Detection of KRAS mutations in colorectal cancer with Fast COLD-PCR.
Int J Oncol
; 40(2): 378-84, 2012 Feb.
Article
en En
| MEDLINE
| ID: mdl-21971641
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.
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Análisis Mutacional de ADN
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Neoplasias Colorrectales
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Reacción en Cadena de la Polimerasa
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Proteínas Proto-Oncogénicas
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Proteínas ras
Tipo de estudio:
Diagnostic_studies
/
Prognostic_studies
Límite:
Humans
Idioma:
En
Revista:
Int J Oncol
Asunto de la revista:
NEOPLASIAS
Año:
2012
Tipo del documento:
Article
País de afiliación:
Italia