Pilot study evaluating duplex sequencing of urinary DNA as a biomarker for recurrence in non-muscle invasive bladder cancer.

Abstract

865 Background: The detection of tumor-associated urinary DNA (uDNA) as a biomarker for non-muscle invasive bladder cancer (NMIBC) has shown promise, but is significantly limited in sensitivity due to replication errors inherent in next-generation sequencing (NGS). Duplex sequencing is an ultrasensitive/accurate sequencing technology which may provide a solution to this problem. We present a pilot study applying duplex sequencing technique to uDNA in NMIBC for use as a biomarker of recurrence. Methods: Urine samples from a total of 30 patients with NMIBC were collected following complete transurethral resection of bladder tumor and prior to intravesical therapy (pre-tx) and following six weeks of intravesical therapy (post-tx). Detection of ten commonly mutated NMIBC genes was carried out using next generation duplex sequencing. Results were correlated with recurrence of tumor. Results: Median age for our cohort was 71 [41, 88] and 80% were male. A total of 16 (53.3%) of the cohort experienced a recurrence within the first year, with a median time to recurrence of 10 months. Tumor-associated uDNA was successfully detected to variant allele fraction (VAF) below 0.001. Presence of any pathogenic variant in the pre-tx urine >0.006 VAF was associated with a 72.7% sensitivity and 78.9% specificity for recurrence within one year. The highest VAF of pathogenic variant in uDNA trended with time-to-relapse. Post-tx uDNA assessment did not correlate with recurrence. Further analysis is limited by small sample size. Conclusions: This pilot study demonstrates potential utility of ultrasensitive duplex sequencing on NMIBC uDNA in the detection of recurrence.