title: Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic and clinical implications.
abstract: 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 wild-type 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.
authors.affiliation: The Johns Hopkins Medical Institutions, Department of Pathology, 600 N. Wolfe Street, Park SB202, Baltimore, MD 21287, USA. atsiati1@jhmi.edu
authors.forename: Athanasios C
authors.initials: AC
authors.lastname: Tsiatis
authors.forename: Alexis
authors.initials: A
authors.lastname: Norris-Kirby
authors.forename: Roy G
authors.initials: RG
authors.lastname: Rich
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Hafez
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Gocke
authors.forename: James R
authors.initials: JR
authors.lastname: Eshleman
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Murphy
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000235:genetics*; D005260:Female; D006801:Humans; D057230:Limit of Detection; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics*; D009691:Nucleic Acid Denaturation; D009928:Organ Specificity / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D013696:Temperature; D018631:ras Proteins / Q000235:genetics*
chemical_list: D003062:Codon; D004273:DNA, Neoplasm; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins