Does Primary Systemic Therapy Erradicate Disseminated and Circulating Tumor Cells?
Authors: Hoffmann O. , Solomayer E. , Kuemmel S. , Hahn M. , Kasimir-Bauer S. , Kimmig R. , Wallwiener D. , Fehm T. 
Published: December 2010

Abstract
Background: It has been widely accepted that breast cancer is a systemic disease For patients with large breast cancer tumors, primary systemic chemotherapy is considered to convert inoperable to operable primary tumors and is undertaken to improve surgical options. Here, we analyzed disseminated tumor cells (DTC) and circulating tumor cells (CTC) before and after primary systemic therapy to a) prove the effectiveness of treatment on DTC and CTC and b) to characterize CTC in blood by molecular profiling including the stem cell marker ALDH1 and markers involved in epithelial mesenchymal transition (EMT) which allows cells to travel to the site of metastasis formation without getting affected by conventional treatment.Materials and Methods: Two x 5 ml blood samples from breast cancer patients were collected before (n=85) and after (n=75) primary systemic therapy. CTC were analyzed using the AdnaTest BreastCancer (AdnaGen AG) for the detection of EpCAM, MUC-1 and HER-2 transcripts. The recovered c-DNA was additionally multiplex tested for three EMT markers (Twist1, Akt2, PI3Kα) and separately for ALDH1. The analysis of PCR products was performed by capillary electrophoresis on the Agilent Bioanalyzer 2100. The identification of EMT markers was considered positive if at least one marker was detected in the sample. Healthy donor samples were used to determine assay specificity. Two BM aspirates from 47 patients before and after treatment were analyzed by immunocytochemistry using the pan-cytokeratin antibody A45-B/B3. Evaluation of DTC was performed with either the ACIS system (Chromavision) or the ARIOL system (Applied Imaging).Results: Primary systemic therapy resulted in therapy response (partial and complete response) in 75% of all cases. DTC were found in 13/47 (28%) BM samples before and in 11/47 (23%) BM samples after therapy. CTC were detected in 15/85 (18%) of the patients before therapy expressing EpCAM (53%), MUC-1 (67%) and HER-2 (60%), respectively. After therapy, 7/75 (9%) were still positive for CTC expressing EpCAM (43%), MUC-1 (43%) and HER-2 (71%), respectively. When blood samples were analyzed for EMT markers and ALDH1 before therapy, 17% were positive for at least one of the EMT markers and 4% for ALDH1, respectively. Interestingly, the percentages after neoadjuvant treatment were 35% and 41%, respectively.Conclusion: The identification of patients at increased risk for recurrence after completion of systemic chemotherapy is of high clinical relevance. Although we achieved clinical response in 75% of all cases our data indicate that 1) DTC in the BM are weakly effected by primary systemic treatment, 2) CTC in the blood stream are erradicated more effectively but 3) CTC found in the blood after treatment show EMT and tumor stem cell characteristics. These markers may serve as an indicator for therapy resistant tumor cell populations and the evaluation of more patients will prove whether these patients might benefit from an additional 'second-line' treatment protocols including bisphosphonates.
Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3001.