Abstract P4-01-04: ESR1 mutation in cell free DNA (cfDNA) is associated with significantly increased circulating tumor cell (CTC)-clusters and progress in stage III/IV breast cancer after systemic treatments
Authors: Zhang Q , Gerratana L , Zhang Y , Flaum L , Shah A , Davis A , Behdad A , Gradishar W , Platanias L , Cristofanilli M 
Published: May 2019

Introduction: CTCs play a critical role in the process of tumor metastasis, and a portion of CTCs may form clusters that contain two or more CTCs bound together which were reported to have up to 50-fold of potential of forming distant metastasis in breast cancer (MBC) as compared to individual CTCs. However, molecular and genomic characterization of CTCs cluster remain largely unknown. Here we report a highly significant correlation between ESR1 mutation in cfDNA, CTCs count and CTC-cluster, which may help to understand MBC metastasis and predict treatment benefit, especially for metastatic or recurrent disease.

Methods: A total of 80 whole blood samples (7.5ml/each) were collected from 80 patients with stage III/IV BCa after informed consent under IRB-approved trial at the RHLCCC at Northwestern University before and after systemic therapies. Among these 80 patients, 41 patients received chemotherapy and 23 patients received endocrine therapy, among which 20 patients received combo treatments (16 plus Palpociclib, 1 plus Ribociclib, 2 plus Everolimus, and 1 plus trastuzumab). CTC enrichment and enumeration were performed in CELLTRACKS ANALYZERII® System (Menarini) by using CTC Kit Meanwhile, we detected the ESR1 hotspot mutations (Y537S and D538G) in plasma cfDNA from all 80 patients by Droplet digital PCR (ddPCR) assay using the QX200 ddPCR System (Bio-Rad). cfDNA was isolated from 2 mL of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen) and the MAF was analyzed using QuantaSoft software (Bio-Rad).Database of CTCs and ESR1 mutation was linked with clinical database. Kruskal-Wallis test was used for statistics.

Results: Of the 80 samples analyzed, there were 57 samples without ESR1 mutations (Group 1), and 23 samples that had ESR1 mutations (8 Y537S mutations and 23 D538G mutations, Group 2). CTC positive (≥5) were detected in 13/57 samples (Group 1) and 15/23 samples, and the average amounts of CTCs were 21.77 CTCs/each sample and 59.86 CTCs/each sample in Group 1 and Group 2 respectively. There was a significant association between ESR1 mutations and high level of CTCs (P=0.000088). More important, CTC-clusters were found in 3 samples in Group 1 (5.26%) and in 5 samples in Group 2 (21.74%) respectively. There was a significant correlation between ESR1 mutations and CTC-clusters (P=0.026). Furthermore, there were 18/57 patients in group 1 and 5/23 in group 2 receiving chemotherapy. Moreover, 26/57 in group 1 and 15/23 in group 2 that received chemotherapy. Our results also confirmed that both endocrine therapy and chemotherapy benefited more patients without ESR1 mutations in compared with patients with ESR1 mutations (P&lt;0.05).

Conclusion:We first elucidated the association between ESR1 mutations in ctDNA and CTC-cluster in MBC patients, and provides new insights on the molecular mechanisms associated with the metastasis process. In addition with the highly significant association between ctDNA ESR1 mutations and endocrine resistance we describe a new association allowing to expand the prognostic and predictive role of both tests enabling monitoring the metastatic prognosis and endocrine resistance for clinical decision-making.

Citation Format: Zhang Q, Gerratana L, Zhang Y, Flaum L, Shah A, Davis A, Behdad A, Gradishar W, Platanias L, Cristofanilli M. ESR1 mutation in cell free DNA (cfDNA) is associated with significantly increased circulating tumor cell (CTC)-clusters and progress in stage III/IV breast cancer after systemic treatments [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-04.