Table S1 shows the associations between the different clinicopathological parameters and the detection of CTCs and CTC phenotypes at baseline. In the HER2-positive setting, the detection of CK+ CTCs was associated with Grade III tumor status; no correlation was observed between the presence of different CTC subsets and clinicopathological parameters. Among HER2-negative patients, the detection of CK+CTCs was correlated to LN and lung metastases; patients with LN metastases more frequently harbored non-CSC+/epithelial-like CTCs, whereas in those with lung metastases, CSC+/partial-EMT+ CTCs were mainly identified. All the associations were investigated by Chi-square two-sided tests.
ARTICLE ABSTRACT
Circulating tumor cells (CTCs) bearing phenotypes related to cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT) have been identified in breast cancer; however, their clinical significance is not clear. In the current study, we investigated the prognostic relevance of single CSC+/partial-EMT+ CTCs in patients with metastatic breast cancer and the effect of first-line chemotherapy on their incidence. For this purpose, triple immunofluorescence against cytokeratin, ALDH1, and TWIST1 was performed in peripheral blood mononuclear cell (PBMC) cytospins from 130 patients before and after first-line chemotherapy. CSC+/partial-EMT+ CTCs were characterized as cells co-expressing cytokeratin, high levels of ALDH1, and nuclear TWIST1. CSC+/partial-EMT+ CTCs were evident in 27.7% of patients at baseline and were correlated to lung metastases (P = 0.010) and decreased progression-free survival [PFS; median 10.2 (8.9–11.6) vs. 13.5 (11.3–15.7) months; P = 0.024]. Their detection was an independent factor predicting for increased risk of relapse [multivariate analysis; HR (95% confidence interval (CI)): 1.785 (1.171–2.720); P = 0.007]. In HER-2–negative patients, CSC+/partial-EMT+ CTCs were additionally associated with reduced overall survival (OS) [median 39 (26.2–51.9) vs. 51 (15.7–86.4) months; P = 0.020] and increased risk of death [multivariate analysis; HR (95% CI): 2.228 (1.066–4.655); P = 0.033]. Chemotherapy resulted in a significant increase in the incidence of CSC+/partial-EMT+ CTCs (mean CTC% per patient: 59.4% post vs. 39.5% pre; P = 0.018), which was subsequently confirmed only in HER2-negative patients (P = 0.040) and in non-responders at the end of treatment (P = 0.020). In conclusion, CSC+/partial-EMT+ CTCs represent a chemoresistant subpopulation, which independently predicts for unfavorable outcome in metastatic breast cancer. Efficient targeting of these CTCs could potentially increase patient survival.
