Paired urine and plasma ctDNA profiling to enable longitudinal molecular monitoring during HER2 ADC bladder preserving therapy in muscle-invasive bladder cancer (HOPE03).

e16608 Background: Although trimodal therapy is the standard organ-preserving approach for muscle-invasive bladder cancer (MIBC), outcomes remain suboptimal, underscoring the need for noninvasive biomarkers to guide real-time decisions. We evaluated a bladder-sparing regimen of HER2 ADC plus PD-1 antibody and radiotherapy in localized HER2-positive MIBC, using NGS-based liquid biopsy to longitudinally track molecular dynamics in paired urine and plasma. Methods: HOPE03 is a multicenter, open-label, single-arm phase Ib/II trial (ChiECRCT20210564) enrolling patients with pathologically and radiographically confirmed HER2-positive MIBC. Patients received disitamab vedotin (RC48) in combination with toripalimab and radiotherapy. In this exploratory biomarker analysis, paired urine and plasma samples were collected serially from baseline through neoadjuvant treatment, radiotherapy, and follow-up to evaluate longitudinal molecular response. Tumor fraction and somatic alterations in circulating tumor DNA (ctDNA) and urinary tumor DNA (utDNA) were quantified using the PredicineCARE targeted NGS assay. Results: In the study cohort, 21 patients were eligible for NGS testing, of whom 18 had paired baseline urine and blood samples available. At baseline, urine demonstrated a substantially higher mutational burden than blood (246 vs. 41 mutations), with partial concordance between compartments (26 shared mutations). The most frequently altered genes in urine were TP53 (50%), TERT (44%), ARID1A (33%), KDM6A (33%), and RB1 (33%). In plasma, the most prevalent alterations were TP53 (33%), ATM (11%), BRAF (11%), BRCA2 (11%), and PIK3CA (11%). Baseline genomic alterations in both urine and blood were associated with clinicopathologic features. Patients harboring RB1 alterations in urine had significantly larger baseline tumor size (p = 0.012), and baseline tumor size was also significantly larger in patients with plasma ATM (p < 0.001), RB1 (p < 0.001), or TP53 (p = 0.009) alterations. Plasma TP53 alterations were additionally associated with higher Ki-67 levels (p = 0.009). Older age correlated with higher tumor fraction in both urine (p = 0.011) and plasma (p = 0.002). Notably, plasma PIK3CA alterations were associated with inferior progression-free survival (median PFS 9.3 months vs. not reached; p = 0.011). Conclusions: Serial NGS profiling of paired urine and plasma in HOPE03 demonstrated the feasibility of noninvasive molecular monitoring during RC48 plus toripalimab and radiotherapy for localized HER2-positive MIBC. These findings support integrated urine–plasma liquid biopsy as a tool for longitudinal response assessment and risk stratification in bladder-preserving therapy, warranting validation in larger prospective cohorts. Clinical trial information: ChiECRCT20210564.