Advancing cancer MRD monitoring through tumor whole-genome informed, duplex ctDNA sequencing.

e15044 Background: Circulating tumor DNA (ctDNA), a cell-free DNA (cfDNA) present in the plasma of cancer patients, originates from cancer cells and serves as a crucial biomarker during cancer treatment. Despite its importance, the current limit of detection (LoD) of conventional ctDNA assays is suboptimal (0.01%; 10 -4 ) for detecting ctDNA from microscopic residual and recurrent cancer tissues. To address this, we integrated two cutting-edge techniques: whole-genome sequencing (WGS) of primary cancer tissue (a mutation capture step) and duplex DNA sequencing-based cfDNA sequencing (mutation recapture steps). Methods: Thirteen colorectal cancer patients participated in this study. Primary tumor tissues and matched normal blood tissues were collected for WGS in the mutation capture step, while plasma samples for cfDNA sequencing in the mutation recapture steps were obtained just prior to colorectal cancer surgery and 1-month post-surgery follow-up. Tumor DNA and germline DNA were analyzed using CancerVision, a CLIA-certified clinical-grade cancer WGS platform, in the mutation capture step. In the mutation recapture steps, somatically acquired mutations from the previous step were tracked in cfDNA samples using the Concatenating Original Duplex for Error Correction (CODEC) technique, a duplex DNA sequencing technique that examines both Watson and Crick strands of double-stranded DNA molecules to discern true mutations from sequencing noise. Results: The median WGS depth of tumor and germline tissues was 40x and 20x, respectively. The mean depth of cfDNA WGS with CODEC was 25x, with a median duplex sequencing rate of 70%. Overall, 4,828-301,434 somatic base substitutions (SBSs) per sample were discovered in the capture step. Four of the thirteen tumors exhibited hypermutator characteristics with microsatellite instability features. The CODEC technique demonstrated excellent capability in tracing cancer-specific mutations in cfDNA sequencing with a technical sequencing error rate of 4 x 10 -7 , defining the maximum technical LoD, which is 250-fold lower errors than conventional sequencing. In our sample cohort, tumor fractions in cfDNA sequencing were robustly estimated from 0.001% (10 -5 ), below the LoD of conventional MRD methods. Conclusions: Our integration of tumor-WGS-informed duplex cfDNA sequencing methods significantly enhances the sensitivity of sequencing-based MRD assays, achieving a technical LoD limit of 10 -7 for detecting ctDNA in cancer patients. These approaches represent a breakthrough in monitoring MRD in real-world cancer patients.