544 Background: Circulating ctDNA is associated with disease progression, worse overall survival, and recurrence in pts with bladder cancer. This study correlated targeted exome ctDNA sequencing with pathologic response to NAC and metastatic recurrence in pts with N+ MIBC undergoing radical cystectomy (RC). Methods: We prospectively identified pts with cT2-3N1-2M0 bladder cancer who underwent NAC prior to RC. Node positivity was determined radiographically and/or by node biopsy. Plasma samples were collected pre-NAC, mid-treatment, after NAC completion, and 3 months after RC. Samples were analyzed using MSK-ACCESS, an ultrasensitive ctDNA platform designed to identify somatic mutations in 129 cancer associated genes. Primary bladder tumors were sequenced using targeted exome sequencing. Results: Samples (31) from 9 pts (6 men) were analyzed. Median age was 63 years (IQR 58-69). NAC regimens included gemcitabine and cisplatin alone (78%) or with paclitaxel (22%). Seven (78%) pts had >1 detectable mutation pre-NAC. The most altered genes detected in both tissue and ctDNA included: TERT (88% vs 28%), TP53 (76% vs 40%), ARID1A (43% vs 17%), RB1 (43% vs 15%), KDM6A (43% vs 15%), and ATM (17% vs 17%). Four (45%) pts had complete response (ypT0N0), 1 (11%) had a partial response (ypT1N0), and 4 (33.3%) were non-responders (ypT2N0-3). All non-responders had disease recurrence after RC (median 3 months, range 2-11 months). Of 7 pts with on-treatment MSK-ACCESS, 2 (29%) had detectable ctDNA (1 ypT2N0, 1 ypT0N0). The pt with a PR had detectable ctDNA post-NAC with 1 mutation identified that was not detected at 3 months. All pts with >ypT2N0 had detectable ctDNA post-NAC. Pts with recurrence/non-responders had a significantly higher mutation count 3 months after RC compared to responders, with a median of 7 vs. 0 mutations, respectively. Two pts with CR had detectable ctDNA at 3 months post-RC but have not recurred (Table). Conclusions: Clearance of ctDNA post-NAC correlated with pathologic complete response. Approximately 70% of pts had ctDNA clearance on NAC. All pts with residual disease at RC had detectable ctDNA post-NAC. [Table: see text]