Predictive Value Of The Tumor-infiltrating Immune Cells In Patients With Colorectal Cancer

BackgroundPeripheral blood neutrophil-to-lymphocyte ratio(p NLR)as an systemic inflammatory response evaluation has been linked to clinical outcome of several malignancies.However,local immune/inflammatory responses were better reflected by tumor-infiltrating neutrophils,lymphocytes and neutrophil-to-lymphocyteratio in tumor microenvironments.These immune cells had been considered as the prognostic factors for various malignant tumors.The immune score(immunoscore)is first described in patients with CRC for the prediction of tumor recurrence and survival.The aim of this study is to investigate the clinical significances of tumor-infiltrating(both intratumor and petitumor)CD66b+ neutrophils,CD3+ T lymphocytes,CD8+ T lymphocytes,CD66b+ neturophil-to-CD3+ T lymphocyte ratio(NLR)and immunoscore,and to analyze the distribution of tumor-infiltrating CD66b+ neutrophils,CD3+ T lymphocytes and CD8+ T lymphocytes in patients with CRC following resection.Material and methods1.Patients: A total of 210 patients between January 2005 to April 2006 who were diagnosed with CRC and received resection in the Department of General Surgery of the First Affiliated Hospital of Anhui Medical University(Hefei,China)were included in this research.The pathology of each patient was confirmed,and patients did not receive any anticancer therapy before the samples were obtained.Individuals with an autoimmune disease,HIV or syphilis were excluded.2.Methods and Evaluation of Immunostaining: The expression of CD66 b,CD3 and CD8 in tumor-infiltrating areas was detected by SP immunohistochemistry.The positive cells exhibited distinct cell membranes brown staining.The numbers of CD66b+ neutrophils,CD3+ T lymphocytes and CD8+ T lymphocytes in tumor-infiltrating areas were counted.3.Statistical Analysis: SPSS statistical software(version 19.0,IBM)was used for all the statistical analysis.Correlations between clinicopathologic features and immunostaining parameters were analyzed by the chi-square test(for categorical variables)and the t test(for continuous variables).Associations between continuous variables were examined by calculating Pearson’s correlation coefficient.Kaplan-Meier method estimates of survival were used to illustrate the survival curves,and survival was measured in months from the resection to either recurrence or the last review.Univariate and multivariate analysis of the prognostic factors for disease-free survival(DFS)and overall survival(OS)were performed using the Cox proportional hazards model.Receiver operating characteristic(ROC)curve analysis was performed to evaluate the predictive value of the obtained model.All tests were two-sided,and P < 0.05 were considered statistically significant.Results1 Part One1.1 The density of the intratumoral CD66b+ neutrophils was not significantly associated with that of the intratumoral CD3+ T lymphocytes(r =﹣0.015,P = 0.832),and there were no correlations between the density of either peritumoral CD66b+ neutrophils or CD3+ T lymphocytes(r =﹣0.106,P = 0.124).The density of intratumoral CD66b+ neutrophils was correlated with that of peritumoral CD66b+ neutrophils(r = 0.877,P < 0.001),and the density of intratumoral CD3+ T lymphocytes was significantly correlated with that of peritumoral CD3+ T lymphocytes(r = 0.903,P < 0.001).1.2 We analysed the correlations between tumor-infiltrating immune cells and the clinicopathological variables of CRC.The results demonstrated that the tumor-infiltrating CD66b+ neutrophils had no association with any of the clinicopathological parameters.The expression of the intratumoral CD3+ T lymphocytes was significantly associated with lymph node metastasis(P = 0.016)and advanced TNM stages(P = 0.025).Furthermore,the decreased peritumoral CD3+ T lymphocytes were correlated with an advanced depth of invasion(P = 0.045),lymph node metastasis(P = 0.013)and an advanced TNM stages(P = 0.006).The results also showed that increased tumor-infiltrating NLR were associated with tumor lymph node metastasis and advanced TNM stages(P < 0.05).In addition,decreased intratumoral NLR was more likely to be well correlated with the tumor differential grade(P = 0.027).1.3 In univariate analysis,decreased tumor-infiltrating CD3+ T lymphocytes,increased CD66b+ neutrophils and NLR were equally significantly associated with poor DFS and OS(P < 0.05 for both),except for the DFS of intratumoral CD3+ T lymphocytes(P = 0.051).Positive lymph node metastasis advanced differential grade,depth of invasion and TNM stages were significantly associated with poor FDS and OS(P < 0.05).Multivariate analysis together with conventional clinicopathological variables that had been found to be significant by univariate analysis revealed that increased peritumoral NLR,advanced differential status and TNM stages were independent prognostics for OS and DFS with higher hazard ratio values.Moreover,positive lymph node metastasis was independently associated with the decreased OS.1.4 In the sub-group analysis of the TNM stage II patients,a high intratumoral and peritumoral NLR was linked to a short survival time(P < 0.05).2 Part Two2.1 The density of CD8+ T lymphocytes was significantly correlated with the density of CD3+ T lymphocytes(r = 0.406,P < 0.001).Meanwhile,the density of CD8+ T lymphocytes was significantly negative correlated with the density of CD66b+ neutrophils(r =-0.259,P < 0.001).The density of CD66b+ neutrophils was not associated with the density of CD3+ T lymphocytes(r =-0.020,P = 0.770).2.2 We analysed the correlations between the intratumoral immune cells and the clinicopathological variables of CRC.The results demonstrated that the CD66b+ neutrophils was only significantly associated with tumor differential grade(P = 0.044).The decreased CD3+ T lymphocytes and CD8+ T lymphocytes were correlated with an advanced depth of invasion,lymph node metastasis and an advanced TNM stages(P < 0.05).Furthermore,CD8+ T lymphocytes were correlated with tumor differential grade(P < 0.001).The results also showed that decreased immunoscore was associated with an advanced depth of invasion,tumor lymph node metastasis and advanced TNM stages(P < 0.05).2.3 In univariate analysis,increased CD66b+ neutrophils,decreased CD8+ T lymphocytes and immunoscore were equally significantly associated with poor DFS and OS(P < 0.05 for both).Decreased CD3+ T lymphocytes were equally significantly associated with poor OS(= 0.003).However,CD3+ T lymphocytes had no associated with DFS(P﹥0.05).Positive lymph node metastasis,advanced differential grade and p TNM stages were significantly associated with poor FDS and OS(P < 0.05).The depth of invasion was significantly associated with OS(P = 0.015,CI:1.177-4.611).2.4 We then performed Cox multivariate regression analysis by adding immunoscore and TNM stages into a model.The result showed that both of them were significantly associated with DFS and OS.Moreover,immunoscore was an independent prognostic factor with a higher hazard ratio.The predicting values of the immunoscore and the TNM stages were further evaluated using ROC analysis.The areas under curve of the immunoscore and the TNM stages were 0.750(95% CI,0.630-0.771)versus 0.701(95% CI,0.684-0.817)for 5-year DFS and 0.772(95% CI,0.707-0.836)versus 0.754(95% CI,0.688-0.821)for 5-year OS,respectively.Conclusions1 Tumor-infiltrating CD66b+ neutrophils,CD3+ T lymphocytes,CD8+ T lymphocytes were prognosises of colorectal cancer patients playing important role in local immune/inlflammatory responses.2 The tumor-infiltrating NLR was an independent prognosticator with a higher hazard ratio,which may help substantially to identify high-risk patients with CRC following resection.3 Immunoscore was a poor prognostic marker in predicting outcome of patient with CRC following resection.Immunoscore is a prognostic factor superior to the TNM stages.