Prospective evaluation of BCG unresponsive bladder cancer carcinoma in situ identifies genetic mechanisms of immunotherapy resistance and targeted therapy using an ultra-sensitive next generation sequencing minimal residual disease (MRD) assay

BackgroundWhile there are multiple emerging treatments for BCG unresponsive CIS bladder cancer (BCa), the early detection of recurrence and targeting of immunotherapy resistance is a significant unmet need. The genomic profile of BCG unresponsive CIS has not been described. Therefore, developing ultra-sensitive, bespoke, urine-based genomic assays could transform BCa surveillance and identify new therapeutic strategies. We performed a prospective profiling of BCG unresponsive CIS and described the early response to salvage therapy.MethodsPatients with BCG unresponsive CIS were prospectively enrolled before starting salvage therapy. All sequencing was performed using the PredicineBEACON MRD assay, including WES of 20,000 genes and boosted sequencing of 600 cancer genes at baseline using tissue and/or urine. Bespoke personalized MRD panels include 16-50 personalized mutations and 500 hotspot mutations. Mutations were annotated, and allele frequency (AF) was calculated. MRD was + if 2 or more mutations were detected.Results30/31 were MRD + before salvage treatment. While 29/30 MRD+ had a decrease in tumor fraction (TF) at the first evaluation, only 2 converted to MRD- at 3 months. Because all tumors were BCG unresponsive, we evaluated for mutations associated with immunotherapy resistance. The median urinary TMB was 7 (range 0.1 to 40). The most frequent mutations were TERT (82%), TP53 (71%), RB1 (32%), FGFR3 (32%), KMT2D (29%) and ERBB3 (21%). We evaluated for alterations in the PDL1 pathway and identified mutations in 26% of CD274/PDL1 and CNVs of 16% of PDL2. Mutations in the IFN-gamma pathway were found in 26% (10% SNVs and 16% CNVs of JAK1/2). Of the 10 FGFR3 alterations, the VAFs ranged from 0.1 to 60%. ERCC2, associated with response to chemotherapy, was found in 21%.ConclusionsNearly all patients with BCG unresponsive CIS begin salvage therapy MRD + and have a quantifiable decline in TF with treatment. We identify genomic alterations previously reported with immune escape and actionable mutations in DNA damage repair and the FGFR3 pathway, now targetable with intravesical therapy.Legal entity responsible for the studyThe authors.FundingHas not received any funding.DisclosureP. Du: Financial Interests, Project Lead: Predicine. Y. Huang, S. Jia: Financial Interests, Personal, Full or part-time Employment: Predicine. All other authors have declared no conflicts of interest.