title: Droplet digital polymerase chain reaction assay for screening of ESR1 mutations in 325 breast cancer specimens.
abstract: Droplet digital polymerase chain reaction (ddPCR), which could perform thousands of PCRs on a nanoliter scale simultaneously, would be an attractive method to massive parallel sequencing for identifying and studying the significance of low-frequency rare mutations. Recent evidence has shown that the key potential mechanisms of the failure of aromatase inhibitors-based therapy involve identifying activating mutations affecting the ligand-binding domain of the ESR1 gene. Therefore, the detection of ESR1 mutations may be useful as a biomarker predicting an effect of the treatment. We aimed to develop a ddPCR-based method for the sensitive detection of ESR1 mutations in 325 breast cancer specimens, in which 270 primary and 55 estrogen receptor-positive (ER+) metastatic breast cancer (MBC) specimens. Our ddPCR assay could detect the ESR1 mutant molecules with low concentration of 0.25 copies/μL. According to the selected cutoff, ESR1 mutations occurred in 7 (2.5%) of 270 primary breast cancer specimens and in 11 (20%) of 55 ER+ MBC specimens. Among the 11 MBC specimens, 5 specimens (45.5%) had the most common ESR1 mutation, Y537S, 4 specimens (36.3%) each had D538G, Y537N, and Y537C. Interestingly, 2 patients had 2 ESR1 mutations, Y537N/D538G and Y537S/Y537C, and 2 patients had 3 ESR1 mutations, Y537S/Y537N/D538G. Biopsy was performed in heterochrony in 8 women twice. In 8 women, 4 women had primary breast cancer and MBC specimens and 4 women had 2 specimens when treatment was failure. Four of these 8 women acquired ESR1 mutation, whereas no ESR1 mutation could be identified at first biopsy. ddPCR technique could be a promising tool for the next-generation sequencing-free precise detection of ESR1 mutations in endocrine therapy resistant cases and may assist in determining the treatment strategy.
authors.affiliation: Department of Breast and Endocrine Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Takeshita
authors.affiliation: Department of Molecular-Targeting Therapy for Breast Cancer, Kumamoto University Hospital, Kumamoto, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Yamamoto
authors.affiliation: Department of Breast and Endocrine Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.
authors.forename: Mutsuko
authors.initials: M
authors.lastname: Yamamoto-Ibusuki
authors.affiliation: Department of Breast and Endocrine Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.
authors.forename: Toko
authors.initials: T
authors.lastname: Inao
authors.affiliation: Department of Molecular-Targeting Therapy for Breast Cancer, Kumamoto University Hospital, Kumamoto, Japan.
authors.forename: Aiko
authors.initials: A
authors.lastname: Sueta
authors.affiliation: Department of Breast and Endocrine Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan.
authors.forename: Saori
authors.initials: S
authors.lastname: Fujiwara
authors.affiliation: Department of Breast and Endocrine Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan; Department of Endocrinological and Breast Surgery, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Omoto
authors.affiliation: Department of Breast and Endocrine Surgery, Graduate School of Medical Science, Kumamoto University, Kumamoto, Japan. Electronic address: hiwase@kumamoto-u.ac.jp.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Iwase
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D047628:Estrogen Receptor alpha / Q000235:genetics*; D005260:Female; D006801:Humans; D008875:Middle Aged; D009154:Mutation; D009362:Neoplasm Metastasis; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: C506487:ESR1 protein, human; D047628:Estrogen Receptor alpha