Sensitive detection of highly expressed genes in lung cancer for diagnostic application using real-time PCR.

本研究旨在建立一种高灵敏度检测肺癌细胞的新方法，利用实时聚合酶链式反应(PCR)扩增肺癌中表达的基因。在第一年，我们优化了有效检测肺癌细胞中m-RNA表达的聚合酶链式反应条件。为此，我们使用癌胚抗原(CEA)作为肺癌细胞高表达的代表性抗原。我们比较了肺癌患者CEA的血清浓度、定位和mRNA表达，以确定这三个因素之间的关系。对10例手术患者的肿瘤进行了分析。在开始治疗前，采用定量乳胶凝集反应测定血清CEA浓度。采用免疫组织化学方法对手术切除的肿瘤标本进行CEA定位。采用实时荧光定量RT-PCR方法检测新鲜冰冻肿瘤组织中CEA基因的表达。…血清CEA阳性率为20%，免疫组化阳性率为60%，荧光定量RT-PCR法阳性率为80%。因此，肺癌组织中CEA的mRNA和蛋白阳性百分率明显高于血清CEA浓度。未发现血清CEA浓度与mRNA表达量之间存在显著的相关性。实时定量RT-PCR可以定量检测一小块组织中特定的mRNA表达。在接下来的一年里，我们选择了HER2、Ki-67、VEGF、细胞角蛋白19、EGFR等基因作为PCR扩增的目标。采集肺癌患者治疗前的外周血样。收集这些样本中的细胞，并提取RNA。当检测这6个遗传标记的表达时，我们检测到6例患者外周血中有癌细胞循环。疾病分期越高，阳性率越高。我们没有发现EGFR基因(外显子18、19和21)突变与外周血中EGFR阳性之间的任何关系。我们可以用实时荧光定量PCR检测外周血中的肺癌细胞。然而，为了更灵敏地检测癌细胞，还需要进一步的研究。较少

This study was designed to develop a new method for highly sensitive detection of lung cancer cells using real-time polymerase chain reaction (PCR) to amplify genes expressed in lung cancer. In the first year, we optimised the conditions of PCR for efficient detection of expressed m-RNA in lung cancer cells. For this purpose, we used carcinoembryonic antigen(CEA) as a representative antigen that was highly expressed in lung cancer cells. We compared the serum concentration, localization, and mRNA expression of CEA to determine the relationship between these three factors in patients with lung cancer. Tumors from ten patients who underwent surgery were analyzed. The serum concentration of CEA was measured before initiating therapy using a quantitative latex agglutination reaction. Immunohistochemical staining of pathologic specimens of resected tumors was performed to localize CEA. Real-time RT-PCR to detect mRNA of CEA was performed for isolated RNA from a piece of fresh-frozen tumor. … More Positivity for CEA production of was 20% for serum, 60% for immunohistochemistry, and 80% for real-time RT-PCR. Thus, the percentage of mRNA and protein positivity of CEA in lung cancer was much higher than for the serum CEA concentration. No statistically significant correlation between the serum CEA concentration and the amount of mRNA expression was found. Real-time RT-PCR is useful to quantify specific mRNA expression from a small piece of tissue.In the next year, we selected the genes, HER2,Ki-67,VEGF,cytokeratin 19,EGFR, in addition to CEA as targets for PCR amplification. Peripheral blood samples from pre-treated patients with lung cancers were collected. Cells in these samples were gathered, and RNAs were extracted. When expression of these 6 genetic markers were examined, we detected 6 patients having cancer cells circulating in peripheral blood. The positivity was higher in patients with higher disease stages. We did not find any relationship between mutations of EGFR genes (Exons 18,19, and 21) and EGFR positivity in peripheral blood.We can detect lung cancer cells in peripheral blood using real-time PCR. However, further studies are required for more sensitive detection of the cancer cells. Less