Abstract P4-01-10: Predictive impact of circulating tumor cells with an epithelial-to-mesenchymal transition phenotype in patients with primary breast cancer treated with primary systemic therapy
Authors: Le Du Fanny , Duose Dzifa Y , Dettman Elisha J , Summer Jackson A , Chavez-MacGregor Mariana , Barcenas Carlos H , Brewster Abenaa M , Ricardo Alvarez H , Valero Vincente , Gonzalez-Angulo Ana M , Reuben James M , Ueno Naoto T 
Published: May 2015

Abstract
Background: Tumor cells with a mesenchymal phenotype include cancer stem-like cells (CSCs), which are known to contribute to metastasis. Circulating tumor cells (CTCs) are epithelial cells in peripheral blood that are detected using an anti-EpCAM antibody; however, CTCs undergoing epithelial-mesenchymal transition (EMT) may not be detected using this method. We have developed an antibody-independent CTC enrichment platform, Apostream®, which does not rely on EpCAM-based capture. We used this instrument to determine the clinical relevancy and feasibility of measuring EMT-CTCs in breast cancer patients.
Methods: Blood samples from newly diagnosed breast cancer patients were prospectively collected at baseline (T0), after primary systemic therapy (T1), and after definitive surgery (T2) and processed using the Apostream® system. Isolated cells were stained with antibodies to cytokeratin (anti-CK), leukocytes (anti-CD45), and the nuclear stain, 4,6-diamidino-2-phenylindole (DAPI), to identify CTCs. These CTCs were also stained with additional markers and examined on a laser scanning cytometer to measure protein expression levels of epithelial (EpCAM, E-cadherin), mesenchymal (β-catenin, vimentin) and CSC-markers (CD44, CD24). Pathological complete response (pCR) and residual cancer burden (RCB) statuses after preoperative treatment were obtained to correlate baseline CTCs and marker expression with treatment response.
Results: The study enrolled 33 patients (10 with early-stage, 19 with locally advanced, and 4 with metastatic breast cancer); 32, 12, and 10 patients provided samples at T0, T1, and T2, respectively. Of the 20 patients who underwent surgery, 6 patients achieved pCR. CTCs were detected (≥ 1 cell in at least one of the three samples) in 47%, 75%, and 80% of the T0, T1, and T2 samples, respectively. EMT markers (either vimentin or β-catenin) were detected in 67%, 11%, and 38% of these CTCs, respectively. The mean number of CTCs per mL detected at T0 was 0.43 (range, 0-3.7). CTC detection was correlated with a high histological Nottingham index (P=0.039). Conversely, vimentin-positive CTC detection was inversely correlated with clinical stage at T0 (P=0.034). No significant correlation was observed between CTCs detected at baseline and breast cancer subtypes. However, there was a trend for CTCs detected at T0 to be predictive of chemotherapy response, as 62% of patients with CTCs at T0 achieved a pCR, whereas only 11% of patients without CTCs at T0 had a pCR (P=0.057). The other EMT and CSC markers we tested did not predict response.
Conclusions: Apostream® was successful in detecting EMT-CTCs in this prospective study. There was a trend for the presence of CTCs at baseline to predict pCR. We will present our final data analysis of 50 patients.
pCR (n=6)†RCB-I (n=2)RCB-II (n=5)RCB-III (n=4)P-valueCK+CD45- CTCsNegative (n=9)1(11)2(22)3(33)3(33)0.146 0.057*Positive (n=8)5(62)0(0)2(25)1(13)† All data are no. of patients (%) Two did not have an RCB status available, and 1 did not have a T0 sample. * Comparing pCR status (pCR vs no-pCR) between CTCs negative and positive.
Citation Format: Fanny Le Du, Dzifa Y Duose, Elisha J Dettman, Jackson A Summer, Mariana Chavez-MacGregor, Carlos H Barcenas, Abenaa M Brewster, Alvarez H Ricardo, Vincente Valero, Ana M Gonzalez-Angulo, James M Reuben, Naoto T Ueno. Predictive impact of circulating tumor cells with an epithelial-to-mesenchymal transition phenotype in patients with primary breast cancer treated with primary systemic therapy [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-01-10.