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BioMed Research International
Volume 2018, Article ID 8727941, 13 pages
Research Article

Comprehensive Validation of Snapback Primer-Based Melting Curve Analysis to Detect Nucleotide Variation in the Codon 12 and 13 of KRAS Gene

1Department of Clinical Laboratory, Huashan Hospital, Fudan University, 200040, China
2Baoshan Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai, 201999, China
3Central Laboratory, Huashan Hospital, Fudan University, Shanghai 200040, China
4Department of Clinical Laboratory, Huashan Hospital North, Fudan University, Shanghai, 201907, China

Correspondence should be addressed to Ming Guan; moc.oohay@88gnimnaug and Zhiyuan Wu; moc.liamtoh@111nauyihzuw

Received 10 November 2017; Accepted 13 August 2018; Published 10 October 2018

Academic Editor: Salvatore Gallone

Copyright © 2018 Qunfeng Zhang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


Background. KRAS genotyping in tumor samples is a decisive clinical test for the anti-EGFR therapy management. However, the complexity of KRAS mutation landscape across different cancer types and the mosaic effect caused by cancer cellularity and heterogeneity make the choice of KRAS genotyping method a challenging topic in the clinical practice. Methods. We depicted the landscape of somatic KRAS mutation in 7,844 primary tumors and 10,336 metastatic tumors across over 30 types of cancer using the Cancer Genome Atlas (TCGA) and Integrated Mutation Profiling of Actionable Cancer Targets (MSKCC-IMPACT) databases, respectively. A snapback primer assay based on melting curve analysis was developed to detect the most common somatic mutations in KRAS codons 12 and 13. The sensitivity and accuracy of the method was validated by genotyping 100 colorectal cancer (CRC) samples, in comparison with Sanger sequencing and T-A cloning sequencing. Results. Pancreas adenocarcinoma (somatic mutation frequency 90.6%), colorectal adenocarcinoma (42.5%), and lung adenocarcinoma (32.6%) are the top three most KRAS mutant primary cancer types. The metastatic tumors showed a higher prevalence (90.99% versus 66.31%) and diversity of KRAS mutation compared with the primary tumors. Mutations in codons 12 and 13 are the predominant genetic alteration in KRAS (84.15% for TCGA and 86.13% for MSK-IMPACT). Moreover, KRAS mutation is highly correlated with the overall survival of patients with metastatic cancer. The snapback primer assay showed a more favorable performance in enriching and detecting the KRAS codon 12 and 13 mutation (1% mutation load) compared with Sanger sequencing (20% mutation load and 7% false-negative rate). Conclusions. KRAS mutation pattern is highly diverse among different cancer types and is associated with the survival of patients with metastatic cancers. The snapback primer assay is a reliable, sensitive method to detect the major mutant KRAS alleles, which might facilitate the effective cancer treatment decisions.