Detection of circulating tumor DNA by tumor-informed whole-genome sequencing enables prediction of recurrence in stage III colorectal cancer patients.

Introduction: Circulating tumor (ctDNA) can be used to detect residual disease after cancer treatment. Detecting low-level ctDNA is challenging, due to the limited number of recoverable ctDNA fragments at any target loci. In response, we applied tumor-informed whole-genome sequencing (WGS), leveraging thousands of mutations for ctDNA detection.

Methods: Performance was evaluated in serial plasma samples (n = 1283) from 144 stage III colorectal cancer patients. Tumor/normal WGS was used to establish a patient-specific mutational fingerprint, which was searched for in 20x WGS plasma profiles. For reproducibility, paired aliquots of 172 plasma samples were analyzed in two independent laboratories. De novo variant calling was performed for serial plasma samples with a ctDNA level > 10 % (n = 17) to explore genomic evolution.

Results: WGS-based ctDNA detection was prognostic of recurrence: post-operation (Hazard ratio [HR] 6.75, 95 %CI 3.18-14.3, p < 0.001), post-adjuvant chemotherapy (HR 28.9, 95 %CI 10.1-82.8; p < 0.001), and during surveillance (HR 22.8, 95 %CI 13.7-37.9, p < 0.0001). The 3-year cumulative incidence of ctDNA detection in recurrence patients was 95 %. ctDNA was detected a median of 8.7 months before radiological recurrence. The independently analyzed plasma aliquots showed excellent agreement (Cohens Kappa=0.9, r = 0.99). Genomic characterization of serial plasma revealed significant evolution in mutations and copy number alterations, and the timing of mutational processes, such as 5-fluorouracil-induced mutations.

Conclusion: Our study supports the use of WGS for sensitive ctDNA detection and demonstrates that post-treatment ctDNA detection is highly prognostic of recurrence. Furthermore, plasma WGS can identify genomic differences distinguishing the primary tumor and relapsing metastasis, and monitor treatment-induced genomic changes.