Correlation Between Immune-related Gene Polymorphisms And Preoperative Inflammation-related Indicators And Gastric Cancer Susceptibility And Prognosis

Part Ⅰ The susceptibility of SNPs in inflammatory genes to risk of gastric cancerBackground:The Helicobacter pylori(H.pylori)causes sustained host chronic inflammatory response,and it is recognized as a group I carcinogen of gastric cancer.The genetic background of inflammatory genes has an important impact on the inflammatory response,resulting in different gastric disease progression of individuals with H.pylori infection.Interleukin(IL)is the main mediator of inflammatory response and the single nucleotide polymorphisms(SNPs)of these genes are potentially associated with susceptibility to gastric cancer.IL-4 and IL-6 are two key cytokines play regulatory role in the inflammatory response gastric mucosal of host with of H.pylori infection and polymorphisms in these two genes were potentially associated with riks of gastric cancer.Previous studies have suggested that"IL-23-IL-23R-STAT3-Thl7-IL-17/IL-17F "axis(IL-23/IL-17 axis)involved in the inflammatory responses and the polymorphisms in the IL-23/IL-23R and Thl 7 gene families are also regarded as the risk of gastric cancer.Toll-like receptor-4(TLR-4),as a representative of the main pattern recognition receptor(PRR)of the innate immune system,is capable of recognizing invasive pathogens,initiating inflammatory responses that are resistant to microbial invasion,and also plays an anti-tumor effect by stimulating the body to produce Thl-type T cell immune responses through intracellular signal transduction.Transforming growth factor-beta(TGF-P),function as a pluripotent cytokine,play a variety of effects on the inhibition of the early stage of tumor growth,progression and metastasis.Polymorphisms in TLR-4 and TGF-B are potentially associated with susceptibility to gastric cancer.Objective:The aim of this study was to investigate the susceptibility of SNPs in the inflammation genes to the risk of gastric cancer.The interaction between environmental factors and genetic factors and the susceptibility of gastric cancer were also discussed.Methods:A case-control study involved 494 cases of gastric cancer and 494 healthy controls were performed.The clinical characteristics of cases and basic information of all participants were collected.The genotypes of all SNPs for participants were detected by Mass-array platform.The H.pylori infection of all participants was determined by using a commercial H.pylori immunogold testing kit.Results:The results showed that IL-6 rs 180079G allele was associated with the increased risk of gastric cancer(GG vs.CC:adjusted OR = 1.87,95%CI = 1.16-3.03;GG/GC vs,CC:adjusted OR =1.31,95%CI = 1.01-1.68).The stratified analysis showed that the rs1800796GG genotype was associated with the increased risk of gastric cancer in the older age group and the female group,and the group of cases in the later clinical stage(T3-T4).IL6 rs1800796GG and CG/GG genotype were associated with the risk of NCGC.IL17A rs8193036CT(adjusted OR = 1.44,95%CI=1.04-1.99),CT/TT(adjusted OR=1.48,95%CI=1.08-2.02)genotype and IL17A rs3748067CT(adjusted OR = 1.55,.95%CI=1.08-2.22),CT/TT(adjusted OR = 1.60,95%CI=1.13-2.26)genotype was associated with the increased risk of gastric cancer in older groups;the rs2275913AA genotype was associated with increased gastric cancer amonge younger group(adjutstedOR = 2.04,95%CI=1.05-3.98)and the female subgroup(adjusted OR =2.08,95%CI = 1.02-4.23).The IL17F rs9382084GG genotype was associated with a risk of CGC(adjusted OR = 1.83,95%CI=1.02-3.30).IL-23R rs10889677CA and CA/CC genotype were associated with increased risk of gastric cancer among younger group(CA vs.AA:adjusted OR =1.61,95%CI =1.01-2.58;CA/CC vs.AA:adjusted OR= 1.57,95%CI=1.00-2.46,P=0.048)and female group(CC vs.AA:adjusted OR = 1.68,95%CI = 1.06-2.97;CA/CC vs.AA:adjusted OR= 1.68,95%CI = 1.02-2.77),respectively.In addition,the rsl0889677CC genotype was associated with increased risk of gastric cancer in negative H.pylori infection subgroup(adjusted OR = 2.26,95%CI=1.06-4.78).TGF-BR1 rs334348GG genotype was associated with a risk of gastric cancer(adjusted OR = 1.03,95%CI = 1.03-2.12).Stratified analysis revealed that the rs334348GG genotype was associated with a risk of gastric cancer in the younger age group(adjusted OR = 1.98,95%CI=1.02-3.83).Subgroup analysis showed that rs334348GG genotype was associated with gastric cancer with poor differentiation(adjusted OR = 1.71,95%Cl = 1.06-2.75)and T1-T2 stage(adjusted OR-1.71,95%CI=1.02-2.86);rs6478974AA was associated with decreased gastric cancer risk among male group(Adjusted OR = 0.51,95%CI=0.29-0.91).The rs6478974AA(adjusted OR = 0.54,95%CI = 0.33-0.89)and AT/AA(adjusted OR = 0.67,95%CI＝0.48-0.94)genotype was a low risk factor for gastric cancer with poor differentiation.TGF-BR1 rs10512263CC was associated with a decreased risk of gastric cancer(adjusted OR = 0.52,95%CI=0.29-0.93).Subgroup analysis found that the rs 10512263CC genotype was associated with decreased risk of gastric cancer among older age group,male group and gastric cancer with low differentiation.Conclusion:IL16 rs1800796GG genotype is associated with the risk of gastric cancer.The rs1800796G allele carriers(GG/GC)have a higher risk of gastric cancer,especially for individuals with age than 60 and female,and it was associated with gastric cancer with later clinical stage and the type of NCGC.IL17A rs8193036C and rs3748067C alleles are associated with gastric cancer risk for older individuals.IL-23R rs10889677C allele is associated with a decreased risk of gastric cancer for individuals younger than 60 years old and female.TGF-BR1 rs334348GG genotype is associated with the risk of gastric cancer,especially for individuals younger than 60 years old,and the genotype was associated with gastric cancer with poor differentiation and early clinical stage.TGF-BR1 rs6478974AA and rs10512263CC genotypes were associated with decreased risk of gastric cancer for male.Part Ⅱ Study on the relationship between preoperative inflammations related factor and prognosis of gastric cancerBackground:Emerging evidence indicates that inflammation plays a critical role in the initiation and progression of numerous cancers,including gastric cancer.Generally,systemic inflammatory response was the characteristic of changes in the relative levels of circulating white blood cells(WBC),largely resulting from the change of neutrophils and lymphocytes.Those biomarkers in the peripheral blood that can exhibit the status of inflammation are considered as potential predictive markers for cancer prognosis.In recent years,several indicators derived from the peripheral blood such as the neutrophil to lymphocyte ratio(NLR),derived neutrophil to lymphocyte ratio(dNLR),platelet to lymphocyte ratio(PLR)and lymphocyte to monocyte ratio(LMR)have been widely investigated as useful prognostic indicators in various kinds of cancers;However,prognostic role of these value was remain unclear and the optimal cut-off values of the biomarkers from these studies were still inconsistent.Serum bilirubin,which is an end product of heme metabolism,was not thought to serve any physiological function.Nevertheless,recent studies have demonstrated that serum bilirubin has potent antioxidant,anti-inflammatory,and anticancer effects in colorectal cancer.The protective effects of serum bilirubin have been reported in several cancers.Additionally,decreased levels of albumin,a factor commonly used as an indicator of nutritional status,are associated with worse outcomes in gastric cancer patients.Although they might improve prognostic predictions in gastric cancer,no measurements or indices that combine serum bilirubin and albumin levels have been developed.In this study,we examined whether serum bilirubin and albumin levels were predictive of survival outcomes in gastric cancer patients.We then evaluated the predictive value of a nomogram based on serum bilirubin and albumin levels in these patients.Objective:To investigate predictive value of composition of circulating white blood cells(WBC)and the serum bilirubin and albumin level for patients with gastric cancer.Methods:Medical records of all newly diagnosed GC patients between January 2007 and January 2009 were collected and retrospectively analyzed in the present study.At last,389 patients were enrolled in the present study.After operation,each patient was followed up regularly for the overall survival(OS),disease-free survival(DFS),cancer specific survival(CSS).Pre-operative blood cell counts were detected by Sysmex XT-1800i Automated Hematology System for counting neutrophil to lymphocyte ration(NLR),derived neutrophil to lymphocyte ratio(dNLR),platelet to lymphocyte ratio(PLR)and lymphocyte to monocyte ratio(LMR).The concentrations of serum CEA and CA199 were measured using electrochemiluminescence The optimal cut-off levels of NLR,dNLR,PLR,and LMR were determined by receiver operating curve(ROC)analysis based on end-point of CSS,OS or DFS.Chi-square test or Fisher’s exact test was used to compare categorical variables,and Mann-Whitney U or Kruskal-Wallis test was applied to compare continuous variables between groups.Continuous variables were expressed as mean ± SD.Survival rates were calculated by Kaplan-Meier survived analysis and the significance was evaluated by Log-rank test.The predictors for on OS,DFS and CSS determined by univariate analysis were evaluated by multivariate analysis using Cox’s proportional hazards model.Nomograms for DFS and CSS were established by R 3.0.3 software(Institute for Statistics and Mathematics,Vienna,Austria),and the predictive accuracy was evaluated by Harrell’s concordance index(c-index).All statistical analyses were conducted using IBM SPSS 20.0 software(IBM,USA).P values less than 0.05 were considered statistically significant.To investigate predictive value of the serum bilirubin and albumin level for patients with gastric cancer,a total of 778 patients with gastric cancer were divided among two cohorts.The prospective training cohort consisted of 479 patients who underwent surgical resection or chemotherapy and the retrospective validation cohort consisted of 299 patients who underwent surgical resection or chemotherapy at the same institution.Serum albumin levels of all patients were measured using a Roche Modular D/P automated analyzer(Roche,USA).Serum bilirubin levels were determined using the vanadium oxidation method.For the statistical analysis,the optimal cutoff values for total bilirubin(TBIL),direct bilirubin(DBIL),indirect bilirubin(IBIL),and albumin levels were determined using X-tile 3.6.1 software.Survival analysis was performed using the Kaplan-Meier curve and log-rank test.Significant prognostic predictors from the survival analysis were included in a multivariate analysis using the Cox proportional hazards model.The nomogram for significant factors associated with 5-year overall survival(OS)was constructed using R software via a stepwise algorithm,and Harrell’s concordance index(c-index)was used to compare the performance of the nomogram.Results:The ROC curves,using CSS as the end-point for NLR,dNLR,PLR and LMR,introduced the areas under curve(AUC)for NLR,dNLR,PLR and LMR were 0.703(P=0.000),0.683(P=0.000),0.585(P=0.010)and 0.695(P=0.000),respectively.The optimal cut-off levels based on CSS were determined to be 2.36 for NLR,1.85 for dNLR,132 for PLR and 4.95 for LMR by ROC curves analysis.The results revealed that NLR,dNLR,PLR and LMR were significantly correlated with tumor stage,depth of invasion,lymph node,distant metastasis,CEA and CA199(Pall<0.05),separately.Moreover,NLR,dNLR and LMR were closely associated with the age of patients,and PLR had significant correlation with the sex of patients and histological types.Kaplan-Meier survival analysis and log-rank tests revealed that NLR(_>2.36),dNLR(≥1.85),PLR(≥ 132),LMR(<4.95),age of patients(≥ 65 years),tumor stage III-IV,depth of invasion T3-T4,lymph node N1-N3,distant metastasis M1,CEA(>5 ng/ml)and CA199(>37 U/ml)were significantly associated with decreased OS,CSS and DFS,and GC family history of patients was also associated with decreased DFS as well as histological types.In addition,a significant relationship between the inflammatory biomarkers(NLR,dNLR,PLR and LMR)and clinical prognosis(OS,CSS and DFS)was observed.The univariate analysis with Cox regression model revealed that age,tumor stage,lymph node and distant metastasis were significantly associated with reduced OS and CSS(Pall<0.01).More importantly,elevated NLR(HR=1.53,P=0.010)and dNLR(HR=1.42,P=0.012)were also associated with reduced CSS and OS,respectively.Meanwhile,NLR(HR=1.38,P= 0.025),GC family history(HR=2.13,P= 0.007),tumor stage(HR=2.04,P=0.000),distant metastasis(HR=2.06,P=0.000)and CA199(HR=1.33,P= 0.028)were considered to be independent indicators for DFS of GC patients.The prognostic nomograms were depicted by Cox regression model analysis using all the significant independent indicators for CSS and DFS,which can predict the probability of recurrence or death for GC patients within 3 or 5 years after initial surgery with c-index for CSS and DFS prediction were 0.89 and 0.85,respectively.The optimal cutoff values of 5.3 μmol/L for TBIL,7.3μmol/L for DBIL,6.7 μmol/L for IBIL,and 33.2 g/L for albumin based on OS in training cohort patients.The markedly more patients in the high DBIL group had T4 and N3 stage disease compared to the low DBIL group,and T1 stage and NO stage disease were more common in patients in the high IBIL group than in those in the low IBIL group.Patients with the high albumin were younger and more likely to have T1-T2,NO,or M0 stage disease than those in low albumin group.Five-year overall survival rates and univariate log-rank test revealed that 5-year OS was shorter in patients with low TBIL,IBIL,and albumin levels(P<0.01).T,N,and M stages as well as TBIL and albumin levels were independently prognostic factors for OS in both the training and validation cohorts.The Harrell’s c-index values for the nomogram in the training and the validation cohorts were 0.774 and 0.760,respectively,compared to 0.727 and 0.702,respectively,for the TNM staging system.Collectively,the predictive accuracy of the nomogram based on TBIL and albumin levels was better than that of the TNM staging system in both patient cohorts(P<0.01).Conclusion:Pre-operative NLR and dNLR may serve as potential prognostic biomarkers in patients with GC who underwent surgical resection.The proposed nomograms can be used for the prediction of CSS and DFS in patients with GC who have undergone gastrectomy.The serum TBIL and albumin levels are independent predictors of OS in gastric cancer patients,and that an index that combines TBIL and albumin levels with the TNM staging system might have more predictive value than any of these measures alone.