Purpose: Investigate and compare the use of plasma- and urine DNA mutation analysis for predicting neoadjuvant chemotherapy (NAC) response and long-term oncological outcome in patients with muscle invasive bladder cancer. Experimental Design: Whole exome sequencing of tumor and germline DNA was performed for 92 patients treated with NAC followed by radical cystectomy (RC). A custom NGS panel capturing approx. 50 mutations per patient was designed and utilized to track tumor derived DNA (tdDNA) in liquid biopsies. A total of 447 plasma samples, 281 urine supernatants and 123 urine pellets collected before, during and after treatment were analyzed. Patients were enrolled from 2013-2019 with a median follow-up time of 41.3 months after RC. Results: We identified tdDNA before initiation of NAC in 89% of urine supernatants, 85% of urine pellets and 43% of plasma samples. tdDNA levels were higher in urine supernatants and urine pellets compared to plasma samples (p<0.001). In plasma, detection of tdDNA before NAC was associated with a lower NAC response rate (p<0.001). Detection of tdDNA after NAC was associated with lower response rates in plasma, urine supernatant and urine pellet (p<0.001, p=0.03, p=0.002). tdDNA dynamics during NAC was predictive of NAC response and outcome in urinesupernatant and plasma (p=0.006, p=0.002). A combined measure from plasma and urine supernatant tdDNA dynamics stratified patients by outcome (p=0.003). Conclusions: Analysis of tdDNA in plasma and urine samples both separately and combined has potential to predict treatment response and outcome.

Lars Dyrskjot has sponsored research agreements with C2i, AstraZeneca, Natera, Photocure, and Ferring; has an advisory/consulting role at Ferring and UroGen; and is Chairman of the Board in BioXpedia A/S. Jorgen Bjerggaard Jensen is proctor for Intuitive Surgery; is a member of advisory board for Olympus Europe, Ambu, Cepheid, and Ferring; and has sponsored research agreements with Medac, Photocure ASA, Cepheid, and Ferring.

This work was funded by AstraZeneca, Natera Inc., the Danish Cancer Society, the Novo Nordisk Foundation and Aarhus University.

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 in Denmark (#1302183).

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.

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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).

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I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.

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The raw sequencing data generated in this study are not publicly available as this compromise patient consent and ethics regulations in Denmark. Processed nonsensitive data are available upon reasonable request from the corresponding author.