The Study Of The Tumor Infiltrating Lymphocyte And Tertiary Lymphoid Structure In Gastric Carcinoma Microenvironment

The First Part: The Prognostic Value of Tumor Infiltrating Lymphocytes in Patients with Gastric CarcinomaBackground:Gastric cancer is a common malignant tumor in digestive system.The incidence and mortality increase by years.Currently,the mainly treatment is surgical resection and adjuvant chemotherapy.With the deepening research of tumor immunology,tumor immunotherapy become more important.The tumor infiltrating lymphocytes(TIL)which is the main immune cell in the tumor microenvironment(TME)is particularly important role.Notably,the expression of TIL is heterogeneous and relates to the prognosis of patients.The prognostic value of TIL in patients with has not been established.Objective:The aim of this study is to evaluate the correlations between TIL and clinicopathological parameters,and assess the prognostic value of TIL in gastric cancer patients.Methods:H&E staining slides were used to evaluate the TIL expression in 1033 specimens from the patients with gastric cancer in the Third Affiliated Hospital of Soochow University from 2002 to 2008.Chi-squar test,Kaplan-Meier estimates,Cox proportional hazard regression analyses and nomogram model were performed to analyse the influencing factors of the risk of tumor progression on the collected data.Results:H&E staining results showed that,in gastric cancer patients,high level of TIL expression accounted for 51.5%.Tumor size,TNMstage,pathological grade,lymph node metastasis,nerve infiltration and intravascular thrombosis had statistical significance to TIL expression.TIL expression was a possitive predictor.The mean OS of patients with TILhi was 78.73 months,and the mean OS of patients with TILlow expression was 42.81 months(χ2=174.77,P<0.001).TIL had the posstive protective effect in patients form TNM I to III staging(P<0.001).Cox regression analysis revealed that compare with the patients less than 50 years old,the patients beyond 50 years old had higher tumor progression risk(HR=1.897,95% CI: 1.437-2.504,P<0.001);compare with tumor size less than 5cm,the tumor size large than 5cm had higher tumor progression risk(HR=1.224,95% CI: 1.014-1.478,P=0.035);compare with no lymph node metastasis,the patients with lymph node metastasis had higher tumor progression risk(HR=1.509,95% CI: 1.080-2.110,P=0.016);compare with no nerve infiltration,the patients with nerve infiltration had higher tumor progression risk(HR=1.308,95% CI: 1.073-1.595,P=0.008);compare with TNM I staging,the patients with TNM II staging or TNM III staging had higher tumor progression risk(TNM II stage: HR=2.519,95% CI: 1.583-4.009,P<0.001,TNM III stage:HR=4.702,95% CI: 2.769-7.984,P<0.001);compare with high TIL expression,the patients with low TIL expression had higher tumor progression risk(HR=0.464,95% CI: 0.384-0.561,P<0.001).The concordance index(C-index)of the nomogram was 0.77,which was significantly higher than that of TNM stage(0.72)and the expression of TILs(0.65).The nomogram displayed more accuracy for predict the prognosis in the GC patients.Conclusion:There is negative correlation between TIL expression and clinicopathological parameters.TIL expression is a positive predictive factor for the prognosis of patients with gastric cancer in Kaplan-Meier analysis and Cox regression analysis.Compare with TNM stage or TIL respectively,the nomogram model displays more accuracy for predict the prognosis in the GC patients.The second part: The Prognostic Value of Tertiary Lymphoid Structure in Patients with Gastric CarcinomaBackground:Tumor microenvironment(TME)is the internal environment for tumor development,including tumor cell and tumor stroma.Its function status reflects the host immune response against the tumor progression.Tertiary lymphoid structure(TLS)has the organ-like function such as recruits naive T and B cells to the tumor site through chemokines or cytokines in TME.However,the formation of TLS and the relationship between TLS and prognosis of patients remain unclear in the realm of gastric cancer.Objective:The aim of this study is to evaluate the expression of TLS,analysis the relationship between TLS expression and clinicopathological parameters and assess the prognostic value of TLS in gastric cancer patients.Methods:H&E staining slides were reviewed to evaluate the TLS expression in 1033 specimens from the patients with gastric cancer in the Third Affiliated Hospital of Soochow University from 2002 to 2008.Chi-squar test,Kaplan-Meier estimates,Cox proportional hazard regression analyses were performed to analyze the influencing factors of the risk of tumor progression on the collected data.Results:H&E staining results showed that high level of TLS was in 61.1% gastric cancer patients.TLS expression had statistical significance to tumor size and lymph node metastasis(P<0.05).TLS expression was a possitive predictor,the mean OS of patients with TLShi was 62.27 months,and the mean OS of patients with TLSlow was 55.70 months(χ2=11.272,P=0.001).TLS had the posstive protective effect in patients form TNM I to II staging(P=0.045,P =0.003),and TNM III staging patient had non-statistical significant to TLS expression(P=0.165).Cox regression analysis revealed that compare with the patients less than 50 years old,the patients beyond 50 years old had higher tumor progression risk(HR=1.859,95% CI: 1.408-2.454,P<0.001);compare with low-medium grade tumors,the high grade tumor had higher tumor progression risk(HR=1.344,95% CI: 1.092-1.654,P=0.005);compare with no lymph node metastasis,the patients with lymph node metastasis had higher tumor progression risk(HR=1.570,95% CI: 1.127-2.189,P=0.008);compare with no nerve infiltration,the patients with nerve infiltration had higher tumor progression risk(HR=1.415,95% CI: 1.161-1.726,P=0.001);compare with TNM I staging,the patients with TNM II staging or TNM III staging had higher tumor progression risk(TNM II stage: HR=2.158,95% CI: 1.580-4.011,P<0.001,TNM III stage:HR=5.132,95% CI: 3.039-8.666,P<0.001);compare with high TLS expression,the patients with low TLS expression had higher tumor progression risk(HR=0.755,95% CI: 0.634-0.900,P=0.002).Conclusion:TLS expression was a positive prognostic factor for gastric cancer patients in Kaplan-Meier survival analysis and Cox regression analysis.The expression of TLS had the negative correlation with clinicopathological parameters in χ2 text.TLS mainly affected the OS for TNM I-II staging patients.The third part: The Combination Expression and The Prognostic Value of TIL-TLS in Gastric Carcinoma PatientsBackground:The expression of TIL or TLS in various of tumors have the positive relationship to patient outcome.Our previous study proved that the expression of TIL or TLS was prositive prognostic factor and the independent prognostic foctor in Cox regression model.TIL reflects the status of immunity cell infiltrating and TLS reflects the formation of the lymphoid structure.The cellular components in TIL can promote the formation of TLS and TLS can recruit immune cell ectopic infiltrating into the TME.However,the relationship between TIL and TLS,and the combination of them impact on patient remain unclear.Objective:The aim of this study is to analysis the differentiation and correlation between the expression of TIL and TLS.Patients were sub-gourped according to the conbination of TIL-TLS.The relationship between TIL-TLS and clinicopathological parameters and the prognostic value of TIL-TLS were analysised.Methods:Correlation test was used to analysis the correlation between the expression of TIL and TLS.According to the previous study of TIL and TLS expression,we devided the patient into TIL-TLS high expression and TIL-TLS low expression.Chi-squar test,Kaplan-Meier estimates,Cox proportional hazard regression analysis were performed to analyse the influencing factors of the risk of tumor progression on the collected data.Results:The correlation between the expression of TIL and TLS was statistically significant differentiation and correlation in gastric cancer patients(χ2=34.547,P<0.001,r=0.192,P<0.001).The TIL-TLShi for 35.9% gastric cancer patients,and had statistical significance to tumor size,TNMstage,pathological grade,lymph node metastasis,nerve infiltration and intravascular thrombosis.TIL-TLS expression was a possitive predictor,the mean OS of patients with TIL-TLShi was 80.72 months,and the mean OS of patients with TILlow expression was 50.78 months(χ2=101.484,P<0.001).TIL-TLS showed the posstive protective effect to patients form TNM I to III staging(P=0.001,P<0.001).Cox regression analysis revealed that compare with the patients less than 50 years old,the patients beyond 50 years old had higher tumor progression risk(HR=1.023,95% CI: 1.014-1.032,P<0.001);compare with low-medium grade tumors,the high grade tumor had higher tumor progression risk(HR=1.232,95% CI: 1.002-1.516,P=0.048);compare with no lymph node metastasis,the patients with lymph node metastasis had higher tumor progression risk(HR=1.522,95% CI: 1.088-2.129,P=0.014);compare with no nerve infiltration,the patients with nerve infiltration had higher tumor progression risk(HR= 1.322,95% CI: 1.084-1.613,P=0.006);compare with TNM I staging,the patients with TNM II staging or TNM III staging had higher tumor progression risk(TNM II stage: HR=2.461,95% CI: 1.526-3.967,P<0.001,TNM III stage : HR=5.021,95% CI: 2.918-8.639,P<0.001);compare with high TIL-TLS expression,the patients with low TIL-TLS expression had higher tumor progression risk(HR=0.467,95% CI: 0.378-0.578,P<0.001).Conclusion:The expression of TIL and TLS have the significant relationship in GC patients.The combination expression of TIL-TLS has predictive value for the prognosis of gastric cancer patients and is an ideal combination of predictive factors.The expression of TIL-TLS reflects the host immunity response protect the patients in TME.In multivariate Cox regression model,TIL-TLS is the only one protective independent prognostic factors.The fourth part: The Analysis of Lymphocyte Subsets in Tumor MicroenvironmentBackground:The subsets of lymphocytes in TIL and TLS directly affect their immune function.The previously study of the subsets in TIL and TLS have been reported,but the composition and distribution of lymphocyte subtypes in gastric cancer remain unclear.Objective:The previous score of TIL and TLS obtained from H&E staining slides were validated by immunohistochemistry staining.Evaluate the expression of the germinal center CD8+ T cell(gc CD8+ TIL)in gastric cancer.The relationship among gc CD8+ TIL,clinicopathological parameters and the patients’ prognosis were analysised.TIL,TLS,TIL-TLS,gc CD8+ TIL were defined as immune infiltrating characteristics and assessed its prognostic value of gastric cancer patients.Methods:H&E staining slides were reviewed to evaluate the TIL and TLS expression in 63 specimens from the patients with gastric cancer in the Third Affiliated Hospital of Soochow University from 2006 to 2008.Immunohistochemistry was used to detect the expression of CD3,CD8 and MECA79 in this cohort of patients.The score of TIL and TLS obtained from H&E staining slides were validated by CD3 and MECA79.Chi-squar test,Kaplan-Meier estimates,Cox proportional hazard regression analyses were performed to analyse the influencing factors of the risk of tumor progression on the collected data.Results:The subgroup status of 63 cases is: TILhi 44 cases(69.8%);TLShi 43cases(68.3%);TIL-TLShi 32 cases(51.8%);MECA79hi 40 cases(63.5%);CD3+ TIL hi 43 cases(68.3%);gc CD8+ TILhi 23 cases(35.9%).The score of TIL that evalued by H&E and immunohistochemistry showed significant agreement(κ value=0.666,P<0.001).The correlation between the expression of MECA79 and TLS was statistically significant differentiation and correlated in gastric cancer patients(χ2=10.262,P=0.001,r=0.389,P=0.002).TIL expression had statistical significance to tumor size,TNM stage,pathological grade,lymph node metastasis and nerve infiltration.TIL-TLS expression had statistical significance to pathological grade,lymph node metastasis,intravascular thrombosis and nerve infiltration.The gc CD8+ TIL expression had statistical significance to patients age,TNM stage and pathological grade.The immune infiltrating parameter(TIL,TLS,TIL-TLS and gc CD8+ TIL)was a positive predictor of gastric cancer patients(P=0.001,P=0.011,P<0.001,P<0.001).The gc CD8+ TIL had the posstive protective effect to patients from TNM I to III staging(P=0.002).Multivariate Cox regression model was designed to compare the combination effects of immune infiltrating parameter on patients.The multivariate Cox regression model of TIL,TLS,gc CD8+ TIL and clinicopathological characteristics showed that gc CD8+ TIL was an independent prognostic factor(HR=0.087,95% CI: 0.011-0.692,P=0.021).The multivariate Cox regression model of TIL-TLS,gc CD8+ TIL and clinicopathological characteristics showed that TIL-TLS(HR=0.247,95% CI: 0.069-0.882,P=0.031)and gc CD8+ TIL(HR=0.067,95% CI: 0.008-0.561,P=0.013)were independent prognostic factors.The multivariate Cox regression model of TIL,TLS and gc CD8+ TIL showed that TIL(HR=0.322,95% CI: 0.124-0.835,P=0.020)and gc CD8+ TIL(HR=0.059,95% CI: 0.008-0.451,P=0.006)were independent prognostic factors.Conclusion:The results of consistency tests of CD3 and MECA79 data showed that the expression of TIL and TLS assessed by H&E staining slides was accurate.The immune infiltrating characteristics had predictive value for the prognosis of gastric cancer patients.In multivariate Cox regression model,immune infiltrating parameter was independent protective factors.The gc CD8+ TIL and TIL-TLS reflected the host immunity response protect the patients in TME.