Evaluation of sanger sequencing method for ESR1 mutation detection in primary breast tumor

Objective: Exon 8 estrogen receptor 1 (ESR1) mutations in the ligand-binding domain play an important role in mechanisms of hormonal therapy resistance in breast cancer. Identification of ESR1 mutations is very important in determining the appropriate steps of therapy. In this study, we evaluate Sanger sequencing technique to detect the ESR1 mutation. Methods: This retrospective study was conducted using 49 advanced breast cancer patients with estrogen receptor positive. Deoxyribonucleic acid (DNA) material was extracted from primary breast tumor samples. Exon 8 ESR1 gene mutation was analyzed by Sanger sequencing method using BigDye Direct Sequencing Kit (Applied Biosystem) with gBlock synthesis gene fragment (Integrated DNA Technologies) D538G as a positive control. Results: The mean age of patients was 46.14 (±9.6) years, and 61.2% were in stage 4. There are no exon 8 ESR1 gene mutations detected in 49 primary tumor samples, whereas the gBlock-positive control showed base substitution in 1613A>G (D538G) indicating the success of sequencing reaction. Conclusion: Sanger sequencing has failed to detect ESR1 mutation in primary tumor breast samples. Other advanced molecular techniques should be performed for diagnosis of primary breast tumors.