EpCAM-independent isolation of circulating tumor cells with epithelial-to-mesenchymal transition and cancer stem cell phenotypes using ApoStream^® in patients with breast cancer treated with primary systemic therapy

Background Tumor cells with a mesenchymal phenotype and/or cancer stem-like cells (CSCs) are known to contribute to metastasis and drug resistance. Circulating tumor cells (CTCs) undergoing epithelial-mesenchymal transition (EMT) and CTCs reflecting a dedifferentiated CSC phenotype may not be detected using only an anti-EpCAM antibody to capture them. We used an antibody-independent CTC enrichment platform, ApoStream^®, which does not rely on any antibody, including anti-EpCAM, to capture EMT- and CSC-CTCs in breast cancer patients who received neoadjuvant chemotherapy and correlated them to pathological complete response (pCR). Methods Blood samples from newly diagnosed breast cancer patients were prospectively collected before neoadjuvant chemotherapy (T₀), after chemotherapy but before surgery (T₁), and after surgery (T₂) and processed using ApoStream. CTCs detected were stained with additional markers to define 3 CTC subsets with the following phenotypes: epithelial CTCs (CK +, EpCAM+ or E-cadherin+), EMT-CTCs (β-catenin+ or vimentin+), and CSC-CTCs (CD44 + and CD24^low). Results We enrolled 55 patients, 47 of which had data for analysis. EMT-CTCs were detected in 57%, 62%, and 72% and CSC-CTCs in 9%, 22%, and 19% at the T₀, T₁, and T₂ time points, respectively. Counts of epithelial (P = 0.225) and EMT (P = 0.522) phenotypes of CTCs at T₀ did not significantly predict pCR. Moreover, no correlation between CTC count change and pCR was demonstrated. Conclusions ApoStream was successful in detecting EMT-CTCs among patients after neoadjuvant chemotherapy. However, EMT-/CSC-CTC counts did not correlate with pCR. Due to the small sample size and heterogeneity of this patient population, further study in a larger cohort of molecularly homogeneous patients is warranted.