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

Pietro Carotenuto, Cristin Roma, Salvatore Cozzolino, Francesca Fenizia, Anna Maria Rachiglio, Fabiana Tatangelo, Alessia Iannaccone, Luigi Baron, Gerardo Botti, Nicola Normanno

Research output: Contribution to journalArticle

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 C to A>T changes in the KRAS gene, which represent >90% of the mutations of this oncogene in CRC.

Original languageEnglish
Pages (from-to)378-384
Number of pages7
JournalInternational Journal of Oncology
Volume40
Issue number2
DOIs
Publication statusPublished - Feb 2012

Fingerprint

Colorectal Neoplasms
Polymerase Chain Reaction
Mutation
Temperature
DNA
Oncogenes
Epidermal Growth Factor Receptor
Paraffin
Colonic Neoplasms
Formaldehyde
Genes
Real-Time Polymerase Chain Reaction
Alleles
Monoclonal Antibodies

Keywords

  • Anti-EGFR therapy
  • Co-amplification-at-lower denaturation-temperature PCR
  • Colorectal carcinoma
  • Epidermal growth factor receptor
  • KRAS
  • Mutations

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

Cite this

Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR. / Carotenuto, Pietro; Roma, Cristin; Cozzolino, Salvatore; Fenizia, Francesca; Rachiglio, Anna Maria; Tatangelo, Fabiana; Iannaccone, Alessia; Baron, Luigi; Botti, Gerardo; Normanno, Nicola.

In: International Journal of Oncology, Vol. 40, No. 2, 02.2012, p. 378-384.

Research output: Contribution to journalArticle

Carotenuto, P, Roma, C, Cozzolino, S, Fenizia, F, Rachiglio, AM, Tatangelo, F, Iannaccone, A, Baron, L, Botti, G & Normanno, N 2012, 'Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR', International Journal of Oncology, vol. 40, no. 2, pp. 378-384. https://doi.org/10.3892/ijo.2011.1221
Carotenuto, Pietro ; Roma, Cristin ; Cozzolino, Salvatore ; Fenizia, Francesca ; Rachiglio, Anna Maria ; Tatangelo, Fabiana ; Iannaccone, Alessia ; Baron, Luigi ; Botti, Gerardo ; Normanno, Nicola. / Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR. In: International Journal of Oncology. 2012 ; Vol. 40, No. 2. pp. 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 C to A>T changes in the KRAS gene, which represent >90{\%} of the mutations of this oncogene in CRC.",
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AU - Tatangelo, Fabiana

AU - Iannaccone, Alessia

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AU - Normanno, Nicola

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AB - 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 C to A>T changes in the KRAS gene, which represent >90% of the mutations of this oncogene in CRC.

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