Exploring PSMA heterogeneity and alternative targets expression in PSMA-negative prostate cancer.

5075 Background: This study aimed to systematically characterize the heterogeneity of PSMA expression in hormone-sensitive prostate cancer (HSPC) and metastatic castration-resistant prostate cancer (mCRPC), and to explore the expression profiles of alternative well-established tumor-associated antigens (TAAs) in PSMA-negative cases. Methods: Formalin-fixed paraffin-embedded (FFPE) real-world clinical samples were retrospectively collected from prostate biopsies and bone metastasis surgeries at Peking University First Hospital from 2013 to 2023. Standard immunohistochemistry (IHC) was performed to evaluate PSMA expression, quantified using membranous H-score (MHscore) and cytoplasmic H-score (CHscore).Intra-patient PSMA expression heterogeneity was assessed by Shannon diversity index (SDI) . The normalized membrane ratio (NMR) was defined as: MHscore / (MHscore + CHscore). PSMA-negative PCa was defined as PSMA MHscore ≤ 20. PSMA-negative samples were further assessed the expression of alternative TAAs: HER2, NECTIN4, TROP2, TF, B7H3 and STEAP1. Results: A total of 127 HSPC and 76 mCRPC cases were identified, including 27 pairs of matched samples. High PSMA expression heterogeneity (SDI >1) was observed in 86 (67.7%) HSPC and 23 (30.1%) mPCa, while moderate heterogeneity (0.5<SDI<1) was found in 20 (15.7%) HSPC and 25 (32.9%) mPCa. PSMA-negative cases were identified in 15.0% of HSPC and 36.8% of mCRPC. PSMA MHscore in HSPC was significantly higher than in mPCa (p < 0.001). The expression profiles of alternative TAAs in PSMA-negative cases are shown in Table 1. Additionally, the NMR of PSMA in HSPC was significantly higher than in mPCa (p < 0.001). STEAP1 and B7H3 exhibited consistently high NMR in PSMA-negative cases. No significant correlations were observed between PSMA alteration and novel anti-androgen therapy, chemotherapy, or radiation history in matched samples. Conclusions: PSMA expression exhibit notable inter- and intra-patient heterogeneity in both HSPC and mCRPC. B7H3 and TROP2 may have relatively more advantageous expression levels in PSMA-negative HSPC, while B7H3 and STEAP1 may potentially show better complementarity in PSMA-negative mCRPC. The expression profiles of alternative TAAs in PSMA-negative prostate cancer. TAA PSMA-negative HSPC (n=19) MHscoremedian(Q1-Q3) PSMA-negative mCRPC (n=28) MHscoremedian(Q1-Q3) p value HER2 TROP2 NECTIN4 TF B7H3 STEAP1 HER2 3.2(2.8-3.6) 29.3(11.6-70.3) - NS # ### NS ## TROP2 84.8(51.2-137.1) 9.9(3.5-56.1) *** - NS # ## ### NECTIN4 7.1(6.2-12.9) 3.7(2.8-26.8) *** *** - NS ## ### TF 12.5(9.0-72.0) 3.2(2.6-11.0) *** NS * - ### ### B7H3 110.0(98.9-133.0) 63.5(40.4-96.0) *** NS *** *** - NS STEAP1 25.7(9.4-103.1) 66.6(23.4-104.0) *** NS * NS ** - *for HSPC #for mCRPC. */# p<0.05, **/## p<0.01,***/### p<0.001; NS: not significant.