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Clinical Significance of PD-L1⁺ Exosomes in Plasma of Head and Neck Cancer Patients.

ABSTRACT: Purpose: The microenvironment of head and neck squamous cell carcinomas (HNSCC) is highly immunosuppressive. HNSCCs expressing elevated levels of PD-L1 have especially poor outcome. Exosomes that carry PD-L1 and suppress T-cell functions have been isolated from plasma of patients with HNSCC. The potential contributions of PD-L1⁺ exosomes to immune suppression and disease activity are evaluated.Experimental Design: Exosomes isolated from plasma of 40 HNSCC patients by size exclusion chromatography were captured on beads using anti-CD63 Abs, stained for PD-1 and PD-L1 and analyzed by flow cytometry. The percentages and mean fluorescence intensities (MFI) of PD-L1⁺ and PD-1⁺ exosome/bead complexes were correlated with the patients' clinicopathologic data. PD-L1^high or PD-L1^low exosomes were incubated with activated CD69⁺ human CD8⁺ T cells ± PD-1 inhibitor. Changes in CD69 expression levels on T cells were measured. Patients' plasma was tested for soluble PD-L1 (sPD-L1) by ELISA.Results: Levels of PD-L1 carried by exosomes correlated with patients' disease activity, the UICC stage and the lymph node status (P = 0.0008-0.013). In contrast, plasma levels of sPD-L1 or exosome PD-1 levels did not correlate with any clinicopathologic parameters. CD69 expression levels were inhibited (P < 0.03) by coincubation with PD-L1^high but not by PD-L1^low exosomes. Blocking of PD-L1⁺ exosome signaling to PD-1⁺ T cells attenuated immune suppression.Conclusions: PD-L1 levels on exosomes, but not levels of sPD-L1, associated with disease progression in HNSCC patients. Circulating PD-L1⁺ exosomes emerge as useful metrics of disease and immune activity in HNSCC patients.

Significance

SUBMITTER: Theodoraki MN

PROVIDER: S-EPMC6126905 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Clinical Significance of PD-L1+ Exosomes in Plasma of Head and Neck Cancer Patients.

Theodoraki Marie-Nicole MN Yerneni Saigopalakrishna S SS Hoffmann Thomas K TK Gooding William E WE Whiteside Theresa L TL

Clinical cancer research : an official journal of the American Association for Cancer Research 20171212 4

Purpose: The microenvironment of head and neck squamous cell carcinomas (HNSCC) is highly immunosuppressive. HNSCCs expressing elevated levels of PD-L1 have especially poor outcome. Exosomes that carry PD-L1 and suppress T-cell functions have been isolated from plasma of patients with HNSCC. The potential contributions of PD-L1+ exosomes to immune suppression and disease activity are evaluated.Experimental Design: Exosomes isolated from plasma of 40 HNSCC patients by siz ...[more]

PMID: 29233903

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Project description:BackgroundExosomes are 50-150 nm endocytic vesicles secreted by almost all type of cells that carry bioactive molecules from host. These small vesicles are considered to be novel cross-talk circuits established by tumor cells and tumor microenvironment. Previous studies have shown certain biological influence of exosomal programmed cell-death ligand 1 (Exo-PD-L1) on immune suppression and dysfunction. The aim of the current study was to investigate the impact of Exo-PD-L1 and soluble PD-L1 (sPD-L1) on non-small cell lung cancer (NSCLC) and explore the concordance between Exo-PD-L1 and PD-L1 expression in matched tumor tissues in NSCLC patients.Methods85 consecutive patients from April 2017 to December 2017 at General Hospital of Eastern Command Theatre who were primarily diagnosed with NSCLC and 27 healthy individuals were enrolled in this study. Two milliliters of whole blood samples were collected from each participant and further centrifuged. Exosomes were derived from serum using the commercial kit (Total Exosome Isolation Kit), which was further identified by Western blotting analysis (CD63/TSG101), transmission electron microscope analysis (TEM) and nanoparticle tracking analysis (NTA). Exosomes were next solubilized for Exo-PD-L1 detection by enzyme-linked immuno-sorbent assay (ELISA). PD-L1 expression in matched tissue were assessed by PD-L1 immunohistochemistry (IHC) (clone 28-8) assay. Tumor proportion score (TPS) ≥ 1% was deemed as "positive" in this study and TPS < 1% was deemed as "negative". Written informed consent were obtained before acquisition of all data and biological sample. Data were analyzed using SPSS 20.0 and Graphpad Prism 5 software. Chi square test was conducted to estimate the correlation between Exo-PD-L1 levels, sPD-L1 levels, PD-L1 IHC profiles and clinicopathological features. For all analysis, a two-sided p < 0.05 was considered significant statistically.ResultsExo-PD-L1 levels were higher in NSCLC patients with advanced tumor stage, larger tumor size (> 2.5 cm) (p < 0.001), positive lymph node status (p < 0.05) and distant metastasis (p < 0.05). In contrast, sPD-L1 levels were not different between NSCLC patients and healthy donors, it was not correlated with any clinicopathologic features except for tumor size (> 2.5 cm) (p < 0.05). In addition, Exo-PD-L1 levels showed slight correlation with sPD-L1 levels (Spearman's correlation at r = 0.3, p = 0.0027) while no correlation with PD-L1 IHC profiles was detected.ConclusionsIn conclusion, Exo-PD-L1, but not sPD-L1, was correlated with NSCLC disease progression, including tumor size, lymph node status, metastasis and TNM stage. However, Exo-PD-L1 was not associated with PD-L1 IHC status.

Project description:Although checkpoint inhibitors (CPI) have recently extended the treatment options and improved clinical response of advanced stage head and neck squamous cell carcinoma (HNSCC), treatment success remains unpredictable. Programmed cell death ligand‑1 (PD‑L1) is a key player in immunotherapy. Tumor cells, and exosomes derived therefrom, are carriers of PD‑L1 and efficiently suppress immune responses. The aim of the present study was to analyze the influence of established therapies on PD‑L1 expression of HNSCC cell lines and their exosomes. The HNSCC cell lines, UM‑SCC‑11B, UM‑SCC‑14C and UM‑SCC‑22C were treated with fractionated radiotherapy (RT; 5x2 Gy), cisplatin (CT) and cetuximab (Cetux) as monotherapy, or combined therapy, chemoradiotherapy (CRT; RT and CT) or radioimmunotherapy (RT and Cetux). The expression of PD‑L1 and phosphorylated (p)ERK1/2 as a mediator of radioresistance were assessed using western blotting, immunohistochemistry and an ex vivo vital tissue culture model. Additionally, exosomes were isolated from concentrated supernatants of the (un‑)treated HNSCC cell lines by size exclusion chromatography. Exosomal protein expression levels of PD‑L1 were detected using western blotting and semi‑quantitative levels were calculated. The functional impact of exosomes from the (un‑)treated HNSCC cell lines on the proliferation (MTS assay) and apoptosis (Caspase 3/7 assay) of the untreated HNSCC cell lines were measured and compared. The HNSCC cell lines UM‑SCC‑11B and UM‑SCC‑22B showed strong expression of pERK1/2 and PD‑L1, respectively. RT upregulated the PD‑L1 expression in UM‑SCC‑11B and UM‑SCC‑14C and in exosomes from all three cell lines. CT alone induced PD‑L1 expression in all cell lines. CRT induced the expression of PD‑L1 in all HNSCC cell lines and exosomes from UM‑SCC‑14C and UM‑SCC‑22B. The data indicated a potential co‑regulation of PD‑L1 and activated ERK1/2, most evident in UM‑SCC‑14C. Exosomes from irradiated UM‑SCC‑14C cells protected the unirradiated cells from apoptosis by Caspase 3/7 downregulation. The present study suggested a tumor cell‑mediated regulation of PD‑L1 upon platinum‑based CRT in HNSCC and in exosomes. A co‑regulation of PD‑L1 and MAPK signaling response was hypothesized.

Project description:ObjectiveThe monoclonal antibodies anti-programmed death protein-1 (anti-PD-1) nivolumab and pembrolizumab are the first immune checkpoint inhibitors (ICIs) approved for treatment of recurrent/metastatic head and neck carcinoma R/M HNSCC in first line and in platinum refractory disease. This network meta-analysis aims to investigate the efficacy of anti-PD-1- vs anti-PD-L1-based therapy in R/M HNSCC cancer patients through a systematic review of the literature to provide support for evidence-based treatment decisions. In particular, the effectiveness of ICIs for R/M HNSCC is analyzed according to the different mechanisms of action of the check-points inhibitory drugs in different subgroups of patients.MethodsWe did a systematic literature review and network meta-analysis (NMA) of randomized controlled trials (RCTs) in PubMed, ClinicalTrials.gov, Embase, Medline, the Cochrane Central Register of Controlled Trials, Web of Science. Our search identified a total of five randomized controlled trials: Keynote 040, Keynote 048, Eagle, Condor, Checkmate 141. These trials included 3001 patients. Treatment was sub-categorized into PD-L1-based, PD-1-based, and standard chemotherapy. Treatments were indirectly compared with anti-PD-L1-based therapy.ResultsThe network meta-analysis demonstrated no significant differences in OS between different subgroups except for the metastatic patients in which anti-PD-1-based therapy was associated with significantly less risk of death. Furthermore, anti-PD-1-based therapy appeared to be effective in smoker patients and in human papilloma-negative (HPV) patients. Conversely, anti-PD-L1-based therapy seems to be better efficient in female patients, in locally recurrent setting and in HPV positive patients.ConclusionThis is the first NMA study that aimed to indirectly compare anti-PD-1- and anti-PD-L1-based therapy in HNSCC patients. The results of our NMA could help define a profile of patient responder or resistant to specific classes of immune drugs and can be used to guide/design future studies in the novel scenario of precision immune-oncology.

Dataset Information

Clinical Significance of PD-L1⁺ Exosomes in Plasma of Head and Neck Cancer Patients.

Significance

Publications

Clinical Significance of PD-L1<sup>+</sup> Exosomes in Plasma of Head and Neck Cancer Patients.

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