Assessment of TROP2, CEACAM5 and DLL3 in metastatic prostate cancer: Expression landscape and molecular correlates

Abstract

Therapeutic approaches targeting proteins on the surface of cancer cells have emerged as an important strategy for precision oncology. To capitalize on the potential impact of drugs targeting surface proteins, detailed knowledge about the expression patterns of the target proteins in tumor tissues is required. In castration-resistant prostate cancer (CRPC), agents targeting prostate-specific membrane antigen (PSMA) have demonstrated clinical activity. However, PSMA expression is lost in a significant number of CRPC tumors. The identification of additional cell surface targets is necessary to develop new therapeutic approaches. Here, we performed a comprehensive analysis of the expression heterogeneity and co-expression patterns of trophoblast cell-surface antigen 2 (TROP2), delta-like ligand 3 (DLL3), and carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in CRPC samples from a rapid autopsy cohort. We show that DLL3 and CEACAM5 exhibit the highest expression in neuroendocrine prostate cancer (NEPC), while TROP2 is expressed across different CRPC molecular subtypes, except for NEPC. We further demonstrated that AR alterations were associated with higher expression of PSMA and TROP2. Conversely, PSMA and TROP2 expression was lower in RB1-altered tumors. In addition to genomic alterations, we show a tight correlation between epigenetic states, particularly histone H3 lysine 27 methylation (H3K27me3) at the transcriptional start site and gene body of TACSTD2 (encoding TROP2), DLL3, and CEACAM5, and their respective protein expression in CRPC patient-derived xenografts. Collectively, these findings provide insights into patterns and determinants of expression of TROP2, DLL3, and CEACAM5 with implications for the clinical development of cell surface targeting agents in CRPC.

Fig. 1. Distribution and co-expression patterns of DLL3, CEACAM5, PSMA, and TROP2 expressions across different molecular subtypes of mCRPC.

a Representative images of cell surface antigen expression (determined by IHC) across different molecular subtypes (AR + /NE- [green], AR-/NE+ [yellow], AR + /NE+ [red], and AR-/NE- [blue]). Molecular subtypes were defined by expression of AR signaling markers (AR, NKX3.1) and NE markers (SYP, INSM1) as described previously. Box plots show the distribution of (b) DLL3, (c). CEACAM5, and d. TROP2 expressions based on H-score in the UW-TAN cohort (N = 753). Box and dot colors indicate molecular phenotypes as above. e Top, micrographs of PSMA and TROP2 in AR + /NE- tumors. Bottom, donut chart shows the distribution of PSMA and TROP2 reactivity. f Top, micrographs of DLL3 and CEACAM5 in AR-/NE+ tumors. Bottom, donut chart shows the distribution of DLL3 and CEACAM5 reactivity. (See Supplementary Table 2 for all co-expression profiles). Scale bars denote 50 μm.

Fig. 2. Anatomic site distribution and inter- and intra-tumoral heterogeneity of DLL3, TROP2 and CEACAM5 expression in mCRPC.

a Distribution of DLL3, TROP2, and CEACAM5 protein expression across different organ sites based on IHC H-scores. Dot colors indicate molecular phenotypes. Each dot represents a tumor sample; the color codes indicate the molecular subtype (AR + /NE- [green], AR-/NE+ [yellow], AR + /NE+ [red], and AR-/NE- [blue]). b Inter- and intra-tumoral heterogeneity of TROP2, PSMA, CEACAM5 and DLL3 expression. Mean (95% confidence interval) hypergeometric expression heterogeneity indices across different metastatic sites in a given patient (inter-tumoral heterogeneity, red) and within a metastatic site (intra-tumoral heterogeneity, gray). Dot and box plots showing the distribution of (c). DLL3, (d). TROP2, and (e). CEACAM5 protein expression IHC H-scores in 52 cases from the UW-TAN cohort. Each dot represents a tumor sample; the color codes indicate the molecular subtype (AR + /NE- [green], AR-/NE+ [yellow], AR + /NE+ [red], and AR-/NE- [blue]). Gray shadings show interquartile ranges. Percentages show the frequencies of cell surface antigens in cases with uniformly low/negative expression (all sites H-score < 20), heterogeneous expression (both H-score < 20 and H-score ≥ 20) and uniformly high expression (all sites H-scores ≥ 20).

Fig. 3. Genetic and epigenetic determinants of TROP2, PSMA, DLL3 and CEACAM5 expression in CRPC.

a Mosaic plots show the frequencies of TROP2, PSMA, DLL3, and CEACAM5 protein expression determined by IHC as a function of the genomic status of AR, BRCA2, CHD1, PTEN, RB1, and TP53 (WT, not altered; ALT, altered) in 44 cases of the UW-TAN cohort. b Mosaic plots show the frequencies of TACSTD2, FOLH1, DLL3, and CEACAM5 mRNA expression determined by RNA-seq as a function of the genomic status of AR, BRCA2, CHD1, PTEN, RB1, SPOP, and TP53 (WT, not altered; ALT, altered) in 99 cases of the SU2C-WCDT. c Representative H3K27ac (gray) and H3K27me3 (blue) ChIP-seq tracks from AR + /NE- (LuCaP 77 and LuCaP 78) and AR-/NE+ (LuCaP 93 and LuCaP 145.1) PDX lines. Note the inverse differential enrichment pattern of H3K27ac and H3K27me3 (yellow box) in the upstream regulatory regions of TACSTD2, DLL3, and CEACAM5.