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

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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