Detection of Somatic Mutations with ddPCR from Liquid Biopsy of Colorectal Cancer Patients
1
Molecular Biology Laboratory, BIA Separations CRO, Labena Ltd, 1000 Ljubljana, Slovenia
2
Department of Colorectal Diseases, Clinic for Digestive Surgery, First Surgical Clinic, Clinical Center of Serbia, 11000 Belgrade, Serbia
3
Medical Faculty, University of Belgrade, 11000 Belgrade, Serbia
4
Serbian Academy of Sciences and Arts, 11000 Belgrade, Serbia
5
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, 11000 Belgrade, Serbia
*
Author to whom correspondence should be addressed.
Academic Editor: Yannick D. Benoit
Genes 2021, 12(2), 289; https://doi.org/10.3390/genes12020289
Received: 8 January 2021
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Revised: 12 February 2021
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Accepted: 16 February 2021
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Published: 19 February 2021
(This article belongs to the Special Issue Colorectal Cancer Genetics, Epigenetics, and Emerging Therapies)
Liquid biopsy and cell-free DNA (cfDNA) show great promise in cancer diagnostics. In this study, we designed a custom droplet digital PCR (ddPCR) assay for the quantification and quality control of cfDNA isolated from serum. The assay was validated on a group of locally advanced colorectal cancer (CRC) patients and two control groups—patients with hemorrhoids and healthy individuals. The assay shows a high correlation with Qubit measurement (r = 0.976) but offers a higher dynamic range. Mean concentrations of cfDNA were 12.36 ng/µL, 5.17 ng/µL, and 0.29 ng/µL for CRC, hemorrhoid patients, and healthy controls, respectively. The quality of cfDNA was assessed with the measurement of B-cell DNA contamination. On a subset of CRC patients, we compared the mutation status on KRAS (G12A, G12D, G12V, G13D) and BRAF (V600E) genes in the primary tumor and cfDNA isolated from the serum. A total of 70.6% of primary tumor samples were mutated, and the mean fractional abundance of mutations was 9.50%. The matching serum samples were mutated in 38% cases with an average fractional abundance of 0.23%. We conclude that any decisions based solely on the amount of cfDNA present in patient serum must be interpreted carefully and in the context of co-morbidities. This study explores the potential of ddPCR somatic mutations detection from liquid biopsy as a supplement to tissue biopsy in targeted personalized CRC patient management.
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Keywords:
colorectal cancer; liquid biopsy; cell-free DNA; somatic mutations; KRAS; BRAF; ddPCR; hemorrhoids
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MDPI and ACS Style
Zmrzljak, U.P.; Košir, R.; Krivokapić, Z.; Radojković, D.; Nikolić, A. Detection of Somatic Mutations with ddPCR from Liquid Biopsy of Colorectal Cancer Patients. Genes 2021, 12, 289. https://doi.org/10.3390/genes12020289
AMA Style
Zmrzljak UP, Košir R, Krivokapić Z, Radojković D, Nikolić A. Detection of Somatic Mutations with ddPCR from Liquid Biopsy of Colorectal Cancer Patients. Genes. 2021; 12(2):289. https://doi.org/10.3390/genes12020289
Chicago/Turabian StyleZmrzljak, Uršula P., Rok Košir, Zoran Krivokapić, Dragica Radojković, and Aleksandra Nikolić. 2021. "Detection of Somatic Mutations with ddPCR from Liquid Biopsy of Colorectal Cancer Patients" Genes 12, no. 2: 289. https://doi.org/10.3390/genes12020289
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