| Background:With the widespread application of lung cancer screening programs and the extensive development of high-resolution chest CT,more and more patients with lung cancer were diagnosed and treated at an early stage,but lung cancer still remains the main cause of cancer-related deaths worldwide.Lung cancer causes more than 1.6 million deaths every year,accounting for about 1/3of cancer-related deaths in the world.This disease not only seriously threatens human health but also increases the social and economic burden.According to the pathological type,lung cancer can be divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).Among them,NSCLC accounts for more than 80%of lung cancer cases,which is the main pathological type of lung cancer.NSCLC can be divided into adenocarcinoma,squamous cell carcinoma,and large cell carcinoma,of which lung adenocarcinoma accounts for about50%-60%of NSCLC.For patients with early-stage NSCLC,curative resection is the most commonly employed treatment at present.However,about30%of NSCLC patients diagnosed with stage I will have tumor recurrence or metastasis within 5 years after curative resection.With the extensive development of lung cancer screening,it is expected that more and more patients with early-stage lung cancer will be diagnosed,and more and more patients with stage I lung cancer will have recurrence after surgery.Therefore,exploring the mechanisms of postoperative recurrence of stage I NSCLC and exploring predictive biomarkers with high sensitivity and specificity is particularly important to improve the prognosis of patients with stage I lung cancer.Previous studies have reported that tumor microenvironmental immune responses are closely related to the clinical prognosis of patients with malignant tumors.Tumor-infiltrating immune cells can form an“ecosystem”in the tumor microenvironment,regulate the occurrence and progression of cancer,and show potential prognostic value.For example,it has been observed that high levels of activated CD8+T cells are positively associated with better disease prognosis in many types of cancer.Cytotoxic CD8+T cells and CD4+helper T cells can target antigenic tumor cells,thus inhibiting tumor progression.On the other hand,other infiltrating immune cells,such as regulatory T cells,macrophages,and myeloid suppressor cells,can inhibit T cell response by secreting immunosuppressive cytokines or directly inhibit the anti-tumor effect of T cells,resulting in immune escape of tumor cells.It has been found that myeloid suppressor cells are the critical factor in forming a microenvironment before metastasis after curative resection of NSCLC.The decrease in the number of myeloid suppressor cells in the lung can prolong disease-free survival and increase the overall survival rate.However,most of these studies focus on survival time,and there is a lack of exploration of the mechanism of disease recurrence after curative operation.Based on the above research background,we take the tumor immune microenvironment characteristics of patients with early postoperative recurrence of stage I NSCLC as the starting point to explore the potential molecular mechanism of early postoperative recurrence of stage I NSCLC from the perspective of immunomics,and to explore immune-related molecular markers closely related to early recurrence of the disease.Furthermore,we will verify it in multiple clinical data sets in order to provide new clues and ideas for the study of the mechanism of early postoperative recurrence of stage I NSCLC.Objective:1.To observe the tumor immune microenvironment characteristics of early postoperative recurrence of stage I NSCLC,to explore the molecular mechanism of early tumor recurrence from the point of view of immunomics,and to study the relationship between molecular characteristics of immune microenvironment and early postoperative recurrence of stage I NSCLC.2.To explore and verify the biomarkers of early postoperative recurrence of stage I NSCLC based on immunological characteristics and to establish a predictive model of early postoperative recurrence of lung cancer,which provides a new direction for improving the prognosis of patients with stage I lung cancer.Chapter 1 Characteristics of tumor immune microenvironment in early postoperative recurrence of stage I NSCLCObjective:To compare and observe the difference in tumor immune microenvironment between early recurrence and no recurrence after surgery of stage I NSCLC,and to explore the molecular characteristics of immune microenvironment in early recurrence after surgery.Materials and methods:1.The stage I NSCLC patients from TCGA database were included as the training cohort,and the GSE32863 data set,GSE37745 data set,GSE116959 data set,and GSE31210 data set from the GEO database were included as the validation cohort.The tumor tissues of patients with stage I NSCLC from Xiangya Second Hospital were collected,and RNA-seq data or gene expression profile data were obtained as the local verification set.2.In patients from TCGA training cohort,GSEA,GO,KEGG,and CIBERSORT methods will be used to investigate the differences in immune-related gene expression,immune-related pathways,and infiltrating immune cells in the tumor microenvironment between early recurrence group and non-recurrence group.Multivariate analysis was used to observe the relationship between immune-related characteristics and early tumor recurrence.3.Validate TCGA data analysis results in each GEO dataset.4.The number of infiltrating immune cells in the tumor microenvironmentwasfurtherverifiedbythe immunohistochemical method in XYEYY data set,and the above results were verified.Results:1.Abnormal expression of immune-related genes was found in the early recurrence group in TCGA cohort.GO and KEGG analysis showed abnormal regulation of several immune-related pathways,suggesting a correlation between the differential expression of immune-related genes and early postoperative recurrence.2.CIBERSORT results showed that regulatory T cells,M0type macrophages,and M1 type macrophages were significantly enriched in the recurrence group.In contrast,the number of memory B cells increased dramatically in the non-recurrence group.Multivariate analysis showed a strong correlation between regulatory T cell infiltration and early postoperative recurrence of stage I NSCLC in TCGA cohort,XYEYY data set,and GSE37745data set.3.Immunohistochemical examination confirmed that regulatory T cells were significantly enriched in the early recurrence group,while there was no significant difference in CD4~+T and CD8~+T lymphocyte infiltration between the early recurrence group and the non-recurrence group.Conclusion:There was differential expression of immune-related genes and abnormal regulation of several immune-related pathways in the tumor microenvironment of patients with early postoperative recurrence of stage I NSCLC,indicating a correlation between the differential expression of immune-related genes and early postoperative recurrence.Regulatory T cells,M0 type macrophages,and M1 type macrophages were significantly enriched in the recurrence group,while the number of memory B cells increased dramatically in the non-recurrence group.Multivariate analysis confirmed a strong correlation between regulatory T cell infiltration and early postoperative recurrence of I NSCLC.Chapter 2 Exploration and verification of predictive biomarkers for early postoperative recurrence of stage I non-small cell lung cancerObjective:To explore the biomarkers that can effectively predict early postoperative recurrence of I NSCLC,select the most valuable genes and establish a prediction model.Furthermore,the predictive performance of the biomarkers was validated in each data set.Materials and methods:1.Based on the differential expression of immune-related genes between the early recurrence group and non-recurrence group,the most valuable genes for predicting recurrence were screened by LASSO regression model and verified by RT-q PCR in the local data set.2.The prediction efficiency of each gene for early postoperative recurrence of lung cancer was obtained by ROC curve,and different combinations were investigated to establish the optimal prediction model.3.The effectiveness and application value of the recurrence prediction model was validated in multiple databases.Results:1.According to the LASSO Cox regression model,a recurrence prediction model containing five immune-related genes was obtained:RLTPR,SLFN13,HYDIN,MIR4500HG,and TPRG1.RT-q PCR confirmed differential expression of these five genes between the early recurrence group and non-recurrence group.2.ROC curve confirmed that RLTPR,SLFN13,HYDIN,MIR4500HG,and TPRG1 could effectively predict the early recurrence of lung cancer.The combination of these five genes had the largest AUC for predicting the early recurrence of lung cancer.3.Recurrence risk analysis and survival analysis showed a strong correlation between gene expression and tumor recurrence-free survival rate.It is further confirmed that RLTPR,SLFN13,HYDIN,MIR4500HG,and TPRG1 can effectively predict early postoperative recurrence of stage I lung cancer.Conclusion:1.The expression of immune-related genes SLFN13,RLTPR,HYDIN,MIR4500HG,and TPRG1 is closely related to the early postoperative recurrence of stage I NSCLC.2.The expression combination of these five genes has a high predictive efficiency on early postoperative recurrence of stage I NSCLC.Summary:The immune microenvironment is closely related to the early postoperative recurrence of stage I NSCLC.The abnormal infiltration of macrophages and regulatory T cells,and the number of memory B cells,may be closely related to the early postoperative recurrence in patients with stage I NSCLC.Compared with tumor-infiltrating lymphocytes,the expression of SLFN13,RLTPR,HYDIN,MIR4500HG,and TPRG1 can be used as robust biomarkers to predict the early recurrence of stage I NSCLC after curative resection. |
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