| Objectives:Based on the endoscopic screening program in high-risk areas of upper gastrointestinal cancer in China,the present study assessed and quantified the long-term risk of gastric cardia precancerous lesions,evaluated the diagnostic value of"serological biopsies" indicators such as serum pepsinogen(PG),gastrin and Helicobacter pylori(H.pylori)for gastric cardia adenocarcinoma(GCA)and precancerous lesions,and explored novel blood plasma protein biomarkers for GCA by proteomic techniques based on mass spectrometry,in order to provide reference data and new ideas for the optimization of endoscopic screening scheme for GCA in China.Materials and Methods:1.A Prospective cohort study on long-term gastric cardia adenocarcinoma risk of precancerous lesions in a high incidence area of upper gastrointestinal cancer in China.This part was a prospective cohort study based on asymptomatic healthy population.Based on the screening cohort in Linzhou,Henan Province,a high incidence area of upper gastrointestinal cancer in China from 2005 to 2009,the population aged 40-69 years who participated in endoscopic screening and had definite pathological diagnosis was selected as the target population,and the general demographic information,epidemiological exposure information and endoscopic pathological diagnosis of the target population were collected.According to the baseline pathological diagnosis,the target population was divided into Normal,non-atrophic carditis(NAC),atrophic carditis(AC),cardia low-grade dysplasia(CLGD),and cardia high-grade dysplasia(CHGD).The incidence and mortality of GCA in the target population were investigated by active follow-up and passive follow-up.The deadline for follow-up was on December 31st,2017.The standardized incidence and mortality of GCA in the cohort population were expressed by person-year.The incidence of GCA during followup was divided by the total number observed at baseline to calculate the 1-year,3-year,5-year and the total cumulative incidence of GCA.Log-Rank method was used to compare the differences in the risk of morbidity/mortality among different groups.The COX proportional hazards model was used to estimate the hazard ratio(HR)and 95%confidence interval(CI)of baseline mucosal lesion status to cardia cancer incidence/death,and subgroup analyses were performed by sex and age group.2.The diagnostic value of serological tests related to gastric function for GCA and precancerous lesions.This part was a multicenter,population-based case-control study.The subjects were from the general population who participated in the esophageal cancer-specific disease cohort study in Linzhou,Henan Province and Feicheng,Shandong Province,from 2017 to 2019.Subjects with baseline pathological diagnosis of early GCA,CHGD and CLGD were collected from the cohort.With early GCA patients as reference,the healthy population in the cohort was selected as the general control group according to age and sex according to the 1:2 matching principle.The serum levels of PG Ⅰ/Ⅱ and gastrin were quantitatively detected by enzyme-linked immunosorbent assay(ELISA),and the serum antibodies of H.pylori were typed by immunoblotting.To describe the distribution of PGⅠ/Ⅱ,gastrin and H.pylori antibodies typing among each group,and analyze the value of the above-mentioned serum indexes of gastric function in the diagnosis of cardia cancer and precancerous lesions in combination with Logistic regression model and ROC curve.3.Plasma biomarkers in GCA by mass spectrometry-based proteomics.This part was a case-control study.Plasma samples from patients with pathologically confirmed GCA,CLGD,CHGD and Normal cardia were studied.The extracted plasma proteins were separated by LC-MS,and the mass spectrometric data were collected in DIA mode on the basis of establishing a scanned spectrum library in DDA mode.The overall classification trend of protein expression profiles among the groups was obtained by principal component analysis(PCA)and partial least squares discriminant analysis(PLS-DA).The differential proteins of Normal,GCA,CHGD and CLGD were analyzed by volcano plots.The key modules related to the pathogenesis of GCA were identified by weighted correlation network analysis(WGCNA).Functional GO analysis and KEGG/WikiPathways pathway analysis were performed on the key modules.Random forest(RF)and the least absolute shrinkage and selection operator(LASSO)were used to regress the characteristic proteins in the key modules.In combination with the tissue specificity of the protein,potential protein markers were screened,and a diagnostic Panel was constructed,and its ability to diagnose cardia cancer and precancerous lesions were evaluated by area under the ROC curve AUC.Results:1.Between 2005-2009 and 2017,a total of 9,740 subjects were included in the final cohort follow-up.During the follow-up period,there were 123 patients diagnosed with GCA and 31 died from GCA.During a median follow-up time of 10.0 years,the age-standardized incidence and mortality rates of GCA in the target population were 128.71 and 35.69 per 100,000 person-years,respectively.The cumulative incidences of GCA were 0.57%(27/4728),0.71%(22/3099),1.58%(20/921),3.05%(27/884)and 25%(27/108)in the Normal,AC,CLGD and CHGD groups,respectively.Compared with the Normal group,the cumulative incidence in the AC group showed an increasing trend after the fifth year of follow-up,whereas the CLGD group had a significant difference in the first 3 years of follow-up.The risk of long-term morbidity and mortality of GCA increased with the increase of lesion grade.In addition,the results of subgroup analysis showed that the cumulative incidence of GCA in the 50-69 years age group was 4.4 times higher than that in the 40-49 age group,and the mortality rate was 4.7 times higher than that in the 40-49 age group;the cumulative incidence of GCA in male subjects was 1.8 times higher than that in women,and the mortality rate was 2 times higher than that in women.2.The levels of PGII in CHGD and GCA groups were higher than those in the control group(P<0.05).and the disease-related risk increased with the increase of PGII levels in the CHGD group(P for trend=0.13),GCA group(P for trend<0.05)and the whole disease group(P for trend<0.05).PGR levels were inversely associated with CHGD(OR=2.42,95%CI:1.19-4.92),GCA(OR=2.04,95%CI:1.00-4.15),and total disease(OR=1.89,95%CI:1.08-3.29).The levels of PGI and G-17 in each case group were not significantly different from those in the control group(P>0.05).The infection rates of H.pylori in control group,CLGD group,CHGD group and GCA group were 53.33%,71.67%,71.88%and 71.67%,respectively.H.pylori infection was associated with the occurrence of CLGD(OR=2.23,95%CI:1.11-4.47),CHGD(OR=2.27,95%CI:1.17-4.39),GCA(OR=2.21,95%CI:1.14-4.32),and the trend analysis showed that there was a positive correlation between the degree of H.pylori infection(P for trend<0.05).Type Ⅰ and Type Ⅱ H.pylori infection was closely related to the occurrence of cardia precancerous lesions(OR=2.67,95%CI:1.57-4.54).There were 66.7%of the patients with precancerous lesions in the population could be detected by the combined diagnosis of five gastric mucosal "serological biopsy" indicators.The combined diagnosis had the highest diagnostic effect for CLGD(AUC=0.72,95%CI:0.65-0.79)and the lowest for GCA(AUC=0.64,95%CI:0.56-0.71).After excluding PGI and G-17,the diagnostic effect did not change significantly.3.PC A and PLS-DA analysis showed that there was a significant classification trend in serum proteins among the four groups,and the main source of variation could be explained by group differences;there were some differences in protein expression profiles between any two groups,which provided a priori evidence for the screening of different proteins.WGCNA was used to analyze the correlation between protein expression profile and disease progression.Forty-two proteins were positively correlated with the development of GCA,and positive correlation protein genes were enriched in trace element selenium-related signaling pathway,ubiquitin-proteasome pathway,Staphylococcus aureus infection and androgen receptor signaling pathway;165 protein molecules were inversely associated with GCA development,and protein genes were enriched in myosin regulation,autophagy signaling pathway,focal adhesion signaling pathway,lipid metabolism,viral carcinogenesis,estrogen-related signaling pathway,and glutathione signaling pathway.RF-derived Top200 protein markers were crossed with those associated with GCA pathogenesis in WGCNA analysis,resulting in 67 key protein molecules.Five potential protein biomarkers,such as LY6G6F,CSRP1,PRDX1,GSTP1 and RAB27B,were identified by LASSO regression combined with tissue specificity.Cross-validation results showed that the constructed Panel had a good diagnostic effect on GCA and precancerous lesions,with an AUC of 0.96(95%CI:0.77-1.00).Conclusion:1.Based on the above results,we suggest that patients with CHGD should receive treatment once detected,and those who refuse treatment should be followed up every six months;patients with CLGD should be reexamined within 1-3 years of initial endoscopy;the endoscopic follow-up interval for patients with AC/CIM was extended to 5 years.Our results also suggest that the age of endoscopy initiation in high-incidence areas can be postponed to 50 years from the perspective of screening costs.The results of this study provide the absolute and relative risks of gastric GCA at all levels of precancerous lesions,provide evidence-based medical evidence for the optimization of endoscopic screening programs in high-risk areas of upper gastrointestinal cancer in China,and provide data support for the popularization of endoscopic screening programs in the next step.2.Compared with PGI and G-17,serum PGII and PGR levels could reflect the progression of gastric cardia mucosa.Type Ⅰ H.pylori infection,especially VagA H.pylori infection,was most closely associated with the occurrence of precancerous lesions of the cardia.PGII,PGR combined with serum H.pylori infection has a good prospect in the diagnosis of early GCA and precancerous lesions involving only the cardia,but more research evidence is still needed to support it.3.Muscle loss,selenium levels and sex hormone-related pathways caused by tumor cachexia are associated with the mechanism of GCA progression.The 5 protein markers found in the longitudinal viewpoint of disease onset have significant effects on the diagnosis of GCA and precancerous lesions.The discovery of potential markers by proteomics provides a new idea for identifying high risk group of GCA,but the biomarkers found based on small populations need to be validated in further large-scale,prospective studies before they are applied to population screening. |
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