A novel liquid-liquid cfDNA extraction method for targeted sequencing with colorectal cancer patient samples: a pilot...

Cell-free DNA (cfDNA) in the bloodstream released from tumor, also known as circulating tumor DNA (ctDNA), has emerged as a promising biomarker for cancer detection and monitoring. Extraction of ctDNA from plasma liquid biopsies is an emerging approach with minimal invasiveness to detect gene mutations associated with colorectal cancer. One hurdle to ctDNA mutational analyses is obtaining sufficient quantity and good quality of DNA which is compatible with downstream diagnostic testing, especially when testing samples with limited volumes of plasma.

We developed and evaluated a novel liquid-liquid extraction method optimized to concentrate and purify ctDNA from plasma samples. Analytical performance was assessed using reference materials and compared to a gold standard commercially available extraction kit under targeted panel next-generation sequencing. A pilot study was conducted on plasma samples from 16 colorectal cancer patients. Additionally, an independent, blinded external validation study was included to confirm the analytical validity of this workflow.

In analytical testing using reference materials, the novel extraction kit achieved higher DNA yield and higher tumor mutation detection rate compared to a gold standard commercially available extraction kit under targeted panel next-generation sequencing. In the pilot study of 16 colorectal cancer patient samples using this workflow with the liquid-liquid extraction method, an average of 73.4% (262/357) of the tumor mutations could be detected in low volumes of plasma, which is higher than the 50% pooled tumor mutation detection rate of plasma cfDNA in a previous meta-analysis. Parallel testing of plasma and tumor samples can enhance the detection of potentially actionable genes. Meanwhile, the workflow achieved full concordance across all samples, providing objective, third-party verification of its diagnostic precision and sensitivity.

These results demonstrate the promising performance of integrating liquid-liquid plasma extraction with targeted panel next-generation sequencing to identify cancer-associated mutations in colorectal cancer (CRC) patients.