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Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer — Evaluation of Several Markers with Real-Time Reverse Transcription-PCR
Klinik und Poliklinik für Frauenheilkunde und Geburtshilfe Ludwig-Maximilians-Universitaet Muenchen, Campus Innenstadt, Maistraße 11, 80337 Munich, Germany
Frauenklinik, Klinikum Traunstein, Cuno-Niggl-Straße 3, 83278 Traunstein, Germany
Department of Molecular Pathology, University of Texas MD Anderson Cancer Center, 7435 Fannin Street, Houston, TX 77054, USA
* Author to whom correspondence should be addressed.
Received: 12 November 2012; in revised form: 3 December 2012 / Accepted: 31 December 2012 / Published: 8 January 2013
Abstract: It is widely known that cells from epithelial tumors, e.g., breast cancer, detach from their primary tissue and enter blood circulation. We show that the presence of circulating tumor cells (CTCs) in samples of patients with primary and metastatic breast cancer can be detected with an array of selected tumor-marker-genes by reverse transcription real-time PCR. The focus of the presented work is on detecting differences in gene expression between healthy individuals and adjuvant and metastatic breast cancer patients, not an accurate quantification of these differences. Therefore, total RNA was isolated from blood samples of healthy donors and patients with primary or metastatic breast cancer after enrichment of mononuclear cells by density gradient centrifugation. After reverse transcription real-time PCR was carried out with a set of marker genes (BCSP, CK8, Her2, MGL, CK18, CK19). B2M and GAPDH were used as reference genes. Blood samples from patients with metastatic disease revealed increased cytokine gene levels in comparison to normal blood samples. Detection of a single gene was not sufficient to detect CTCs by reverse transcription real-time PCR. Markers used here were selected based on a recent study detecting cancer cells on different protein levels. The combination of such a marker array leads to higher and more specific discovery rates, predominantly in metastatic patients. Identification of CTCs by PCR methods may lead to better diagnosis and prognosis and could help to choose an adequate therapy.
Keywords: breast cancer; circulating tumor cells; reverse transcription real-time PCR; marker genes
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Andergassen, U.; Hofmann, S.; Kölbl, A.C.; Schindlbeck, C.; Neugebauer, J.; Hutter, S.; Engelstädter, V.; Ilmer, M.; Friese, K.; Jeschke, U. Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer — Evaluation of Several Markers with Real-Time Reverse Transcription-PCR. Int. J. Mol. Sci. 2013, 14, 1093-1104.
Andergassen U, Hofmann S, Kölbl AC, Schindlbeck C, Neugebauer J, Hutter S, Engelstädter V, Ilmer M, Friese K, Jeschke U. Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer — Evaluation of Several Markers with Real-Time Reverse Transcription-PCR. International Journal of Molecular Sciences. 2013; 14(1):1093-1104.
Andergassen, Ulrich; Hofmann, Simone; Kölbl, Alexandra C.; Schindlbeck, Christian; Neugebauer, Julia; Hutter, Stefan; Engelstädter, Verena; Ilmer, Matthias; Friese, Klaus; Jeschke, Udo. 2013. "Detection of Tumor Cell-Specific mRNA in the Peripheral Blood of Patients with Breast Cancer — Evaluation of Several Markers with Real-Time Reverse Transcription-PCR." Int. J. Mol. Sci. 14, no. 1: 1093-1104.