Characterizing PSMA heterogeneity in prostate cancer and identifying clinically actionable tumor associated antigens in PSMA negative cases

Abstract

Prostate-specific membrane antigen (PSMA)-targeted theranostics have profoundly reshaped prostate cancer (PCa) management. However, patterns of PSMA expression heterogeneity and the identification of alternative targets for PSMA-negative cases remain insufficiently understood. To address this gap, this study investigates PSMA expression heterogeneity in 127 primary lesions (PL) from hormone-sensitive PCa (HSPC) cohort and 76 bone metastatic lesions (BML) from metastatic castration-resistant PCa (mCRPC) cohort through immunohistochemical analysis, including 27 matched PL-BML samples. Notable inter-patient variability in PSMA expression was observed, with H-scores ranging from 1.42 to 197.16 overall. Among matched samples, six cases exhibited HSPC-/mCRPC + and seven cases showed HSPC+/mCRPC- PSMA expression patterns. Intra-tumoral heterogeneity was significant, with 67.7% of PL and 30.1% of BML showing high variability in PSMA staining intensity. Membranous PSMA expression and the normalized membrane ratio were significantly higher in PL compared to BML (both p < 0.001). PSMA-negative cases (membranous staining ≤ 20) were found in 15.0% of HSPC and 36.8% of mCRPC cases. PSMA expression levels differed significantly among androgen receptor (AR) expression groups (p < 0.001), with lower PSMA expression associated with lower AR expression levels. Alternative tumor-associated antigens (TAAs) were identified in PSMA-negative cases: B7H3 and TROP2 expression were prominent in HSPC, and STEAP1 and B7H3 expression dominated in mCRPC. These findings highlight the dynamic nature of PSMA expression and support the rationale for exploring alternative theranostic strategies in PSMA-negative PCa.

Keywords: Heterogeneity; Prostate cancer; Prostate-specific membrane antigen; Tumor-associated antigen.

Fig. 1

Membranous PSMA expression of PL is heterogeneous in clinical samples obtained from patients with HSPC at initial diagnosis of PCa. (A) Scatter plot of PSMA MHscore and age, showing no correlation was observed. (B) Scatter plot of PSMA MHscore and log(tPSA + 1). Due to the wide distribution of tPSA, a mathematical transformation was applied. However, no significant correlation was observed before or after the transformation. (C) Box plot of PSMA MHscore distribution across ISUP groups. Significant differences across subgroups are marked in the plot. (D) Membranous PSMA expression at the initial HSPC status. Expression of membranous PSMA quantified by H-score and presented in order of increasing MHscore. Degree of PSMA heterogeneity was measured by Shannon’s diversity index (SDI) and depicted as heat map ranging from low heterogeneity (light green) to high heterogeneity (dark green). PSMA-negative cases are indicated with a dashed line in the plot with the definition as MHscore ≤ 20. (E) Head-to-head comparison bar plots showing the expression profiles of other well-established TAAs in PSMA-negative HSPC cases. (F) Box plot showing the comparison of MHscore for well-established TAAs in PSMA-negative HSPC cases. Significant differences across subgroups are marked in the plot, *represents p < 0.05, **represents p < 0.01, ***represents p < 0.001.

Fig. 2

Membranous PSMA expression of BML is heterogeneous in clinical samples obtained from patients with mCRPC. (A) Membranous PSMA expression in mCRPC status. Expression of membranous PSMA quantified by H-score and presented in order of increasing MHscore. Degree of heterogeneity in mPSMA was measured by SDI and depicted as heat map ranging from low heterogeneity (light green) to high heterogeneity (dark green). PSMA-negative cases are indicated with a dashed line in the plot with the definition as MHscore ≤ 20. (B) Box plot of PSMA MHscore distribution across bone metastatic sites, with no significant difference was observed. (C) Head-to-head comparison bar plots showing the expression profiles of other well-established TAAs in PSMA-negative mCRPC cases. (D) Box plot showing the comparison of MHscore for well-established TAAs in PSMA-negative mCRPC cases. Significant differences across subgroups are marked in the plot, *represents p < 0.05, **represents p < 0.01, ***represents p < 0.001. (E) Heatmap of NMR classification across TAAs in PSMA-negative mCRPC cases. NMR > 50% indicates a membranous dominant expression pattern, while NMR < 50% indicates a cytoplasmic dominant expression pattern.

Fig. 3

Comparison of PSMA expression profile between HSPC-PL and mCRPC-BML status. (A) Box plot showing the comparison of PSMA MHscore between PL and BML, PSMA expression level in BML was significant lower than PL. (B) Violin plot showing the comparison of PSMA NMR between PL and BML, PSMA NMR in BML was also significant lower than PL. (C) Waterfall plot showing the alteration of membranous PSMA expression from PL to BML, along with the corresponding treatment history heatmap (second generation androgen receptor antagonist (SGARA), chemotherapy and radiotherapy), no significant correlation was observed between PSMA alteration and any of the various treatment history. (D, E,F, G) Different patterns of intra-patient PSMA expression heterogeneity in matched samples. (D) Heterogeneously low expression in different regions of the same HSPC sample (①③: within same regions, even adjacent tumor tissues can exhibit heterogeneous PSMA expression level, the tumor tissue in the red dashed area shows absent PSMA expression;②completely absent PSMA expression tumor region), with high and uniform PSMA expression in matched mCRPC. (E) High PSMA expression in HSPC, low expression in matched mCRPC. (F) Heterogeneously low expression in both HSPC and matched mCRPC. (G) High expression in primary HSPC, heterogeneously low expression in both matched synchronously mHSPC and metachronously mCRPC. All the black scale bars in the IHC image represent 100 μm. Brown scale bars in the IHC image represent 500 μm.