Purpose: The CellSearch system (Veridex,Warren, NJ) is designed to enrich and enumerate circulating tumor cells (CTCs) from peripheral blood. Here, we validated the analytic performance of this system for clinical use in patients with metastatic breast cancer. Experimental Design: This prospective multicenter study conducted at three independent laboratories involved samples from 92 patients with metastatic breast cancer. Intra-and interassay variability using controls containing defined numbers of cells (average, 50 and 1,000, respectively), cell stability based on varying storage and shipment conditions, recovery precision from samples spiked with 4 to 12 tumor cells, inter-instrument variability, and positivity of samples from metastatic breast cancer patients were tested. Results: Intra-and inter-assay precision for two sites were high: All eight positive controls analyzed in the same run and >95% of the run to run control values (n = 299) were within the specified ranges. Recovery rate of spiked samples averaged between 80% and 82%. CTCs were detected in f70 % of metastatic breast cancer patients. CTC values of identical samples processed either immediately after blood drawing or after storage for 24, 48, or 72 h at room temperature or at 4jC did not differ significantly. Shipment of samples had no influence on CTC values. When analyzing identical samples in different centers, inter-instrument accordance was high. Conclusions: The CellSearch system enables the reliable detection of CTCs in blood and is suitable for the routine assessment of metastatic breast cancer patients in the clinical laboratory. Blood samples should be shipped at room temperature and CTC counts are stable for at least 72 h.Distant metastasis is the leading cause of breast carcinomarelated death. However, factors enabling cancer cells to move and grow outside of the primary site and the timing of tumor cell dissemination are still not well understood. Current models of metastasis support the detachment of cells from primary tumors and their movement to distant sites through the blood and lymphatic system (1). A large number of studies have documented disseminated tumor cells (DTCs) in bone marrow or circulating tumor cells (CTCs) in peripheral blood from patients with most types of epithelial cancers (for review, see refs. 1 -3). Within the last 10 years, several studies have shown that detection of tumor cells in bone marrow of cancer patients is accompanied by a substantially worse prognosis for these patients. Particularly, Braun et al. have reported that f30% of women with primary breast cancer have DTCs in bone marrow, and a 10-year follow-up of these patients revealed a significantly decreased disease-free survival and overall survival when compared with patients without DTCs (4, 5). However, aspiration of bone marrow is time consuming and, in many cases, uncomfortable for the patients precluding multiple samplings for therapy monitoring studies. Therefore, recent efforts have concentrated on the detection of CTCs in per...

Purpose: This study was aimed at detecting and characterizing circulating tumor cells (CTC) before and after neoadjuvant therapy (NT) in the peripheral blood of patients with breast cancer.Experimental Design: The clinical trial GeparQuattro incorporated NT approaches (epirubicin/cyclophosphamide prior to randomization to docetaxel alone, docetaxel in combination with capecitabine, or docetaxel followed by capecitabine) and additional trastuzumab treatment for patients with HER2-positive tumors. We used the Food and Drug Administration-approved CellSearch system for CTC detection and evaluation of HER2 expression and developed HER2 immunoscoring for CTC.Results: We detected ≥1 CTC/7.5 mL in 46 of 213 patients (21.6%) before NT and in 22 of 207 patients (10.6%) after NT (P = 0.002). Twenty (15.0%) initially CTC-positive cases were CTC-negative after NT, whereas 11 (8.3%) cases were CTC-positive after NT, although no CTC could be found before NT. CTC detection did not correlate with primary tumor characteristics. Furthermore, there was no association between tumor response to NT and CTC detection. HER2-overexpressing CTC were observed in 14 of 58 CTC-positive patients (24.1%), including 8 patients with HER2-negative primary tumors and 3 patients after trastuzumab treatment. CTC scored HER2-negative or weakly HER2-positive before or after NT were present in 11 of 21 patients with HER2-positive primary tumors. HER2 overexpression on CTC was restricted to ductal carcinomas and associated with high tumor stage (P = 0.002).Conclusion: CTC number was low in patients with primary breast cancer. The decrease in CTC incidence during treatment was not correlated with standard clinical characteristics and primary tumor response. Information on the HER2 status of CTC might be helpful for stratification and monitoring of HER2-directed therapies. Clin Cancer Res; 16(9); 2634-45. ©2010 AACR.Neoadjuvant treatment (NT) strategies allow the assessment of therapeutic efficacy of chemotherapy and novel targeted approaches in patients with breast cancer without the need for long follow-up periods, which are required in the adjuvant setting. The German Breast Group has conducted successful clinical trials in the neoadjuvant setting. The GeparQuattro study is a phase III trial program that incorporated different NT chemotherapy approaches with the addition of trastuzumab into current NT regimes for primary breast cancer ( Fig. 1; refs. 1,2). This provides an opportunity for translational research projects to examine biomarkers that might elucidate mechanisms underlying these therapies.Disseminated tumor cells in the bone marrow of patients with breast cancer are a significant independent predictor of poor prognosis in patients with nonmetastatic Authors' Affiliations: 1 Institute of Tumor Biology, 2 Department of G y n e c o l o g y , a n d 3 I n s t i t u t e of E x p er i m e n t al an d C l i n i c al Pharmacology and Toxicology, University Medical Center HamburgEppendorf, Hamburg, Germany; 4 German Breast Group, Neu Isenburg...

Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered heart tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30–40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5–10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 µN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for heart research.

Purpose: The incidence and biological characteristics of circulating tumor cells in the blood of patients with breast cancer were examined and subgroups were evaluated in the context of systemic treatment and the presence of disseminated tumor cells in bone marrow. Experimental Design: Circulating tumor cells were isolated from the peripheral blood of patients with breast cancer using a gradient system designed for the enrichment of circulating tumor cells (OncoQuick). Circulating tumor cells were identified with the anti-cytokeratin antibody, A45-B/ B3. In subsets of patients, expression of the proliferation-associated Ki-67 antigen in circulating tumor cells and the concomitant presence of micrometastases in bone marrow were examined.

Exposure to infective larvae of the filarial nematode Onchocerca volvulus (Ov) either results in patent infection (microfilaridermia) or it leads to a status called putative immunity, characterized by resistance to infection. Similar to other chronic helminth infections, there is a T cell proliferative hyporesponsiveness to Ov antigen (OvAg) by peripheral blood mononuclear cells (PBMC) from individuals with patent infection, i.e. generalized onchocerciasis (GEO), compared to PBMC from putatively immune (PI) individuals. In this study, mechanisms mediating this cellular hyporesponsiveness in GEO were investigated: the low proliferative response in PBMC from GEO individuals was associated with a lack of IL-4 production and significantly lower production of IL-5 compared to those from PI individuals, arguing against a general shift towards a T(h)2 response being the cause of hyporesponsiveness. In contrast, IL-10 and transforming growth factor (TGF)-beta, two cytokines associated with a T(h)3 response, seemed to mediate hyporesponsiveness: PBMC from individuals with GEO produced significantly more IL-10, and T cell proliferative hyporesponsiveness in this group could be reversed by the addition of anti-IL-10 and anti-TGF-beta antibodies. Hyporesponsiveness was specific for OvAg and not observed upon stimulation with related nematode antigens, arguing for a T cell-mediated, Ov-specific down-regulation. Ov-specific T cells could be cloned from GEO PBMC which have a unique cytokine profile (no IL-2 but high IL-10 and/or TGF-beta production), similar to the T cell subsets known to suppress ongoing inflammation (T(h)3 and T(r)1), indicating that this cell type which has not been found so far in infectious diseases may be involved in maintaining Ov-specific hyporesponsiveness.

Purpose:The bone marrow is a frequent and clinically important homing site for early disseminated breast cancer cells. Here, we aimed to profile the protein expression of these cells using unique cell line models and to evaluate the prognostic relevance of candidate gene expression for breast cancer patients. Experimental Design: To identify expression patterns characteristic for micrometastatic cells, three different cell lines (BC-K1, BC-P1, and BC-S1) established by SV40 immortalization of cancer cells isolated from the bone marrow of patients with breast cancer were compared with MCF-7 breast cancer and SV40 immortalized normal breast ductal cells (MTSV-1.7) using twodimensional gel electrophoresis followed by MALDI-ToF analysis.The prognostic significance and clinicopathologic associations of selected differentially expressed proteins were evaluated using high-density breast cancer tissue microarrays. Results: In contrast to MCF-7 and MTSV1-7 reference cell lines, all micrometastatic cancer cell lines displayed loss of epithelial cytokeratins (CK8, CK18, and CK19) and ectopic expression of vimentin commonly present in mesenchymal cells. Immunohistochemical analysis of 2,517 samples of breast cancer further showed that loss of cytokeratin and ectopic vimentin expression were significantly associated with a higher tumor grade, high mitotic index, and negative estrogen/ progesterone-receptor status. Although in univariate analyses significantly related to clinical outcome, none of the cytokeratins analyzed were independently associated with either overall or cancer-specific survival. Conclusions: Micrometastatic cancer cells exhibit marked changes in the expression pattern of cytoskeletal proteins indicative of an epithelial-mesenchymal transition. This phenotypical change could already be detected in primary tumors and is associated with the aggressive behavior of breast cancer cells in vivo.Breast cancer, the most common malignancy in females, kills f300,000 women worldwide every year and is the main reason for cancer-related death in the postoperative development of distant metastasis in secondary organs (1). Many of the patients who are primarily diagnosed as having apparently localized or regional breast cancer eventually develop distant metastases. Clinical experience showed that in early clinical stages, cells could already dissociate from the tumor's parenchyma via the hematogenous route and colonize the bone marrow, the clinically most relevant site of metastatic disease in patients with solid epithelial tumors (2). Often, many years after resection of the primary tumor and treatment of cancer patients, micrometastatic cancer cells can grow out to form overt metastases. Indeed, evidence is accumulating that the presence of occult carcinoma cells of epithelial origin in the bone marrow is an independent risk factor in breast cancer (3 -5). Understanding the biology of these dormant tumor cells in the bone marrow is therefore pivotal for the development of novel therapeutic approaches for the t...

These data showed that CYP2D6 poor metabolizers had a 5-fold higher risk for development of adverse effects during metoprolol treatment than patients who were not poor metabolizers. Because the absolute risk of adverse effects of metoprolol is unknown, the clinical relevance of the CYP2D6 genotype for metoprolol therapy has to be determined in a prospective manner.

The data suggest a hitherto unknown gender-specific impact of the -24C>T ABCC2 gene polymorphism on high-dose methotrexate pharmacokinetics. Whereas a nonfunctional MRP2 variant has been described in a patient with severe impairment of methotrexate excretion, our study is the first to suggest that a frequent ABCC2 polymorphism contributes to variability of methotrexate kinetics.