Comparison of Sanger Sequencing, Pyrosequencing, and Melting Curve Analysis for the Detection of KRAS Mutations Diagnostic and Clinical Implications

被引:368
作者
Tsiatis, Athanasios C. [1 ]
Norris-Kirby, Alexis [1 ]
Rich, Roy G. [1 ]
Hafez, Michael J. [1 ]
Gocke, Christopher D. [1 ]
Eshleman, James R. [1 ]
Murphy, Kathleen M. [1 ]
机构
[1] Johns Hopkins Med Inst, Dept Pathol, Baltimore, MD 21287 USA
关键词
K-RAS MUTATIONS; CELL LUNG-CANCER; METASTATIC COLORECTAL-CANCER; CARCINOMAS; RESISTANCE; CETUXIMAB; GENES;
D O I
10.2353/jmoldx.2010.090188
中图分类号
R36 [病理学];
学科分类号
100104 ;
摘要
Mutations in codons 12 and 13 of the KRAS oncogene are relatively common in colorectal and lung adenocarcinomas. Recent data indicate that these mutations result in resistance to anti-epidermal growth factor receptor therapy. Therefore, we assessed Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS codon 12/13 mutations in formalin-fixed paraffin-embedded samples, including 58 primary and 42 metastatic colorectal adenocarcinomas, 63 primary and 17 metastatic lung adenocarcinomas, and 20 normal colon samples. Of 180 tumor samples, 62.2% were KRAS mutant positive, and 37.8% were negative. Melting curve analysis yielded no false positive or false negative results, but had 10% equivocal calls. Melting curve analysis also resulted in 4 cases with melting curves inconsistent with either wildtype or codon 12/13 mutations. These patterns were generated from samples with double mutants in codons 12/13 and with mutations outside of codons 12/13. Pyrosequencing yielded no false positive or false negative results as well. However, two samples from one patient yielded a pyrogram that was flagged as abnormal, but the mutation subtype could not be determined. Finally, using an electronic cutoff of 10%, Sanger sequencing showed 11.1% false positives and 6.1% false negatives. In our hands, the limit of detection for Sanger sequencing, pyrosequencing, and melting curve analysis was approximately 15 to 20%, 5%, and 10% mutant alleles, respectively. (J Mol Diagn 2010, 12:425-432; DOI: 10.2353/jmoldx.2010.090188)
引用
收藏
页码:425 / 432
页数:8
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