Abstract PS2-06: The detection and enumeration of circulating tumor cells (CTCs) and circulating endothelial cells (CECs) in metastatic breast cancer
Authors: Zhang Qiang , D'Amico Paolo , Donahue Jeannine , Gerratana Lorenzo , Davis Andrew A. , Jacob Saya Liz , Cai Zheng , Vagia Elena , Qiang Wenan , Shah Ami N. , Kerby Katy , Flaum Lisa , Zhang Youbin , Wehbe Firas , Behdad Amir , Gradishar William , Platanias Leonidas , Cristofanilli Massimo 
Published: February 2021

Abstract
Introduction: Circulating tumor cells (CTCs) are the roots of metastasis which is the main cause for death in metastatic breast cancer (MBC). CTCs enumeration is strongly prognostic in advanced disease and can stratify patients in two distinct disease, Stage IV aggressive and Stage IV indolent. In the former disease, the detection of CTC clusters and HER2-expression increase prognostic and predictive value. The metastatic cascade is a complex, regulated process involving immune cells and endothelial cells for progression and neoangiogenesis. Circulating endothelial cells (CECs) from the inner wall of blood vessels are shed into the blood stream during formation of blood vessels which is considered a sensitive marker of endothelial damage in pathological conditions such as cancer. CECs have been also studied as a biomarker for tumor progression and monitoring anti-angiogenic therapeutic effects in MBC. We evaluated the concomitant detection of CTCs and CECs in MBC patients, along with expression of HER2 in CTCs that may offer an interesting clue to elucidate the metastasis mechanisms. Methods: Whole blood samples (7.5ml/each) were collected from 14 stage IV MBC patients before systemic therapy. CTCs enumeration was performed in FDA approved CELLTRACKS System (Menarini) by using CTC Kit contains specific antibodies targeting the EpCAM for capturing CTCs, anti-CK-PE (for epithelial cells), DAPI (for nucleus), anti-CD45-APC (for leukocytes), and anti-HER-2/neu-FLU. The CTCs were classified as CK+, EpCAM+, DAPI+ and CD45-. Meanwhile, the same patients’ blood samples (4.0ml/each) were processed for CEC analyzed by using CEC kit which immunomagnetically captures CD146+ cells, and then stains the cells for CD105-PE (specific for protein endolgin), CD45-APC, nucleus-DAPI. The positive CECs were classified as CD 146+, CD105+, DAPI+ and CD45-. The associations between CTCs, HER2 expression and CECs were evaluated. Results: The average age of patients was 53.1. Subtypes of Luminal, HER2 positive and TNBC were 64.2% 7.2% and 28.6% respectively. Distant metastasis were found in 13 out of 14 patients, including bone (7), liver (5), Lymph nodes (5) and Pleura (2). CTCs were found positive (≥5, Stage IV aggressive) in 5 patients (range: 5-47, mean=24), and HER expression was identified in all 5 of these cases with a range of numbers between 1 and 7 (mean=4.2). The ratios of HER+ CTC/total ratios were 8.51%, 17.95%, 20%, 30.77%, and 33.33%. HER2 expression were defined officially in our lab according to the percentile of positive HER2 CTCs/Total CTCs and the expression intensity as - (&lt;20%), + (20-39%), ++ (40-59%) and +++ (≥60%) respectively. There were 9 patients (%) were identified as CTCs negative (&lt;5, Stage IV indolent) with the mean=1, and HER+ CTCs were found in only 2 patients with Stage IV indolent. Meanwhile, CECs were found in all 14 patients with a range of numbers between 4 to 115. There were an average of 33 CECs in Stage IV aggressive disease, compared to 53 CECs in Stage IV indolent. The average of CECs were 53.44, 12 and 37.25 in Luminal, HER2 positive and TNBC groups respectively. On the other hand, patients with HER2+ CTCs had an average of 50 CECs which is significantly higher than average of 41 CECs in patients without HER2+ CTCs. Moreover, there were average of 94.5, 56 and 40.22 CECs were found in groups when HER2 expression was ++/+++, above + and - respectively. The results demonstrated that although CTC enumeration have a reverse correlation with CECs numbers, HER2 expression in CTCs was significantly related with high CECs numbers. Conclusions: Our data provides the first evidence of potential association between CTCs and CECs in metastatic breast cancer. The association between HER2 expression and CECs offers a potential new insight to mechanism connections between CECs and disease metastasis in MBC.
Citation Format: Qiang Zhang, Paolo D'Amico, Jeannine Donahue, Lorenzo Gerratana, Andrew A. Davis, Saya Liz Jacob, Zheng Cai, Elena Vagia, Wenan Qiang, Ami N. Shah, Katy Kerby, Lisa Flaum, Youbin Zhang, Firas Wehbe, Amir Behdad, William Gradishar, Leonidas Platanias, Massimo Cristofanilli. The detection and enumeration of circulating tumor cells (CTCs) and circulating endothelial cells (CECs) in metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS2-06.