液体活检在非小细胞肺癌中的临床应用

发布时间：2018-01-03 22:00

本文关键词：液体活检在非小细胞肺癌中的临床应用　出处：《北京协和医学院》2017年博士论文　论文类型：学位论文

【摘要】：背景与目的肺癌是目前发病率和死亡率最高的恶性肿瘤,而非小细胞肺癌(NSCLC)占到其中85%。此外,NSCLC的早期诊断和对患者转移的预测以及肺部结节的鉴别诊断,目前都缺乏一个效能高、创伤小而又方便快捷的的临床手段。新兴的液体活检,包括ctDNA、CTC和外泌体,由于其无创、简便和可重复的优势,被越来越多的应用到了各类肿瘤的临床应用中。但由于其方法缺乏统一性,目前在NSCLC中尚缺乏一个良好的液体活检的技术指标来完成上述亟待解决的临床需求。本研究第一部分旨在探究作为液体活检的CTC对肺部结节良恶性的鉴别诊断的能力,并与PET/CT进行对比。第二部分探索了外泌体在肺腺癌的诊断和预测转移的潜能,并进行了外泌体与原发肿瘤蛋白表达及瘤负荷关系的探索性研究。通过这两部分研究,本文希望对液体活检在NSCLC中的临床应用潜能进行较全面的探索和分析。方法第一部分通过招募肺部结节患者,利用基于EpCAM特异性识别多肽的CTC捕获分离技术,对其进行CTC检测和计数。将CTC计数作为肺部结节良恶性的鉴别诊断指标,进行ROC曲线分析并确定其对肺部结节鉴别诊断的灵敏度与特异度,同时与PET/CT的鉴别诊断进行对比。此外,按照胸部CT的影像特征对上述分析进行进一步的亚组分析,以确定该技术的CTC检测技术最佳的应用范围。第二部分则通过招募肺腺癌患者和健康受试者,利用基于醛基化乳胶微珠联合流式细胞术的外泌体蛋白定量分析技术,对受试者外周血的外泌体进行EGFR蛋白和CXCR4蛋白的定量检测。将外泌体EGFR蛋白表达量作为肺腺癌诊断指标,进行差异分析和ROC曲线分析,并确定其对肺癌诊断的灵敏度与特异度。另外,将外泌体CXCR4蛋白表达量作为肺腺癌转移预测的指标,进行上述相同的分析,并探究其与肺腺癌侵袭性之间的相关性。最后通过将外泌体蛋白表达与腺癌组织免疫组化染色对比,以及患者术前术后外泌体蛋白表达对比,探究外泌体与原发肿瘤蛋白表达及瘤负荷关系。结果第一部分的研究纳入了 104例肺部结节患者(97例肺腺癌、25例良性病变)。分析发现CTC计数在良恶性两组间的差异有用统计学意义,分别为0.5个/mL和2.0个/mL(P0.05)。CTC计数鉴别诊断肺部结节良恶性的ROC曲线AUC为0.651(P0.05),以0.5个/mL为阈值时,其灵敏度和特异度分别为45.6%和80.0%。亚组分析发现,CTC计数对于在CT影像中表现为纯磨玻璃影(pGGO)结节具有良好的鉴别诊断效能:在良恶性两组的差异有统计学意义,其中位数分别为0个/mL和1.0个/mL(P0.01);而在pGGO组中鉴别诊断的ROC曲线AUC为0.863(P0.01),以0个/mL为阈值时其鉴别诊断的灵敏度度和特异度分别为87.1%和83.3%。而通过与PET/CT进行比较发现CTC计数对肺部结节的鉴别诊断在pGGO组显著优于PET/CT,后者的灵敏度和特异度仅为54.8%和66.7%,显著低于CTC计数(P=0.039)。第二部分的研究纳入了 68例受试者(38例肺腺癌、30例健康受试者)。分析发现外泌体浓度、外泌体EGFR蛋白表达量在两组人群没有表现出差异。而外泌体CXCR4蛋白表达与肺腺癌侵袭性都显著正相关,流式细胞分析中外泌体CXCR4荧光强度与与肺腺癌病理侵袭性评分的相关系数为0.413(P0.01)。此外,外泌体CXCR4蛋白表达量在转移组和未转移组间有显著差异(P0.04)。通过ROC曲线分析,外泌体CXCR4蛋白表达率和外泌体CXCR4荧光强度预测肺腺癌转移的AUC分别为 0.697(P=0.04)和 0.770(P0.01);分别以7.4%和1429.39 为阈值,外泌体CXCR4蛋白表达率和荧光强度预测肺癌转移的灵敏度均为100%,而特异度分别为40.9%和59.1%。对比肺腺癌组织免疫组化染色和外泌体蛋白表达,没有观察到一致性。此外,患者术前和术后的外泌体相关指标也没有一致的变化趋势,统计分析发现手术前后外泌体相关指标变化没有统计学意义。结论本研究通过对比CTC计数与PET/CT在肺部结节良恶性的鉴别诊断能力,发现该技术的CTC计数具有良好的鉴别诊断能力。对纯磨玻璃影结节,其鉴别诊断效能甚至优于PET/CT。最后,利用流式细胞术对外周血外泌体的蛋白定量,发现外泌体EGFR蛋白的表达量不具有诊断肺癌的能力;而外泌体CXCR4蛋白表达与肺癌的侵袭性有显著相关性,同时具有较高的预测肺腺癌转移的潜能。该技术的外泌体蛋白定量表达并不能反映原发肿瘤蛋白表达量,也不具有监测肺腺癌患者瘤负荷的能力。
[Abstract]:Background and objective: lung cancer is the highest morbidity and mortality of malignant tumors, and non-small cell lung cancer (NSCLC) accounted for 85%. in the early diagnosis of NSCLC and prediction of patients with metastasis and differential diagnosis of pulmonary nodules, the lack of a high efficiency, small trauma and convenient clinical approach. A new liquid biopsy, including ctDNA, CTC and exosomes, because of its non-invasive, simple and repeatable advantage is more and more applied to the clinical application of all kinds of tumors. But because of its lack of unity, now in NSCLC there is no technical indicators of a good liquid biopsy to complete the clinical needs to be solved. The first part of this study is to explore the CTC as a liquid biopsy for pulmonary nodule benign and malignant ability, and compared with PET/CT. The second part explores the exosomes in the lung The diagnosis and prediction of cancer metastasis, and explored exosomes and primary tumor protein expression and tumor load. Through this two part study, this paper hopes to explore and a comprehensive analysis of the potential clinical application of liquid biopsy in NSCLC. Methods: the first part through the recruitment of patients with lung nodules, EpCAM was used to identify peptide CTC capture and separation technology based on CTC detection and counting the. CTC counts as pulmonary nodules and differential diagnosis of benign and malignant index, ROC curve analysis and determine the sensitivity for the diagnosis of pulmonary nodules with different and specificity, and compared with the differential diagnosis of PET/CT. In addition, according to the above analysis of subgroup analysis further imaging features of chest CT, to determine the optimum technology of CTC detection technology application. The second part is through the recruitment of lung adenocarcinoma Cancer patients and healthy subjects, using the quantitative analysis technique of exosome protein combined with flow cytometry based on aldehydized latex beads, quantitative detection of exosomes from peripheral blood of EGFR protein and CXCR4 protein. The exosome expression of EGFR protein as a diagnostic indicator of lung adenocarcinoma cancer, analysis of variance analysis and ROC curve, and determine its sensitivity and specificity for the diagnosis of lung cancer. In addition, the exosome protein expression of CXCR4 in lung adenocarcinoma metastasis as the prediction index, the same analysis, and explore the invasion of lung adenocarcinoma and the correlation between the last. The exosome protein expression and adenocarcinoma were compared, and the patients with preoperative and postoperative exosome protein expression comparison, explore exosomes and primary tumor protein expression and tumor load. The first part of the study included 104 patients suffering from pulmonary nodules Patients (25 cases of benign lesions and 97 cases of adenocarcinoma of the lung.) analysis showed that CTC count useful in benign and malignant and the difference between the two groups was statistically significant, respectively 0.5 /mL and 2 /mL (P0.05).CTC in the differential diagnosis of benign and malignant pulmonary nodule count ROC curve of AUC was 0.651 (P0.05), with 0.5 /mL as the threshold, the sensitivity and specificity for subgroup analysis found that 45.6% and 80.0%., CTC counts for pure ground glass opacity in the CT image (pGGO) nodules have good differential diagnostic efficacy: there was significant difference in the benign and malignant group two, the figures were 0 /mL and 1 /mL (P0.01); in the pGGO group of ROC curve in the differential diagnosis of AUC was 0.863 (P0.01), with 0 /mL as the threshold differential diagnostic sensitivity and specificity were 87.1% and 83.3%. by PET/CT were compared with that of CTC count to identify lung nodules in pGGO group were significantly better than the diagnosis PET/CT, the sensitivity and specificity were 54.8% and 66.7%, significantly lower than the CTC count (P=0.039). The second part of the study included 68 subjects (38 cases of adenocarcinoma, 30 cases of healthy subjects). The result showed that exosomes secreted expression concentration, the amount of EGFR protein in two group group showed no difference. The outer body of urinary CXCR4 protein expression and invasion of lung adenocarcinoma has a significant positive correlation, correlation coefficient and flow cytometric analysis of urinary CXCR4 fluorescence intensity and the invasion of lung adenocarcinoma and pathological score was 0.413 (P0.01). In addition, exosomes CXCR4 protein expression in metastasis transfer group and no significant difference between the groups (P0.04). Through ROC analysis, the expression of CXCR4 protein exosome exosome prediction rate and fluorescence intensity of CXCR4 in the metastasis of lung adenocarcinoma AUC were 0.697 (P=0.04) and 0.770 (P0.01); respectively to 7.4% and 1429.39 as the threshold, and the rate of exosomes the expression of CXCR4 protein The fluorescence intensity of the predictive sensitivity of lung cancer metastasis was 100%, and specificity were 40.9% and 59.1%. expression of lung adenocarcinoma by immunohistochemical staining and exosome protein, observed no consistent. In addition, exosomes related indicators of patients before and after surgery had no consistent trend, statistical analysis found before and after the operation changes of exosomes was not statistically significant. Conclusion this study by comparing the count of CTC and PET/CT in the differential diagnosis of benign and malignant lung nodules ability, found the technology CTC count has a differential diagnostic ability of pure ground glass opacity nodules, the differential diagnosis efficiency is even better than the last PET/CT., using quantitative protein flow cytometry of peripheral blood exosomes, found the expression of exosomes EGFR protein has no ability to diagnose lung cancer; the outer urinary body CXCR4 protein expression with invasion of lung cancer significantly Meanwhile, the quantitative expression of exocrine protein in the technology does not reflect the expression level of primary tumor protein, nor does it have the ability to monitor tumor burden in patients with lung adenocarcinoma.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

【相似文献】