新型外周血分子标志物组合在非小细胞肺癌早期诊断中的研究

The poor prognosis of non-small cell lung cancer (NSCLC) can be greatly improved by novel early detection strategy. In order to develop such a new detection system, in this project, we will combinedly analyze tumor-associated autoantibodies along with cell-free DNA hypermethelation profiles as biomarkers for NSCLC early detection. Since tumor-associated autoantibodies originate from overexpression of tumor-associated proteins during the process of immune surveillance and DNA hypermethylation triggers the cancer development by.inhibiting tumor-suppressor genes, we could achieve much more sensitivity by measuring these two key complementary factors. The PI of this project has successfully developed a platform using phage-display and protein microarray that is capable in identifying autoantibodies for detection of NSCLC, and also mastered the MIRA technique for DNA methylation profiles using for NSCLC detection as well. In our preliminary study, combined analysis of these two types of biomarkers has achieved sensitivity of 92% and specificity of 97% in detection of 50 NSCLC and 50 normal serum samples, which was much more accurate than using any single type of biomarkers. Therefore, in this study, we will further identify and validate autoantibody and DNA methylation biomarkers for combined use in early detection of NSCLC through testing a large cohort of existing clinical serum samples. Meanwhile, we will also test our novel biomarkers for the ability in distinguishing different stages of NSCLC, and risk factors.

非小细胞肺癌（NSCLC）的早期诊断是提高患者生存率的关键。申请人拟利用血清中的肿瘤相关自身免疫抗体和游离DNA甲基化构象进行联合检测，开发出全新的早期NSCLC血液诊断体系。自身免疫抗体可对高表达的癌基因蛋白进行检测，而DNA高甲基化构象则是抑癌基因失活的标志物，通过对二者联合检测的互补性来提高对癌症早期诊断的准确率。申请人前期运用噬菌体展示及蛋白芯片技术开发出了NSCLC自身抗体诊断体系，同时也掌握了领先的DNA甲基化MIRA芯片检测技术。预实验表明通过对这两类分子标志物的联合检测分析，对50份NSCLC和50份正常血清样本诊断的敏感性达到92%特异性达97%，准确率高于任何单一方法。本项目中，我们将对大量已存的临床血清样本进行蛋白免疫学和表观遗传学的高量筛选，并利用统计学方法对新型的自身抗体和DNA甲基化联合构象进行整合分析，从而实现对NSCLC精准的早期诊断，降低患者的死亡率。

肺癌是我国发病率和死亡率最高的恶性肿瘤，早期诊断被认为是治疗肺癌及提高患者生存率的关键。本项目利用肿瘤基因高表达所引起的自身免疫抗体与DNA高甲基化所引起的抑癌基因低表达存在的互补性，在前期自身免疫抗体标志物对NSCLC诊断的研究基础上结合血清中游离DNA甲基化的构像来构建新型分子标志物联合诊断体系，以实现对NSCLC精准的早期诊断，同时探索新型血液分子标志物在NSCLC筛查、分期及风险评估等方面的应用价值。通过对NSCLC噬菌体展示文库的筛查得到了LHX2, LHX4, PAX7, HBXOB13, DLEC1, GAGE7六种肿瘤相关蛋白，通过血清中自身免疫抗体对这6种蛋白的反应，我们获到了敏感性86.3%、特异性87.5%的诊断率。进一步筛查血清中NSCLC相关DNA甲基化标志物，我们得到了血清中LHX2、 LHX4、DLECI、CDH13、RASSFIA、 SEPT96种甲基化组合标志物，对肺癌的诊断效能达到敏感性86.3%，特异性87.5%准确性。通过使用600份血清样本对这两类标志物的进行双盲联合检测，我们最终得到了敏感性 92%，特异性93.5% （正常人样本）和特异性86%（非肺癌肺病样本）的诊断准确度，高于目前临床上所应用的诊断标志物。接下来我们将与多家医院进行合作，结合传统的影像学检测方法，进一步展开临床验证，同时进一步完善我们的肿瘤标志物组合，为尽快推广到临床应用打好基础。