Circulating tumour cells (CTCs) serve as valuable biomarkers. However, EpCAM positive CTCs are less frequently detected in NSCLC patients compared to other epithelial tumours. First, EpCAM protein expression was analysed in primary and metastatic lung cancer tissue. In both groups 21% of the samples were EpCAM negative. Second, the CellSearch system identified 15% of patients (n = 48) as CTC positive whereas a multiplex RT-PCR for PIK3CA, AKT2, TWIST, and ALDH1 following EGFR, HER2 and EpCAM based enrichment detected CTCs in 29% of the patients. Interestingly, 86% of CTC positive patients were found to express ALDH1. Only 11% of the patients were CTC-positive by both techniques. CTC positivity was associated with patient disease state when assessed by the multiplex RT-PCR assay (p = 0.015). Patients harbouring tumours with an altered EGFR genotype were more frequently CTC-positive compared to patients with EGFR wildtype tumours. In subsets of patients, CTCs were found to express genes involved in resistance to therapy such as HER3 and MET. In conclusion, using multiple targets for CTC capture and identification increases the sensitivity of CTC detection in NSCLC patients, which can be explained by the presence of different CTC subtypes with distinct molecular features.

BackgroundCirculating tumor cells (CTC) are discussed to be an ideal surrogate marker for individualized treatment in metastatic breast cancer (MBC) since metastatic tissue is often difficult to obtain for repeated analysis. We established a nine gene qPCR panel to characterize the heterogeneous CTC population in MBC patients including epithelial CTC, their receptors (EPCAM, ERBB2, ERBB3, EGFR) CTC in Epithelial-Mesenchymal-Transition [(EMT); PIK3CA, AKT2), stem cell-like CTC (ALDH1) as well as resistant CTC (ERCC1, AURKA] to identify individual therapeutic targets.ResultsAt TP0, at least one marker was detected in 84%, at TP1 in 74% and at TP2 in 79% of the patients, respectively. The expression of ERBB2, ERBB3 and ERCC1 alone or in combination with AURKA was significantly associated with therapy failure. ERBB2 + CTC were only detected in patients not receiving ERBB2 targeted therapies which correlated with no response. Furthermore, patients responding at TP2 had a significantly prolonged overall-survival than patients never responding (p = 0.0090).Patients and Methods2 × 5 ml blood of 62 MBC patients was collected at the time of disease progression (TP0) and at two clinical staging time points (TP1 and TP2) after 8–12 weeks of chemo-, hormone or antibody therapy for the detection of CTC (AdnaTest EMT-2/StemCell Select™, QIAGEN Hannover GmbH, Germany). After pre-amplification, multiplex qPCR was performed. Establishment was performed using various cancer cell lines. PTPRC (Protein tyrosine phosphatase receptor type C) and GAPDH served as controls.ConclusionsMonitoring MBC patients using a multimarker qPCR panel for the characterization of CTC might help to treat patients accordingly in the future.

Summary Immunohistological detection of P-glycoprotein (P-gp) with monoclonal antibody C219 was performed on serial sections of 37 neuroblastoma specimens representative of the different forms of the disease, from stage 1 ganglioneuroma to stage 4 neuroblastoma. Malignant cells, irrespective of their degree of maturation varying from neuroblasts to ganglion cells, were negative on all specimens. The expression of P-glycoprotein was detected in nine specimens, but it was restricted to normal cells within the tumour. In four specimens, C219 reacted with normal infiltrating cells in the stroma (i.e. monocytes, histiocytes or fibroblasts) representing 5 to 10% of the total population within the section; in three specimens, the residual adrenal gland was strongly positive, and in two ganglioneuromas, a weak reactivity of C219 was observed on a few satellite cells and schwann cells. Three of 15 biopsies obtained at diagnosis contained normal P-gp positive cells: two were classified as stage 1 ganglioneuromas; one was a typical stage 4 composite tumours with positive histiocytes and fibroblasts in the well-differentiated counterpart. Six of 22 biopsies obtained after patients had received our current protocol of chemotherapy contained normal P-gp positive cells: five were partially differentiated and necrotic under the effect of chemotherapy; only one positive specimen was classified as undifferentiated neuroblastoma. Among negative specimens from previously treated patients, one was obtained from a patient in relapse after high-dose chemotherapy and ABMT, two were obtained from patients who had not responded to induction therapy, and six from patients in partial remission after induction therapy. The clinical evolution was very similar in both groups of patients with P-gp negative or positive biopsies.These findings suggest that the quantitative assessment of MDR RNA by northern blotting on fresh homogenates is likely to overestimate its expression on neuroblastoma cells, and that the mechanism of chemoresistance in widespread neuroblastoma is less likely to be associated with P-gp expression.The relationship between the overexpression of the 170 kD cell membrane glycoprotein (P-glycoprotein) and the phenomenon of multidrug resistance (MDR) has been clearly demonstrated in human tumour cell lines; P-glycoprotein (P-gp) functions as a drug-efflux pump which can be reversed in vitro by calcium channel blockers such as Verapamil (Gerlach et al., 1986;Rothenberg & Ling, 1989). The overexpression of P-gp has been well documented in a number of tissues, including liver, colon, kidney and adrenal gland, in untreated human malignancies arising from these tissues, as well as in a variety of tumours after treatment (Bell et al., 1985;Gerlach et al., 1987;Ma et al., 1987;Epstein et al., 1989;Lai et al., 1989;Merkel et al., 1989;Thiebaut et al., 1989; CordonCardo et al., 1990;Weinstein et al., 1990;Miller et al., 1991). However, a meaningful relationship between the overexpression of P-gp and the resistance of these tumours to ch...

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