Beyond Binary MRD: Quantitative ctDNA Interpretation After Curative-Intent Surgery for Colorectal Cancer

BackgroundCirculating tumor DNA (ctDNA) detection after curative-intent surgery is being used to identify minimal residual disease (MRD) in colorectal cancer (CRC). However, MRD classification is dependent on analytical sensitivity, and the impact of detection threshold on observed post-operative positivity remains incompletely characterized. We evaluated MRD positivity in stage I-III CRC using a CRISPR-based plasma sequencing assay, MUTE-Seq. MethodsPatients were prospectively enrolled and analyzed using customized tumor-informed panels applied to baseline and post-operative plasma samples collected at 4-week and 3-month. We report preliminary results from 39 plasma samples obtained from the first 14 patients. MRD positivity was assessed across multiple hypothetical detection thresholds (1-100 ppm). ResultsAll 14 patients (100%) had detectable mutations at baseline. Mutation-positive call number significantly decreased after surgery (baseline vs 4-week, p = 0.006; baseline vs 3-month, p = 0.004), and ctDNA concentration likewise declined (baseline vs 4-week, p = 0.002; baseline vs 3-month, p = 0.003). Among stage II-III patients, MRD positivity at 4-week was 20% at a 100-ppm threshold but increased to 70% at 10 ppm and 100% at 1 ppm. At 3-month, MRD positivity was 11% at a 100-ppm threshold and 78% at 1 ppm. At both time points, approximately 80% of MRD-positive stage II-III patients harbored ctDNA levels below 100 ppm, and half of these cases were below 15 ppm. Two patients (one stage I and one stage II) developed recurrence; both were MRD-positive at 4-week and demonstrated increasing mutation-positive calls at 3-month, with a median radiologic lead time of 4 months. ConclusionsPost-operative MRD classification in CRC is strongly influenced by analytical sensitivity. A substantial proportion of residual disease signals reside below the conventional ctDNA detection threshold of 100 ppm, supporting the clinical relevance of ultrasensitive ctDNA detection.