Programmed Death Ligand 1 Protein Expression Research Articles

Prognostic effect of programmed death-ligand 1 (PD-L1) in ovarian cancer: a systematic review, meta-analysis and bioinformatics study

BackgroundThe expression of PD-L1 has been reported in ovarian cancer. However, the prognostic role of PD-L1 expression in ovarian carcinoma remained controversial. This study was performed to assess the prognostic value of PD-L1 expression on ovarian cancer.MethodsThe PubMed, Embase, EBSCO, and Cochrane Library databases were searched to identify available publications. The pooled odds ratio (OR) or hazard ratios (HRs: multivariate analysis) with their 95% confidence intervals (95% CIs) were calculated in this analysis. A bioinformatics study based on The Cancer Genome Atlas (TCGA) sequencing and microarray datasets was used to further validate the results of PD-L1 mRNA expression. Kaplan-Meier (KM) survival curves were performed to evaluate the prognostic effect of PD-L1 mRNA expression.ResultsTwelve studies with 1630 ovarian cancers regarding PD-L1 immunohistochemical expression were identified. Meta-analysis showed that PD-L1 protein expression was not associated with tumor grade, clinical stage, lymph node status, tumor histology, overall survival (OS), and progression-free survival (PFS). TCGA data showed no association between PD-L1 mRNA expression and ovarian cancer. Further validation using microarray data suggested that no association between PD-L1 mRNA expression and OS was found in large independent patient cohorts (1310 cases). PD-L1 mRNA expression was significantly linked to worse PFS in 1228 patients with ovarian cancer (227458_at: HR = 1.55, 95% CI = 1.28–1.88, P < 0.001; 223834_at: HR = 1.41, 95% CI = 1.14–1.75, P = 0.0015).ConclusionsMeta-analysis showed that PD-L1 may not be a prognostic factor for ovarian cancer. But a bioinformatics study showed that PD-L1 expression was significantly associated with worse PFS of ovarian cancer. More clinical studies are needed to further validate these findings.

Predictive and prognostic value of PDL1 protein expression in breast cancer patients in neoadjuvant setting

ABSTRACT Objective Programmed death-ligand-1 (PDL1) is a molecule involved in immune evasion in various kinds of tumors. Here, we aim to determine whether the expression of PDL1 protein is related to the response of patients to neoadjuvant therapy and survival outcome. Methods Immunohistochemistry (IHC) was performed on paraffin-embedded tumor samples from core needle biopsy before neoadjuvant therapy (NAT). Univariate and multivariate logistic regression were used to analyze the associations between PDL1 protein expression and pathological complete response (pCR) outcome. Kaplan-Meier plot and log-rank test were used to compare disease-free survival (DFS) between groups. A cox proportional hazards model was used to calculate the adjusted hazard ratio (HR) with 95% confidential interval (95%CI). Results A total of 94 patients were included for IHC testing. PDL1 protein expression on tumor cells was associated with better pCR rate in both univariate (OR = 2.621, p = 0.043) and multivariate (OR = 3.595, p = 0.029) logistic regression analysis. It was also associated with shorter DFS both by log-rank test (p = 0.015) and cox hazard model (HR = 22.824, 95%CI 1.621–321.284, p = 0.020). In hormone receptor (HR)-positive patients, PDL1 protein expression was also associated with better pCR (OR = 2.362, p = 0.022). It was also associated with poor DFS (HR = 18.821, 95%CI 1.645–215.330, p = 0.018). Conclusions Our results show that PDL1 protein expression is a predictive biomarker of pCR and a prognostic factor of DFS in breast cancer patients and HR-positive subgroups.

Expressions of programmed death-ligand 1 and 2 and phosphorylated protein kinase B in diffuse large B-cell lymphoma and their clinical significances

Objective To investigate the expressions of programmed death-ligand 1 (PD-L1) and PD-L2 and phosphorylated protein kinase B (p-AKT) in diffuse large B-cell lymphoma (DLBCL) patients and their correlations with clinicopathological features and prognosis. Methods A total of 68 paraffin-embedded specimens of DLBCL patients diagnosed in Shanxi Provincial Cancer Hospital with detailed follow-up record from January 2010 to December 2012 were included in the study. The expressions of PD-L1, PD-L2 and p-AKT proteins in DLBCL were detected by using immunohistochemistry (IHC). Results The positive rate of PD-L1 protein in DLBCL patients was 22.1% (15/68), which was related to germinal center B-cell (GCB) subtype or not (χ2=5.591, P = 0.018), clinical stage (χ2=3.969, P = 0.046), international prognostic index (IPI) grades (χ2=4.178, P = 0.041) and treatment remission rate (χ2=6.587, P = 0.010). The positive rate of PD-L2 protein in DLBCL patients was 14.7% (10/68), which was related to extranodal metastasis or not (χ2=6.772, P = 0.009). The positive rate of p-AKT for DLBCL patients was 61.8% (42/68), which was correlated with age (≥60 years old) or not (χ2=6.227, P = 0.013), Eastern Cooperative Oncology Group (ECOG) grades (χ2=4.005, P = 0.045), B symptoms (χ2=10.187, P = 0.001) and treatment remission rate (χ2=4.096, P = 0.043). Univariate survival analysis showed that the overall survival (OS) rate and progression free survival (PFS) rate of PD-L1 protein positive expression group were lower than those of PD-L1 protein negative expression group (both P < 0.05). In the patients with non-GCB subtype, OS rate and PFS rate of PD-L1 protein positive expression group were lower than those of PD-L1 protein negative expression group (both P < 0.05). p-AKT protein positive expression group had poorer OS rate and PFS rate compared to p-AKT negative expression group (both P < 0.05). Correlation analysis showed that PD-L1 protein expression was correlated with PD-L2 and p-AKT proteins expressions (r = 0.380, P = 0.001; r = 0.273, P = 0.025). The prognosis was worse when p-AKT and PD-L1 proteins was co-expressed (P < 0.05). Multivariate analysis suggested high expressions of PD-L1 and p-AKT proteins were independent prognosis risk factors in DLBCL (both P < 0.05). Conclusions The expressions of PD-L1 and p-AKT proteins may be involved in the occurrence and development of DLBCL. Blocking PD-1 and PD-L1 access or combined blocking could provide a promising future for the clinical therapy. Key words: Lymphoma, large B-cell, diffuse; Programmed death-ligand 1; Programmed death-ligand 2; Protein kinase B; Prognosis

Abstract A153: Targeting DNA damage response upregulates PD-L1 level and promotes antitumor immunity in small-cell lung cancer

Abstract Purpose of the Study: Despite recent advances in the use of immunotherapy, only a minority of small cell lung cancer (SCLC) patients respond to immune checkpoint blockade (ICB) with programmed cell death protein 1 (PD-1) or programmed death ligand 1 (PD-L1) antibodies as monotherapy or combination. Therefore, there is a critical need to develop strategies to enhance the efficacy of ICB in SCLC, an otherwise immunosuppressed disease with dismal 5-year survival rate of &amp;lt;5%, which has remained unchanged for &amp;gt;30 years. We have previously shown that prexasertib and olaparib treatment enhanced the protein (but not mRNA) and surface expression of PD-L1 in SCLC cell lines and tumor models. Here we elucidate the previously unexplored mechanism of how DNA damage response targeting enhances antitumor immunity in SCLC. Methods: SCLC cell lines and immune competent murine models were treated with inhibitors targeting DNA damage repair (DDR) proteins, checkpoint kinase 1 (CHK1) (by prexasertib), poly (ADP-ribose) polymerase (PARP) (by olaparib) either as single agents or in combination with anti-PD-L1. End point analyses were done by Western blot, real-time RT-PCR, multicolor flow cytometry and reverse phase protein array (RPPA). Result: We observed increased PD-L1 glycosylation post-prexasertib and olaparib treatment. Prexasertib and/or olaparib +/- anti-PD-L1 combination activates mTOR and inactivates GSK3β pathway thus providing mechanistic insight into DDR-targeting mediated PD-L1 glycosylation and stabilization. We treated tumor-bearing immune-competent B6129F1 mice with either IgG (control), prexasertib (10mg/kg, 2/7), anti-PD-L1 (300ug, 1/7) or combination (n=10/group). At Day 21, anti-PD-L1 did not cause tumor regression and prexasertib treatment delayed tumor growth [T/C=0.31(p&amp;lt;0.001)]. However, most notably, 60% mice treated with anti-PD-L1+ prexasertib had a complete tumor regression within 15 days of treatment and the other 40% had no further increase in tumor growth beyond baseline [T/C=0.05(p&amp;lt;0.001)]. Co-targeting CHK1+PD-L1 significantly increases the level of CD8+ cytotoxic-T-cell tumor infiltration and decreases exhausted T-cell population in SCLC models. CD8 depletion was able to reverse the antitumor effect of the combination demonstrating the role of cytotoxic T-cell infiltration in CHK1i+PD-L1 response. Next, we treated the tumor-bearing B6129F1 mice with either IgG (control), olaparib (100mg/kg, 4/7), anti-PD-L1 (300ug, 1/7) or combination (n=10/group). At Day 21, anti-PD-L1 or olaparib did not cause tumor regression. However, there was a significant delay in tumor growth when the mice were treated with olaparib+anti-PD-L1 combination [T/C=0.26(p&amp;lt;0.001)]. Immune profiling demonstrated increased CD8+ cytotoxic-T-cell tumor infiltration, decreases exhausted T-cell population in the olaparib+anti-PD-L1 combination group. Conclusions: Expression of PD-L1 is associated with tumors deficient in DNA damage repair and we, for the first time, provide rationale for investigating the role of immunotherapy in the context of DDR targeting in SCLC. This study further demonstrates the novel mechanism of DDR-mediated PD-L1 regulation in SCLC via GSK3β inactivation and stabilization. Moreover, our results demonstrating the remarkable efficacy of the combination of PD-L1 blockade with PARP and CHK1 inhibition provide a strong scientific rationale for combining these modalities in clinical trials for SCLC patients. Combining DDR inhibition with ICB increases CD8+ cytotoxic T-cell infiltration in SCLC models. The immune profiling and correlative biomarker data from our study further provide valuable mechanistic insight and indicate the subset of the patient population who are most likely to respond to these treatments. Because prexasertib, olaparib and other PARP inhibitors are already in clinical trials for SCLC, we expect that this hypothesis has the potential for rapid translation into the clinic. Citation Format: Triparna Sen, Bertha Leticia Rodriguez, Limo Chen, Naoto Morikawa, Junya Fujimoto, Lixia Diao, Youhong Fan, Jing Wang, Bonnie S. Glisson, Ignasio Wistuba, Julien Sage, John V. Heymach, Don L. Gibbons, Lauren A. Byers. Targeting DNA damage response upregulates PD-L1 level and promotes antitumor immunity in small-cell lung cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A153.

PD-L1 protein expression in non-small-cell lung cancer and its relationship with the hypoxia-related signaling pathways: A study based on immunohistochemistry and RNA sequencing data

ObjectivesTherapies that target programmed cell death protein-1 (PD-1)/programmed death-ligand 1 (PD-L1) have shown promising efficacy in non-small-cell lung cancer (NSCLC). Hypoxia-related genes are also important regulators of PD-L1, and the role of PD-L1 in NSCLC is still not clear. The objective of this study was to investigate PD-L1 expression and its correlation with hypoxic-inducible factor 1α (HIF1A), vascular endothelial growth factor A (VEGFA), glucose transporter 1 (GLUT1), and carbonic anhydrase 9 (CAIX) expression in NSCLC patients. The association between PD-L1 expression and survival was also determined. Materials and methodsPD-L1/protein expression was evaluated in 295 resected NSCLCs and its correlation with HIF1A, VEGFA, GLUT1, CAIX expression and survival was determined based on immunohistochemical and RNA sequencing data obtained from The Cancer Genome Atlas (TCGA) database. ResultsPD-L1 protein expression was significantly correlated with HIF1A, VEGFA, GLUT1, and CAIX expression only in adenocarcinoma when a 10% or a 50% cut-off was used. PD-L1 mRNA expression was also significantly correlated with HIF1A, VEGFA, GLUT1, and CAIX expression in adenocarcinoma. Univariate analysis revealed that HIF1A expression was associated with poor recurrence-free survival (RFS), and GLUT1 was associated with poor overall survival (OS) and RFS. GLUT1 was an independent prognostic factor for OS in multivariate analysis of immunohistochemical and TCGA data (p = 0.024 and 0.029, respectively). Patients with low expression of both PD-L1 and GLUT1 had longer OS than other patterns in immunohistochemical and TCGA data (p = 0.003 and 0.051, respectively). ConclusionsPD-L1 protein and mRNA expression were correlated with HIF1A, VEGFA, GLUT1, and CAIX expression in adenocarcinoma alone. Low expression of GLUT1 and low expression of both PD-L1 and GLUT1 were associated with improved prognosis. Our findings support the rationale for co-targeting hypoxia-related genes and PD-L1 in cancer therapy. Expression of hypoxia-related genes may be helpful in selecting patients appropriate for PD-L1 therapy.

Integrative analysis of PD-L1 DNA status, mRNA status and protein status, and their clinicopathological correlation, in diffuse large B-cell lymphoma.

The protein expression of programmed death-ligand 1 (PD-L1) has been recognised as being a biomarker for poor prognosis in diffuse large B-cell lymphoma (DLBCL). The aims of this study were to determine PD-L1 DNA status and mRNA status, and to explore whether they contribute to protein expression and their clinicopathological correlation in DLBCL. In this study, we used fluorescence in-situ hybridisation, RNA in-situ hybridisation and immunohistochemistry to determine PD-L1 status at three different levels in 287 DLBCL samples with follow-up. Their correlation and clinical pathological relevance were also analysed. Our results showed that 1.7% (3/175) of patients had PD-L1 DNA amplification, 19.9% (57/287) had high PD-L1 mRNA expression, and 11.8% (34/287) had high PD-L1 protein expression. Both mRNA and protein expression of PD-L1 were significantly higher in non-germinal centre B-cell-like (GCB) DLBCL than in GCB DLBCL (P<0.05). In addition, the patients with high PD-L1 mRNA or high PD-L1 protein expression but no PD-L1 DNA amplification had significantly poorer overall survival (OS) than those with low PD-L1 expression (P<0.05). Furthermore, we found that PD-L1 mRNA and PD-L1 protein expression were highly correlated (P=0.012), which was observed in all three samples with DNA amplification. PD-L1 DNA amplification is a rare event, PD-L1 mRNA is the main contributor to the high PD-L1 protein expression, and the latter two will serve as important biomarkers for predicting the prognosis of DLBCL patients and selecting them for immunotherapy.

PD-L1 Gene Alterations Identify a Unique Subset of Diffuse Large B Cell Lymphoma That Harbors a T Cell Inflamed Phenotype

Background: Programmed death - ligand 1 (PD-L1) expression is a dominant immune escape mechanism across human cancers. Ubiquitous copy gains of chromosome 9p24.1 - a region containing the PD-L1 and PD-L2 loci - lead to PD-L1 expression on Hodgkin-Reed-Sternberg cells in classical Hodgkin lymphoma. PD-L1 gene alterations also occur in diffuse large B cell lymphoma (DLBCL), albeit less commonly. Hypothesizing that PD-L1 gene alterations identify DLBCLs in which potent anti-lymphoma immune responses have been activated, we aimed to characterize the immune landscape of PD-L1 gene-altered DLBCLs, describe the key clinical features of these patients, and determine the degree to which PD-L1 gene alterations predict for response to PD-1 blockade therapy in DLBCL.Methods: FISH was performed on 105 formalin-fixed, paraffin-embedded (FFPE) DLBCL specimens with DNA probes to PD-L1, a region centromeric to PD-L2, and centromere 9. Lymphoma-infiltrating T cell numbers (68 cases), HLA class I/II expression (74 cases), and PD-L1 protein expression (93 cases) were assessed by IHC. RNA sequencing (seq) was performed on FFPE samples utilizing the Illumina HiSeq 2000 platform. Differentially-expressed genes were identified between PD-L1-altered and not altered DLBCLs using a false discovery rate (FDR) of 0.05 and log2 fold change of >1.5. Whole exome seq (WES) was performed on DNA from DLBCL specimens and 22 matched blood samples using the Illumina HiSeq exome kit in collaboration with Theragen Etex Bio Institute.Results:PD-L1 gene alterations were identified by FISH in 28/105 samples (27% - 16% relative PD-L1 copy gains, 7% PD-L1 amplifications, 2% PD-L1 translocations, 2% chromosome 9 polysomy), were enriched in non-germinal center (GC) DLBCLs (75% non-GC vs 25% GC; p=0.001), and were associated with robust PD-L1 protein expression (Fig 1A-B). PD-L1 gene-altered DLBCLs were more heavily infiltrated by CD8+ T cells compared to PD-L1 not altered DLBCLs (22.6 vs 13.8 CD8+ T cells/hpf; p<0.05), and TCR-β seq revealed a more clonal TCR repertoire among PD-L1-amplified DLBCLs (Fig 1C). Consistent with heightened T cell surveillance, HLA class I expression was absent or decreased more frequently among DLBCLs with vs without PD-L1 gene alterations (42% vs 20%; p<0.05). Furthermore, RNAseq of 12 PD-L1 gene-altered and 12 not altered DLBCLs (controlled for COO) identified differential expression of ~140 genes (Fig 1D). Gene ontology (GO) terms enriched in PD-L1 gene-amplified DLBCLs (p<0.05) included those related to negative regulation of T cell activation, while ingenuity pathway analysis (IPA) identified that a TH1 response and NF-κB activation were significantly enriched in PD-L1 gene-altered vs not altered DLBCLs (z-score p-value <0.05). Pathway-level up-regulation of NF-κB-associated genes in PD-L1 gene-altered DLBCLs was confirmed by GSEA (NES=1.65; p=0.009). WES performed on the same 24 cases revealed a 2-3-fold increase in somatic mutations in genes involved in antigen presentation (HLA, B2M, CIITA) and T cell co-stimulation (CD70, CD83) in PD-L1 gene-altered vs not altered DLBCLs (p=0.02), consistent with the activation of additional genetic mechanisms of immune escape in these lymphomas. Somatic TP53 and TNFAIP3 (A20) mutations, among others, were enriched in DLBCLs with PD-L1 gene alterations (p<0.05) (Fig 1E). Total tumor mutational burden and predicted neo-antigen numbers were similar in PD-L1 gene-altered and not altered DLBCLs (p=ns), arguing that PD-L1 gene-altered DLBCLs are not inherently more antigenic. Patients with PD-L1 gene-altered DLBCLs had higher-risk clinical features and inferior progression-free survival following initial treatment (Fig 1F). Finally, in the relapsed/refractory setting, PD-L1 gene alterations in an exploratory retrospective analysis were associated with response to PD-1 blockade therapy with pembrolizumab among 29 DLBCL patients enrolled in the KEYNOTE-013 study with tissue available (50% objective responses in PD-L1 gene-altered DLBCLs vs 8% in not altered cases), suggesting that PD-L1 gene alterations may be a useful predictive biomarker of response to anti-PD-1 antibody therapy in DLBCL.Conclusions: Our results indicate that PD-L1 gene alterations identify a unique biological subset of DLBCL in which endogenous anti-lymphoma immune responses have been activated, and which is associated with responsiveness to PD-1 blockade therapy. DisclosuresKline:Merck: Honoraria, Research Funding; iTeos: Research Funding. Orlowski:Merck & Co., Inc.: Employment. Smith:Portola: Honoraria; BMS: Consultancy.

CRISPR/Cas9-Mediated Disruption of PD-L1 Reduces the T Cell Suppressive Effect of Wharton's Jelly Mesenchymal Stromal Cells and Their Extracellular Vesicles

BackgroundMesenchymal stem/stromal cells (MSCs) are a promising treatment for acute graft-versus-host disease and other inflammatory diseases. MSCs express programmed death-ligand 1 (PD-L1) that has been reported to contribute to MSC-mediated immune suppression through T-cell receptor (TCR)-mediated inhibitory signaling. MSCs actively secrete extracellular vesicles (EVs) that have immune modulatory properties resembling their cell of origin. Although the release of EVs by MSCs has been extensively reported, the presence and functional impact of immune inhibitory checkpoint molecules in MSC-derived EVs is still being determined. We sought to determine if PD-L1 and other immune inhibitory checkpoint molecules are enriched in MSC-derived EVs. In this study we report that PD-L1 is enriched in MSC-EVs, and that PD-L1(+) EVs and PD-L1(-) EVs differ in their T cell immune suppressive capacity.MethodsUmbilical cords and healthy donor blood samples were collected on separate IRB-approved protocols after written informed consent (HSC#1546/HSC#5929). Umbilical cord Wharton's jelly-MSCs (WJMSC) were isolated from umbilical cords under GMP conditions and characterized by morphology, multiparameter flow cytometry (MPFC), and differentiation capacity. Three clinical-grade WJMSC cell lines were cultured at low passage (2-5) in EV-depleted media. EVs were isolated from the media by differential ultracentrifugation and the 100K pellets enriched with exosomes were characterized by Western blot (WB), nanoparticle tracking analysis (NTA), and electron microscopy (EM). To further identify EV-enriched immune proteins, the WJMSC and EV proteomes were characterized by Tandem Mass Tag (TMT) labeling and LC MS/MS, using isobaric labels to compare protein quantities between samples. Gene disruption of PD-L1 was performed through site-specific CRISPR/Cas9 introducing a single cysteine nucleotide deletion in exon 3; the genetic mutation was confirmed by Sanger sequencing. The absence of PD-L1 transcript was confirmed by RT-PCR, and the absence of PD-L1 protein was confirmed by WB, immunofluorescence, MPFC, and IFNγ-response. PBMC were isolated from healthy donor blood samples; T cells were activated by CD3/CD28 beads and induced CD154 was measured on CD4(+) T cells by MPFC. Alternatively, CMV-experienced T cells were activated with CMV-pp65 peptide and induced IFNγ was measured on CD8(+) T cells by MPFC. Activated T cells were treated with PD-L1(+) and PD-L1(-) WJMSCs or their derived EVs. Phospho-Zap-70 (Tyr319)/Syk (Tyr352) MPFC was used to analyze T-cell receptor (TCR)-mediated signaling.ResultsWJMSCs were CD73(+), CD90(+), CD105(+), and CD34(-), CD45(-), CD11b(-), CD19(-) and HLA-DR(-); and inhibited proliferation in a mixed lymphocyte reaction. WJMSCs-derived EVs were cup-shaped 100-200nm nanoparticles by EM and were CD9(+), CD81(+), CD73(+), CD90(+), and CD105(+) by WB. Quantitative proteomic profiling revealed a total of 1,634 proteins across WJMSC-derived EV preparations. Proteins that were highly enriched in MSC-derived EVs and that were classified under “negative regulation of lymphocyte activation” (Gene Ontology Biological Processes; GOBP) included PDL1, CD276, DLG1, TNR6, ERBB2, TGFβ1, LYN, and HMGB3. Both WJMSCs and EVs suppressed CD4(+) T cell activation by CD3/CD28 beads and CD8(+) T cell activation by CMV peptide. CRISPR/Cas9 edited WJMSC exhibited normal morphology and differentiation characteristics. PD-L1 gene disruption efficiently reduced PD-L1 protein expression on WJMSC and their derived EVs, and reduced the ability of both to suppress T cell activation. Similarly, blocking PD-L1 with a specific internalization antibody decreased WJMSC and EV-mediated T cell activation.ConclusionProteomic analyses reveal that PD-L1 is enriched in WJMSC-derived EVs. CRISPR/Cas9-mediated disruption of PD-L1 in WJMSCs alone did not affect the release of EVs in vitro. Gene-edited WJMSCs stably generate PD-L1(-) EVs. The PD-L1(-) EVs generated from PD-L1(-) WJMSC did not suppress T cell activation in co-culture. These findings indicate that PD-L1 is enriched in WJMSC EVs and is biologically active. WJMSC-EVs can deliver membrane-bound PD-L1 to T cells, resulting in attenuated TCR signaling. DisclosuresMcGuirk:Fresenius Biotech: Research Funding; Bellicum Pharmaceuticals: Research Funding; Astellas Pharma: Research Funding; Pluristem Ltd: Research Funding; Gamida Cell: Research Funding; Kite Pharma: Honoraria, Other: travel accommodations, expenses, speaker ; Novartis Pharmaceuticals Corporation: Honoraria, Other: speaker, Research Funding.

Differential Prognostic Impact of Strong PD-L1 Expression and 18F-FDG Uptake in Triple-negative Breast Cancer.

Triple-negative breast cancers (TNBC) is an aggressive disease and often associated with early distant metastases, which negate the role of adjuvant radiotherapy. We studied the clinical utility of programmed death ligand-1 (PD-L1) and other available factors in predicting clinical outcome in TNBC. Of the 539 patients with newly diagnosed TNBC between 2004 and 2011, we analyzed 117 patients who had both tumor samples which PD-L1 protein expression could be evaluated using immunohistochemistry and initial staging F-fluorodeoxyglucose (FDG) positron emission tomography (PET) data to find available immunologic or metabolic factors. Median follow-up duration was 53 months. Strong PD-L1 expression was significantly associated with increased risk of recurrence along with tumor hypermetabolism. The systemic recurrence rate was significantly higher in the strong PD-L1 group than the weak PD-L1 group (35% vs. 11%; P=0.002); whereas there was no difference in locoregional failures (8% vs. 8%). Meanwhile, tumor hypermetabolism seemed to relate with an increase in overall recurrences (26% vs. 8%; P=0.019), not with specific type (locoregional, 9% vs. 3% [P=0.289]; systemic, 22% vs. 8% [P=0.051]). The relationship between PD-L1 expression and survival outcomes retained significance even after adjusting potential risk factors. PD-L1 and tumor metabolism might have role of predicting an increase in treatment failures. Especially, strong PD-L1 expression status was related to distant metastasis-dominant recurrence pattern which needs for intensive systemic therapy.

B-cell receptor–mediated NFATc1 activation induces IL-10/STAT3/PD-L1 signaling in diffuse large B-cell lymphoma

AbstractKnowledge of programmed death ligand 1 (PD-L1) expression and its regulation in B-cell lymphoma cells is limited. Investigating mechanisms that control PD-L1 expression in B-cell lymphoma cells might identify biomarkers that predict the efficacy of immunotherapy with anti–programmed death-1/PD-L1 antibodies. In addition, identification of mechanisms that regulate PD-L1 may identify molecules that can be targeted to improve the clinical efficacy of immune checkpoint inhibitors. In this study, we used proteomic approaches and patient-derived B-cell lymphoma cell lines to investigate mechanisms that regulate PD-L1 expression. We found that PD-L1 expression, particularly in nongerminal center B cell–derived diffuse large B-cell lymphoma (DLBCL), is controlled and regulated by several interactive signaling pathways, including the B-cell receptor (BCR) and JAK2/STAT3 signaling pathways. We found that that BCR-mediated NFATc1 activation upregulates IL-10 chemokine expression in PD-L1+ B-cell lymphoma cells. Released IL-10 activates the JAK2/STAT3 pathway, leading to STAT3-induced PD-L1 expression. IL-10 antagonist antibody abrogates IL-10/STAT3 signaling and PD-L1 protein expression. We also found that BCR pathway inhibition by BTK inhibitors (ibrutinib, acalabrutinib, and BGB-3111) blocks NFATc1 and STAT3 activation, thereby inhibiting IL-10 and PD-L1 expression. Finally, we validated the PD-L1 signaling network in 2 primary DLBCL cohorts consisting of 428 and 350 cases and showed significant correlations among IL-10, STAT3, and PD-L1. Thus, our findings reveal a complex signaling network regulating PD-L1 expression in B-cell lymphoma cells and suggest that PD-L1 expression can be modulated by small molecule inhibitors to potentiate immunotherapies.

PD-1 blockade inhibits lymphocyte apoptosis and restores proliferation and anti-viral immune functions of lymphocyte after CP and NCP BVDV infection in vitro

Bovine viral diarrhea virus (BVDV) is an important virus that can cause extensive economic losses in both dairy and beef industry worldwide. Acute infection with BVDV results in peripheral blood lymphopenia, apoptosis and immunosuppression. Up-regulated programmed death-1 (PD-1) expression induces functional exhaustion of lymphocytes, inhibition of proliferation and apoptosis of lymphocytes during acute and chronic viral infections, such as HIV and HCV. However, there are no reports showing the role of PD-1 in peripheral blood lymphopenia, apoptosis and immunosuppression after acute BVDV infection. Accordingly, we measured the mRNA and protein expression of PD-1 and programmed death-ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMCs) infected with BVDV, and analyzed the effects of PD-1 blockade on immune-associated function and activity in peripheral blood lymphocytes (PBLs). The results showed that both cytopathic (CP) BVDV (strain NADL) and non-cytopathic (NCP) BVDV (strain KD) infection stimulated the mRNA and protein expression of PD-1 and PD-L1 significantly. The upregulation of PD-1/PD-L1 was accompanied by the decreased PBLs proliferation and increased apoptosis. Additionally, PD-1 blockade restored proliferation, inhibited apoptosis, increased IFN-γ production and decreased BVDV load. Remarkably, the PD-1/PD-L1 interaction has a more substantial effect on the immunoregulation of inhibiting proliferation induced by CP BVDV infection. Our findings confirm that PD-1 plays a vital role in peripheral blood lymphopenia and apoptosis caused by acute BVDV infection, and provide new insights into exploring the immunopathological mechanisms of BVDV or other members of the Flaviviridae family, and a potential therapeutic strategy to control BVDV infection.

Correlation of PD-L1 Expression with Tumor Mutation Burden and Gene Signatures for Prognosis in Early-Stage Squamous Cell Lung Carcinoma

ObjectivesAnti–programmed cell death 1 (PD-1)/programmed death ligand 1 (PD-L1) immunotherapy has demonstrated success in the treatment of advanced NSCLC. Recently, PD-1/PD-L1 blockade also has demonstrated interesting results in small trials of neoadjuvant treatment in stage IB to IIIA NSCLC. In addition, several clinical trials using anti–PD-1/PD-L1 immunotherapy as an adjuvant or neoadjuvant treatment in patients with resectable stage NSCLC are ongoing. However, few analyses of anti–PD-1/PD-L1 immunotherapy–related biomarkers in early-stage squamous cell lung carcinoma (SqCLC) have been reported. In this study, we evaluated PD-L1 protein expression, tumor mutation burden, and expression of an immune gene signature in early-stage SqCLC, providing data for identifying the potential role for patients with anti–PD-1/PD-L1 treatment in early-stage SqCLC. MethodsA total of 255 specimens from patients with early-stage SqCLC were identified within participating centers of the Strategic Partnering to Evaluate Cancer Signatures program. PD-L1 protein expression by immunohistochemistry was evaluated by using the Dako PD-L1 22C3 pharmDx kit on the Dako Link 48 auto-stainer (Dako, Carpinteria, CA). Tumor mutation burden (TMB) was calculated on the basis of data from targeted genome sequencing. The T-effector and interferon gamma (IFN-γ) gene signature was determined from Affymetrix gene chip data (Affymetrix, Santa Clara, CA) from frozen specimens. ResultsThe prevalence of PD-L1 expression was 9.8% at a tumor proportion score cutoff of at least 50%. PD-L1 mRNA and programmed cell death 1 ligand 2 mRNA positively correlated with PD-L1 protein expression on tumor cells (TCs) and tumor-infiltrating immune cells. PD-L1 protein expression on tumor-infiltrating immune cells was correlated with the T-effector and IFN-γ gene signature (p < 0.001), but not with TMB. For TCs, all of these biomarkers were independent of each other and neither PD-L1 protein expression, TMB, or T-effector and IFN-γ gene signatures were independently prognostic for patient outcomes. ConclusionsEvaluation of PD-L1 expression, TMB, and T-effector and IFN-γ gene signatures in the cohort with early-stage SqCLC found them to be independent of each other, and none was associated with overall survival. Our results also support the hypothesis that PD-L1 expression is regulated by an intrinsic mechanism on TCs and an adaptive mechanism on immune cells.

ERα is a negative regulator of PD-L1 gene transcription in breast cancer

The programmed death-ligand 1 (PD-L1) expression by tumors results in potent antitumor immune suppression through binding to programmed death-1 (PD-1) on T cells and subsequent inhibition of T cells activity. Although recent pathological studies have shown that PD-L1 is actively expressed in certain ERα-negative breast cancer, little is known about whether ER signaling regulates PD-L1 gene expression. Here, we investigated the relationship between ERα and PD-L1 in breast cancer. Analysis of Comprehensive Cell Line Encyclopedia (CCLE) data showed that the average mRNA level of PD-L1 in ERα-positive breast cancer cell lines was significantly lower than that in ERα-negative breast cancer cell lines. E2 treatment inhibited PD-L1 mRNA expression in hormone-depleted ERα-positive MCF7 cells. Moreover, ectopic expression of ERα in triple-negative MDA-MB-231 cells reduced PD-L1 mRNA and protein expression. Consistently, analysis of The Cancer Genome Atlas (TCGA) data revealed an inverse correlation between ERα and PD-L1 expression in ERα-positive breast cancer. Taken together, our results identify ERα as a negative regulator of PD-L1 gene transcription in breast cancer cells, suggesting that ERα loss-of-function may facilitate the immune evasion of breast cancer cells via up-regulation of PD-L1.

The Protein Expression of PDL1 Is Highly Correlated with Those of eIF2α and ATF4 in Lung Cancer.

Introduction The expression of programmed death 1 (PD1) and programmed death ligand 1 (PDL1) can be induced by the interferon (IFN)/signal transducer and activator of transcription (STAT) pathway. The PD1/PDL1 reverse signaling can activate the eukaryotic translation initiation factor 2 (eIF2α)/activating transcription factor 4 (ATF4) pathway which in turn regulates the expression of IFN regulatory factor (IRF) 7 and IFNα. The eIF2α/ATF4 pathway is responsible for the integrated stress response (ISR) of unfolded protein response (UPR) which can affect immune cell function in tumor microenvironment. Materials and Methods The protein levels of PDL1, IRF1, IRF7, STAT1, STAT2, IFNAR1, eIF2α, and ATF4 in the normal and tumor tissues of 27 subjects with lung cancer were determined by Western blot. Results The protein level of PDL1 was significantly correlated with those of IRF1, eIF2α, and ATF4 in the tissues of all subjects and the subgroup of squamous cell carcinoma but not in the normal tissue of adenocarcinoma. The protein levels of IRF1, eIF2α, and ATF4 were consistently correlated in the tumor tissues but to various extents in the normal ones. The protein level of PDL1 was not correlated with those of STAT1 and STAT2 in all the tissues. Conclusion The PDL1 expression in lung cancer may be independent of STAT1 and STAT2. The PD1/PDL1 axis and UPR/ISR may be closely associated in the tumor tissues of lung cancer.

Abstract 5740: PD-L1 overexpression induced by oncogenic KRAS and BRAF mutations through MAPK pathway activation in non-small cell lung cancer

Abstract Mutant KRAS and BRAF are important oncogenic drivers in non-small cell lung cancer (NSCLC), however, effective treatment strategies for most of these mutants have not been established. Immuno-oncology clinical trials have shown promising results of anti-programmed death 1 (PD-1) antibodies for a subset of NSCLC patients and tumor expression of programmed-death ligand 1 (PD-L1) is regarded as a predictive marker for anti-PD-1 therapy. We found that CD274 mRNA and PD-L1 protein expression were reduced in NSCLC lines by small interfering RNAs (siRNAs) targeting mutant KRAS, but not wild-type KRAS, and also by pharmacologic inhibitors of MEK and ERK. In addition, there was a positive correlation between CD274 expression and KRAS expression in NSCLC lines, and analyses of The Cancer Genome Atlas (TCGA) database revealed that NSCLCs with high KRAS expression exhibited higher levels of CD274 compared to the tumors with low KRAS expression among KRAS-mutant NSCLCs. Similarly, siRNA-mediated BRAF knockdown and inhibitors of BRAF and MEK reduced PD-L1 expression in BRAF-mutant NSCLC cells, suggesting that PD-L1 expression was also upregulated by oncogenic BRAF. Mechanistically, these results suggest that oncogenic KRAS and BRAF induce PD-L1 overexpression in NSCLCs by activation of the MEK-ERK pathway. For clinical translation, our results suggest that: 1. in NSCLCs the combination of KRAS and PD-L1 overexpression could be a surrogate predictive marker for anti-PD-1 treatment; and 2. that since the goal of checkpoint inhibitor immuno-oncology therapy is to inhibit the PD-L1 axis, that a combination of anti-PD-1 and targeted therapy to inhibit the MEK-ERK pathway could be beneficial. Citation Format: Yosuke Miura, Noriaki Sunaga, Pinjie Bao, Luc Girard, Yusuke Tsukagoshi, Tomomi Masuda, Norimitsu Kasahara, Reiko Sakurai, Kyoichi Kaira, Takeshi Hisada, John Minna, Masanobu Yamada. PD-L1 overexpression induced by oncogenic KRAS and BRAF mutations through MAPK pathway activation in non-small cell lung cancer [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 5740.

Abstract 4847: Evaluation of the combination of olaparib and cediranib in small-cell lung cancer cells

Abstract Metastatic Small cell lung cancer (SCLC) is exceptionally lethal, having a median survival of 9-10 months from the time of diagnosis and a 5 year survival rate of less than 2 percent. More effective treatment approaches to SCLC are desperately needed. Poly ADP-ribose polymerase (PARP) inhibitor such as olaparib exploit the concept of synthetic lethality by selectively targeting cancer cells with defective DNA repair pathways. Preclinical data indicates that SCLC cell lines overexpress PARP and are sensitive to PARP inhibition, and PARP inhibitors show promise in the clinical setting of SCLC. The identification of combination regimens that potentiate the efficacy of PARP inhibition would be of great clinical value. Recent findings indicate that olaparib and the vascular endothelial growth factor receptor (VEGFR) inhibitor, cediranib, is an effective therapeutic combination in patients with BRCA wild-type ovarian cancer. Therefore, we evaluated the efficacy of the olaparib and cediranib combinations in SCLC models. Human SCLC cell lines, NCI-H841, H526, COR-L24 and murine SCLC cell line TKOmTmG were sensitive to olaparib or cediranib therapy in vitro as determined by Cell Titer Glo® assay. Moreover, a significantly greater effect on cell viability was observed when olaparib and cediranib were used in combination. Likewise, in H841 xenograft models, single agent treatment of olaparib or cediranib inhibited tumor growth, and combination treatment of olaparib plus cediranib induced tumor regression. Western blot analysis indicated that treatment with cediranib or the combination of olaparib and cediranib induced PD-L1 protein expression both in vivo and in vitro. In TKOmTmG xenograft model in syngeneic mice, programmed death-ligand 1 (PD-L1) inhibitor plus olaparib or olaparib/cediranib combination resulted in superior combined efficacy compared to the respective single agent therapies in terms of tumor growth inhibition. These data indicate that olaparib plus cediranib may be an effective therapeutic strategy for the treatment of SCLC. Further study is warranted to understand the mechanism of action and clinical activity. Citation Format: Naoto Morikawa, Monique B. Nilsson, Irene Guijarro, You-Hong Fan, Alissa Poteete, John V. Heymach. Evaluation of the combination of olaparib and cediranib in small-cell lung 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 4847.

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

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

Upregulation of programmed death ligand 1 and epidermal growth factor receptor is associated with poor prognosis in gastric cancer

Background: Programmed death ligand-1 (PD-L1) and epidermal growth factor receptor (EGFR) are both expressed on the surface of gastric cancer cells. We aimed to evaluate the relationships between protein expression levels and patient clinicopathologic characteristics as well as their prognostic impacts. Methods: The expression levels of PD-L1 and EGFR on tumor tissues and adjacent normal tissues were measured by immunohistochemistry in 90 cases of human gastric adenocarcinoma. The relationships of protein expression with clinicopathologic characteristics and prognosis were calculated by SPSS version 21. Results: PD-L1 and EGFR protein expression were upregulated in tumor tissues compared with adjacent normal tissues (P=0.036, P Conclusions: Our findings suggest that elevated expressions of PD-L1 and EGFR are prognostic factors for shorter overall survival respectively. Patients with co-expression of both tended to have worse prognosis.

48P Clinical characteristics of Korean lung cancer patients with programmed death-ligand 1 expression

Background: Programmed death-ligand 1 (PD-L1) is a transmembrane protein binds to the programmed death-1 (PD-1) receptor and anti-PD-1 therapy enables the immune response against tumors. The aim of this study was to assess the clinical and pathologic characteristics of PD-L1 positive lung cancer patients in Korea. Methods: We retrospectively reviewed the clinical data of pathologically proven lung cancer patients, and collected 267 cases of formalin-fixed, paraffin-embedded tissue sample from single institution. PD-L1 expression was detected by immunohistochemical assay using Monoclonal Mouse Anti-PD-L1, Clone 22C3. PD-L1 protein expression is determined by using Tumor Proportion Score (TPS), the percentage of viable tumor cells showing partial or complete membrane staining. The specimen should be considered PD-L1 positive as TPS ≥ 50% of tumor cells. We categorized according to the percentage of TPS; more than 1% or 50%. Results: A total of 267 patients were enrolled and major histologic types were adenocarcinoma (ADC)(69.3%). The majority was smoker (67.4%) and clinical stage IV (60.7%). 31 (11.6%) cases of EGFR mutation and 17 (6.4%) cases of ALK FISH positive were included. The patients who showed TPS ≥ 1% and 50% were 116 (42%) and 58 (21%), respectively. More than 1% of TPS group was older than below (64.83 ± 9.38 vs. 61.73 ± 10.78, p = 0.014). More than 1% of TPS group was consisted of ADC (67.8%) and squamous cell carcinoma (SqC) (29.6%) histology. And more than 50% of TPS group was composed of ADC (72.4%), SqC (22.4%). More than 1% of TPS group was significantly older than less (64.83 ± 9.38 years vs. 61.73 ± 10.78 years, p = 0.014). The rate of poorly differentiated pathology was significantly higher in TPS ≥ 1% group (42(40.8%) vs. 32(25.8%)) and TPS ≥ 50% group (25(53.2%) vs. 49 (27.2%)). There was no difference in smoking, EGFR mutation, ALK rearrangement status or biopsy site. Conclusions: In Korean lung cancer patients, PD-L1 positive group defined as TPS ≥ 1% was older than negative group. And major histology was poorly differentiated non-small cell lung cancer in both TPS ≥ 1% and 50% groups. Legal entity responsible for the study: Chonnam National University Hospital, Hwasun Funding: Has not received any funding Disclosure: The author has declared no conflicts of interest.

MP84-13 PROGRAMMED DEATH LIGAND-1 (PD-L1) EXPRESSION STATUS IN PENILE SQUAMOUS CELL CARCINOMA TUMOR CELLS AND TUMOR INFILTRATING IMMUNE CELLS

You have accessJournal of UrologySexual Function/Dysfunction: Penis/Testis/Urethra: Benign Disease & Malignant Disease II1 Apr 2018MP84-13 PROGRAMMED DEATH LIGAND-1 (PD-L1) EXPRESSION STATUS IN PENILE SQUAMOUS CELL CARCINOMA TUMOR CELLS AND TUMOR INFILTRATING IMMUNE CELLS Jennifer Rider, Alyssa Harlow, Francesca Giunchi, Jessica Carlsson, Peter Kirrander, Michelangelo Fiorentino, Ove Andren, and Sabina Davidsson Jennifer RiderJennifer Rider More articles by this author , Alyssa HarlowAlyssa Harlow More articles by this author , Francesca GiunchiFrancesca Giunchi More articles by this author , Jessica CarlssonJessica Carlsson More articles by this author , Peter KirranderPeter Kirrander More articles by this author , Michelangelo FiorentinoMichelangelo Fiorentino More articles by this author , Ove AndrenOve Andren More articles by this author , and Sabina DavidssonSabina Davidsson More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2781AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Studies of PD-L1 expression in penile squamous cell carcinoma (SCC) have been small or have used only one anti-PD-L1 antibody for immunohistochemical (IHC) staining, despite evidence of variability in antibody specificity. Using a large penile SCC archival tumor tissue cohort, our objective was to compare PD-L1 protein expression status in tumor and tumor infiltrating immune cells (TIICs) using two different antibodies, and relate expression to clinical and tumor characteristics. METHODS Among 220 patients treated for penile SCC in Orebro, Sweden between 1984-2008, PD-L1 IHC staining was undertaken on three tumor cores per patient using two anti-PD-L1 rabbit monoclonal antibodies (SP142, Roche; 28-8, Abcam). Specific membranous and/or cytoplasmic staining was independently evaluated by two uro-pathologists (F.G and M.F) blinded to all clinical data. The percentage of PD-L1 staining in tumor cells and TIICs were evaluated separately. Immunostaining was scored using 3 categories of % positive cells: <5%; >5% to <50%; and >50%. RESULTS Positive staining most often occurred in <5% of cells, so subsequent analyses only considered any vs. no staining in at least one core. For both antibodies, PD-L1 expression was observed more frequently in TIICs than in tumor cells (45.0% vs. 7.3% for SP-142; 64.2% vs 32.1% for 28-8). Positive TIIC staining with SP142 was associated with more poorly differentiated tumors (54.3% vs 34.2% grade 2/3; p=0.02), advanced T-stage (24.2% vs 10.2% stage T3; p=0.004), advanced pT stage (16.1% vs. 8.5% stage pT3; p=0.03), and a lower frequency of HPV-positive tumors (52.5% vs 72.5%; p=0.01). Positive staining in tumor with 28-8 was also associated with higher grade (65.2% vs. 39.9% grade 2/3; p=0.002), advanced T-stage (25.4% vs. 12.5% stage T3; p<0.0001), and advanced pT stage (18.5% vs 9.0% stage pT3; p=0.02). PD-L1 expression in tumor was marginally associated with increased penile cancer-specific mortality (18.8 vs. 6.2%, p=0.06 for SP142; 12.5% vs 4.9%, p=0.05 for 28-8). CONCLUSIONS PD-L1 expression in tumor and TIICs has potential prognostic value for penile cancer. In TIICs, the SP142 antibody was more prognostic, while positive tumor staining using the 28-8 antibody was more strongly associated with clinicopathological variables. The results provide further rationale for targeting immune checkpoint inhibitor pathways in advanced penile SCC. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1129-e1130 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Jennifer Rider More articles by this author Alyssa Harlow More articles by this author Francesca Giunchi More articles by this author Jessica Carlsson More articles by this author Peter Kirrander More articles by this author Michelangelo Fiorentino More articles by this author Ove Andren More articles by this author Sabina Davidsson More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...