A K Boysen1, B S Sørensen2, A C Lefevre3, R Abrantes2, J S Johansen4, B V Jensen4, J V Schou4, F O Larsen4, D Nielsen4, H Taflin5, B Gustavson5, Y Wettergren5, B S Sorensen6, A H Ree7, S Dueland8, N Pallisgaard9, K L Spindler10. 1. Department of Experimental Clinical Oncology, Aarhus University Hospital, Nørrebrogade 44, 8000 Aarhus, Denmark; Department of Oncology, Aarhus University Hospital, Denmark. Electronic address: andboy@rm.dk. 2. Department of Experimental Clinical Oncology, Aarhus University Hospital, Nørrebrogade 44, 8000 Aarhus, Denmark. 3. Department of Oncology, Aarhus University Hospital, Denmark. 4. Departments of Oncology and Medicine, Copenhagen University Hospital, Herlev, Denmark. 5. Surgical Oncology Laboratory, Department of Surgery, Sahlgrenska University Hospital, Gothenburg, Sweden. 6. Department of Clinical Biochemistry, Aarhus University Hospital, Denmark. 7. Department of Oncology, Akershus University Hospital, Norway. 8. Department of Oncology, Oslo University Hospital, Norway. 9. Department of Pathology, Zealand University Hospital, Roskilde, Denmark. 10. Department of Experimental Clinical Oncology, Aarhus University Hospital, Nørrebrogade 44, 8000 Aarhus, Denmark; Department of Oncology, Aarhus University Hospital, Denmark.
Abstract
BACKGROUND: Cell free DNA (cfDNA) has shown promising utility as prognostic biomarker for patients with colorectal cancer (CRC), with an ongoing need to optimize and validate the laboratory methodology. Here, we report our optimization and validation of a direct fluorescent assay and display the potential utility in patients with colorectal cancer. METHODS: Plasma cfDNA was analyzed by a direct fluorescent assay (DFA) and compared to quantification by droplet digital PCR (ddPCR). For clinical validation, baseline blood samples were available for a total of 273 patients from six different Nordic trials, covering patients with locally advanced rectal cancer (n = 176, cohorts A + B), liver limited metastatic CRC (n = 75C + D) and wide spread metastatic CRC (n = 22 E + F). RESULTS: Validating the DFA analysis with ddPCR revealed a strong correlation with an R2 of 0.81. For the clinical cohorts, the levels of cfDNA were: 0.8 ng/uL (95%CI 0.75-0.83) (A + B), 0.93 ng/uL (95%CI 0.86-1.02) (C + D) and 1.2 ng/uL (95%CI 0.85-1.47) (E + F), respectively (p < 0.01). All cohorts of colorectal cancer had higher levels of cell free DNA than healthy individuals (n = 94) (p < 0.01). CONCLUSION: Analysis of cell free DNA by a direct fluorescent assay could be an attractive laboratory option for a rapid inexpensive quantification of cell free DNA.
BACKGROUND: Cell free DNA (cfDNA) has shown promising utility as prognostic biomarker for patients with colorectal cancer (CRC), with an ongoing need to optimize and validate the laboratory methodology. Here, we report our optimization and validation of a direct fluorescent assay and display the potential utility in patients with colorectal cancer. METHODS: Plasma cfDNA was analyzed by a direct fluorescent assay (DFA) and compared to quantification by droplet digital PCR (ddPCR). For clinical validation, baseline blood samples were available for a total of 273 patients from six different Nordic trials, covering patients with locally advanced rectal cancer (n = 176, cohorts A + B), liver limited metastatic CRC (n = 75C + D) and wide spread metastatic CRC (n = 22 E + F). RESULTS: Validating the DFA analysis with ddPCR revealed a strong correlation with an R2 of 0.81. For the clinical cohorts, the levels of cfDNA were: 0.8 ng/uL (95%CI 0.75-0.83) (A + B), 0.93 ng/uL (95%CI 0.86-1.02) (C + D) and 1.2 ng/uL (95%CI 0.85-1.47) (E + F), respectively (p < 0.01). All cohorts of colorectal cancer had higher levels of cell free DNA than healthy individuals (n = 94) (p < 0.01). CONCLUSION: Analysis of cell free DNA by a direct fluorescent assay could be an attractive laboratory option for a rapid inexpensive quantification of cell free DNA.
Authors: Barbara Kinga Barták; Tamás Fodor; Alexandra Kalmár; Zsófia Brigitta Nagy; Sára Zsigrai; Krisztina Andrea Szigeti; Gábor Valcz; Péter Igaz; Magdolna Dank; István Takács; Béla Molnár Journal: Int J Mol Sci Date: 2022-03-29 Impact factor: 5.923