Methodological development and biological observations of cell free DNA with a simple direct fluorescent assay in colorectal cancer

TY - JOUR

T1 - Methodological development and biological observations of cell free DNA with a simple direct fluorescent assay in colorectal cancer

AU - Boysen, A K

AU - Sørensen, B S

AU - Lefevre, A C

AU - Abrantes, R

AU - Johansen, J S

AU - Jensen, B V

AU - Schou, J V

AU - Larsen, F O

AU - Nielsen, D

AU - Taflin, H

AU - Gustavson, B

AU - Wettergren, Y

AU - Sørensen, B S

AU - Ree, A H

AU - Dueland, S

AU - Pallisgaard, N

AU - Spindler, K L

N1 - Copyright © 2018 Elsevier B.V. All rights reserved.

PY - 2018/12

Y1 - 2018/12

N2 - 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.

AB - 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.

KW - Cell-Free Nucleic Acids/blood

KW - Cohort Studies

KW - Colorectal Neoplasms/blood

KW - DNA, Neoplasm/blood

KW - Fluorescent Antibody Technique, Direct

KW - Humans

KW - Polymerase Chain Reaction

U2 - 10.1016/j.cca.2018.09.029

DO - 10.1016/j.cca.2018.09.029

M3 - Journal article

C2 - 30240586

SN - 0009-8981

VL - 487

SP - 107

EP - 111

JO - Clinica Chimica Acta

JF - Clinica Chimica Acta

ER -