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• W2890899992 abstract "Background: Lung cancer is the most common cause of cancer death worldwide and is estimated to account for more than 1,380,000 deaths per year. Lung cancer can be separated into two major histological types: Small Cell Lung Cancer (SCLC) and Non-Small Cell Lung Cancer (NSCLC), accounting for approximately 15% and 85% of cases respectively. Epidermal Growth Factor Receptor (EGFR) is a tyrosine-kinase receptor. In NSCLC EGFR overexpression is found in over 80% of cases, and EGFR copy number gain (CNG) or amplification is found in nearly 60% of them. Tumours with EGFR mutations can be treated using anti-EGFR drugs; however currently genetic analysis has to be performed on tissue which is obtained by biopsy.Aims: This project aims to investigate alternative methods of obtaining tumour DNA for genetic analysis, to potentially improve or support the current diagnostic process. This project will investigate both new testing methods (molecular assays) and new sources of tumour DNA (cell free DNA from plasma).Methods: A number of methods were employed during this project. Initially EGFR and KRAS mutation detection was attempted using a novel Peptide Nucleic Acid Polymerase Chain Reaction (PNA-PCR) assay devised by GeneFirst Ltd (Oxford, UK). The second approach utilised custom designed TaqMan Array 384 well plate assays for the detection of EGFR, KRAS, NRAS and BRAF mutations. 40 clinical EDTA blood samples were obtained for the investigation of the use cfDNA for oncogenic mutation detection. Plasma DNA extracted using two automated platforms (Qiagen EZ1 and Promega Maxwell). The extracted DNA was analysed using the Ion Torrent Next Generation Sequencing (NGS) platform.Results: The GeneFirst novel PNA PCR assays appeared to tolerate low concentration FFPE DNA samples but had a very high false positive rate and the endogenous control assay failed regularly (0- 33.3% failure rate over different assay versions). The TaqMan Array assay was very successful at detecting EGFR, KRAS, NRAS and BRAF mutations from FFPE tissue, displaying 97.62% and 94.74% concordance with previously used diagnostic assays (Qiagen Therascreen EGFR RGQ PCR and Thermo Fisher KRAS castPCR). For the automated isolation of cfDNA, the Promega Maxwell instrument gave consistently superior results to the Qiagen EZ1. CfDNA was successfully used to detect oncogenic mutations using both PCR and NGS assays.Conclusion: This project has utilised a number of approaches in order to investigate new approaches for the detection of clinically actionable oncogenic mutations, both in FFPE tissue (obtained through surgery or biopsy) and the relatively new cfDNA analyte. Two PCR techniques were compared using DNA from FFPE tissue, and the TaqMan Array assay was shown to be vastly superior. The TaqMan Array was subsequently adopted as the primary diagnostic assay in UHCW Pathology. CfDNA (despite the limited number of samples) showed great potential as an alternative for tissue for detection actionable cancer mutations. The Ion Torrent Next Generation Sequencing system proved to be the most sensitive and powerful technique of the ones utilised here, and will prove an invaluable asset for future development of this work." @default.
• W2890899992 created "2018-09-27" @default.
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• W2890899992 date "2018-04-01" @default.
• W2890899992 modified "2023-09-26" @default.
• W2890899992 title "Cancer gene mutation detection in circulating cell-free DNA in blood" @default.
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