Non-invasive molecular analysis of
circulating tumor DNA (ctDNA) is a promising application in personalized
cancer management, although there is still much to learn about the biological characteristics of ctDNA. The present study compared absolute amounts of KRAS mutated ctDNA and total circulating
cell-free DNA (
cfDNA) in
colorectal cancer (CRC) patients (n=50) from various stages and healthy controls (n=8) by Intplex allele-specific and digital droplet PCR. In addition, the impact of two prominent extraction techniques (
silica-based membrane vs. magnetic beads) on
cfDNA and ctDNA recovery was analyzed in 38 paired samples from CRC patients and specific spike-in
DNA controls.
CfDNA fragment size was assessed using the Agilent 2100 Bioanalyzer. Relative quantities of total
cfDNA quantities were measured using the Qubit fluorometer. Statistical analysis on total
cfDNA yield revealed a strong correlation (r=0.976) between Qubit and absolute Intplex allele-specific PCR measurements in
cancer patients and healthy controls. Total
cfDNA was significantly increased in
cancer patients compared to healthy controls, with the highest yield in distant metastatic disease. In line, the highest amount of ctDNA (1.35 ng/μL) was found in patients with distant organ
metastasis. Of great interest, the
silica-based membrane method significantly promoted extraction of long
cfDNA fragments. In contrast, the magnetic bead system more efficiently recovered short
cfDNA fragments in serum of
cancer patients. Further, a decreased KRAS allele frequency was observed in serum compared to plasma. This study suggests that the source of
cfDNA and choice of pre-analytical extraction systems needs to be more carefully validated in routine clinical practice.