Programmed Death-ligand 1 mRNA Expression Research Articles

MicroRNA-378a-3p regulates the progression of hepatocellular carcinoma by regulating PD-L1 and STAT3

ABSTRACT Programmed death ligand 1 (PD-L1) plays an essential role in the development or progression of hepatocellular carcinoma (HCC). MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression during normal and pathophysiological events. Here, we explored the functions and detailed mechanisms of miR-378a-3p and PD-L1 in HCC progression. First, miR-378a-3p was selected by analyzing miRNA levels in two HCC Gene Expression Omnibus datasets. We found that miR-378a-3p levels exhibited a downward trend in HCC and were negatively correlated with PD-L1 levels. Additionally, a dual luciferase assay predicted that miR-378a-3p directly targets PD-L1. Moreover, the transfection of miR-378a-3p mimics into Li-7 and HuH-7 cells effectively decreased the PD-L1 mRNA and protein expression levels, and inhibited Treg differentiation in co-culture models by modulating the expression levels of certain cytokines. Furthermore, the overexpression of miR-378a-3p hindered cell proliferation and migration but facilitated apoptosis by repressing STAT3 signaling in HCC cells. In conclusion, miR-378a-3p appears to inhibit HCC tumorigenesis by regulating PD-L1 and STAT3 levels. Thus, miR-378a-3p may be a potential target for HCC therapy.

The effect of hypoxia on PD-L1 expression in bladder cancer

IntroductionRecent data has demonstrated that hypoxia drives an immunosuppressive tumour microenvironment (TME) via various mechanisms including hypoxia inducible factor (HIF)-dependent upregulation of programmed death ligand 1 (PD-L1). Both hypoxia and an immunosuppressive TME are targetable independent negative prognostic factors for bladder cancer. Therefore we sought to investigate whether hypoxia is associated with upregulation of PD-L1 in the disease.Materials and methodsThree human muscle-invasive bladder cancer cell lines (T24, J82, UMUC3) were cultured in normoxia (20% oxygen) or hypoxia (1 and 0.1% oxygen) for 24 h. Differences in PD-L1 expression were measured using Western blotting, quantitative polymerase chain reaction (qPCR) and flow cytometry (≥3 independent experiments). Statistical tests performed were unpaired t tests and ANOVA. For in silico work an hypoxia signature was used to apply hypoxia scores to muscle-invasive bladder cancers from a clinical trial (BCON; n = 142) and TCGA (n = 404). Analyses were carried out using R and RStudio and statistical tests performed were linear models and one-way ANOVA.ResultsWhen T24 cells were seeded at < 70% confluence, there was decreased PD-L1 protein (p = 0.009) and mRNA (p < 0.001) expression after culture in 0.1% oxygen. PD-L1 protein expression decreased in both 0.1% oxygen and 1% oxygen in a panel of muscle-invasive bladder cancer cells: T24 (p = 0.009 and 0.001), J82 (p = 0.008 and 0.013) and UMUC3 (p = 0.003 and 0.289). Increasing seeding density decreased PD-L1 protein (p < 0.001) and mRNA (p = 0.001) expression in T24 cells grown in both 20 and 1% oxygen. Only when cells were 100% confluent, were PD-L1 protein and mRNA levels higher in 1% versus 20% oxygen (p = 0.056 and p = 0.037). In silico analyses showed a positive correlation between hypoxia signature scores and PD-L1 expression in both BCON (p = 0.003) and TCGA (p < 0.001) cohorts, and between hypoxia and IFNγ signature scores (p < 0.001 for both).ConclusionTumour hypoxia correlates with increased PD-L1 expression in patient derived bladder cancer tumours. In vitro PD-L1 expression was affected by cell density and decreased PD-L1 expression was observed after culture in hypoxia in muscle-invasive bladder cancer cell lines. As cell density has such an important effect on PD-L1 expression, it should be considered when investigating PD-L1 expression in vitro.

Evaluation of the programmed death-ligand 1 mRNA expression and immunopositivity and their correlation with survival outcomes in Indian lung cancer patients.

The presence of membranous immunopositivity of programmed death-ligand 1 (PD-L1) in tumors serves as a key determinant of response to immune checkpoint inhibitors. However, there are very limited studies on the evaluation of the PD-L1 mRNA expression and immunopositivity and their correlation with therapeutic response and survival outcomes, especially in Indian lung cancer patients. In this prospective study, conducted between 2017 and 2020, we collected biopsies and surgically resected tumors from 173 lung cancer patients. PD-L1 immunopositivity and mRNA expression were determined by immunohistochemistry using SP263 assay and qRT-PCR, respectively. PD-L1 expression was correlated with various clinicopathological variables, response to therapy, and survival outcomes using appropriate statistical methods. The median age was 60years (range 33-81years) with the majority of patients being male (86.5%) and smokers (83%). Histologically, the majority of patients were non-small cell lung cancer (89.4%) and of squamous cell carcinoma histology (64.3%). PD-L1 immunopositivity in tumor cells (tumor proportion score (TPS) ≥ 1%) was detected in 37.6%, while high immunopositivity (TPS ≥ 50%) was detected in 16.8% of lung cancer patients. Almost 76% of lung cancer patients with PD-L1 TPS ≥ 50% belonged to PD-L1 mRNA high-expression group. PD-L1 mRNA expression and immunopositivity did not correlate with response to therapy and survival outcomes. We conclude that PD-L1 immunopositivity and mRNA expression do not seem to serve as a prognostic biomarker for lung cancer patients treated with chemotherapy. More prospective studies should be planned to evaluate the predictive and prognostic relevance of PD-L1 expression in Indian lung cancer patients being treated with immune checkpoint inhibitors.

Discordance of PD-L1 Expression at the Protein and RNA Levels in Early Breast Cancer.

Simple SummaryDespite the increasing use of checkpoint inhibitors for early and metastatic breast cancer, Programmed Death Ligand 1 (PD-L1) remains the only validated albeit imperfect predictive biomarker. Significant discordance in PD-L1 protein expression depending on the antibody used has been demonstrated, while the weak correlation and discordant prognostic information between protein and gene expression underscore its biologic heterogeneity. In this study, we use material from two patient cohorts of early breast cancer and multiple methodologies (immunohistochemistry, RNA fluorescent in situ hybridization, immunofluorescence, bulk gene expression, and multiplex fluorescent immunohistochemistry) to demonstrate the significant discordance in PD-L1 expression among various methods and between different areas of the same tumor, which hints toward the presence of spatial, intratumoral and biological heterogeneity.We aimed to assess if the discrepant prognostic information between Programmed Death Ligand 1 (PD-L1) protein versus mRNA expression in early breast cancer (BC) could be attributed to heterogeneity in its expression. PD-L1 protein and mRNA expression in BC tissue microarrays from two clinical patient cohorts were evaluated (105 patients; cohort 1: untreated; cohort 2: neoadjuvant chemotherapy-treated). Immunohistochemistry (IHC) with SP142, SP263 was performed. PD-L1 mRNA was evaluated using bulk gene expression and RNA-FISH RNAscope®, the latter scored in a semi-quantitative manner and combined with immunofluorescence (IF) staining for the simultaneous detection of PD-L1 protein expression. PD-L1 expression was assessed in cores as a whole and in two regions of interest (ROI) from the same core. The cell origin of PD-L1 expression was evaluated using multiplex fluorescent IHC. IHC PD-L1 expression between SP142 and SP263 was concordant in 86.7% of cores (p < 0.001). PD-L1 IF/IHC was weakly correlated with spatial mRNA expression (concordance 54.6–71.2%). PD-L1 was mostly expressed by lymphocytes intra-tumorally, while its stromal expression was mostly observed in macrophages. Our results demonstrate only moderate concordance between the various methods of assessing PD-L1 expression at the protein and mRNA levels, which may be attributed to both analytical performance and spatial heterogeneity.

A secondary role for hypoxia and HIF1 in the regulation of (IFN\u03b3-induced) PD-L1 expression in melanoma

Cancer cells are able to escape immune surveillance by upregulating programmed death ligand 1 (PD-L1). A key regulator of PD-L1 expression is transcriptional stimulation by the IFNγ/JAK/STAT pathway. Recent studies suggest that hypoxia can induce PD-L1 expression. As hypoxia presents a hallmark of solid tumor development, hypoxic control of PD-L1 expression may affect the efficacy of cancer immunotherapy. This study aims to explore the hypoxic regulation of PD-L1 expression in human melanoma, and its interaction with IFNγ-induced PD-L1 expression. Analysis of the cutaneous melanoma dataset from the cancer genome atlas revealed a significant correlation of the HIF1-signaling geneset signature with PD-L1 mRNA expression. However, this correlation is less pronounced than other key pathways known to control PD-L1 expression, including the IFNγ/JAK/STAT pathway. This secondary role of HIF1 in PD-L1 regulation was confirmed by analyzing single-cell RNA-sequencing data of 33 human melanoma tissues. Interestingly, PD-L1 expression in these melanoma tissues was primarily found in macrophages. However, also in these cells STAT1, and not HIF1, displayed the most pronounced correlation with PD-L1 expression. Moreover, we observed that hypoxia differentially affects PD-L1 expression in human melanoma cell lines. Knockdown of HIF1 expression indicated a minor role for HIF1 in regulating PD-L1 expression. A more pronounced influence of hypoxia was found on IFNγ-induced PD-L1 mRNA expression, which is controlled at a 952 bp PD-L1 promoter fragment. These findings, showing the influence of hypoxia on IFNγ-induced PD-L1 expression, are relevant for immunotherapy, as both IFNγ and hypoxia are frequently present in the tumor microenvironment.

Membrane-Associated RING-CH 8 Functions as a Novel PD-L1 E3 Ligase to Mediate PD-L1 Degradation Induced by EGFR Inhibitors.

Expression of programmed death-ligand 1 (PD-L1) on cancer cells is a critical mechanism contributing to immunosuppression and immune escape. PD-L1 expression may also affect therapeutic outcomes of epidermal growth factor receptor (EGFR)-targeted therapy (e.g., with osimertinib/AZD9291) against EGFR-mutant non-small cell lung cancers (NSCLC) and can even be altered during the treatment albeit with largely undefined mechanisms. This study primarily focuses on elucidating the mechanism by which osimertinib induces PD-L1 degradation in addition to validating osimertinib's effect on decreasing PD-L1 expression in EGFR-mutant NSCLC cells and tumors. Osimertinib and other EGFR inhibitors effectively decreased PD-L1 levels primarily in EGFR-mutant NSCLCs and xenografted tumors. Osimertinib not only decreased PD-L1 mRNA expression, but also prompted proteasomal degradation of PD-L1 protein, indicating both transcriptional and posttranslational mechanisms accounting for osimertinib-induced reduction of PD-L1. Knockdown of β-TrCP or inhibition of GSK3 failed to prevent PD-L1 reduction induced by osimertinib. Rather, knockdown of membrane-associated RING-CH 8 (MARCH8) that encodes a membrane-bound E3 ubiquitin ligase rescued osimertinib-induced PD-L1 reduction. Furthermore, manipulation of MARCH8 expression accordingly altered PD-L1 degradation rate. Critically, MARCH8 interacted with PD-L1 through its N-terminal region and also ubiquitinated PD-L1 in cells. Collectively, these results strongly suggest that MARCH8 is a previously undiscovered E3 ubiquitin ligase responsible for PD-L1 degradation including osimertinib-induced PD-L1 degradation, establishing a novel connection between MARCH8 and PD-L1 regulation. IMPLICATIONS: This study has demonstrated a previously undiscovered function of MARCH8 in mediating PD-L1 degradation induced by EGFR inhibitors in EGFR-mutant NSCLC cells, establishing a novel connection between MARCH8 and PD-L1 regulation.

LPS stimulates gingival fibroblasts to express PD-L1 via the p38 pathway under periodontal inflammatory conditions

ObjectiveThe overall aim of this research was to investigate the differences in the expression of programmed death ligand 1 (PD-L1) in human gingival fibroblasts (HGFs) between a periodontal healthy group and a periodontal inflammatory group. and explore the possible mechanism involved. MethodsDifferences in PD-L1 mRNA and protein expression in HGFs from a periodontal healthy group and a periodontal inflammatory group were examined by qPCR and western blotting, respectively, and were further tested after lipopolysaccharide (LPS) stimulation in both groups. The effects of a p38 pathway inhibitor on the changes in p38 phosphorylation levels and PD-L1 expression after LPS stimulation were investigated in both groups. ResultsPD-L1 mRNA and protein levels in HGFs in the periodontal inflammatory group were significantly higher than those in the periodontal healthy group (p < 0.05). After 10 μg/mL LPS stimulation, PD-L1 mRNA levels in HGFs from both groups increased significantly (p < 0.05), peaking at 4 h, and the peak was significantly higher in the periodontal inflammatory group than in the periodontal healthy group (p < 0.05). However, PD-L1 protein expression was upregulated only in the inflammatory group (p < 0.05). Inhibition of the p38 pathway in HGFs decreased p38 phosphorylation in both groups (p < 0.05) but this treatment reversed the LPS-induced increase in PD-L1 mRNA and protein levels only in the inflammatory group (p < 0.05). ConclusionIn the periodontal inflammatory state, the expression of PD-L1 in HGFs is more easily activated, and may be influenced by the p38 pathway.

MicroRNA-124-3p suppresses PD-L1 expression and inhibits tumorigenesis of colorectal cancer cells via modulating STAT3 signaling.

Programmed death ligand 1 (PD-L1) plays a significant role in colorectal tumorigenesis through induction of regulatory T cells (Tregs) and suppression of antitumor immunity. Furthermore, microRNAs (miRNAs) as the posttranscriptional regulators of gene expression show considerable promise as a therapeutic target for colorectal cancer (CRC) treatment. Considering this, in vitro effects of miRNA-124 (miR-124-3p) on CRC cell tumorigenesis and Tregs differentiation via targeting PD-L1were investigated in the current study. Functional analysis showed that miR-124 is significantly downregulated in CRC tissues as compared with marginal normal samples (p < .0001), and its downregulation was negatively correlated with PD-L1 expression. Moreover, a specific region in PD-L1 3'-untranslated region was predicted as the miR-124 target and validated using the luciferase assay. Further investigation showed that transfection of HT29 and SW480 cells with miR-124 mimics significantly reduced PD-L1 mRNA, protein, and cell surface expression, and inhibited Tregs in coculture models via modulating interleukin[IL]-10, IL-2, tumor necrosis factor α, transforming growth factor beta, and interferon gamma expression levels. Besides, miR-124 overexpression decreased CRC cell proliferation and arrested cell cycle at the G1 phase through downregulation of c-Myc and induced apoptosis in CRC cells via upregulation of both intrinsic and extrinsic pathways. Also, miR-124 exogenous overexpression could reduce colony and spheroid formation ability of CRC cells via downregulating CD44 mRNA expression. miR-124 also diminished MMP-9 expression and subsequently suppressed cell migration and invasion. We also illustrated that STAT3 signaling was repressed by miR-124 in CRC cells. Taken together, our findings imply that considering the involvement of miR-124 in the regulation of PD-L1 through colorectal tumorigenesis and its remarkable antitumor effects, this miRNA could be regarded as the promising target for the development of therapeutic approaches for colorectal cancer.

Profile of PD-L1 mRNA Expression in Childhood Acute Leukemia

Background: Programmed Death Ligand 1 (PD-L1) expression was used to determine type of patients who respond to immunotherapy using immune checkpoint inhibitor in several solid malignancies. However, the role of PD-L1 in hematological malignancies is less explored. Therefore, we aim to investigate PD-L1 mRNA expression in childhood leukemia patients. Association between PD-L1 expression and clinicopathological features was also analyzed. Method: Blood samples of 17 childhood leukemia patients and 12 healthy individuals as control were used in this study. The samples were collected in Dharmais Cancer Hospital. Real time PCR was used to analyze PD-L1 expression with GAPDH as internal control. Result: PD-L1 mRNA expression is significantly lower in childhood leukemia patients (medians: 0.0012) compared to healthy individuals (medians: 0.0041) (p=0.017). PD-L1 mRNA expressions were not correlated with age (p=1.000), sex (p=1.000), outcome (p=1.000), type of leukemia (p=1.000), hyperleukocytosis (p=1.000), and syndrome down (p=0.426). Conclusion: PD-L1 expression is lower in leukemia patients compared to healthy controls. PD-L1 expressions were not correlated with all clinicopathological features. Our result provides preliminary data of PD-L1 expression in Indonesian childhood leukemia patients.

PD-L1 amplification is associated with an immune cell rich phenotype in squamous cell cancer of the lung

Gene amplification is considered to be one responsible cause for upregulation of Programmed Death Ligand-1 (PD-L1) in non-small cell lung cancer (NSCLC) and to represent a specific molecular subgroup possibly associated with immunotherapy response. Our aim was to analyze the frequency of PD-L1 amplification, its relation to PD-L1 mRNA and protein expression, and to characterize the immune microenvironment of amplified cases. The study was based on two independent NSCLC cohorts, including 354 and 349 cases, respectively. Tissue microarrays were used to evaluate PD-L1 amplification by FISH and PD-L1 protein by immunohistochemistry. Immune infiltrates were characterized immunohistochemically by a panel of immune markers (CD3, CD4, CD8, PD-1, Foxp3, CD20, CD138, CD168, CD45RO, NKp46). Mutational status was determined by targeted sequencing. RNAseq data was available for 197 patients. PD-L1 amplification was detected in 4.5% of all evaluable cases. PD-L1 amplification correlated only weakly with mRNA and protein expression. About 37% of amplified cases were negative for PD-L1 protein. PD-L1 amplification did not show any association with the mutational status. In squamous cell cancer, PD-L1 amplified cases were enriched among patients with high tumoral immune cell infiltration and showed gene expression profiles related to immune exhaustion. In conclusion, PD-L1 amplification correlates with PD-L1 expression in squamous cell cancer and was associated with an immune cell rich tumor phenotype. The correlative findings help to understand the role of PD-L1 amplification as an important immune escape mechanism in NSCLC and suggest the need to further evaluate PD-L1 amplification as predictive biomarker for checkpoint inhibitor therapy.

Identifying the prognostic significance of B3GNT3 with PD-L1 expression in lung adenocarcinoma.

BackgroundAs a novel treatment, programmed cell death protein 1 (PD-1) inhibitor appears to be less effective in tumors of lung adenocarcinoma patients with epidermal growth factor receptor (EGFR) mutation. Beta-1,3-N-acetylglucosaminyltransferase 3 (B3GNT3) has reported to be associated with programmed death ligand 1 (PD-L1)/PD-1 interaction. However, the relationship between B3GNT3 and PD-L1 and its prognostic significance inEGFRmutant status are still unknown.MethodsB3GNT3 was identified through transcriptome sequencing and The Cancer Genome Atlas Lung Adenocarcinoma (TCGA-LUAD) database. Flow cytometry and real-time polymerase chain reaction were performed to investigate the association between B3GNT3, PD-L1, and EGFR. Then, B3GNT3 and PD-L1 expression were evaluated by immunohistochemical analysis in 145 surgically resected primary lung adenocarcinomas. The relationships between survival and B3GNT3, PD-L1, and EGFR status were assessed, and the potential prognostic factors in patients with B3GNT3 expression were identified.ResultsWe found that EGFR activation induced PD-L1 expression, and EGFR tyrosine kinase inhibitor (TKI) could reduce PD-L1 protein in EGFR-TKI-sensitive HCC827 and PC9 cell lines. Subsequent analysis showed that EGFR inhibitor could also lead to both decreased PD-L1 and B3GNT3 mRNA expression. A total of 145 lung adenocarcinoma patients were included. PD-L1 >1% and B3GNT3-positive expression in patients might contribute to worse prognosis in both overall survival (OS) [hazard ratio (HR), 2.63; 95% confidence interval (CI), 0.98–7.06; P=0.048] and disease-free survival (DFS) (HR, 3.04; 95% CI, 1.13–8.14; P=0.019), especially in the PD-L1 ≥50% group. However, when patients were negative for B3GNT3, PD-L1, and EGFR (or “triple negative”), there were significant decreases in OS (HR, 5.44; 95% CI, 0.99–29.83; P=0.029) and DFS (HR, 7.24; 95% CI, 1.32–39.73; P=0.008). Positive B3GNT3 expression was a significant risk factor associated with lower DFS (HR, 3.30; P=0.043).ConclusionsOur results indicate that the B3GNT3 expression is tightly correlated with PD-L1 expression and EGFR mutation status. B3GNT3 is associated with poor prognosis in lung adenocarcinoma patients. Collectively, these findings may offer new insight into enhancing immune therapy efficacy for lung adenocarcinoma patients.

Z-guggulsterone induces PD-L1 upregulation partly mediated by FXR, Akt and Erk1/2 signaling pathways in non-small cell lung cancer

Programmed death-ligand 1 (PD-L1) is an immune checkpoint molecule, that is overexpressed in non-small cell lung cancer (NSCLC) and has been associated with the response to anti-PD-1/PD-L1 immunotherapy. Z-guggulsterone (Z-GS), an active compound extracted from the gumresin of the Commiphora mukul tree, has been shown to have anti-tumor effects in NSCLC in our previous study. However, whether Z-GS could affect PD-L1 expression levels in tumor cells remains unknown. In this study, we verified the inhibitory effects of Z-GS on NSCLC cell viability and cell cycle progression in vitro, and mouse Lewis lung carcinoma (LLC) tumor growth in vivo. Notably, Z-GS treatment increased PD-L1 surface and mRNA expression levels, and gene transcription in NSCLC cells, in a dose- and time-dependent manner. Mechanistic experiments showed that the upregulation of PD-L1 was mediated, partly by farnesoid X receptor inhibition, and partly by the activation of the Akt and Erk1/2 signaling pathways in Z-GS-treated NSCLC cells. In vivo, Z-GS treatment dose-dependently increased PD-L1 expression levels in mouse LLC tumor models. Overall, our findings demonstrated a promoting role for Z-GS in PD-L1 expression in NSCLC and provided mechanistic insights, that may be used for further investigation into synergistic combined therapies.

PKM2-Induced the Phosphorylation of Histone H3 Contributes to EGF-Mediated PD-L1 Transcription in HCC.

High expression of programmed death-ligand-1 (PD-L1) in hepatocellular carcinoma (HCC) cells usually inhibits the proliferation and functions of T cells, leading to immune suppression in tumor microenvironment. However, very little has been described regarding the mechanism of PD-L1 overexpression in HCC cells. In the present study, we found epidermal growth factor (EGF) stimulation promoted the expression of PD-L1 mRNA and protein in HCC cells. Inhibition of epidermal growth factor receptor (EGFR) could reverse EGF-induced the expression of PD-L1 mRNA and protein. Subsequently, we also observed that the phosphorylation level of Pyruvate kinase isoform M2 (PKM2) at Ser37 site was also increased in response to EGF stimulation. Expression of a phosphorylation-mimic PKM2 S37D mutant stimulated PD-L1 expression as well as H3-Thr11 phosphorylation in HCC cells, while inhibition of PKM2 significantly blocked EGF-induced PD-L1 expression and H3-Thr11 phosphorylation. Furthermore, mutation of Thr11 of histone H3 into alanine abrogated EGF-induced mRNA and protein expression of PD-L1, Chromatin immunoprecipitation (ChIP) assay also suggested that EGF treatment resulted in enhanced H3-Thr11 phosphorylation at the PD-L1 promoter. In a diethylnitrosamine (DEN)-induced rat model of HCC, we found that the expression of phosphorylated EGFR, PKM2 nuclear expression, H3-Thr11 phosphorylation as well as PD-L1 mRNA and protein was higher in the livers than that in normal rat livers. Taken together, our study suggested that PKM2-dependent histone H3-Thr11 phosphorylation was crucial for EGF-induced PD-L1 expression at transcriptional level in HCC. These findings may provide an alternative target for the treatment of hepatocellular carcinoma.

Evodiamine suppresses non-small cell lung cancer by elevating CD8+ T cells and downregulating the MUC1-C/PD-L1 axis

BackgroundAccumulating evidence showed that regulating tumor microenvironment plays a vital role in improving antitumor efficiency. Programmed Death Ligand 1 (PD-L1) is expressed in many cancer cell types, while its binding partner Programmed Death 1 (PD1) is expressed in activated T cells and antigen-presenting cells. Whereas, its dysregulation in the microenvironment is poorly understood. In the present study, we confirmed that evodiamine downregulates MUC1-C, resulting in modulating PD-L1 expression in non-small cell lung cancer (NSCLC).MethodsCell viability was measured by MTT assays. Apoptosis, cell cycle and surface PD-L1 expression on NSCLC cells were analyzed by flow cytometry. The expression of MUC1-C and PD-L1 mRNA was measured by real time RT-PCR methods. Protein expression was examined in evodiamine-treated NSCLC cells using immunoblotting or immunofluorescence assays. The effects of evodiamine treatment on NSCLC sensitivity towards T cells were investigated using human peripheral blood mononuclear cells and Jurkat, apoptosis and IL-2 secretion assays. Female H1975 xenograft nude mice were used to assess the effect of evodiamine on tumorigenesis in vivo. Lewis lung carcinoma model was used to investigate the therapeutic effects of combination evodiamine and anti-PD-1 treatment.ResultsWe showed that evodiamine significantly inhibited growth, induced apoptosis and cell cycle arrest at G2 phase of NSCLC cells. Evodiamine suppressed IFN-γ-induced PD-L1 expression in H1975 and H1650. MUC1-C mRNA and protein expression were decreased by evodiamine in NSCLC cells as well. Evodiamine could downregulate the PD-L1 expression and diminish the apoptosis of T cells. It inhibited MUC1-C expression and potentiated CD8+ T cell effector function. Meanwhile, evodiamine showed good anti-tumor activity in H1975 tumor xenograft, which reduced tumor size. Evodiamine exhibited anti-tumor activity by elevation of CD8+ T cells in vivo in Lewis lung carcinoma model. Combination evodiamine and anti-PD-1 mAb treatment enhanced tumor growth control and survival of mice.ConclusionsEvodiamine can suppress NSCLC by elevating of CD8+ T cells and downregulating of the MUC1-C/PD-L1 axis. Our findings uncover a novel mechanism of action of evodiamine and indicate that evodiamine represents a potential targeted agent suitable to be combined with immunotherapeutic approaches to treat NSCLC cancer patients. MUC1-C overexpression is common in female, non-smoker, patients with advanced-stage adenocarcinoma.

Lymphoma Microenvironment Deconvolution Links M1 Macrophage Infiltration to Clinical Outcome in Diffuse Large B-Cell Lymphoma

Introduction: The lymphoma microenvironment is increasingly recognized as crucial to sustaining lymphoma cell growth and an important contributor to treatment outcome, especially in the context of immunotherapies. CD20-targeted monoclonal antibodies (e.g. obinutuzumab [G] and rituximab [R]) function by several mechanisms, including antibody-dependent cellular cytotoxicity/phagocytosis (ADCC/ADCP). Immune effector cells, such as natural killer (NK) cells and phagocytes (i.e. macrophages and dendritic cells), and the Fc gamma receptor (FcγR) found on the surface of these cells, are critical to antibody treatment efficacy. Here we evaluated how the lymphoma microenvironment may affect clinical outcome in patients (pts) with previously untreated diffuse large B-cell lymphoma (DLBCL) receiving immunochemotherapy. Methods: We leveraged two large Phase III clinical trials of pts with previously untreated DLBCL (GOYA [NCT01287741] and MAIN [NCT00486759]) to produce comprehensive lymphoma immune microenvironment profiles from 604 tissue biopsies from pts treated with R plus cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP) or G plus CHOP (G-CHOP) using the RNA-Seq deconvolution and marker gene methods: quanTIseq and xCell. The infiltration scores in each pt for various immune and stromal cell types were assessed, and their contribution to disease biology and treatment outcome was examined. Results: The extent of lymphoma microenvironment heterogeneity highlighted by the deconvolution analyses was consistent with previous studies (Figure A). Of the infiltrating cell types analyzed, the M1 macrophage signature quantified by either quanTIseq or xCell was most strongly associated with lower risk of progression (progression-free survival [PFS]; quanTIseq: HR, 0.596; 95% CI: 0.441-0.805; 24-month PFS: 82% [M1 high] vs 68% [M1 low] and xCell: HR, 0.627; 95% CI: 0.465-0.844; 24-month PFS: 80% [M1 high] vs 70% [M1 Low]; Figure B, C, D) and improved overall survival (OS; quanTIseq: HR, 0.465; 95% CI: 0.318-0.679; and xCell: HR, 0.527; 95% CI: 0.365-0.762). This finding was confirmed by both algorithms. This prognostic trend was stronger amongst G-treated pts than R-treated pts, consistent with the previous finding that G exhibits higher ADCC versus R (Mössner, et al. Blood 2010). Pts with PFS &amp;gt;24 months had significantly higher levels of M1 macrophage scores than pts with PFS &amp;lt;24 months. Despite the correlation with delayed disease progression, there was no differential enrichment of M1 macrophages in pts with complete response versus non-responders at end of treatment, or depending on International Prognostic Index. M1 scores did not significantly differ depending on cell of origin, although there was a trend for higher M1 macrophage scores in germinal center B-cell DLBCL. Aside from M1 macrophages, CD4+Th2 cells showed the strongest prognostic trend in DLBCL (PFS; HR, 0.745; 95% CI: 0.553-1.000; Figure C). In contrast to M1 macrophages, pts with M2 macrophage infiltration tended to have shorter PFS and OS although relatively low levels were observed for these signatures (Figure B, C). This suggests that lymphoma-infiltrating macrophages more commonly resemble the classically activated M1 polarization phenotype and are linked to prolonged PFS, while alternatively activated M2 macrophages, although their frequency is lower in DLBCL, are associated with shorter PFS. Consistent with previous work showing that programmed death-ligand 1 (PD-L1) levels correlate with a macrophage signature in DLBCL (McCord, et al. Blood Adv 2019), M1, but not M2, macrophage infiltration correlated with PD-L1 mRNA expression. M1 enrichment was highly correlated with CD8+ T cell signatures (including central and effector memory CD8+ T cells) in DLBCL. Conclusions: Data suggest macrophage polarization may be an important contributor to immunochemotherapy outcome in DLBCL. Previous studies aiming to link tumor-associated macrophages to R-CHOP outcome have yielded conflicting results, perhaps as most relied on CD68/CD163 staining alone as markers. Although R and G are thought to function via NK cell-mediated ADCC, FcγR-dependent stimulation of M1 macrophage-mediated ADCP may be key to sustaining their anti-lymphoma activity. Strategies facilitating the recruitment of M1 macrophages or macrophage repolarization may augment responses to immunochemotherapy in DLBCL. Disclosures Yan: F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company. Chang:F. Hoffmann-La Roche: Current Employment, Current equity holder in private company. Raghavan:F. Hoffmann-La Roche: Current Employment. Dong:In graduate school: University of Toronto, MSc Biostatistics: Ended employment in the past 24 months; F. Hoffmann-La Roche, Mississauga, Biometrics: Current Employment. Klein:Roche: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Nielsen:F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company. Paulson:Genentech, Inc: Current Employment; F. Hoffmann-La Roche: Current equity holder in private company, Current equity holder in publicly-traded company. Hatzi:F. Hoffmann-La Roche: Current equity holder in publicly-traded company; Genentech, Inc.: Current Employment.

Profile of PD-1 and PD-L1 mRNA Expression in Peripheral Blood of Nasopharyngeal Carcinoma

Background: Programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1) expression is associated with prognostic and respond to immunotherapy with immune checkpoint inhibitor in several solid malignancies. However, the prognostic roles of PD-1 and PD-L1 expression in nasopharyngeal carcinoma (NPC) are less clear. This study aims to investigate PD-1 and PD-L1 mRNA expression levels in peripheral blood of Indonesian NPC patients and its association with clinicopathological features.Materials and Methods: This study used blood samples of 21 NPC patients and 10 healthy volunteers as controls. Real-time polymerase chain reaction (PCR) was used to measure mRNA expression of PD-1 and PD-L1.Results: PD-1 mRNA expression levels were significantly lower in NPC patients (∆CT mean: 9.65±2.04) compared to healthy individuals (∆CT mean: 8.04±1.51) (p=0.031). In contrast, PD-L1 mRNA expression levels were higher in NPC patients (∆CT mean: 6.96±1.32) compared to healthy individuals (∆CT mean: 7.11±0.55), but the difference was not statistically significant (p=0.554). The expression of PD-1 was associated with tumour-node-metastasis (TNM) stage (p=0.030) but not associated with age (p=1.000), sex (p=1.000), body mass index (p=0.350), tumor stage (p=0.338), nodal stage (p=0.579), metastasis stage (p=0.371), and Eastern Cooperative Oncology Group (ECOG) status (p=0.228). Meanwhile PD-L1 expression was not associated with all clinicophatological features.Conclusion: The PD-1 mRNA expression levels were significantly lower, while PD-L1 expression levels were higher in NPC patients compared to healthy controls. PD-1 expression was correlated with TNM stage.Keywords: nasopharyngeal carcinoma, immune checkpoint inhibitors, PD-1, PD-L1

Loss of tumor intrinsic PD-L1 confers resistance to drug-induced apoptosis in human colon cancer.

Colorectal cancer (CRC) with BRAF (V600E) is associated with microsatellite instability (MSI) that predicts response to immune checkpoint inhibitors. We demonstrated the interrogation of TCGA RNA-seq human datasets revealed that BRAFV600E tumors had significantly higher Programmed Death Ligand 1 (PD-L1) mRNA compared to non-mutated BRAF CRCs. Also, MSI-H tumors were evaluated as higher PD-L1 than MSS CRCs. Inhibition of MEK/ERK by cobimetinib or CDK inhibitor dinaciclib was shown to attenuate mutant BRAF-induced PD-L1 coincident with reduced c-JUN and YAP expression whose combined knockdown reduced PD-L1. Using TCGA datasets, PD-L1 mRNA expression in human colon cancers was significantly associated with YAP expression. The deletion of PD-L1 can reduce tumor cell growth shown by clonogenic assay. Analysis of the role of PD-L1 as a mediator of chemosensitivity was then performed. Knockout of PD-L1 was shown to attenuate the induction of DNA double-strand breaks (pH2AX) and caspase-3 cleavage by 5-fluorouracil (5-FU) and paclitaxel compared to parental CRC cells. Results were confirmed in PD-L1 knockout MC38 murine CRC cells where re-expression of wild-type PD-L1 promoted DNA damage and apoptosis. We also performed the clonogenic assay and flow cytometry to prove that loss of PD-L1 attenuated DNA damage and apoptosis induced by diverse anti-cancer drugs that could be reversed by restoration of wild-type PD-L1. Mechanistically, knockout of PD-L1 reduced chemosensitivity in association with reductions in p-AKT and in BH3-only proteins BIM and BIK, rather than STAT3 in CRC cells. However, STAT3 had a significant role in melanoma, which shows the heterogeneity of cancers. In summary, BRAFV600E can upregulate PD-L1 expression that was induced by c-jun and YAP to enhance chemotherapy-induced apoptosis. Together, we demonstrate a potential role for PD-L1 as a regulator of chemotherapy-induced apoptosis whose deletion or suppression confers chemoresistance. These findings expand the understanding of PD-L1 functions to include nonimmune mechanisms and suggest the potential use of PD-L1 as a biomarker of response to cytotoxic chemotherapy.

Association of PD-L1 and IDO1 expression with JAK-STAT pathway activation in soft-tissue leiomyosarcoma.

Therapies targeting the immune checkpoint molecules programmed death ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1) have been explored in various malignant tumours. In this study, we examined the relationship between PDL-1, IDO1 and JAK2 expression and the roles of these signal pathways in soft tissue leiomyosarcoma (LMS). The next-generation sequencing data of 53 patients with LMS were obtained from an online public database and were used to assess PD-L1, IDO1 and JAK2 gene amplification and mRNA expression. Then, we determined the relationship between JAK-STAT pathway activation and PD-L1 and IDO1 expression in a LMS cell line. In addition, immunohistochemical staining of 69 cases of LMS was performed for PD-L1, IDO1, TDO2 and phosphorylated JAK2 (pJAK2). Comprehensive gene expression analysis using microarray and RNA-Seq data revealed that PD-L1 and IDO1 mRNA expression positively correlated with JAK2 and STAT1 mRNA expression. Two of the 53 cases exhibited PD-L1 and JAK2 gene amplification; however, they were not related to their gene expression. LMS cell line analysis revealed that IFN-γ supplementation induced IDO1 and PD-L1 expression; these effects were suppressed by JAK inhibition. Immunohistochemical analysis of the resected specimens revealed that TDO2 expression positively correlated with pJAK2 (P = 0.0490) and IDO1 expression (P < 0.0001). PD-L1-positive specimens tended to express pJAK2; however, the relationship did not reach statistical significance (P = 0.1477). The results suggest the possible feasibility of the combined inhibition of PD-1/PD-L1 or IDO1 with IFN-γ-JAK-STAT pathway inhibition to treat soft tissue LMS.

Influence of genetic variation of programmed death-ligand 1 (PD-L1) on the prognosis of patients with non-small cell lung cancer who received platinum-based adjuvant chemotherapy

Objective: The aim of present study was to investigate the influence of genetic variation of programmed death-ligand 1 (PD-L1) on the prognosis of patients with non-small cell lung cancer (NSCLC) who received platinum-based adjuvant chemotherapy. Methods: This study was designed as a retrospective analysis, and a total of 278 patients with postoperative NSCLC who received platinum-based adjuvant chemotherapy from January 2012 to December 2018 in the Department of Respiratory Medicine of the First affiliated Hospital of Zhengzhou University were included in this study. Biological specimens of the patients were collected during hospitalization. Recurrence status and adverse reactions were evaluated in the hospital during adjuvant chemotherapy. Survival data of the patients were obtained through telephone follow-up after completing the fixed cycle of adjuvant chemotherapy. DNA extracted from the collected hematological specimens was genotyped for PD-L1 gene polymorphism. Additionally, postoperative cancer tissue specimens from 68 patients were collected for RNA extraction in order to perform the PD-L1 mRNA expression analysis. The univariate analysis of genotypes and prognosis was carried out by Kaplan-Meier survival analysis. Results: Prognostic results indicated that the median disease-free survival (DFS) of the 278 patients with NSCLC was 3.2 years and the median overall survival (OS) was 4.9 years. The prevalence of -1813G>C polymorphism were: GG genotype 173 cases (62.23%), GC genotype 92 cases (33.09%), CC genotype 13 cases (4.68%), the minor allele frequency was 0.21, the distribution of the three genotypes was in accordance with Hardy-Weinberg Equilibrium (P=0.864). In view of the rare frequency of CC genotype, GC and CC genotype were merged in the following analysis. The survival analysis results of the two genotype groups suggested that the median DFS of patients with GG and GC/CC genotype was 2.7 and 4.0 years, which was statistically significant (P=0.013). Furthermore, the median OS of patients with GG and GC/CC was 4.0 and 5.4 years respectively, which was statistically significant as well (P=0.009). However, the safety analysis failed to find the significant association between the polymorphism and adverse events (P>0.05). Interestingly, expression analysis of RNA extracted from cancer tissues specimens indicated that the PD-L1 mRNA expression of the patients with GG genotype were significantly higher than those of the GC/CC genotype (3.67±0.65 vs 2.69±0.78, P<0.001). Conclusion: The prognosis of patients with postoperative non-small cell lung cancer who received platinum-based adjuvant chemotherapy is influenced by -1813G>C polymorphism of PD-L1 gene.

Association between immunotherapy biomarkers and glucose metabolism from F-18 FDG PET.

To assess associations between parameters derived from F-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) and mRNA expression levels of immune checkpoint biomarkers such as programmed death receptor 1 (PD-1), programmed death-ligand 1 (PD-L1), cytotoxic T-lymphocyte antigen 4 (CTLA-4) as well as tumor mutation burden (TMB) in non-small cell lung cancer (NSCLC) patients. Integrated data were downloaded from Genomic Data Common Data Portal. Clinical, mRNA-seq, and whole exome-seq data of lung adenocarcinoma and squamous cell carcinoma from The Cancer Genome Atlas (TCGA) database were analyzed. TMB was defined as the total number of somatic missense mutations per megabase of the genome examined. Expression levels of PD-1, PD-L1, CTLA4 mRNA and TMB were collected. Correlations between imaging parameters of glucose metabolism and the expression levels of genomic biomarkers from cancers were evaluated. Bonferroni correction (adjusted p<0.0027) was applied to reduce type 1 error. Of 31 NSCLC cases, 11 cases were adenocarcinoma (LUAD) and 20 were squamous cell carcinoma (LUSC). In linear regression analysis, texture parameters such as low gray-level run emphasis (LGRE, R2=0.48, p<0.0001), short run low gray-level emphasis (SRLGE, R2=0.45, p<0.0001) and long run low gray-level emphasis (LRLGE, R2=0.41, p=0.0001) derived from gray-level run length matrix (GLRLM) showed remarkable correlation with PD-L1 mRNA expression. Expression of PD-1, CTLA-4, and TMB failed to show any significant correlation with parameters of the F-18 FDG PET/CT. Texture parameters derived from PET, known to indicate glucose uptake distribution, were correlated with expression of PD-L1 mRNA but not with expression of PD-1, CTLA-4 and TMB. Thus, tumoral heterogeneity could be a surrogate marker for the identification of PD-L1 level in NSCLC.