Detection and Characterization of Oncogene Mutations in Preneoplastic and Early Neoplastic Lesions

  • Toshinari MinamotoEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2102)


While it has been more than 30 years since its discovery, the ras family of genes has not yet lost its impact on basic and clinical oncology. These genes remain central to the field of molecular oncology as tools for investigating carcinogenesis and oncogenic signaling, as powerful biomarkers for the identification of those who have or are at high risk of developing cancer, and as oncogene targets for the design and development of new chemotherapeutic drugs. Mutational activation of the K-RAS proto-oncogene is an early event in the development and progression of the colorectal, pancreatic, and lung cancers that are the major causes of cancer death in the world. The presence of point mutational “hot spots” at sites necessary for the activation of this proto-oncogene has led to the development of a number of highly sensitive PCR-based methods that are feasible for the early detection of K-RAS oncogene mutations in the clinical setting. In light of these facts, mutation at the K-RAS oncogene has the potential to serve as a useful biomarker in the early diagnosis and risk assessment of cancers with oncogenic ras signaling. This chapter describes a highly sensitive method for detecting mutant K-RAS, enriched PCR, and its application to early detection of alterations in this oncogene in preneoplastic and early neoplastic lesions of the colon and rectum.

Key words

Oncogene K-RAS Enriched PCR Molecular diagnosis Risk assessment Biomarker Colorectal cancer Preneoplastic lesion Aberrant crypt foci 


  1. 1.
    Hanahan D, Weinberg RA (2011) Hallmarks of cancer: the next generation. Cell 144:646–674CrossRefGoogle Scholar
  2. 2.
    Vogelstein B, Papadopoulos N, Velculescu VE, Zhou S, Daiz LA Jr, Kinzler KW (2013) Cancer genome landscape. Science 339:1546–1558CrossRefGoogle Scholar
  3. 3.
    Martincorena I, Campbell PJ (2015) Somatic mutation in cancer and normal cells. Science 349:1483–1489CrossRefGoogle Scholar
  4. 4.
    Garber JE, Offit K (2005) Hereditary cancer predisposition syndromes. J Clin Oncol 23:276–292CrossRefGoogle Scholar
  5. 5.
    Jinesh GG, Sambandam V, Vijayaraghavan S, Balaji K, Mukherjee S (2018) Molecular genetics and cellular events of K-Ras-driven tumorigenesis. Oncogene 37:839–846CrossRefGoogle Scholar
  6. 6.
    Grabocka E, Commisso C, Bar-Sagi D (2015) Molecular pathways: targeting the dependence of mutant RAS cancers on the DNA damage response. Clin Cancer Res 21:1243–1247CrossRefGoogle Scholar
  7. 7.
    Carvalho PD, Guimarães CF, Cardoso AP, Mendonça S, Costa ÂM, Oliveira MJ, Velho S (2018) KRAS oncogenic signaling extends beyond cancer cells to orchestrate the microenvironment. Cancer Res 78:7–14CrossRefGoogle Scholar
  8. 8.
    Prior IA, Levis PD, Mattos C (2012) A comprehensive survey of Ras mutations in cancer. Cancer Res 72:2457–2467CrossRefGoogle Scholar
  9. 9.
    Minamoto T, Mai M, Ronai Z (2000) K-ras mutation: early detection in molecular diagnosis and risk assessment of colorectal, pancreas, and lung cancers–a review. Cancer Detect Prev 4:1–12Google Scholar
  10. 10.
    van Mansfeld ADM, Bos JL (1992) PCR-based approaches for detection of mutated ras genes. PCR Methods Appl 1:211–216CrossRefGoogle Scholar
  11. 11.
    Ronai Z, Yakubovskaya M (1995) PCR in clinical diagnosis. J Clin Lab Anal 9:269–283CrossRefGoogle Scholar
  12. 12.
    Kahn SM, Jiang W, Culbertson TA et al (1991) Rapid and sensitive nonradioactive detection of mutant K-ras genes via ‘enriched’ PCR amplification. Oncogene 6:1079–1083PubMedGoogle Scholar
  13. 13.
    Jiang W, Kahn S, Guillem J, Lu S, Weinstein IB (1989) Rapid detection of ras oncogenes in human tissues: applications to colon, esophageal, and gastric cancer. Oncogene 4:923–928PubMedGoogle Scholar
  14. 14.
    Minamoto T, Ronai Z, Yamashita N et al (1994) Detection of Ki-ras mutation in non-neoplastic mucosa of Japanese patients with colorectal cancers. Int J Oncol 4:397–401PubMedGoogle Scholar
  15. 15.
    Carethers JM, Jung BH (2015) Genetics and genetic biomarkers in sporadic colorectal cancer. Gastroenterology 149:1177–1190CrossRefGoogle Scholar
  16. 16.
    Amaro A, Chiara S, Pfeffer U (2016) Molecular evolution of colorectal cancer: from multistep carcinogenesis to the big bang. Cancer Metastasis Rev 35:63–74CrossRefGoogle Scholar
  17. 17.
    Huang D, Sun W, Zhou Y, Li P, Chen F, Chen H, Xia D, Xu E, Lai M, Wu Y, Zhang H (2018) Mutations of key driver genes in colorectal cancer progression and metastasis. Cancer Metastasis Rev 37:173–187CrossRefGoogle Scholar
  18. 18.
    Minamoto T, Ronai Z (2001) Gene mutation as a target for early detection in cancer diagnosis. Crit Rev Oncol Hematol 40:195–213CrossRefGoogle Scholar
  19. 19.
    Ronai Z, Luo FC, Gradia S, Hart WJ, Butler R (1994) Detection of K-ras mutation in normal and malignant colonic tissues by an enriched PCR method. Int J Oncol 4:391–396PubMedGoogle Scholar
  20. 20.
    Minamoto T, Yamashita N, Ochiai A et al (1995) Mutant K-ras in apparently normal mucosa of colorectal cancer patients. Its potential as a biomarker of colorectal cancer patients. Cancer 75:1520–1526CrossRefGoogle Scholar
  21. 21.
    Ronai Z, Minamoto T, Butler R et al (1995) Sampling method as a key factor in identifying K-ras oncogene mutations in preneoplastic colorectal lesions. Cancer Detect Prev 19:512–517PubMedGoogle Scholar
  22. 22.
    Minamoto T, Esumi H, Ochiai A et al (1997) Combined analysis of microsatellite instability and K-ras mutation increases detection incidence of normal samples from colorectal cancer patients. Clin Cancer Res 3:1413–1417PubMedGoogle Scholar
  23. 23.
    Tobi M, Luo F-C, Ronai Z (1994) Detection of K-ras mutation in colonic effluent samples from patients without evidence of colorectal carcinoma. J Natl Cancer Inst 86:1007–1010CrossRefGoogle Scholar
  24. 24.
    Zhang B, Ougolkov A, Yamashita K, Takahashi Y, Mai M, Minamoto T (2003) β-catenin and ras oncogenes detect most human colorectal cancers. Clin Cancer Res 3:3073–3079Google Scholar
  25. 25.
    Alrawi SJ, Schiff M, Carroll RE et al (2006) Aberrant crypt foci. Anticancer Res 26:107–119PubMedGoogle Scholar
  26. 26.
    Gupta AK, Pretlow TP, Schoen RE (2007) Aberrant crypt foci: what we know and what we need to know. Clin Gastroenterol Hepatol 5:526–533CrossRefGoogle Scholar
  27. 27.
    Yamashita N, Minamoto T, Ochiai A, Onda M, Esumi H (1995) Frequent and characteristic K-ras activation and absence of p53 protein accumulation in aberrant crypt foci of the colon. Gastroenterology 108:434–440CrossRefGoogle Scholar
  28. 28.
    Takayama T, Katsuki S, Takahashi Y et al (1998) Aberrant crypt foci of the colon as precursors of adenoma and cancer. N Engl J Med 339:1277–1284CrossRefGoogle Scholar
  29. 29.
    Gupta AK, Schoen RE (2009) Aberrant crypt foci: are they intermediate endpoints of colon carcinogenesis in humans? Curr Opin Gastroenterol 25:59–65CrossRefGoogle Scholar
  30. 30.
    Khare S, Chaudhary K, Bissonnette M, Carroll R (2009) Aberrant crypt foci in colon cancer epidemiology. Methods Mol Biol 472:373–386CrossRefGoogle Scholar
  31. 31.
    Otori K, Sugiyama K, Hasebe T, Fukushima S, Esumi H (1995) Emergence of adenomatous aberrant crypt foci (ACF) from hyperplastic ACF with concomitant increase in cell proliferation. Cancer Res 55:4743–4746PubMedGoogle Scholar
  32. 32.
    Konstantakos AK, Siu I-M, Pretlow TG, Stellato TA, Pretlow TP (1996) Human aberrant crypt foci with carcinoma in situ from a patient with sporadic colon cancer. Gastroenterology 111:772–777CrossRefGoogle Scholar
  33. 33.
    Orlando FA, Tan D, Baltodano JD et al (2008) Aberrant crypt foci as precursors in colorectal cancer progression. J Surg Oncol 98:207–213CrossRefGoogle Scholar
  34. 34.
    Pretlow TP, Brasitus TA, Fulton NC, Cheyer C, Kaplan EL (1993) K-ras mutation in putative preneoplastic lesions in human colon. J Natl Cancer Inst 85:2004–2007CrossRefGoogle Scholar
  35. 35.
    Takayama T, Ohi M, Hayashi T et al (2001) Analysis of K-ras, APC, and β-catenin in aberrant crypt foci in sporadic adenoma, cancer, and familial adenomatous polyposis. Gastroenterology 121:599–611CrossRefGoogle Scholar
  36. 36.
    Suehiro Y, Hinoda Y (2008) Genetic and epigenetic changes in aberrant crypt foci and serrated polyps. Cancer Sci 99:1071–1076CrossRefGoogle Scholar
  37. 37.
    Ahmed D, Eide PW, Eilertsen IA, Danielsen SA, Eknæs M, Hektoen M, Lind GE, Lothe RA (2013) Epigenetic and genetic features of 24 colon cancer cell lines. Oncogene 2:e71CrossRefGoogle Scholar
  38. 38.
    Moore DD, Strauss WM (1995) Preparation of genomic DNA from mammalian tissue. In: Ausubel F, Brent R, Kingston RE et al (eds) Short protocols in molecular biology, 3rd edn. Wiley, Hoboken, pp 2–8Google Scholar
  39. 39.
    Wolff R, Gemmill R (1997) DNA from mammalian sources. In: Birren B, Green ED, Klapholz S, Myers RM, Roskams J (eds) Genome analysis: a laboratory manual, vol 1. Cold Spring Harbor Laboratory Press, Plainview, pp 4–16Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Division of Translational and Clinical Oncology, Cancer Research Institute and Cancer CenterKanazawa University and HospitalKanazawaJapan

Personalised recommendations