非小细胞肺癌患者手术前后循环肿瘤DNA检测研究

发布时间：2018-03-27 22:27

本文选题：非小细胞肺癌　切入点：二代测序　出处：《中国人民解放军医学院》2017年博士论文

【摘要】：研究背景及目的:近年,靶向治疗已成为非小细胞肺癌(non-small lung cancer,NSCLC)重要的治疗手段,通过对非小细胞肺癌患者肿瘤组织标本进行基因检测,根据基因检测结果选择有效的靶向药物进行有针对性的治疗已经被普遍接受,然而,要发现患者的靶向用药驱动基因就必须进行多基因检测,这样可以让患者获得更多的治疗机会,但是,一代测序因其复杂的检测过程过高的成本要实现多基因检测并不现实。二代基因检测技术的成熟为多基因检测提供了技术强有力的保障,用外周血中的循环肿瘤DNA(circulating tumor DNA,ctDNA)进行“液体活检”,样本相对于组织活检容易取得,而且可重复取样。ctDNA包含患者的基因突变信息,通过对外周血中的ctDNA进行重复检测可以监测患者的肿瘤进展、用药疗效、耐药突变等,对那些肿瘤无法切除或者穿刺无法取到的肺癌患者更有意义。用二代测序技术进行ctDNA液体活检,已开始进入临床应用。但目前,组织的多基因检测依然是金标准,ctDNA多基因检测用于临床的可靠性以及ctDNA能否作为非小细胞肺癌患者诊断和手术效果的判断指标,有待广泛的临床数据验证。本研究通过对非小细胞肺癌患者组织和血浆ctDNA检测,对两者的一致性、患者手术前后的ctDNA突变丰度变化等进行分析,来确证对血浆ctDNA测序选择靶向药物治疗是否可靠、血液ctDNA突变丰度变化是否与肿瘤变化程度相关、监测血液ctDNA变化判断肿瘤进展与目前监测肺癌标志物判断肺癌患者肿瘤进展是否更具有优势。方法:①收集41例非小细胞肺癌患者手术切除的原发肿瘤组织以及每位患者手术前、后的外周血,对上述标本进行基于Ion Proton二代测序平台的包括BRAF、EGFR、KRAS、TP53、ERBB2、PIK3CA等在内的50基因检测,观察上述肿瘤驱动基因的突变情况,对组织和血浆DNA的一致性进行比较分析;②取患者手术前的血清,用化学发光免疫分析方法检测6种血清标志物:CA125,CA19-9,CYFRA21-1,CEA,NSE和SCC,对比分析血清标志物数值与肿瘤的关系,并与手术前ctDNA检出率进行比较。结果:①外周血ctDNA与原发肿瘤组织驱动基因突变的一致性为78.1%,敏感性和特异性分别为69.2%和93.3%,阳性预测值(PPV)为94.7%。②手术后2天即有91.7%患者血中ctDNA突变的频率较手术前明显下降。③外周血ctDNA比目前临床应用的6种肿瘤生物标志物具有更高的阳性预测值。结论:靶向测序检测ctDNA可以检出非小细胞肺癌患者的突变基因,并反映患者手术治疗的效果,具有一定的可靠性,这种监测外周血中ctDNA变化的方法对NSCLC患者的临床管理具有一定的应用价值。
[Abstract]:Background and objective: in recent years, targeted therapy has become an important therapy for non-small lung lung cancer (NSCLC). It has been widely accepted to select effective targeted drugs for targeted therapy based on the results of gene detection. However, to find the driving genes of targeted drugs in patients, we must carry out multi-gene tests. This will give patients more access to treatment, but, Because of the high cost of the complex detection process, it is not realistic for a generation of sequencing to realize multi-gene detection. The maturity of the second-generation gene detection technology provides a strong technical guarantee for multi-gene detection. Using circulating tumor DNA(circulating tumor DNA in peripheral blood to perform "fluid biopsy", samples are easily obtained than tissue biopsies, and repeatable samples contain mutational information about the patient's genes. Repeated detection of ctDNA in peripheral blood can monitor tumor progression, drug efficacy, drug resistance mutation, and so on. It makes more sense for lung cancer patients whose tumors are unresectable or can't be punctured. CtDNA fluid biopsies using second-generation sequencing techniques have begun to be used clinically. Tissue polygenic detection is still the gold standard ctDNA polygene test for clinical reliability and whether ctDNA can be used as a diagnostic and surgical outcome in patients with non-small cell lung cancer (NSCLC). In this study, the consistency of ctDNA in tissues and plasma of patients with non-small cell lung cancer (NSCLC) and the changes of ctDNA mutation abundance before and after operation were analyzed. To confirm the reliability of selective drug therapy for plasma ctDNA sequencing, and whether the change in blood ctDNA mutation abundance is related to the degree of tumor change. Monitoring the changes of ctDNA in blood to judge the progress of tumor was superior to monitoring the markers of lung cancer. Methods 41 patients with non-small cell lung cancer were collected from 41 patients with non-small cell lung cancer and each patient was treated before surgery. In the peripheral blood of the patients, 50 genes, including BRAFFN EGFRX, TP53, ERBB2PIK3CA and so on, were detected on the basis of Ion Proton second generation sequencing platform, and the mutation of the tumor driving genes was observed. The consistency between tissue and plasma DNA was compared and analyzed. The serum samples were collected from patients before operation. Six serum markers, CYFRA21-9, CYFRA21-1 and SCC, were detected by chemiluminescence immunoassay, and the relationship between serum markers and tumor was compared and analyzed. The detection rate of ctDNA was compared with that before operation. Results the consistency between the mutation of ctDNA and the driving gene of primary tumor tissue was 78.1, the sensitivity and specificity were 69.2% and 93.33.The positive predictive value was 94.72.The positive predictive value was 91.7% 2 days after operation. The frequency of ctDNA mutation in human blood was significantly lower than that before operation. 3. 3 ctDNA in peripheral blood was significantly lower than that of 6 tumor biomarkers in clinical use. Conclusion: the detection of ctDNA by targeted sequencing can detect non-small cell lung cancer. The patient's mutant gene, The method of monitoring the changes of ctDNA in peripheral blood is valuable for clinical management of patients with NSCLC.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R734.2

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