Abstract P1-02-02: ESR1 mutations in circulating tumor cell versus circulating cell-free DNA of metastatic breast cancer patients before first-line endocrine therapy and at progression
Authors: Sieuwerts AM , Beije N , Kraan J , Van M , Onstenk W , Vitale SR , van der Vlugt – Daane M , Hamberg P , Dirix LY , Brouwers A , de Jongh FE , Jager A , Seynaeve CM , Jansen MPHM , Foekens JA , Martens JWM , Sleijfer S 
Published: March 2017

Abstract
Background
Mutations in ESR1, the gene encoding the estrogen receptor, have been linked to endocrine resistance in metastatic breast cancer (MBC). It is thought that these mutations are selected during endocrine treatment (ET), but direct evidence that these ESR1 mutations (mESR1) emerge during treatment with endocrine agents is scant. We set out to evaluate mESR1 in circulating tumor cells (CTCs) and matched plasma cell-free DNA (cfDNA) of MBC patients before start of 1st line ET and at progression.
Materials & Methods
CellSearch-enriched CTCs (≥ 5 CTC/7.5 mL) of 37 MBC patients before start of 1st line ET (baseline cohort; BL) and 38 MBC patients who had progressed on any line of ET for metastatic disease (progressive disease cohort; PD) were evaluated. 52% of the PD patients received one line of ET and 48% more lines, of which 92% contained an aromatase inhibitor. In addition, 10 CellSearch-enriched fractions from healthy blood donors (HBDs) and 46 matched plasma samples (7xHBD, 15xBL, 24xPD) were included. DNA was isolated using the AllPrep kit and cfDNA with the QIAamp CNA kit (Qiagen). Hotspot mutations for ESR1 (D538G, Y537S, Y537C and Y537N) were evaluated with mutation-specific Taqman assays by chip-based digital PCR (QuantStudio 3D). mESR1 status was assessed after target-specific ESR1 amplification capturing all 4 mutations, with thresholds for positivity based on the highest variant allele frequencies in HBDs.
Results
Of all the CTC samples in the BL cohort, 1 patient had mutated Y537N copies, while this mutation was not detected in the matched cfDNA. This patient had received adjuvant treatment with tamoxifen. Also none of the other 14 BL cfDNA samples analyzed harbored mESR1. Three PD patients (8%) were positive for mESR1 in their CTCs (2x D538G and 1x Y537S). These D538G variants identified in CTCs were also detected in the corresponding cfDNA of these patients; for the Y537S mutation no matched cfDNA was available. Seven additional mESR1 carriers were identified in the other 22 matched cfDNA PD samples, resulting in 38% mESR1 positivity of the PD plasma samples (7x D538G, 1x Y537C and 1x Y537C).
Conclusion
Sensitivity for detecting mESR1 in CTC fractions (identified in 8% of the PD patients) was lower than for cfDNA samples. Using cfDNA for mESR1 detection, we found an higher prevalence of mESR1 variants in samples obtained at progression to ET (38%) compared to baseline (0%). These findings further substantiate the role of mESR1 in endocrine resistance.
Citation Format: Sieuwerts AM, Beije N, Kraan J, Van M, Onstenk W, Vitale SR, van der Vlugt – Daane M, Hamberg P, Dirix LY, Brouwers A, de Jongh FE, Jager A, Seynaeve CM, Jansen MPHM, Foekens JA, Martens JWM, Sleijfer S. ESR1 mutations in circulating tumor cell versus circulating cell-free DNA of metastatic breast cancer patients before first-line endocrine therapy and at progression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-02-02.