Postoperative cfDNA levels and ctDNA detection rates in patients with stage II colon cancer screened for CIRCULATE (AIO-KRK-0217, ABCSG).

3610 Background: Postoperative circulating tumor DNA (ctDNA) has emerged as a prognostic biomarker for disease recurrence in patients (pts) with resected colorectal cancer (CRC) and may potentially guide adjuvant treatment decisions. The timing of ctDNA screening is critical, as postop. plasma cell-free DNA (cfDNA) levels may vary due to factors such as tissue disruption from the surgical resection. The CIRCULATE trial (NCT04089631) investigates ctDNA guided adjuvant therapy in stage II CRC pts in > 140 centres in Germany and Austria. Methods: To investigate the impact of blood sampling time points on cfDNA concentrations and the ctDNA positivity rates, we analyzed the postop. plasma samples of 1439 pts with stage II CRC screened for CIRCULATE between 2020 and 2024. Blood samples were collected within 5-60 days post tumor resection in stabilizing tubes (Streck or PaxGene). Samples were analyzed for postop. cfDNA concentration and tumor-informed ctDNA in plasma samples by an error-reduced Next-Generation Sequencing (NGS) approach [Stasik S Front Genet. 2022]. Results: Plasma cfDNA concentrations (measured by qPCR for beta-globin gene) ranged from 0.02 to 20.32 ng/µL (mean: 0.689 ng/µL; median: 0.360 ng/µL). The highest cfDNA levels were observed within 2 weeks after surgery (mean: 1.079 ng/µL; median: 0.540 ng/µL), with a significant decrease in samples collected > 3 weeks postop. (mean: 0.631 ng/µL; median: 0.355 ng/µL; p < 0.0001), suggesting an impact of surgical trauma and subsequent cfDNA release from normal tissue. In ctDNA-neg. pts, cfDNA concentrations stabilized between 0.405 and 0.449 ng/µL during weeks 4-8. In contrast, ctDNA-pos. pts had significantly elevated cfDNA levels at 2 months post-surgery (mean: 0.972 ng/µL; p = 0.419), indicating ongoing tumor-specific DNA shedding associated with recurrent disease. Variant allele frequencies (VAFs) in ctDNA-pos samples were negatively correlated with cfDNA concentrations, particularly in early postop. samples (Spearman r = -0.508), suggesting a dilution effect of ctDNA post-surgery. This correlation diminished at later time points, supporting the potential advantage of later sampling to improve ctDNA sensitivity. Despite temporal variations in cfDNA concentrations, ctDNA positivity was consistent across all sampling intervals (i.e. week 1: 4.1%; week 6-8: 5.64%), demonstrating the assay's robust sensitivity. Conclusions: We observed a significant variation in cfDNA levels depending on the timing of postop. sampling. Different kinetics, such as cfDNA release from normal tissue and tumor shedding, may influence cfDNA levels and the sensitivity to ctDNA detection. Nonetheless, our assay demonstrates consistent and reliable sensitivity for ctDNA detection across all postop. sampling time points. Clinical trial information: NCT04089631 .