Expression Level Of Programmed Death-ligand 1 Research Articles

B7-H3 is widely expressed in soft tissue sarcomas

PurposeTargeted therapy development in soft tissue sarcoma (STS) has been burdened by the heterogeneity of this group of rare tumors. B7 homolog 3 protein (B7-H3) is a molecule in the same family as programmed death-ligand 1 (PD-L1). It has limited expression in noncancerous tissues and is overexpressed in many cancers, making it an attractive target for cancer therapy, and clinical trials targeting B7-H3 are actively underway. While available data demonstrate high expression levels of B7-H3 in individual sarcoma subtypes, its expression patterns across STS subtypes are not well described. The purpose of this study was to characterize the expression patterns of B7-H3 in STS.Patients and methodsThis retrospective analysis evaluated STS tumor specimens from patients with a variety of different subtypes. Specimens were evaluated by immunohistochemistry (IHC) for expression and staining pattern of B7-H3 both in tumors and in associated vasculature.ResultsSpecimens from 153 sarcoma patients included 15 different STS subtypes. B7-H3 was broadly expressed in 97% of samples (95% CI 0.93–0.99) and 69.2% demonstrated high levels of B7-H3 expression (95% CI 0.61–0.76). No significant association between B7-H3 positivity or expression level and prior treatment(s), tumor size, tumor grade, or patient age. B7-H3 positivity in vessels was found in 94.7% (145/153) of samples. In tumors that had been previously assessed for PD-L1 and PD-1, there was no correlation between B7-H3 positivity or expression and the positivity or expression level of PD-L1 or PD-1.ConclusionThese data show high levels of B7-H3 positivity across soft tissue sarcoma subtypes, suggesting its feasibility as a therapeutic target for future sarcoma treatments. Future clinical trials are needed to evaluate whether targeting B7-H3 can provide clinical benefit to help patients with sarcoma.

Effect of an artificial intelligence–powered programmed death-ligand 1 combined positive score analyzer in urothelial cancer on inter-observer and inter-site variability.

e13546 Background: Programmed death-ligand 1 (PD-L1) is a predictive marker for immune checkpoint inhibitors treatment response in urothelial carcinoma (UC). The combined positive score (CPS) is a representative method to evaluate the expression level of PD-L1 in UC. However, inter-observer and inter-institute variations can disrupt accurate CPS evaluation. The purpose of this study is to assess the role of an artificial intelligence (AI)-powered PD-L1 CPS analyzer on UC in reducing inter-observer and inter-institute variability. Methods: Lunit SCOPE PD-L1 CPS was developed with 4.94 x 105 tumor cells and 4.17 x 105 immune cells from 360 PD-L1 stained whole-slide images (WSIs) of UC from multiple institutions. The algorithm consisted of tissue area segmentation and cell detection AI models. The AI models calculated the CPS by detecting tumor cells over the tumor area and immune cells over the tumor and adjacent area. Three uropathologists from different university hospitals evaluated the CPS classification (≥10 or <10) of 543 PD-L1 stained WSIs of UC from each hospital. The result with concordant CPS classification of each slide across ≥ 2 pathologists was considered the consensus. Each pathologist revisited to evaluate WSIs by referencing the AI model inference, after a washout period, if there was a discrepancy between the pathologist and the AI model. Results: Of 543 WSIs, 446 (82.1%) were classified as the same CPS subgroup by all three uropathologists. Also, pathologists had a high degree of concordance with the consensus in WSIs from their own hospitals. They re-evaluated 64, 73, and 75 WSIs with AI assistance, respectively, and changed the CPS classification for 47, 48, and 48 WSIs. After re-evaluation with AI assistance, three uropathologists agreed on the same CPS classification in 510 WSIs (93.9%). The overall percentage agreement (OPA) of each pathologist with the consensus increased from 95.0%, 94.8%, and 92.3% to 98.7%, 98.3%, and 96.9% after AI assistance, and the OPA for WSIs of other institutions increased more compared to the OPA for WSIs of their own hospital. Conclusions: This study shows that an AI-powered PD-L1 CPS analyzer in UC can reduce inter-observer and inter-site variability. This result suggests that the AI model will help evaluate CPS in UC more accurately and reduce variation in situations where pathologists analyze WSIs from unfamiliar institutions.[Table: see text]

Regulation of programmed death ligand 1 expression by interferon-γ and tumour necrosis factor-α in canine tumour cell lines.

Expression of programmed death ligand 1 (PD-L1) on tumour cells provides an immune evasion mechanism by inducing suppression of cytotoxic T cells. Various regulatory mechanisms of PD-L1 expression have been described in human tumours, however, little is known in canine tumours. To investigate whether inflammatory signalling is involved in PD-L1 regulation in canine tumours, the effects of interferon (IFN)-γ and tumour necrosis factor (TNF)-α treatment were examined in canine malignant melanoma cell lines (CMeC and LMeC) and an osteosarcoma cell line (HMPOS). The protein level of PD-L1 expression was upregulated by IFN-γ and TNF-α stimulation. Upon IFN-γ stimulation, all cell lines showed an increase in expression of PD-L1, signal transducer and activator of transcription (STAT)1, STAT3 and genes regulated by STAT activation. Upregulated expression of these genes was suppressed by the addition of a JAK inhibitor, oclacitinib. Contrastingly, upon TNF-α stimulation, all cell lines exhibited higher gene expression of the nuclear factor kappa B (NF-κB) gene RELA and genes regulated by NF-κB activation, whereas expression of PD-L1 was upregulated in LMeC only. Upregulated expression of these genes was suppressed by the addition of an NF-κB inhibitor, BAY 11-7082. The expression level of cell surface PD-L1 induced by IFN-γ and TNF-α treatment was reduced by oclacitinib and BAY 11-7082, respectively, indicating that upregulation of PD-L1 expression by IFN-γ and TNF-α stimulation is regulated via the JAK-STAT and NF-κB signalling pathways, respectively. These results provide insights into the role of inflammatory signalling in PD-L1 regulation in canine tumours.

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.

Abstract 5990: Targeted nanoparticles reduce tumor progression and lung metastasis by limiting DNA damage repair and immune suppression in metastatic triple-negative breast cancer

Abstract Background: Metastatic triple-negative breast cancer (TNBC), especially with BRCA1/2 mutations, is a deadly subtype of breast cancer with ~12% of 5-year survival. Despite their FDA approval, the therapeutic benefits of poly(adenosine diphosphate-ribose) polymerase inhibitors (PARPi) are limited to BRCA1/2 mutated malignancies. Moreover, the PARPi olaparib has been shown to upregulate the expression of programmed death-ligand 1 (PD-L1) leading to immune suppression. The purpose of this study is to investigate if a novel nanoparticle formulation of oligomer hyaluronic acid (oHA) and doxorubicin (DOX) co-loaded in an iRGD-conjugated polymer-lipid nanocarrier (iRGD-DOX-oHA-PLN) can inhibit DNA damage repair and suppress PD-L1 in both mutant and non-mutant TNBC cells and reduce tumor progression and lung metastasis compared to the PARPi olaparib. We postulate that the co-administered oHA will block the signaling pathways of native hyaluronic acid (HA) receptors, the cluster of differentiation 44 (CD 44) and receptor for HA mediated motility (RHAMM), that regulate DNA damage repair and immunosuppression and enhance DOX efficacy. Methods: In vitro cellular uptake of various DOX formulations by TNBC MDA-MB-231-luc-D3H2LN cells and MDA-MB-436 (BRCA1 mutant) cells was examined using confocal laser scanning microscopy or spectrophotometer. The expression level of RHAMM, PD-L1, and PARP1 parylation (PAR) was evaluated after treatment with iRGD-DOX-oHA-PLN or olaparib in both cell lines using western blot or confocal laser scanning microscopy. The therapeutic efficacy of iRGD-DOX-oHA-PLN compared to olaparib was determined by monitoring the tumor progression and lung metastasis development in orthotopic breast tumor models of MDA-MB-231-luc-D3H2LN or MDA-MB-436 cell line. Results: The expression of native HA receptor RHAMM was profoundly reduced by ~5 folds in vivo and the DNA DSB was significantly increased by the iRGD-DOX-oHA-PLN treatment with 40% of γH2AX positive cells in tumor tissue sections. The iRGD-functionalized PLN enhanced DOX cellular uptake compared to DOX free drug or non-targeted NPs by ~3- and ~1.5-fold, in MDA-MB-231 and MDA-MB-436 cells, respectively. While olaparib upregulated PD-L1 and free DOX increased PAR level, the iRGD-DOX-oHA-PLN reduced the parylation and PD-L1 expression in both TNBC cell lines. The iRGD-DOX-oHA-PLN treatment (two dose of 10 mg/kg i.v. biweekly) outperformed olaparib (twenty doses of 50 mg/kg i.p 5x/week) in preventing tumor progression and lung metastasis in vivo over a 4-week period. Conclusion: The results suggest that the iRGD-DOX-oHA-PLN can effectively inhibit DNA damage repair and immunosuppression of cancer cells and could be a promising multitargeted nanomedicine for the treatment of both BRCA1-mutant and non-mutant metastatic TNBC. Citation Format: Ibrahim Alradwan, Pei Zhi, Tian Zhang, HoYin Lip, Abdulmutalib Zetrini, Chunsheng He, Jeffery Henderson, Andrew Rauth, Xiao Yu Wu. Targeted nanoparticles reduce tumor progression and lung metastasis by limiting DNA damage repair and immune suppression in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5990.

Isoform Selectivity of HDAC Inhibitors Has a Significant Effect on PD-L1 Expression in the Triple-negative Cancer Cell Line MDA-MB-231

Various reports have been published in recent years on the effects of histone deacetylase (HDAC) inhibitors on programmed death ligand 1 (PD-L1) expression in cancer cells. The combination therapy of immune checkpoint inhibitors and HDAC inhibitors utilizing these effects has attracted attention as a new clinical treatment of triple-negative breast cancers. We investigated how the expression level of PD-L1 changes depending on the type of HDAC inhibitor exposed to triple-negative breast cancer cell line MDA-MB-231. We found that the mRNA expression level of PD-L1 was significantly decreased by Vorinostat and K-32 (pan-HDAC inhibitors) at high concentrations exhibiting low cell viability, while it was increased by high concentrations of K-560 (HDAC1,2 inhibitor) and Entinostat (HDAC1,3 inhibitor). On the other hand, the mRNA level of PD-L1 was increased by all of these HDAC inhibitors at low concentrations showing high cell viability. Of particular note, K-32 induced more PD-L1 mRNA than all the other HDAC inhibitors at the lowest concentration of 0.5 μM. This finding might suggest the usefulness of pan-HDAC inhibitors in clinical treatment in combination with immune checkpoint inhibitors.

MicroRNA-20a promotes non-small cell lung cancer proliferation by upregulating PD-L1 by targeting PTEN.

Non-small cell lung cancer (NSCLC) remains one of the most common malignant tumors worldwide. The aim of the present study was to investigate the possibility of microRNA-20a (miR-20a) as a biomarker and therapeutic target for the diagnosis and treatment of NSCLC. Bioinformatics prediction, together with functional validation, confirmed miR-20a bound to programmed death ligand-1 (PD-L1) 3′-untranslated region to upregulate PD-L1 expression. Both miR-20a and PD-L1 could promote the proliferation of NSCLC cells. The expression level of PD-L1 was controlled by PTEN; however, further upstream regulation of PD-L1 expression was largely unknown. The present study showed that miR-20a could not restore the inhibition of PD-L1 expression levels by PTEN. Knockdown of PTEN expression upregulated the expression level of PD-L1 and promoted the proliferation of NSCLC cells. PTEN negatively regulated the Wnt/β-catenin signaling pathway by inhibiting β-catenin and Cyclin D1. Interestingly, PTEN could reverse miR-20a-mediated proliferation of NSCLC cells and the inhibitory effect was similar to that of XAV-939. miR-20a promotes the proliferation of NSCLC cells by inhibiting the expression level of PTEN and upregulating the expression level of PD-L1. It is suggested that miR-20a could be used as a biomarker and therapeutic target for the treatment of NSCLC.

The relationship between different subtypes of KRAS and PD-L1 & tumor mutation burden (TMB) based on next-generation sequencing (NGS) detection in Chinese lung cancer patients

BackgroundKRAS gene mutations are the most common driver oncogenes in non-small cell lung cancer (NSCLC). We conducted an analysis of the immunological characteristics including tumor mutation burden and programmed death-ligand 1 (PD-L1) expression of different subtypes of KRAS in 2880 KRAS-mutant NSCLC patients.MethodsA total of 2,880 patients with NSCLC were included in the study. Somatic mutation data were provided by Berry Oncology (Fujian, China), Geneplus BioTech (Beijing, China), Nanjing Geneseeq Technology Inc (Nanjing, China), and Burning Rock Biotech (Guangzhou, China). Z-scores were used to unify all data. SPSS 20.0 (SPSS, Chicago, IL, USA) software was used for statistical analyses. All scatter plots and boxplot maps were drawn using GraphPad Prism 8. Tumor mutation burden (TMB) expression was defined by the number of somatic mutations. The PD-L1 clone 22C3 pharmDx kit was used to measure the expression level of PD-L1. Mann-Whitney U test was used for statistical analysis. P value <0.05 was considered statistically significant.ResultsWe identified 2,880 patients with KRAS-mutant NSCLC. The percentage level of TMB and expression of PD-L1 was significantly decreased in KRAS Q61X-mutant lung cancer tissue and blood samples (n=162). The percentage level of TMB and expression of PD-L1 in KRAS G13X-mutant lung cancer specimens was significantly increased (n=190).ConclusionsThe findings demonstrate a decreased level of TMB and expression of PD-L1 in KRAS Q61X-mutant lung cancer and the increased level of TMB and expression of PD-L1 in KRAS G13X-mutant lung cancer. Further work is needed to identify if the subtype of KRAS mutation could be a potential therapeutic biomarker in lung cancer patients with KRAS mutation. TMB data was consistently verified in tissue and blood samples and confirmed the feasibility of next-generation sequencing (NGS) verification in plasma samples. Our research may help to provide more individualized treatment options for NSCLC patients.

Predicting programmed death-ligand 1 expression level in non-small cell lung cancer using a combination of peritumoral and intratumoral radiomic features on computed tomography

In this study, we investigated the possibility of predicting expression levels of programmed death-ligand 1 (PD-L1) using radiomic features of intratumoral and peritumoral tumors on computed tomography (CT) images. We retrospectively analyzed 161 patients with non-small cell lung cancer. We extracted radiomic features for intratumoral and peritumoral regions on CT images. The null importance, least absolute shrinkage, and selection operator model were used to select the optimized feature subset to build the prediction models for the PD-L1 expression level. LightGBM with five-fold cross-validation was used to construct the prediction model and evaluate the receiver operating characteristics. The corresponding area under the curve (AUC) was calculated for the training and testing cohorts. The proportion of ambiguously clustered pairs was calculated based on consensus clustering to evaluate the validity of the selected features. In addition, Radscore was calculated for the training and test cohorts. For expression level of PD-L1 above 1%, prediction models that included radiomic features from the intratumoral region and a combination of radiomic features from intratumoral and peritumoral regions yielded an AUC of 0.83 and 0.87 and 0.64 and 0.74 in the training and test cohorts, respectively. In contrast, the models above 50% prediction yielded an AUC of 0.80, 0.97, and 0.74, 0.83, respectively. The selected features were divided into two subgroups based on PD-L1 expression levels≥50% or≥1%. Radscore was statistically higher for subgroup one than subgroup two when radiomic features for intratumoral and peritumoral regions were combined. We constructed a predictive model for PD-L1 expression level using CT images. The model using a combination of intratumoral and peritumoral radiomic features had a higher accuracy than the model with only intratumoral radiomic features.

Camrelizumab monotherapy or in combination with apatinib for PD-L1-positive advanced pulmonary sarcomatoid carcinoma: A multicenter, open-label, single-arm, phase II study.

TPS9130 Background: Pulmonary sarcomatoid carcinoma (PSC) is a rare subtype of non-small cell lung cancer (NSCLC) with a fairly poor prognosis. As a highly aggressive malignancy, PSC is insensitive to conventional chemotherapy or radiotherapy and no optimal treatment for PSC has been established yet. Immune checkpoint inhibitors (ICIs) were documented to possess encouraging therapeutic efficacy in PSC patients, which is demonstrated to be associated with high expression of programmed death-ligand 1 (PD-L1) in PSC. However, because of the rarity of PSC, most of the existed data were derived from case repots which could not reflect the true clinical efficacy of ICIs for PSC treatment. This clinical trial was designed to investigate the clinical outcomes of camrelizumab in treating PD-1-positive PSC. Apatinib may be used simultaneously based on the expression level of PD-L1 in PSC as the combination of camrelizumab and apatinib exhibited treatment potential in NSCLC in previous researches. Methods: In this multicenter, open-label, single-arm, phase II study conducted in 44 sites in China, 30 patients with an age of 18-80 years old, an Eastern Cooperative Oncology Group (ECOG) performance score of 0–2, positive PD-L1 expression, no EGFR, ALK, ROS1, and MET gene mutations, and histologically or cytologically confirmed stage IIIB-IV PSC regardless of prior lines of standard therapy will be enrolled. Patients with PD-L1 expression 1-49% will receive Camre (200 mg, IV, Q3W) and Apa (250mg, QD). Patients with PD-L1 expression≥50% receive Camre (200 mg IV Q3W) alone. The treatment will continue until a maximum treatment duration of 36 months, disease progression, intolerable toxicity, death or consent withdrawal. The primary endpoint is objective response rate (ORR). The secondary endpoints are progression-free survival (PFS), overall survival (OS), disease control rate (DCR), time to objective response (TTR), duration of response (DOR) and safety. This study is ongoing. Clinical trial information: ChiCTR2000032649, China.

Clinical performance of artificial intelligence-powered annotation of tumor cell PD-L1 expression for treatment of immune-checkpoint inhibitor (ICI) in advanced non-small cell lung cancer (NSCLC).

9026 Background: Programmed death ligand 1 (PD-L1) expression is the standard biomarker for first line ICI in advanced NSCLC. However, manual evaluation of tumor proportion score (TPS) by pathologists has practical limitations including intra/inter-observer bias, variation in subjectivity on area of interest and intensive labor. We developed an artificial intelligence (AI)-powered TPS analyzer, namely Lunit SCOPE PD-L1, for objective annotation of tumor cell PD-L1 expression for prediction of ICI response in advanced NSCLC. Methods: Lunit SCOPE PD-L1 was developed by a total of 393,565 tumor cells annotated by board-certified pathologists for PD-L1 expression in 802 whole-slide images (WSI) stained by 22C3 pharmDx immunohistochemistry. A After excluding the in-house control tissue regions, the WSI were divided into patches, from which a deep learning-based model detected the location and PD-L1 positivity of tumor cells. The patch-level cell predictions were aggregated for TPS estimation. Clinical performance of the model was validated in an external cohort of 430 NSCLC tumor slides from patients treated with ≥ ICI at Seoul National University Bundang Hospital and Samsung Medical Center. Independent control TPS annotation of this external validation cohort was performed by three pathologists, and their consensus TPS was calculated by mean value of such. Results: AI-model (Lunit SCOPE PD-L1) predicts PD-L1-positive tumor cell with the area under the curves of 0.889 and PD-L1-negative tumor cells with that of 0.809 at cell-level analysis. At WSI-level, significant positive correlation was observed between TPS by AI model and control TPS by pathologists (Spearman coefficient = 0.9247, P &lt; 0.001). Concordance rate between AI-model and control TPS by pathologists according to expression level of PD-L1 ≥ 50%, 1-49%, and &lt; 1% status was 85.7%, 89.3%, and 52.4%, respectively. Median progression-free survival (mPFS) according to TPS by AI model ≥ 1% vs. &lt; 1% were 2.8 vs. 1.7 months (hazard ratio, HR, 0.52, 95% confidence interval, CI, 0.38-0.71, P &lt; 0.001). In contrast, mPFS according to control TPS was 2.8 vs. 2.1 months (HR 0.70, 95% CI 0.55-0.91, P &lt; 0.001). Forty out of 84 patients (47.6%) annotated as control TPS &lt; 1% by pathologists were considered as TPS ≥ 1% by AI-model and mPFS of this subgroup was 2.7 months. Conclusions: PD-L1 expression by AI-model correlates with PD-L1 expression by pathologists. Clinical performance of AI-model in WSI-level is comparable with assessment by pathologists. The AI-model can accurately predict tumor response and progression-free survival of ICI in advanced NSCLC.

Interferon-τ regulates the expression and function of bovine leukocyte antigen by downregulating bta-miR-204.

IFN-τ is a pregnancy recognition factor that regulates embryo implantation in ruminants. IFN-τ has been suggested to be involved in the expression of microRNA (miRNA/miR) and bovine leukocyte antigen (BoLA), which is an analog of the human major histocompatibility complex class I. However, little is known about whether the miRNAs are involved in the expression of BoLA in ruminants. The present study firstly verified that bta-miR-204 was downregulated and that BoLA was upregulated in the uterine tissues of dairy cows during early pregnancy. Subsequently, luciferase reporter assays, reverse transcription-quantitative PCR and western blot analysis were used to validate BoLA as the target gene of bta-miR-204. Moreover, BoLA was markedly upregulated and bta-miR-204 was downregulated in bovine endometrial epithelial cells (bEECs) treated with IFN-τ. In addition, the results indicated that when the expression level of BoLA was increased by IFN-τ, the expression level of programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2) was also increased. Furthermore, when BoLA was silenced in bEECs by small interfering RNA, the expression of PD-L1 and PD-L2 was not affected by IFN-τ. The expression level of PD-L1 and PD-L2 was also increased in the uterine tissues of pregnant dairy cattle. In conclusion, IFN-τ may function by suppressing the expression of bta-miR-204 to increase the expression of BoLA during the embryo implantation period in cattle. IFN-τ may induce PD-L1 and PD-L2 transcription by regulating BoLA, which may influence the T cell immune response, thereby regulating pregnant cattle immunization.

Immuno-PET imaging of 68Ga-labeled nanobody Nb109 for dynamic monitoring the PD-L1 expression in cancers.

The checkpoint blockade immunotherapy has become a potent treatment strategy for cancers, and programmed death ligand-1 (PD-L1) is a prominent checkpoint ligand that is highly expressed in some cancers. The identification of immune checkpoint marker PD-L1 is critical for improving the success of immunotherapy. Accordingly, the binding specificity and dynamic monitoring property of a non-blocking nanobody tracer 68Ga-NOTA-Nb109 to PD-L1 were assessed in this study. The endogenous expression level of PD-L1 in several cancer cells was measured by flow cytometry, Western blot, and cellular uptake assay. Sensitivity and specificity of 68Ga-NOTA-Nb109 in monitoring the expression of PD-L1 in vivo were evaluated by PET imaging of different tumor-bearing models (U87, high PD-L1 expression; HCT 116, medium PD-L1 expression; and NCI-H1299, low PD-L1 expression). In vivo PET imaging results agreed well with those detected in vitro. In addition, PET imaging of PD-L1 expression in U87 and NCI-H1299 xenografts using 18F-FDG was also performed for comparison. The maximum tumor-to-muscle uptake ratio of 68Ga-NOTA-Nb109 was more than twofold that of 18F-FDG in U87 xenograft. The change of PD-L1 expression in NCI-H1299 cells and xenografts induced by cisplatin (CDDP) was sensitively monitored by 68Ga-NOTA-Nb109. This study demonstrated the feasibility of tracer 68Ga-NOTA-Nb109 for specifically targeting endogenous PD-L1 and dynamic monitoring the change of PD-L1 expression, and could guide the immunotherapy and immunochemotherapy for refractory cancers.

Serum level of programmed death-ligand 1 in patients with gastric cancer in Mazandaran Province as a High-Risk Region in Iran

Background: Gastric cancer is one of the most common cancers in the world. Programmed death-ligand 1 (PD-L1) is the main ligand of the programmed death-1 receptor that plays a key role in apoptosis in patients with gastric cancer. Materials and Methods: In this study, forty gastric cancer patients and forty healthy controls were enrolled. Serum PD-L1 was measured by enzyme-linked immunosorbent assay. Results: The mean (interquartile range) value of expression level of PD-L1 was 70.6 (36.6–127.3) in patients and 47.7 (29.9–92.1) in controls. There were no significant differences in PD-L1 expression between the two groups and tumor characteristics, but there was a statistically significant difference between the high expression level of PD-L1 and gastric cancer development (P = 0.044, odds ratio = 2.77, 95% confidence interval = 1.12–6.86). Conclusions: PD-L1 level increases in gastric cancer patients, so it could be used as a predictor factor.

Effect of PD-L1 and tumor regression rate on prognosis of nasopharyngeal carcinoma.

To explore the expression level of programmed death ligand-1 (PD-L1) and the tumor regression rate after chemoradiotherapy, and to investigate their influence on the prognosis of patients with nasopharyngeal carcinoma. Eighty-six patients, who were initially diagnosed with non-metastatic nasopharyngeal carcinoma in the Second Xiangya Hospital of Central South University from January 2014 to June 2014, were recruited. Positive expression of PD-L1 in prepared sections of nasopharyngeal carcinoma tissues was detected by immunohistochemical staining. The clinical data of recruited patients and the tumor regression rate were calculated before chemoradiotherapy and 1 month after chemoradiotherapy. In addition, the potential influence of PD-L1 level and the tumor regression rate after chemoradiotherapy on therapeutic efficacy and the prognosis of nasopharyngeal carcinoma were assessed. Among the 86 patients with nasopharyngeal carcinoma, there were 30 patients with high expression of PD-L1 in nasopharyngeal carcinoma sections (34.88%), while there were 56 patients with low expression (65.12%). Expression levels of PD-L1 in nasopharyngeal carcinoma tissues were not related with sex, age, smoking history, concurrent chemoradiotherapy, pathological type, N staging, tumor staging, and pre-treatment EBV-DNA load in peripheral blood in the affected patients (all P>0.05). But the expression level of PD-L1 was related to the tumor regression rate and T staging in the patients with nasopharyngeal carcinoma (both P<0.05). The expression levels of PD-L1 in nasopharyngeal carcinoma tissues was a risk factor for tumor regression rate (OR=0.226, P<0.05). Compared with the patients with low expression levels of PD-L1, there was shorter disease-free survival in the patients with high expression levels of PD-L1 (P<0.05). Moreover, compared with the patients with low tumor regression rate, the nasopharyngeal carcinoma patients with high tumor regression rate showed a favorable disease-free survival (P<0.05). A high expression level of PD-L1 and a low tumor regression rate after chemoradiotherapy are unfavorable to the prognosis of nasopharyngeal carcinoma.

Histone deacetylase 6 inhibition in urothelial cancer as a potential new strategy for cancer treatment.

Histone deacetylases (HDACs) are enzymes that remove acetyl groups from histones and have attracted attention as potential targets for cancer therapy. Several small molecule inhibitors have been developed to target HDACs; however, clinical trials of pan-HDAC inhibitors have found these types of inhibitors to be inefficient and to be relatively highly toxic. In the present study, the role of one HDAC isozyme, HDAC6, in urothelial cancer was investigated. Protein expression levels and subcellular localization of HDAC6 was identified in surgically resected bladder tumors using immunohistochemistry. The antitumor effects of 12 small molecule HDAC6 inhibitors were also examined in vitro using cultured urothelial cancer cells. The HDAC6 inhibitors decreased cell viability, with IC50 values in the low µM range, as low as 2.20 µM. HDACi D, E and F had the lowest IC50 values. HDAC6 has been previously reported to regulate programmed death-ligand 1 (PD-L1) and PD-L1 expression was found to be a predictor of decreased overall survival time. There was no association between the protein expression level of HDAC6 and PD-L1 in tumor tissues; however, HDAC6 inhibition by specific small molecule inhibitors resulted in decreased expression levels of membranous PD-L1 in cultured urothelial cancer cell lines. The results suggested that inhibition of HDAC6 could be a promising novel approach for the treatment of urothelial cancer.

Local Activator and T Cell Engager (αBCMA x αPD-L1 x αCD3) with Enhanced Tumor Killing and Minimal Cytokine Release for the Treatment of Multiple Myeloma

BCMA-targeting bispecific T-cell engagers in clinical development have demonstrated encouraging preclinical efficacy. The most advanced of these is AMG 420, which showed significantly improved response rates in relapsed/refractory multiple myeloma (MM) patients. Nevertheless, median duration until relapse is currently limited to approximately 12 months, highlighting the need for new drugs with novel MoA. Recently, we reported on a Local Activator and T cell Engager (LocATE) antibody that targets BCMA and selectively blocks programmed death-ligand 1 (PD-L1) on malignant cells (ASCO, June 2019). LocATE induced superior T cell activation and cancer cell killing, in vitro and ex vivo, compared to a BCMAxCD3 BiTE alone or in combination with a PD-L1 inhibitor. Here, we sought to further characterize the novel MoA of our LocATE. To assess the therapeutic potential of the LocATE, we first investigated whether potent cytotoxicity is uncoupled from high levels of cytokine release. We evaluated three LocATE molecules with different PD-L1 affinities (low, medium, high). Using BCMA-expressing MM cell lines (U-266, MM.1S, RPMI-8226 and H929) with distinct PD-L1 surface expression levels (3 - 53%), we determined the cytokine profile (IL-2, IL-6, IFN-γ, TNF-α) and target cell lysis induced by each candidate in the presence of CD3-positive human T cells. All three candidates exhibited comparable killing potency, however, low-affinity PD-L1 LocATE antibodies induced significantly less cytokine release (up to 10-fold) than its higher PD-L1 affinity counterparts across all cell lines investigated. Notably, using the low-affinity PD-L1 LocATE, we observed a 2-fold increase in tumor cell killing compared to bispecific BCMAxCD3 targeting controls in cell lines expressing high PD-L1 levels (53%), underlining the contribution of PD-L1 inhibition. Accordingly, phenotypic profiling of effector cells showed that the LocATE more potently induced dose-dependent upregulation of the activation markers CD69, CD25 and HLA-DR compared to bispecific controls. Importantly, cytotoxic activity, T cell activation and cytokine release were not induced when BCMA-negative cells expressing high levels of PD-L1 were treated with LocATE, underlining the BCMA-selective killing mechanism. Since the superior efficacy of LocATE was found to correlate with the expression level of PD-L1 on MM cell lines and upregulation of PD-L1/PD-1 has been reported as one of the major myeloma cell escape mechanisms during treatment with BiTEs, we subsequently investigated the efficacy of LocATE using primary bone marrow samples and peripheral blood mononuclear cell (PBMCs) obtained from MM patients. Six bone marrow mononuclear cell (BMMC) and eight PBMC samples from MM donors of different disease stages were characterized for PD-1/PD-L1 expression levels; analysis of T cell frequency and level of activation/exhaustion was performed based on CD4, CD8, CD25, CD69, Tim-3, Lag-3 and PD-1 marker expression. Using patient samples with high frequencies of PD-1 expressing T cells prior to treatment, LocATE induced superior MM tumor cell lysis and T cell activation compared to BCMAxCD3 bispecific antibodies. No activity was observed on healthy cells, underlining the safe and selective killing mechanism through tumor-local reactivation of exhausted T cells. Collectively, these findings demonstrate that the simultaneous T cell redirection and tumor-specific checkpoint inhibition with the LocATE leads to an improved therapeutic index with robust tumor cell killing and low levels of cytokine release. Capable of counteracting adaptive immune resistance caused by increased PD-1/PD-L1 signaling, the LocATE antibody has the prospect to significantly improve survival for multiple myeloma patients. Disclosures Vrohlings: CDR-Life: Current Employment. Jungmichel:CDR-Life: Current Employment, Other: current option holder. Senn:CDR-Life: Current Employment. Schleier:CDR-Life: Current Employment, Current equity holder in private company. Scheifele:CDR-Life: Current Employment, Current equity holder in private company. Wendelspiess:CDR-Life: Current Employment. Leisner:CDR-Life: Current Employment, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees. Jaeger:CDR Life AG: Consultancy, Research Funding; Miltenyi: Consultancy, Honoraria; Karyopharm: Honoraria; BMS/Celgene: Consultancy, Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; AbbVie: Honoraria; F. Hoffmann-La Roche: Honoraria, Research Funding. Borras:CDR-Life: Current Employment, Current equity holder in private company.

Expert consensus on PD-L1 expression testing in non-small-cell lung cancer in China

Cancer immunotherapy, especially the programmed death 1 (PD-1) and (or) programmed death ligand 1 (PD-L1) checkpoint inhibitors, has ushered in the modern oncology era and has achieved impressive success in the treatment of different tumors, including non-small-cell lung cancer (NSCLC). Yet, only a subset of patients achieved clinical benefit. Therefore, how to effectively screen the potential beneficiaries of PD-1 and (or) PD-L1 inhibitors has become a new challenge in the era of cancer immunotherapy. Clinical studies have shown that the expression level of PD-L1 in NSCLC is positively correlated with the efficacy of PD-1 and (or) PD-L1 inhibitors, and is one of the important predictive biomarkers. However, there are many challenges in PD-L1 testing. With the approving of various PD-1 and (or) PD-L1 inhibitors for immunotherapy setting of NSCLC, expert consensus of important issues regarding clinical significance of PD-L1 testing, testing time-point, as well as the standardization of PD-L1 testing procedure, including the PD-L1 testing operation steps, results' interpretation, quality control and so on, is of great significance for improving the accuracy of detection and reducing the difference between laboratories. On account to the practical questions of PD-L1 testing, this consensus was finally reached, which is based on the combination of literature, expert experience and internal discussion among committee members, in the hope of providing the guide for standardizing the PD-L1 testing and providing accurate and reliable result to screen the potential patients and predict clinical efficacy. It must be pointed out that in view of the existence of objective and complex factors of PD-L1 testing and the fact that the clinical application of PD-L1 testing is still at the early stage in China, there are still many issues need to be updated in near future based on further practice experience and research data.

Relationship between IL10 and PD-L1 in Liver Hepatocellular Carcinoma Tissue and Cell Lines

Background Despite the large-scale clinical application of programmed death-ligand 1 (PD-L1) monoclonal antibody, reduction in its clinical response rate has become a gradual problem. As such, use of PD-L1 monoclonal antibody in combination with other anticarcinoma drugs has been the main strategy in improving its efficacy. Interleukin 10 (IL10) is a recognized inflammatory and immunosuppressive factor. Previous studies have suggested that there is a link between PD-L1 and IL10. Objective This study was aimed at clarifying the relationship between PD-L1 and IL10 in liver hepatocellular carcinoma (LIHC) and whether IL10 enhances the efficacy of PD-L1 inhibitor. Methods Expression levels of PD-L1 and IL10 in carcinoma and adjacent tissues were tested by immunochemistry, Western blotting, and RT-PCR. Survival duration and follow-up data of each patient were recorded. LIHC cell lines Bel7405 and MHCC 97-H were used for in vitro experiments. Exogenous IL10 and anti-IL10 were added to cell supernatant. Expression level of PD-L1 in the LIHC cell lines was determined using Western blotting and ELISA. CCK8 and transwell assays were adopted to examine the effect of PD-L1 combined with IL10 on proliferation, invasion, and metastasis of LIHC cells. Results The survival period of patients with low expression of IL10 was longer than that of patients with high expression (P = 0.01). Overexpression of PD-L1 increased the IL10 and Met levels in LIHC tissues and cell lines. IL10 downregulated the expression level of PD-L1 and enhanced the efficacy of crizotinib via the Met signaling pathway in the LIHC cells. Conclusions A combination of IL10 and PD-L1 inhibitor holds great promise as an effective treatment for LIHC.

Programmed Death Ligand 1 Indicates Pre-Existing Adaptive Immune Response by Tumor-Infiltrating CD8+ T Cells in Non-Small Cell Lung Cancer.

Aberrant expression of programmed death ligand 1 (PD-L1) on tumor cells impedes antitumor immunity and instigates immune evasion. The remarkable efficacy of immune checkpoint blockade has been confirmed in various solid tumors. However, the correlation between PD-L1 expression and host immunological landscape remains of considerable controversy in non-small cell lung cancer (NSCLC). In the present study, PD-L1 expression and CD8+ tumor-infiltrating lymphocyte (TIL) infiltration levels were determined by immunohistochemistry (IHC) in tumor sections of 138 NSCLC patients. The expression level of PD-L1 was positively correlated with the abundance of CD8 + TILs (p < 0.0001). Furthermore, no constitutive expression of PD-L1 was observed in the majority of six NSCLC cell lines detected by Western blot; but exposure to interferon-γ (IFN-γ), a primary cytokine secreted by activated CD8+ T cells, prominently increased PD-L1 expression. Notably, a significantly positive association was determined within PD-L1, CD8 and IFN-γ gene expression by qRT-PCR, which was corroborated by RNA-sequencing from TCGA lung cancer dataset. These findings demonstrate that PD-L1 expression indicates an adaptive immune resistance mechanism adopted by tumor cells in the aversion of immunogenic destruction by CD8+ TILs. Both higher expression of PD-L1 and infiltration of CD8+ TILs were correlated with superior prognosis (p = 0.044 for PD-L1; p = 0.002 for CD8). Moreover, Cox multivariate regression analysis showed that the combination of PD-L1 and CD8 were independent prognostic factors, which was more accurate in prediction of prognosis in NSCLC than individually. Finally, we found that IFN-γ induced the upregulation of PD-L1 in NSCLC cells, mainly through the JAK/STAT1 signaling pathway. In conclusion, PD-L1 expression is mainly induced by activated CD8+ TILs via IFN-γ in the immune milieu and indicates pre-existing adaptive immune response in NSCLC.