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.

Figure 1

Melting curve, Sanger sequencing, and pyrosequencing data from a wild-type KRAS specimen and mutant KRAS specimen (G12D) are shown. Arrows indicate the mutation.

Figure 2

Results of SS, MC, and PS for a G12F mutation. A: SS demonstrated G to T transversions at positions 1 and 2 of codon 12. Arrows indicate the mutation. B: MC showed a Tm significantly lower than expected for a single mutation. Arrows indicate the mutation. C: PS generated a pyrogram that was flagged as abnormal, but the pattern could not be definitively interpreted with regard to the specific mutation subtype. D: A hypothetical PS pyrogram that would have resulted had the G to T transversions at positions 1 and 2 of codon 12 been on separate alleles.

Figure 3

Dilution series data presented here include 100%, 20%, 10%, 5%, and 0% KRAS mutant cell mixtures and the corresponding predicted percentage of mutant alleles in each mixture. The dilutions representing the limit of detection for a KRAS mutation at codons 12/13 by SS, MC, and PS are designated by the arrow.