Study On Risk Factors,Serum Biomarkers,and Salivary Microbiota Of Upper Gastrointestinal Cancers

AimsBased on the prospective cohort data of the Nutrition Intervention Trial(NIT)in Linxian,Henan Province,China,we conducted studies on risk factors,serum biomarkers,and salivary microbiota of upper gastrointestinal cancers to provide data support and evidence for the prevention and control of upper gastrointestinal cancers in Linxian.Materials and Methods1.In 1984,29,553 residents aged 40-69 years old were recruited in the trial in Linxian,a high-risk area of esophageal cancer in China,and baseline demographic characteristics,lifestyles,family history,physical examination,comorbidities,tooth loss,oral cavity,mucosal leukoplakia,and other information were collected.The cohort was followed up through March 2016.The outcomes were total upper gastrointestinal cancers,including esophageal squamous cell carcinoma,gastric cardia cancer,gastric non-cardia cancer and liver cancer mortality.Hazards Ratio(HR)and 95%Confidence Interval(CI)of each risk factor was calculated using Cox proportional regression model and Cox time-dependent regression model.The restricted cubic spline graph was drawn to depict the dose-response curve and Kaplan-Meier survival curve was used to describe the survival of upper gastrointestinal cancers.2.We performed two nested case-control studies in the NIT population.We quantitated serum androgens,estrogens,and SHBG from 328 non-cardia gastric cancer men and matched controls by age at the start of pills,date of blood collection and trials in sex hormones study.Besides,baseline serum ferritin was measured for 226 incident primary liver cancer cases,281 chronic liver disease deaths diagnosed,and 1,061 matched controls.We used multivariable logistic regression models to calculate odds ratio(OR)and 95%CI.We explored interactions with body mass index(BMI),age,smoking,alcohol drinking,and follow-up time in sex hormones study.Besides,subgroup analysis and interaction tests were performed by age,gender,alcohol drinking,Hepatitis B Virus seropositivity(HBV+)/Hepatitis C Virus seropositivity(HCV+)and trials in ferritin study.3.The pedigree inclusion criteria are at least two or more patients with esophageal squamous cell carcinoma or gastric cardia cancer in three consecutive generations,or a family with two esophageal squamous cell carcinoma or gastric cardia cancer patients of the same generation.Total genome DNA from samples was extracted,and the V4 region of the 16S ribosomal DNA gene was amplified by PCR,and sequencing library was sequenced on an Illumina NovaSeq platform.Sequences with?97%similarity were assigned to the same OTUs.The Wilcoxon rank-sum test was used to test Alpha diversity differences,and permutational multivariate analysis of variance was used to test the differences in Beta diversity.Results1.Through March 2016,there were 2,603 deaths from esophageal squamous cell carcinoma,1,410 deaths from gastric cardia cancer,560 deaths from gastric non-cardia cancer,and 341 deaths from liver cancer.In general,middle-aged,male,family history of upper gastrointestinal cancer,and oral mucosal leukoplakia were risk factors for upper gastrointestinal cancers mortality,and the effect of age gradually increased with time.Specifically,middle-aged and elderly people,family history of upper gastrointestinal cancer,smoking,tooth loss,oral leukoplakia increased the risk of esophageal squamous cell carcinoma mortality;education,overweight and obesity,drinking,and fresh fruit consumption reduced the risk of esophageal squamous cell carcinoma mortality.Middle-aged and elderly people,family history of upper gastrointestinal cancer,and tooth loss were risk factors for gastric cardia cancer mortality.Higher education level,eating fresh fruits more than once a week,eating red meat more than once a week,and monthly egg consumption were protective factors for gastric cardia cancer mortality.Middle-aged and elderly,male,tooth loss were risk factors for gastric non-cardia cancer mortality,and attended middle school or more was found to be protective factors for death from gastric non-cardia cancer.Middle-aged and elderly people,men,and family history of cancer were risk factors for liver cancer mortality.2.Highest quartile of SHBG at baseline serum was associated with 87%increased incidence of gastric cancer(OR=1.87,95%CI=1.01,3.44),particularly if combined with lower BMI.Our study found a novel association suggesting that higher serum concentrations of SHBG may be associated with the risk of gastric cancer for males.3.Participants with serum ferritin in the highest quartile,as compared to those in the lowest quartile,had an increased risk of CLD mortality(OR=1.72,95%CI=1.12,2.64;P-trend<0.01).Moreover,the association with higher serum ferritin was stronger among alcohol drinkers and those who were HCV+(P-interaction<0.05).For incident liver cancer,risk estimates were above one but were not statistically significant.4.There were 45 sequenced saliva samples from 6 qualified high-risk families,including 9 cases of esophageal cancer,7 cases of gastric cardia cancer,and 29 cases of healthy controls in the family.The average relative abundance of Prevotella of the Bacteroides phylum in the overall case group was significantly reduced lower than that of the overall control group.In esophageal cancer family,the average relative abundance of esophageal cancer patients from Rothia of Actinobacteriota,Fusobacterium and Solobacterium of Fusobacteriota was significantly lower than that of the control group.In gastric cardia cancer family,patients with gastric cardia cancer have higher average relative abundances of Burkholderia-Caballeronia-Paraburkholderia of Proteobacteria and Peptococcus of Firmicutes than healthy controls.Conclusion1.We found that the elderly,male,family history,oral leukoplakia were risk factors for the mortality of upper gastrointestinal cancer,and higher education level was a protective factor for the mortality of upper gastrointestinal cancers.The risk factors for gastrointestinal tumors should be verified in cancer prevention and control practices to provide evidence for improving anti-cancer strategies in high-risk areas.2.Our research found that higher concentration of SHBG was associated with increased risk of gastric cancer and there were pieces of evidences for associations of sex steroid hormones in men with lower BMIs.Higher levels of serum ferritin at baseline were associated with subsequent mortality from chronic liver disease,particularly if combined with alcohol drinking or viral hepatitis.Further work is warranted to confirm our findings.3.The saliva microbiome research of high-risk families showed that the lower abundance of Rothia,Fusobacterium,and Solobacterium were seen in case group in esophageal cancer family.The abundance of Burkholderia-Caballeronia-Paraburkholderia and Peptococcus in case group were significantly higher in gastric cardia cancer family,which is novel as similar research has not been reported.As for the total case group,the abundance of Prevotella was lower than that of total control group.The correlation between microbiota and gastrointestinal tumors is worthy of in-depth study which provides new ideas for cancer prevention and control.