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DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS
Citation:
Keohavong, P., W. Gao, K. Zheng, Q. Lan, J L. Mumford, H. Mady, AND M. Melhem. DETECTION OF K-RAS AND P53 MUTATIONS IN SPUTUM SAMPLES OF LUNG CANCER PATIENTS USING LASER CAPTURE MICRODISSECTION MICROSCOPE AND MUTATION ANALYSIS. ANALYTICAL BIOCHEMISTRY. Elsevier Science BV, Amsterdam, Netherlands, 324(1):92-99, (2004).
Description:
Detection of K-ras and p53 Mutations in Sputum Samples of Lung Cancer Patients Using Laser Capture Microdissection Microscope and Mutation Analysis
Phouthone Keohavong a,*, Wei-Min Gao a, Kui-Cheng Zheng a, Hussam Mady b, Qing Lan c, Mona Melhem b, and Judy Mumford d.
a Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA 15260
b Department of Pathology, School of Medicine, University of Pittsburgh and Veterans Administration Health Care System, Pittsburgh, PA 15240
c Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, Bethesda, Maryland 20892
d National Health and Environmental Effects Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711
* Correspondence to: Phouthone Keohavong, Department of Environmental and Occupational Health, 3343 Forbes Avenue, Pittsburgh, PA 15260. Telephone: 412-383-2087. Telefax: 412-383-2123. E-mail: pho1@pitt.edu.
Category: DNA recombination technique and nucleic acids
Short title: Analysis of K-ras and p53 mutations in sputum
Keywords: lung cancer, sputum, laser capture microdissection, K-ras and p53 mutations
Abstract
Mutations in the p53 tumor suppressor gene and K-ras oncogene have been frequently found in sputum and bronchoalveolar lavage (BAL) samples of lung cancer patients and also in those of patients prior to presenting clinical symptoms of lung cancer, suggesting they may provide useful biomarkers for early lung cancer diagnosis. However, the detection of these gene mutations in sputum and BAL samples has been complicated by the fact that they often occur in only a small fraction of epithelial cells among sputum cells and, in the case of p53 gene, at many codons. In this study, sputum cells were collected on a filter membrane by sputum cyto-centrifugation and morphologically analyzed. Epithelial cells were selectively taken by using a laser capture microdissection microscope and analyzed by polymerase chain reaction (PCR) and single-stranded conformational polymorphism (SSCP), for p53 mutations, and PCR and denaturing gradient gel electrophoresis (DGGE), for K-ras mutations. This method was applied to analyze sputum of 15 Chinese women with lung cancer from Xuan Wei County, China, and detected mutations in sputum of 7 (46.7%) patients, including 5 patients with each a p53 mutation, one patient with a K-ras mutation, and one patient with a K-ras and a p53 mutations. For comparison, only two of the mutations were detected by conventional methods. Therefore, the laser capture/mutation analysis method is sensitive and facilitates the detection of low fraction mutations occurring in throughout the p53 gene and K-ras gene in sputum of lung cancer patients. This method may be applicable to analyze epithelial cells from clinically normal sputum or BAL samples from individuals with a high risk for developing lung cancer.
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