Clinical Relevance of Immune Checkpoints on Circulating Tumor Cells in Breast Cancer

Abstract

The role of CD47 and PD-L1 expression on circulating tumor cells (CTCs) remains unclear, and it is currently unknown whether their distribution varies between the blood and tumor tissue in breast cancer (BC). In this study, CD47 and PD-L1 expression was investigated a) on peripheral blood mononuclear cell (PBMC) cytospins from early (n = 100) and metastatic (n = 98) BC patients, by triple immunofluorescence for CD47/PD-L1/Cytokeratins, and b) on matched primary and/or metastatic tumor tissue from CTC-positive patients using immunohistochemistry. CD47+and/orPD-L1+ CTCs were detected in 11%, 16.9%, and 29.6% of early, recurrent, and de novo metastatic patients (p = 0.016). In metastatic disease, CD47highand/orPD-L1high CTCs were associated with disease progression (p = 0.005) and shorter progression-free survival (PFS) (p = 0.010), and independently predicted for an increased risk of relapse (HR: 2.719; p = 0.008) and death (HR: 2.398; p = 0.034). PD-L1 expression rates differed between CTCs and tissue tumor cells and between peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) (positive concordance of 3.8% and 4%, respectively). CD47 expression also differed between CTCs and tumor cells (positive concordance of 11.5%). In conclusion, CTCs expressing CD47 and PD-L1 have independent poor prognostic implications in metastatic BC, indicating a potential role of innate and adaptive immune evasion mechanisms in their metastatic potential. The clinical value of the parallel assessment of the peripheral and local immune response merits further evaluation in BC.

Optimization of the Enrichment of Circulating Tumor Cells for Downstream Phenotypic Analysis in Patients with Non-Small Cell Lung Cancer Treated with Anti-PD-1 Immunotherapy

“…This is potentially related to the fact that spiking experiments may overestimate the performance of methods since cancer cell lines tend to be more homogenous compared to CTCs, which present significant individual heterogeneity and distinct profiles compared to cancer cell lines [ 53 ]. This hypothesis is in line with a recent finding showing a reduction in the cell mass and size of tumor cells undergoing epithelial-to-mesenchymal transition (EMT) [ 54 ], a process commonly identified on CTCs [ 38 , 39 , 40 ].…”

“…Our findings, albeit preliminary, suggest that CTC characterization according to IDO and PD-L1 may promote our understanding of the mechanisms underlying immunotherapy resistance and may provide valuable prognostic information for NSCLC patients treated with anti-PD1 ICIs. Similarly, we recently demonstrated that the parallel assessment of innate (CD47) and adaptive (PD-L1) immune checkpoints on CTCs has significant prognostic and predictive implications in breast cancer [ 39 ]. Overall, the above observations highlight the importance of the phenotypic characterization of single CTCs in the effort to obtain a broader view of the systemic antitumor immune response and suggest that CTC analysis in real time might improve the stratification of patients treated with different immunotherapy strategies.…”

“…Following filtration, membranes were washed with PBS and WFI, air-dried at RT, and stored at −20 °C. The membrane spots ( n = 10), each one corresponding to 1 mL whole blood, were individually cut out and immobilized on 10 glass slides using adhesive ribbon to be further stained and analyzed by microscopy, as previously described by Pailler et al [ 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 ]. Although the entire ISET membrane can be stained and/or immobilized on a single slide, we did not select this procedure since it required larger volumes of antibody solutions compared to the staining of individual spots (a minimum of 2 mL vs 100 μL per spot to a final volume of 1 mL, respectively).…”

“…The triple CK/EpCAM/CD45 staining was developed and optimized using ficoll-enriched H1975 spiked cells, as controls. For this purpose, positive controls (including all three primary and the corresponding secondary antibodies) and negative controls, one for each marker (including the secondary Immunoglobulin G (IgG) isotype antibody only and omitting the corresponding primary antibody) were prepared as previously described [ 38 , 39 ].…”

“…In NSCLC, CTC analysis has mainly focused on the identification of targetable molecular alterations [ 22 , 33 , 34 , 35 ]. However, further characterization of the protein expression profile of single CTCs may provide additional prognostic information and could significantly contribute to the understanding of mechanisms of disease progression and resistance to current therapies [ 36 , 37 , 38 , 39 , 40 ].…”