Methods For Detection Of Hot-spot Mutations Of Critical Genes Involved In EGFR Signaling Pathway Based On Fluorescent ARMS Technique

EGFR signaling pathway is a complex system mediated by epidermal growth factor receptor and integrated with various cell factors. The cytobiology effects of EGFR signaling pathway include cellular proliferation, growth, metastasis and angiogenesis, of which plays an essential role in tumorigenesis and development. According to plenty of clinical researches, the mutant status of several genes which encode critical signaling-transduction factors of EGFR signaling pathway, including EGFR, KRAS, BRAF, PIK3CA are firmly associated with the clinical outcome of patients receiving relevant targeted therapies. By detecting the hot-spot mutations of critical genes of EGFR signaling pathway, it is not only feasible to help doctors selecting patients but reducing the risk of adverse response of targeted therapies to some extent.Therefore, to meet the clinical demand for detection of hot-spot mutations of critical genes of EGFR signaling pathway, establishing a sensitive, simple-handled and robust method is highly required. Presently, a lot of methods, both laboratorial or commercial, have been used for this purpose. However, many of these methods are either costly or time-consumed, which is not suit for clinical application. In this work, we established and improved a system based on fluorescent ARMS technique to detecting the hot-spot mutations of critical genes of EGFR signaling pathway.Based on the system established in this work, we screened hot-spot mutations of critical genes of EGFR signaling pathway in30paraffin-embedded non-small cell lung cancer samples and30paraffin-embedded colorectal cancer samples. And part of results were further detected by CFDA-approved commercial kit as validation. By setting pyrosequencing as comparison, our method showed obvious advantages in many aspects. The established system shows very good sensitivity and specificity, and is simple-handled and analyzed that is suitable for clinical application.