EGFR promoter methylation detection in cervical cancer by a hybridization-fluorescence polarization assay

The methylation status of the epidermal growth factor receptor (EGFR) promoter is of potential predictive value for benefitting from EGFR inhibition therapy. Stratified therapy assignment for cervical cancer patients based on the EGFR promoter methylation status requires a simple detection method in the daily practice of diagnosis. A novel assay detecting the EGFR promoter methylation status in cervical cancer tissue samples using a hybridization-fluorescence polarization (FP) technique was developed. A pair of primers was used to amplify a 156 bp fragment in the promoter region of EGFR. Two probes specific for either methylated or unmethylated EGFR promoter DNA labeled with different fluorophores hybridized, respectively, with their target amplicons. The EGFR promoter methylation status was determined by the FP values. A total of 273 cervical cancer tissue samples were simultaneously analyzed using the new assay technique and combined bisulfite restriction analysis. The new assay was more sensitive compared with the combined bisulfite restriction analysis, and it allowed the discrimination of the EGFR promoter methylation status directly in solution without the restriction enzyme digestion. Sensitivity, specificity, and stability of the hybridization-FP assay had been recorded. The minimum detection level established with the new assay was 50 copies/μL, and it was able to detect the minor population of the EGFR promoter methylation status even when its contents were as low as 10%. No cross-reaction was observed in the assay when the amount of plasmids used accounted for no more than 10(9) copies/μL. The coefficient of variation of the reproducibility for the assay was <10%.