Detection of KRAS Mutations in Colorectal Carcinoma Patients with an Integrated PCR/Sequencing and Real-Time PCR Approach

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.

KEYWORDS::

• colorectal carcinoma
• EGFR
• EGFR
• KRAS
• mutations
• therapy

Acknowledgements

The following pathologists collaborated for this study: G Antinolfi, Anatomia ed Istologia Patologica, AO MONALDI, Napoli, Italy; L Baron and F Quarto, Anatomia ed Istologia Patologica e Citopatologia, Azienda Sanitaria ASL Napoli 5, PO ‘S Leonardo‘, Castellammare di Stabia (NA), Italy; A Di Blasi, Anatomia ed Istologia Patologica, AO ‘G RUMMO‘, Benevento; F Ferraraccio, Anatomia ed Istologia Patologica, Seconda Università degli Studi di Napoli, Italy; A Mirabella, Anatomia Patologica e Citopatologia, Ospedale Pubblico ‘M Scarlato‘, Scafati (SA), Italy; A Pollio, Anatomia Patologica, PO ‘A Cardarelli‘, Campobasso, Italy; A Santarsiere, Anatomia e Istologia Patologica, Ospedale ‘S Giovanni Moscati‘, Aversa, Italy; D Tomaselli, Anatomia e Istologia Patologica, Ospedale ‘Sacro Cuore di Gesù‘, Benevento, Italy.

Financial & competing interests disclosure

This work was in part supported from a grant from the Research Department of the Campania Region. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Additional information

Funding

This work was in part supported from a grant from the Research Department of the Campania Region. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.