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Article

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 / Revised: 12 February 2021 / Accepted: 16 February 2021 / 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. View Full-Text
Keywords: colorectal cancer; liquid biopsy; cell-free DNA; somatic mutations; KRAS; BRAF; ddPCR; hemorrhoids 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 Style

Zmrzljak, 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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