Multiplexed real-time polymerase chain reaction cell-free DNA assay as a potential method to monitor stage IV colorectal cancer.

Abstract

Liquid biopsy is a new area in cancer diagnostics that measures cell-free DNA in plasma from tumor that may serve as a monitoring tool in colorectal cancer patients. Multiplexed real-time polymerase chain reaction based on multicopy retro-transposable elements (targeting 80 base pair and 265 base pair sequences and an internal-positive-control) was used to evaluate the ability of cell-free DNA concentration and DNA Integrity Index to discriminate cancer from healthy patients. A cohort of 40 healthy controls and 39 stage IV colorectal patient's plasma were interrogated. The potency of each biomarker was measured by using receiver operating characteristic curves and derived area under the curve measures. Significant differences in cell-free DNA concentration and DNA integrity index were observed between controls and stage IV patients with a limit of detection <0.1 pg/μL. Investigation of the ability of both biomarker candidates to differentiate cancer from healthy patients showed an area under the curve of 0.9891 and 0.9859 for 80 base pair and 265 amplicons respectively and 0.8603 for DNA integrity index-265/80. After establishing differences in cell-free DNA levels between healthy and treated and untreated stage IV patients, the multiplexed real-time polymerase chain reaction measurements of retro-transposable elements in cancer patient plasma potentially possess the ability to monitor therapy responsiveness in near real time.