Dickkopf-1自身抗体亚型在非小细胞肺癌诊断和预后判断中的应用

发布时间：2018-06-22 05:23

本文选题：非小细胞肺癌 + 自身抗体　；参考：《北京市结核病胸部肿瘤研究所》2017年硕士论文

【摘要】：背景:肺癌的早期诊断和筛查对预防肺癌远期进展和改善预后具有重要意义。LDCT(Low-dose Computed Tomography,胸部低剂量计算机断层扫描)是目前临床用于肺癌高危人群筛查的主要手段,但是存在的一些问题不可忽视,如假阳性率高、过度诊断、辐射暴露风险等。寻找高灵敏度和特异度的非侵入性的检查方法是肺癌筛查和早期诊断研究的新趋势。血清标志物如肿瘤相关抗原和自身抗体成为肺癌早期诊断领域的研究热点。在肿瘤早期,机体的免疫系统能够识别异常表达的肿瘤抗原,激活免疫应答,产生大量自身抗体。自身抗体在血液中半衰期较长,在肿瘤早期就能检测到较高滴度的自身抗体,因此可能作为早期诊断标志物。自身抗体在肿瘤发生、发展过程中同样具有免疫监视的作用。研究发现在多种类型肿瘤中,自身抗体可能与疾病预后有关,但目前研究结论尚不统一,作用机制也并不清楚。因此自身抗体对肿瘤的早期诊断和预后判断的作用值得进一步深入研究。目的:本研究利用多肽阵列技术检测NSCLC(Non-small cell lung cancer,非小细胞肺癌)患者和健康者血清中DKK1(Dickkopf-1)自身抗体识别的抗原表位,再根据识别不同的抗原表位将DKK1自身抗体分为不同的亚型,探索不同的DKK1自身抗体亚型在NSCLC诊断和预后判断中的作用。方法:应用多肽合成仪合成DKK1肽库,分别使用9组(n=72)NSCLC患者混合血清和2组(n=16)健康人血清与肽库进行免疫学筛选,筛选出抗原表位中的高频位点。利用固相合成技术把筛选出的优势抗原表位合成多肽。应用竞争性ELISA(Enzyme Linked Immunosorbent Assay,酶联免疫吸附试验)验证血清中的DKK1自身抗体能够与合成的多肽特异性结合,再利用间接多肽ELISA法检测206例NSCLC患者和99例健康人血清中DKK1自身抗体的水平。分析DKK1自身抗体亚型与临床病理因素之间的关系,以及在NSCLC中的诊断价值和预后价值。结果:1、多肽阵列结果显示,有四个高频位点被Dickkopf-1自身抗体所识别,分别为aa67-84(Pep A)、aa37-54(Pep B)、aa145-156(Pep C)、aa247-261(Pep D)。通过比对自身抗体识别的高频位点和DKK1的氨基酸序列,确定四种自身抗体识别的高频位点在DKK1中的空间位置分布,为进一步研究DKK1自身抗体的功能提供新的线索。2、间接ELISA结果显示,四种DKK1自身抗体亚型在NSCLC患者血清中的水平明显高于正常人(P0.001),在M0期患者中Pep B、Pep C、Pep D三种DKK1自身抗体亚型明显高于M1期(P=0.007、P=0.007、P=0.001),而Pep A亚型的DKK1自身抗体在M0和M1期中的差异无统计学意义(P0.05)。3、在多个临床病理因素中,Pep B、Pep C、Pep D三种DKK1自身抗体亚型均与NSCLC的TNM分期(P=0.001;P=0.001;P=0.002)和远处转移(P=0.007;P=0.007;P=0.001)相关。4、DKK1自身抗体作为NSCLC的诊断标记物,四种DKK1自身抗体亚型的AUC(Area Under ROC Curve,受试者工作特征曲线下面积)分别是0.744(95%CI=0.684-0.799),0.809(95%CI=0.756-0.854),0.740(95%CI=0.684-0.791),0.767(95%CI=0.712-0.814),四者之间的AUC无统计学差异(P0.05)。四种DKK1自身抗体亚型联合检测的AUC为0.821(95%CI=0.764-0.868),敏感性和特异性分别是58.1%和85.3%。在I期和II期NSCLC中,四种亚型的DKK1自身抗体AUC分别是0.706(95%CI=0.611-0.790)、0.808(95%CI=0.732-0.871)、0.723(95%CI=0.640-0.796)、0.780(95%CI=0.704-0.843),四种亚型的DKK1自身抗体联合检测的AUC为0.818(95%CI=0.719-0.894),敏感性和特异性分别为76.9%和75.9%。5、生存分析显示,Pep B自身抗体阳性的患者OS(Overall Survival,总生存期)和PFS(Progression-free survival,无进展生存期)明显高于自身抗体阴性的患者(P=0.004;P=0.006)。而其他自身抗体亚型与患者预后不相关。COX比例风险回归模型提示,Pep B自身抗体是肺癌患者的独立预后因素(OS:P=0.008,HR=0.435,95%CI:0.236-0.802;PFS:P=0.032,HR=0.533,95%CI:0.322-0.950)。结论:四种DKK1自身抗体亚型在NSCLC中的表达明显上调,有可能成为NSCLC的诊断标记物。Pep B自身抗体与远处转移和临床分期有关,与NSCLC的总生存期和无进展生存期呈正相关,是NSCLC良好的独立预后因素,提示Pep B自身抗体可能在NSCLC的发生和发展中发挥重要作用。
[Abstract]:Background: early diagnosis and screening of lung cancer is of great significance for the prevention of long term progress and improvement of the prognosis of lung cancer.LDCT (Low-dose Computed Tomography, low dose computed tomography) is the main method for screening high risk population of lung cancer at present, but there are some problems that can not be ignored, such as high false positive rate and overdiagnosis. Detection of high sensitivity and specificity is a new trend in screening and early diagnosis of lung cancer. Serum markers such as tumor related antigens and autoantibodies have become a hot spot in the field of early diagnosis of lung cancer. In the early stage of the tumor, the immune system of the body can identify abnormal expression. The tumor antigen, which activates the immune response, produces a large number of autoantibodies. The autoantibody has a longer half life in the blood and can detect the high titer autoantibody at the early stage of the tumor. Therefore, it may be used as an early diagnostic marker. The autoantibodies are also immune monitoring during the development of the tumor. In the tumor, the autoantibodies may be related to the prognosis of the disease, but the research conclusions are not uniform and the mechanism of action is not clear. Therefore, the role of autoantibodies in the early diagnosis and prognosis of tumor should be further studied. Objective: to detect NSCLC (Non-small cell lung cancer, non small cell lung) by using polypeptide array technique. The antigen epitopes identified by DKK1 (Dickkopf-1) autoantibodies in the cancer patients and healthy persons, and then the differentiation of DKK1 autoantibodies into different subtypes according to the identification of different epitopes, explore the role of different DKK1 autoantibody subtypes in the diagnosis and prognosis of NSCLC. Methods: the peptide library was synthesized by the peptide synthesizer, and 9 groups were used respectively. N=72) NSCLC patients' mixed serum and 2 groups of (n=16) healthy human serum and peptide libraries were screened to screen out high frequency loci in the epitopes. The dominant antigen epitopes were synthesized by solid phase synthesis technique. The competitive ELISA (Enzyme Linked Immunosorbent Assay, ELISA test) was used to verify the serum DKK1 The autoantibody could be combined with the specific peptide of synthetic peptide, and then the level of DKK1 autoantibody in 206 cases of NSCLC and 99 healthy human serum was detected by indirect polypeptide ELISA. The relationship between the DKK1 autoantibody subtype and the clinicopathological factors, and the diagnostic value and prognostic value in NSCLC were analyzed. Results: 1, the peptide array results showed. Four high frequency loci were identified by Dickkopf-1 autoantibodies, including aa67-84 (Pep A), aa37-54 (Pep B), aa145-156 (Pep C), aa247-261 (Pep). The spatial location of the high frequency loci of the four kinds of anti body recognition was determined by comparing the high frequency loci of the autoantibodies and the amino acid sequences. The function of DKK1 autoantibody provides a new clue.2. The indirect ELISA results show that the level of the four DKK1 autoantibody subtypes in the serum of NSCLC patients is significantly higher than that of normal people (P0.001), Pep B, Pep C in M0 phase patients, and three types of autoantibodies in the M0 phase. The differences in M0 and M1 were not statistically significant (P0.05).3. Among the multiple clinicopathological factors, Pep B, Pep C and Pep D were all associated with NSCLC's stages and its antibodies as diagnostic markers, and four kinds of autoantibodies. Type AUC (Area Under ROC Curve, the area under the working characteristic curve of the subjects) was 0.744 (95%CI=0.684-0.799), 0.809 (95%CI=0.756-0.854), 0.740 (95%CI=0.684-0.791), 0.767 (95%CI=0.712-0.814), and there was no statistical difference between the four (P0.05). Four kinds of DKK1 autoantibody subtypes were 0.821 (0.821). The sensitivity and specificity were 58.1% and 85.3%. in I and II NSCLC respectively. The DKK1 autoantibody AUC of the four subtypes was 0.706 (95%CI=0.611-0.790), 0.808 (95%CI=0.732-0.871), 0.723 (95%CI=0.640-0.796), 0.780 (95%CI=0.704-0.843), and four subtypes of DKK1 autoantibodies. The sensitivity and sensitivity were 0.818. The specificity was 76.9% and 75.9%.5, and the survival analysis showed that OS (Overall Survival, total survival) and PFS (Progression-free survival, progression free survival) of patients with Pep B autoantibody were significantly higher than those with autoantibody negative (P=0.004; P=0.006). The subtype of his autoantibody was not associated with the prognosis of the patient's prognosis. It is suggested that Pep B autoantibodies are independent prognostic factors of lung cancer patients (OS:P=0.008, HR=0.435,95%CI:0.236-0.802; PFS:P=0.032, HR=0.533,95%CI:0.322-0.950). Conclusion: the expression of four DKK1 autoantibody subtypes in NSCLC is obviously up-regulated, and may become a diagnostic marker for NSCLC.Pep B autoantibody and distant metastasis and clinical classification. It is related to the total survival and progression free survival of NSCLC, which is a good independent prognostic factor for NSCLC, suggesting that Pep B autoantibodies may play an important role in the development and development of NSCLC.
【学位授予单位】：北京市结核病胸部肿瘤研究所
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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