Abstract P6-07-07: Clinical significance of ESR1 mutations using droplet digital polymerase chain reaction assay in 325 breast cancer samples
Authors: Takeshita T , Yamamoto Y , Yamamoto-Ibusuki M , Inao T , Sueta A , Fujiwara S , Iwase H 
Published: May 2016

Abstract
Purpose: We aimed to develop a droplet digital Polymerase Chain Reaction (ddPCR)-based method for the sensitive detection of estrogen receptor (ER) α (ESR1) mutations in the primary and recurrent/metastatic tumor tissues of breast cancer.
Experimental Design: We studied a total of 325 tumor specimens (270 primary breast cancer specimens and 55 ER-positive recurrent/metastatic tumor specimens). Because the recurrent/metastatic tumor specimens had much inflammatory and stromal cells, we captured only tumor cells using laser microdissection. We investigated the quantification of rare ESR1 mutations, four representative types, Y537S, Y537N, Y537C, and D538G in extracted genomic DNA using ddPCR system that simultaneously performed thousands of PCRs on a nanoliter scale.
Results: In 270 primary breast cancer samples, we analyzed each ESR1 alteration percentage in each subtype. ESR1 Y537C tended to be higher in hormone receptor-positive (HR+)/ human epidermal growth factor receptor 2-negative (HER2-) group (P = 0.06) and higher percentage of ESR1 D538G was statistically significant in HR+/ HER2- group (P = 0.027), compared with HER2+ group. There was no statistically different in each ESR1 alteration percentage between HR+/ HER2- group and HR-/ HER2- group. Whether each ESR1 alteration was dichotomized as positive or not, we used the percentage which HER2+ group and HR-/ HER2- were not identified, as a cutoff point. ESR1 mutations occurred in 7 samples (2.5%) out of 270 primary samples, but ESR1 mutations occurred in 11 samples (20%) out of 55 metastatic/ recurrent breast cancer samples.
Table 1 Patients characteristics of 11 metastatic ER-positive breast cancer cases with ESR1 mutationsCaseAge (years)HER2 statusKi67 LIER HSPgR HSBiopsy siteMutation472-102051Lymph nodeY537C 1610 A&gt;G only665-518218Lymph nodeY537S 1610 A&gt;C Y537N 1609 T&gt;A D538G 1613 A&gt;G1555-201105SkinY537C 1610 A&gt;G only1863-10162130Lymph nodeD538G 1613 A&gt;G only3354-417015Lymph nodeY537S 1610 A&gt;C Y537N 1609 T&gt;A D538G 1613 A&gt;G4268-516950SkinY537C 1610 A&gt;G only4466-24270159Lymph nodeY537N 1609 T&gt;A only4673-20224110Lymph nodeY537N 1609 T&gt;A and D538G 1613 A&gt;G4952-20275138SkinY537S 1610 A&gt;C and Y537C 1610 A&gt;G5040-101740Lymph nodeY537S 1610 A&gt;C only5140-101895IBTRY537S 1610 A&gt;C onlyAbbreviations: HER2, human epidermal growth factor receptor 2; LI, labeling index; ER, estrogen receptor; HS, histoscore; PgR, progesteron receptor; ET, endocrine therapy; IBTR, ipsilateral breast tumor recurrence; SD, stable disease; PD, progressive disease; MPA, medroxyprogesterone acetate
Two biopsies were performed in 8 women, in which four women had primary and recurrent/metastatic samples. Four out of these 8 women acquired ESR1 mutation, whereas no ESR1 mutation could be identified at first biopsy.
Conclusions: We demonstrated the sensitive detection and accurate quantification of low frequency ESR1 mutations in 270 primary breast cancer samples and 55 recurrent/metastatic samples using ddPCR assay. This technique could prove a useful method for the precise detection of ESR1 mutations in endocrine therapy resistant cases.
Citation Format: Takeshita T, Yamamoto Y, Yamamoto-Ibusuki M, Inao T, Sueta A, Fujiwara S, Iwase H. Clinical significance of ESR1 mutations using droplet digital polymerase chain reaction assay in 325 breast cancer samples. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-07-07.