Abstract P2-02-04: Distinct clinical and biological values of subpopulations of circulating tumor cells (CTCs) in primary breast cancer
Authors: Mego M , Jurisova S , Karaba M , Minarik G , Benca J , Sedlackova T , Manasova D , Malejcikova M , Sieberova G , Macuch J , Gronesova P , Sufliarsky J , Pindak D , Cristofanilli M , Reuben JN , Mardiak J 
Published: May 2016

Background: CTCs represent a heterogeneous population of cells with different phenotypes and biological values. Epithelial to mesenchymal transition (EMT) gives rise to cells with stem cell-like properties with increased resistance to chemotherapy that may be under detected by currently approved assays. The aim of this study was to characterize CTCs based on the expression of epithelial and mesenchymal markers in primary breast cancer (BC) and to correlate them with patients'/tumor characteristics.

Methods: This prospective translational study included 422 patients with primary BC enrolled from March 2012 to February 2015. Blood for CTC detection was drawn before surgery (422 patients), before 1st cycle (95 patients) and before 2nd cycle (53 patients) of adjuvant therapy. Isolated peripheral blood mononuclear cells (PBMC) were depleted of cells of hematopoietic origin (CD45+) using RossetteSep kit (StemCell Technologies) negative selection with anti-CD45 antibody. RNA extracted from CD45-depleted (CD45) PBMC was interrogated for expression of EMT-inducing transcription factors (TWIST1, SNAIL1, SLUG, ZEB1) and epithelial (CK19) gene transcripts by quantitative reverse transcription-PCR. Expressions of gene transcripts in CD45- PBMC from patients were compared to those of CD45- PBMC of 60 healthy donors.

Results: Totally, CTCs were detected in 116/422 (27.5%) patients before surgery, in 21/95 (22.1%) patients after surgery and before 1st cycle and in 19/53 (35.8%) of patients before 2nd cycle of adjuvant therapy. Before surgery, CTCs exhibited only epithelial markers in 38 (9.0%) patients, only EMT markers in 68 (16.1%) of patients, while in 10 (2.4%) patients CTCs with both epithelial and EMT markers were detected. Epithelial CTCs were more often detected before surgery compared to after surgery (11.4% vs. 2.1%; p = 0.003), while mesenchymal CTCs were more often detected after the 1st cycle of chemotherapy as opposed to detection before surgery (30.2% vs. 18.2%; p = 0.05). Patients with N2-3 disease had more often detectable CTCs compared to patients with N0-1 disease (41.4% vs. 24.9%, p = 0.01) and this was mainly driven by mesenchymal CTCs (31.0% for N2-3 vs. 16.0% for N0-1; p = 0.007). Similarly, patients that lacked p53 expression (wild type TP53) in primary tumor had more often CTCs with EMT phenotype opposite to patients with p53 expression (p = 0.02). Presence of epithelial CTCs was significantly associated with lower absolute lymphocyte (p = 0.02) and neutrophil (p = 0.02) counts in peripheral blood.

Conclusions: Our results support the concept of CTCs phenotypic heterogeneity in breast cancer patients. These results support the role of EMT in cancer pathogenesis and suggest that CTCs with EMT phenotype are involved in tumor dissemination while their increase after chemotherapy might be a mechanism of treatment resistance. Moreover, these data suggest inverse relationship between immune cells and epithelial CTCs which stress the role of immune cells in tumor dissemination.

Citation Format: Mego M, Jurisova S, Karaba M, Minarik G, Benca J, Sedlackova T, Manasova D, Malejcikova M, Sieberova G, Macuch J, Gronesova P, Sufliarsky J, Pindak D, Cristofanilli M, Reuben JN, Mardiak J. Distinct clinical and biological values of subpopulations of circulating tumor cells (CTCs) in primary breast cancer. [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 P2-02-04.