Abstract P5-05-07: Assessment of immune checkpoint expression on the peripheral blood mononuclear cells (PBMCs) of patients with breast cancer (BC)
Authors: Papadaki Maria A. , Monastirioti Alexia , Apostolopoulou Christina Α , Agelaki Sofia , Mavroudis Dimitrios 
Published: March 2023

Abstract
Background: Growing body of evidence highlights the role of the peripheral anti-tumor immune response in patients with solid tumors. Peripheral blood mononuclear cells (PBMCs) comprise all the key circulating immune cell subsets, and their analysis may inform on the peripheral anti-tumor immune response status in real-time. Programmed death-ligand 1 (PD-L1), toll-like receptor 4 (TLR4) and signal transducer and activator of transcription 3 (STAT3) hold a key role in the cancer-associated inflammation and tumor immune evasion. We herein aimed to investigate the distribution of these molecules on PBMCs and their prognostic value among patients with breast cancer (BC). Methods: Peripheral blood (PB) was obtained from patients with early (n=99) and metastatic (n=99) BC, prior to the initiation of adjuvant and first-line treatment, respectively. PBMCs were isolated through ficoll density gradient centrifugation and PBMC cytospins were immunofluorescently stained using PD-L1, TLR4 and phosphorylated STAT3 (pSTAT3) antibodies. MDA.MB.231 BC cells served as controls to define the positivity of PBMCs for the respective markers via fluorescence microscopy. Results: PD-L1, TLR4 and pSTAT3 expression was identified on PBMCs of 27.8%, 27.1%, and 83.9% of all patients, respectively. The mean (± standard error of mean, SEM) percentage of positive PBMCs per patient was 13.8% ±1.8%, 10.5% ±1.6% and 37.1% ±1.9, respectively. A positive correlation was shown between the PD-L1pos, TLR4pos and pSTAT3pos PBMC proportions (PD-L1*TLR4; p=0.000, PD-L1*pSTAT3; p=0.001, TLR4*pSTAT3; p=0.002, Spearman’s rho analysis). Patients with metastatic disease displayed increased TLR4pos PBMC percentages as compared to early disease (mean: 15.8% versus 5.2%; p=0.008, Mann Whitney U test). In the early BC setting, the detection of TLR4pos PBMCs was associated with reduced disease-free survival (DFS; median: not reached; p=0.020), while PD-L1pos PBMCs were correlated to shorter overall survival (OS; median: not reached; p=0.009). Early BC patients with PD-L1pos/TLR4pos PBMCs showed significantly reduced survival times (median DFS: not reached; p=0.016; median OS: not reached; p=0.004, Kaplan Meier analysis). Conclusions: PD-L1, TLR4 and pSTAT3 molecules are frequently expressed on PBMCs of patients with BC and provide significant prognostic information for early-stage BC patients. The role of the immune-phenotyping of PBMCs as a source for biomarker discovery merits further investigation in BC.
Citation Format: Maria A. Papadaki, Alexia Monastirioti, Christina Α Apostolopoulou, Sofia Agelaki, Dimitrios Mavroudis. Assessment of immune checkpoint expression on the peripheral blood mononuclear cells (PBMCs) of patients with breast cancer (BC) [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-05-07.