Tumor Immunological Characteristics Of Gastric Cancer And Their Relationship With Patient's Postoperative Clinical Outcomes

Background:Gastric cancer(GC)is one of the commonest tumors in the human digestive system,often with high morbidity and mortality.Surgery still remains the only radical treatment for GC.With the advancement of surgical techniques,radiotherapy,chemotherapy and neoadjuvant therapy,the 5-year survival rate of patients with GC has significantly improved.The successful rate in diagnosing early GC,however,is low,mainly due to the lack of specific symptoms at the early stage of GC.Most patients with GC are diagnosed at the late stage and are often inoperable or relapse after radical gastrectomy.Therefore,early detection of GC is the key to proper management and ideal outcomes of the patients.Many recent work focused on identifying molecular markers for early GC:little success has been seen in their translation into clinical application.Studies to identify biomarkers that are conventional,non-invasive,highly specific and can be detected in the early stage are still needed.Advances in immunotherapy in the past decade have seen a rapid development of anti-tumor immunotherapy strategies such as oncolytic viruses,chimeric antigen receptor T cells(chimeric antigen receptor T cells,CAR-Ts),bi-specific antibodies and immunocheckpoint inhibitors.Factors regulating these immunotherapies have yet been fully elucidated.Studies suggested that the overall immune-competency of patients with tumor and the characteristics of tumor local infiltration of immune cells directly affect the efficacy of tumor immunotherapy and the prognosis of the patients.The overall immunological characteristics of patients with GC and the characteristics of infiltrating immune cells in the immune microenvironment,however,have not been systematically described before.This study intended to explore and verify the immune characteristics of patients with GC from three aspects:peripheral blood immune indexes,radiomic features of the immune organ spleen and infiltrating immune cells in tumor tissues.In addition,we aimed to screen the immunological indexes that had a great impact on the clinical outcome of patients with GC,expecting to identify a potential target for the immunotherapy of GC and lay a solid foundation for the immunotherapy of GC.Specific aims:1.To establish and validate a scoring system using quantitative data of lymphocytes,platelets,neutrophils and other immune cells in the peripheral blood of patients with GC,and to determine the power of the scoring system in predicting the clinicopathological characteristics and prognosis of patients with GC.2.To explore the correlation between the radiomic features of the spleen and the prognosis of patients with GC and to establish an accurate prognosis prediction model for patients with GC using radiomic data of the spleen.3.To examine the characteristics of infiltrating immune cells in GC by using immunohistochemistry assays;to examine the relationship between immunohistopathological characteristics of GC tissue and the prognosis of patients with GC.Material and Methods:1.A retrospective study was conducted on 542 patients with GC who received elective radical gastrectomy at The Second Affiliated Hospital of Wenzhou Medical University from January 2012 to December 2016.All patients were diagnosed with gastric adenocarcinoma by gastroscopy and histopathology before surgery.Kolmogorov-Smirnov test was used to determine the normal distribution of continuous data such as neutrophil counts,lymphocyte counts,platelet counts,platelet/lymphocyte ratio(PLR),neutrophil/lymphocyte ratio(NLR),etc.Chi square test,independent t-test or a non-parametric Mann-Whitney method was performed to analyze differences in clinicopathological characteristics and PLR and NLR of peripheral blood before surgery.In addition,we used chi-square test to examine the correlation between the clinical characteristics of tumor patients and the incidence of postoperative complications above grade ?.On the basis of single factor analysis,multiple logistic regression analysis was used to calculate the odds ratio(OR)and 95%confidence interval(95%CI)of each variable,in order to determine the independent risk factors for postoperative complications above grade ? of patients with GC.The predictive scoring system was constructed by summing the calculated values of each variable.Receiver operating curve(ROC)was used to compare the accuracy of the scoring system and other independent risk factors in predicting postoperative complications above grade ? of patients with GC.At last,COX survival analysis was used to analyze the clinicopathological factors of postoperative survival of patients with GC.2.Clinical Data of 243 patients with GC who were admitted to The Second Affiliated Hospital of Wenzhou Medical University for feasible radical surgery between December 2014 and December 2016 were used for this study.Pyradiomics was used to extract radiomic features of spleen,and during the screening of spleen-related features and establishment of the prognostic model,243 patients were randomly divided into the primary group and the validation group(124 and 119 patients,respectively).COX regression analysis was performed for 833 features in the primary group and according to the order of P-value,the feature with P-value less than 0.1 was selected to further establish the survival risk model.After the establishment of the spleen prognostic model,patients from the primary and the validation groups were employed for the univariate and multivariate analysis of prognostic factors in order to establish the visual prognostic prediction model.3.The clinicopathological data of 475 patients with GC who underwent gastrectomy at The Second Affiliated Hospital of Wenzhou Medical University between December 2006 and December 2011 were retrospectively collected.Tissue microarrays containing 475 GC samples were used.Immunohistochemical analysis of 6 lymphocyte markers(CD3+,CD4+,CD8+,CD20+,CD57+,FOXP3+)was used to detect Tumor-infiltrating lymphocytes(TILs)in GC.In order to analyze the association between the densities of TILs and clinicopathologic characteristics,we used ROC analysis to categorize the 6 lymphocyte markers and their ratios into high and low expression groups according to the survival status of the patient.To predict the survival of patients with GC,a LASSO logistic regression model was used to identify indicative lymphocyte markers and construct a comprehensive index TIL-Score in the training cohort.ROC was used to categorize lymphocyte factors and the TIL-Score into two different levels.Chi-square test was used to analyze the correlation between the TIL-Score and clinicopathological characteristics of patients with GC.Survival analysis was performed by Kaplan-Meier Method using a log-rank test.Cox proportion hazards model was used to perform multivariate regression analysis and construct a nomogram.The predictive efficiency of the nomogram was determined by using a calibration curve and the ROC.Results:1.To the best of my knowledge,this was the first study that identify whether preoperative PLR and NLR in peripheral blood can be used to predict postoperative complications of patients with GC.According to ROC curve calculation,the cutoff values of PLR and NLR for postoperative complications above grade ? were set as 158.7 and 2.71,respectively.When patients were divided into high PLR and NLR groups and low PLR and NLR groups,it was found that PLR and NLR were closely related to age and some other clinicopathological features of tumors as listed below.Increased PLR and NLR were associated with larger tumor size,deeper tumor infiltration,ulcerative type,and higher densities of TILs.The ROC curve showed that the combined complication prediction model based on PLR had the highest sensitivity and specificity in predicting complications among all preoperative predictive indicators.Based on the survival analysis,significant differences in survival were found between high and low PLR group,as well as high and low NLR groups.Both PLR and NLR were significantly correlated with age.Comparative analysis of PLR and NLR and other clinicopathological features showed that the increase of PLR and NLR was statistically significant in terms of larger tumor size,higher TNM stage,higher Charlson comorbidity index(CCI),more lymph node metastasis and deeper invasion depth,and it can better reflect the aggressive behavior of gastric cancer.Age,PLR,CCI,tumor location,laparoscopic surgery and combined visceral resection were all significant parameters in the logistic regression model.In this model,for the first time we identify peripheral immune blood index PLR as an independent predictor of short-term postoperative complications of patients with GC.In terms of long-term survival,we found that PLR,NLR,combined visceral resection,total gastrectomy,age and TNM stage were independent risk factors for disease-free survival of patients with GC after surgery.Our scoring system combined common factors such as age,PLR,CCI,tumor location,laparoscopic surgery and combined visceral resection to develop a visual combined predictive model which might provide a reliable predictor of short-term postoperative complications.2.Pyradiomics was used to extract the radiomic features of spleen,and patients were divided into two groups according to the characteristics of spleen.There were no statistical differences in the base data(BMI,age,gender,hypertension,diabetes,NRS-2002 score,abdominal surgery history,tumor differentiation)between the two groups with different spleen characteristics.However,a significant difference in TNM stage was noticed between the high-risk group and the low-risk group(P=0.03),and the TNM stage of the group with poor prognosis was more advanced.Univariate analysis suggested that spleen characteristics,age,NRS-2002 score,ASA grade,CCI,anastomotic method,and TNM stage were associated with long-term survival.Multivariate analysis of survival suggested that the risk of death was 70%higher in patients with spleen characteristics at high-risk than in the low-risk group(HR=1.7,P=0.021).TNM stage also showed good prognostic value,and the risk of death of patients in stage ? was 6.6 times higher than that of patients in stage ?(HR=6.6,P<0.001).The risk of death of patients in stage ? was 2.1 times higher than that of patients in stage ?,however,the difference was not statistically significant(HR=2.1,P=0.144).Other independent risk factors in multivariate COX analysis,such as age(P<0.001)and anastomotic method(P=0.007),also had a significant impact on postoperative survival.Visual nomogram developed from independent risk factors can accurately predict the 1-3 years survival of patients with GC after surgery.3.CD3+,CD4+,CD8+,CD20+,CD57+,and FOXP3+TILs in a tissue microarray containing 475 GC tissues were detected.K-M analysis showed that a high density of CD3+,CD4+,CD8+,CD20+,and CD5 7+TILs was significantly associated with the favorable prognosis.There is no significant correlation between FOXP3+ cell infiltration and survival in patients with GC.To further develop and validate the model,patients were randomly divided into the primary and validation groups.Six lymphocyte markers and their ratios were used to determine a comprehensive biomarker TIL-Score using the LASSO logistic regression model in the primary cohort.In the formula,CD8+,CD3+,and CD57+cells and the CD4+:CD8+ ratio,CD4+:CD3+ ratio,CD4+:CD20+ ratio,CD8+:CD57+ratio,and CD3+:FOXP3+ are protective factors for survival,while FOXP3+ cells,CD4+:CD20+ ratio,and CD8+:CD20+ ratio are negative factors.Further analysis showed that the TIL-Score was associated with smaller tumor size,shallower depth of tumor invasion,less lymph node metastasis,and lower TNM stage in both the primary and validation cohorts.However,the association between the TIL-Score and distant metastasis did not conform in the primary and validation cohorts.Both Kaplan-Meier survival analysis and distribution analysis of the TIL-Score revealed a favorable correlation with the prognosis of patients with GC.To better predict the prognosis of patients with GC,a nomogram integrating age,tumor size,differentiation status,TNM stage,and TIL-Score was developed.The area under the curve value for the nomogram was more than 0.8,indicating high accuracy.The nomogram was validated by ROC analysis and using a calibration curve,which showed high agreement in the primary and validation cohort.Decision curve analysis showed that the nomogram for predicting the probability of survival at 5 years was more beneficial than treat-all group and treat-none group in patients with a threshold probability of 0.1-0.76.Conclusion:1.PLR and NLR can be used to predict short-term complications and long-term survival after GC surgery and then guide individualized treatment.However,PLR combined with age,CCI,tumor location,laparoscopic surgery and combined visceral resection and other factors can significantly improve the prediction accuracy.Moreover,this new scoring system is an economical and convenient tool for predicting postoperative complications,which is of great significance for the precaution and treatment of postoperative complications of GC.2.Spleen characteristics from pyradiomics predicts the postoperative long-term survival of patients with GC,and its accuracy was validated both in primary and validation groups.Spleen characteristics is associated with TNM stage to some extent.Univariate and multivariate analyses suggested that age,group of spleen characteristics,TNM stage,and anastomotic method were independent risk factors for survival.Nomogram developed from independent risk factors can accurately predict the 1-3 years survival of patients with GC after surgery,with an accuracy of 78.7%,providing a value for prognosis prediction and treatment plan formulation of patients with GC.3.We examined six lymphocyte markers and their ratios to determine a comprehensive biomarker TIL-Score,and then developed a nomogram integrating TIL-Score and clinical risk factors,which can be used to better predict the prognosis of patients with GC.