Investigate The Immune Mechanism Of Different Primary Tumor Location Of CRC Possess Different Outcome

Objectives:Colorectal cancer（CRC）is a malignant tumor of the digestive tract which originates from the cecum to the rectum.It is one of the leading causes of cancer death in worldwide.According to statistics,more than half（about 55%）of colorectal cancer occurs in developed countries,but with the development of economy and the Westernization of life style in developing countries（mainly China,Brazil and India）,the incidence of colorectal cancer is also increasing year by year.So it is urgent to break through in diagnosis and treatment.For early diagnosis,there are various early diagnosis methods,but they have not found the greatest balance between sensitivity and specificity.Finding an ideal early diagnosis method is one of the important contents in the prevention and treatment of colorectal cancer.For treatment,especially immunotherapy,has become the hope of radical cure cancer.Tumor-specific and non-specific immunocyte therapy and molecular targeted therapy are in the ascendant,but still need to be verified and studied from mechanism to efficacy.In the 1990s,Bufill first proposed that colorectal cancer should not be regarded as the same disease,because different parts of colorectal cancer have many differences in tumor biology and clinical aspects.Bufill and subsequent studies divided the colorectum into the right colon（cecum to transverse colon）and the left colon（colon to spleen）.A number of multicenter clinical studies have also shown that the primary site of the tumor has a significant impact on the prognosis and treatment outcomes of patients with primary colorectal tumors,and the response to cetuximab and bevacizumab is also significantly different.Based on the present situation and the immunomodulation effect of immunotherapy drugs,this study proposes the following research assumptions:Take the perspective of tumor immune microenvironment,we hope to describe and analyse the immune characteristics of left and right colorectal cancer with large public data,and use clinical specimens to verify the results of the analysis.Thus,we can improve the understanding of the heterogeneity of left and right colon cancer from the perspective of tumor immunity,and suggest the possible mechanism of different prognosis and treatment response.Methods:1.Analyze the immune characteristics of colorectal cancer and compare the immune microenvironment of bilateral colorectal cancer.In order to examine the immune spectrum characteristics of colorectal cancer from a holistic perspective,we analyzed the standardized RNA expression profiles,primary sites,clinical TNM stages,TP53/KRAS/BRAF/EGFR gene mutation status and follow-up information of 638 colorectal cancer samples in TCGA database.Cluster analysis and correlation analysis were used to analyze the data of 27 immune cell gene clusters.Then according to the degree of infiltration of immune cells,colorectal cancer was divided into high,medium and low infiltration,and the differences among the three types were compared.Finally,to further clarify the heterogeneity of left and right colorectal cancer,we evaluated the immune microenvironment of left and right colorectal cancer in detail by distinct primary tumor locations.2.Focus on the good response of left colorectal cancer to cetuximab,and analyze the immune mechanism behind it.Several multicenter clinical studies have shown that cetuximab has a greater survival benefit than bevacizumab for left colorectal cancer.So what are the immune mechanisms behind left colorectal cancer that affect the clinical efficacy?In view of such clinical problems,we continue to search for the underlying immune mechanism.The specific method is using to deep bioinformatics analysis,such as single-sample Gene Set Enrichment Analysis（ssGSEA）,to find the key factors affecting the final outcome of patients with left-sided colorectal cancer,and KEGG pathway annotation analysis to find the immune system different from right-sided colorectal cancer.3.Reasons for good response to bevacizumab in right colon cancer were analyzed and verified by clinical specimens.Studies have shown that the survival benefit of right half colon cancer in bevacizumab therapy is more obvious.To further clarify the underlying mechanism of this phenomenon,we searched for immune-related factors in right colon cancer through correlation analysis.The premise is that this difference does not play a dominant role in left-sided colorectal cancer,in order to find a clear indication of the right-sided colorectal cancer treatment outcome of the relevant indicators.On the basis of data analysis,we used clinical samples to verify the results obtained by us.By means of immunohistochemical staining with tissue microarray,the tumor foci and adjacent tissues of 90 patients were analyzed to explore the role of immune related factors in the real world of tumor.Results:1.The left and right colorectal cancers have their own specific immune microenvironment.To facilitate further characterization,unsupervised clustering was applied to categorize the cohort into three infiltration subgroups–termed high（H,n=214）,median（M,n=261）,and low（L,n=163）infiltration.Considering the concomitant infiltration of activating and suppressive immune cell types,we investigated whether higher immune cell infiltration correlated with elevated levels of cytotoxic function.It was found that CYT and IFN-gamma were positively correlated with immune infiltration.We further compared the differences in immune infiltration and immune activity between left and right colorectal cancer.It was found that the infiltration of immune cells in right colon cancer was significantly higher than that in left colon cancer（H=81;M=82;L=36 vs H=102;M=160;L=97,p=0.0052）.The influence of cytotoxicity score（CYT,p=7.73e-6）,antigen presentation machinery（APM,p=4.16e-5）,T cell infiltration score（TIS,p=6.942e-10）,interferon-γsignature（p=0.0004）and CD8⁺T-cell/Treg ratio（p=7.51e-11）on T cell function were stronger in the right side than the left side.2.Left colorectal cancer has a higher number of CD56^bright NK infiltration.Our results suggest that CD56 ^bright NK cells in left colorectal cancer are up-regulated（p=0.012）compared with those in right colon cancer and are positively correlated with survival in left colorectal cancer.Activation of 4-1BB signaling pathway（left:p=0.000424;right:p=0.428）and interferon-αsignaling pathway（left:p=0.000973;right:p=0.594）were found to have a good prognosis only in left patients.That is to say,CD56^bright NK has a significant effect on the outcome of colorectal cancer in the left colon cancer.3.VEGFA blocks CD8⁺T cell infiltration and function in right colon cancerThe survival benefit of right colon cancer after using bevacizumab is obvious.Our results showed that compared with left colorectal cancer,VEGFA blocks CD8⁺T cell infiltration in right colon cancer（right side:r=-0.267,p=0.0001;left side:r=-0.115,p=0.029）,VEGFA blocks Th1 cell infiltration（right side:r=-0.239,p=0.0007;left side:r=-0.111,p=0.034）and VEGFA blocks PRF1 expression（right side:r=-0.187,p=0.007;left side:r=-0.02,r=-0.007）.p=0.702).At the same time,we also found that elevated VEGFA blocked the function of T cells,and elevated VEGFA decreased the survival prognosis of patients.Conclusions:1.Both left and right colorectal cancer have specific immune microenvironment,which may be the immunological basis of individual prognosis and treatment response.2.In consideration of cetuximab-mediated ADCC effect,CD56^brightNK cells can be used as an immune marker to predict the prognosis of patients with left colorectal cancer,and can explain the mechanism of good response to cetuximab.3.VEGFA blocks the infiltration and function of CD8⁺T cells.The combination of VEGFA and CD8a can be used as an immune marker to predict the prognosis of patients with right colon cancer,and can also explain the mechanism of good response to bevacizumab.