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


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).


CONCLUSIONS
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.
Carotenuto
Pietro
P
Pharmacogenomic Laboratory, Centro Ricerche Oncologiche di Mercogliano, Avellino, Italy.
Roma
Cristin
C
Rachiglio
Anna Maria
AM
Tatangelo
Fabiana
F
Pinto
Carmine
C
Ciardiello
Fortunato
F
Nappi
Oscar
O
Iaffaioli
R Vincenzo
RV
Botti
Gerardo
G
Normanno
Nicola
N
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D015331:Cohort Studies; D015179:Colorectal Neoplasms / Q000235:genetics* / Q000473:pathology; D006801:Humans; D009154:Mutation; D009362:Neoplasm Metastasis; D020411:Oligonucleotide Array Sequence Analysis; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics*
C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins