Development of a multichannel TaqMan assay to simultaneously evaluate EGFR expression level and mutation status

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Epidermal Growth Factor Receptor (EGFR) is one of a family of receptors that regulates cell growth, division, and death. An increased amount of EGFR protein has been observed in a variety of cancers. Small molecule tyrosine kinase inhibitors (TKI) are used to target EGFR in non small cell lung cancer (NSLC) patients. Unfortunately many NSLC patients treated with TKIs do not have a significant response. Studies have shown that many of the patients that respond contain either a δ746-750 or L858R somatic mutation.Here we describe the development of an assay that can simultaneously detect both nucleotide mutations and gene expression level in a single tube using multichannel detection on the COBAS® TaqMan® 48. TaqMan probes were developed on four channels, Fam was used to detect the δ746-750 mutation, Hex for the L858R mutation, JA270 for overall EGFR level and Cy5.5 for the housekeeping gene beta-glucuronidase (βGUS).We show that one can quantify EGFR expression and detect δ746-750 and L858R mutations with total RNA extracted from tumors. Determining both in a single standardized assay may resolve the dilemma about which function contributes most to clinical response in treatment with TKIs. This is important not only for this study, but also as a model for a unique approach to detect a dynamic range of somatic mutations.