Abstract 3340: Bladder field cancerization impacts tumor development and T-cell exhaustion, and is reflected in urinary tumor DNA
Authors: Strandgaard Trine , Nordentoft Iver , Birkenkamp-Demtröder Karin , Salminen Liina , Prip Frederik , Christensen Emil , Lamy Philippe , Andreasen Tine Ginnerup , Lindskrog Sia Viborg , Knudsen Michael , Rasmussen Julie , Steiniche Torben , Jensen Jørgen Bjerggaard , Dyrskjøt Lars 
Published: June 2023

Abstract
Introduction: Patients with high-risk non-muscle invasive bladder cancer (NMIBC) are treated with Bacillus Calmette-Guérin (BCG), which targets altered normal-appearing urothelium (field cancerization). Field cancerization may affect treatment response and outcome. High urinary tumor DNA (utDNA) levels in patients with NMIBC have been associated with worse clinical outcomes, and utDNA may be used for real-time assessment of residual disease. The prognostic and predictive values of field cancerization and utDNA need further investigation.
Materials and methods: We analyzed samples procured from 134 patients with NMIBC - including 210 tumors, 751 selected site biopsies (SSBs) and 187 urine samples. The median follow-up time was 7.8 years. Samples were collected before and after BCG-treatment. Sixty-four patients (48%) progressed to muscle-invasive bladder cancer or experienced high-grade NMIBC recurrence within two years after BCG treatment, indicating BCG-failure. Tumor samples were analyzed using whole-exome and RNA-sequencing. DNA from SSBs and urine samples was subjected to tumor-informed deep-targeted sequencing. SSB DNA was sequenced to a mean coverage of 1,357X, and urinary DNA to a mean coverage of 2,153X. Unique molecular identifiers were applied and the deepSNV pipeline was used for error-robust mutation calling. The level of field cancerization in each patient was estimated by the highest number of called mutations adjusted by the number of mutations on the sequencing panel.
Results: We detected mutations in 458 out of 751 analyzed SSBs. Mutations in normal-appearing SSBs were detected at a mean frequency of 0.04 (range: 0.002-0.39). The level of field cancerization increased with age (p=0.0027). utDNA levels reflected the bladder disease status, increasing with higher tumor stage and multiplicity and was shed from both bladder field cancerization and tumors. High utDNA levels after BCG-treatment were associated with high recurrence rates (p&lt;0.001), high risk of progression (p=0.0014) and worse high-grade recurrence-free survival (p=0.047). Field cancerization level was not associated with response to BCG treatment. High levels of field cancerization and utDNA were associated with high tumor-associated CD8 T-cell exhaustion. Additionally, a high field cancerization level correlated with a high tumor neoantigen load (p=0.029), indicating a possible link to the observed CD8 T-cell exhaustion.
Conclusion: A high level of field cancerization was associated with an immune exhausted phenotype of the tumor. Measurements of utDNA reflected the overall disease status of the bladder, including field cancerization. Collectively, these results suggest that field cancerization may impact the tumor microenvironment and utDNA levels may be used for assessing clinical risk in NMIBC and for guiding surveillance and early treatment interventions.
Citation Format: Trine Strandgaard, Iver Nordentoft, Karin Birkenkamp-Demtröder, Liina Salminen, Frederik Prip, Emil Christensen, Philippe Lamy, Tine Ginnerup Andreasen, Sia Viborg Lindskrog, Michael Knudsen, Julie Rasmussen, Torben Steiniche, Jørgen Bjerggaard Jensen, Lars Dyrskjøt. Bladder field cancerization impacts tumor development and T-cell exhaustion, and is reflected in urinary tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3340.