Epithelial-mesenchymal Transition and Stem Cell Markers in Patients with HER2-positive Metastatic Breast Cancer

Currently, there is extensive information about circulating tumor cells (CTC) and their prognostic value; however, little is known about other characteristics of these cells. In this prospective study, we assessed the gene transcripts of epithelial-to-mesenchymal transition-inducing transcription factors (EMT-TF) and cancer stem cell (CSC) features in patients with HER2(+) metastatic breast cancer (MBC). Epithelial cells were enriched from peripheral blood mononuclear cells (PBMC) using antibody-coated anti-CD326 antibody (CD326(+)) magnetic beads, and the residual CD326(-) PBMCs were further depleted of leukocytes using anti-CD45 antibody-coated magnetic beads (CD326(-)CD45(-)). RNA was extracted from all cell fractions, reverse transcribed to cDNA, and subjected to quantitative reverse transcription PCR to detect EMT-TFs (TWIST1, SNAIL1, ZEB1, and TG2) as a measure of CTCs undergoing EMT (EMT-CTCs). In addition, PBMCs were analyzed using multiparameter flow cytometry for ALDH activity and CSCs that express CD24, CD44, and CD133. Twenty-eight patients were included in this study. At least one EMT-TF mRNA was elevated in the CTCs of 88.2% of patients and in the CD326(-)CD45(-) cell fraction of 60.7% of patients. The CD326(-)CD45(-) fraction of patients with elevated SNAIL1 and ZEB1 transcripts also had a higher percentage of ALDH(+)/CD133(+) cells in their blood than did patients with normal SNAIL1 and ZEB1 expression (P = 0.038). Our data indicate that patients with HER2(+) MBCs have EMT-CTCs. Moreover, an enrichment of CSCs was found in CD326(-)CD45(-) cells. Additional studies are needed to determine whether EMT-CTCs and CSCs have prognostic value in patients with HER2(+) MBCs treated with trastuzumab-based therapy.