Whole genome mutational analysis for accurate tumor-informed ctDNA based MRD surveillance, treatment monitoring and biological characterization of urothelial carcinoma

Circulating tumor (ctDNA) can be used for sensitive detection of minimal residual disease (MRD). However, the probability of detecting ctDNA at low tumor burden is limited by the number of mutations analyzed and available plasma volume. Here we applied a tumor-informed WGS approach for ctDNA-based MRD detection (91% sensitivity, 92% specificity) and treatment response evaluation in 916 longitudinally collected plasma samples from 112 patients with localized muscle-invasive bladder cancer. We show that WGS-based ctDNA detection is prognostic of patient outcomes with a median lead time of 131 days over radiographic imaging. We performed genomic characterization of post-treatment plasma samples to study tumor evolution and observed acquisition of the platinum therapy-associated mutational signatures and copy number variations not present in the primary tumors. Our results support the use of WGS for ultra-sensitive ctDNA detection, and highlight how tracking of tumor evolution using WGS of plasma samples opens opportunities to refine precision oncology. ### Competing Interest Statement Lars Dyrskjoet has sponsored research agreements with C2i Genomics, Natera, AstraZeneca, Photocure, and Ferring and has an advisory/consulting role at Ferring, MSD and UroGen. Lars Dyrskjoet has received speaker honoraria from AstraZeneca, Pfizer and Roche and received travel support from MSD. Lars Dyrskjoet is board member at BioXpedia. Joergen Bjerggaard Jensen is proctor for Intuitive Surgery, is a member of advisory board for Olympus Europe, Ambu, Cepheid, Janssen, and Ferring and has sponsored research agreements with Medac, Photocure ASA, Cepheid, Olympus, and Ferring. Asaf Zviran is the co-founder and a member of the board of directors of C2i Genomics. Boris Oklander is the co-founder and CTO of C2i Genomics. Santiago Gonzalez, Maja Kuzman, Jurica Levatic, Dunja Glavas, Ryan Ptashkin, James Smadbeck, Danielle Afterman, Tomer Lauterman, Yarin Cohen, Zohar Donenhirsh, Iman Tavassoly, Ury Alon are employees of C2i Genomics. Paz Polak is a former employee of C2i Genomics. ### Funding Statement This work was funded by research grants to L.D. from C2i Genomics, The Danish Cancer Society, Novo Nordisk Foundation, Independent Research Fund Denmark, Aarhus University and Aarhus University Hospital. We would like to thank technical personnel at the Departments of Molecular Medicine, Urology and Oncology, Aarhus University Hospital, for sample handling and processing. Sample collection was supported by the Danish Cancer Biobank. ### 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 patients provided informed written consent, and the study was approved by The National Committee on Health Research Ethics (#1302183). Study data were collected and managed using REDCap hosted at Aarhus University I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The raw sequencing data generated in this study are not publicly available as this compromises patient consent and ethics regulations in Denmark. Processed non-sensitive data are available upon request from the corresponding author.