464 Background: Genomic alterations in FGFR3, PIK3CA, and CDKN2A are common actionable targets in urothelial cancer (UC). We aimed to determine the efficacy of alpelisib, a PIK3CA inhibitor, and abemaciclib, a CDK4/6 inhibitor in bladder cancer patient-derived organoid using single-cell targeted DNA sequencing. Methods: We established a patient-derived UC organoid (PDO) harboring the FGFR3 mutation (p.Y375C), the PIK3CA (p. E452K) mutation, and CDKN2A deletion, which we characterized using whole-genome sequencing. We generated dose-response curves of alpelisib, abemaciclib, and erdafitinib (an FGFR3 inhibitor) in PDO cells to determine the IC50 concentrations. The scDNA-seq (Tapsteri) platform was used to measure changes in the variant allele frequencies (VAF) and clonal fractions post-treatment. The Chi-square test for trend was used to test for linear trend across ordered categories. Results: scDNA-seq was performed after treating PDO cells with 3uM erdafitinib or DMSO. A total of 7000 single cells were obtained (4179 cells treated with erdafitinib vs. 2821 cells treated with DMSO). After removing variants mutated in <50% of cells, we identified 94 clonal variants. As expected, cells harboring FGFR3 Y375C have significantly decreased post erdafitinib treatment compared to DMSO-treated cells (66.05% vs. 82.36%, p<0.0001). We identified mutations in two genes ( RAB31 and SMAD4) that were associated with clonal expansion following FGFR3 inhibition (12% vs. 53% and 13% vs. 26%, respectively). We treated PDO cells with 3uM abemaciclib. We identified three genes harboring SNVs ( RB1, GNAQ, and SMAD4). The SNVs harboring cells were significantly decreased after abemaciclib compared to DMSO (p<0.0001). Then, we treated the cells with 1uM alpelisib for 72 hours alone or combined with abemaciclib (0.1uM). We identified that 100% of cells harbored the PIK3CA mutation E452K at pre-treatment, which limited our ability to detect significant changes in VAF post-treatment. Instead, we analyzed the effect on the FGFR3 Y375C clone. Using trend analysis, there was a significant reduction of FGFR3-mutant cells observed across the three conditions, abemaciclib + alpelisib vs. alpelisib alone vs. DMSO (74% vs. 85% vs. 92%, trend test p<0.0001), suggesting in vitro efficacy of alpelisib alone and significant synergism with the addition of abemaciclib. Conclusions: This study established the feasibility of using scDNA-seq as a promising tool to study the clonal evolution patterns in patient-derived UC organoids. Combined pharmacologic inhibition of CDK4/6 and PIK3CA showed more in vitro sensitivity than PIK3CA inhibition alone.
Minor psychiatric disorders and their associations in family caregivers of people with mental disorders