PPARG protein expression in advanced urothelial carcinoma and selection for response to FX-909, a first-in-class PPARG-targeting agent.

Abstract

856 Background: FX-909, a PPARG inverse agonist, is being evaluated in a first-in-human, dose escalation and expansion study in patients with advanced urothelial carcinoma (UC; NCT05929235). PPARG drives development of UC and its expression is associated with the luminal lineage, which accounts for ~65% of all advanced UC patients 2 . An IHC prototype assay was developed to detect PPARG expression in formalin-fixed paraffin-embedded UC tissues. Our data show that PPARG protein expression correlated with PPARG RNA expression in a retrospective cohort of archival tissue from high-grade localized UC. Methods: Commercial antibodies, clones C26H12, K.242.9 and E-8, were tested and validated using UC and control normal tissues. The E-8 clone was selected for further development based on demonstration of good specificity, sensitivity, range, and linearity. Specificity of the PPARG staining was demonstrated in cell lysates, where PPARG expression level was confirmed by Western blot (HEK293, BC3C, UMUC9, and RT112). Assay performance was evaluated in a cohort of 25 high-grade localized, stage III-IV UC patient samples. Semi-quantitative analysis [percent (%) tumor score and H-score] was conducted by a pathologist. Gene expression values from RNAseq were normalized by transcripts-per-million (TPM). Molecular classification was performed using non-negative matrix factorization rank 5 following the Robertson method 3 derived from RNAseq. Results: The E-8 clone detected a broad range of PPARG expression, with a predominantly nuclear staining pattern at staining intensities from 0 to +3. Each sample exhibited different staining intensities, highlighting the heterogeneity of PPARG expression. Eighty-eight percent of cases showed PPARG expression in more than 50% of their tumor cells (22 out of 25). H-scores varied from 0 to 270 (Median H-Score = 146), where 64% of the cases showed a moderate PPARG expression with H-scores ranging between 100 and 200. PPARG protein expression quantitated based upon the % tumor area with 2+ or greater staining correlated with RNA expression levels (Spearman correlation coefficient r = 0.66; p-value <0.01). Similarly to previous data 2,5 , 52% of the cases in this cohort were of luminal lineage. The median RNA expression level of PPARG was significantly higher in the luminal subgroup compared to the non-luminal subgroup (6.0 Log2[TPM+1] vs 3.53 Log2[TPM+1], p <0.01). Conclusions: These data show the IHC assay is sensitive and specific for the detection of PPARG expression in advanced UC with utility to identify patients with potential to respond to FX-909, a first-in-class PPARG-targeting agent 4 . 1. Sims R, AACR 2023, Fl; 2. Motley W, et al. EORTC 2022; 3. Robertson AG, et al. Cell 2017; 4. Iyer G, et al. ASCO GU 2024, CA; 5. Kirov S, AACR-NCI-EORTC 2023, MA.