The Clinical Significance Of Immune Microenvironment And Classification Of Lung Adenocarcinoma(LUAD)

ObjectiveAt present,lung cancer has become one of the most common malignant tumors,the most common pathological type of lung cancer is lung adenocarcinoma（LUAD）.Immunotherapy has become an irreplaceable method in the treatment of LUAD,but even after positive selection of immune checkpoints,immunotherapy still has poor efficacy for a majority of patients,one of the main reasons is the heterogeneity of tumor immune microenvironment（TIME）.Therefore,in order to give full play to the value of immunotherapy in the comprehensive treatment of LUAD,it is necessary to formulate the corresponding immunotherapy strategy according to the TIME type.The purpose of this study was to analyze the characteristics of the immune microenvironment of LUAD,and to further explore the methods,mechanisms of the TIME classification of LUAD,and create a more practical and accurate LUAD tumor immune microenvironment classification method,so as to provide new basis and ideas for the precise selection of combination therapy strategy of LUAD.MethodsA total of 254 patients with newly treated lung adenocarcinoma who received surgical treatment and confirmed pathology were included in this study from July2013 to October 2015 in Tianjin Medical University Cancer Hospital.1.The multi-color immunofluorescence technique was used to study the distribution characteristics of single-indicator and multi-indicator immune cell subsets in the immune microenvironment of lung adenocarcinoma and the relationship between the characteristics of immune microenvironment and prognosis of lung adenocarcinoma,and to determine the independent factors of single-indicator and multi-indicator immune cell subsets affecting the prognosis of lung adenocarcinoma.2.These lung adenocarcinoma patients were preliminarily classified by hierarchical cluster analysis,and each type was corresponding to a tumor immune microenvironment type.Then,through in-depth analysis,the distribution characteristics of immune cell subsets in each TIME type and their relationship with clinicopathological factors and prognosis were summarized,so providing a new basis and idea for understanding and creating a new TIME classification of lung adenocarcinoma.3.Based on the results of the previous two parts and the clinicopathological characteristics of LUAD,a more practical and accurate TIME classification method for LUAD was proposed,providing a new basis for the accurate selection of combined treatment strategy of LUAD.Results1.After the distribution characteristics of single-indicator immune cell subsets in the immune microenvironment of LUAD were obtained by multicolor immuno-fluorescence technique,univariate analysis results showed that 13 factors including TNM stage,CD4_CT、⁺Foxp3_CT、⁺CD73_CT、⁺TIM-3_IM、⁺CD39_CT、⁺LAG-3_IM、⁺CD8_CT、⁺CD68_CT、⁺CD11c_IM、⁺IDO_IM、⁺PD-L1_CT、⁺PD-L1_IM⁺were influencing factors of OS in patients with LUAD（all P value<0.05）.Multivariate analysis showed that TNM stage,CD4_CT⁺,Foxp3_IM⁺,CD8_IM⁺,IDO_IM⁺,PD-L1_CT⁺and PD-L1_IM⁺were independent prognostic factors of the LUAD overall survival.2.After the distribution characteristics of multi-indicator immune cell subsets in the immune microenvironment of LUAD were obtained by multicolor immuno-fluorescence technique,univariate analysis results showed that another 13multi-indicator immune cell subsets were the influencing factors of OS in patients with LUAD（all P value<0.05）.Further multivariate analysis showed that CD8⁺CD73_CT^-、CD4⁺Foxp3_CT、^-PD-L1⁺IDO_IM、⁺CD68⁺PD-L1_IM⁺、CD163⁺IDO_IM、⁺CD163⁺PD-L1_IM⁺six multi-indicator immune cell subsets were also independent prognostic factors of LUAD overall survival.3.The hierarchical cluster analysis was used to divide the LUAD patients into four categories（TYPEⅠ,TYPEⅡ,TYPEⅢ,TYPEⅣ）,and each type corresponds to a TIME type.The results of survival analysis indicated that there was a statistical difference in PFS among the four types of patients（P=0.042）.Patients grouped into TYPEⅠhad the best prognosis,while those grouped into TYPEⅡhad the worst prognosis.The comparison of OS among four types of patients showed the same trend as PFS survival analysis,but there was no statistical difference（P=0.054）.The distribution of single and multi-indicator immune cell subsets in the tumor core area（CT）and tumor infiltrating edge（IM）in these four TIME types showed obvious specificity and regularity.The comparative advantage of the expression of positive regulatory immune cell subsets with anti-tumor activity and negative regulatory immune cell subsets with pro-tumor activity constitutes the main characteristics of each TIME type,and influences the selection of immunotherapeutic strategies for patients with LUAD.4.According to the results of the previous two parts and the clinicopathological characteristics of LUAD,a more operable TIME classification of LUAD was further proposed,that is,according to the expression of CD8⁺CD73_CT^-and PD-L1_CT⁺,the TIME of lung adenocarcinoma was divided into four types:type 1(CD8⁺CD73_CT^-highPD-L1_CT^+low),type2(CD8⁺CD73_CT^-highPD-L1_CT^+high),type3(CD8⁺CD73_CT^-lowPD-L1_CT^+low),type4(CD8⁺CD73_CT^-lowPD-L1_CT^+high).Survival analysis showed that there were significant differences in PFS and OS among the four types of patients（P=0.000,P=0.001）.These results indicated that this new method of TIME classification of LUAD has strong feasibility and accuracy,and provided a new basis for the precise selection of combined treatment strategy of LUAD.Conclusions1.In the immune microenvironment of LUAD,the type,density and location of immune cell subsets are important component components,which directly determine the characteristics of the TIME.Among them,CD4_CT、⁺Foxp3_CT、⁺CD8_CT、⁺IDO_IM、⁺PD-L1_CT、⁺PD-L1_IM、⁺CD8⁺CD73_CT^-、CD4⁺Foxp3_CT^-、PD-L1⁺IDO_IM⁺、CD68⁺PD-L1_IM⁺、CD163⁺IDO_IM⁺、CD163⁺PD-L1_IM⁺were independent prognostic factors of LUAD.2.Further by in-depth analysis of TIME classification of LUAD using the method of cluster analysis,It prompted that the TIME classification of LUAD can be based on the type and relative density characteristics of the single and multiple indices immune cell subsets in the core region（CT）and tumor infiltrating edge（IM）,then the survival analysis was used to verify the feasibility of the classification.It provided a new basis and idea for understanding and creating a new TIME classification of LUAD.3.Based on the distribution characteristics of immune cell subsets in LUAD immune microenvironment and the TIME typing characteristics based on hierarchical clustering analysis,this study proposed a more clinically operable TIME classification method of LUAD based on CD8⁺CD73_CT^-and PD-L1_CT⁺,which had stronger clinical operability and accuracy,and provided a new basis for precise selection of combined treatment strategy of LUAD.