Development of a methylation-based, tissue-free test for the detection of molecular residual disease by circulating tumor DNA.

3048 Background: Clinical validation studies support tumor-informed molecular residual disease (MRD) as a prognostic biomarker for disease recurrence across multiple solid tumor types. However, these tests are not always feasible due to the occasional lack of tumor tissue for next-generation sequencing. Here, we discuss the design of a test for tissue-free (tf)MRD detection and its application to a cohort of patients with colorectal cancer (CRC). Methods: A targeted panel composed of differentially methylated regions was developed. A machine-learning model was trained on differential methylation patterns in order to classify plasma samples as MRD-positive or MRD-negative. Performance of the trained classifier was assessed in an independent cohort of 246 patients enrolled in the Bespoke CRC trial (NCT04264702). These patients had MRD results available using a tumor-informed circulating tumor DNA (ctDNA) assay (SignateraTM), of whom 163 were persistently MRD-negative without clinical progression, and 83 had MRD-positive results. Tissue-free MRD results were compared to the tumor-informed results by calculating the percent positive agreement (PPA) and negative percent agreement (NPA). Clinical outcomes (recurrence-free survival [RFS]) were evaluated based on tfMRD results in all patients and stratified based on whether the patient received adjuvant chemotherapy (ACT). Results: In this clinical cohort from Bespoke CRC (72% non-Hispanic White, 54% male, mean age 61.4±12.3 years), 71 (28%) patients had stage II CRC, and 146 (59%) had stage III CRC. Overall, PPA was 86% (95% CI: 77-93%) and NPA was 98% (95% CI: 95-100%). Patients with tfMRD-positive status showed inferior RFS compared to tfMRD-negative patients (p < 0.001). Significant benefit from ACT was observed among tfMRD-positive (p < 0.001) but not among tfMRD-negative patients (p = 0.19). For patients who did not receive ACT in this cohort, we observed 100% PPV and 100% specificity. Conclusions: This is the first study of its kind demonstrating a high concordance between a tfMRD test and a clinically validated tumor-informed ctDNA assay. Similar to recently reported data using a tumor-informed ctDNA assay, patients with tfMRD-positive results appeared to derive benefit from ACT treatment. These findings demonstrate that in cases where tissue is not available or of inadequate quality, a methylation-based tissue-free assay may serve as a potential alternative for MRD detection.