Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: Diagnostic and clinical implications

Athanasios C. Tsiatis, Alexis Norris-Kirby, Roy G. Rich, Michael J. Hafez, Christopher Gocke, James Eshleman, Kathleen M. Murphy

Research output: Contribution to journalArticle

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 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.

Original languageEnglish (US)
Pages (from-to)425-432
Number of pages8
JournalJournal of Molecular Diagnostics
Volume12
Issue number4
DOIs
StatePublished - Jul 1 2010

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Freezing
Codon
Mutation
Oncogenes
Epidermal Growth Factor Receptor
Paraffin
Formaldehyde
Limit of Detection
Colon
Adenocarcinoma
Alleles
Neoplasms
Adenocarcinoma of lung

ASJC Scopus subject areas

  • Molecular Medicine
  • Pathology and Forensic Medicine
  • Medicine(all)

Cite this

Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations : Diagnostic and clinical implications. / Tsiatis, Athanasios C.; Norris-Kirby, Alexis; Rich, Roy G.; Hafez, Michael J.; Gocke, Christopher; Eshleman, James; Murphy, Kathleen M.

In: Journal of Molecular Diagnostics, Vol. 12, No. 4, 01.07.2010, p. 425-432.

Research output: Contribution to journalArticle

Tsiatis, Athanasios C. ; Norris-Kirby, Alexis ; Rich, Roy G. ; Hafez, Michael J. ; Gocke, Christopher ; Eshleman, James ; Murphy, Kathleen M. / Comparison of Sanger sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations : Diagnostic and clinical implications. In: Journal of Molecular Diagnostics. 2010 ; Vol. 12, No. 4. pp. 425-432.
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