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

Authors Boysen AK, Sørensen BS, Lefevre AC, Abrantes R, Johansen JS, Jensen BV, Schou JV, Larsen FO, Nielsen D, Taflin H, Gustavson B, Wettergren Y, Sorensen BS, Ree AH, Dueland S, Pallisgaard N, Spindler KL

Source Clin Chim Acta. 2018 Dec;487:107-111. Publicationdate 18 Sep 2018

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.