Abstract P4-01-08: Anatomical staging and value of CTCs in locally advanced breast cancer (LABC) and metastatic breast cancer (MBC)
Authors: Zhang Qiang , Gerratana Lorenzo , Flaum Lisa Flaum , Shah Ami Shah , Davis Andrew , Behdad Amir , Gradishar William , Platanias Leon , Cristofanilli Massimo 
Published: March 2020

Abstract
Introduction: The stage of a metastatic breast cancer (MBC) is determined by the cancer’s characteristics based on AJCC TNM system, including the size of the cancer tumor and invasion into nearby tissue (T), lymph nodes (N), and other parts of the body beyond the breast (M). Additional information including hormone-receptor status, HER2 status, and possibly Oncotype DX score has contributed to include biological characteristics and improve both, prognostic and predictive value of staging. Metastatic breast cancer (MBC) is a clinical and biologically heterogeneous condition poorly defined by current staging system. We recently report that CTCs enumeration can classify MBC in two distinct prognostic groups independently of clinical and molecular characteristics (Crit Rev Oncol Hematol. 2019). Moreover, our group reported that CTCs is associated with HER2 expression in MBC which may indicate more aggressive tumor (2019 AACR #1919). Here we compared CTCs enumeration of Stage III and Stage IV, and it would be helpful to evaluate the MBC metastasis capability and treatment in clinic.
Methods: The study included 64 specimens prospectively collected under IRB-approved protocol from 36 patients with Stage III BCa, and 424 specimens from 203 patients with stage IV BCa who received standard systemic treatments based on disease subtypes at NMH (2016-2019). Duplicate whole blood samples (7.5ml/each) were collected in EDTA tubes from these patients who were longitudinally characterized for CTCs before therapy (baseline, BL), and 3 months (first evaluation, FE) or 6 months (second evaluation, SE) after systemic therapies respectively. CTCs enrichment and enumeration were performed in FDA approved semi-automated fluorescence CELLTRACKS ANALYZERII® System (Menarini) by using CELLSEARCH® CXC Kit contains antibodies targeting the Epithelial Cell Adhesion Molecule (EpCAM) antigen for capturing CTCs, anti-CK-PE which is specific for the intracellular protein cytokeratin in epithelial cells, DAPI for staining the cell nucleus, anti-CD45-APC is specific for leukocytes, and anti-HER-2/neu-FLU is specific for HER-2/neu antigen (2019 ASCO #1036). The CTCs were classified based on morphology and correct phenotype as CK+, EpCAM+, DAPI+ and CD45−. Kruskal-Wallis test was used for statistics.
Results: There were median of 6.0 CTCs found in baseline level of Stage III patients, including average of 3. HER2− CTCs and 2.66 HER2+ CTCs. On the other hand, there were median of CTCs as 96.16 were detected at baseline level of Stage IV patients including average of 56.45 HER2− CTCs and 39.8 HER2+ CTCs respectively, which is about 15.9 folds higher than CTCs in Stage III patients (P&lt;0.001). Moreover, median of 14.51 CTCs (including 7.92 HER+ CTCs) were found in all 64 Stage III specimens, which is significantly lower than average of 62.25 CTCs (including 24.8 HER2+ CTCs) in total 419 Stage IV specimens. Meanwhile, CTC-clusters were found in 8.33% (3 out of 36) patients with average of 10.66 clusters in each positive specimen in Stage III group, in compared to 13.33% (27 out of 203) patients with average of 11.44 clusters in each positive specimen respectively in Stage IV group. Furthermore, within the Stage IV group, there were 117 out of 203 patients with FE CTCs test, among which 60 had also SE CTC tests. The total average CTCs decreased from 96.16 to 33 from BL to FE level, and then decreased to 15.86 in SE level after systemic therapies.
Conclusions: In this study, we showed that the total CTCs, HER2+ CTCs, and CTC-clusters is dramatically higher in Stage IV patients compared to Stage III patients, which provides an additional “staging tool” associated with a new measure of metastasis capability. Enumeration of CTCs can offer a strong supplemental criteria for TNM system for clinical decision-making.
Citation Format: Qiang Zhang, Lorenzo Gerratana, Lisa Flaum Flaum, Ami Shah Shah, Andrew Davis, Amir Behdad, William Gradishar, Leon Platanias, Massimo Cristofanilli. Anatomical staging and value of CTCs in locally advanced breast cancer (LABC) and metastatic breast cancer (MBC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P4-01-08.