Abstract 3066: Establishment of a new method for the selection and detection of circulating tumor cells in metastatic breast cancer patients

Abstract

Background: Circulating tumor cells (CTCs) are discussed to be an ideal surrogate marker for individualized treatment options in metastatic breast cancer (MBC) since metastatic tissue may be difficult to obtain for repeated analysis. Here we established a new method for selection and detection of the heterogeneous CTC population using immunomagnetic enrichment followed by multi-marker profiling of genes related to different CTC phenotypes. Materials and Methods: Establishment of a nine gene qPCR panel was performed using various cancer cell lines for the markers EpCAM (epithelial); PI3K, AKT2 [epithelial-mesenchymal-transition (EMT)]; ALDH1 (stem cell); ERCC1, Aurora kinase (resistance markers); HER2, HER3, EGFR (receptors); CD45 (leucocyte control) and GAPDH (housekeeping gene) as well as the synthetic EpCAM fragment as an internal reference. 2x5 ml blood of 20 MBC patients was collected at the time of disease progression (T0) and at clinical staging (T1) after 8-12 weeks of chemo-, hormone or antibody therapy for the detection of CTCs applying a new positive immunomagnetic selection using anti-EpCAM, anti-HER2 and anti-EGFR coated magnetic beads (AdnaTest EMT-2/Stem Cell Select, AdnaGen AG Germany). cDNA was gene specifically pre-amplified using the TATAA Multiplex Grand Master Mix according to in-house designed assays. qPCR was performed using Bio-Rad iTaq Universal Supermix SYBR Green Mix. The cutoff was calculated for each gene separately in a way that the false positive rate in all healthy donors (n=18) was lower than 10% (specificity >90%). Subsequently, delta delta Ct was calculated as Ct (cutoff)-Ct(sample) - [Ct(CD45 cutoff)-Ct(CD45sample)]. Results were correlated with clinical response to a given therapy. Results: At T0, at least one of the studied markers was detected in 19/20 patients and at T1 in 16/20 patients, respectively. The distribution of the markers across all patients was highly variable at both time points. However, EpCAM, Aurora kinase as well as EGFR and HER3 were observed most frequently. Interestingly, some patients expressed only one CTC-subtype. Clinically, we observed an increased frequency for epithelial, EMT, stem cell and resistance markers in therapy resistant patients as compared to responders. Whereas EMT and stem cell like CTCs were only detected in the resistant group, CTCs still detectable in the responder group mainly represented an epithelial phenotype showing HER2, HER3 or EGFR expression. Interestingly, some of these persisting CTCs in responders were also positive for ERCC1 and Aurora kinase. Conclusion: We successfully established a new method for the detection of the heterogeneous CTC population which might help to match the right therapy to the right patient in the future. Despite these promising preliminary findings, the qPCR panel has to be further optimized and needs to be verified in a larger patient population. Citation Format: Maren Bredemeier, Bahriye Aktas, Jenny Wagner, Doreen Schellbach, Rainer Kimmig, Sabine Kasimir-Bauer. Establishment of a new method for the selection and detection of circulating tumor cells in metastatic breast cancer patients. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3066. doi:10.1158/1538-7445.AM2014-3066

Molecular characterization of circulating tumor cells from patients with metastatic breast cancer reflects evolutionary changes in gene expression under the pressure of systemic therapy

“…Both kits employ immunomagnetic capturing of CTCs but different antigens are used, the EMT1 kit has antibodies against EpCAM and MUC1 whereas the EMT2 kit has antibodies against EpCAM, HER2, and EGFR. It has been suggested that the EMT2 kit can identify more mesenchymal CTCs that have passed through EMT [ 78 ]. The enriched CTCs were purified and lysed to retrieve mRNA.…”