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利用实时聚合酶链反应（PCR）扩增肺癌中表达基因的高灵敏度检测肺癌细胞的新方法。在第一年，我们优化了高效检测肺癌细胞中表达的m-RNA的PCR条件。为此，我们选用癌胚抗原（CEA）作为在肺癌细胞中高表达的代表性抗原。我们比较了肺癌患者血清中CEA的浓度、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。在开始治疗前用定量胶乳凝集反应测定血清CEA浓度。对切除肿瘤病理标本进行免疫组化染色定位CEA。采用实时RT-PCR法对新鲜冷冻肿瘤分离RNA进行CEA mRNA检测。血清CEA阳性率为20%，免疫组化阳性率为60%，实时RT-PCR阳性率为80%。因此，肺癌组织中CEA mRNA和蛋白的阳性比例远高于血清中CEA浓度的阳性比例。血清CEA浓度与mRNA表达量无统计学意义。实时RT-PCR可用于定量小块组织中特定mRNA的表达。在接下来的一年里，我们选择了HER2、Ki-67、VEGF、细胞角蛋白19、EGFR以及CEA作为PCR扩增的靶点。收集治疗前肺癌患者外周血样本。收集这些样本中的细胞，提取rna。当检测这6种基因标记的表达时，我们检测到6例外周血中有癌细胞循环的患者。疾病分期越高，阳性率越高。我们没有发现EGFR基因突变（外显子18、19和21）与外周血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，需要进一步的研究来更灵敏地检测癌细胞。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