Real-time PCR panels for detection of mutations responsible for failure of EGFR-targeted therapy in colorectal cancer

• Main Authors: Chia Yu Chang, 張家瑜
• Other Authors: C. C. Chiou
• Format: Others
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/29195640825955655361

碩士 === 長庚大學 === 醫學生物技術暨檢驗學系 === 99 === Cetuximab and panitumumab are major drugs against epidermal growth factor receptor (EGFR) in colorectal cancer. However, mutations in downstream of EGFR signaling pathway, including KRAS, BRAF, and PIK3CA, abolish the response of anti-EGFR therapy. Therefore, these gene aberrations are predictive markers for failure of EGFR-targeted therapy. In this study, we established a high-resolution melting (HRM) analysis-based panel and a single probe fluorescence resonance energy transfer (FRET) analysis-based panel to detect mutations in these genes. DNA samples from five colorectal cancer cell lines and two blood samples of healthy volunteer were used to establish the panels. Mutant DNA is differentiated from wild-type DNA through unique HRM pattern or melting temperature. In optimal condition, both assays could detect 5%-10% mutant alleles in wild-type background. Using these panels to screen clinical samples 48% (24/50) patients harbored at least one mutation. 34% KRAS mutation, 10% BRAF mutation, and 14% PIK3CA mutation were detected in these samples. In summary, we have established two screening panel for detecting gene mutations responsible for failure of EGFR-targeted therapy. These panels will be useful for evaluating responses of EGFR-targeted therapy in colorectal cancer.