Influence of immunomagnetic enrichment on gene expression of tumor cells

BACKGROUND: Metastasis is the leading cause of cancer-related death. Bone marrow (BM) is a frequent site for the settlement of disseminated tumor cells which occurs years before overt metastases signal incurability. METHODS: Here we describe a new method to assess the initial stage of metastasis development in cancer patients. By immunomagnetic selection with HER2/neu and EpCAM as catcher antigens single disseminated tumor cells can be enriched from BM samples. To examine whether the immunomagnetic enrichment technique may change gene expression in the selected tumor cells, we performed differential expression profiling with the breast cancer cell lines MCF-7 and BT474 as models. The profiles were performed using 1.2 Cancer Arrays (Clontech) containing 1176 cDNAs that can be grouped into different functional categories, such as signal transduction, cell cycle, adhesion, cytoskeleton plasticity, growth factors and others. RESULTS: The reproducibility of the gene expression profiling between duplicate cDNA-array experiments was assessed by two independent experiments with MCF-7 breast cancer cells. Scatter blot analysis revealed a good reproducibility of the cDNA array analysis (i.e. less than 10% difference in the gene expression between the arrays). Subsequent comparative cDNA-array analyses of immunobead-selected and unselected MCF-7 and BT474 cancer cells indicated that the antibody incubation during the immunomagnetic selection procedure did not considerably alter the gene expression profile. CONCLUSION: The described method offers an excellent tool for the enrichment of micrometastatic tumor cells in BM without largely changing the gene expression pattern of these cells

Circulating microRNAs as blood-based markers for patients with primary and metastatic breast cancer

Introduction: MicroRNAs (miRs) are interesting new diagnostic targets that may provide important insights into the molecular pathogenesis of breast cancer. Here we evaluated, for the first time, the feasibility and clinical utility of circulating miRs as biomarkers for the detection and staging of breast cancer. Methods: The relative concentrations of breast cancer-associated miR10b, miR34a, miR141 and miR155 were measured in the blood serum of 89 patients with primary breast cancer (M0, n = 59) and metastatic disease (M1, n = 30), and 29 healthy women by a TaqMan MicroRNA Assay. Results: The relative concentrations of total RNA (P = 0.0001) and miR155 (P = 0.0001) in serum significantly discriminated M0-patients from healthy women, whereas miR10b (P = 0.005), miR34a (P = 0.001) and miR155 (P = 0.008) discriminated M1-patients from healthy controls. In breast cancer patients, the changes in the levels of total RNA (P = 0.0001), miR10b (P = 0.01), miR34a (P = 0.003) and miR155 (P = 0.002) correlated with the presence of overt metastases. Within the M0-cohort, patients at advanced tumor stages (pT3 to 4) had significantly more total RNA (P = 0.0001) and miR34a (P = 0.01) in their blood than patients at early tumor stages (pT1 to 2). Conclusions: This pilot study provides first evidence that tumor-associated circulating miRs are elevated in the blood of breast cancer patients and associated with tumor progression

Stromal Caveolin-1 and Caveolin-2 Expression in Primary Tumors and Lymph Node Metastases

The expression of caveolin-1 (CAV1) in both tumor cell and cancer-associated fibroblasts (CAFs) has been found to correlate with tumor aggressiveness in different epithelial tumor entities, whereas less is known for caveolin-2 (CAV2). The aim of this study was to investigate the clinicopathological significance and prognostic value of stromal CAV1 and CAV2 expression in lung cancer. The expression of these two genes was investigated at protein level on a tissue microarray (TMA) consisting of 161 primary tumor samples. 50.7% of squamous cell lung cancer (SCC) tumors showed strong expression of CAV1 in the tumor-associated stromal cells, whereas only 15.1% of adenocarcinomas (AC) showed a strong CAV1 expression (p <0 01). A strong CAV2 stromal expression was found in 46.0% of the lung tumor specimens, with no significant difference between the subtypes. Neither CAV1 nor CAV2 stromal expression was associated with any other clinicopathological factor including survival. When the stromal expression in matched primary tumors and lymph node metastases was compared, both CAV1 and CAV2 expressions were frequently found lost in the corresponding stroma of the lymph node metastasis (40.6%, p = 0 003 and 38.4%, p = 0 001, resp.). Loss of stromal CAV2 in the lymph node metastases was also significantly associated with earlier death (p = 0 011). In conclusion, in contrast to the expression patterns in the tumor tissue of lung cancer, stromal expression of CAV1 in primary tumors was not associated with clinical outcome whereas the stromal expression of especially CAV2 in the metastatic lymph nodes could be associated with lung cancer pathogenesis.Peer reviewe

Apoptosis-related deregulation of proteolytic activities and high serum levels of circulating nucleosomes and DNA in blood correlate with breast cancer progression

<p>Abstract</p> <p>Background</p> <p>As cell-free circulating DNA exists predominantly as mono- and oligonucleosomes, the focus of the current study was to examine the interplay of circulating nucleosomes, DNA, proteases and caspases in blood of patients with benign and malignant breast diseases.</p> <p>Methods</p> <p>The concentrations of cell-free DNA and nucleosomes as well as the protease and caspase activities were measured in serum of patients with benign breast disease (n = 20), primary breast cancer (M0, n = 31), metastatic breast cancer (M1, n = 32), and healthy individuals (n = 28) by PicoGreen, Cell Death Detection ELISA, Protease Fluorescent Detection Kit and Caspase-Glo^®3/7 Assay, respectively.</p> <p>Results</p> <p>Patients with benign and malignant tumors had significantly higher levels of circulating nucleic acids in their blood than healthy individuals (p = 0.001, p = 0.0001), whereas these levels could not discriminate between benign and malignant lesions. Our analyses of all serum samples revealed significant correlations of circulating nucleosome with DNA concentrations (p = 0.001), nucleosome concentrations with caspase activities (p = 0.008), and caspase with protease activities (p = 0.0001). High serum levels of protease and caspase activities associated with advanced tumor stages (p = 0.009). Patients with lymph node-positive breast cancer had significantly higher nucleosome levels in their blood than node-negative patients (p = 0.004). The presence of distant metastases associated with a significant increase in serum nucleosome (p = 0.01) and DNA levels (p = 0.04), and protease activities (p = 0.008).</p> <p>Conclusion</p> <p>Our findings demonstrate that high circulating nucleic acid concentrations in blood are no indicators of a malignant breast tumor. However, the observed changes in apoptosis-related deregulation of proteolytic activities along with the elevated serum levels of nucleosomes and DNA in blood are linked to breast cancer progression.</p

Functional characterization of circulating tumor cells (CTCs) from metastatic ER+/HER2− breast cancer reveals dependence on HER2 and FOXM1 for endocrine therapy resistance and tumor cell survival: Implications for treatment of ER+/HER2− breast cancer

Mechanisms of acquired endocrine resistance and late recurrence in patients with ER+/HER2− breast cancer are complex and not fully understood. Here, we evaluated mechanisms of acquired resistance in circulating tumor cells (CTCs) from an ER+/HER2− breast cancer patient who initially responded but later progressed under endocrine treatment. We found a switch from ERα-dependent to HER2-dependent and ERα-independent expression of FOXM1, which may enable disseminated ER+/HER2− cells to re-initiate tumor cell growth and metastasis formation in the presence of endocrine treatment. Our results also suggest a role for HER2 in resistance, even in ER+ breast cancer cells that have neither HER2 amplification nor activating HER2 mutations. We found that NFkB signaling sustains HER2 and FOXM1 expression in CTCs in the presence of ERα inhibitors. Inhibition of NFkB signaling blocked expression of HER2 and FOXM1 in the CTCs, and induced apoptosis. Thus, targeting of NFkB and FOXM1 might be an efficient therapeutic approach to prevent late recurrence and to treat endocrine resistance. Collectively our data show that CTCs from patients with endocrine resistance allow mechanisms of acquired endocrine resistance to be delineated, and can be used to test potential drug regimens for combatting resistance

Prognostic impact of circulating tumor cells assessed with the CellSearch System (TM) and AdnaTest Breast (TM) in metastatic breast cancer patients: the DETECT study

Introduction: There is a multitude of assays for the detection of circulating tumor cells (CTCs) but a very limited number of studies comparing the clinical relevance of results obtained with different test methods. The DETECT trial for metastatic breast cancer patients was designed to directly compare the prognostic impact of two commercially available CTC assays that are prominent representatives of immunocytochemical and RT-PCR based technologies. Methods: In total, 254 metastatic breast cancer patients were enrolled in this prospective multicenter trial. CTCs were assessed using both the AdnaTest Breast Cancer and the CellSearch system according to the manufacturers' instructions. Results: With the CellSearch system, 116 of 221 (50%) evaluable patients were CTC-positive based on a cut-off level at 5 or more CTCs. The median overall survival (OS) was 18.1 months in CTC-positive patients. (95%-CI: 15.1-22.1 months) compared to 27 months in CTC-negative patients (23.5-30.7 months; p&lt;0.001). This prognostic impact for OS was also significant in the subgroups of patients with triple negative, HER2-positive and hormone receptor-positive/HER2-negative primary tumors. The progression free survival (PFS) was not correlated with CTC status in our cohort receiving different types and lines of systemic treatment (p = 0.197). In multivariate analysis, the presence of CTCs was an independent predictor for OS (HR: 2.7, 95%-CI: 1.6-4.2). When the AdnaTest Breast was performed, 88 of 221 (40%) patients were CTC-positive. CTC-positivity assessed by the AdnaTest Breast had no association with PFS or OS. Conclusions: The prognostic relevance of CTC detection in metastatic breast cancer patients depends on the test method. The present results indicate that the CellSearch system is superior to the AdnaTest Breast Cancer in predicting clinical outcome in advanced breast cancer