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Background  Circulating tumour DNA (ctDNA) is known as a tumour-specific personalised biomarker, but the mutation-selection criteria from heterogeneous tumours remain a challenge.    Methods  We conducted multiregional sequencing of 42 specimens from 14 colorectal tumours of 12 patients, including two double-cancer cases, to identify mutational heterogeneity to develop personalised ctDNA assays using 175 plasma samples.    Results  “Founder” mutations, defined as a mutation that is present in all regions of the tumour in a binary manner (i.e., present or absent), were identified in 12/14 tumours. In contrast, “truncal” mutations, which are the first mutation that occurs prior to the divergence of branches in the phylogenetic tree using variant allele frequency (VAF) as continuous variables, were identified in 12/14 tumours. Two tumours without founder and truncal mutations were hypermutators. Most founder and truncal mutations exhibited higher VAFs than “non-founder” and “branch” mutations, resulting in a high chance to be detected in ctDNA. In post-operative long-term observation for 10/12 patients, early relapse prediction, treatment efficacy and non-relapse corroboration were achievable from frequent ctDNA monitoring.    Conclusions  A single biopsy is sufficient to develop custom dPCR probes for monitoring tumour burden in most CRC patients. However, it may not be effective for those with hypermutated tumours.

Frequent post-operative monitoring of colorectal cancer using individualised ctDNA validated by multiregional molecular profiling