Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
Bachmann, S.
Identification of driver mutations in plasma DNA from lung cancer patients
Humanmedizin; [ Diplomarbeit ] Graz Medical University; 2017. pp. 53
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- Autor*innen der Med Uni Graz:
- Betreuer*innen:
- Heitzer Ellen
- Altmetrics:
- Abstract:
- Lung cancer remains one of the deadliest tumor entities and currently various targeted therapies are under investigation to improve overall and progression-free survival. Knowledge of the genetic alterations affecting tumor progression in individual patients is urgently needed throughout the course of the disease to readily respond if actionable mutations for targeted therapies emerge. To date mutation detection mainly relies on tumor biopsies which impose a burden to patients. A novel approach for mutation detection in cancer patients is termed liquid biopsy. Among other things, it refers to the analysis of circulating tumor DNA (ctDNA) from blood and could offer a fast and minimal-invasive alternative to tissue biopsies. The focus of this thesis was to address several issues which affect robustness of mutation detection in plasma DNA. Especially preanalytical considerations were investigated such as the selection of blood tubes and different extraction methods of cell-free DNA (cfDNA) from plasma. Yield, size distribution and tumor content of the plasma DNA were compared to establish standards for further research. Furthermore, a targeted gene panel was tested using a cancer cell line in order to assess the resolution limit for mutation detection and subsequently several lung cancer samples were sequenced using this panel. To this end, blood of two healthy controls and two metastasized colon carcinoma patients was drawn into three different blood tubes and cfDNA was extracted from plasma using three different protocols including a manual, a vacuum-based and a fully-automated version. No significant differences were found between the different blood tubes. However with respect to DNA yield, the vacuum extraction method outperformed the two other methods. The resolution limit for the QIAGEN GeneRead Lung Cancer Panel was established using a dilution series with the cancer cell line HCT116 and additionally the panel was tested on six lung cancer samples. It was shown that the detection of mutations below a mutant allele frequency (MAF) of about 5% was not possible with the tested panel. A total of 51 variants were identified after sequencing and manual variant prioritization of the six lung cancer samples. However, the variants were not confirmed with independent methods. In conclusion, the three blood tubes performed equally well in preserving cfDNA if processed in a timely manner. The vacuum extraction proved to be superior to the other methods concerning DNA yield and this suggests its preferential use for further extractions taking the low abundance of ctDNA into account. The established resolution limit of about 5% MAF for the QIAGEN GeneRead Lung Cancer Panel proved not to be sufficient for sensitive mutation detection in plasma DNA where ctDNA fraction can be as low as 0.01%. This was confirmed when testing it on the lung cancer samples which showed a high number of variants and certain mutations that were seen across the sample range, indicating a high number of false-positives or artifacts.