【作者】 王强；

【导师】 陈晨；

【作者基本信息】 中南大学 ， 外科学， 2022， 硕士

【摘要】 随着肺癌筛查项目的广泛开展和胸部CT的普及应用,越来越多的肺癌患者可以得到早期诊断及治疗,但肺癌仍然是目前全球癌症相关死亡的主要原因之一。肺癌每年造成160多万人死亡,约占全世界癌症死亡人数的三分之一。这种疾病不仅严重威胁着人类健康,同时还加重了社会经济负担。依据病理学类型,肺癌可分为小细胞肺癌(Small Cell Lung Cancer,SCLC)和非小细胞肺癌(Non-Small Cell Lung Cancer,NSCLC)。其中NSCLC占肺癌病例的80%以上,是肺癌的主要病理类型。NSCLC又可以分为腺癌,鳞癌和大细胞癌等,其中肺腺癌约占NSCLC的50%-60%。对于早期NSCLC患者,根治性手术切除是目前最常用的治疗方法。然而,大约30%被诊断为Ia-b期的NSCLC患者在根治性切除后的5年内会出现肿瘤复发或转移。随着肺癌筛查的广泛开展,预计将有越来越多的早期肺癌患者得到诊断,由此也会有越来越多的I期肺癌术后出现复发的患者。因此,探索I期NSCLC术后肿瘤复发的致病机制,并探索具有高灵敏度和特异性的生物预测标记物,对于改善I期肺癌预后尤为重要。先前已有研究证实了肿瘤免疫微环境特征与恶性肿瘤患者的临床预后密切相关。肿瘤浸润性免疫细胞在肿瘤微环境中可以形成一个“生态系统”,调节癌症的发生和进展,并显示出潜在的预后判断价值。例如,在多种类型的癌症中均已经观察到高水平的活化CD8~+T细胞与较好的疾病预后正相关。细胞毒性CD8~+T细胞和CD4~+辅助T细胞可以靶向抗原性肿瘤细胞,从而抑制肿瘤进展。另一方面,另外一些浸润的免疫细胞,如调节性T细胞、巨噬细胞和髓系抑制细胞,可以通过分泌免疫抑制细胞因子来抑制T细胞反应,或直接抑制T细胞的抗肿瘤作用,导致肿瘤细胞发生免疫逃逸。研究发现,髓系抑制细胞是NSCLC根治性切除后转移前微环境形成的关键因素。肺中髓系抑制细胞数量的减少可以延长无病生存期并提高总存活率。然而,这些研究大多以生存时间为研究终点,对于根治术后疾病复发的机制尚缺乏探索。基于以上研究背景,我们以I期NSCLC术后早期复发患者的肿瘤免疫微环境特征为研究切入点,从免疫组学的角度探索I期NSCLC术后早期复发的潜在分子机制,并探索与疾病早期复发密切相关的免疫相关分子标记物,进而在多个临床病例数据集中进行验证,以期为I期NSCLC术后早期复发的机制研究提供全新的线索和思路。目的:1.观察I期NSCLC根治术后早期复发的肿瘤免疫微环境特征,从免疫组学的角度探索肿瘤早期复发的分子机制,研究免疫微环境分子特征与I期NSCLC术后早期复发之间的关系。2.探索并验证基于免疫学特征的I期NSCLC术后早期复发的生物预测标记物,建立肺癌术后早期复发的预测模型,为改善I期肺癌患者预后提供新方向。第一章I期NSCLC术后早期复发的肿瘤免疫微环境特征研究目的:对比观察I期NSCLC术后早期复发和无复发的肿瘤免疫微环境的差异,探索术后肿瘤早期复发的免疫微环境分子特征。材料与方法:1.采用TCGA数据库I期NSCLC患者为训练集,GEO数据库的GSE32863数据集、GSE37745数据集、GSE116959数据集、GSE31210数据集为验证集。收集湘雅二医院I期NSCLC患者肿瘤组织,获取RNA-seq数据或基因表达谱数据,作为本地验证集。2.在TCGA训练集患者中采用GSEA、GO和KEGG、CIBERSORT方法分别对比观察早期复发组和无复发组肿瘤微环境免疫相关基因表达的差异、免疫相关通路差异以及浸润性免疫细胞的差异,采用多因素变量分析观察免疫微环境与肿瘤早期复发的关系。3.在GEO各个数据集中验证TCGA数据分析结果。4.在XYEYY数据集中采用免疫组织化学法进一步验证肿瘤微环境中浸润性免疫细胞数量,并验证上述结果。结果:1.在TCGA患者队列早期复发组中发现了多种免疫相关基因表达异常,GO分析和KEGG分析发现多条免疫相关通路调控异常,提示免疫相关基因的差异表达与术后早期复发存在相关性。2.CIBERSORT结果显示调节性T细胞、M0型巨噬细胞和M1型巨噬细胞在复发组中显著富集,而记忆B细胞在无复发组中的数量明显增加。多因素变量分析显示在TCGA训练集、XYEYY数据集和GSE37745数据集中,调节性T细胞浸润与I期NSCLC术后早期复发有很强的相关性。3.免疫组化检测也证实在早期复发组中调节性T细胞明显富集,而CD4~+T和CD8~+T淋巴细胞浸润在早期复发组和无复发组之间无统计学差异。结论:I期NSCLC术后早期复发患者肿瘤微环境中存在免疫相关基因的差异表达,以及多条免疫相关通路调控异常,表明免疫相关基因的差异表达与术后早期复发存在相关性。调节性T细胞、M0型巨噬细胞和M1型巨噬细胞在复发组中显著富集,而记忆B细胞在无复发组中的表达明显增加。多因素变量分析验证了调节性T细胞浸润与I期NSCLC术后早期复发具有很强的相关性。第二章I期NSCLC术后早期复发预测标记物的探索及验证目的:为了探索能够有效预测I期NSCLC术后早期复发的生物标记物,本章节从早期复发组与无复发组的差异表达基因出发,通过LASSO Cox回归模型筛选最有价值的预测早期复发的基因,并采用实时荧光定量PCR进行验证,建立预测模型。进而在各个数据集中验证生物标记物对肺癌术后早期复发的预测性能。材料与方法:1.以早期复发组与无复发组差异表达的免疫相关基因为基础,使用LASSO Cox回归模型筛选最有价值的复发预测基因,通过RT-q PCR在本地数据集样本中进行验证。2.通过计算ROC曲线获取各个基因对肺癌术后早期复发的预测效能,尝试不同组合,建立最优的预测模型。3.在多个数据库中验证复发预测模型的效能和应用价值。结果:1.根据LASSO Cox回归模型得到一个包含5个免疫相关基因的复发预测模型:RLTPR、SLFN13、HYDIN、MIR4500HG和TPRG1。RT-q PCR结果验证了这5个基因在早期复发组与无复发组中存在表达差异。2.ROC曲线验证了RLTPR、SLFN13、HYDIN、MIR4500HG和TPRG1这5个基因能有效预测肺癌的早期复发;这5个基因联合使用时预测肺癌早期复发的AUC最大。3.复发风险分析及生存分析表明基因表达与肿瘤无复发存活率之间有很强的相关性。更进一步证实了RLTPR、SLFN13、HYDIN、MIR4500HG和TPRG1这5个基因能有效预测I期肺癌术后早期复发。结论:1免疫相关基因SLFN13、RLTPR、HYDIN、MIR4500HG和TPRG1的表达与I期NSCLC术后早期复发密切相关。2.这5个基因的表达组合对于I期NSCLC术后早期复发有较高的预测效能。总结:本研究以I期NSCLC患者根治性切除后40个月内肿瘤复发的患者为研究对象,探索I期NSCLC术后早期复发的肿瘤免疫微环境特征以及肿瘤早期复发免疫相关生物标记物,发现免疫微环境特征与I期NSCLC术后早期复发密切相关。异常浸润的巨噬细胞和调节性T细胞,以及记忆B细胞的数量,都可能与I期NSCLC术后早期复发密切相关。与肿瘤浸润性淋巴细胞相比,SLFN13、RLTPR、HYDIN、MIR4500HG和TPRG1这5个免疫相关基因的表达可作为预测I期NSCLC根治切除后早期复发的强有力的生物标记物。更多还原

【Abstract】 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.更多还原