PD-L1 Expression In Tissue Research Articles

Adipocytes promote tumor progression and induce PD-L1 expression via TNF-\u03b1/IL-6 signaling

BackgroundObesity confers increased risk for various types of cancer. PD-L1 is a key molecule in tumor immune evasion by inducing T cell exhaustion. The relationship between obesity and PD-L1 is still ambiguous. This study was designed to reveal the development of hepatocellular carcinoma and melanoma in obese mice and to investigate if adipocytes regulate PD-L1 expression and the underlying mechanism.MethodsMonosodium glutamate-induced obese mice were inoculated with H22 tumor cells and High fat diet (HFD)-induced obese mice were inoculated with B16-F1 mouse melanoma cells. Human hepatoma HepG2 cells and B16-F1 cells were treated with conditional media from 3T3-L1 adipocytes (adi-CM). Neutralized anti-TNF-α and anti-IL-6 antibodies and inhibitor of NF-κB or STAT3 were used to reveal the mechanism of effect of adi-CM.ResultsIn obese mice, H22 and B16-F1 tumor tissues grew faster and PD-L1 expression in tumor tissue was increased. Adi-CM up-regulated PD-L1 level in HepG2 and B16-F1 cells in vitro. Differentiated 3T3-L1 adipocytes secreted TNF-α and IL-6, and neutralizing TNF-α and/or IL-6 reduced PD-L1 expression in adi-CM-treated cells. p-NF-κB/NF-κB level was downregulated in HepG2 and B16-F1 cells, and p-STAT3/STAT3 level was also decreased in HepG2 cells. In addition, inhibitor of NF-κB or STAT3 reversed the effect of adi-CM on PD-L1 expression.ConclusionsTNF-α and IL-6 secreted by adipocytes up-regulates PD-L1 in hepatoma and B16-F1 cells, which may be at least partially involved in the role of obesity in promoting tumor progression.

NEXT: A phase II, open-label study of nivolumab adjuvant to chemoradiation in patients (pts) with localized muscle invasive bladder cancer.

TPS605 Background: In pts with localized muscle invasive bladder cancer (MIBC), trimodality bladder preserving therapy (TMT), with transurethral resection of bladder tumor, radio-sensitizing chemotherapy and definitive radiation, has up to a 50% risk of local and systemic relapse during the 2-year post treatment phase. We hypothesize that by inhibiting immune checkpoints, chemo-radiation induced tumor specific immune response will be enhanced both locally and abscopally, resulting in better failure-free survival (FFS). In the NEXT trial, we evaluate the efficacy of nivolumab after completion of the TMT in this setting. Methods: Pts with localized MIBC who have completed standard TMT are eligible. Pts receive nivolumab 480 mg intravenously every 4 weeks for up to 12 doses. Treatment with nivolumab begins within 90 days of completion of TMT. Subjects undergo surveillance cystoscopic and scan based assessments on study. Archived tumor tissue at baseline and at relapse is obtained for correlative studies. The primary endpoint is 2-year FFS. Secondary endpoints include FFS at 2 years in patients with intact bladder, rate of radical cystectomy (RC), cystoscopic local control, distant FFS in patients with intact bladder and those that undergo RC, overall survival, toxicity and quality of life. Exploratory endpoints include to characterize changes in immune cell subsets that can be correlated with clinical outcome and to assess the correlation of response to PD-L1 expression in pretreatment tumor tissue in the study subjects. The planned sample size for this single arm, open label trial is 28 pts. Kaplan-Meier methods will be used to plot survival endpoints and cystoscopic local control rates. Exact binomial methods will be used to provide 6 months, 1-year and 2-year estimates for these endpoints. The sample size justification is based on the maximum width of a two-sided 95% binomial confidence interval for 2 year FFS. With 28 evaluable subjects, a 95% exact binomial confidence interval, estimated using the method of Clopper-Pearson, will extend no more than 20% from the observed FFS. Clinical trial information: NCT03171025.

PD-L1 profiling of circulating tumor cells for immune checkpoint inhibitor therapy in gastroesophageal cancers.

29 Background: Selection of Immune Checkpoint Inhibitor (ICI) therapies in Gastroesophageal cancers are based on IHC based detection of PD-L1 expression in tumor tissue. Invasive biopsy to obtain tumor tissue for IHC is associated with procedural risks, sequelae and expenses. Prior efforts at PD-L1 profiling of Circulating Tumor Cells (CTCs) have been constrained by low CTC yields. We employed a novel approach for harvesting sufficient CTCs from Gastroesophageal cancers that permit PD-L1 profiling by Immunocytochemistry (ICC). Methods: 15 ml peripheral blood was collected from 106 patients among whom 20 were diagnosed with gastric adenocarcinomas (AD) and 86 with either esophageal AD or esophageal squamous cell carcinomas (SCC). CTCs were enriched from PBMCs via an epigenetically acting stabilization process which induced apoptosis in non-malignant cells and conferred survival privilege on apoptosis-resistant CTCs, which were harvested and confirmed by immunostaining for EpCAM and pan-CK positivity. Deep ICC profiling of CTCs was performed with organ-specific markers as well as PD-L1:22c3 and PD-L1:28-8 clones. Results: Viable CTCs could be enriched and harvested in 103 out of 106 samples (97.2%) regardless of metastatic or treatment status. Deep ICC and PD-L1 profiling could be performed in all 103 samples (100%). PD-L1 expression was quantitatively assigned as ‘Low’, ‘Moderate’ or ‘High’. Among the 19 gastric ADs, 5 (26.3%) samples were positive for PD-L1:22c3 and 2 (10.5%) were positive for PD-L1:28-8; all gastric ADs were ‘Low’ for either PD-L1 subtype. Among the 84 esophageal carcinomas (AD+SCC), 32 samples (38.1%) were positive for PD-L1:22c3 (26 Low + 5 Moderate + 1 High) and 16 (19.1%) were positive for PD-L1:28-8 (11 Low + 5 Moderate). Conclusions: CTCs in Gastroesophageal cancers can be considered for evaluating PD-L1 expression in patients where ICI therapies are otherwise viable.

PD-L1 profiling of circulating tumor cells for immune checkpoint inhibitor therapy in head and neck cancers.

36 Background: Selection of Immune Checkpoint Inhibitor (ICI) therapies in Head and Neck cancers are based on IHC based detection of PD-L1 expression in tumor tissue. Invasive biopsy to obtain tumor tissue for IHC is associated with procedural risks, sequelae and expenses. Prior efforts at PD-L1 profiling of Circulating Tumor Cells (CTCs) have been constrained by low CTC yields. We employed a novel approach for harvesting sufficient CTCs from Head and Neck cancers that permit PD-L1 profiling by Immunocytochemistry (ICC). Methods: 15 ml peripheral blood was collected from 95 patients with head and neck squamous cell carcinomas (HNSCC). CTCs were enriched from PBMCs via an epigenetically acting stabilization process which induced apoptosis in non-malignant cells and conferred survival privilege on apoptosis-resistant CTCs, which were harvested and confirmed by immunostaining for EpCAM and pan-CK. Deep ICC profiling of CTCs was performed with organ-specific markers as well as PD-L1:22c3 and PD-L1:28-8 clones. Results: Viable CTCs could be enriched and harvested in 90 out of 95 samples (95.3%) regardless of metastatic or treatment status. Deep ICC and PD-L1 profiling could be performed in all 90 samples (100%). PD-L1 expression was quantitatively assigned as ‘Low’, ‘Moderate’ or ‘High’. 28 (31.1%) samples were positive for PD-L1:28-8 (27 Low + 1 Moderate) and 26 (28.9%) samples were positive for PD-L1:22c3 (24 Low + 2 Moderate). 15 samples were positive for both PD-L1 clones. Conclusions: CTCs in Head and Neck squamous cell carcinomas can be considered for evaluating PD-L1 expression in patients where ICI therapies are otherwise viable.

Anti-PD-1 Therapy Response Predicted by the Combination of Exosomal PD-L1 and CD28

Background: Anti-PD-1 therapy has been approved for cancer treatment. However, the response rate is unsatisfactory. The expression of PD-L1 in tumor tissues affected by chemotherapy, radiotherapy, and activity of signal transduction pathways, is unreliable to predict the treatment response. Recent studies suggested that exosomal PD-L1 not only exerts immunosuppressive effects but also plays a significant role in the development of tumor microenvironment. Thus, the present study aimed to investigate exosomal PD-L1 in improving its predictive value and treatment efficacy. Methods: A total of 44 patients with several types of advanced tumors, treated by anti-PD-1 therapy, were enrolled. Exosomes were collected and purified from plasma using the exosome isolation kit. The exosomal PD-L1 and exosomal PD-1 were detected by ELISA. The cytokines were measured by the MILLIPLEX magnetic bead assay. Results: Compared to the responders, exosomal PD-L1 and exosomal PD-1 of the non-responders were significantly higher than those of the responders (P=0.010 and P=0.022, respectively) before the treatment. Concurrently, exosomal PD-L1, exosomal PD-1, and tumor burden decreased when the therapy was effective. Also, the baseline expression of CD28 was higher in the responders than non-responders (P=0.005). Univariate and multivariate analysis validated with 1000 times bootstrapping suggested that high exosomal PD-L1 and low CD28 expression were negative factors for progression-free survival (PFS) in patients who underwent anti-PD-1 treatment. The combination of exosomal PD-L1 and CD28 obtained more area under the curve (AUC) of receiver operating characteristic (ROC) (AUC 0.850 vs. 0.784 vs. 0.678) and showed a higher probability without progression via nomograph. Conclusions: Baseline low levels of exosomal PD-L1 and high expression of CD28 screened out the potential beneficiary populations of anti-PD-1 therapy in a variety of solid tumors. These findings suggested that the expression of exosomal PD-L1 and CD28 could serve as the predictive biomarkers for clinical responses to anti-PD-1 treatment. Funding Statement: This study was funded by The National Key Research and Development Program of China (NO.2017YFC1308900); National Science and Technology Major Project of the Ministry of Science and Technology of China (No. 2017ZX09304025); National Natural Science Foundation of China (NO.81602098, NO.31770963); The Key Research and Development Program of Liaoning Province (NO.2018225060); Science and Technology Plan Project of Liaoning Province (NO.2016007010). Declaration of Interests: All authors have declared no conflicts of interest. Ethics Approval Statement: The study was approved by the local Ethics Committee and all the procedures were conducted in accordance with the ethical principles of China Medical University (Scientific Ethics NO. 2019-142-2). Clinical information of all patients was retrieved from the Hospital Information System.

High expression of PKM2 synergizes with PD-L1 in tumor cells and immune cells to predict worse survival in human lung adenocarcinoma.

Background: Immunotherapy targeting PD-1/PD-L1 represents a breakthrough in the treatment of lung cancer. Pyruvate kinase M2 (PKM2) is not only a critical player in glycolysis, but also conducive to tumor progression and immune response. While both have been linked to lung adenocarcinoma (AC), the correlation and clinical significance of PKM2 and PD-L1 expression in human lung AC tissues remains not entirely explored.Methods: Expression of PKM2 and PD-L1 proteins were detected by immunohistochemistry in 74 lung AC cases and the corresponding noncancerous tissues. Simultaneously, multiplex immunofluorescence was used to detect PKM2, PD-L1, CK, CD3, and CD68 in the lung AC tissues. We measured expression patterns and co-localization of these markers, evaluating their association with clinicopathological features and overall survival. Validation of findings was conducted using mRNA expression data from The Cancer Genome Atlas (TCGA) of 515 lung AC cases.Results: High expression of PKM2 in tumor cells was significantly related with lymph node metastasis and TNM stage (p=0.035, p=0.017, respectively). Moreover, PKM2 expression in tumor cells was positively correlated with tumor PD-L1 expression. High expression of PKM2, PD-L1 in tumor cells and immune cells predicted high mortality rate and poorer survival rates, respectively. Additionally, multivariate Cox regression models indicated that high expression of PKM2 in tumor cells was an independent prognostic factor. Based on TCGA genomic data, high PKM2 mRNA expression was significantly associated with poorer survival (p=0.001).Conclusion: High expression of PKM2 synergizes with PD-L1 in tumor cells and immune cells to predict poorer survival rates in patients with lung AC.

LRP11 activates β-catenin to induce PD-L1 expression in prostate cancer

Prostate cancer (PRAD) is associated with abnormal cholesterol metabolism and low-density lipoprotein (LDL) receptor-related protein (LRP) family is essential for the homeostasis of cholesterol. Immune check points like PD-L1 are vital for tumour cells to evade immune attack. However, the potential cross-talk between these two pathways has not been explored before in PRAD. Insight from the regulation mechanism of PD-L1 in PRAD may help to optimise PD-L1 based immunotherapy. In this study, we investigated a regulation network of LRP11/β-catenin/PD-L1 in PRAD. We showed that the expression of LRP11 and PD-L1 was up-regulated in PRAD compared to paired normal tissues. LRP11 expression was positively correlated to PD-L1 expression in PRAD tissues. Further experiments in two PRAD cell lines with LRP11 over-expression and knockdown showed that LRP11 induced PD-L1 expression through β-catenin signalling. In addition, LRP11 over-expression in PRAD cell line induced immunosuppression of Jurkat cell in in-vitro co-culture system. The effects of LRP11 could be blocked by neutralising LRP11 or PD-L1 antibody. Our results provide evidence for a novel regulation mechanism of PD-L1 expression in PRAD and LRP11 may be a potential therapeutic target in PRAD.

PD-L1 profiling of circulating tumour cells is a viable companion diagnostic for checkpoint inhibitor therapy in lung cancer

Abstract Background Selection of Checkpoint Inhibitor therapies in several cancers are based on PD-L1 expression in tumor tissue determined by IHC. Invasive biopsy to obtain tumor tissue is associated with procedural risks, sequelae and expenses. Though PD-L1 profiling of Circulating Tumor Cells (CTCs) has been attempted previously, limitations arise due to low yields of CTCs in conventional methods. We describe a novel approach for harvesting sufficient CTCs that can be used for PD-L1 profiling by Immunocytochemistry (ICC). Methods 15 ml peripheral blood was collected from 95 patients with histopathologically confirmed diagnosis of lung cancer. PBMCs were isolated by centrifugation. CTCs were enriched from PBMCs via an epigenetically acting stabilization process which induces apoptosis in normal (non-cancerous) cells while simultaneously conferring survival privilege on apoptosis-resistant cells of tumorigenic origin (CTCs). Surviving CTCs were confirmed by immunostaining for EpCAM and pan-CK. CTCs were further profiled for Napsin and TTF-1 (Lung-Adenocarcinoma specific), p40 (Squamous Cell Carcinoma specific), CK7 (Adenocarcinoma specific), Synaptophysin and Chromogranin (Neuroendocrine carcinoma specific) and PD-L1 (22c3). Results Viable CTCs could be enriched and harvested in all 95 samples regardless of treatment status and extent of disease (metastatic status). Deep ICC profiling including all organ- and subtype-specific markers as well as PD-L1 could be conducted in 71 out of the 95 samples (74.7%). Among the 71 samples which were evaluable, PD-L1 positivity was observed in 33 samples (46.5%). PD-L1 expression in these 33 samples was quantitatively estimated and assigned as ‘Low’, ‘Moderate’ or ‘High’. Among the 33 PD-L1 positive samples, 25 (75.8%) had low PD-L1 expression, 7 (21.2%) had moderate expression, and 1 (3%) had high expression. Conclusion ICC profiling of CTCs is a viable approach for determining suitability of patients for checkpoint inhibitor therapies. This method permits quantitative determination of PD-L1 expression which appears to have clinical relevance in determining the probability of favourable response to checkpoint inhibitor therapies. Legal entity responsible for the study The authors. Funding Datar Cancer Genetics Limited. Disclosure R. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. D. Akolkar: Full / Part-time employment: Datar Cancer Genetics Limited. D. Patil: Full / Part-time employment: Datar Cancer Genetics Limited. V. Datta: Full / Part-time employment: Datar Cancer Genetics Limited. P. Devhare: Full / Part-time employment: Datar Cancer Genetics Limited. S. Patel: Full / Part-time employment: Datar Cancer Genetics Limited. A. Srinivasan: Full / Part-time employment: Datar Cancer Genetics Limited. R. Datar: Shareholder / Stockholder / Stock options: Datar Cancer Genetics Limited. All other authors have declared no conflicts of interest.

TFEB Mediates Immune Evasion and Resistance to mTOR Inhibition of Renal Cell Carcinoma via Induction of PD-L1.

Despite the FDA approval of mTOR inhibitors (mTORi) for the treatment of renal cell carcinoma (RCC), the benefits are relatively modest and the few responders usually develop resistance. We investigated whether the resistance to mTORi is due to upregulation of PD-L1 and the underlying molecular mechanism. The effects of transcription factor EB (TFEB) on RCC proliferation, apoptosis, and migration were evaluated. Correlation of TFEB with PD-L1 expression, as well as effects of mTOR inhibition on TFEB and PD-L1 expression, was assessed in human primary clear cell RCCs. The regulation of TFEB on PD-L1 was assessed by chromatin immunoprecipitation and luciferase reporter assay. The therapeutic efficacies of mTORi plus PD-L1 blockade were evaluated in a mouse model. The function of tumor-infiltrating CD8+ T cells was analyzed by flow cytometry. TFEB did not affect tumor cell proliferation, apoptosis, and migration. We found a positive correlation between TFEB and PD-L1 expression in RCC tumor tissues, primary tumor cells, and RCC cells. TFEB bound to PD-L1 promoter in RCCs and inhibition of mTOR led to enhanced TFEB nuclear translocation and PD-L1 expression. Simultaneous inhibition of mTOR and blockade of PD-L1 enhanced CD8+ cytolytic function and tumor suppression in a xenografted mouse model of RCC. These data revealed that TFEB mediates resistance to mTOR inhibition via induction of PD-L1 in human primary RCC tumors, RCC cells, and murine xenograft model. Our data provide a strong rationale to target mTOR and PD-L1 jointly as a novel immunotherapeutic approach for RCC treatment.

Plasma Soluble Programmed Death Ligand 1 Levels Predict Clinical Response in Peripheral T-Cell Lymphomas

Background: Immune checkpoints, including PD-1/PD-L1, play an important role in immunosuppression in various malignancies. Elevated levels of soluble programmed death-ligand1 (sPD-L1) are associated with worse prognosis in multiple myeloma and diffuse large B cell lymphoma. Herein, the purpose of this study is to investigate the relationships between plasma sPD-L1 levels and clinical response in peripheral T-cell lymphomas (PTCLs) patients. Methods: Data from three cohorts, including 11 ALCL patients, 28 PTCL-NOS patients and 81 matched normal control tissues, were also obtained from the ONCOMINE database (https://www.oncomine.org) for PD-L1 gene expression array. The comparison dataset analysis of PD-L1 mRNA levels among diverse PTCL subtypes and matched normal control tissues was performed. Other 37 PTCLs patients and 20 healthy volunteers were also enrolled in this study. Peripheral blood from patients was collected prior to systemic therapy. Plasma levels of sPD-L1 and IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA). PD-L1 expression in tissues was detected by immunohistochemistry (IHC). Clinical response for patients was evaluated. Results: ONCOMINE database analyses showed that the PD-L1 mRNA expression was upregulated in PTCLs, which were significantly higher in PTCLs compared to matched normal control tissues (1 cohort, P=0.029; 2 cohort, P=0.020 and 3 cohort, P=0.021). Among another cohort of 37 PTCLs patients and 20 healthy volunteers, the median sPD-L1 level was respectively 0.729 ng/ml for 20 healthy volunteers and 1.696 ng/ml for PTCLs patients, which was significantly higher than that in healthy volunteers (P=0.000). The median IFN-γ level of PTCLs patients was 4.555 pg/ml, and that the levels of sPD-L1 was positively correlated with the level of IFN-γ (P=0.000, r=0.849) for PTCLs patients. We found that patients with elevated LDH level, advanced stage and elevated β2-MG level had higher sPD-L1 levels than those with normal LDH level, early stage and normal β2-MG level. The sPD-L1 level was also positively correlated with LDH levels (P=0.003), clinical staging (P=0.045) and β2-MG level (P=0.045). Patients with high sPD-L1 level had lower overall response rate than those with low sPD-L1 level (88.9% vs 50.0%, P= 0.022), suggesting that sPD-L1 levels was an underlying plasma biomarker to predict the clinical response in PTCL patients. The median PFS for high and low sPD-L1 level groups was 42.7 months (95% CI, 27.9-57.6) and 53 months (95% CI, 33.7-72.3), respectively. As well, the median OS for high and low sPD-L1 level groups was 48.3 months (95% CI, 35.2-61.2) and 71 months (95% CI, 51.0-90.9), respectively. Survival data suggested that patients with high sPD-L1 levels tended to have shorter PFS and OS than those with low sPD-L1 levels. We also found that plasma sPD-L1 level appeared to have a positive relationship with tissue PD-L1 expression in PTCLs patients, and both of them had a high matched rate each other (90.9%). Conclusions: PTCLs patients had higher sPD-L1 level compared with healthy volunteers. High sPD-L1 level was correlated with worse clinical response, suggesting that sPD-L1 level was an underlying plasma biomarker to predict the prognosis for PTCLs patients. Disclosures No relevant conflicts of interest to declare.

PD-L1 Expression in Patients with Primary Central Nervous System Diffuse Large B-Cell Lymphoma: Its Correlation with Serum Levels of Soluble Programmed Death-Ligand 1 (sPD-L1)

Background: The genetic evaluation of primary central nervous system lymphoma (PCNSL) demonstrated frequent copy-number alterations of 9p24.1/PD-L1/PD-L2 increasing the expression of programmed cell death protein 1 (PD-1) ligands, PD-L1 and PD-L2 in PCNS. Given the role of immune escape in the development of neoplasm, the expression of PD-L1/PD-L2 might play a role in the biology of PCNSL. Indeed, the PD-1 blockade by nivolumab, an immune checkpoint inhibitor has shown a single-agent activity in relapsed and refractory patients with PCNSL, implying its role as a salvage treatment for relapsed or refractory PCNSL. Moreover, tissue biopsy of brain is not always possible due to the risk of post-biopsy complications including bleeding or the occurrence of neurologic sequelae. Thus, liquid biopsy such as blood sampling might be more feasible and useful if a marker obtained from liquid biopsy could reflect biologic characteristics of tumor cells. Thus, serum sPD-L1 could be potentially used as a biomarker for representing tissue expression of PD-L1. Therefore, we measured the level of sPD-L1 in patients with PCNSL and analyzed its clinical relevance as a prognostic marker. Also, its correlation with PD-L1 expression of tumor cells are evaluated. Material and Methods: The study population was from patients who were diagnosed with PCNSL between January 2009 and February 2017. 68 patients who had archived serum samples available for measurement of sPD-L1 were analyzed. The measurement of sPD-L1 was performed using archived serum samples at diagnosis, and the sPD-L1 values in blood serum specimens of healthy controls were determined by the same method. Also, tissue expression of PD-L1 was also analyzed using archived paraffin-embedded tissue blocks. The extent of PD-L1 expression on tumor cell was defined as the proportion of tumor cells showing PD-L1 expression with any intensity in tumor area. Macrophages and lymphocytes infiltrating tumor area were considered non-tumor immune cells and the proportion of PD-L1 expression was assessed in a same manner as tumor cells. PD-L1-positive tumor cell was defined as positively stained for PD-L1 with distinct pattern of membranous and/or cytoplasmic or punctate/granular of any intensity because the immunostaining pattern of PD-L1 was reported to be membranous, cytoplasmic, or/and punctate/granular. Disease relapse, progression-free survival (PFS) and overall survival (OS) were analyzed according to the extent of sPD-L1 in serum and PD-L1 expression in tissue. Results: The median level of patients' serum sPD-L1 (n = 68) was 0.429 ng/mL (range: 0.324 - 0.757 ng/mL), and it was significantly higher than that of healthy control group (0.364 ng/mL; range: 0.329 - 0.390 ng/mL, P < 0.01). The optimal cutoff for predicting OS was determine as 0.432 ng/mL (the area under curve of sPD-L1 for OS was 0.739). The OS and PFS of high group were significantly lower than that of low group. The occurrence of relapse or progression was more frequent in the high sPD-L1 group (78%, 25/32) than low sPD-L1 (50%, 18/36) group (P = 0.023). PD-L1-positive tumor cells were found in 35 patients (67%), the median percentage was 0.7% and 20 patients (57%) had less than 1% of positivity. the extent of PD-L1-postive tumor cells showed modest associated with serum levels of sPD-L1 (r = 0.299, P = 0.031). However, the median percentage of positively stained immune cells including macrophages was 2.7% (Q1: 1.2% - Q3: 6.3%), and the percentage of PD-L1-postive immune cells was not correlated with serum levels of sPD-L1. Survival analysis revealed a trend of poor OS and PFS in patients with ≥ 1% of PD-L1-positive tumor cells. Conclusion: Our study demonstrated serum levels of sPD-L1 could reflect the expression of PD-L1 in tumor cells of PCNSL, and predict poor survival outcomes of patients. The studied results suggest the importance of PD-L1-positive tumor cells contributing to serum level of sPD-L1 compared to immune cells' PD-L1 expression. In line with these findings, the survival analysis based on the extent of PD-L1 expression of tumor cells showed better association with tumor cells' PD-L1 expression rather than immune cells. Thus, sPD-L1 in serum could be a feasible biomarker for determining the risk-adapted treatment strategy of PCNSL patients. Figure Disclosures No relevant conflicts of interest to declare.

Stat3-/- Alloreactive Donor T Cells Reduce FAO but Increase Leakage of ROS in Response to PD-1 Signaling Triggered By Host-Tissue PD-L1, Leading to Prevention of Gvhd While Preserving GVL Effect

The GVHD pathogenicity of alloreactive T cells is associated with enhanced glycolysis or enhanced fatty acid oxidation (FAO). PD-1 signaling in T cells inhibits glycolysis but augments FAO. Host tissue PD-L1 interaction with PD-1 on activated alloreactive T cells is not able to effectively prevent acute GVHD mediated by donor CD4+ and CD8+ T cells together, although it can reduce the severity of the disease. Using a murine HCT model of C57BL/6 donor to BALB/c recipient, we show that although WT T cells (CD4+ and CD8+ T cells together) induced lethal GVHD at 1-5 x106/mouse, the same dose of Stat3-/- donor T cells induced little signs of acute or chronic GVHD because of induction of T cell anergy, exhaustion and apoptosis, but with retention of a strong GVL effect. This induction of tolerance depends on donor T cell PD-1 interaction with host-tissue PD-L1, because Stat3-/- T cells induce severe lethal GVHD in the PD-L1-/- recipients and in WT recipients treated with blocking anti-PD-1 mAb. The tolerance of Stat3-/- donor T cells induced by PD-1 signaling is associated with lower FAO and mitochondria membrane potential (MMP) but higher ROS leakage, with little difference in glycolysis, as compared to non-tolerant WT T cells. In the absence of PD-1 signaling, Stat3-/- T cells manifest markedly enhanced glycolysis and loss of tolerance. These results indicate that activated donor T cell PD-1 interaction with host-tissue PD-L1 reduces glycolysis and reduces their GVHD capacity. In addition, absence of mitochondrial pStat3 in Stat3-/- donor T cells leads to reduced FAO and MMP but increased ROS leakage, resulting in effective anergy, exhaustion, and apoptosis of tissue infiltrating donor T cells and prevention of GVHD. At the same time, the reduced expression of PD-L1 in recipient lymphoid tissues allows donor T cell expansion and strong GVL activity. Our studies have revealed novel roles of mitochondria pStat3 in regulating PD-1 signaling, T cell metabolism and tolerance. (Grant support: R01AI066008 to Zeng) . Disclosures No relevant conflicts of interest to declare.

Concordance of PD-L1 expression detected by two immunohistochemical platforms and antibodies

Objective: To evaluate the concordance of PD-L1 expression in various tissues using antibodies 28-8 and SP263 on their respective detection platforms. Methods: Three hundred seventy four specimens of surgical resection of pulmonary diseases in the First Affiliated Hospital of Nanjing Medical University from January 1, 2012 to January 31, 2017 were collected. Totally 374 cases were tested for PD-L1 expression using the two antibodies, 28-8 and SP263, by respective detection platforms (Dako and Ventana). Finally, 336 cases were used for further evaluation, and the results were statistically analyzed for concordance. Results: For non-small cell lung carcinoma (NSCLC), the positive rate of PD-L1 was 57.5% (177/308) using SP263, and 57.5% (177/308) using 28-8 antibody. The correlation coefficient was 0.97 (P<0.01). The positive rate of both benign lung diseases and paracancerous tissues was about 10.7% (3/28), and the positive concordance rate was 100.0%. The distribution of both antibodies was also relatively consistent. Conclusions: The expression levels of 28-8 and SP263 antibodies in NSCLC and other tissues are relatively consistent, suggesting both antibodies may be complementary and substitute for each other, which may be useful in guiding clinical management.

Role of immune checkpoint inhibitors in bone sarcomas and future aspects

Abstract Background The most common subtypes of bone sarcomas are osteosarcoma and Ewing sarcoma. They are rare and heterogeneous form of cancers of mainly mesenchymal origin. Neo-adjuvant chemotherapy followed by wide surgical resection followed by adjuvant chemotherapy and radiotherapy are the main treatment options for these tumors. But despite all measures, mortality rates have not come down in last few decades. Immunotherapies with various vaccines and cytokines had limited success in clinical trials. This has led to the need of novel therapies like PD-L1 immune checkpoint inhibitors for the treatment of bone and soft tissue sarcomas, which have shown promising results in other malignancies like metastatic melanoma, renal cell carcinoma, lung cancer, and breast cancer. Methods PubMed was searched to identify relevant clinical studies of PD-1/PD-L1-targeted therapies in soft tissue and bone sarcomas. A review of data from the current trials on clinicaltrial.gov was incorporated, as well as data presented in abstracts in annual meetings of American Society of Clinical Oncology and European Society for Medical Oncology were included in the study and analysed. Results 23.7% of osteosarcomas demonstrate high expression and 50% show intermediate expression of PD-L1 at tissue level which is clinically significant, as it correlates with tumor-infiltrating lymphocytes (TILs), which often mark metastasis and poorer outcomes. Other studies and clinical trials support these findings. In a systematic meta-analysis of 11 studies with 661 total patients, osteosarcoma had 14–75% higher PD-L1 expression in tumor tissues and significantly correlated with high mortality rate, metastasis and poor overall survival. Other studies have also shown higher PD-L1 expression in metastatic tumor tissues, especially within the lung, compared to primary tumor tissues. Conclusions Immune checkpoint blockade therapy has demonstrated remarkable clinical results in other malignancies but they have yet to show positive results in bone and soft tissue sarcomas. Though early clinical trials have shown promising results in using anti-PD-1/PD-L1 therapy either alone or with other treatment options, continuous clinical trials and studies with long term results are necessary to have conclusive results. Legal entity responsible for the study Barkatullah University. Funding Has not received any funding. Disclosure All authors have declared no conflicts of interest.

Clinical significance of PD-L1 expression in serum-derived exosomes in NSCLC patients

BackgroundExosomes are 50–150 nm endocytic vesicles secreted by almost all type of cells that carry bioactive molecules from host. These small vesicles are considered to be novel cross-talk circuits established by tumor cells and tumor microenvironment. Previous studies have shown certain biological influence of exosomal programmed cell-death ligand 1 (Exo-PD-L1) on immune suppression and dysfunction. The aim of the current study was to investigate the impact of Exo-PD-L1 and soluble PD-L1 (sPD-L1) on non-small cell lung cancer (NSCLC) and explore the concordance between Exo-PD-L1 and PD-L1 expression in matched tumor tissues in NSCLC patients.Methods85 consecutive patients from April 2017 to December 2017 at General Hospital of Eastern Command Theatre who were primarily diagnosed with NSCLC and 27 healthy individuals were enrolled in this study. Two milliliters of whole blood samples were collected from each participant and further centrifuged. Exosomes were derived from serum using the commercial kit (Total Exosome Isolation Kit), which was further identified by Western blotting analysis (CD63/TSG101), transmission electron microscope analysis (TEM) and nanoparticle tracking analysis (NTA). Exosomes were next solubilized for Exo-PD-L1 detection by enzyme-linked immuno-sorbent assay (ELISA). PD-L1 expression in matched tissue were assessed by PD-L1 immunohistochemistry (IHC) (clone 28-8) assay. Tumor proportion score (TPS) ≥ 1% was deemed as “positive” in this study and TPS < 1% was deemed as “negative”. Written informed consent were obtained before acquisition of all data and biological sample. Data were analyzed using SPSS 20.0 and Graphpad Prism 5 software. Chi square test was conducted to estimate the correlation between Exo-PD-L1 levels, sPD-L1 levels, PD-L1 IHC profiles and clinicopathological features. For all analysis, a two-sided p < 0.05 was considered significant statistically.ResultsExo-PD-L1 levels were higher in NSCLC patients with advanced tumor stage, larger tumor size (> 2.5 cm) (p < 0.001), positive lymph node status (p < 0.05) and distant metastasis (p < 0.05). In contrast, sPD-L1 levels were not different between NSCLC patients and healthy donors, it was not correlated with any clinicopathologic features except for tumor size (> 2.5 cm) (p < 0.05). In addition, Exo-PD-L1 levels showed slight correlation with sPD-L1 levels (Spearman’s correlation at r = 0.3, p = 0.0027) while no correlation with PD-L1 IHC profiles was detected.ConclusionsIn conclusion, Exo-PD-L1, but not sPD-L1, was correlated with NSCLC disease progression, including tumor size, lymph node status, metastasis and TNM stage. However, Exo-PD-L1 was not associated with PD-L1 IHC status.

Clinicopathologic and microenvironmental analysis of primary cutaneous CD30-positive lymphoproliferative disorders: a 26\u2009year experience from an academic medical center in Brazil

BackgroundPrimary cutaneous CD30+ lymphoproliferative disorders (pc-CD30-LPD) are a group of clonal T cell lymphoproliferative disorders that despite very similar tumor histology follow different and characteristic clinical courses, suggesting a homeostatic role of the tumor microenvironment. Little is known about tumor microenvironment and there is almost no literature about PD-L1 expression in pc-CD30-LPD.MethodsThis retrospective study presents a fully clinicopathologically characterized series of pc-CD30-LPDs from an academic medical center in Brazil, including 8 lymphomatoid papulomatosis (LyP), 9 primary cutaneous anaplastic large cell lymphoma (pcALCL) and 4 borderline lesions. All the cases were scored for FOXP3+ regulatory T-cells (Treg) and CD8+ cytotoxic tumor infiltrating lymphocytes (TIL) densities, as well as PD-L1 expression in tumor cells and tissue associated macrophages. The CD8+/FOXP3+ ratio was also evaluated.ResultsAmong the 21 cases of pc-CD30-LPD, PD-L1 expression is frequent in both tumor cells and tissue associated macrophages in pc-CD30-LPD across categories, suggesting that the PD-L1 axis may be a common feature of pc-CD30-LPDs. While reactive T cell infiltrates vary widely from case to case, a common feature across pc-CD30-LPDs is higher density of CD8 than FOXP3 + T cells. The distribution of T cells within the lesions however differed between LyP and pcALCL: we found that LyP lesions tend to be permeated by CD8+ and FOXP3+ T cells, whereas pcALCL tend to be surrounded by a rim of CD8+ TIL and FOXP3+ Tregs with relatively lower density infiltrates in the center of the lesion.ConclusionsLyP has a trend to have denser immune cells throughout the lesion, with higher FOXP3+ Treg and CD8+ TIL in the center than the edge comparing with pcALCL. PD-L1+ is frequent in tumor cells and tissue associated macrophages in pc-CD30-LPD. The differential distribution of CD8+ and FOXP3+ TILs in LyP as compared to pcALCL could provide a clue to the relapsing/remitting course of LyP as compared to the less frequent spontaneous regression of pcALCL.

138P - Expression of PD-L1 in plasma exosomes of NSCLC patients and its associations with PD-L1 expression of corresponding tumour tissues

BackgroundThe aim of this study was to research whether there was expression of PD-L1 in plasma exosome of NSCLC patients and whether its expression can partly represent PD-L1 expression of corresponding lung tumor tissues. MethodsPlasma was collected before and after surgery of NSCLC patients with stage II to stage III. Plasma of NSCLC patients with stage IV was also collected before and after anti-PD-1/PD-L1 therapy. Digital PCR was used to detect PD-L1 and β-actin mRNA expression of plasma exosomes. Expression of PD-L1 in tumor tissues was determined by immunohistochemical staining (22C3). Pearson correlation analysis and wilcoxon rank sum test was performed by SPSS 22.0. ResultsA total of 126 patients (54 patients with surgery and 72 patients with non-surgery therapy) were participated in this study. Exosomal PD-L1 expression can be detected in all plasma samples. Exosomal expression of PD-L1 calculated by both absolute quantification method and relative quantification method showed a positive correlation with PD-L1 expression of tumor tissues (absolute quantification method: r2=0.289, P=0.001; relative quantification method: r2=0.547, P=0.000). However, expression of exosomal PD-L1 calculated by relative quantification calculation showed higher correlation. Exosomal PD-L1 expression significantly decreased after surgery (P<0.001) and three patients who showed good response (PR) to anti-PD-1/PD-L1 therapy also showed significantly decreasing exosomal PD-L1 expression after anti-PD-1/PD-L1 therapy (P<0.001). ConclusionsPlasma exosomes of NSCLC patients contains substantial expression of PD-L1. Plasma exosomal PD-L1 expression showed high correlation with PD-L1 expression of tumor tissues. Exosomal PD-L1 expression could represent PD-L1 expression of tumor tissues and might be a potential predictive marker for anti-PD-1/PD-L1 therapy. Legal entity responsible for the studyJifeng Feng. FundingThe National Natural Science Foundation of China. DisclosureAll authors have declared no conflicts of interest.

1211P - Induction of tumour-infiltrating functional CD8 positive cells and PD-L1 expression in esophageal cancer by S-588410

BackgroundS-588410 is a cancer peptide vaccine composed of five HLA-A*24:02-restricted epitope peptides derived from five cancer-testis antigens, DEPDC1, MPHOSPH1, URLC10, CDCA1 and KOC1, all of which are overexpressed in esophageal cancer. The aim of this study is to evaluate the effect of S-588410 on CD8 positive (+) T-lymphocytes in both blood and tumor tissue and PD-L1 expression in tumor tissue, by comparing of the specimens from before and after vaccination. MethodsHLA-A*24:02 positive patients (pts) with esophageal cancer who can receive the treatment more than 30 days before the surgery were eligible. S-588410 (1mg each of 5 peptides mixed with Montanide ISA 51 VG) was weekly injected subcutaneously. Blood and tumor tissue were collected for T cell receptors (TCR) repertoire analysis, multi-parameter immunohistochemistry and flow cytometry. Peptide-specific CTLs in PBMC were evaluated using ELISpot assay and tetramer assay. ResultsA total 15 pts were enrolled from Sep./2016 to Dec./2017. Pts received a median of 5 injections (range, 3-14) of S-588410. After vaccination, peptide-specific CTLs activity in PBMC were induced in all pts at least for 1 of 5 peptides. Multifunctional CD8+ T cells in PBMC were increased in 7 out of 12 pts and maintained in the other pts after vaccination. The densities of CD8+, CD8+GranzymeB+, CD8+PD1+ and PD-L1+ cell in tumor tissue after vaccination were higher than those before vaccination. Furthermore, in 6 out of 7 pts, TCRs identified from peptide-specific CTLs were present in both tumor tissue and PBMC after vaccination. ConclusionsVaccination of S-588410 induces CD8+/ CD8+PD1+ tumor-infiltrating cells and PD-L1 expression in esophageal cancer. These results suggest that S-588410 enhances tumor immunity and PD1/PD-L1 axis. Thus a combination of S-588410 with anti-PD1/PD-L1 antibody is expected to be more effective than monotherapy. Clinical trial identificationUMIN000023324. Legal entity responsible for the studyShionogi & Co., Ltd. FundingShionogi & Co., Ltd. DisclosureT. Kojima: Research grant / Funding (institution), Travel / Accommodation / Expenses, Sponsored initiated study: Shionogi; Research grant / Funding (institution), Sponsored initiated study: Ono Pharmaceutical; Research grant / Funding (institution), Sponsored initiated study: MSD; Research grant / Funding (institution), Sponsored initiated study: Oncolys BioPharma; Research grant / Funding (institution), Sponsored initiated study: Astellas Amgen BioPharma; Research grant / Funding (institution), Sponsored initiated study: Chugai; Research grant / Funding (institution), Sponsored initiated study: Parexel. T. Nakatsura: Advisory / Consultancy, Compensation for IDMC: Shionogi. K. Kato: Research grant / Funding (institution): Ono Pharmaceutical; Research grant / Funding (institution): Merck; Research grant / Funding (institution): Merck Serono. T. Hikichi: Full / Part-time employment: Cancer Precision Medicine. S. Yoshimura: Full / Part-time employment: Cancer Precision Medicine. T. Nakagawa: Full / Part-time employment: Shionogi. M. Furukawa: Full / Part-time employment: Shionogi. K. Stoeber: Full / Part-time employment: Shionogi. M. Nagira: Full / Part-time employment: Shionogi. N. Ide: Full / Part-time employment: Shionogi. All other authors have declared no conflicts of interest.

LBA89 - PD-L1 expression, patterns of progression and patient-reported outcomes (PROs) with durvalumab plus platinum-etoposide in ES-SCLC: Results from CASPIAN

LBA89 - PD-L1 expression, patterns of progression and patient-reported outcomes (PROs) with durvalumab plus platinum-etoposide in ES-SCLC: Results from CASPIAN

Prognostic significance of PD-1 and PD-L1 positive tumor-infiltrating immune cells in ovarian carcinoma

IntroductionOvarian carcinoma is associated with the highest mortality of all gynecologic malignancies. Even after optimal treatment, prognosis remains poor. There is no established biomarker to predict individual patient outcome.ObjectiveTo evaluate...