Abstract 1725: Detection of ESR1 D538G mutation in circulating tumor cells (CTCs) and paired circulating tumor DNA (ctDNA) samples of breast cancer patients
Authors: Tzanikou Eleni , Markou Athina , Politaki Eleni , Koytsodontis Giorgos , Psyrri Amanda , Georgoulias Vassileios , Lianidou Evi 
Published: July 2017

AIMS: Molecular characterization of CTCs and ctDNA analysis holds promise as an extremely powerful tool for the molecular profiling of cancer patients in real time. Estrogen receptor alpha (ERα) is expressed in approximately 70% of all breast cancers and endocrine therapy represents a major treatment modality in ERα-positive disease. Recently, somatic mutations in the ERα gene (ESR1) were linked to acquired resistance to endocrine therapies in breast cancer. In this study, we analyzed the most frequent ERS1 mutation (D538G) in CTCs (DNA samples isolated from CellSearch cartridges), corresponding ctDNA from early and metastatic breast cancer patients and healthy donors.

METHODS: We first developed a highly sensitive and specific methodology for the detection of ESR1 D538G hotspot mutation, based on a combination of allele-specific PCR, asymmetric rapid PCR and high resolution melting analysis. We analyzed DNAs isolated from CTCs (CellSearch) and the corresponding ctDNA before and/or after therapy in: a) 25 patients with ER+ operable breast cancer, b) 11 patients with ER+ metastatic breast cancer, c) 13 patients with ER- early breast cancer, d) 5 patients with ER- metastatic breast cancer and e) 80 healthy female volunteers. In all cases ctDNA (extracted from 2 ml plasma) and DNA from CTCs were first examined for their DNA quality before analysis.

RESULTS: The assay is highly sensitive (analytical sensitivity: 0.05%) and specific (0/80 healthy donors). ERS1 D538G hotspot mutation was identified in ctDNA in 4/18 (22.2%) of ER+ metastasis-verified and in 5/33 (15.2%) of ER+ early breast cancer. In CTCs, ERS1 D538G mutation was identified in 6/18 (33.3%) of ER+ metastasis-verified and 5/33 (15.2%) of ER+ early breast cancer. In ER-pos metastasis-verified breast cancer, the concordance for D538G mutation between CTCs and ctDNA was 10/18 (55.6%), whereas the corresponding concordance for ER+ operable breast cancer was 25/33 (75.8%). Moreover, ERS1 D538G hotspot mutation was identified in ctDNA in 2/9 (22.2%) of ER- metastasis-verified breast cancer and 2/16 (12.5%) of ER- early breast cancer. ERS1 D538G hotspot mutation was identified in CTCs in 3/16 (18.8%) of ER- operable breast cancer, whereas none of ER- metastasis-verified breast cancer patients (0/9) were positive. In ER- metastasis-verified breast cancer patients, the concordance between CTCs and ctDNA for D538G mutation was 7/9(77.8%), whereas the corresponding concordance for ER- early breast cancer was 15/16 (93.8%).

CONCLUSIONS: We developed and validated an ultrasensitive and highly specific methodology for the detection of ERS1 D538G hotspot mutation. This mutation was detected not only in the ER+ group, but also in the ER- group of breast cancer patients. We will further evaluate our findings in a large cohort of patients before and after treatment, to evaluate response to endocrine therapies in breast cancer.

Citation Format: Eleni Tzanikou, Athina Markou, Eleni Politaki, Giorgos Koytsodontis, Amanda Psyrri, Vassileios Georgoulias, Evi Lianidou. Detection of ESR1 D538G mutation in circulating tumor cells (CTCs) and paired circulating tumor DNA (ctDNA) samples of breast cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1725. doi:10.1158/1538-7445.AM2017-1725