Zmrzljak, Uršula Prosenc (52265076000)Uršula Prosenc (52265076000)ZmrzljakKošir, Rok (24179270900)Rok (24179270900)KoširKrivokapić, Zoran (55503352000)Zoran (55503352000)KrivokapićRadojković, Dragica (6602844151)Dragica (6602844151)RadojkovićNikolić, Aleksandra (57194842918)Aleksandra (57194842918)Nikolić2025-07-022025-07-022021https://doi.org/10.3390/genes12020289https://www.scopus.com/inward/record.uri?eid=2-s2.0-85101875555&doi=10.3390%2fgenes12020289&partnerID=40&md5=f273b5b275b94a0181db1823ec4fbaa5https://remedy.med.bg.ac.rs/handle/123456789/12346Liquid 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 inter-preted 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 per-sonalized CRC patient management. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.BRAFCell-free DNAColorectal cancerDdPCRHemorrhoidsKRASLiquid biopsySomatic mutations