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

Purpose Circulating 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. Conclusions Personalized 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 relevance Circulating 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. ### Competing Interest Statement Satoshi Nishizuka Array Jet: Grant/Research Support Taiho Pharmaceuticals: Grant/Research Support Boehringer-Ingelheim: Grant/Research Support Chugai Pharmaceutical: Honorarium Geninus: Grant/Research Support CLEA Japan: Advisor/Board Member Mills Institute for Personalized Care (MIPCC): Consultation Nomura Jimusyo: Consultation Gordon Mills AstraZeneca: Consultant/Scientific Advisory Board Companies, Sponsored Research Companies Chrysalis: travel reimbursement only ImmunoMET: Consultant/Scientific Advisory Board Companies, Stock/Options/Financial Companies Ionis: Consultant/Scientific Advisory Board Companies Lilly USA, LLC: Consultant/Scientific Advisory Board Companies Mills Institute for Personalized Care (MIPCC): travel reimbursement only Nuevolution: travel reimbursement only PDX Pharma: Consultant/Scientific Advisory Board Companies Signalchem Lifesciences: Consultant/Scientific Advisory Board Companies Symphogen: Consultant/Scientific Advisory Board Companies Tarveda: Consultant/Scientific Advisory Board Companies, Stock/Options/Financial Companies Catena Pharmaceuticals: Stock/Options/Financial Companies SignalChem: Stock/Options/Financial Companies Spindletop Ventures: Stock/Options/Financial Companies HRD assay to Myriad Genetics: Licensed Technology Companies DSP to Nanostring: Licensed Technology Companies Karus Therapeutics: Sponsored Research Companies Nanostring: Sponsored Research Companies Pfizer: Sponsored Research Companies Tesaro: Sponsored Research Companies ### Funding Statement This study is supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers and other Grants. Mizunori Yaegashi: JSPS 16K19951, JSPS Fujita Memorial Fund for Medical Research Takeshi Iwaya: JSPS 17K10605 and 19K09130 Fumitaka Endo: JSPS 19K09224 Kei Sato: JSPS 18K15326 Satoshi Nishizuka: JSPS 15KK0317, Keiryoukai Research Grant #136, Ministry of Education, Culture, Sports, Science, and Technology 16H01578, and Iwate Prefectural Regional Innovation Grant. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All samples in this study were acquired after obtaining written informed consent and approval by the Institutional Review Board of Iwate Medical University School of Medicine according to the Helsinki Declaration(HGH27-29). All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. 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