Minimal Residual Disease Detection with Urine-derived DNA Is Prognostic for Recurrence-free Survival in Bacillus Calmette-Guérin-unresponsive Non-muscle-invasive Bladder Cancer Treated with Nadofaragene Firadenovec.

BACKGROUND AND OBJECTIVE: Urinary tumor DNA (utDNA) profiling identifies mutations associated with urothelial carcinoma and can be used to detect minimal residual disease (MRD). We evaluate the utility of utDNA profiling to predict treatment failure in bacillus Calmette-Guérin-unresponsive high-grade (HG) non-muscle-invasive bladder cancer (NMIBC) treated with nadofaragene firadenovec. METHODS: Urine was collected from participants prior to induction (n = 32) and at their 3-mo evaluation (n = 18) in the parallel-arm, phase 2 study (NCT01687244) of nadofaragene firadenovec. The UroAmp MRD assay (Convergent Genomics, South San Francisco, CA, USA) was used to perform utDNA testing. Risk of HG NMIBC recurrence was determined using two algorithm versions, and recurrence-free survival (RFS) was assessed using a Kaplan-Meier analysis. KEY FINDINGS AND LIMITATIONS: TP53, TERT, PIK3CA, ARID1A, PLEKHS1, ELF3, and ERBB2 were the most prevalently mutated genes. With pretreatment urine, the validated MRD algorithm resulted in 12-mo RFS of 56% for negative and 22% for positive patients (p = 0.097). The experimental, enhanced algorithm classified two additional patients as positive, giving RFS of 71% for negative and 20% for positive patients (p = 0.012). With 3-mo urine, both algorithms gave RFS of 100% for negative and 38% for positive patients (p = 0.038). Longitudinal utDNA testing classified patients as negative (7%), complete responders (13%), partial responders (27%), unresponsive (20%), and expanding (33%). CONCLUSIONS AND CLINICAL IMPLICATIONS: Urinary MRD testing after nadofaragene firadenovec induction provided statistically significant prognostication of recurrence among phase 2 trial participants. PATIENT SUMMARY: By analyzing urine-borne tumor DNA, we can help determine which patients with high-grade non-muscle-invasive bladder cancer are at the greatest risk of recurrence when receiving second-line therapy.

Development and Multicenter Case-Control Validation of Urinary Comprehensive Genomic Profiling for Urothelial Carcinoma Diagnosis, Surveillance, and Risk-Prediction.

PURPOSE: Urinary comprehensive genomic profiling (uCGP) uses next-generation sequencing to identify mutations associated with urothelial carcinoma and has the potential to improve patient outcomes by noninvasively diagnosing disease, predicting grade and stage, and estimating recurrence risk. EXPERIMENTAL DESIGN: This is a multicenter case-control study using banked urine specimens collected from patients undergoing initial diagnosis/hematuria workup or urothelial carcinoma surveillance. A total of 581 samples were analyzed by uCGP: 333 for disease classification and grading algorithm development, and 248 for blinded validation. uCGP testing was done using the UroAmp platform, which identifies five classes of mutation: single-nucleotide variants, copy-number variants, small insertion-deletions, copy-neutral loss of heterozygosity, and aneuploidy. UroAmp algorithms predicting urothelial carcinoma tumor presence, grade, and recurrence risk were compared with cytology, cystoscopy, and pathology. RESULTS: uCGP algorithms had a validation sensitivity/specificity of 95%/90% for initial cancer diagnosis in patients with hematuria and demonstrated a negative predictive value (NPV) of 99%. A positive diagnostic likelihood ratio (DLR) of 9.2 and a negative DLR of 0.05 demonstrate the ability to risk-stratify patients presenting with hematuria. In surveillance patients, binary urothelial carcinoma classification demonstrated an NPV of 91%. uCGP recurrence-risk prediction significantly prognosticated future recurrence (hazard ratio, 6.2), whereas clinical risk factors did not. uCGP demonstrated positive predictive value (PPV) comparable with cytology (45% vs. 42%) with much higher sensitivity (79% vs. 25%). Finally, molecular grade predictions had a PPV of 88% and a specificity of 95%. CONCLUSIONS: uCGP enables noninvasive, accurate urothelial carcinoma diagnosis and risk stratification in both hematuria and urothelial carcinoma surveillance patients.

Urinary Comprehensive Genomic Profiling Correlates Urothelial Carcinoma Mutations with Clinical Risk and Efficacy of Intervention.

The clinical standard of care for urothelial carcinoma (UC) relies on invasive procedures with suboptimal performance. To enhance UC treatment, we developed a urinary comprehensive genomic profiling (uCGP) test, UroAmplitude, that measures mutations from tumor DNA present in urine. In this study, we performed a blinded, prospective validation of technical sensitivity and positive predictive value (PPV) using reference standards, and found at 1% allele frequency, mutation detection performs at 97.4% sensitivity and 80.4% PPV. We then prospectively compared the mutation profiles of urine-extracted DNA to those of matched tumor tissue to validate clinical performance. Here, we found tumor single-nucleotide variants were observed in the urine with a median concordance of 91.7% and uCGP revealed distinct patterns of genomic lesions enriched in low- and high-grade disease. Finally, we retrospectively explored longitudinal case studies to quantify residual disease following bladder-sparing treatments, and found uCGP detected residual disease in patients receiving bladder-sparing treatment and predicted recurrence and disease progression. These findings demonstrate the potential of the UroAmplitude platform to reliably identify and track mutations associated with UC at each stage of disease: diagnosis, treatment, and surveillance. Multiple case studies demonstrate utility for patient risk classification to guide both surgical and therapeutic interventions.