Table S2 shows the associations between the type of chemotherapy and the presence and phenotype of CTCs before and after treatment. Among HER2-positive patients, there was no correlation between the type of therapy and the detection of CTC phenotypes before or after treatment. In the HER2-negative cohort, no associations were observed regarding the baseline detection of distinct CTC subpopulations. Post-treatment, monotherapy with Taxanes or Anthracyclines was strongly associated with negative CTC status (p=0.000) and with absence of CSC+/partial-EMT+ CTCs (p=0.002). In contrast, combination treatment without Taxanes or Anthracyclines resulted in a 100% positivity for CTCs and a higher incidence of CSC+/partial-EMT+ CTCs. All the associations were investigated Exact two-sided Chi-square 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.
