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Liu Z.,Charite - Medical University of Berlin | Fusi A.,Charite - Medical University of Berlin | Klopocki E.,Charite - Medical University of Berlin | Schmittel A.,Charite - Medical University of Berlin | And 3 more authors.
Journal of Translational Medicine | Year: 2011

Background: A limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction.Methods: The 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed.Results: CTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84).Conclusion: Enriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined. © 2011 Liu et al; licensee BioMed Central Ltd.

Liu Z.,Charite - Medical University of Berlin | Fusi A.,Charite - Medical University of Berlin | Schmittel A.,Charite - Medical University of Berlin | Tinhofer I.,Translational Radiobiology and Radiooncology Research Laboratory | And 2 more authors.
Cancer Biology and Therapy | Year: 2010

For breast cancer patients, treatment decisions based on the molecular profile of the primary tumor have been used for many years. In Her2-overexpressing tumors, trastuzumab is a key component of therapy. However, despite persistent expression of Her2, most tumors eventually become resistant to trastuzumab. When this happens, the patients benefit from a regime containing lapatinib, a dual EGFR and Her2 tyrosine kinase inhibitor. We report on a patient affected by chemo-refractory metastatic Her2-positive breast cancer enrolled in a translational research program for the detection and characterization of circulating tumor cells (CTCs). Depletion of the EGFR-positive CTC pool in the blood was associated with tumor response, whereas disease progression was related to a recurrence in CTCs, which were both EGFR and Her2 negative. Although a proof for the clinical significance of EGFR-positive circulating tumor cells is currently lacking, expression of EGFR may predict response to lapatinib-based treatments as in the case presented. © 2010 Landes Bioscience.

Sinn B.,Translational Radiobiology and Radiooncology Research Laboratory | Tallen G.,Charite - Medical University of Berlin | Schroeder G.,Translational Radiobiology and Radiooncology Research Laboratory | Grassl B.,Translational Radiobiology and Radiooncology Research Laboratory | And 3 more authors.
Molecular Cancer Therapeutics | Year: 2010

PTEN mutations are frequently found in malignant glioma and can result in activated phosphatidylinositol-3-kinase/Akt survival signaling associated with resistance to radiotherapy. Strategies to interfere with aberrant PI3K/Akt activity are therefore being developed to improve the therapeutic efficacy of radiotherapy in patients with malignant glioma. The methylxanthine caffeine has been described as a PI3K inhibitor and is also known to sensitize cells to ionizing radiation. However, a direct association between these two caffeine-mediated effects has not been reported yet. Therefore, we asked whether caffeine or its derivative pentoxifyl-line differentially affect the radiosensitivity of malignant gliomas with different PTEN status. As models, we used the radiosensitive EA14 malignant glioma cell line containing wild-type PTEN and the radioresistant U87MG malignant glioma cell line harboring mutant PTEN. Our study revealed that caffeine and pentoxifylline radiosensitized PTEN-deficient but not PTEN-proficient glioma cells. Radiosensitization of PTEN-deficient U87MG cells by caffeine was significantly correlated with the activation of the G1 DNA damage checkpoint that occurred independently of de novo synthesis of p53 and p21. The p53 independency was also confirmed by a significant caffeine-mediated radiosensitization of the glioma cell lines T98G and U373MG that are deficient for both PTEN and p53. Furthermore, caffeine-mediated radiosensitization was associated with the inhibition of Akt hyperphosphorylation in PTEN-deficient cells to a level comparable with PTEN-proficient cells. Our data suggest that the methylxanthine caffeine or its derivative pentoxifylline are promising candidate drugs for the radiosensitization of glioma cells particularly with PTEN mutations. ©2010 AACR.

Hristozova T.,Translational Radiobiology and Radiooncology Research Laboratory | Konschak R.,Translational Radiobiology and Radiooncology Research Laboratory | Stromberger C.,Translational Radiobiology and Radiooncology Research Laboratory | Fusi A.,Charite - Medical University of Berlin | And 6 more authors.
Annals of Oncology | Year: 2011

Background: The mechanisms regulating tumor cell dissemination in locally advanced squamous cell carcinoma of the head and neck region (SCCHN) are largely unresolved. We assessed the frequency of circulating tumor cells (CTCs), their association with clinicopathologic parameters and their kinetics during radiochemotherapy. Patients and methods: Peripheral blood samples from 42 patients with locally advanced SCCHN were included. CTCs were detected using flow cytometric analysis of CD45-epithelial cell adhesion molecule+cytokeratin+ cells and results were validated by nested RT-PCR analysis of circulating epidermal growth factor receptor transcripts. The association between the presence of CTCs and T stage, tumor volume, N stage and human papillomavirus status was evaluated. The influence of radiochemotherapy on CTC numbers was determined. Results: CTCs were detected in 18 of 42 SCCHN patients (43%), with a mean ± standard deviation of 1.7 ± 0.9 CTCs per 3.75 ml blood. We observed no significant correlation between the presence of CTCs and T stage or tumor volume. However, a nodal stage of N2b or higher was associated with higher frequency of CTCs. Though concurrent radiochemotherapy reduced their frequency, CTCs persisted during treatment in 20% of cases. Conclusions: Detection of CTCs correlates with regional metastasis in inoperable SCCHN. Further follow-up is needed to evaluate the prognostic significance of CTC detection, in addition to clinical staging of lymph nodes, for regional or distant recurrence. © The Author 2011. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved.

Sinn B.,Translational Radiobiology and Radiooncology Research Laboratory | Schulze J.,Translational Radiobiology and Radiooncology Research Laboratory | Schroeder G.,Translational Radiobiology and Radiooncology Research Laboratory | Konschak R.,Translational Radiobiology and Radiooncology Research Laboratory | And 3 more authors.
Radiation Research | Year: 2010

Activation of p53 has been causally linked to normal tissue damage after irradiation. Pifithrin-(PFT-), a specific inhibitor of p53, has been suggested as a combinatory agent in the treatment of p53-deficient tumors in which inhibition of p53 would not compromise therapeutic efficacy but would decrease p53-mediated side effects in normal tissue. We tested this concept for radiotherapy of p53-deficient and-proficient glioma. We observed significant interaction of PFT-with radiation-induced G1 checkpoint activation and plating efficiency only in glioma cells expressing at least one wild-type allele of p53. This interaction was correlated with PFT-mediated inhibition of radiation-induced expression of the p53 target gene p21 Waf1. Despite inhibition of p53 function we did not observe significant changes in radiosensitivity after treatment with PFT-in either p53-deficient or p53-proficient tumor cells. We confirmed these results in p53-proficient lung cancer cells. In contrast, PFT-significantly increased the fraction of normal astrocytes and fibroblasts surviving irradiation; this was accompanied by improved DNA damage repair, speaking against an accumulation of cells with genetic lesions after PFT-treatment. In conclusion, PFT-might prove useful in protecting normal tissue from the side effects of radiotherapy without reducing the efficacy of treatment for both p53-proficient and-deficient tumors. © 2010 by Radiation Research Society.

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