A comparison of Direct sequencing, Pyrosequencing, High resolution melting analysis, TheraScreen DxS, and the K-ras StripAssay for detecting KRAS mutations in non small cell lung carcinomas
- Equal contributors
1 Laboratory of Experimental Medicine, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic
2 IntellMed Ltd, The Science and Technology Park of Palacky University, Olomouc, Czech Republic
3 Departments of Experimental Medicine and Human Genetics, McGill University, Montreal, Quebec, Canada
4 Institute of Applied Biotechnologies, Prague, Czech Republic
5 1st Department of Surgery, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic
6 Department of Pneumology and Tuberculosis, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic
7 Laboratory of Molecular Pathology, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University and University Hospital in Olomouc, Olomouc, Czech Republic
Journal of Experimental & Clinical Cancer Research 2012, 31:79 doi:10.1186/1756-9966-31-79Published: 20 September 2012
It is mandatory to confirm the absence of mutations in the KRAS gene before treating metastatic colorectal cancers with epidermal growth factor receptor inhibitors, and similar regulations are being considered for non-small cell lung carcinomas (NSCLC) and other tumor types. Routine diagnosis of KRAS mutations in NSCLC is challenging because of compromised quantity and quality of biological material. Although there are several methods available for detecting mutations in KRAS, there is little comparative data regarding their analytical performance, economic merits, and workflow parameters.
We compared the specificity, sensitivity, cost, and working time of five methods using 131 frozen NSCLC tissue samples. We extracted genomic DNA from the samples and compared the performance of Sanger cycle sequencing, Pyrosequencing, High-resolution melting analysis (HRM), and the Conformité Européenne (CE)-marked TheraScreen DxS and K-ras StripAssay kits.
Results and conclusions
Our results demonstrate that TheraScreen DxS and the StripAssay, in that order, were most effective at diagnosing mutations in KRAS. However, there were still unsatisfactory disagreements between them for 6.1% of all samples tested. Despite this, our findings are likely to assist molecular biologists in making rational decisions when selecting a reliable, efficient, and cost-effective method for detecting KRAS mutations in heterogeneous clinical tumor samples.