Open Access

Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR

  • Authors:
    • Pietro Carotenuto
    • Cristin Roma
    • Salvatore Cozzolino
    • Francesca Fenizia
    • Anna Maria Rachiglio
    • Fabiana Tatangelo
    • Alessia Iannaccone
    • Luigi Baron
    • Gerardo Botti
    • Nicola Normanno
  • View Affiliations

  • Published online on: October 4, 2011     https://doi.org/10.3892/ijo.2011.1221
  • Pages: 378-384
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Abstract

Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-epidermal growth factor receptor (EGFR) monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting for the choice of the most appropriate therapy. Co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) is a novel modification of the conventional PCR method that selectively amplifies minority alleles from a mixture of wild-type and mutant sequences irrespective of the mutation type or position within the sequence. In this study, we compared the sensitivity of a COLD-PCR method with conventional PCR/sequencing and the real-time PCR-based Therascreen kit to detect KRAS mutations. By using dilutions of KRAS mutant DNA in wild-type DNA from colon cancer cell lines with known KRAS status, we found that Fast COLD-PCR is more sensitive than the conventional PCR method, showing a sensitivity of 2.5% in detecting G>A and G>T mutations. The detection of G>C transversions was not improved by either Fast COLD-PCR or Full COLD-PCR. We next analyzed by COLD-PCR, conventional PCR and Therascreen 52 formalin-fixed paraffin-embedded samples from mCRC patients. Among 36 samples with >30% tumor cells, 8 samples were negative by conventional PCR, Therascreen and Fast COLD-PCR; 20 mutations identified by conventional PCR were confirmed by Therascreen and Fast COLD-PCR; 8 cases undetermined by conventional PCR were all confirmed to carry KRAS G>A or G>T mutations by using either Therascreen or Fast COLD-PCR. Conventional PCR was able to detect only 2 KRAS mutations among 16 samples with <30% tumor cells (12.5%), whereas Therascreen and Fast COLD-PCR identified 6 mutants (37.5%). These data suggest that Fast COLD-PCR has a higher clinical sensitivity as compared with conventional PCR in detecting G>C to A>T changes in the KRAS gene, which represent >90% of the mutations of this oncogene in CRC.

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February 2012
Volume 40 Issue 2

Print ISSN: 1019-6439
Online ISSN:1791-2423

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Copy and paste a formatted citation
APA
Carotenuto, P., Roma, C., Cozzolino, S., Fenizia, F., Rachiglio, A.M., Tatangelo, F. ... Normanno, N. (2012). Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR. International Journal of Oncology, 40, 378-384. https://doi.org/10.3892/ijo.2011.1221
MLA
Carotenuto, P., Roma, C., Cozzolino, S., Fenizia, F., Rachiglio, A. M., Tatangelo, F., Iannaccone, A., Baron, L., Botti, G., Normanno, N."Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR". International Journal of Oncology 40.2 (2012): 378-384.
Chicago
Carotenuto, P., Roma, C., Cozzolino, S., Fenizia, F., Rachiglio, A. M., Tatangelo, F., Iannaccone, A., Baron, L., Botti, G., Normanno, N."Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR". International Journal of Oncology 40, no. 2 (2012): 378-384. https://doi.org/10.3892/ijo.2011.1221