Abstract 3326: The clinical validity of urinary pellet DNA monitoring of recurrent bladder cancer
Authors: Abe Masakazu , Hiraki Hayato , Tsuyukubo Takashi , Ono Sadahide , Maekawa Shigekatsu , Tamura Daichi , Ikarashi Daiki , Kato Renpei , Matsuura Tomohiko , Kanehira Mitsugu , Takata Ryo , Fujisawa Hiromitsu , Iwaya Takeshi , Idogawa Masashi , Obara Wataru , Nishizuka Satoshi S. 
Published: June 2023

Introduction: Approximately half of high-risk non-muscle-invasive bladder cancers (NMIBCs) recur after transurethral resection of the bladder tumor (TURBT), whereas conventional urological testing for bladder cancer (BC) patients has been considered invasive and with low sensitivity. Our previous reports have shown that circulating tumor DNA (ctDNA) monitored by digital PCR (dPCR) can predict relapse and evaluate treatment efficacy in digestive tract cancers. In BC patients, tumor-derived genetic mutations detected in urinary DNA are expected to be a diagnostic biomarker. This study evaluates the clinical validity of monitoring tumor-specific gene mutations in ctDNA and urine pellet DNA (upDNA) using dPCR as a biomarker for early relapse prediction and determination of treatment efficacy.

Materials and Methods: Thirty-two previously treated and untreated BC patients were enrolled. Tumor DNA was extracted from archived TURBT tissues, and gene mutation analysis by Next Generation Sequencing (NGS) of tumors was performed to select patient-unique somatic mutations for both ctDNA and upDNA monitoring by dPCR. Longitudinal variant allele frequencies (VAFs) of ctDNA and upDNA were monitored by dPCR for up to two years. While dPCR is rapid and has a high sensitivity of less than 0.1% VAF, a mutation-specific primer/probe set is required. Therefore, we established over 1,000 primer/probe sets for frequent mutations in human cancer. This large-scale dPCR primer/probe library allow us to select patient specific mutations for immediate ctDNA monitoring. Clinical recurrence (Crec) based on imaging and urinary cell findings are compared with VAF dynamics of ctDNA and upDNA.

Results: The median observation period was 516 days (30-733), and a total of 230 urine samples were collected. The selection of monitoring mutations based on NGS analysis was performed in 93.8% (30/32) cases, with 2.3 (range: 1-6) mutations monitored per case. The primer/probe sets covered 87.5% (28/32) of cases and 87.5% (42/48) of mutations. The VAFs of the upDNA of cases started showing constantly increasing trends 3-12 months earlier than the diagnosis of Crec in 71.4% (5/7) of the cases. The other two Crec cases, which did not show an elevated upVAF, were both pyuria cases. After local treatment, the upDNA VAFs remained high in the Crec cases. Conventional Crec did not seem to be fully reflected by ctDNA alone.

Conclusion: Frequent VAF monitoring using upDNA by dPCR enables the prediction of local relapse and the evaluation of treatment efficacy in the management of BC patients.

Citation Format: Masakazu Abe, Hayato Hiraki, Takashi Tsuyukubo, Sadahide Ono, Shigekatsu Maekawa, Daichi Tamura, Daiki Ikarashi, Renpei Kato, Tomohiko Matsuura, Mitsugu Kanehira, Ryo Takata, Hiromitsu Fujisawa, Takeshi Iwaya, Masashi Idogawa, Wataru Obara, Satoshi S. Nishizuka. The clinical validity of urinary pellet DNA monitoring of recurrent bladder cancer [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 3326.