HER-2 V777L mutation detection using HRMA based Real-Time PCR in Breast Cancer

Introduction: The development of targeted therapies has created a need for the rapid molecular characterization of cancers. We describe here the usefulness of high-resolution melting analysis (HRM) to screen for HER-2 mutations in clinical cancer samples. In Breast cancer, HER-2 mutations have been shown to identify a group of patients that do not respond to SERMs and the identification of these mutations is thus clinically important. Methods: HER-2 DNA sequence was extracted from NCBI Gene data bank. Primer set specific for HER-2 gene was designed by Beacon designer software. Then, the specificity of primers was confirmed using BLAST tool. We developed ahigh-resolution melting (HRM) assay to detect somatic mutations in exon 20, notably codon 777 of the HER-2 gene using the intercalating dye EVA Green. We tested 20 Breast paraffin-embedded tissue samples and then examined the sensitivity of mutation detection with bidirectional DNA sequencing. Results: We found that three samples were positive for V777L mutation which was readily detectable using HRM. The validation of HRMA assay with DNA sequencing and dye-probe based technologies showed that the sensitivity and specificity of PCR/HRMA were 100% and 100%, respectively. Discussion: These findings show that HRMA is a highly sensitive, simple and low-cost designed assay to detect human disease-associated mutations, especially for samples with mutations of low incidence. As a result, the use of DNA sequencing and dye-probe based technologies could be significantly declined by the implementation of HRMA.

Keywords:

HER-2 , Breast cancer , HRMA , PCR