BACKGROUND: Somatic mutations in the KRAS gene have been reported to confer drug resistance to epidermal growth factor receptor tyrosine kinase inhibitors and some monoclonal antibodies. However, current DNA mutation detection technologies are primarily DNA sequencing-based and not high throughput, nor sensitive enough to meet clinical needs. METHODS: A mutant-enriched PCR method was designed by introducing a unique restriction enzyme site to the PCR product. This allowed the wild-type KRAS sequence to be selectively removed by restriction enzyme digestion before application to the Luminex liquidchip system. RESULTS: A total of 100 copies of mutant KRAS DNA fragment mixed with 1x10(5) copies of the wild-type KRAS DNA could be detected to achieve a sensitivity of 0.1%. This technology is currently used for clinical testing of KRAS somatic mutations for the purpose of pharmacogenomic evaluation. Serum samples from 109 patients with non-small cell lung cancer were tested and 34 mutations were detected (34/109). The formalin-fixed and paraffin-embedded samples from 60 patients with colorectal cancer were tested and 19 mutations were detected (19/60). CONCLUSIONS: A novel, qualitative, sensitive, reliable and high throughput liquidchip technology has been developed for detecting KRAS mutations using clinical serum and formalin-fixed and paraffin-embedded samples.
BACKGROUND: Somatic mutations in the KRAS gene have been reported to confer drug resistance to epidermal growth factor receptor tyrosine kinase inhibitors and some monoclonal antibodies. However, current DNA mutation detection technologies are primarily DNA sequencing-based and not high throughput, nor sensitive enough to meet clinical needs. METHODS: A mutant-enriched PCR method was designed by introducing a unique restriction enzyme site to the PCR product. This allowed the wild-type KRAS sequence to be selectively removed by restriction enzyme digestion before application to the Luminex liquidchip system. RESULTS: A total of 100 copies of mutant KRAS DNA fragment mixed with 1x10(5) copies of the wild-type KRAS DNA could be detected to achieve a sensitivity of 0.1%. This technology is currently used for clinical testing of KRAS somatic mutations for the purpose of pharmacogenomic evaluation. Serum samples from 109 patients with non-small cell lung cancer were tested and 34 mutations were detected (34/109). The formalin-fixed and paraffin-embedded samples from 60 patients with colorectal cancer were tested and 19 mutations were detected (19/60). CONCLUSIONS: A novel, qualitative, sensitive, reliable and high throughput liquidchip technology has been developed for detecting KRAS mutations using clinical serum and formalin-fixed and paraffin-embedded samples.
Authors: S Li; L Li; Y Zhu; C Huang; Y Qin; H Liu; L Ren-Heidenreich; B Shi; H Ren; X Chu; J Kang; W Wang; J Xu; K Tang; H Yang; Y Zheng; J He; G Yu; N Liang Journal: Br J Cancer Date: 2014-04-17 Impact factor: 7.640