Mohammad Amin Kerachian1,2,3, Marjan Azghandi4,5, Ali Javadmanesh4,5, Kamran Ghaffarzadegan6,7, Sina Mozaffari-Jovin8,9,10. 1. Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. amin.kerachian@mail.mcgill.ca. 2. Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Azadi Square, Mashhad, 917794-8564, Iran. amin.kerachian@mail.mcgill.ca. 3. Cancer Genetics Research Unit, Reza Radiotherapy and Oncology Center, Mashhad, Iran. amin.kerachian@mail.mcgill.ca. 4. Cancer Genetics Research Unit, Reza Radiotherapy and Oncology Center, Mashhad, Iran. 5. Department of Animal Science, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. 6. Department of Pathology and Laboratory Medicine, Mayo Clinic, Arizona, Scottsdale, AZ, USA. 7. Razavi Cancer Research Center, Razavi Hospital, Imam Reza International University, Mashhad, Iran. 8. Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran. Sina.Mozaffari-Jovin@mpibpc.mpg.de. 9. Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Azadi Square, Mashhad, 917794-8564, Iran. Sina.Mozaffari-Jovin@mpibpc.mpg.de. 10. Department of Cellular Biochemistry, Max-Planck-Institute for Biophysical Chemistry, Am Faßberg 11, D-37077, Goettingen, Germany. Sina.Mozaffari-Jovin@mpibpc.mpg.de.
Abstract
PURPOSE: Recently, 'solid tumor biopsies' have been challenged by the emergence of 'liquid biopsies', which are aimed at the isolation and detection of circulating cell-free tumor DNA (ctDNA) in body fluids. Here, we developed and optimized a method for selective capture of ctDNA on magnetic beads (SCC-MAG) for mutation detection in plasma of patients with colorectal cancer (CRC). METHODS: Blood and tissue samples from 28 CRC patients were included for the detection of KRAS mutations. For the tissue samples, mutation analysis was conducted by high resolution melting (HRM) analysis and sequencing. For the SCC-MAG method, ctDNA was isolated from 200 µl plasma from patients with a mutant KRAS gene. For comparison, ctDNA extraction was carried out using a silica membrane-based method, after which mutations were detected using Intplex allele-specific PCR. RESULTS: The mean ctDNA integrity index in plasma samples of cancer patients was 1.03, comparable with that of silica membrane-derived ctDNA (1.011). Notably, the limit of detection for the SCC-MAG approach was lower than that of the silica membrane method and measured 2.25 pg/ml ctDNA in plasma. Our analyses showed that while the silica membrane-based approach was capable of collecting ctDNA from two out of six CRC patient samples (average Cq 34.23), the SCC-MAG captured ctDNA from all samples with an average Cq of 29.76. CONCLUSIONS: We present a robust, reproducible, and highly sensitive method for the analysis of mutation statuses in liquid biopsies. The SCC-MAG method can readily be applied to any nucleic acid target for diagnostic purposes upon careful design of the specific capture probes, and can be multiplexed by several probes to identify multiple targets.
PURPOSE: Recently, 'solid tumor biopsies' have been challenged by the emergence of 'liquid biopsies', which are aimed at the isolation and detection of circulating cell-free tumor DNA (ctDNA) in body fluids. Here, we developed and optimized a method for selective capture of ctDNA on magnetic beads (SCC-MAG) for mutation detection in plasma of patients with colorectal cancer (CRC). METHODS: Blood and tissue samples from 28 CRCpatients were included for the detection of KRAS mutations. For the tissue samples, mutation analysis was conducted by high resolution melting (HRM) analysis and sequencing. For the SCC-MAG method, ctDNA was isolated from 200 µl plasma from patients with a mutant KRAS gene. For comparison, ctDNA extraction was carried out using a silica membrane-based method, after which mutations were detected using Intplex allele-specific PCR. RESULTS: The mean ctDNA integrity index in plasma samples of cancerpatients was 1.03, comparable with that of silica membrane-derived ctDNA (1.011). Notably, the limit of detection for the SCC-MAG approach was lower than that of the silica membrane method and measured 2.25 pg/ml ctDNA in plasma. Our analyses showed that while the silica membrane-based approach was capable of collecting ctDNA from two out of six CRCpatient samples (average Cq 34.23), the SCC-MAG captured ctDNA from all samples with an average Cq of 29.76. CONCLUSIONS: We present a robust, reproducible, and highly sensitive method for the analysis of mutation statuses in liquid biopsies. The SCC-MAG method can readily be applied to any nucleic acid target for diagnostic purposes upon careful design of the specific capture probes, and can be multiplexed by several probes to identify multiple targets.
Authors: Mohammad Amin Kerachian; Marjan Azghandi; Sina Mozaffari-Jovin; Alain R Thierry Journal: Clin Epigenetics Date: 2021-10-18 Impact factor: 6.551
Authors: Panagiota M Kalligosfyri; Sofia Nikou; Sofia Karteri; Haralabos P Kalofonos; Vasiliki Bravou; Despina P Kalogianni Journal: Biosensors (Basel) Date: 2022-02-04