Expression of immune checkpoints on circulating tumor cells in men with metastatic prostate cancer

Abstract

Background: A subset of men with metastatic prostate cancer (mPC) responds to immune checkpoint inhibitors, and there is an unmet need to predict those most likely to benefit. We characterized circulating tumor cells (CTCs) for expression of immune checkpoint ligands in men with mPC as a non-invasive biomarker of immune evasion and immunotherapy benefit.

Methods: Three cohorts of patients were enrolled: 1) men with mCRPC starting abiraterone acetate/prednisone or enzalutamide (pre-ARSI), 2) men with mCRPC who were progressing on enzalutamide or abiraterone acetate/prednisone (post-ARSI), and 3) men with newly diagnosed metastatic hormone sensitive prostate cancer (mHSPC) starting androgen deprivation therapy. CTCs were captured using the CellSearch® system and stained for PD-L1, PD-L2, B7-H3, and CTLA-4 at baseline, on treatment, and disease progression. Summary statistics on mean CTCs per cohort, as well as rates of ligand positivity were used to analyze CTCs by cohort and by timepoint.

Results: Men in all cohorts and timepoints had prevalent CTC B7-H3 expression (> 80%). We found evidence for CTC PD-L1 expression across disease states, in which > 1 positive CTC or > 50% of CTCs were positive for PD-L1 in 40 and 30% of men with mHSPC, respectively, 60 and 20% of men with mCRPC pre-ARSI, and 70 and 30% of men with mCRPC post-ARSI. CTC PD-L2 expression was present in 20-40% of men in each disease state, while CTC CTLA-4 expression was rare, present in 20% of men with mCRPC pre-ARSI and 10% of men with mCRPC post-ARSI or with mHSPC. CTC immune checkpoint expression was heterogeneous within/between men and across disease states.

Conclusions: We have identified that CTCs from men with mPC heterogeneously express immune checkpoints B7-H3, PD-L1, PD-L2, and CTLA-4, and the detection of these immune checkpoints may enable monitoring on immunotherapy.

Keywords: CTLA-4; Circulating tumor cells; Metastatic prostate cancer; PD-L1; PD-L2.

Fig. 1

Healthy volunteer spiking experiments for (A) PD-L1, (B) PD-L2, (C) B7-H3, and (D) CTLA-4 checkpoint ligands on CTCs. Cell lines known to be positive or negative for expression of each immune checkpoint biomarker were spiked into healthy volunteer whole blood. Control samples were spiked with approximately 500 cells (variation due to random error). Each panel shows unspiked, negative and positive control cells captured from these spiking experiments, with total # of CTCs captured below. Panels of CTCs depicted below are the raw CTC output from Cellearch – from left to right, merged image of captured cell, cytokeratin (CK) stain, the DAPI nuclear stain, CD45 (leukocyte marker, negative in tumor cells), and the checkpoint ligand of interest

Fig. 2

CONSORT diagram of men with metastatic prostate cancer with circulating tumor cells examined in this study. Cohort A: men with mCRPC starting abiraterone acetate/prednisone or enzalutamide (pre-ARSI), Cohort B: men with mCRPC who were progressing on enzalutamide or abiraterone acetate/prednisone (post-ARSI), and Cohort C: men with newly diagnosed metastatic hormone sensitive prostate cancer (mHSPC) starting androgen deprivation therapy (ADT) or ADT/docetaxel chemotherapy

Fig. 3

Representative patient CTCs stained for cytokeratin (CK), 4′,6-diamidino phenylindole (DAPI), CD45 (marker of leukocytes), and the immune checkpoint ligand of interest ([A] PD-L1, [B] PD-L2, [C] B7-H3, or [D] CLTA-4). CTCs are defined as being CK-positive, DAPI-positive, and CD45-negative. Orange boxes are indicators from the CellTracks Analyzer II that a cell was identified as marker-positive

Fig. 4

Expression of immune checkpoint biomarkers over time in each cohort. (A) PD-L1, (B) PD-L2, (C) B7-H3, (D) CTLA-4. Histograms made to depict percentage of total CTCs from each patient. Percentage of patients in each cohort with at least one marker-positive CTC at each timepoint (E). Percentage of patients in each cohort with at least 50% marker-positive CTCs at each timepoint (F). ARSI: Androgen receptor signaling inhibitors