Programmed Cell Death Ligand 1 mRNA Research Articles

Escherichia coli K12 Upregulates Programmed Cell Death Ligand 1 (PD-L1) Expression in Gamma Interferon-Sensitized Intestinal Epithelial Cells via the NF-κB Pathway.

Programmed cell death ligand-1 (PD-L1) is an immune checkpoint protein which is used by tumor cells for immune evasion. PD-L1 is upregulated in inflamed intestinal tissues. The intestinal tract is colonized by millions of bacteria, most of which are commensal bacterial species. We hypothesized that under inflammatory conditions, some commensal bacterial species contribute to increased PD-L1 expression in intestinal epithelium and examined this hypothesis. Human intestinal epithelial HT-29 cells with and without interferon (IFN)-γ sensitization were incubated with six strains of four enteric bacterial species. The mRNA and protein levels of PD-L1 in HT-29 cells were examined using quantitative real-time PCR and flow cytometry, respectively. The levels of interleukin (IL)-1β, IL-18, IL-6, IL-8, and tumor necrosis factor (TNF)-α secreted by HT-29 cells were measured using enzyme-linked immunosorbent assay. Apoptosis of HT-29 cells was measured using a caspase 3/7 assay. We found that Escherichia coli K12 significantly upregulated both PD-L1 mRNA and protein in IFN-γ-sensitized HT-29 cells. E. coli K12 induced the production of IL-8 in HT-29 cells, however, IL-8 did not affect HT-29 PD-L1 expression. Inhibition of the nuclear factor-kappa B pathway significantly reduced E. coli K12-induced PD-L1 expression in HT-29 cells. The other two E. coli strains and two enteric bacterial species did not significantly affect PD-L1 expression in HT-29 cells. Enterococcus faecalis significantly inhibited PD-L1 expression due to induction of cell death. Data from this study suggest that some gut bacterial species have the potential to affect immune function under inflammatory conditions via upregulating epithelial PD-L1 expression.

RNA N6-methyladenosine demethylase FTO regulates PD-L1 expression in colon cancer cells

Fat mass and obesity-associated protein (FTO) is an enzyme that demethylates N6-methyladenosine (m6A), the most abundant RNA modifications in a cell. The upregulated expression of FTO promotes the progression of various types of cancer by modulating cell-intrinsic genes which relate to malignant potential. However, the impact of FTO on the expression of immune-checkpoint molecules in the tumor cells, which are important for immune escape, has not been well understood. We examined the relevance of FTO to programmed cell death-ligand 1 (PD-L1) expression in colon cancer cells. HCT-116 cells showed high expression of both FTO and PD-L1 proteins. The knockdown of FTO by small interfering RNA decreased mRNA and protein levels of PD-L1 in HCT-116 cells. To elucidate the underlying mechanism by which FTO regulates the expression of PD-L1, we depleted FTO in HCT-116 in the presence of IFN-γ, which is a major stimulus to upregulate PD-L1 expression. Depletion of FTO reduced PD-L1 expression in an IFN-γ signaling-independent manner. RNA immunoprecipitation assay revealed the m6A modification of the PD-L1 mRNA and the binding of FTO to the PD-L1 mRNA in HCT-116. Taken together, our results indicated that FTO could regulate PD-L1 expression in colon cancer cells and provides new insights into the regulation of PD-L1 expression by RNA modification.

Lentinan, a Shiitake Mushroom ß-Glucan, Downregulates the Enhanced PD-L1 Expression Induced by Platinum Compounds in Gastric Cancer Cells -

Background: Despite recent therapeutic improvements, the prognosis of unresectable gastric cancer remains poor. Upregulation of programmed cell death ligand 1 (PD-L1) in tumor cells is believed to be an important mechanism to escape from the host immune response. The expression of PD-L1 in tumors is regulated in a highly complex manner by various upstream signaling molecules, depending on the cell type. Given that the efficacy of chemotherapeutic agents for metastatic gastric cancer is limited due to immune escape caused by enhanced PD-L1 expression, PD-1/PD-L1 targeted immunotherapy may be a promising alterative for chemotherapy. However, immune checkpoint inhibitor monotherapy has shown clinical benefits in less than 20% of patients with gastric cancer and its underlying mechanism remains to be elucidated. On the other hand, lentinan, a glucan purified from Shiitake mushrooms, has significant immune-stimulating effects and has been reported to improve survival in patients with metastatic gastric cancer receiving chemotherapy. In the current study we investigated the mechanism by which lentinan increases the chemotherapeutic efficacy by focusing on the expression of PD-L1. Methods: To evaluate the effects of lentinan as well as antineoplastic agents, the expression of PD-L1 and associated molecules was analyzed by real-time polymerase chain reaction and western blotting using the human gastric cancer cell lines, NUGC3, MKN1, and MKN45. Results: Treatment with either cisplatin or oxaliplatin dose-dependently enhanced PD-L1 mRNA and protein expression through the mitogen-activated protein kinase (MAPK) pathway in gastric cancer cells. However, lentinan treatment inhibited the platinum drug-stimulated expression of PD-L1 in gastric cancer cells mainly by suppressing MAPK signaling without affecting the phosphatidylinositol-3 kinase/AKT pathway or transcription factors. Conclusions: Platinum-based drugs enhanced the expression of PD-L1 via the MAPK pathway in gastric cancer cells. Lentinan downregulated PD-L1 expression induced by either cisplatin or oxaliplatin, suggesting that a combination of this glucan and platinum-based chemotherapy could restore the chemosensitivity of cells.

662 Targeting the α9-nAChR/PD-L1 axis in antiproliferative effects of Allium cepa L. var. proliferum Regel extract on melanoma cells

Programmed cell death ligand-1 (PD-L1) is critical for melanoma development, progression and treatment. The α9 nicotinic acetylcholine receptor (nAChR) is shown to affect melanoma cell proliferation. Allium cepa L. var. proliferum Regel extract possesses anticancer properties. However, the mechanism of Allium cepa L. var. proliferum Regel extract in the antiproliferative effects of melanoma cells remains unclear. Using UPLC-ESI-MS/MS method, a total of 42 compounds were identified in Allium cepa L. var. proliferum Regel extract classified into five categories, including organosulfur compounds (962.20 ± 34.55 μg/g), polyphenols (100.40 ± 12.55 μg/g), flavonoids (58.36 ± 10.75 μg/g), organic acids (54.04 ± 2.69 μg/g), and alkaloids (15.08 ± 3.10 μg/g) where we found various bioactive components targeted to nAChRs. We analyzed the genetic dependency of α1-10 nAChRs from Achilles_CRISPR dataset (n=38) using CRISPR-Cas9 technology. α9-nAChR contributed significantly to the survival of melanoma cells (dependency scores < 0). We evaluated α1-10 nAChRs and PD-L1 mRNA expression from CCLE_Expression dataset (n=50). α9-nAChR was the only gene that strongly correlated to PD-L1 expression (r=0.6, p<0.001). There was also a strong correlation between α9-nAChR and PD-L1 expression (r=0.7, p<0.001) in the tissue microarray (n=192) using the IHC staining. Knockdown or overexpression of α9-nAChR significantly down- or upregulated the expression of PD-L1 via the transcription factor STAT3 binding to the PD-L1 promoter, respectively, and affected melanoma cell proliferation. Allium cepa L. var. proliferum Regel extract inhibited melanoma cell proliferation through inactivation of α9-nAChR/PD-L1 axis. Our results suggest that α9-nAChR/ PD-L1 axis regulates the cell proliferation of melanoma and Allium cepa L. var. proliferum Regel extract is considered as a potential candidate to prevent melanoma.

Abstract 1773: The tyrosine phosphatase, SHP-2, is involved in regulating PD-L1 expression in non-small cell lung cancer

Abstract Immune checkpoint inhibitors (ICIs), specifically those which target the T-cell surface receptor Programmed Cell Death-1 (PD-1) and its ligand, Programmed cell death ligand-1 (PD-L1), have demonstrated substantial clinical benefit in patients with non-small cell lung cancer (NSCLC). Tumoral PD-L1 expression may be important for response to therapy, but the precise mechanism of regulation has yet to be fully elucidated. It is known that PD-L1 expression is elevated in cancers harboring mutations in the RAS family of genes, specifically KRAS. Mutations in the KRAS gene are found in up to 30% of NSCLC tumors, and there are no targeted treatment options available for this subset of patients. The tyrosine phosphatase, SHP-2, has been shown to be a critical regulator of the KRAS signaling cascade. Therefore, genes regulated by the RAS/MAPK signaling cascade, including PD-L1, may be modulated by SHP-2 inhibition. Thus, this study aims to investigate the impact of SHP-2 activity on PD-L1 expression in NSCLC. The allosteric inhibitor of SHP-2, SHP099, was used to ablate SHP-2 activity in KRAS-mutant NSCLC cell lines, H460, A549, H2122, and EGFR-mutant NSCLC cell line PC9. PD-L1 levels were quantified by western blot and flow cytometry analyses. PD-L1 mRNA was measured by quantitative real time PCR (qRT-PCR) analysis. SHP099 activity was confirmed by SHP-2, siRNA transfection in KRAS active cell lines. Inhibition of SHP-2 led to increased levels of PD-L1 expression in the KRAS-mutant cell lines, but not in the EGFR-mutant cells. Analysis of PD-L1 surface expression by flow cytometry confirmed that surface PD-L1 levels increased following exposure to SHP099. To determine whether SHP-2 controlled expression at the transcriptional level, mRNA quantification by qRT-PCR displayed increased levels of PD-L1 mRNA following SHP099 treatment. Together, these results suggest that SHP-2 regulates PD-L1 expression in KRAS-active NSCLC at the level of PD-L1 gene expression. It is presently unclear whether SHP-2 also has a role in controlling protein turnover. Using SHP-2 mutant constructs that mimic a constitutively active phosphatase, we hope to gain further insight into the roles of both SHP-2 catalytic and scaffolding functionality on PD-L1 expression. Citation Format: Keller Toral. The tyrosine phosphatase, SHP-2, is involved in regulating PD-L1 expression in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1773.

Abstract LB-011: Fit-for-purpose quantitative programmed cell death ligand -1 (PD-L1) droplet digital PCR assay development using PAXgene RNA blood samples

Abstract Purpose Programmed death ligand receptor 1, (PD-L1) is a key immune checkpoint inhibitory receptor expressed on activated tumor specific immune cells. While immunohistochemistry assays have been developed for the detection of PD-L1 expression in several cancer types for patient treatment decision, a blood-based test for PD-L1 expression evaluation would be beneficial in providing complimentary evidence for potential clinical application. This study developed a fit-for-purpose clinical biomarker assay for the detection of PD-L1 mRNA expression in blood using droplet digital polymerase chain reaction (ddPCR) methods. Study Procedure Taqman primer/probe assays were selected based on coverage of PD-L1 gene and were tested for linearity and efficiency using a cDNA construct. Four housekeeping genes were analyzed in positive control cell line (A549 treated with interferon gamma), negative control cell line (A549 untreated) and PAXgene blood from healthy volunteers. Real-time quantitative PCR method was tested first, following initial experiments there were concerns with the performance of our qPCR assay prompting the switch to ddPCR which offers absolute quantification and a lower limit of detection. Assay performance characteristics of the ddPCR assay were evaluated including linearity, limit of blank (LOB), limit of detection (LOD), and precision. Thirty-five healthy volunteer samples were evaluated to establish a baseline level of PD-L1 expression. Data Summary Conducting linearity comparison between Taqman primer/probe kits using cDNA titrations showed no significant difference in linearity (r2=0.99) or efficiency (~95%) among the three primer/probe kits. Housekeeping genes B2M, GUSB, RPLP0, and TBP were compared and GUSB was most consistently expressed between IFN gamma treated and untreated cells; it was selected for use thereafter. In ddPCR, the LOB was determined to be 0 copies and the LOD was determined to be 19 copies of PD-L1. The average intra-run coefficient of variation (CV) was 7.44% and average inter-run CV was 7.70%. Analysis of healthy control samples yielded an average of 2415 PD-L1 copies with a range of 768-7510 copies. Treatment of A549 cells with IFN gamma resulted in a 6.7-fold increase in PD-L1 expression (21,580 copies in untreated cDNA versus 145,000 copies in treated cDNA). Conclusion Our fit-for purpose clinical biomarker assay development efforts have demonstrated that the ddPCR assay is capable of reliably and accurately detecting PD-L1 in PAXgene blood with low limit of detection and minimal inter-and-intra run variability. Compared to real-time PCR, ddPCR is a purely quantitative and offers a much broader dynamic range and provides more sensitive detection of PD-L1 in blood. Further evaluation and comparison of cancer to healthy volunteer samples is underway for more information on assay performance and disease biology. Citation Format: Dennis O'Rourke, Danyi Wang, Maria Cusano, Jorge F. Sanchez-Garcia, Ti Cai, Juergen Scheuenpflug, Zheng Feng. Fit-for-purpose quantitative programmed cell death ligand -1 (PD-L1) droplet digital PCR assay development using PAXgene RNA blood samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-011.

Programmed death-ligand 1 (PD-L1) expression in pheochromocytoma.

537 Background: Programmed cell death ligand-1 (PD-L1) is a key target molecule of immunotherapy that is frequently overexpressed in several neoplasms. But there were few reports about PD-L1 expression of pheochromocytoma. In the present study, we examined PD-L1 expression in pheochromocytoma. Methods: PD-L1 mRNA expression was compared across 184 pheochromocytoma, 492 prostate cancer cases and 404 bladder cancer cases based on The Cancer Genome Atlas (TCGA). Furthermore, we enrolled 32 pheochromocytoma patients treated with surgery at our hospital between June 2005 and February 2016. We conducted an immunohistochemistry (IHC) of PD-L1 using the SP142 assay. PD-L1 expression was scored at three diagnostic levels (0/1/2). Results: Comparison of PD-L1 mRNA expression based on the TCGA revealed that PD-L1 expression was significantly higher in pheochromocytoma than in bladder cancer and in prostate cancer (p &lt; 0.001). In the SP 142 assay of our 32 pheochromocytoma, the prevalence of positive PD-L1 expression (IHC score 1 or 2 (1/2)) in tumor-infiltrating immune cells (TICs) was 8 patients (25%). The prevalence of positive PD-L1 expression in tumor cells (TCs) was 9 patients (28.1%). Tumor diameter of PD-L1 (+) in TICs patients was 3.36±0.35 cm and that of PD-L1 (-) in TCs patients was 5.37±0.50cm, there was statistically significance between two groups. (unpaired t test: p=0.044) In our cohort, there were two malignant pheochromocytoma. But there were not PD-L1 positive cases in malignant pheochromocytoma. Conclusions: PD-L1 expression is relatively high in pheochromocytoma compared to bladder cancer and prostate cancer based on TCGA. In the SP142 assay of our 32 pheochromocytoma cases, tumor diameter of PD-L1 (-) in TICs cases was larger than that of PD-L1 (+) cases. In our cohort, there were not PD-L1 (+) cases in malignant pheochromocytoma. These findings suggest that PD-L1 expression of pheochromocytoma was comparatively common and PD-L1 positive expression of pheochromocytoma may not be associated with tumor aggressiveness.

MRNA expression of programmed cell death ligand 1 and components of the phosphatidylinositol 3-kinase/AKT/phosphatase and tensin homolog pathway in epidermal growth factor receptor mutation-positive lung adenocarcinoma.

The objective of this study is to investigate the relationship between programmed cell death ligand 1 (PD-L1) expression and the mutation status of epidermal growth factor receptor (EGFR) in lung adenocarcinoma, as well as the correlation between the clinical features of patients and mRNA levels of PD-L1 and components of the phosphatidylinositol 3-kinase (PI3K)/ protein kinase B (AKT)/phosphatase and tensin homolog (PTEN) pathways. mRNA levels of PD-L1, PI3K, AKT, and PTEN in tumor and matched normal tissues of patients with EGFR mutation-positive lung adenocarcinoma were determined by real-time polymerase chain reaction. Twenty-three patients with EGFR mutation-positive lung adenocarcinoma were enrolled, and 46 samples (23 pairs of tumor and matched normal lung tissues) were collected. PD-L1 and AKT mRNA levels were higher in EGFR mutation-positive lung adenocarcinoma than in matched normal lung tissues (P = 0.047 and P = 0.046, respectively), whereas PI3K and PTEN mRNA levels were significantly lower in the cancerous tissues (P = 0.009 and P = 0.039, respectively). PD-L1 expression was positively correlated with PI3K/AKT signaling pathway activation. PD-L1 upregulation in lung adenocarcinoma was positively correlated with EGFR exon 19 mutations (P = 0.034). AKT mRNA upregulation was correlated with lymph node metastasis (P = 0.034). PTEN mRNA downregulation was correlated with high tumor staging and lymph node metastasis (P = 0.035 and P = 0.014, respectively). Elevated PD-L1 mRNA expression in lung adenocarcinoma is associated with EGFR mutation and may be mediated through the PI3K-AKT pathway. EGFR exon 19 mutations closely correlate with increased PD-L1 mRNA expression. Increased AKT mRNA expression correlates with lymph node metastasis, while decreased PTEN mRNA levels correlate with advanced tumor stage and lymph node metastasis.

EBV-positive diffuse large B-cell lymphoma features PD-L1 protein but not mRNA overexpression

Programmed cell death ligand 1 (PD-L1) is upregulated in various types of haematological malignancies and is associated with immunosuppression. This study aimed to investigate the expression pattern of PD-L1 in Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL). We retrospectively analysed clinicopathological characteristics in 30 cases of EBV-positive DLBCL and immunohistochemically evaluated the level of membrane bound PD-L1 protein. Twenty-eight cases expressed PD-L1 protein, 15 of which showed an intense positive staining. In addition, we investigated the relationships between PD-L1 protein and PD-L1 mRNA and MYC, respectively. The expression level of PD-L1 protein was not fully parallel with PD-L1 mRNA, and no significant correlation was observed between PD-L1 protein and MYC. Notably, PD-L1 mRNA was at a low dosage, which indicated that there might be other mechanisms inducing the overexpression of membrane bound PD-L1 protein apart from genetic alterations. Furthermore, the low expression level of MYC may not interfere with the PD-L1 protein expression in EBV-positive DLBCL. In conclusion, overexpression of PD-L1 protein can be observed in EBV-positive DLBCL, and the level was non-parallel with both PD-L1 mRNA and MYC. Moreover, we emphasise that immunohistochemistry is a clinically reasonable method for screening formalin fixed, paraffin embedded (FFPE) tumour samples in this entity.

Programmed cell death ligand 1 expression is upregulated in the skeletal muscle of patients with myasthenia gravis

We investigated the expression patterns of programmed cell death-ligand 1 (PD-L1) in the skeletal muscle of 15 patients with myasthenia gravis (MG). In 11 of the patients, immunohistochemistry confirmed the localization of PD-L1 in the muscle cell membrane with cytoplasmic integrity. PD-L1 mRNA expression levels were higher in the muscles of MG patients as compared to those of non-myogenic patients (p < .05). Moreover, a unimodal relationship between PD-L1 mRNA levels and quantitative MG scores for disease severity was identified by a generalized additive model, indicating reciprocal effects. Together, these results suggest that MG symptoms might influence PD-L1 expression in muscle cells, and upregulated PD-L1 expression might inhibit autoimmune reactivity.

Green Tea Catechin Is an Alternative Immune Checkpoint Inhibitor that Inhibits PD-L1 Expression and Lung Tumor Growth.

The anticancer activity of immune checkpoint inhibitors is attracting attention in various clinical sites. Since green tea catechin has cancer-preventive activity in humans, whether green tea catechin supports the role of immune checkpoint inhibitors was studied. We here report that (−)-epigallocatechin gallate (EGCG) inhibited programmed cell death ligand 1 (PD-L1) expression in non–small-cell lung cancer cells, induced by both interferon (IFN)-γ and epidermal growth factor (EGF). The mRNA and protein levels of IFN-γ–induced PD-L1 were reduced 40–80% after pretreatment with EGCG and green tea extract (GTE) in A549 cells, via inhibition of JAK2/STAT1 signaling. Similarly, EGF-induced PD-L1 expression was reduced about 37–50% in EGCG-pretreated Lu99 cells through inhibition of EGF receptor/Akt signaling. Furthermore, 0.3% GTE in drinking water reduced the average number of tumors per mouse from 4.1 ± 0.5 to 2.6 ± 0.4 and the percentage of PD-L1 positive cells from 9.6% to 2.9%, a decrease of 70%, in lung tumors of A/J mice given a single intraperitoneal injection of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). In co-culture experiments using F10-OVA melanoma cells and tumor-specific CD3+ T cells, EGCG reduced PD-L1 mRNA expression about 30% in F10-OVA cells and restored interleukin-2 mRNA expression in tumor-specific CD3+ T cells. The results show that green tea catechin is an immune checkpoint inhibitor.

Abstract 4551: Thyroid hormone induces PD-L1 expression in oral cancer cells

Abstract Oral cancer is a fatal disease, which accounts for the fourth highest incidence of malignancy in males and the seventh highest in the general population of Taiwan. Oral cancer is increasing in Taiwan. About 95% of oral cancer in Taiwan is oral squamous cell carcinoma (OSCC). With the development of cancer molecular biology and immunology, targeted therapy for immune checkpoints of programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) has shown enormous development prospects for treatment of head and neck cancer. The PD-1/PD-L1 checkpoint is a critical regulator of activated T cell-cancer cell interactions which defend tumor cells against immune surveillance. Thyroid hormone induces PD-L1 expression in human oral cancer cells. Human oral cancer OEC-M1 and SCC-25 cells were treated with different concentrations of T4 (10-8 to 10-6M) for 24 h and cells were harvested and total RNA was extracted. qPCR of PD-L1 revealed that PD-L1 mRNA was significantly induced by thyroid hormone on a concentration-dependent basis. Parallel studies were conducted to study the effect of thyroid hormone on PD-L1 protein accumulation. Cancer cells were treated with different concentrations of T4 for 24 h. Total proteins were extracted and western blot analysis of PD-L1 was conducted. Thyroid hormone-activated ERK1/2 and STAT3 were companied with PD-L1 expression. Inhibition of ERK1/2 and consequently STAT3 activation also blocked PD-L1 induced by thyroid hormone. Knockdown of PD-L1 expression by siRNA also inhibits thyroid hormone-induced proliferation of oral cancer cells which indicated that PD-L1 expression is involved in thyroid hormone-induced cancer growth via an ERK1/2-STAT3 signal transduction pathway in oral cancer cells. Citation Format: Yu-Tang Chin, Kwan-Wei Su, Yee-Tang Lim, Ya-Jung Shih, Yi-Ru Chen, Sheng-Yang Lee, Paul J. Davis, Hung-Yun Lin, Earl Fu. Thyroid hormone induces PD-L1 expression in oral cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4551.

Quantification and prognostic value of programmed cell death ligand-1 expression in dogs with diffuse large B-cell lymphoma

OBJECTIVE To measure programmed cell death ligand-1 (PD-L1) mRNA expression in archived primary nodal diffuse large B-cell lymphoma (DLBCL) specimens of dogs and determine whether that expression was associated with progression-free survival time (PFST). SAMPLE Archived tumoral lymph node specimens from 42 dogs with DLBCL and lymph node specimens from 10 healthy dogs (controls). PROCEDURES Archived tumoral and control lymph node specimens underwent multiplex qPCR analysis with probes and primers against canine PD-L1 and glyceraldehyde 3-phosphate dehydrogenase (housekeeping gene) to determine PD-L1 mRNA expression. The 2-ΔΔCt method was used to calculate the fold change in PD-L1 expression in DLBCL specimens relative to that in control lymph nodes. Kaplan-Meier and Cox proportional hazard analyses were used to evaluate the association of various tumoral and clinical factors with PFST. RESULTS The fold change in PD-L1 mRNA expression in DLBCL specimens relative to control specimens ranged from 0.21 to 7.44. Twenty-one of 42 (50%) DLBCL specimens had a PD-L1-fold change > 1, which suggested PD-L1 was overexpressed in those specimens. Median PFST was 249 days for dogs with DLBCL. The PFST was not associated with PD-L1 mRNA expression but was associated with thrombocytopenia at the time of diagnosis (hazard ratio, 2.56; 95% confidence interval, 1.28 to 5.15). CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that tumoral PD-L1 mRNA expression varied among dogs with DLBCL and that PD-L1 MRNA was overexpressed in half the study population. Therefore, anti-PD-L1 therapies may be clinically beneficial for some dogs with DLBCL.

Programmed Cell Death Ligand 1 Expression in Primary Central Nervous System Lymphomas: A Clinicopathological Study.

Programmed cell death ligand 1 (PD-L1)/programmed cell death 1 (PD-1) have been shown to predict response to PD-L1/PD-1-targeted therapy. We analyzed PD-L1 expression in primary central nervous system lymphomas (PCNSLs). PD-L1 protein and mRNA expression were evaluated in 64 PCNSL tissue samples. IFN-γ, IL-10, CD4, and CD8 mRNA expression was also evaluated. PD-L1 protein was detected in tumor cells in 2 (4.1%) cases and in tumor microenvironments in 25 (52%) cases. PD-L1 mRNA positively correlated with IFN-γ (p=0.0024) and CD4 (p=0.0005) mRNA expression. IFN-γ mRNA positively correlated with CD8 mRNA expression (p=0.0001). Furthermore, tumor cell PD-L1 expression correlated positively with overall survival (p=0.0177), whereas microenvironmental PD-L1 expression exhibited an insignificant negative trend with overall survival (p=0.188). PD-L1 was expressed on both tumor and/or tumor-infiltrating immune cells in PCNSL. The biological roles of this marker warrant further investigation.

A Radiosensitivity Gene Signature and PD-L1 Status Predict Clinical Outcome of Patients With Lower Grade Glioma in the Cancer Genome Atlas Dataset

A radiosensitivity gene signature that included 31 genes was identified using microarray data from NCI-60 cancer cells. This study was aimed to validate 31 genes signature in lower grade glioma (LGG) data set and also investigate the relationship between this gene signature, programmed cell death ligand 1 (PD-L1) status, and clinical outcome in order to identify a group of patients that would be beneficial for radiotherapy (RT) combined with anti-PD1/PD-L1 therapy. We validated the 31-gene signature related to radiosensitivity and evaluated the PD-L1 status of lower grade glioma in The Cancer Genome Atlas (TCGA) dataset. First, we downloaded TCGA lower grade glioma (LGG) gene expression datasets from 516 samples that were analyzed with the RNA Sequencing platform (IlluminaHiSeq RNASeqV2). To validate the gene signature, 516 samples were divided into two clusters using a consensus clustering algorithm based on expression profile of gene signature. According to their prognosis, two clusters were designated as their radiosensitive (RS) or radioresistant (RR). Patients were also stratified as PD-L1-high or PD-L1-low based on the median value of CD274 mRNA expression level as surrogates of PD-L1. Survival outcomes were compared between RR with PD-L1-high (PD-L1-high-RR) versus the others using log-rank test and Cox proportional hazard models. Patents assigned to the RS group had better 5-year overall survival (OS) rate than patients in the RR group by univariate analysis (63% vs. 52%, P=0.019) only when patients were treated with primary RT. The RR group was independently associated with the PD-L1-high group, and PD-L1 mRNA expression was significantly higher in the RR group (P<0.001) compared to that of the RS group. ESTIMATE immune score, representing the immunogenicity of tumors, was positively correlated with PD-L1 mRNA expression level (Pearson’s correlation=0.46. P=<0.001). PD-L1-high-RR group had worse 5-year OS rate compared to the others (50% vs. 60%, P=0.010). Cox multivariate analysis demonstrated that IDH-wild type and RR signature with high PD-L1 expression demonstrated detrimental effect on overall survival (HR =1.68, 95% CI=1.00-2.81, P=0.048). Gene enrichment analysis showed that differentially expressed genes in the PD-L1-high-RR group were involved in immune and inflammatory response. KEGG pathway analysis revealed that these genes were mapped into T-cell receptor signaling pathway as well. We first validated the predictive value of the radiosensitivity gene signature and PD-L1 in the TCGA dataset for the LGG. The patients having RR signature with high PD-L1 expression demonstrated worse outcome and association with T-cell receptor signaling suggesting that PD-L1-high-RR group could be benefited from anti-PD1/PD-L1 therapy combined with RT.

Validated programmed cell death ligand 1 immunohistochemistry assays (E1L3N and SP142) reveal similar immune cell staining patterns in melanoma when using the same sensitive detection system.

Tumour cell and/or immune cell programmed cell death ligand 1 (PD-L1) expression is considered as a potential biomarker for anti-PD1 and anti-PD-L1 immunotherapy. Currently, different PD-L1 assays are used. This study aims to compare the staining patterns of two PD-L1 antibody clones in melanoma metastases and correlate them with PD-L1 mRNA expression. The immunohistochemistry assays were optimized and validated independently on a Ventana Benchmark Ultra (Ventana Medical Systems Inc., Tucson, AZ, USA) (E1L3N) and XT (SP142), using the same detection system. In total, 46 melanoma metastases were stained with both validated immunohistochemistry assays. Stained slides were digitized for qualitative and semi-quantitative evaluation; done by pathologist and semi-automated software analysis. A subset of 21 melanoma metastases was selected for quantification of the PD-L1 mRNA expression. Accuracy and precision criteria were met for both assays. PD-L1 protein and mRNA expression showed remarkably good Spearman's coefficients of 0.90 (E1L3N) and 0.87 (SP142). Despite the remarkable correlation between both PD-L1 assays in expression patterns and quantification values (ρ > 0.90), E1L3N showed significantly more tumour cell staining than SP142. E1L3N and SP142 IHC assays were optimized and validated successfully and independently for sensitive and accurate PD-L1 detection. Concordance was best for immune cell scoring, while E1L3N tended to detect more tumour cells. Determination of the clinically relevant cut-off values for immune cell versus tumour cell detection requires further research.

Abstract 4021: The class I HDAC inhibitor, mocetinostat, induces expression of PD-L1 and tumor antigen presentation machinery and modifies tumor immune cellular subsets providing a rationale for immune checkpoint inhibitor combinations

Abstract Immunotherapy has led to major treatment breakthroughs for a number of cancers including non-small cell lung cancer (NSCLC). Although initial responses to immune checkpoint inhibitors are promising, a significant percentage of patients do not respond or rapidly acquire resistance. Although the mechanisms underlying intrinsic and acquired resistance remain largely unexplained; the expression of programmed cell death-ligand 1 (PD-L1), lack of tumor cell capacity to effectively present neoantigens, and presence of immunosuppressive cellular subsets have been implicated as potential mechanisms. Histone deacetylase (HDAC) inhibitors have emerged as a class of agents that may combat checkpoint inhibitor resistance by reversing immune evasion and eliciting an anti-tumor activity through a multi-faceted immuno-stimulatory mechanism of action. Mocetinostat is a spectrum-selective Class I/IV HDAC inhibitor specifically targeting HDAC-1, -2, -3 and -11. The present studies were designed to explore mocetinostat's effect as an immune-enhancer and ultimately, to evaluate its potential to be used in combination with immune checkpoint inhibitors (e.g., PD-1/PD-L1 antagonists). Specifically, we assessed mocetinostat's effect on the expression of various immunomodulatory factors by tumor cells as well as its effect on immune cell sub-populations in the tumor microenvironment in vivo. Mocetinostat elicited a concentration-dependent increase in PD-L1 mRNA expression which translated into increased PD-L1 surface protein expression in a panel of NSCLC cell lines. In addition, mocetinostat elicited a concentration-dependent increase in expression of MHC-class I related polypeptide-related sequence A (MIC-A) and MIC-B, and cluster of differentiation 86 (CD86). Furthermore, mocetinostat induced expression of several human leukocyte antigen (HLA) gene complex family members including HLA-A, -B, -DRA, and -DPA among others. To determine the effect of mocetinostat on systemic and tumor immune cell subpopulations we treated CT26 tumor-bearing mice. Mocetinostat increased splenic CD4-positive T effector cells and tumor mature cytolytic CD8-postive T cells and at the same time decreased tumor FoxP3-positive T regulatory cells and CD11b/Gr1-positive myeloid-derived suppressor cells (MDSC). These data provide evidence that mocetinostat modulates key immune regulators both in tumor cells as well as in relevant immune cell types in the tumor microenvironment and provides strong rationale for combination with immune checkpoint inhibitors. Citation Format: David Briere, Niranjan Sudhakar, Lars Engstrom, Jill Hallin, Ruth Tang, Harrah Chiang, Maryland Rosenfeld-Franklin, Peter Olson, James Christensen. The class I HDAC inhibitor, mocetinostat, induces expression of PD-L1 and tumor antigen presentation machinery and modifies tumor immune cellular subsets providing a rationale for immune checkpoint inhibitor combinations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4021.

PD-L1 marks a subset of melanomas with a shorter overall survival and distinct genetic and morphological characteristics

Programmed cell death ligand 1 (PD-L1) is a cell surface molecule that plays a critical role in suppressing immune responses, mainly through binding of the PD-1 receptor on T lymphocytes. PD-L1 may be expressed by metastatic melanoma (MM). However, its clinical and biological significance remains unclear. Here, we investigated whether expression of PD-L1 in MM identifies a biologically more aggressive form of the disease, carrying prognostic relevance. PD-L1 expression was analyzed by immunohistochemistry using two different antibodies in primary tumors and paired metastases from 81 melanoma patients treated at a single institution. Protein expression levels were correlated with PD-L1 mRNA, BRAF mutational status and clinical outcome. PD-L1(+) and PD-L1(-) subsets of the A375 cell line were stabilized in vitro and compared using gene expression profiling and functional assays. Results were confirmed using xenograft models. PD-L1 membrane positivity was detected in 30/81 (37%) of patients. By multivariate analysis, Breslow thickness and PD-L1 membrane positivity were independent risk factors for melanoma-specific death {PD-L1 5% cutoff [hazard ratio (HR) 3.92, confidence interval (CI) 95% 1.61-9.55 P < 0.003], PD-L1 as continuous variable (HR 1.03, 95% CI 1.02-1.04 P < 0.002)}. PD-L1 expression defined a subset of the BRAF-mutated A375 cell line characterized by a highly invasive phenotype and by enhanced ability to grow in xenograft models. PD-L1 is an independent prognostic marker in melanoma. If confirmed, our clinical and experimental data suggest that PD-L1(+) melanomas should be considered a disease subset with distinct genetic and morpho-phenotypic features, leading to enhanced aggressiveness and invasiveness.

Abstract 3750: Targeting PD-L1 sensitizes head and neck squamous cell carcinoma to cisplatin

Abstract Platinum-based chemotherapy agents such as Cisplatin (CDDP) are commonly used as first-line treatment for several types of human cancers including ovarian, lung, testicular, and head and neck squamous cell carcinomas (HNSCC). Unfortunately, CDDP monotherapy is often ineffective due to inherent or acquired tumor resistance to therapy, also known as chemoresistance. This contributes to a need for the administration of larger, more toxic doses, as well as failure of chemotherapy and tumor recurrence. Despite the clinical significance of chemoresistance, the mechanisms driving this phenomenon are not completely understood. Programmed cell death ligand 1 (PD-L1), a membrane-bound glycoprotein responsible for modulating the immunological response through the induction of T-Cell anergy and apoptosis, has been shown to be overexpressed in many cancers, including HNSCC. While PD-L1 overexpression has been directly related to the immunoevasion of cancer cells allowing for tumor progression, recent reports suggest that PD-L1 also plays an important role in the tumor response to doxorubicin-based chemotherapy independent of its immune function. The present study investigates the role of PD-L1 in response to CDDP in HNSCC. In vitro experiments were performed using four well-characterized human HNSCC cell lines. Quantitative polymerase chain reaction (qPCR) and western blot were conducted to determine the baseline and post-CDDP-treatment PD-L1 gene expression. The results demonstrated that PD-L1 mRNA transcription and protein expression are upregulated in response to CDDP, suggesting that PD-L1 is involved in the chemoresponse. Conversely, knockdown of PD-L1 using siRNA resulted in chemosensitization of HNSCC cells to CDDP. Together, these findings implicate PD-L1 in the CDDP-chemoresponse in HNSCC. In conclusion, we propose a role for PD-L1 that is distinct from the classic immunoevasion pathway. Our findings demonstrate that disruption of this protein leads to increased sensitivity of HNSCC cells to CDDP, suggesting a potential combination therapeutic strategy for patients with CDDP-resistant HNSCC. Citation Format: Peter Qiao, Shayanne A. Lajud, Danish A. Nagda, Nobuaki Tanaka, Alyssa Civantos, Bert W. O'Malley, Daqing Li. Targeting PD-L1 sensitizes head and neck squamous cell carcinoma to cisplatin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3750. doi:10.1158/1538-7445.AM2014-3750

Clinical significance of programmed death ligand-1 expression in non-small cell lung cancer (NSCLC).

11075 Background: Programmed cell death ligand-1 (PDL1) is expressed on various human cancers and is a major mechanism for immune evasion. Preliminary evidence suggests that PDL1 expression on cancer cells predicts response to anti-PD-1 therapy. Here we report our findings using automated quantitative immunofluorescence (QIF) to determine the frequency and prognostic value of PDL1 in two independent NSCLC cohorts. Methods: Five antibodies against PDL1 were screened for sensitivity and specificity using PDL1 transfected cells, normal human placenta and tonsil. Only one monoclonal antibody (clone 5H1) met criteria for specificity. This antibody was used to assess two cohorts of NSCLC cases in TMAs. The cohorts represented 204 cases from Yale University and 340 cases from hospitals in Greece. PDL1 protein was measured using QIF analysis using AQUA method. In addition, in-situ PDL1 mRNA levels were measured in the Greek cohort using the RNAscope paired-primer assay with AQUA method. Results: PDL1 protein expression was detected in 25% and 36% of the studied cohorts, respectively. PDL1 positivity was significantly associated with the presence of tumor-infiltrating lymphocytes (TIL) in both series. Patients with PDL1 protein positivity had better outcome in both series (Log Rank: p=0.036 for Yale cohort, p=0.027 for Greek cohort). Multivariate analysis showed that PDL1 expression was associated with better outcome independent of histology, presence of TILs, and stage in the Greek cohort and marginally significant in the Yale cohort. These findings were further validated by the PDL1 in-situ mRNA measurement in the Greek cohort. PDL1 protein and mRNA levels showed a positive non-linear relationship (R2=0.14, P&lt;0.001). Positive PDL1 mRNA was found in 53% of patients with NSCLC and was also associated with the presence of TILs and longer overall survival (31 vs. 43 months, p=0.017). Conclusions: Tumor PDL1 protein positivity is detected in 25-30% of NSCLCs and associates with increased TILs and better outcome. Elevated PDL1 mRNA occurs in a higher proportion of NSCLC cases and provides comparable prognostic information. Measurement of in situ PDL1 protein and mRNA could be of more clinical value than either method alone.