J Pathol Transl Med.  2017 Jan;51(1):24-31. 10.4132/jptm.2016.10.03.

KRAS Mutation Test in Korean Patients with Colorectal Carcinomas: A Methodological Comparison between Sanger Sequencing and a Real-Time PCR-Based Assay

Affiliations
  • 1Department of Hospital Pathology, College of Medicine, The Catholic University of Korea, Seoul, Korea. esjung@catholic.ac.kr

Abstract

BACKGROUND
Mutations in the KRAS gene have been identified in approximately 50% of colorectal cancers (CRCs). KRAS mutations are well established biomarkers in anti-epidermal growth factor receptor therapy. Therefore, assessment of KRAS mutations is needed in CRC patients to ensure appropriate treatment.
METHODS
We compared the analytical performance of the cobas test to Sanger sequencing in 264 CRC cases. In addition, discordant specimens were evaluated by 454 pyrosequencing.
RESULTS
KRAS mutations for codons 12/13 were detected in 43.2% of cases (114/264) by Sanger sequencing. Of 257 evaluable specimens for comparison, KRAS mutations were detected in 112 cases (43.6%) by Sanger sequencing and 118 cases (45.9%) by the cobas test. Concordance between the cobas test and Sanger sequencing for each lot was 93.8% positive percent agreement (PPA) and 91.0% negative percent agreement (NPA) for codons 12/13. Results from the cobas test and Sanger sequencing were discordant for 20 cases (7.8%). Twenty discrepant cases were subsequently subjected to 454 pyrosequencing. After comprehensive analysis of the results from combined Sanger sequencing-454 pyrosequencing and the cobas test, PPA was 97.5% and NPA was 100%.
CONCLUSIONS
The cobas test is an accurate and sensitive test for detecting KRAS-activating mutations and has analytical power equivalent to Sanger sequencing. Prescreening using the cobas test with subsequent application of Sanger sequencing is the best strategy for routine detection of KRAS mutations in CRC.

Keyword

KRAS mutation test; Sanger sequencing; Cobas test; 454 pyrosequencing

MeSH Terms

Biomarkers
Codon
Colorectal Neoplasms*
Humans
Biomarkers
Codon

Figure

  • Fig. 1. Study design and specimen selection. Two hundred sixty-four formalin-fixed, paraffin-embedded (FFPE) colorectal cancer (CRC) specimens were selected and processed using Sanger sequencing and cobas test.

  • Fig. 2. Electropherogram from Sanger sequencing. Representative sample shows mutant codon 12 with GGT>GAT (arrow).


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