| Background and purpose:Renal cell carcinoma(RCC)is a malignant tumor originating from the proximal tubular epithelium of the renal parenchyma.According to the latest report by China National Cancer Center(NCC),about 76,000 new kidney cancers were diagnosed and 27,000 deaths were recorded in China in 2016.Clear cell renal cell carcinoma(ccRCC)is the most common pathological subtype of Renal cell carcinoma,accounting for approximately 70%of RCC.Tumor patients often have more active coagulation pathway compared to normal people.Deep vein thrombosis and pulmonary embolism are common complications,and venous thromboembolism(VTE)is the second leading cause of death in patients with tumors,after tumor progression.Renal cancer often progresses to an advanced stage with tumor thrombus(TT),The unique clinical feature of renal tumor thrombus is that it can invade through the renal vein into the inferior vena cava and even grow into the right heart cavity,Tumor thrombus in the renal vein or vena cava is present in approximately 15%of kidney cancer cases.Tumor invasion can activate coagulation by disrupting the vessel wall or increasing vascular permeability and plasma extravasation,thus coagulation pathway is actived during tumor thrombosis.It has been found that coagulation tissue can interact with the tumor microenvironment to coordinate or inhibit tumor progression and even influence the tumor’s immune response.Also,several recent studies have found a significant reduction in tumor thrombus size after treatment with Immune Checkpoint Blocker(ICBs)in renal tumor patients.what is the role of coagulation-related pathways in the development of renal cancer,especially in renal tumor thrombosis progression,and whether there is some association with the immune microenvironment.In this study,we propose to investigate the role of coagulation pathways in the development of kidney cancer and the relationship with the immune microenvironment of kidney cancer,to identify key coagulation-related genes,and to explore new prognostic assessment tools for risk stratification of kidney cancer patients through bioinformatic approaches to the expression profiling data of kidney cancer.Methods1.ThecBioPortal database was used to visualize copy number variants and mutations in 451 patients with renal clear cell carcinoma in the TCGA-KIRC cohort.2.The R package "limma" was used to screen for differentially expressed coagulation-associated genes in cancer tissues and identify coagulation subtypes in renal clear cell carcinoma using consensus clustering with K=2 1000 iterations.The impact of different subgroups on survival and correlation with clinical features were investigated by KM analysis and Percentage stacking diagram.3.Pathway enrichment of coagulation subgroups was analyzed by GSVA,and GO/KEGG.Immune AI investigated the differences in immune cells infiltration between subgroups.major histocompatibility complexes,immune checkpoints,ESTIMATE score,between the two groups between subgroups were explored.4.GSEA enrichment analysis to explore the differential pathways between T3b stage tumors and T1,T2 tumors.PPI(protein-protein interaction)network to search for coagulation-related genes with high association in protein level.Pan-cancer differential expression of 4 candidate genes in the TCGA,pan-cancer prognostic analysis,methylation analysis were studied using GSCA database.5.Validation of MMP9 gene using renal clear cell carcinoma-specimens.qPCR analysis of differential expression of 4 candidate genes,correlation analysis of MMP9 expression with 3 immune cell markers.Immunohistochemical staining were used to search MMP9 differential expression between carcinoma and normal tissue,and the correlation of MMP9 with 3 immune cell markers.6.Coagulation risk score was constructed by Lasso regression and stepwise multifactor COX regression and validated in an external cohort."pRRophetic",an R package,was used to analyze the differences in semi-inhibitory concentrations(IC50)of commonly used targeted drugs between risk score groups.7.Incorporate clinically relevant characteristics to construct clinically relevant prognostic models.Results1.coagulation pathway activation was associated with worse prognosis,advanced clinical stage,and grading.2.Activation of the coagulation pathway is accompanied by an altered immune microenvironment with increased infiltration of immune cells such as CD8,CD4,exhausted T cells,Treg cells and cytotoxic T cells.3.The MMP9 gene may play an important role in the formation of thrombus,influenced immune microenvironment in renal cancer tumors which contribute to poor prognosis by increasing the infiltration of immune cells such as CD8 and Treg cells.4.The prognosis model of renal clear cell carcinoma based on clinical characteristics combined with risk score has clinical application value.ConclusionWe analyzed the clinical significance of coagulation-related gene pathways in renal clear cell carcinoma and their relevance to the immune cell infiltration characteristics.Combining the significance of clinical stage and protein-protein interactions in renal cancer,the gene MMP9,which plays a key role,was screened and its relationship with immune cell infiltration was verified by external experiments.Finally,a coagulation-related risk score model was constructed with an external cohort to validate and its correlation with response to drug therapy was further studied.In summary,our systematic study facilitates to understanding the role of coagulation-related genes in renal clear cell carcinoma and the value its further application. |
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