Abstract
Mutations in the p53 tumor-suppressor gene and K-ras oncogene have been frequently found in sputum and bronchoalveolar lavage samples of lung cancer patients and also in those of patients prior to presenting clinical symptoms of lung cancer, suggesting that they may provide useful biomarkers for early lung cancer diagnosis. However, the detection of these mutations 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 the p53 gene, inactivating mutations may occur at many codons. This chapter describes methods to identify p53 and K-ras mutations present in low fractions of epithelial cells among the excess of other cell types in sputum samples from lung cancer patients.
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References
Jemal A, Siegel R, Xu J, Ward E (2010) Cancer statistics, 2010. CA Cancer J Clin 60:277–300
Cancer Facts and Figures 2013 (2013) American Cancer Society
Birrer MJ, Brown PH (1992) Application of molecular genetics to the early diagnosis and screening of lung cancer. Cancer Res 52: 2658s–2664s
Scott A, Salgia R (2008) Biomarkers in lung cancer: from early detection to novel therapeutics and decision-making. Biomark Med 2: 577–586
Maldonado F, Jett JR (2010) Advances in the diagnosis of lung cancer: contribution of molecular biology to bronchoscopic diagnosis. Curr Opin Pulm Med 16:315–320
Vogelstein B, Lane B, Levine AJ (2000) Surfing the p53 network. Nature 408:307–310
Loeb LA, Harris CC (2008) Advances in chemical carcinogenesis: a historical review and prospective. Cancer Res 68:6863–6872
Suzuki Y, Orita M, Shiraishi M, Hayashi K, Sekiya T (1990) Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products. Oncogene 5: 1037–1043
Sugio K, Ishida T, Yokoyama H, Inoue T, Sugimachi K, Sasazuki T (1992) ras gene mutation as a prognostic marker in adenocarcinoma of the human lung without lymph node metastasis. Cancer Res 52:2903–2906
Rodenhuis S, Slebos RJ (1992) Clinical significance of ras oncogene activation in human lung cancer. Cancer Res 52:2665s–2669s
Husgafvel-Pursianen K, Hackman P, Ridanpaa M, Anttila S, Karjalainen A, Partanen T et al (1993) K-ras mutations in human adenocarcinoma of the lung: association with smoking and occupational exposure to asbestos. Int J Cancer 53:250–256
Keohavong P, DeMichele MA, Melacrinos AC, Landreneau RJ, Weyant RJ, Siegfried JM (1996) Detection of K-ras mutations in lung carcinomas: relationship to prognosis. Clin Cancer Res 2:411–418
Hollstein M, Sidransky D, Vogelstein B, Harris CC (1991) p53 mutations in human cancers. Science 253:49–53
Greenblatt MS, Bennett WP, Hollstein M, Harris CC (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 54: 4855–4878
Yokota J, Wada M, Shimosato Y, Terada M, Sugimura T (1987) Loss of heterozygosity on chromosomes 3, 13, and 17 in small-cell carcinoma and on chromosome 3 in adenocarcinoma of the lung. Proc Natl Acad Sci U S A 84: 9252–9256
Chiba I, Takahashi T, Nau MM, D’Amico D, Curiel DT, Mitsudomi T et al (1990) Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Oncogene 5:1603–1610
Kishimoto Y, Murakami Y, Shiraishi M, Hayashi K, Sekiya T (1992) Aberrations of the p53 tumor suppressor gene in human non-small cell carcinomas of the lung. Cancer Res 52:4799–4804
Takahashi T, Nau MM, Chiba I, Birrer MJ, Rosenberg RK, Vinocour M et al (1989) p53: a frequent target for genetic abnormalities in lung cancer. Science 246:491–494
Cho Y, Gorina S, Jeffrey PD, Pavletich NP (1994) Crystal structure of a p53 tumor suppressor-DNA complex: understanding tumorigenic mutations. Science 265:346–355
Hainaut P, Hollstein M (2000) p53 and human cancer: the first ten thousand mutations. Adv Cancer Res 77:81–137
Jassem E, Niklinski J, Rosell R, Niklinska W, Jakobkiewicz J, Monzo M et al (2001) Types and localisation of p53 gene mutations: a report on 332 non-small cell lung cancer patients. Lung Cancer 34:47s–51s
Caron de Fromentel C, Soussi T (1992) TP53 tumor suppressor gene: a model for investigating human mutagenesis. Genes Chromosomes Cancer 4:1–15
de Anta JM, Jassem E, Rosell R, Martinez-Roca M, Jassem J, Martinez-Lopez E et al (1997) TP53 mutational pattern in Spanish and Polish non-small cell lung cancer patients: null mutations are associated with poor prognosis. Oncogene 15:2951–2958
Cha RS, Zarbl H, Keohavong P, Thilly WG (1992) Mismatch amplification mutation assay (MAMA): application to the c-H-ras gene. PCR Methods Appl 2:14–20
Mitsudomi T, Viallet J, Mulshine JL, Linnoila RI, Minna JD, Gazdar AF (1991) Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines. Oncogene 6:1353–1362
Jiang W, Kahn SM, Guillem JG, Lu SH, Weinstein IB (1989) Rapid detection of ras oncogenes in human tumors: application to colon, esophageal, and gastric cancer. Oncogene 4:923–928
Kumar R, Dunn LL (1989) Designed diagnostic restriction fragment length polymorphism for the detection of point mutations in ras oncogenes. Oncogene Res 1:235–241
Levi S, Urbano-Ispizua A, Gill R, Thomas DM, Gilbertson J, Foster C et al (1991) Multiple K-ras 12 mutations in cholangiocarcinomas demonstrated with a sensitive polymerase chain reaction technique. Cancer Res 51: 3497–3502
Kahn SM, Jiang W, Culbertson TA, Weinstein IB, Williams GM, Tomita N et al (1991) Rapid and sensitive non-radioactive detection of mutant K-ras genes via enriched PCR amplification. Oncogene 6:1079–1083
Yakubovskaya MS, Spiegelman V, Luo FC, Malaev S, Salnev A, Zborovskaya I et al (1995) High frequency of K-ras mutations in normal appearing lung tissues and sputum of patients with lung cancer. Int J Cancer 63:810–814
Mills NE, Fishman CL, Scholes J, Anderson SE, Rom WN, Jacobson DR (1995) Detection of K-ras oncogene mutations in bronchoalveolar lavage fluid for lung cancer diagnosis. J Natl Cancer Inst 87:1056–1060
Mao L, Hruban RH, Boyle JO, Tockman M, Sidransky D (1994) Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer Res 54:1634–1637
Scott FM, Modali R, Lehman TA, Seddon M, Kelly K, Dempsey EC et al (1997) High frequency of K-ras codon 12 mutations in bronchoalveolar lavage fluid of patients at high risk for second primary lung cancer. Clin Cancer Res 3:479–482
Keohavong P, Zhu D, Whiteside TL, Swalsky P, Bakker A, Elder EM et al (1997) Detection of infrequent and multiple K-ras mutations in human tumors and tumor-adjacent tissues. Anal Biochem 247:394–403
Zhang LF, Gao WM, Gealy R, Weissfeld J, Elder E, Whiteside TL et al (2003) Comparison of K-ras gene mutations in tumor and sputum DNA of patients with lung cancer. Biomarkers 8:156–161
Acknowledgments
This work was supported by grants from the American Cancer Society (RPG-99-61-01-CNE and RSG-99-161-04-CNE).
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Gao, W., Keohavong, P. (2014). Detection of Point Mutations of K-ras Oncogene and p53 Tumor-Suppressor Gene in Sputum Samples. In: Keohavong, P., Grant, S. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 1105. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-739-6_26
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DOI: https://doi.org/10.1007/978-1-62703-739-6_26
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