Abstract Purpose: Genomic profiling in urothelial carcinoma (UC) conventionally uses tissue DNA. However, tissue biopsy is so invasive that it can be difficult in some cases. Cell-free DNA (cfDNA) assay is a minimally invasive tool obtaining a comprehensive genomic information reflecting a tumor heterogeneity. Recently, the investigation of cfDNA assay in UC is widely conducted, but its clinical utility remains largely unknown. We aimed to establish plasma cfDNA assay and clarify its clinical role in UC. Method: Blood and matched tissue were obtained from 48 cases with advanced UC before 1st line systemic therapy or radical surgery. For genomic profiling of all samples, we applied a customed targeted DNA-sequencing strategy capturing 54 UC-relevant genes. Somatic mutations were required to be supported by a minimum variant allele frequency (VAF) of 0.5% in plasma cfDNA and 5% in tissue DNA. All mutation calls were filtered against matched leukocyte DNA. The result of mutational analysis was validated with digital PCR. The proportion of tumor-derived cfDNA (circulating tumor DNA (ctDNA) fraction) was estimated. We investigated the association between genomic profile and molecular subtyping with RNA sequencing and immunohistochemistry (IHC). Result: Fifty-five somatic mutations were detected in 22 cfDNA samples (22/48, 45.8%). Twenty-six of all somatic mutations identified in cfDNA (26/55, 47.3%) were concurrently present in tumor tissues. In 14 mutations randomly selected from all of those detected in cfDNA, VAF of digital PCR was statistically consisted with the result of NGS (p<0.001, R2=0.95). Furthermore, digital PCR showed that mutations detected in cfDNA alone were cleared via radical surgery. And those were found in another tissue sample from the same patient. Of the 22 cases, 6 had a ctDNA fraction greater than 2% of total cfDNA. High ctDNA fraction was associated with advanced clinical tumor stage, high PD-L1 expression in tumor tissue by IHC and basal molecular subtype (p = 0.04, 0.02, <0.01, respectively). Conclusions: We established cfDNA assay for genome profiling in advanced UC. The result of validation using digital PCR suggested that cfDNA assay can identify the mutation which cannot be detected in tissue sample as a tumor heterogeneity. Additionally, cfDNA assay generate the information for classifying UC cases by molecular subtypes and predicting the expression of immune checkpoint molecules. Further investigation is needed as a promising minimally invasive assay to predict an immunotherapy responsiveness against metastatic UC. Citation Format: Yuki Kita, Takayuki Sumiyoshi, Takeshi Sano, Akihiro Hamada, Toru Sakatani, Kenji Nakamura, Hideaki Takada, Ryousuke Ikeuchi, Takayuki Goto, Atsuro Sawada, Shusuke Akamatsu, Takashi Kobayashi. Establishing clinical utility for genomic profiling with plasma cell free DNA in urothelial carcinoma [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 235.