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.

The current study aimed at the optimization of circulating tumor cell (CTC) enrichment for downstream protein expression analyses in non-small cell lung cancer (NSCLC) to serve as a tool for the investigation of immune checkpoints in real time. Different enrichment approaches—ficoll density, erythrolysis, their combination with magnetic separation, ISET, and Parsortix—were compared in spiking experiments using the A549, H1975, and SKMES-1 NSCLC cell lines. The most efficient methods were tested in patients (n = 15) receiving immunotherapy targeting programmed cell death-1 (PD-1). Samples were immunofluorescently stained for a) cytokeratins (CK)/epithelial cell adhesion molecule (EpCAM)/leukocyte common antigen (CD45), and b) CK/programmed cell death ligand-1 (PD-L1)/ indoleamine-2,3-dioxygenase (IDO). Ficoll, ISET, and Parsortix presented the highest yields and compatibility with phenotypic analysis; however, at the patient level, they provided discordant CTC positivity (13%, 33%, and 60% of patients, respectively) and enriched for distinct CTC populations. IDO and PD-L1 were expressed in 44% and 33% and co-expressed in 19% of CTCs. CTC detection was associated with progressive disease (PD) (p = 0.006), reduced progression-free survival PFS (p = 0.007), and increased risk of relapse (hazard ratio; HR: 10.733; p = 0.026). IDO-positive CTCs were associated with shorter PFS (p = 0.039) and overall survival OS (p = 0.021) and increased risk of death (HR: 5.462; p = 0.039). The current study indicates that CTC analysis according to distinct immune checkpoints is feasible and may provide valuable biomarkers to monitor NSCLC patients treated with anti-PD-1 agents.

e14045 Background: CTCs expressing innate (CD47) and adaptive (PD-L1) immune checkpoints may have enhanced potential to escape immunosurveillance. In the current study, we evaluated the incidence and clinical relevance of CTCs expressing CD47 and/or PD-L1 in patients with mBC. Methods: Blood was obtained from 98 mBC patients before the initiation of first-line therapy. Triple immunofluorescence staining for cytokeratins (CK), CD47 and PD-L1 was performed on peripheral blood mononuclear cells (PBMC) cytospins and 1*106 PBMCs per patient were analyzed using the Ariol microscopy system. CD47 and PD-L1 expression levels on CK+ CTCs were normalized using MDA.MB.231 breast cancer cells as controls. Results: CK+ CTCs were detected in 22 out of 98 patients (22.4%) (total CTC No: 43; range: 1-12). High CD47 and PD-L1 expression was identified in 41.9% and 11.6% of CTCs, respectively, whereas in 9.1% of CTCs, high expression was observed for both markers. CTCs with high expression of at least one marker ( CD47high and/or PD-L1high) were identified in 11.2% of patients, albeit with a differential distribution among patients with triple-negative, hormone receptor-positive and HER2-positive primary tumors (27.3%, 12.5% and 0%, respectively, p = 0.040; Chi-square test). CD47high and/or PD-L1high CTCs were associated with disease progression (27.8% vs 5.6%, p = 0.005; Chi-square test) and shorter PFS [median: 5.8 (4.1-7.5) vs 13.3 (11.4-15.2) months, p = 0.010; Kaplan-Meier], whereas the detection of PD-L1high CTCs only was correlated with reduced OS [median: 23.8 (5.8-41.8) vs 35.7 (29.9-41.4) months, p = 0.043]. Multivariate Cox-regression analysis revealed that CD47high and /or PD-L1high CTCs predicted for increased risk of relapse [HR: 2.7 (95% CI: 1.3-5.7); p = 0.009], whereas PD-L1high CTCs were associated with high risk of death [HR: 4.8 (95% CI: 1.4-16.6); p = 0.011]. Conclusions: The immune checkpoints CD47 and PD-L1 are co-expressed in a subset of CTCs in mBC patients. The detection of high CD47 and/or PD-L1 expression on CTCs is associated with triple-negative tumors, disease progression and poor patient outcome and could serve for the refinement of prognosis in mBC. Patients bearing this CTC population may benefit from anti-CD47 and anti-PD-L1 immunotherapy strategies.