Timing of ctDNA Analysis Aimed at Guiding Adjuvant Treatment in Colorectal Cancer

Multiple clinical trials are investigating ctDNA to guide adjuvant chemotherapy (ACT) in colorectal cancer. Timely ACT initiation necessitates early ctDNA testing, but the impact of postoperative cell-free DNA (cfDNA) and ctDNA dynamics remains unclear, particularly with cost-reducing input caps employed in some assays. This study investigates ctDNA detection at day 14 versus day 30, comparing whole-sample analysis with capping the cfDNA input, and evaluates single and dual timepoint assessments for ACT allocation.

From 2019 to 2023, 611 patients with stage I to III colorectal cancer were enrolled. Blood was collected preoperatively and postoperatively on ∼day 14 and ∼day 30. The cfDNA levels were assessed using digital PCR, and ctDNA was assessed using tumor-informed digital PCR or targeted sequencing analyzing all cfDNA from 8 mL of plasma.

Despite elevated cfDNA in 85% of day 14 samples, performance was comparable between the two timepoints (sensitivity, 31% vs. 32% and specificity, both 98%). A 50-ng cfDNA input cap reduced ctDNA detection probability, affecting 78% of day 14 samples and 65% of day 30 samples. At both timepoints, ctDNA detection was prognostic of recurrence (day 14; HR, 9.0, 95% confidence interval, 5.5-14.8 and day 30: HR, 12.5, 95% confidence interval, 7.6-20.4). In 74% of ctDNA-positive recurrence patients, both samples had ctDNA detected. An increase in the ctDNA level from day 14 to day 30 was associated with a shorter time to recurrence (Pearson R = -0.63, P = 0.003). Combining the timepoints would increase sensitivity (36%) and allow earlier ACT start in 80% of patients.

Early ctDNA sampling is feasible and highly prognostic. Supplemental later testing may improve sensitivity while allowing early ACT initiation for most ctDNA-positive patients.