Abstract P2-02-11: Detection of activating estrogen receptor 1 (ESR1) mutation on single circulating tumor cells from metastatic breast cancer patients
Authors: Paolillo C , Mu Z , Austin L , Nguyen T , Capoluongo E , Fortina P , Cristofanilli M 
Published: May 2016

Abstract
Background: 65% of primary breast cancers express the estrogen receptor α (ERα) and the mainstay of treatment are therapies that result in selective estrogen receptor modulation (SERM) of estrogen deprivation (aromatase inhibitors, AIs). Even thought endocrine therapy resulted in reduced recurrence and mortality, a significant portion of patients relapse with a metastatic disease and subsequently progress while of therapy for advanced disease (endocrine resistance). Recent evidence showed that activating hot spot mutation in the ligand binding domain of the ERα are acquired on treatment (frequency of 20%) and can drive resistance to endocrine therapy. Circulating tumor cells (CTCs) provide a non-invasive accessible source of tumor material and the molecular profiling of these rare cells might lead to insight on disease progression and therapeutic strategies. These features suggest that the detection of ESR1 mutation on single CTC may be a useful biomarker for therapy guidance.
Purpose: Investigate the incidence and heterogeneity of ESR1 mutational status within single CTCs isolated from individual metastatic breast cancer patients (mBCs), combining the FDA approved CellSearch® system for enumeration of CTCs with the DEPArrayTM technologies.
Methods: CTCs were enriched and enumerate by CellSearch® in 7.5 ml blood samples collected from 21 mBCs according to standard protocol. Each CTC-enriched sample with at least 20 CTCs was recovered from Veridex cartridge and loaded into the DEPArrayTM A300K chip, since the DEPArrayTM analyzed only the 66% of the sample volume loaded, according to the manufacturer's instructions. The chip scanning was performed by automated fluorescence microscope. The loaded cells were recovered as single cell and subdivided in tree different group: Cytokeratin (CK) positive ( Dapi+, CK+, ER-, CD45-); ER positive (Dapi+, ER+, CK+, CD45-); White Blood cells (WBCs) (Dapi+, CD45+, CK-, ER-). Single CTCs and WBCs were then submitted to whole genome amplification (WGA) using the Single Cell WGA kit (Yikon Genomics) according the manufacturer's instructions. Detection of target 14 ESR1 hot spot mutations was performed on ABI PRISM® 3700 genetic analyzer by target Sanger sequencing.
Results: 3 out of 21 mBCs with ≥20 CTCs were sorted and a total of 65 cells were recovered. WGA and ESR1 mutational status were performed on a total of 25 cells (respectively 11 ER+, 6 CK+ and 8 WBCs). In 1 of the 3 patients, that failed 2 lines of chemotherapy and previous single agent endocrine therapy, molecular heterogeneity was detected among its ER+ cells. 4 of 5 ER+ cells were heterozygote for the Y537S while one cell was homozygous, maybe due to a loss of heterozygosity. Y537S is one of the most common mutations that leads to a ligand independent ER transcriptional activity that does not respond to endocrine manipulation. No mutations were reported in all the CK+ and WBC cells analyzed.
Conclusions: This study demonstrates the feasibility of a non-invasive approach based on liquid biopsy in mBCs. Evaluation of ER status and early identification of ESR1 mutation in ER+ CTCs might allow to predict effect of the endocrine therapies and switching to other treatments before the emergence of metastatic disease.
Citation Format: Paolillo C, Mu Z, Austin L, Nguyen T, Capoluongo E, Fortina P, Cristofanilli M. Detection of activating estrogen receptor 1 (ESR1) mutation on single circulating tumor cells from metastatic breast cancer patients. [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 P2-02-11.