Programmed Death-ligand 1 mRNA Expression Research Articles

Development of a PD-L1 Overexpression System in HEK293T Cells: A Platform for Studying Immune Evasion Mechanisms

Programmed death-ligand 1 (PD-L1) is a key immune checkpoint protein that facilitates tumor immune evasion by interacting with PD-1 receptors on T cells, making the PD-1/PD-L1 axis a critical target in cancer immunotherapy. However, resistance to these therapies remains a challenge, necessitating further exploration of PD-L1’s functions and regulatory mechanisms. In this study, we amplified the PD-L1 coding sequence (CDS) from human cDNA, cloned it into a pcDNA3.1 vector, and transfected it into HEK293T cells to establish a robust in vitro PD-L1 overexpression system. Quantitative RT-qPCR demonstrated a 300-400 fold increase in PD-L1 mRNA expression compared to controls. These results confirm the successful construction of the PD-L1 overexpression plasmid and validate its application as a tool for studying PD-L1’s role in immune modulation and its potential implications in overcoming resistance to immunotherapy. This system provides a valuable platform for future investigations into PD-L1-mediated mechanisms of immune evasion and cancer progression.

PD-L1 mRNA derived from tumor-educated platelets as a potential immunotherapy biomarker in non-small cell lung cancer.

To date, the role of programmed death ligand-1 (PD-L1) messenger RNA (mRNA) derived from tumor-educated platelets (TEPs) has not been well investigated in patients with advanced non-small cell lung cancer (NSCLC). A few reports have examined whether mRNA in TEPs can predict the clinical responses of patients with advanced NSCLC following immunotherapy. This study aimed to identify novel biomarkers to improve the clinical benefits and outcomes of NSCLC patients. Advanced NSCLC patients receiving a combination of immunotherapy and chemotherapy, or immunotherapy alone as a first- or second-line treatment at the Fudan University Shanghai Cancer Center were enrolled in this study. All the patients had wild-type epidermal growth factor receptor/anaplastic lymphoma kinase. The patients were enrolled in clinical trials for immune checkpoint inhibitors (ICIs), including nivolumab, pembrolizumab, atezolizumab, durvalumab, tremelimumab, and camrelizumab. Tumoral PD-L1 expression was tested by immunohistochemistry (PD-L1 22C3 pharmDx kit, Agilent, Santa Clara, CA, USA) in archived tissue samples, when available, to calculate the tumor proportion scores (TPSs). RNA and exosomal RNA of blood were isolated before immunotherapy using the Yunying RNA extraction kit (Yunying Medicine, Shanghai, China). The concentration and quality of the RNA was determined using a Qubit fluorometer (Life Technologies, Carlsbad, CA, USA). Finally, we analyzed the predictive value of TEP-derived PD-L1 mRNA expression and association with the level of the tumoral PD-L1 expression. In total, 72 patients were enrolled in this study. Most of the patients were male (n=54, 75.0%), had adenocarcinoma (n=49, 68.1%). We found there was no significant correlation between the TEP-derived mRNA of PD-L1 and tumoral PD-L1 expression based on the results of the Pearson Correlation test (r=-0.19, P=0.233). Based on the median of PD-L1 mRNA, 72 patients were divided into a high PD-L1 group and a low PD-L1 group. We found that 19 patients (44.4%) responded to immunotherapy [partial response or progression-free survival (PFS) >6 months] in the high PD-L1 group, but only five patients (13.9%) responded to immunotherapy in the low PD-L1 group (P<0.01). The median PFS of the low PD-L1 group was lower than that of the high PD-L1 group (2.8 vs. 8.3 months, P<0.001). For the patients who were treated with immunotherapy alone (n=64), a similar PFS advantage was observed in the high PD-L1 group (2.8 vs. 8.0 months, P=0.002). This article presented the first data on TEP-derived PD-L1 mRNA in advanced NSCLC patients following immunotherapy and showed the potential advantage of using it as the surrogate biomarker for predicting the PFS and overall survival of patients following immunotherapy.

PD-L1 mRNA and protein expression in canine mammary carcinomas: Correlation with histopathological grade and molecular markers.

Programmed death ligand 1 (PD-L1) is an immune checkpoint molecule that plays a crucial role in regulating antitumor immune responses. Canine mammary carcinomas (CMCs) are common tumors of dogs. Despite extensive studies on the heterogeneity of CMCs, there is still a lack of effective precision therapies for the treatment of CMCs. In this study, we aimed to investigate the correlation between PD-L1 mRNA and protein expression in CMCs and explore its association with histopathological grade and molecular markers, including the estrogen receptor, epidermal growth factor receptor 2, and cytokeratin 5/6 (CK5/6). Formalin-fixed paraffin-embedded samples were evaluated for PD-L1 mRNA expression using RNA in situ hybridization and PD-L1 protein expression using immunohistochemistry. We observed no substantial correlation between PD-L1 mRNA and protein expression in CMCs; however, PD-L1 mRNA levels were significantly higher in grade 3 than in grade 1 tumors (P = .001). In addition, we observed a positive correlation between PD-L1 protein expression and CK5/6 expression in CMCs (P = .032). These findings suggest that PD-L1 expression in CMCs is heterogeneous and may be regulated post-transcriptionally. Further studies are needed to explore the prognostic and therapeutic implications of PD-L1 expression in different molecular subtypes of CMCs and their potential as predictive biomarkers for immunotherapy.

Predicting PD-L1 Status in Solid Tumors Using Transcriptomic Data and Artificial Intelligence Algorithms

Programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) is routinely used to predict the clinical response to immune checkpoint inhibitors (ICIs); however, multiple assays and antibodies have been used. This study aimed to evaluate the potential of targeted transcriptome and artificial intelligence (AI) to determine PD-L1 RNA expression levels and predict the ICI response compared with traditional IHC. RNA from 396 solid tumors samples was sequenced using next-generation sequencing (NGS) with a targeted 1408-gene panel. RNA expression and PD-L1 IHC were assessed across a broad range of PD-L1 expression levels. AI was used to predict the PD-L1 status. PD-L1 RNA levels assessed by NGS demonstrated robust linearity across high and low expression ranges, and those assessed using NGS and IHC (tumor proportion score and tumor-infiltrating immune cells) had a similar pattern. RNA sequencing provided in-depth information on the tumor microenvironment and immune response, including CD19, CD22, CD8A, CTLA4, and PD-L2 expression status. Subanalyses showed a sustained correlation of mRNA expression with IHC (tumor proportion score and immune cells) across different solid tumor types. Machine learning showed high accuracy in predicting PD-L1 status, with the area under the curve varying between 0.83 and 0.91. Targeted transcriptome sequencing combined with AI is highly useful for predicting PD-L1 status. Measuring PD-L1 mRNA expression by NGS is comparable to measuring PD-L1 expression by IHC for predicting ICI response. RNA expression has the added advantages of being amenable to standardization and avoiding interpretation bias, along with an in-depth evaluation of the tumor microenvironment.

Examination of the Functional Relationship between PD-L1 DNA Methylation and mRNA Expression in Non-Small-Cell Lung Cancer

Immunotherapy targeting the interaction between programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) is a treatment option for patients with non-small-cell lung cancer (NSCLC). The expression of PD-L1 by the NSCLC cells determines treatment effectiveness, but the relationship between PD-L1 DNA methylation and expression has not been clearly described. We investigated PD-L1 DNA methylation, mRNA expression, and protein expression in NSCLC cell lines and tumor biopsies. We used clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) to modify PD-L1 genetic contexts and endonuclease deficient Cas9 (dCas9) fusions with ten-eleven translocation methylcytosine dioxygenase 1 (TET1) and DNA (cytosine-5)-methyltransferase 3A (DNMT3A) to manipulate PD-L1 DNA methylation. In NSCLC cell lines, we identified specific PD-L1 CpG sites with methylation levels inversely correlated with PD-L1 mRNA expression. However, inducing PD-L1 mRNA expression with interferon-γ did not decrease the methylation level for these CpG sites, and using CRISPR-Cas9, we found that the CpG sites did not directly confer a negative regulation. dCas9-TET1 and dCas9-DNMT3A could induce PD-L1 hypo- and hyper-methylation, respectively, with the latter conferring a decrease in expression showing the functional impact of methylation. In NSCLC biopsies, the inverse correlation between the methylation and expression of PD-L1 was weak. We conclude that there is a regulatory link between PD-L1 DNA methylation and expression. However, since these measures are weakly associated, this study highlights the need for further research before PD-L1 DNA methylation can be implemented as a biomarker and drug target for measures to improve the effectiveness of PD-1/PD-L1 immunotherapy in NSCLC.

Classification of Tumor Immune Microenvironment According to Programmed Death-Ligand 1 Expression and Immune Infiltration Predicts Response to Immunotherapy Plus Chemotherapy in Advanced Patients With NSCLC

IntroductionAccording to mechanisms of adaptive immune resistance, tumor immune microenvironment (TIME) is classified into four types: (1) programmed death-ligand 1 (PD-L1)–negative and tumor-infiltrating lymphocyte (TIL)–negative (type I); (2) PD-L1–positive and TIL-positive (type II); (3) PD-L1–negative and TIL-positive (type III); and (4) PD-L1–positive and TIL-negative (type IV). However, the relationship between the TIME classification model and immunotherapy efficacy has not been validated by any large-scale randomized controlled clinical trial among patients with advanced NSCLC. MethodsOn the basis of RNA-sequencing and immunohistochemistry data from the ORIENT-11 study, we optimized the TIME classification model and evaluated its predictive value for the efficacy of immunotherapy plus chemotherapy. ResultsPD-L1 mRNA expression and immune score calculated by the ESTIMATE method were the strongest predictors for the efficacy of immunotherapy plus chemotherapy. Therefore, they were determined as the optimized definition of the TIME classification system. When compared between combination therapy and chemotherapy alone, only the type II subpopulation with high immune score and high PD-L1 mRNA expression was significantly associated with improved progression-free survival (PFS) (hazard ratio = 0.12, 95% confidence interval: 0.06–0.25, p < 0.001) and overall survival (hazard ratio = 0.27, 95% confidence interval: 0.13–0.55, p < 0.001). In the combination group, the type II subpopulation had a much longer survival time, not even reaching the median PFS or overall survival, but the other three subpopulations were susceptible to having similar PFS. In the chemotherapy group, there was no marked association between survival outcomes and TIME subtypes. ConclusionsOnly patients with both high PD-L1 expression and high immune infiltration could benefit from chemotherapy plus immunotherapy in first-line treatment of advanced NSCLC. For patients lacking either PD-L1 expression or immune infiltration, chemotherapy alone might be a better treatment option to avoid unnecessary toxicities and financial burdens.

Abstract P6-14-15: PD-L1 expression regulated by JAK/STAT signaling pathway contributes to cell migration and invasion in breast cancer

Abstract Background: Programmed death-ligand 1 (PD-L1) implicated in tumor immune evasion and predictive biomarker in immunotherapy is widely recognized, however, the role of PD-L1 in modulating tumor invasion remains largely unexplored. PD-L1 expression on tumor tissue is usually limited by the invasiveness, tumor heterogeneity as well as insufficient tumor tissue samples, while PD-L1 expression on circulating tumor cells (CTCs) might overcome the limitation. In the present study, we aimed to investigate the role and mechanism of PD-L1 in regulating the migration and invasion of breast cancer cells and to evaluate PD-L1 expression on CTCs in metastatic breast cancer patients. Methods: The expression level of PD-L1 in MCF-7 cells and MDA-MB-231 cells was assessed by quantitative real-time RT-PCR and Western Blot. Gain-of-function and loss-of-function study on cell migration and invasion abilities were carried out by overexpression of PD-L1 or silencing PD-L1. PD-L1 expression on CTCs in thirty-six metastatic breast cancer patients were detected. A novel staining procedure which included fluorescent glucose analog staining for CTC enumeration and immunostaining targeting CD45, vimentin and PD-L1 were analyzed. Survival curves were estimated by the Kaplan-Meier method and the log-rank test was used to compare between groups. All tests were two-sided, and p values were considered significant at the 0.05 level. Results: Compared with MCF-7 cells, PD-L1 mRNA and PD-L1 protein expression were significantly increased in MDA-MB-231 cells. Down-regulation of PD-L1 expression in MDA-MB-231 cells inhibited the migration and invasion of MDA-MB-231 cells, while overexpression of PD-L1 in MCF-7 cells increased the migration and invasion of MCF-7 cells. In addition, we found that PD-L1 expression was regulated by JAK/STAT signaling pathway. PD-L1 expression on CTCs in metastatic breast cancer patients was associated with triple negative breast cancers subtype (P=0.013). High expression of PD-L1 on CTCs in metastatic breast cancer patients was correlated to poor overall survival (HR=3.165, 95%CI: 1.121-8.938, P=0.029). Conclusion: Our results indicate that high expression of PD-L1 contributes to cell migration and invasion in breast cancer cell possibly partially through JAK/STAT signaling pathway. The PD-L1 expression on CTCs might serve as a promising non-invasive prognostic biomarker in patients with metastatic breast cancer. Citation Format: Junqing Chen. PD-L1 expression regulated by JAK/STAT signaling pathway contributes to cell migration and invasion in breast cancer [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 P6-14-15.

Characterization of expression and prognostic implications of transforming growth factor beta, programmed death-ligand 1, and T regulatory cells in canine histiocytic sarcoma

Histiocytic sarcoma (HS) is an aggressive malignant neoplasm in dogs. Expression and prognostic significance of transforming growth factor beta (TGF-β), programmed death-ligand 1 (PD-L1), and T regulatory cells (Tregs) in HS is unknown. The goal of this study was to investigate the expression and prognostic significance of TGF-β, PD-L1, and FoxP3/CD25 in canine HS utilizing RNA in situ hybridization (RNAscope®). After validation was performed, RNAscope® on formalin-fixed paraffin-embedded (FFPE) patient HS tissue samples was performed for all targets and expression quantified with HALO® software image analysis. Cox proportional hazard model was conducted to investigate the association between survival time and each variable. Additionally, for categorical data, the Kaplan-Meier product-limit method was used to generate survival curves. TGF-β and PD-L1 mRNA expression was confirmed in the DH82 cell line by reverse transcription polymerase chain reaction (RT-PCR) and CD25 + FoxP3 + cells were detected by flow cytometry in peripheral blood. Once the RNAscope® method was validated, TGF-β H-score and dots/cell and FoxP3 dots/cell were assessed in HS samples and found to be significantly correlated with survival. Moderate positive correlations were found between FoxP3 and PD-L1 H-score, percent staining area, and dots/cell, and FoxP3 and TGF-β dots/cell. In summary, RNAscope® is a valid technique to detect TGF-β and PD-L1 expression and identify Tregs in canine HS FFPE tissues. Furthermore, canine HS expresses TGF-β and PD-L1. Increased TGF-β and FoxP3 correlated with worse prognosis. Prospective studies are warranted to further investigate TGF-β, PD-L1, and Tregs effect on prognosis.

&lt;i&gt;Dictyostelium&lt;/i&gt; Differentiation-inducing Factor Derivatives Reduce the Glycosylation of PD-L1 in MDA-MB-231 Human Breast Cancer Cells

Objectives Triple-negative breast cancer (TNBC) is a metastatic and intractable cancer with limited treatment options. Refractory cancer cells often express the immune checkpoint molecules programmed death-ligand 1 (PD-L1) and PD-L2, which inhibit the anticancer effects of T cells. Differentiation-inducing factors, originally found in Dictyostelium discoideum, and their derivatives possess strong antiproliferative activity, at least in part by reducing cyclin D1 expression in various cancer cells, but their effects on PD-L1/PD-L2 have not been examined. In this study, we investigate the effects of six DIF compounds (DIFs) on the expression of PD-L1/PD-L2 and cyclin D1/D3 in MDA-MB-231 cells, a model TNBC cell line.Methods MDA-MB-231 cells were incubated for 5 or 15 h with or without DIFs, and the mRNA expression of cyclin D1, PD-L1, and PD-L2 were assessed by quantitative polymerase chain reaction (qPCR). Whereas, MDA-MD-231 cells were incubated for 12 or 24 h with or without DIFs, and the protein expression of cyclins D1 and D3, PD-L1, and PD-L2 were assessed by Western blotting.Results As expected, some DIFs strongly reduced cyclin D1/D3 protein expression in MDA-MB-231 cells. Contrary to our expectation, DIFs had little effect on PD-L1 mRNA expression or increased it transiently. However, some DIFs partially reduced glycosylated PD-L1 and increased non-glycosylated PD-L1 in MDA-MB-231 cells. The level of PD-L2 was very low in these cells.Conclusion Since PD-L1 glycosylation plays an important role in preventing T cells from attacking cancer cells, such DIFs may promote T cell attack on cancer cells in vivo.

The Effect of Secreted IL-10 from Mesenchymal Stem Cell on Immune Checkpoint Molecules.

Immunosuppression in sepsis is hypothesized to result from the increased expression of the immune checkpoint molecules programmed death-1 (PD-1) and programmed death ligand-1 (PD-L1). PD-1 and PD-L1 blockade therapies have been reported to increase survival in septic animals. Currently, the interleukin (IL)-10 within mesenchymal stem cell (MSC) secretome is known for its immunomodulatory capacity. To study the effect of IL-10 within MSC secretome on the expression of immune checkpoints in the rat model of sepsis. Methods: We used 48 male Rattus norvegicus rats in this research and divided them into four groups: sham (rats without sepsis induction and treatment), control (sepsis-induced rats without treatment), T1 (sepsis-induced rats treated with 150 μL of secreted IL-10 from MSC), and T2 (sepsis-induced rats treated with 300 μL of secreted IL-10 from MSC). Forty-eight hours after sepsis induction, we terminated the rats and collected the blood to examine the PD-1 and PD-L1 expression levels. We found a decrease in the relative expression of PD-1 in the septic rat group given 150 μL and 300 μL of secreted IL-10 from MSC compared to the control group, but the decrease was not significant. We also found a decrease in the relative expression of PD-L1 mRNA in the septic rat group given 150 μL and 300 μL of secreted IL-10 from MSC compared to the control group. Administering secreted IL-10 from MSC reduces the expression of PD-1 and PD-L1 in sepsis. These findings suggest that MSC secretome can improve the immunosuppression in sepsis.

Similar programmed death ligand 1 (PD-L1) expression profile in patients with mild COPD and lung cancer

Programmed Death Ligand 1 (PD-L1) is crucial in regulating the immunological tolerance in non-small cell lung cancer (NSCLC). Alveolar macrophage (AM)-derived PD-L1 binds to its receptor, PD-1, on surveilling lymphocytes, leading to lymphocyte exhaustion. Increased PD-L1 expression is associated with cigarette smoke (CS)-exposure. However, the PD-L1 role in CS-associated lung diseases associated with NSCLC, such as chronic obstructive pulmonary disease (COPD), is still unclear. In two different cohorts of ever smokers with COPD or NSCLC, and ever and never smoker controls, we evaluated PD-L1 expression: (1) via cutting-edge digital spatial proteomic and transcriptomic profiling (Geomx) of formalin-fixed paraffin-embedded (FFPE) lung tissue sections (n = 19); and (2) via triple immunofluorescence staining of bronchoalveolar lavage (BAL) AMs (n = 83). PD-L1 mRNA expression was also quantified in BAL AMs exposed to CS extract. PD-L1 expression was increased in the bronchiolar wall, parenchyma, and vascular wall from mild-moderate (GOLD 1–2) COPD patients compared to severe-very severe (GOLD 3–4) COPD patients and controls. Within all the COPD patients, PD-L1 protein expression was associated with upregulation of genes involved in tumor progression and downregulation of oncosuppressive genes, and strongly directly correlated with the FEV1% predicted, indicating higher PD-L1 expression in the milder vs. more severe COPD stages. In bronchioles, PD-L1 levels were strongly directly correlated with the number of functionally active AMs. In BAL, we confirmed that AMs from patients with both GOLD 1–2 COPD and NSCLC had the highest and similar, PD-L1 expression levels versus all the other groups, independently from active cigarette smoking. Intriguingly, AMs from patients with more severe COPD had reduced AM PD-L1 expression compared to patients with mild COPD. Acute CS extract stimulation increased PD-L1 mRNA expression only in never-and not in ever-smoker AMs. Lungs from patients with mild COPD and NSCLC are characterized by a similar strong PD-L1 expression signature in bronchioles and functionally active AMs compared to patients with severe COPD and controls. Active smoking does not affect PD-L1 levels. These observations represent a new resource in understanding the innate immune mechanisms underlying the link between COPD and lung cancer onset and progression and pave the way to future studies focused on the mechanisms by which CS promotes tumorigenesis and COPD.

PD-L1 on Circulating Tumor Cells Indicates Poor Prognosis in Breast Cancer

Background: In breast cancer, research concerning programmed death-ligand 1 (PD-L1) on circulating tumor cells (CTCs) is limited. Besides, the best cell type for determining the prognostic and predictive values of PD-L1 has not been established. This study investigated associations between PD-L1 on tumor cells (TCs), CTCs, and tumor-infiltrating immune cells (TIICs), respectively, and prognosis and clinicopathological features in breast cancer. Methods: Twenty patients with breast cancer were recruited; one was excluded for confirmed lymphoma. The PD-L1 on TCs and TIICs was determined via immunohistochemistry; PD-L1 mRNA expression on CTCs was analyzed. The chi-squared test and Kaplan-Meier and Cox proportional hazards model analyses were applied. Results: The median follow-up time was 60 months. Of the 19 patients, 14 had &gt;1 CTC/10 mL peripheral blood. Among these, each had ≥1 CTC showing PD-L1. At baseline, there was no difference between groups with or without metastasis regarding CTCs and PD-L1 expression. Patients with high PD-L1 levels on CTCs had poor overall survival (p = 0.034). In the multivariate analysis, PD-L1 levels on CTCs and T stage were independent prognostic factors (p = 0.029, 0.010, respectively). Conclusions: High levels of PD-L1 on CTCs may prognose shorter overall survival in breast cancer.

EP01.01-008 Expression of PD-L1 mRNA and Its Epigenetic Regulators: MicroRNA-17, -93 and -142 in Early Stages NSCLC Patients’ Plasma

One of the leading treatment options for advanced non-small cell lung cancer (NSCLC) patients is immunotherapy based on immune checkpoint inhibitors, including programmed death ligand 1 (PD-L1) inhibitors. PD-L1 molecule expressed on tumor cells causes PD-1+ tumor infiltrating lymphocytes (TILs) exhaustion and thus escape from immune surveillance. Blockade of the PD-1/PD-L1 pathway by monoclonal antibodies enables restoration of the anti-tumor response. PD-L1 expression is regulated by epigenetic factors, e.g.: microRNAs (miRNAs).

Prognostic significance of programmed death-1 and programmed death ligand-1 proteins in breast cancer.

In numerous studies related to tumor prognosis, programmed death-ligand 1 (PD-L1) has been identified as a biomarker. This work aimed to determine the prognostic importance of PD-L1 in breast cancer. We searched electronic databases such as PubMed, Google scholar, home pages of publishing groups, medical, clinical, and pharmaceutical sciences journals, as well as other relevant sources to discover the importance of PD-1 and PD-L1 expression in breast cancer therapies and also recurrence. The keywords used in this search were autoimmunity, programmed cell death, PD-L1 or PD-1, and breast cancer. Our inclusion criteria included studies showing the synergy between the expression of PD-L1 and PD-1 in primary breast cancers as prognostic markers and this research was limited to humans only. We included review articles, original research, letters to the editor, case reports, and short communications in our study, published in English. We focused our work on PD-L1 mRNA expression in breast cancer cell lines. PD-L1 expression has been decisively demonstrated to be a high-risk factor for breast cancer with a bad prognosis.

Fusobacterium is enriched in oral cancer and promotes induction of programmed death-ligand 1 (PD-L1)

Recently, increased number of studies have demonstrated a relationship between the oral microbiome and development of head and neck cancer, however, there are few studies to investigate the role of oral bacteria in the context of the tumor microenvironment in a single head and neck subsite. Here, paired tumor and adjacent normal tissues from thirty-seven oral tongue squamous cell carcinoma (SCC) patients were subjected to 16S rRNA gene sequencing and whole exome sequencing (WES), in addition to RNA sequencing for tumor samples. We observed that Fusobacterium was significantly enriched in oral tongue cancer and that Rothia and Streptococcus were enriched in adjacent normal tissues. A decrease in alpha diversity was found in tumor when compared to adjacent normal tissues. While increased Fusobacterium in tumor samples was not associated with changes in immune cell infiltration, it was associated with increased PD-L1 mRNA expression. Therefore, we examined the effects of Fusobacterium on PD-L1 expression in head and neck SCC cell lines. We demonstrated that infection with Fusobacterium species can increase both PD-L1 mRNA and surface PD-L1 protein expression on head and neck cancer cell lines. The correlation between Fusobacterium and PD-L1 expression in oral tongue SCC, in conjunction with the ability of the bacterium to induce PD-L1 expression in vitro suggests a potential role for Fusobacterium on modulation of the tumor immune microenvironment in head and neck cancer.

Programmed Death-Ligand 1 and Programmed Death-Ligand 2 mRNAs Measured Using Closed-System Quantitative Real-Time Polymerase Chain Reaction Are Associated With Outcome and High Negative Predictive Value in Immunotherapy-Treated NSCLC

IntroductionImmune checkpoint inhibitors (ICIs) have become standard of care in lung cancer management, but only a relatively small percentage of patients treated respond. Current predictive biomarkers, including immunohistochemical detection of programmed death-ligand 1 (PD-L1), are insufficient for determining who will respond or, more importantly in the adjuvant setting, who will not respond to ICI therapy. Here, we investigate an alternative method of assessment of PD-L1 to predict nonresponse. MethodsThis study uses a research use only quantitative real-time reverse transcription polymerase chain reaction assay on the GeneXpert system, to test for the association between four target immune genes, CD274 (PD-L1), PDCD1LG2 (programmed death-ligand 2 [PD-L2]), CD8A, and IRF1, and response to ICI therapy. Tissues were collected from 122 patients with advanced NSCLC before ICI therapy in a retrospective cohort, macrodissected, and analyzed using the GeneXpert. ResultsBoth high PD-L1 and PD-L2 mRNA expression levels were associated with improved long-term benefit at 24 months (p = 0.047 for both PD-L1 and PD-L2) and overall survival (PD-L1, p = 0.048; PD-L2, p = 0.049). Both PD-L1 and PD-L2 mRNA levels were higher in patients with KRAS mutations. Most importantly, low PD-L1 mRNA level had a high negative predictive value of 0.92 for absence of long-term benefit. ConclusionsWith further validation of this assay in low-stage patients, an assessment of PD-L1 mRNA rather than protein, could be a method to determine which low-stage patients that should not be treated with ICIs in the adjuvant setting. This approach may also be a useful objective method for selecting patients for treatment in the advanced setting.

Expression Modulation of Immune Checkpoint Molecules by Ibrutinib and Everolimus Through STAT3 in MCF-7 Breast Cancer Cells

Tumor-targeted therapy with small-molecule inhibitors (SMIs) has been demonstrated to be a highly effective therapeutic strategy for various cancers. However, their possible associations with immune evasion mechanisms remain unknown. This study examined the association of inhibitors of the protein kinase B (AKT), mammalian target of rapamycin (mTOR), and Bruton’s tyrosine kinase (BTK) signaling pathways with the expression of immune checkpoint ligands programmed death-ligand 1 (PD-L1), CD155, and galectin-9 (Gal-9) in a breast cancer cell line. MCF-7 cells were treated with everolimus, MK-2206, and ibrutinib. An MTT assay was used to determine the optimal dose for all drugs. A real-time polymerase chain reaction was utilized to measure the mRNA expression of PD-L1, CD155, and Gal-9. The western blot technique was also employed to evaluate the protein expression of the phosphorylated signal transducer and activator of transcription 3 (STAT3). The optimal doses of everolimus, MK-2206, and ibrutinib were observed to be 200, 320, and 2000 nM, respectively. The PD-L1 and CD155 mRNA expression was significantly decreased following the treatment with everolimus and ibrutinib, but not with MK-2206. There were no differences in Gal-9 expression between the single-treated and control groups; however, combined treatment with everolimus and ibrutinib increased its mRNA expression. Everolimus and ibrutinib both inhibited constitutive STAT3 phosphorylation in MCF-7, which was more pronounced in combination treatment. The findings regarding the modulation of PD-L1, CD155, and Gal-9 molecules by SMIs emphasize the crosstalk between the expression of these immune checkpoint molecules and AKT/mTOR/BTK signaling pathways through STAT3 as a critical transcription factor.

Harmonization of programmed death-ligand 1 immunohistochemistry and mRNA expression scoring in metastatic melanoma: a multicentre analysis.

The aim of this multicentre study was to harmonize programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) and melanoma scoring. To provide a reference for PD-L1 expression independently of the IHC protocol, PD-L1 mRNA expression was compared with IHC. Standardized PD-L1 assays (22C3, 28-8, SP142, SP263) and laboratory-developed tests (QR1, 22C3) were evaluated on three IHC platforms with a training set (seven cases). mRNA expression was determined by RNAscope (CD274/PD-L1 probe) and analysed with image analysis. PD-L1 IHC findings were scored by seven blinded pathologists using the tumour proportion score (TPS), the combined positive score (CPS), and the MELscore. This method was validated by three blinded pathologists on 40 metastatic melanomas. Concordances among various antibody/platforms were high across antibodies [intraclass correlation coefficient (ICC) >0.80 for the CPS], except for SP142. Two levels of immunostaining intensity were observed: high (QR1 and SP263) and low (28-8, 22C3, and SP142). Reproducibilities across pathologists were higher for QR1 and SP263 (ICC ≥0.87 and ICC ≥0.85 for the TPS and the CPS, respectively). QR1, SP263 and 28-8 showed the highest concordance with mRNA expression. We developed a standardized method for PD-L1 immunodetection and scoring, tested on 40 metastatic melanomas. Concordances among antibodies were excellent for all criteria, and concordances among pathologists were better for the MELscore than for other scores. Harmonization of PD-L1 staining and scoring in melanomas with good concordance is achievable with the PD-L1 IHC protocols applied to other cancers; this reproducible approach can simplify daily practice.

Expression of CD274 mRNA Measured by qRT-PCR Correlates With PD-L1 Immunohistochemistry in Gastric and Urothelial Carcinoma.

Programmed death-ligand 1 (PD-L1) immunohistochemistry (IHC) is widely used to predict the clinical responses to immune checkpoint inhibitors (ICIs). However, PD-L1 IHC suffers from the complexity of multiple testing platforms and different cutoff values caused by the current one drug-one diagnostic test co-development approach for ICIs. We aimed to test whether PD-L1 (CD274) mRNA expression levels measured using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) can represent PD-L1 IHC and predict responses to ICI. The FDA-approved PD-L1 IHC results with 22C3 pharmDx (gastric cancer) and SP142 (urothelial carcinoma) were compared with CD274 mRNA expression levels via qRT-PCR using the same formalin-fixed, paraffin-embedded tissue blocks from 59 gastric cancer and 41 urothelial carcinoma samples. CD274 mRNA expression was identified using three independent sets of primers and TaqMan® probes targeting exon 1–2, exon 3–4, and exon 5–6. CD274 mRNA levels in spanning exon 1–2, exon 3–4, and exon 5–6 junctions of CD274 correlated well with PD-L1 expression (r2=0.81, 0.65, and 0.59, respectively). The area under the curve of exon 1–2 was the highest (0.783), followed by exon 3–4 (0.701), and exon 5–6 (0.671) of the CD274 gene against the PD-L1 combined positive score cutoff of 10. When CD274 mRNA expression was matched for response to immunotherapy, the overall response rate was higher in patients with high CD274 mRNA levels with a cutoff of 0.0722 (gastric cancer) and 0.0480 (urothelial carcinoma) than in those with low CD274 mRNA expression (P < 0.001 and P = 0.018, respectively). These results show that CD274 mRNA levels predicted ICI responses in patients with gastric or urothelial carcinomas and could be used as alternatives for PD-L1 IHC.

Ezrin Regulates the Cell Surface Localization of PD-L1 in HEC-151 Cells.

Programmed death ligand-1 (PD-L1) is an immune checkpoint molecule widely expressed on the surface of cancer cells and is an attractive immunotherapeutic target for numerous cancer cell types. However, patients with endometrial cancer derive little clinical benefit from immune checkpoint blockade therapy because of their poor response rate. Despite the increasingly important function of PD-L1 in tumor immunology, the mechanism of PD-L1 localization on endometrial cancer cell surfaces is largely unknown. We demonstrated the contribution of the ezrin, radixin, and moesin (ERM) family, which consists of scaffold proteins that control the cell surface localization of several transmembrane proteins to the localization of PD-L1 on the cell surface of HEC-151, a human uterine endometrial cancer cell line. Confocal immunofluorescence microscopy and immunoprecipitation analysis revealed the colocalization of all the ERM with PD-L1 on the cell surface, as well as their protein–protein interactions. The RNA-interference-mediated knockdown of ezrin, but not radixin and moesin, significantly reduced the cell surface expression of PD-L1, as measured by flow cytometry, with little impact on the PD-L1 mRNA expression. In conclusion, among the three ERM proteins present in HEC-151 cells, ezrin may execute the scaffold function for PD-L1 and may be mainly responsible for the cell surface localization of PD-L1, presumably via the post-translational modification process.