic biopsies specimens(20.9%).Based on the pathological diagnosis,the positive rates of P16 methylation in EC(surgery/biopsy tissue),HGIN,LGIN,andnormal/esophagitis were 62.6%(72/115)and 44.8%(13/29),34.3%(12/35)and 15.0%(25/167),indicating a positive correlation trend with the lesion grade(p<0.01).6.80.0%subjects reported that they have not suffered unbearable pain and anxiety and 50.0%of subjects experienced a sense of suffocation or vomiting during the esophageal sponge examination.The effect of sponge ball examination on the mucosa was mild,which was "superficial mucosal abrasion without bleeding" dominantly.Esophageal sponge balls could effectively obtain esophageal exfoliated cells,and the average DNA concentration was 146.4 ng/uL and the average Ct values of ?-ACT were 26.67 and 27.81,respectively,when the original DNA was 25-fold and 50-fold diluted.Conclusions1.P16 methylation and CND were both significantly prevalent in EC tissues,suggesting a promising prospect in the early diagnosis and screening of EC.2.ITH of EC significantly affected the detection results of P16 methylation in tumor tissues.The central tissue of EC was more representative in pathological diagnosis and P16 methylation detection,and the P16 methylation status of para-cancer tissue was closely related to its distance from the tumor.3.The positive rate of P16 methylation and CND correlated to the grade of esophageal lesions.Detection of P16 methylation in esophageal exfoliated cells could effectively detect EC and precancerous lesions,and the prediction model combined with P16 methylation shown potential value in EC screening.4.Esophageal sponge ball examination could effectively obtain esophageal exfoliated cells,meanwhile had good population acceptance,suggesting it is expected to be used in the primary screening of EC... |
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