Molecular Medicine in Practice Epithelial–Mesenchymal Transition and StemCell 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 knownabout other characteristics of these cells. In thisprospective study,weassessed thegene transcripts of epithelial-to-mesenchymal transition—inducing transcription factors (EMT-TF) and cancer stem cell (CSC) features in patients with HER2þmetastatic breast cancer (MBC). Epithelial cells were enriched from peripheral blood mononuclear cells (PBMC) using antibody-coated anti-CD326 antibody (CD326þ) magnetic beads, and the residual CD326 PBMCswere further depleted of leukocytes using anti-CD45 antibody-coated magnetic beads (CD326 CD45 ). RNAwas 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 ALDHactivity andCSCs that express CD24, CD44, andCD133. Twenty-eight patientswere 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 ALDHþ/CD133þ cells in their blood than did patients with normal SNAIL1 and ZEB1 expression (P 1⁄4 0.038). Our data indicate that patients with HER2þ 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 HER2þ MBCs treated with trastuzumab-based therapy. Mol Cancer Ther; 11(11); 1–9. 2012 AACR.