Programmed Death-ligand 1 mRNA Levels Research Articles

Detection of Exosomal PD-L1 RNA in Saliva of Patients With Periodontitis.

Periodontitis is the most prevalent inflammatory disease of the periodontium, and is related to oral and systemic health. Exosomes are emerging as non-invasive biomarker for liquid biopsy. We here evaluated the levels of programmed death-ligand 1 (PD-L1) mRNA in salivary exosomes from patients with periodontitis and non-periodontitis controls. The purposes of this study were to establish a procedure for isolation and detection of mRNA in exosomes from saliva of periodontitis patients, to characterize the level of salivary exosomal PD-L1, and to illustrate its clinical relevance. Bioinformatics analysis suggested that periodontitis was associated with an inflammation gene expression signature, that PD-L1 expression positively correlated with inflammation in periodontitis based on gene set enrichment analysis (GSEA) and that PD-L1 expression was remarkably elevated in periodontitis patients versus control subjects. Exosomal RNAs were successfully isolated from saliva of 61 patients and 30 controls and were subjected to qRT-PCR. Levels of PD-L1 mRNA in salivary exosomes were higher in periodontitis patients than controls (P < 0.01). Salivary exosomal PD-L1 mRNA showed significant difference between the stages of periodontitis. In summary, the protocols for isolating and detecting exosomal RNA from saliva of periodontitis patients were, for the first time, characterized. The current study suggests that assay of exosomes-based PD-L1 mRNA in saliva has potential to distinguish periodontitis from the healthy, and the levels correlate with the severity/stage of periodontitis.

Epigenetic treatment-mediated modulation of PD-L1 predicts potential therapy resistance over response markers in myeloid malignancies: A molecular mechanism involving effectors of PD-L1 reverse signaling.

Recent evidence suggests an association exists between resistance to epigenetic therapy (EGT) and the expression of programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) in myeloid malignancies. Biomarkers are required to predict resistance to EGT in myeloid malignancies, which together with the delineation of associated molecular mechanisms, may provide additional understanding for novel treatment strategies when investigating resistance to EGT. The present study aimed to investigate the in vivo effects of EGT on the expression of PD-1, PD-L1 and orphan nuclear receptor NUR77 with clinical responses in patients with myeloid malignancies. In addition, in vitro and in vivo characterization of the effects of EGT on the expression of NF-κB and Bcl-xL, potential downstream targets of PD-L1 reverse signaling, were evaluated to determine components of the molecular mechanism responsible for these effects. The in vivo effects of EGT on the expression of PD-1, PD-L1 and a previously identified molecular marker of response to EGT, NUR77 was characterized in peripheral blood mononuclear cells (PBMC) from patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) treated with either azacytidine (Aza) alone or a combination of Aza and the histone deacetylase inhibitor (HDACi) LBH-589 during the first cycle of therapy. The correlation of clinical responses to treatment with EGT with the expression of PD-1, PD-L1 and NUR77 demonstrated that the induction of PD-L1 mRNA levels was associated with resistance to EGT despite the concurrent augmentation of NUR77 expression. The characterization of potential downstream effector molecules of reverse PD-L1 signaling identified EGT-mediated induction of Bcl-xL and NF-κB mRNA expression in vitro and in vivo, suggesting a potential anti-apoptotic molecular mechanism was responsible for PD-L1-mediated resistance to EGT. Taken together, these observations suggest that enhanced PD-L1 expression may confer resistance to EGT over known EGT response markers in myeloid malignancies, and provides a potential molecular mechanism involving the modulation of effectors of PD-L1 reverse signaling, which may in-part, be responsible for these effects.

Frequencies and expression levels of programmed death ligand 1 (PD-L1) in circulating tumor RNA (ctRNA) in various cancer types

BackgroundPrecision medicine and prediction of therapeutic response requires monitoring potential biomarkers before and after treatment. Liquid biopsies provide noninvasive prognostic markers such as circulating tumor DNA and RNA. Circulating tumor RNA (ctRNA) in blood is also used to identify mutations in genes of interest, but additionally, provides information about relative expression levels of important genes. In this study, we analyzed PD-L1 expression in ctRNA isolated from various cancer types. Tumors inhibit antitumor response by modulating the immune checkpoint proteins programmed death ligand 1 (PD-L1) and its cognate receptor PD1. The expression of these genes has been implicated in evasion of immune response and resistance to targeted therapies. MethodsBlood samples were collected from gastric (GC), colorectal (CRC), lung (NSCLC), breast (BC), prostate cancer (PC) patients, and a healthy control group. ctRNA was purified from fractionated plasma, and following reverse transcription, levels of PD-L1 expression were analyzed using qPCR. ResultsPD-L1 expression was detected in the plasma ctRNA of all cancer types at varying frequencies but no PD-L1 mRNA was detected in cancer-free individuals. The frequencies of PD-L1 expression were significantly different among the various cancer types but the median relative PD-L1 expression values were not significantly different. In 12 cases where plasma and tumor tissue were available from the same patients, there was a high degree of concordance between expression of PD-L1 protein in tumor tissues and PD-L1 gene expression in plasma, and both methods were equally predictive of response to nivolumab. ConclusionsPD-L1 mRNA can be detected and quantitated in ctRNA of cancer patients. These results pave the way for further studies aimed at determining whether monitoring the levels of PD-L1 mRNA in blood can identify patients who are most likely to benefit from the conventional treatment.

YAP regulates PD-L1 expression in human NSCLC cells.

Programmed death-ligand 1 (PD-L1) is a membrane protein on tumor cells that binds to the PD-1 receptor expressed on immune cells, leading to the immune escape of tumor cells. Yes-associated protein (YAP) is a main effector of the Hippo/YAP signaling pathway, which plays important roles in cancer development. Here we show that YAP regulates PD-L1 expression in human non-small cell lung cancer (NSCLC) cells. First, we investigated YAP and PD-L1 expression at the protein level in 142 NSCLC samples and 15 normal lung samples. In tumor tissue, immunohistochemistry showed positive staining for YAP and PD-L1, which correlated significantly (n = 142, r = 0.514, P < 0.001). Second, in cell lines that express high levels of PD-L1 (H460, SKLU-1, and H1299), the ratio of p-YAP/YAP was lower and GTIIC reporter activity of the Hippo pathway was higher than those in three cell lines expressing low levels of PD-L1 (A549, H2030, and PC9) (P < 0.05). Third, in the same three cell lines, inhibition of YAP by two small interfering RNAs (siRNAs) decreased the mRNA and protein level of PD-L1 (P < 0.05). Fourth, forced overexpression of the YAP gene rescued the PD-L1 mRNA and protein level after siRNA knockdown targeting 3′UTR of the endogenous YAP gene. Finally, chromatin immunoprecipitation (ChIP) assays using a YAP-specific monoclonal antibody resulted in the precipitation of PD-L1 enhancer region encompassing two putative TEAD binding sites. Our results indicate that YAP regulates the transcription of PD-L1 in NSCLC.

Comparison of PD-L1 mRNA Expression Measured with the CheckPoint Typer® Assay with PD-L1 Protein Expression Assessed with Immunohistochemistry in Non-small Cell Lung Cancer.

Immunohistochemical (IHC) assessment of programmed death-ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC) has become important since the development of anti-PD-1/-PD-L1 directed drugs. Various PD-L1 antibodies and cut-offs have been used in different trials to predict response to these drugs, thus comparison of those studies is difficult. We compared PD-L1 mRNA expression measured by RT-qPCR with PD-L1 protein expression evaluated by IHC. Moreover, we investigated the impact of different tumour tissue acquisition methods on the reliability of PD-L1 measurement techniques. NSCLC cases (N=22), including n=9 mediastinal lymph node biopsies acquired by endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) and n=5 metastases, were evaluated prospectively for PD-L1 protein on tumor cells (TC) and immune cells (IC) using E1L3N and 28-8 antibodies and PD-L1 mRNA using the CheckPoint TYPER® assay. In primary NSCLC tissues, agreement between PD-L1 mRNA and TC staining using the 28-8 antibody was excellent (ĸ=0.85, p=0.0002). Comparing both PD-L1 antibodies against each other showed a kappa value of 0.58 (p=0.0106). In EBUS-TBNA, PD-L1 mRNA correlated perfectly with the 28-8 antibody (ĸ=1.0, p=0.0023). PD-L1 mRNA levels significantly differed when comparing 28-8 TC staining of tumours >49% with 1-49% and 0% (p=0.0040; p=0.0081, respectively). In metastatic lesions, differences between PD-L1 mRNA and IHC became apparent (ĸ=0.2, p=0.2525). Testing of PD-L1 mRNA and 28-8 IHC showed an excellent agreement in NSCLC samples including mediastinal lymph node biopsies. Since PD-L1 expression in >50% TC detected by 28-8 IHC can be reliably detected by RT-qPCR, quantitative PD-L1 mRNA determination should be considered as an alternative to IHC as there is no interobserver variability in RNA results.

Programmed death-ligand 1 (PD-L1) expression in tumour cell and tumour infiltrating lymphocytes of HER2-positive breast cancer and its prognostic value

Immunotherapy targeting PD-1/PD-L1 axis showed benefits in cancer. Prognostic significance of tumour infiltrating lymphocytes (TILs) has been determined. We evaluated PD-L1 protein expression in tumour cells and TILs, PD-L1 mRNA level and various histopathologic factors including TILs using 167 formalin-fixed paraffin embedded tissues and 39 fresh tissue of HER2-positive breast cancer. TILs level and PD-L1 expression in tumour cells and TILs were significantly correlated one another. PD-L1 positivity in tumour cells was associated with high histologic grade and high TILs level (p < 0.001, both). High PD-L1 immunoscore in TILs and high total immunoscore (in tumour cells and TILs) of PD-L1 were correlated with high histologic grade (p = 0.001 and p < 0.001, respectively), absence of lymphovascular invasion (p = 0.012 and p = 0.007, respectively), negative hormone receptor expression (p = 0.044 and p = 0.001, respectively) and high TILs level (p < 0.001, both). High PD-L1 mRNA expression was associated with high TILs level (p < 0.001, both). PD-L1 positivity in tumour cells was associated with better disease-free survival in HR−/HER2+ breast cancer (p = 0.039). PD-L1 expression in tumour cells and TILs are significantly associated with TILs level in HER2-positive breast cancer. PD-L1 expression in tumour cells might be positive prognostic factor in HR−/HER2+ breast cancers.

Effects of programmed death-ligand 1 expression on OK-432 immunotherapy following transurethral resection in non-muscle invasive bladder cancer.

The present study aimed to investigate the effect of the negative costimulatory molecule programmed death-ligand 1 (PD-L1) on immunotherapy with OK-432, following transurethral resection of bladder tumors in non-muscle invasive bladder cancer (NMIBC), and to elucidate the underlying mechanism. PD-L1 was detected by immunohistochemical staining in tumor specimens from 55 cases of NMIBC following postoperative immunotherapy with OK-432. The PD-L1 mRNA and protein expression levels were measured in the bladder cancer T24 cell line and the human uroepithelial SV-HUC-1 cell line, following treatment with interleukin (IL)-2, interferon (IFN)-α and IFN-γ, by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. PD-L1 was widely expressed in the NMIBC tumors, with 56.4% (31/55) of specimens exhibiting positive staining. When compared with PD-L1-negative patients, PD-L1-positive patients exhibited significantly increased recurrence [48.4% (15/31) vs. 16.7% (4/24)] and progression [16.1% (5/31) vs. 4.2% (1/24)] rates (P<0.05). RT-qPCR and western blotting demonstrated that cytokines IL-2, IFN-α and IFN-γ markedly upregulated PD-L1 mRNA expression rates and protein levels in bladder cancer T24 cells (P<0.05), but had no significant effect in non-tumor SV-HUC-1 cells. In conclusion, PD-L1 expression was negatively-associated with the efficacy of OK-432 intravesical immunotherapy in patients with NMIBC. The results indicated that the involved mechanism occurred via upregulation of PD-L1 by immune cytokines, which in turn suppressed the antitumor effectiveness of the immune system, thereby promoting tumor recurrence and progression.

Increased expression of programmed death ligand 1 (PD-L1) in human pituitary tumors.

PurposeSubsets of pituitary tumors exhibit an aggressive clinical courses and recur despite surgery, radiation, and chemotherapy. Because modulation of the immune response through inhibition of T-cell checkpoints has led to durable clinical responses in multiple malignancies, we explored whether pituitary adenomas express immune-related biomarkers that could suggest suitability for immunotherapy. Specifically, programmed death ligand 1 (PD-L1) has emerged as a potential biomarker whose expression may portend more favorable responses to immune checkpoint blockade therapies. We thus investigated the expression of PD-L1 in pituitary adenomas.MethodsPD-L1 RNA and protein expression were evaluated in 48 pituitary tumors, including functioning and non-functioning adenomas as well as atypical and recurrent tumors. Tumor infiltrating lymphocyte populations were also assessed by immunohistochemistry.ResultsPituitary tumors express variable levels of PD-L1 transcript and protein. PD-L1 RNA and protein expression were significantly increased in functioning (growth hormone and prolactin-expressing) pituitary adenomas compared to non-functioning (null cell and silent gonadotroph) adenomas. Moreover, primary pituitary adenomas harbored higher levels of PD-L1 mRNA compared to recurrent tumors. Tumor infiltrating lymphocytes were observed in all pituitary tumors and were positively correlated with increased PD-L1 expression, particularly in the functional subtypes.ConclusionsHuman pituitary adenomas harbor PD-L1 across subtypes, with significantly higher expression in functioning adenomas compared to non-functioning adenomas. This expression is accompanied by the presence of tumor infiltrating lymphocytes. These findings suggest the existence of an immune response to pituitary tumors and raise the possibility of considering checkpoint blockade immunotherapy in cases refractory to conventional management.

PD-L1 Expression by Two Complementary Diagnostic Assays and mRNA In Situ Hybridization in Small Cell Lung Cancer.

Therapeutic antibodies to immune checkpoints show promising results. Programmed death-ligand 1 (PD-L1), an immune checkpoint ligand, blocks the cancer immunity cycle by binding the PD-L1 receptor (programmed death 1). We investigated PD-L1 protein expression and messenger RNA (mRNA) levels in SCLC. PD-L1 protein expression and mRNA levels were determined by immunohistochemistry (IHC) with SP142 and Dako 28-8 PD-L1 antibodies and in situ hybridization in primary tumor tissue microarrays in both tumor cells and tumor-infiltrating immune cells (TIICs) obtained from a limited-disease SCLC cohort of 98 patients. An additional cohort of 96 tumor specimens from patients with extensive-disease SCLC was assessed for PD-L1 protein expression in tumor cells with Dako 28-8 antibody only. The overall prevalence of PD-L1 protein expression in tumor cells was 16.5%. In the limited-disease cohort, the prevalences of PD-L1 protein expression in tumor cells with SP142 and Dako 28-8 were 14.7% and 19.4% (tumor proportion score cutoff ≥1%) and PD-L1 mRNA ISH expression was positive in 15.5% of tumor samples. Increased PD-L1 protein/mRNA expression was associated with the presence of more TIICs (p < 0.05). The extensive-disease cohort demonstrated a 14.9% positivity of PD-L1 protein expression in tumor cells with Dako 28-8 antibody. A subset of SCLCs is characterized by positive PD-L1 and/or mRNA expression in tumor cells. Higher PD-L1 and mRNA expression correlate with more infiltration of TIICs. The prevalence of PD-L1 in SCLC is lower than that published for NSCLC. The predictive role of PD-L1 expression in SCLC treatment remains to be established.

Renal Cell Carcinoma Programmed Death-ligand 1, a New Direct Target of Hypoxia-inducible Factor-2 Alpha, is Regulated by von Hippel–Lindau Gene Mutation Status

BackgroundClear cell renal cell carcinomas (ccRCC) frequently display a loss of function of the von Hippel–Lindau (VHL) gene. ObjectiveTo elucidate the putative relationship between VHL mutation status and immune checkpoint ligand programmed death-ligand 1 (PD-L1) expression. Design, setting, and participantsA series of 32 renal tumors composed of 11 VHL tumor-associated and 21 sporadic RCCs were used to evaluate PD-L1 expression levels after sequencing of the three exons and exon–intron junctions of the VHL gene. The 786-O, A498, and RCC4 cell lines were used to investigate the mechanisms of PD-L1 regulation. Outcome measurements and statistical analysisFisher's exact test was used for VHL mutation and Kruskal–Wallis test for PD-L1 expression. If no covariate accounted for the association of VHL and PD-L1, then a Kruskal–Wallis test was used; otherwise Cochran–Mantel–Haenzsel test was used. We also used the Fligner–Policello test to compare two medians when the distributions had different dispersions. Results and limitationsWe demonstrated that tumors from ccRCC patients with VHL biallelic inactivation (ie, loss of function) display a significant increase in PD-L1 expression compared with ccRCC tumors carrying one VHL wild-type allele. Using the inducible VHL 786-O-derived cell lines with varying hypoxia-inducible factor-2 alpha (HIF-2α) stabilization levels, we showed that PD-L1 expression levels positively correlate with VHL mutation and HIF-2α expression. Targeting HIF-2α decreased PD-L1, while HIF-2α overexpression increased PD-L1 mRNA and protein levels in ccRCC cells. Interestingly, chromatin immunoprecipitation and luciferase assays revealed a direct binding of HIF-2α to a transcriptionally active hypoxia-response element in the human PD-L1 proximal promoter in 786-O cells. ConclusionsOur work provides the first evidence that VHL mutations positively correlate with PD-L1 expression in ccRCC and may influence the response to ccRCC anti-PD-L1/PD-1 immunotherapy. Patient summaryWe investigated the relationship between von Hippel–Lindau mutations and programmed death-ligand 1 expression. We demonstrated that von Hippel–Lindau mutation status significantly correlated with programmed death-ligand 1 expression in clear cell renal cell carcinomas.