Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Hoffmann, E.
Establishment of new methods for detection and isolation of circulating tumor cells and circulating cell-free tumor DNA in cancer patients
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] Medical University of Graz; 2014. pp. 130
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- Autor*innen der Med Uni Graz:
- Hoffmann Eva Maria
- Betreuer*innen:
- Geigl Jochen Bernd
- Speicher Michael
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- Abstract:
- With the growing number of available predictive biomarkers clinical management of cancer is becoming increasingly reliant on the accurate serial monitoring of tumor genotypes. In this thesis project it was tested whether changes in the tumor genome can be inferred from the peripheral blood of patients with cancer. To this end “liquid biopsies”, i.e. the analysis of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs) was of special interest. To elucidate the potential of ctDNA the plasma DNA of 32 patients with stage IV colorectal carcinoma (CRC) was analyzed. In a subset of the patients (34.4%) we found the presence of long (i.e. ~320 bps) DNA fragments, which were associated with increased CTC numbers and elevated concentration of mutated plasma DNA fragments. In these cases we were able to establish genome-wide tumor-specific copy number alterations directly from plasma DNA. When we analyzed plasma DNA from 35 patients with metastatic breast cancer we made similar observations, suggesting that our approach may be applicable to a variety of tumor entities. However, an unexpected finding was that not all patients with progressive metastatic disease appear to release tumor DNA into the circulation in measurable quantities. Furthermore, we sought to facilitate the detection of CTCs. These cells, shed from the primary tumor or metastatic lesions, co-occur with millions of cells from the hematopoetic system and are very rare. Therefore the detection and isolation of these cells pose a technological challenge and most of the currently used systems for CTC-detection lack specificity and sensitivity. We developed a telomerase-specific adenoviral-expression-vector, which is able to transfect blood cells. Due to the inserted expression-cassette - consisting of a human telomerase reverse transcriptase promoter (hTERT) linked to the green fluorescent protein (GFP) - GFP is expressed exclusively in telomerase-active cells. Tests with various tumor cell lines, such as the highly telomerase-active colorectal cancer cell line HCT 116, the weak telomerase-expressing breast cancer cell line MCF 7, and the telomerase-negative osteosarcoma cell line U2-OS, suggested that our constructed adenoviral-vector is able to mediate GFP-expression in telomerase-active cancer cells. Thus, it might be an applicable tool to identify CTCs with ease and to make them amenable for further analyses. In summary, we show in this thesis that we could establish the current copy number status of a tumor genome by non-invasive means from the peripheral blood. Furthermore, we developed a novel detection system, which may greatly facilitate detection of CTCs.