Detection of KRAS mutations in colorectal carcinoma patients with an integrated PCR/sequencing and real-time PCR approach

Pietro Carotenuto, Cristin Roma, Anna Maria Rachiglio, Fabiana Tatangelo, Carmine Pinto, Fortunato Ciardiello, Oscar Nappi, Vincenzo Iaffaioli, Gerardo Botti, Nicola Normanno

Research output: Contribution to journalArticle

Abstract

Aims: Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-EGF receptor monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting to aid in the choice of appropriate therapy. Materials & methods: We developed a cost-effective approach for the determination of KRAS mutations in codons 12 and 13 in clinical practice based on a sensitive PCR/sequencing technique and the commercially available real-time PCR-based Therascreen® kit (DxS Ltd). Results & conclusion: The PCR/sequencing test was able to detect 10% mutant DNA in a background of wild-type DNA. By using this assay, we determined the mutational status of KRAS in 527 out of 540 (97.6%) formalin-fixed paraffin-embedded tissues from mCRC patients. PCR/sequencing was not conclusive in 13 cases, in which low-intensity peaks suggestive of potential mutations were identified. The DxS assay, which showed a sensitivity of 1%, identified mutations in 11 out of 13 inconclusive cases. Interestingly, five of these 11 cases showed high levels of DNA fragmentation. No significant difference was found in the ability of PCR/sequencing and DxS to identify KRAS mutations within 160 cases with more than 30% tumor cells. However, in 24 samples with less than 30% tumor cells, DxS showed an higher sensitivity. In conclusion, our findings suggest that PCR/sequencing can be used for mutational analysis of the majority of tumor samples that have more than 30% tumor cell content, whereas more sensitive and expensive tests should be reserved for inconclusive cases and for samples with a low amount of tumor cells.

Original languageEnglish
Pages (from-to)1169-1179
Number of pages11
JournalPharmacogenomics
Volume11
Issue number8
DOIs
Publication statusPublished - Aug 2010

Fingerprint

Real-Time Polymerase Chain Reaction
Colorectal Neoplasms
Polymerase Chain Reaction
Mutation
Neoplasms
DNA
DNA Fragmentation
Epidermal Growth Factor Receptor
Codon
Paraffin
Formaldehyde
Monoclonal Antibodies
Costs and Cost Analysis
Therapeutics
Genes

Keywords

  • colorectal carcinoma
  • EGFR
  • KRAS
  • mutations
  • therapy

ASJC Scopus subject areas

  • Pharmacology
  • Genetics
  • Molecular Medicine

Cite this

Detection of KRAS mutations in colorectal carcinoma patients with an integrated PCR/sequencing and real-time PCR approach. / Carotenuto, Pietro; Roma, Cristin; Rachiglio, Anna Maria; Tatangelo, Fabiana; Pinto, Carmine; Ciardiello, Fortunato; Nappi, Oscar; Iaffaioli, Vincenzo; Botti, Gerardo; Normanno, Nicola.

In: Pharmacogenomics, Vol. 11, No. 8, 08.2010, p. 1169-1179.

Research output: Contribution to journalArticle

Carotenuto, Pietro ; Roma, Cristin ; Rachiglio, Anna Maria ; Tatangelo, Fabiana ; Pinto, Carmine ; Ciardiello, Fortunato ; Nappi, Oscar ; Iaffaioli, Vincenzo ; Botti, Gerardo ; Normanno, Nicola. / Detection of KRAS mutations in colorectal carcinoma patients with an integrated PCR/sequencing and real-time PCR approach. In: Pharmacogenomics. 2010 ; Vol. 11, No. 8. pp. 1169-1179.
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AU - Pinto, Carmine

AU - Ciardiello, Fortunato

AU - Nappi, Oscar

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AU - Botti, Gerardo

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AB - Aims: Patients with metastatic colorectal carcinoma (mCRC) carrying activating mutations of the KRAS gene do not benefit from treatment with anti-EGF receptor monoclonal antibodies. Therefore, KRAS mutation testing of mCRC patients is mandatory in the clinical setting to aid in the choice of appropriate therapy. Materials & methods: We developed a cost-effective approach for the determination of KRAS mutations in codons 12 and 13 in clinical practice based on a sensitive PCR/sequencing technique and the commercially available real-time PCR-based Therascreen® kit (DxS Ltd). Results & conclusion: The PCR/sequencing test was able to detect 10% mutant DNA in a background of wild-type DNA. By using this assay, we determined the mutational status of KRAS in 527 out of 540 (97.6%) formalin-fixed paraffin-embedded tissues from mCRC patients. PCR/sequencing was not conclusive in 13 cases, in which low-intensity peaks suggestive of potential mutations were identified. The DxS assay, which showed a sensitivity of 1%, identified mutations in 11 out of 13 inconclusive cases. Interestingly, five of these 11 cases showed high levels of DNA fragmentation. No significant difference was found in the ability of PCR/sequencing and DxS to identify KRAS mutations within 160 cases with more than 30% tumor cells. However, in 24 samples with less than 30% tumor cells, DxS showed an higher sensitivity. In conclusion, our findings suggest that PCR/sequencing can be used for mutational analysis of the majority of tumor samples that have more than 30% tumor cell content, whereas more sensitive and expensive tests should be reserved for inconclusive cases and for samples with a low amount of tumor cells.

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