The frequency of KRAS mutation detection in human colon carcinoma is influenced by the sensitivity of assay methodology: a comparison between direct sequencing and real-time PCR.
OBJECTIVE
Kirsten rat sarcoma (KRAS) gene mutations occur early in the progression of colorectal adenoma to carcinoma. The mutation status of the KRAS gene determines the benefits of molecular targeting drugs in patients with advanced colorectal cancer, although many methods are available to detect such mutations. The purpose of this study was to evaluate the influence of assay sensitivity on the detection frequency of mutated genes.


METHODS
Colorectal tumors in 224 colorectal cancer patients were characterized for KRAS mutations using PCR amplification following by direct sequencing as well as a peptide nucleic acid (PNA)-clamp real-time PCR-based assay.


RESULTS
KRAS mutations were observed in 32.1% (72/224) patients by direct sequencing, and 43.3% (97/224) by PNA-clamp PCR. The chi-square test revealed that the difference in the frequency of KRAS mutations determined by direct sequencing and PNA-clamped PCR (threshold for 1% detection) was statistically significant (p<0.015).


CONCLUSIONS
Our data suggest that assay method sensitivity clearly influences the detection frequency of mutated genes. As more sensitive assays detect more mutated genes in clinical samples, this must be taken into consideration when determining KRAS gene status in clinical practice.
Kobunai
Takashi
T
Department of Surgery, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan. azteca_tk@yahoo.co.jp
Watanabe
Toshiaki
T
Yamamoto
Yoko
Y
Eshima
Kiyoshi
K
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002277:Carcinoma / Q000235:genetics*; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); 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