Frequent post-treatment monitoring of colorectal cancer using individualized ctDNA validated by multi-regional molecular profiling

PurposeCirculating tumor DNA (ctDNA) analysis has been proposed as an approach for prediction of post-treatment patient outcomes. However, whether a single platform will provide optimal information in all patients or alternatively a patient-specific monitoring approach based on assessment of mutations in the primary tumor from that patient remains an urgent question. Experimental Design: We conducted multiregional sequencing of 42 specimens of 14 colorectal tumors (Stage III and more) from 12 patients, including two double cancer cases, to identify the full spectrum of mutational heterogeneity and identify aberrations that could be used to develop personalized ctDNA assays.Results“Founder” mutations that occur in all regions of the sample were identified in 12/14 (85.7%) tumors. Subsequent phylogenetic analysis of each tumor showed that 12/14 tumors (85.7%) carried at least one “truncal” mutation. Most founder and truncal mutations exhibited higher variant allele frequency (VAF) than “non-founder” and “branch” mutations. In addition, both founder and truncal mutations were more likely to be detected as ctDNA than non-founder and branch mutations. Synchronized ctDNA dynamics of multiple mutations suggested those mutations from the same clonal origin. For 10/12 patients (83.3%) with nearly 1,000 days of post-operative observation, the validity of frequent personalized ctDNA monitoring was confirmed in terms of early relapse prediction, treatment efficacy, and non-relapse corroboration.ConclusionsPersonalized ctDNA monitoring based on aberrations with a high VAF in the primary tumor site should be explored in larger prospective clinical trials to determine the full clinical validity.Translational relevanceCirculating tumor DNA (ctDNA) has been reported to be a new class of tumor-specific personalized biomarkers, but the selection criteria of index gene mutations from heterogeneous tumors as well as the achievement of sufficient sensitivity remain a challenge. Among mutations detected by multiregional sequencing, we monitored mutations with high variant allele frequencies (VAFs) from advanced colorectal cancers. Clinical validity of longitudinal ctDNA monitoring using highly-sensitive digital PCR was evaluated in terms of: (a) early relapse prediction; (b) treatment efficacy evaluation; and (c) no relapse corroboration. We found that ctDNA from high VAF mutations of a tumor are likely to be founder/truncal mutations. Based on rigorous longitudinal monitoring, our results suggest that sensitivity required the VAF to be 0.01-0.1%. The ctDNA from high VAF mutations strongly reflects tumor burden in a timely manner, thereby establishing clinical validity as a new class of tumor-specific personalized biomarkers.