Skip to main content
SpringerLink
Log in
Book cover

Lung Cancer pp 325–333Cite as

Detection of K-ras and p53 Mutations by “Mutant-Enriched” PCR-RFLP

  • Protocol

  • 704 Accesses

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 75))

Abstract

Early diagnosis of lung cancer is critical, as most cases are already inoperable at the time of diagnosis, and thus bear a grave prognosis. With increasing knowledge of the genetic aspects of lung cancer, the field has also experienced an increasing number of potential markers that might serve in the detection of changes in the lung epithelium that predispose to cancer (reviewed in refs. 1–3). Of these, oncogene mutations were among the first genetic biomarkers to be studied extensively, since they fulfill many criteria that link their de novo appearance to the process of field cancerization (4). Oncogene mutations of the ras- and p53-type are found in chronic smokers and patients with preneoplastic and neoplastic lesions but almost never in normal lung (4). In animal models carcinogens present in tobacco smoke have been shown to induce typical G-T transversions that lead to missense mutations (5). Moreover it has been shown that smoking leads to particular types of mutations, many of which are concentrated in particular hot spots, thus making it plausible that the same spectrum also arises in man on continuous exposure to tobacco carcinogens.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Sekido, Y., Fong, K. M., and Minna, J. D. (1998) Progress in understanding the molecular pathogenesis of human lung cancer. Biochim. Biophys. Acta 1378, F21–F59.

    PubMed  CAS  Google Scholar 

  2. Salgia, R. and Skarin, A. T. (1998) Molecular abnormalities in lung cancer. J. Clin. Oncol. 16, 1207–1217.

    PubMed  CAS  Google Scholar 

  3. Sozzi, G. and Carney, D. (1998) Molecular biology of lung cancer. Curr. Opin. Pulm. Med. 4, 207–212.

    Article  PubMed  CAS  Google Scholar 

  4. Fong, K. M., Sekido, Y., and Minna J. D. (1999) Molecular pathogenesis of lung cancer. J. Thorac. Cardiovasc. Surg. 118, 1136–1152.

    Article  PubMed  CAS  Google Scholar 

  5. Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. (1996) Preferential formation of benzo[a]pyrene adducts at lung cancer mutational hotspots in P53. Science 274, 430–432.

    Article  PubMed  CAS  Google Scholar 

  6. Rodenhuis, S. and Slebos, R. J. (1992) Clinical significance of ras oncogene activation in human lung cancer. Cancer Res. 52, 2665s–2669s.

    PubMed  CAS  Google Scholar 

  7. Husgafvel-Pursiainen, 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.

    Article  PubMed  CAS  Google Scholar 

  8. Li, S., Rosell, R., Urban, A., Font, A., Ariza, A., Armengol, P., et al. (1994) K-ras gene point mutation: a stable tumor marker in non-small cell lung carcinoma. Lung Cancer 11, 19–27.

    Article  PubMed  CAS  Google Scholar 

  9. Mao, L., Hruban, R. H., Boyle, J. O., Tockman, M., and Sidransky, D. (1994) Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer Res. 54, 1634–1637.

    PubMed  CAS  Google Scholar 

  10. Mills, N. E., Fishman, C. L., Rom, W. N., Dubin, N., and Jacobson, D. R. (1995) Increased prevalence of K-ras oncogene mutations in lung adenocarcinoma. Cancer Res. 55, 1444–1447.

    PubMed  CAS  Google Scholar 

  11. Mitsudomi, T., Viallet, J., Mulshine, J. L., Linnoila, R. I,. Minna, J. D., and Gazdar, A. F. (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.

    PubMed  CAS  Google Scholar 

  12. Rodenhuis, S., Slebos, R. J., Boot, A. J., Evers, S. G., Mooi, W. J., Wagenaar, S. S., et al. (1988) Incidence and possible clinical significance of K-ras oncogene activation in adenocarcinoma of the human lung. Cancer Res. 48, 5738–5741.

    PubMed  CAS  Google Scholar 

  13. Rodenhuis, S. and Slebos, R. J. (1990) The ras oncogenes in human lung cancer. Am. Rev. Respir. Dis. 142, 27–30.

    Article  Google Scholar 

  14. Chiba, I., Takahashi, T., Nau, M. M., D’Amico, D., Curiel, D. T., Mitsudomi, T., et al. (1990) Mutations in the p53 gene are frequent in primary, resected non-small cell lung cancer. Lung Cancer Study Group. Oncogene 5, 1603–1610.

    PubMed  CAS  Google Scholar 

  15. Marchetti, A., Buttitta, F., Pellegrini, S., Campani, D., Diella, F., Cecchetti, D., et al. (1993) p53 mutations and histological type of invasive breast carcinoma. Cancer Res. 53, 4665–4669.

    PubMed  CAS  Google Scholar 

  16. Takahashi, T., Suzuki, H., Hida, T., Sekido, Y., Ariyoshi, Y., and Ueda, R. (1991) The p53 gene is very frequently mutated in small-cell lung cancer with a distinct nucleotide substitution pattern. Oncogene 6, 1775–1778.

    PubMed  CAS  Google Scholar 

  17. Top, B., Mooi, W. J., Klaver, S. G., Boerrigter, L., Wisman, P., Elbers, H. R., et al. (1995) Comparative analysis of p53 gene mutations and protein accumulation in human non-small-cell lung cancer. Int. J. Cancer 64, 83–91.

    Article  PubMed  CAS  Google Scholar 

  18. Greenblatt, M. S., Bennett, W. P., Hollstein, M., and Harris, C. C. (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res. 54, 4855–4878.

    PubMed  CAS  Google Scholar 

  19. Sugio, K., Kishimoto, Y., Virmani, A. K., Hung, J. Y., and Gazdar A. F. (1994) K-ras mutations are a relatively late event in the pathogenesis of lung carcinomas. Cancer Res. 54, 5811–5815.

    PubMed  CAS  Google Scholar 

  20. Fontanini, G., Vignati, S., Bigini, D., Merlo, G. R., Ribecchini, A., Angeletti, C. A., et al. (1994) Human non-small cell lung cancer: p53 protein accumulation is an early event and persists during metastatic progression. J. Pathol. 174, 23–31.

    Article  PubMed  CAS  Google Scholar 

  21. Sozzi, G., Miozzo, M., Donghi, R., Pilotti, S., Cariani, C. T., Pastorino, U., et al. (1992) Deletions of 17p and p53 mutations in preneoplastic lesions of the lung. Cancer Res. 52, 6079–6082.

    PubMed  CAS  Google Scholar 

  22. Walker, C., Robertson, L. J., Myskow, M. W., Pendleton, N., and Dixon, G. R. (1994) p53 expression in normal and dysplastic bronchial epithelium and in lung carcinomas. Br. J. Cancer 70, 297–303.

    Article  PubMed  CAS  Google Scholar 

  23. Orita, M., Suzuki, T., and Hayashi, K. (1989) Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5, 874–879.

    Article  PubMed  CAS  Google Scholar 

  24. Takahashi, N., Hiyama, K., Kodaira, M., and Satoh, C. (1990) An improved method for the detection of genetic variations in DNA with denaturing gradient gel electrophoresis. Mutat. Res. 234, 61–70.

    Article  PubMed  CAS  Google Scholar 

  25. Anwar, K., Nakakuki, K., Shiraishi, T., Naiki, H., Yatani, R., and Inuzuka, M. (1992) Presence of ras oncogene mutations and human papillomavirus DNA in human prostate carcinomas. Cancer Res. 52, 5991–5996.

    PubMed  CAS  Google Scholar 

  26. Yashiro, T., Fulton, N., Hara, H., Yasuda, K., Montag, A., Yashiro, N., et al. (1993) Comparison of mutations of ras oncogene in human pancreatic exocrine and endocrine tumors. Surgery 114, 758–763.

    PubMed  CAS  Google Scholar 

  27. Lepage, V., Ivanova, R., Loste, M.N., Mallet, C., Douay, C., Naoumova, E., and Charron, D. (1995) Determination of DQB1 alleles using PCR amplification and allele-specific primers. Eur. J. Immunogenet. 22, 413–422.

    Article  PubMed  CAS  Google Scholar 

  28. Ørum, H., Nielsen, P. E., Egholm, M., Berg, R. H., Buchardt, O., and Stanley, C. (1993) Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Res. 21, 5332–5336.

    Article  PubMed  Google Scholar 

  29. Thiede, C., Bayerdorffer, E., Blasczyk, R., Wittig, B., and Neubauer, A. (1996) Simple and sensitive detection of mutations in the ras proto-oncogenes using PNA-mediated PCR clamping. Nucleic Acids Res. 24, 983–984.

    Article  PubMed  CAS  Google Scholar 

  30. Behn, M., Thiede, C., Neubauer, A., Pankow, W., and Schuermann, M. (2000) Facilitated detection of oncogene mutations from exfoliated tissue material by a PNA-mediated ‘enriched PCR’ protocol. J. Pathol. 190, 69–75.

    Article  PubMed  CAS  Google Scholar 

  31. Rhodes, C. H., Honsinger, C., Porter, D.M., and Sorenson, G. D. (1997) Analysis of the allele-specific PCR method for the detection of neoplastic disease. Diagn. Mol. Pathol. 6, 49–57.

    Article  PubMed  CAS  Google Scholar 

  32. Kahn, S. M., Jiang, W., Culbertson, T. A., Weistein, I. B., Williams, G. M., Tomita, N., and Ronai, Z. (1991) Rapid and sensitive nonradioactive detection of mutant K-ras genes via ‘enriched’ PCR amplification. Oncogene 6, 1079–1083.

    PubMed  CAS  Google Scholar 

  33. Levi, S., Urbano-Ispizua, A., Gill, R., Thomas, D. M., Gilbertson, J., Foster, C., and Marshall, C. J. (1991) Multiple K-ras codon 12 mutations in cholangiocarcinomas demonstrated with a sensitive polymerase chain reaction technique. Cancer Res. 51, 3497–3502.

    PubMed  CAS  Google Scholar 

  34. Jacobson, D. R. and Mills, N. E. (1994) A highly sensitive assay for mutant ras genes and its application to the study of presentation and relapse genotypes in acute leukemia. Oncogene 9, 553–563.

    PubMed  CAS  Google Scholar 

  35. Sugio, K., Kishimoto, Y., Virmani, A. K., Hung, J. Y., and Gazdar, A. F. (1994) K-ras mutations are a relatively late event in the pathogenesis of lung carcinomas. Cancer Res. 54, 5811–5815.

    PubMed  CAS  Google Scholar 

  36. Mills, N. E., Fishman, C. L., Scholes, J., Anderson, S. E., Rom, W. N., and Jacobson, D. R. (1995) Detection of K-ras oncogene mutations in bronchoalveolar lavage fluid for lung cancer diagnosis. J. Natl. Cancer Inst. 87, 1056–1060.

    Article  PubMed  CAS  Google Scholar 

  37. Behn, M., Qun, S., Pankow, W., Havemann, K., and Schuermann, M. (1998) Frequent detection of ras and p53 mutations in brush cytology samples from lung cancer patients by a restriction fragment length polymorphism-based “enriched PCR” technique. Clin. Cancer Res. 4, 361–371.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Hematology, Oncology and Immunology, Phillipps-University of Marburg, Marburg, Germany

    Marcus Schuermann

Authors
  1. Marcus Schuermann
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Children#x2019;s Hospital, Los Angeles, CA

    Barbara Driscoll phd

Rights and permissions

Reprints and permissions

Copyright information

© 2003 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Schuermann, M. (2003). Detection of K-ras and p53 Mutations by “Mutant-Enriched” PCR-RFLP. In: Driscoll, B. (eds) Lung Cancer. Methods in Molecular Medicine™, vol 75. Humana Press, Totowa, NJ. https://doi.org/10.1385/1-59259-324-0:325

Download citation

  • DOI: https://doi.org/10.1385/1-59259-324-0:325

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-0-89603-920-9

  • Online ISBN: 978-1-59259-324-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation