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Selected Publication:
Heidary, M.
ANALYSES OF CIRCULATING CELL FREE TUMOR DNA AND CIRCULATING TUMOR CELLS IN PATIENTS WITH METASTATIC BREAST CANCER
PhD-Studium (Doctor of Philosophy); Humanmedizin; [ Dissertation ] ; 2015. pp. 167
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- Authors Med Uni Graz:
- Heidary Maryam
- Advisor:
- Speicher Michael
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- Abstract:
- Recently, there has been considerable interest into “liquid biopsies”, i.e. analyses of circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), to improve the management of patients with cancer. In this thesis several important issues were addressed in order to make liquid biopsies amenable for routine analysis. To reliably establish the role of CTCs as potential biomarkers hundreds of these cells will have to be analyzed. To rapidly prescreen isolated cells to test whether they are indeed CTCs we developed and tested two approaches, i.e. QF-PCR for copy number changes and SNP genotyping for identification of mutations. To analyze copy number changes in CTCs, marker panels located in chromosomal regions often involved in copy number changes in colorectal, prostate and breast tumors were developed. We used constitutional DNA and whole-genome amplified DNA from single cells of healthy donors and evaluated the marker panels by QF-PCR. We identified no significant changes in QF-PCR profiles of whole genome amplified single cells compared to constitutional DNA suggesting that this assay is suitable to prescreen the copy number status in single cells, such as CTCs. Moreover, we designed tumor genotyping assays consisting of SNaPshot multiplex panels of genes, commonly affected by somatic mutations in mentioned cancers. The results of SNP genotyping for cancer cell lines, FFPE tumor tissues and CTCs of colon cancer patients were consistent with the results of standard Sanger sequencing and next generation sequencing. Importantly, DNA of low quality did not influence sensitivity of the assay. This is a prerequisite to develop highly sensitive and specific multiplex SNaPshot assays to prescreen the mutation spectrum in CTCs in a high-throughput format at affordable costs. Recent studies suggested that a major proportion of CTCs in metastatic breast cancer patients shows epithelial-mesenchymal transition (EMT) and cancer stem cell characteristics. In order to identify CTCs with stem-cell/mesenchymal characteristics, we studied gene expression profiles in claudin-low (SUM159) and luminal (MCF7) breast cancer cell lines. We observed clear differences for the expression of EMT and stem cell markers between these cell lines, whereas there were no significant differences among different subpopulations of each cell line. Whereas the available FACS allowed isolation of MCF7 and SUM159 subpopulations, its resolution was unfortunately not sufficient to isolate tumor cells from blood samples. Recent studies showed that ctDNA is an informative and highly sensitive biomarker of metastatic breast cancer to monitor the tumor burden dynamics and treatment response. However, the dynamic range of ctDNA in these patients is not fully elucidated yet. In an index patient with more than 100,000 CTCs in serial blood analyses, whole genome, exome, or targeted deep sequencing of the primary tumor, metastases, and 551 CTCs were consistent with a genetically homogeneous cancer. However, the allele fractions (AFs) of ctDNA were very low, only 2-3%, in each analysis, which neither reflected the tumor burden nor the dynamics of this progressive disease. Indeed, analyses of 71 further plasma samples from 58 patients demonstrated highly variable AFs of mutant fragments, which frequently did not correspond to the tumor burden. Altogether, our data revealed, in contrast to other studies, highly variable range of ctDNA in the patients with metastatic breast cancer. In summary, in this thesis novel methods for prescreening of CTCs based on copy number changes and mutations were developed. Furthermore, a potential approach for identification of cells with EMT/stem cell characteristics was established. Finally detailed analyses of ctDNA and CTCs revealed an unexpected heterogeneity in patients with metastatic breast cancer. Altogether, the insights gained from this thesis are an important step to introduce liquid biopsies into the clinic.