Programmed Cell Death Ligand 1 Antibody Research Articles

Anti-PD-L1 antibody direct activation of macrophages contributes to a radiation-induced abscopal response in glioblastoma.

Most glioblastomas recur near prior radiation treatment sites. Future clinical success will require achieving and optimizing an "abscopal effect," whereby unirradiated neoplastic cells outside treatment sites are recognized and attacked by the immune system. Radiation combined with anti-programmed cell death ligand 1 (PD-L1) demonstrated modest efficacy in phase II human glioblastoma clinical trials, but the mechanism and relevance of the abscopal effect during this response remain unknown. We modified an immune-competent, genetically driven mouse glioma model (forced platelet derived growth factor [PDGF] expression + phosphatase and tensin homolog loss) where a portion of the tumor burden is irradiated (PDGF) and another unirradiated luciferase-expressing tumor (PDGF + luciferase) is used as a readout of the abscopal effect following systemic anti-PD-L1 immunotherapy. We assessed relevance of tumor neoepitope during the abscopal response by inducing expression of epidermal growth factor receptor variant III (EGFRvIII) (PDGF + EGFRvIII). Statistical tests were two-sided. Following radiation of one lesion, anti-PD-L1 immunotherapy enhanced the abscopal response to the unirradiated lesion. In PDGF-driven gliomas without tumor neoepitope (PDGF + luciferase, n = 8), the abscopal response occurred via anti-PD-L1 driven, extracellular signal-regulated kinase-mediated, bone marrow-derived macrophage phagocytosis of adjacent unirradiated tumor cells, with modest survival implications (median survival 41 days vs radiation alone 37.5 days, P = 0.03). In PDGF-driven gliomas with tumor neoepitope (PDGF + EGFRvIII, n = 8), anti-PD-L1 enhanced abscopal response was associated with macrophage and T-cell infiltration and increased survival benefit (median survival 36 days vs radiation alone 28 days, P = 0.001). Our results indicate that anti-PD-L1 immunotherapy enhances a radiation- induced abscopal response via canonical T-cell activation and direct macrophage activation in glioblastoma.

JQ1-Loaded Polydopamine Nanoplatform Inhibits c-MYC/Programmed Cell Death Ligand 1 to Enhance Photothermal Therapy for Triple-Negative Breast Cancer.

Programmed cell death ligand 1 (PD-L1) blockade has achieved great success in cancer immunotherapy; however, the response of triple-negative breast cancer (TNBC) to PD-L1 antibodies is limited. To address this challenge, we use the bromodomain and extra-terminal inhibitor JQ1 to down-regulate the expression of PD-L1 and thus elicit the immune response to TNBC instead of using antibodies to block PD-L1. JQ1 also inhibits the growth of TNBC as a targeted therapeutic agent by inhibiting the BRD4-c-MYC axis. The polydopamine nanoparticles (PDMNs) are introduced as a biodegradable and adaptable platform to load JQ1 and induce photothermal therapy (PTT) as another synergistic therapeutic modality. Because the JQ1-loaded PDMNs (PDMN-JQ1) are self-degradable and release JQ1 continuously, this synergistic treatment can lead to remarkable activation of cytotoxic T lymphocytes and induce a strong immune-memory effect to protect mice from tumor re-challenge. Taken together, our study demonstrates a compact and simple nanoplatform for triple therapy, including targeted therapy, PTT, and immunotherapy, for TNBC treatment.

Cisplatin contributes to programmed death-ligand 1 expression in bladder cancer through ERK1/2-AP-1 signaling pathway.

Bladder cancer (BC) is the second most common urologic malignancy and the ninth most common malignancy worldwide. Surgical resection is the mainstay of treatment for patients with early-stage disease, whereas therapeutic options are limited for patients with advanced-stage or residual BC. Programmed cell death ligand-1 (PD-L1) is an important target for immunotherapy. It is known that PD-L1 is overexpressed in BC; a clinical trial involving PD-L1 immune checkpoint inhibitors in advanced BC is ongoing. In the present study, we used Western blot and quantitative real-time PCR (qPCR) to define the expression level of PD-L1 after cisplatin treatment in BC-derived cell lines. The signal activation was also evaluated by Western blot in BC-derived cell lines. We found that chemotherapeutic drug cisplatin can induce PD-L1 but not PD-L2 expression in BC-derived cell lines. Furthermore, the expression level of PD-L1 was increased in a dose- and time-dependent manner after cisplatin treatment. The cisplatin-induced PD-L1 expression is mainly mediated by ERK1/2 but not Akt/mTOR signal pathway. Moreover, we found that cisplatin activates transcription factor activator protein-1 (AP-1) to regulate PD-L1 expression. The chemotherapy drug such as cisplatin may trigger resistance of BC through PD-L1 up-regulation. The present study suggests that PD-L1 antibody should be used concomitantly with chemotherapy in the setting of advanced and metastatic BC.

Immune-related adverse events associated with programmed cell death protein-1 and programmed cell death ligand 1 inhibitors for non-small cell lung cancer: a PRISMA systematic review and meta-analysis

BackgroundProgrammed cell death protein-1 (PD-1) and programmed cell death ligand 1 (PD-L1) inhibitors have remarkable clinical efficacy in the treatment of non-small cell lung cancer (NSCLC); however, the breakdown of immune escape causes a variety of immune-related adverse events (irAEs). With the increasing use of PD-1/PD-L1 inhibitors alone or in combination with other therapies, awareness and management of irAEs have become more important. We aimed to assess the incidence and nature of irAEs associated with PD-1 and PD-L1 inhibitors for NSCLC.MethodsArticles from the MEDLINE, EMBASE, and Cochrane databases were searched through December 2017. The incidence of overall and organ-specific irAEs was investigated in all clinical trials with nivolumab, pembrolizumab, atezolimumab, durvalumab, and avelumab as single agents for treatment of NSCLC. We calculated the pooled incidence using R software with package Meta.ResultsSixteen trials were included in the meta-analysis: 10 trials with PD-1 inhibitors (3734 patients) and 6 trials with PD-L1 inhibitors (2474 patients). The overall incidence of irAEs was 22% (95% confidence interval [CI], 17–28) for all grades and 4% (95% CI, 2–6) for high-grade irAEs. The frequency of irAEs varied based on drug type and organ, and patients treated with PD-1 inhibitors had an increased rate of any grade and high-grade irAEs compared with patients who received PD-L1 inhibitors. Organ-specific irAEs were most frequently observed in, in decreasing order, the endocrine system, skin, pulmonary tract, and gastrointestinal tract. The total number of patients whose death was attributed to irAEs was 14 (0.34%), and most (79%) of these patients died because of pneumonitis. The median time to the onset of irAEs after the initiation of treatment was 10 weeks (interquartile range, 6–19.5 weeks) and varied depending on the organ system involved.ConclusionsThe specificity of irAEs was closely associated with the mechanism of PD-1/PD-L1 antibodies involved in restarting anticancer immune attacks. Comprehensive understanding, timely detection, and effective management could improve the compliance of patients and guide the interruption of treatment.

Clinicopathological and prognostic value of PD-L1 in urothelial carcinoma: a meta-analysis.

Objective: Our objective was to conduct a meta-analysis to investigate the clinicopathological features and prognostic value of programmed cell death ligand 1 (PD-L1) expression in patients with urothelial carcinoma (UC).Materials and methods: Twenty-seven studies with 4,032 patients were included in the meta-analysis. Pooled ORs and 95% CIs were used to examine the associations between clinical factors and PD-L1 expression. HRs and 95% CIs were extracted from eligible studies. Heterogeneity was evaluated using the chi-squared-based Q test and I2 statistic.Results: Expression of PD-L1 on tumor cells (TCs) was associated with muscle-invasive disease (OR=3.67, 95% CI: 2.53–5.33), and inversely associated with the history of intravesical bacilli Calmette-Guerin therapy (OR=0.39, 95% CI: 0.18–0.82) in bladder cancer patients. PD-L1 expression on TCs was associated with worse overall survival (HR=2.06, 95% CI: 1.38–3.06) in patients with organ-confined bladder cancer. PD-L1 expression in patients with UC was significantly related to better objective response rate after PD-1/PD-L1 antibody treatment.Conclusions: Expression of PD-L1 on TCs was associated with muscle-invasive disease in patients with bladder cancer. Patients with PD-L1-positive UC had a significantly better response to PD-1/PD-L1 targeted treatment.

Abstract A006: Phase 1 study to evaluate the safety and tolerability of MEDI4736 (durvalumab, durva) + tremelimumab (treme) in patients with advanced solid tumors

Abstract Durvalumab (durva) is a human IgG1 monoclonal antibody (mAb) that blocks programmed cell death ligand-1 (PD-L1). Tremelimumab (treme) is a human IgG2 mAb inhibitor of cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4). Blocking these checkpoints can result in antitumor activity in some solid tumors. The targets for durva and treme are non-redundant, providing sound rationale for clinical testing of the combination.This is an ongoing phase 1, multicenter, open-label study (NCT01975831) with a dose escalation (3+3 design) and subsequent expansion phase. Patients with renal cell carcinoma (RCC), cervical (CC), colorectal (CRC), non-triple-negative breast (NTNBC), or ovarian (OC) cancer are included in the expansion phase. The protocol excludes patients who had prior exposure to anti-CTLA-4 or anti-PD-1/PD-L1 antibodies. Primary objectives are safety/tolerability and identification of maximum tolerated dose (MTD) of the combination. Secondary objectives include tumor response, progression-free survival (PFS), and overall survival (OS). The intent-to treat (ITT) analysis set includes all patients who received at least one dose of durva or treme and had the baseline and at least one post-baseline tumor assessment.As of 11 May 2018, 104 patients were treated (73.1% female; median age: 56 (30 to 80) years. Durva 1500 mg every 4 weeks (Q4W) X 12 and treme 75 mg Q4W X 4 was the regimen used for opening the expansion phase (n = 82 patients). The majority of treatment-related adverse events (TRAEs) for all patients were Grades 1 and 2. TRAEs ≥ Grade 3 were reported in 17 (16.3%) patients; the majority were diarrhea/colitis (n = 6) and abnormal liver function tests (n = 4) and responded to established treatment algorithms. There was 1 Grade 5 TRAE: multi-organ failure. Fifteen (14.4%) patients experienced TRAEs leading to treatment discontinuation; the majority were diarrhea/colitis (n = 6) and abnormal liver function tests (n = 5). No new toxicities were identified. Tumor response by immune-related Response Criteria (irRC) was assessed by tumor type in the ITT analysis set for all cohorts included in the expansion phase; the follow-up period was at least 12 months. Best overall responses (complete response (irCR), partial response (irPR) and stable disease (irSD)) are presented by tumor type. For OC (n = 27): irCR = 0, irPR = 2 (7.4%), irSD = 10 (37%); for CRC (n = 18): irCR = 1 (5.6%), irPR = 1 (5.6%), irSD = 2 (11.1%); for NTNBC (n = 16): irCR = 0, irPR = 1 (6.3%), irSD = 2 (12.5%); for RCC (n = 19): irCR = 0, irPR = 3 (15.8%), irSD = 11 (57.9%); and for CC (n = 16): irCR = 1 (6.3%), irPR = 3 (18.8%), irSD = 4 (25%) patients. PD-L1 status is not yet available. MSI status was collected retrospectively for patients with CRC; in this group, the patient with irPR had MSI-high status, and the MSI status of the patient with irCR is unknown. PFS and OS rates will be presented at the meeting. In conclusion, the durva and treme combination has a manageable safety profile, with evidence of clinical activity. These data support continued study of the combination therapy. Citation Format: Margaret K. Callahan, Kunle Odunsi, Mario Sznol, John Nemunaitis, Patrick A. Ott, Patrick Dillon, Reva Schneider, Andrew Park, Paul Schwarzenberger, Toni Ricciardi, Mary Macri, Aileen Ryan, Ralph Venhaus, Jedd D. Wolchok. Phase 1 study to evaluate the safety and tolerability of MEDI4736 (durvalumab, durva) + tremelimumab (treme) in patients with advanced solid tumors [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 A006.

PD-L1 expression and the prognostic significance in gastric cancer: a retrospective comparison of three PD-L1 antibody clones (SP142, 28\u20138 and E1L3N)

BackgroundImmunohistochemistry (IHC) for programmed cell death ligand 1 (PD-L1) displays staining diversity. We compared IHC staining of PD-L1 in gastric cancer (GC) by using three commercially available antibody clones, and analyzed the correlation with the prognosis.MethodsIHC using PD-L1 antibodies (clones SP142, 28–8 and E1L3N) in 315 formalin-fixed paraffin-embedded samples was qualitatively compared at the 1, 5 and 10% cut-off by two pathologists on total, tumor and immune/stromal cells. We used computer – assisted scoring to quantitatively analyze and compare the “H-score” of PD-L1 expression in 66 samples on total cells. The antibody clone SP142 was selected to investigate the infiltration of PD-L1+CD8+ T cells using automated quantitative immunofluorescence analyses (n = 50) and the prognostic significance. The prognoses were assessed by log-rank test.ResultsPD-L1 clones SP142 and 28–8 displayed great concordance by qualitative (κ = 0.816, 0.810 for total cells and tumor cells at the 5% cut-off) and quantitative analyses (R2 = 0.7991, 0.8187 for positive percentage and “H-score”). PD-L1 clone SP142 showed the highest positivity in immune/stromal cells staining (18.41%) compared to 28–8 (7.62%), while clone E1L3N showed poor staining in both tumor and immune/stromal cells. Clone SP142, but not 28–8 and E1L3N, predicted a worse prognosis at the 5% cut-off (p = 0.0243). Both the clone SP142 and 28–8 had high inter-pathologist correlation for tumor staining (R2 = 0.9805 and R2 = 0.9853), but a moderate correlation for stromal/immune cell staining (R2 = 0.5653 and R2 = 0.5745). Furthermore, a higher density of PD-L1+CD8+ T cells was correlated with a shorter survival time (R2 = 0.0909, p = 0.0352).ConclusionsPD-L1 antibody clone SP142 was superior in cell staining, particularly in immune/stromal cell and prognosis. These findings are important for selection of PD-L1 antibody clones in the future diagnostic test.

Programmed Cell Death Ligand 1 (PD-L1) Signaling Regulates Macrophage Proliferation and Activation.

Tumor-associated macrophages (TAMs) express programmed cell death ligand 1 (PD-L1) and contribute to the immune-suppressive tumor microenvironment. Although the role of the PD-L1 and PD-1 interaction to regulate T-cell suppression is established, less is known about PD-L1 signaling in macrophages and how these signals may affect the function of TAMs. We used in vitro and in vivo models to investigate PD-L1 signaling in macrophages and the effects of PD-L1 antibody treatment on TAM responses. Treatment of mouse and human macrophages with PD-L1 antibodies increased spontaneous macrophage proliferation, survival, and activation (costimulatory molecule expression, cytokine production). Similar changes were observed in macrophages incubated with soluble CD80 and soluble PD-1, and in PD-L1-/- macrophages. Macrophage treatment with PD-L1 antibodies upregulated mTOR pathway activity, and RNAseq analysis revealed upregulation of multiple macrophage inflammatory pathways. In vivo, treatment with PD-L1 antibody resulted in increased tumor infiltration with activated macrophages. In tumor-bearing RAG-/- mice, upregulated costimulatory molecule expression by TAMs and reduced tumor growth were observed. Combined PD-1/ PD-L1 antibody treatment of animals with established B16 melanomas cured half of the treated mice, whereas treatment with single antibodies had little therapeutic effect. These findings indicate that PD-L1 delivers a constitutive negative signal to macrophages, resulting in an immune-suppressive cell phenotype. Treatment with PD-L1 antibodies reverses this phenotype and triggers macrophage-mediated antitumor activity, suggesting a distinct effect of PD-L1, but not PD-1, antibody treatment. Cancer Immunol Res; 6(10); 1260-73. ©2018 AACR.

Prognostic Impact of PD-L2 Expression and Association with PD-L1 in Patients with Small-cell Lung Cancer.

Although some previous studies suggested that programmed cell death-ligand 1 (PD-L1) expression was significantly associated with a favorable postoperative prognosis in patients with small-cell lung cancer (SCLC), the prognostic significance of PD-L2 expression remains unknown. The aim of the current study was to investigate the prognostic significance of PD-L2 expression in patients with SCLC. Thirty-eight patients who underwent resection of SCLC were analyzed. A monoclonal anti-human PD-L1 antibody (clone SP142) and a monoclonal anti-human PD-L2 antibody (clone 176611) were used as the primary antibodies. Cut-off value for PD-L1 and PD-L2 expression was set to 1%. Among 38 patients, 15 (39.5%) were positive for PD-L2 expression. No significant associations between PD-L2-positivity and clinicopathological factors, including PD-L1 positivity or prognosis were identified. No significant differences in disease-free survival and overall survival were observed between PD-L2-positive patients and PD-L2-negative patients (p=0.367 and p=0.726, respectively). PD-L2 expression is not related to clinicopathological factors or postoperative prognosis in patients with SCLC, though this should be further investigated in studies involving larger populations.

Oncogenic activation of the STAT3 pathway drives PD-L1 expression in natural killer/T-cell lymphoma

AbstractMature T-cell lymphomas, including peripheral T-cell lymphoma (PTCL) and extranodal NK/T-cell lymphoma (NKTL), represent a heterogeneous group of non-Hodgkin lymphomas with dismal outcomes and limited treatment options. To determine the extent of involvement of the JAK/STAT pathway in this malignancy, we performed targeted capture sequencing of 188 genes in this pathway in 171 PTCL and NKTL cases. A total of 272 nonsynonymous somatic mutations in 101 genes were identified in 73% of the samples, including 258 single-nucleotide variants and 14 insertions or deletions. Recurrent mutations were most frequently located in STAT3 and TP53 (15%), followed by JAK3 and JAK1 (6%) and SOCS1 (4%). A high prevalence of STAT3 mutation (21%) was observed specifically in NKTL. Novel STAT3 mutations (p.D427H, E616G, p.E616K, and p.E696K) were shown to increase STAT3 phosphorylation and transcriptional activity of STAT3 in the absence of cytokine, in which p.E616K induced programmed cell death-ligand 1 (PD-L1) expression by robust binding of activated STAT3 to the PD-L1 gene promoter. Consistent with these findings, PD-L1 was overexpressed in NKTL cell lines harboring hotspot STAT3 mutations, and similar findings were observed by the overexpression of p.E616K and p.E616G in the STAT3 wild-type NKTL cell line. Conversely, STAT3 silencing and inhibition decreased PD-L1 expression in STAT3 mutant NKTL cell lines. In NKTL tumors, STAT3 activation correlated significantly with PD-L1 expression. We demonstrated that STAT3 activation confers high PD-L1 expression, which may promote tumor immune evasion. The combination of PD-1/PD-L1 antibodies and STAT3 inhibitors might be a promising therapeutic approach for NKTL, and possibly PTCL.

Abstract 3624: Tanshinone IIA could decrease programmed death ligand 1 expression in human breast cancer BT-20 cells

Abstract Breast cancer is the leading cause of cancer-related death in women. Many prescriptions for chemotherapy have been derived from plants. Danshen (Salviae miltiorrhizae radix) has been used in traditional Chinese medicine and appeared in the Chinese book, Shennong Bencao Jing (ca. 100 A.D.). Tanshinone IIA (Tan-IIA) is extracted from danshen and first described by researchers in 1968. Tan-IIA is well known to have antioxidant and anti-inflammatory properties. Tan-IIA could inhibit many human breast cancer cell lines through different molecular mechanisms in vitro and in vivo have been well documented. Our previous study showed Tan-IIA could inhibit human breast cancer cells through increased Bax to Bcl-xL ratios. Tan-IIA inhibits BT-20 (estrogen-, progesterone-, and HER2-negative subtype (TNBC)) human breast cancer cell proliferation through increasing caspase 12, GADD153 and phospho-p38 protein expression. Tan-IIA also inhibits human breast cancer MDA-MB-231 cells (estrogen-, progesterone-, and HER2-negative subtype (TNBC)) by decreasing LC3-II, Erb-B2 and NF-κBp65 in vivo. However, the effects and molecular mechanisms of Tan-IIA in human breast cancer have not been elucidated clearly. Programmed cell death-ligand 1 (PD-L1) is expressed on many cancer cells, which played a protective role against the cytotoxicity. PD-L1 interacts with programmed cell death-1 receptor (PD-1) to inhibit the T cells and block the antitumor immune response. PD-L1 antibodies, has demonstrated the possibility of dismiss immune suppression in many cancer cells. PD-L1 expression is a favorable biomarker for the prognosis of breast cancer; therefore, immune checkpoint blockade agents may be offering the opportunity to be the future treatment for breast cancer. The present study focused to evaluate the efficacy of Tan-IIA to be one of the immune checkpoint blockade agents for human breast cancer cells. Material and Method: In the present study, the human breast cancer BT-20 cells were treated with SJKJT in vitro. The effects of Tan-IIA on the protein expressions of PD-1, PD-L1, Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), B7-1 (CD80) and B7-2 (CD86) were measured by Western blotting. Results: The results showed that Tan-IIA can inhibit the protein expressions of PD-L1, in breast cancer BT-20 cells significantly. Conclusion: Breast cancer BT-20 cells were treated with Tan-IIA could inhibit the activity of PD-L1, CTLA-4, B7-1 (CD80) and B7-2 (CD86) significantly. These results suggest that one of the molecular mechanisms for Tan-IIA to inhibit breast cancer BT-20 cells maybe through inhibiting the protein expression of PD-L1. The use of Tan-IIA may become a feasible novel therapy option. Further studies are warranted to fully elucidate its mechanisms of action. Citation Format: Chin Cheng Su. Tanshinone IIA could decrease programmed death ligand 1 expression in human breast cancer BT-20 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 3624.

Programmed cell death-ligand 1 (PD-L1) expression is associated with RAS/TP53 mutations in lung adenocarcinoma

IntroductionThe systematic assessment of anti-programmed cell death ligand 1 (PD-L1) expression by immunohistochemistry (IHC) in lung adenocarcinomas is becoming standard practice. However, the assessment of PD-L1 expression on small tissue specimens needs to be evaluated and the association with other features more thoroughly analyzed. MethodsThis retrospective single center study evaluated the immunohistochemical expression of the SP263 anti-PD-L1 antibody on tissue microarrays (TMA) of 152 surgically resected lung adenocarcinomas, using a 25% positivity threshold. The positive cases and 50 randomly chosen negative cases in tissue microarray (TMA) were reassessed on whole tissue sections. The results were correlated to clinical, histopathological and to molecular data obtained through the screening of 214 mutations in 26 genes (LungCarta panel, Agena Biosciences). ResultsAmong 152 primary lung adenocarcinomas, 19 cases (13%) showed PD-L1 expression. The agreement between TMA and whole tissue sections was 89%, specificity was 97%. PD-L1 expression was correlated to RAS mutations (p = .04), RAS/TP53 co-mutations (p = .01) and to the solid or acinar subtype (p = .048). ConclusionsWith the SP263 PD-L1 antibody, small samples appear as a reliable means to evaluate the PD-L1 status in lung adenocarcinoma. The association between PD-L1 expression and RAS/TP53 mutations may have clinical relevance to predict the efficacy of PD-1/PD-L1 immune checkpoints inhibitors.

PD-L1 expression according to the EGFR status in primary lung adenocarcinoma

ObjectivesIt was reported that programmed cell death-ligand 1 (PD-L1) expression is associated with smoking and wild-type epidermal growth factor receptor (EGFR) in lung adenocarcinoma. However, the association between PD-L1 expression and EGFR mutation site in EGFR mutation-positive lung adenocarcinoma is unclear. Materials and methodsWe retrospectively examined the relationship between PD-L1 expression and EGFR status in 441 surgically resected primary lung adenocarcinomas. Membrane PD-L1 expression on tumor cells was evaluated by immunohistochemical analysis using a PD-L1 antibody (clone SP142) and defined by tumor proportion scores (TPSs) of 0%, 1–4%, 5–49%, and ≥50%, respectively. ResultsTwo hundred and eighteen (49.4%) patients had wild-type EGFR, and 223 (50.6%) had mutant EGFR—98 (44.0%) with exon 19 deletion, 116 (52.0%) with exon 21 L858R point mutation, and nine (4.0%) with another EGFR mutation. Overall, Fisher’s exact test showed that PD-L1 positivity was associated with wild-type EGFR, and there was only one case with PD-L1 TPS ≥50% among the cases with mutant EGFR. The analysis of cases with mutant EGFR indicated no significant association between EGFR mutation site and PD-L1 expression. However, the prevalence of PD-L1 TPS 5–49% was higher among patients with EGFR exon 19 deletion than with EGFR exon 21 L858R point mutation. ConclusionsPD-L1 expression was significantly associated with wild-type EGFR, and PD-L1 TPS ≥50% seldom overlaps with presence of driver oncogene EGFR. There was no significant difference in PD-L1 expression among the EGFR mutation sites

PD-L1 immunohistochemistry in clinical diagnostics of lung cancer: inter-pathologist variability is higher than assay variability

Assessment of programmed cell death ligand 1 (PD-L1) immunohistochemical staining is used for decision on treatment with programmed cell death 1 and PD-L1 checkpoint inhibitors in lung adenocarcinomas and squamous cell carcinomas. This study aimed to compare the staining properties of tumor cells between the antibody clones 28-8, 22C3, SP142, and SP263 and investigate interrater variation between pathologists to see if these stainings can be safely evaluated in the clinical setting. Using consecutive sections from a tissue microarray with tumor tissue from 55 resected lung cancer cases, staining with five PD-L1 assays (28-8 from two different vendors, 22C3, SP142, and SP263) was performed. Seven pathologists individually evaluated the percentage of positive tumor cells, scoring each sample applying cutoff levels used in clinical studies: <1% positive tumor cells (score 0), 1–4% (score 1), 5–9% (score 2), 10–24% (score 3), 25–49% (score 4), and >50% positive tumor cells (score 5). Pairwise analysis of antibody clones showed weighted kappa values in the range of 0.45–0.91 with the highest values for comparisons with 22C3 and 28-8 and the lowest involving SP142. Excluding SP142 resulted in kappa 0.75–0.91. Weighted kappa for interobserver variation between pathologists was 0.71–0.96. Up to 20% of the cases were differently classified as positive or negative by any pathologist compared with consensus score using ≥1% positive tumor cells as cutoff. A significantly better agreement between pathologists was seen using ≥50% as cutoff (0–5% of cases). In conclusion, the concordance between the PD-L1 antibodies 22C3, 28-8 and SP263 is relatively good when evaluating lung cancers and suggests that any one of these assays may be sufficient as basis for decision on treatment with nivolumab, pembrolizumab, and durvalumab. The scoring of the pathologist presents an intrinsic source of error that should be considered especially at low PD-L1 scores.

Advances in the treatment of non-small cell lung cancer: focus on anti-PD-1/PD-L1 antibody

Immunotherapy is revolutionizing the treatment of non-small cell lung cancer (NSCLC). Programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) monoclonal antibod-ies have recently led to significant and durable improvements in the clinical outcome of NSCLC, and the anti-PD-1 antibody has been approved to use in first-line and second-line treatment of NSCLC. However, there are still many problems to be solved. The role of PD-L1 as a predictive biomarker remains unclear. Combination treatment models are being explored. This review summarizes the clinical efficacy, drug adverse reaction, combined treatment, and potential immune biomarkers of anti-PD-1/PD-L1 antibody research progress in the treatment of NSCLC. Key words: Membrane glycoproteins/TU; Antibodies, monoclonal/TU; Carcinoma, non-small-cell lung/TH; Immunotherapy; Review

Comparison of Different Antibody Clones for Immunohistochemistry Detection of Programmed Cell Death Ligand 1 (PD-L1) on Non–Small Cell Lung Carcinoma

Programmed cell death ligand 1 (PD-L1) is a major immune checkpoint protein that mediates antitumor immune suppression and response. Preliminary data suggest that its detection using immunohistochemistry (IHC) in formalin-fixed and paraffin-embedded tissues may predict clinical response to PD-1/PD-L1 therapy. In diagnostic pathology, it is essential to count with a validated IHC that can reliably detect PD-L1-positive cases. The present study was conducted to compare and validate different PD-L1 commercial clones and identify which ones can be reliably used by surgical pathologist to detect PD-L1 expression in human cancer tissues. Eight commercial available PD-L1 clones were tested and compared with a noncommercial PD-L1 antibody clone 5H1. Western blot and IHC using cell lines and human tissues were used to validate these clones. From all PD-L1 antibodies, only the clones E1L3N, E1J2J, SP142, 28-8, 22C3, and SP263 passed the Western blot and IHC validation, providing similar pattern than the clone 5H1 and then they were tested in 259 non–small cell lung cancer cases placed in 9 tissue microarrays. Among all cases, only those with ≥2 cores were included (185 cases). Positive and significant correlation was found between the median PD-L1 H-score in tumor and stroma compartments, for all selected antibodies. Overall, 56 of 185 cases were detected as positive cases in malignant cells expressing membranous PD-L1 by all the clones. However, the clone SP263 identified more PD-L1-positive cases compared with the other clones. Our results show that clones E1L3N, E1J2J, SP142, 28-8, 22C3, and SP263 provide positive membrane staining pattern comparable with clone 5H1. These commercial clones are comparable, but a careful evaluation by the pathologist is necessary to minimize error of positive misinterpretations.

Abstract 655: Development of IHC staining protocols for assessment of PD-L1 expression in cytological samples

Abstract Immunohistochemistry (IHC) staining of non-small cell lung cancer (NSCLC) samples for programmed cell death ligand 1 (PD-L1) can help identify patients that may benefit from anti-PDL1 therapy. However, resection or biopsy samples cannot be obtained for some patients with NSCLC. Fine needle aspiration (FNA) is a less invasive method for obtaining samples in such patients, and it is yet to be determined if PD-L1 can be assessed using cytological samples. In this study we had three objectives 1. Develop staining protocols on the VENTANA BenchMark ULTRA automated staining platform for cytology samples fixed with the most common methods using cell lines as a model. 2, Determine the optimal cytology fixation method for VENTANA PD-L1 (SP142) IHC Assay (PD-L1 (SP142)) staining, and 3. Assess PD-L1 expression in tumor cells (TC) and tumor-infiltrating immune cells (IC) in cytological samples prepared from NSCLC patients. KARPAS 299 cell line was used as a model system for the fixation studies. Liquid-based preparations (LBPs) fixed in PreservCyt, conventional smears (unstained and pap-stained) fixed in 95% ethanol, and paraffin embedded cell blocks (CBs) fixed in 95% ethanol, 10% neutral buffered formalin (NBF), PreservCyt, and SurePath preservative were optimized on the BenchMark ULTRA using PD-L1 (SP142) antibody and OptiView DAB Detection and Amplification Kits. NSCLC FNA CBs fixed in 10% NBF (N=69) and a subset of FNAs with matched resections (N=20) were stained with the optimized protocol. FNAs and resections were assessed for percentage of TC and IC with PD-L1 staining. Staining parameters were optimized on the BenchMark ULTRA for all sample types tested, and they all produced a range of moderate to strong PD-L1 expression. CBs fixed in 95% ethanol and 10% NBF produced the highest percentage of staining, with expression in 90% of cells. CBs fixed in PreservCyt and SurePath preservative had a lower percentage of staining, 40% and 75% respectively. LBPs had 25% cells staining and smears ranged from 20%-60%. NSCLC FNA CBs fixed in 10% NBF produced interpretable results when stained with PD-L1 (SP142). IC staining was seen in 8.7% (6/69) of samples, TC staining was seen in 8.7% (6/69) of samples, both IC and TC staining was seen in 14.5% (10/69) of samples. The subset of FNAs with matched resections showed concordance with PD-L1 IC staining in 60% (12/20) of samples and TC staining in 75% (15/20) of samples. The discordant cases showed that FNAs were negative for IC when the resection was negative and positive for TC in 5% of cases when the resection was negative. The VENTANA PD-L1 (SP142) Assay staining parameters were found to be optimal for staining NSCLC FNA CB fixed in 10% NBF. PD-L1 staining was detected in both TC and IC in FNA samples and concordance of FNA and matched resections was high for both IC and TC. A larger study is necessary to validate the use of FNAs for assessment of PD-L1 expression in a clinical setting. Citation Format: Christine Boyiddle, Mark Ruboyianes, Ed Del Valle, Lukas Bubendorf, Kerstin Trunzer, Judith Pugh, Jennifer Boone. Development of IHC staining protocols for assessment of PD-L1 expression in cytological samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 655. doi:10.1158/1538-7445.AM2017-655

PD-L1 expression in neuroendocrine tumors of the lung

BackgroundVarious tumors express programmed cell death ligand 1 (PD-L1), an immune checkpoint ligand, the expression of which correlates with certain effects of anti-programmed cell death 1 (PD-1)/PD-L1 drugs. The aim of this study was to assess the frequency of PD-L1 expression in each of the types of neuroendocrine tumors of the lung. MethodsThe subjects enrolled in this study were patients who had been diagnosed with neuroendocrine tumors of the lung and had been treated at the National Cancer Center Hospital (Tokyo, Japan) between 1982 and 2010. We performed immunohistochemical analysis on a tissue microarray (TMA) of the surgical specimens using the validated PD-L1 antibody clone, E1L3N. Tumor PD-L1 expression scores were calculated semiquantitatively (staining intensity [0–3]×stained area [0–100%]). A score of 1 was used as a cut-off to determine the presence or absence of PD-L1 expression. ResultsAmong the 227 patients included in this study, the patient demographics were as follows: median age (range), 65 years (19–84); sex (male/female), 168/59; pStage (IA, IB, IIA, IIB, IIIA, IIIB, IV): 79, 36, 25, 29, 47, 6, 5, respectively; and histology was typical carcinoid (TC), atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNEC), small cell lung cancer (SCLC): 46, 6, 106, 69, respectively. The numbers (proportions) of PD-L1-expression tumors were as follows: TC/AC/LCNEC/SCLC, 0/0/11 (10.4%)/4 (5.8%). ConclusionsPD-L1 expression was apparent in 10.4% of LCNEC and 5.8% of SCLC tumors, and was not observed in carcinoid tumors.

High PD-L1 expression is associated with stage IV disease and poorer overall survival in 186 cases of small cell lung cancers.

BackgroundSmall cell lung cancer (SCLC) is an aggressive malignancy with a distinct natural history and dismal prognosis. SCLC is characterized as a recalcitrant neoplasm with limited therapeutic options and platinum-based chemotherapy is the treatment of choice. Programmed cell death-ligand 1(PD-L1)-mediated immune escape may be a suitable target for specific therapy, but its role in SCLC is unclear.Materials and methodsIn total, 186 SCLC cases were investigated. Paraffin-embedded tumor sections were stained with a PD-L1 antibody. PD-L1 overexpression was denoted by moderate-to-strong PD-L1 membrane staining in ≥ 5% of tumor cells. Tumor cells and infiltrating lymphocytes were scored separately.ResultsThe overall frequency of PD-L1 overexpression, in tumor cells and tumor infiltrating lymphocytes (TILs) was 78.0% and 54.3%, respectively. High tumor PD-L1 expression was significantly correlated with high TIL PD-L1 expression (P=0.001) and stage IV disease (P=0.048). Multivariate analysis revealed that high tumor PD-L1 expression and stage IV disease were two independent risk factors for poor overall survival.ConclusionsHigh PD-L1 expression was observed in SCLCs compared with their expression in conventional NSCLCs. The aggressive behavior of SCLC could be partially related to PD-L1-mediated immune escape. High PD-L1 expression correlated with poor prognosis and may provide a rationale for immunotherapy for high-grade SCLC.

PL04a.03: Durvalumab in ≥3rd-Line Locally Advanced or Metastatic, EGFR/ALK Wild-Type NSCLC: Results from the Phase 2 ATLANTIC Study

Treatment with anti-PD-1/PD-L1 antibodies has demonstrated meaningful clinical benefit in patients with advanced NSCLC. Patients that progress after 2 lines of chemotherapy have few treatment options and poor outcomes. Durvalumab is an engineered human IgG1 mAb targeting programmed cell death ligand-1 (PD-L1). ATLANTIC (NCT02087423) is a Phase 2, open-label, single-arm trial in patients with locally advanced or metastatic Stage IIIB–IV NSCLC (WHO PS 0 or 1; ≥2 prior systemic treatment regimens, including one platinum-based). There was no maximum number of prior treatments. The study initially enrolled all-comers and then was restricted to patients with PD-L1 high tumors (≥25% of tumor cells with membrane staining). The study includes three cohorts; here we report final results in Cohorts 2 and 3 that had EGFR/ALK wild-type or unknown status. Patients enrolled in Cohort 3 had ≥90% of tumor cells with PD-L1 staining. The primary endpoint is ORR (RECIST v1.1), based on independent central review. Secondary endpoints include DCR, DoR, PFS, OS, and safety (CTCAE v4.03). As of 3 June 2016, in Cohorts 2/3, 265/68 patients (median age 62/61 years, 67/72% PS 1, 21/29% squamous histology; mean of 3.2/2.6 prior therapies) had received durvalumab (10 mg/kg i.v. q2w). Responses were durable; in Cohort 2, patients with PD-L1 ≥25%, the ORR was similar in patients with squamous and non-squamous histology.Tabled 1Cohort 2Cohort 3PD-L1 high (≥25%)PD-L1 low/negative (<25%)PD-L1 ≥90%n=146n=93n=68ORR,* % (95%CI)16.4(10.8-23.5)7.5(3.1-14.9)30.9(20.2-43.3)DCR, % (95%CI)28.8(21.6-36.8)20.4(12.8-30.1)38.2(26.7-50.8)mDoR, months (25th, 75th percentile)12.3(7.5-NR)n=149NR(7.2-NR)n=94NR; 18/21 responders progression free at DCOn=67mPFS, months (95%CI)3.3(1.9-3.7)1.9(1.8-1.9)2.4(1.8-5.5)mOS, months (95%CI)10.9(8.6-13.6)9.3(5.9-10.8)NR(5.9-NC)1-year OS, % (95%CI)47.7(39.3-55.5)34.5(25.0-44.1)50.8(36.9-63.2)mFollow-up for OS, months9.49.37.0*Confirmed response per independent central review. DCO=data cutoff; DCR=disease control rate (complete response, partial response or stable disease ≥24 weeks); DoR=duration of response; m=median; NC=not calculated; NR=not reached; ORR=objective response rate; OS=overall survival; PFS=progression-free survival. Open table in a new tab *Confirmed response per independent central review. DCO=data cutoff; DCR=disease control rate (complete response, partial response or stable disease ≥24 weeks); DoR=duration of response; m=median; NC=not calculated; NR=not reached; ORR=objective response rate; OS=overall survival; PFS=progression-free survival. Most AEs were low grade and resolved with treatment delay and/or immunosuppressive interventions. Overall, 10.2% of patients had Grade ≥3 treatment-related AEs and 2.7% had treatment-related AEs leading to discontinuation. Durvalumab was active and led to durable responses in a heavily pretreated metastatic NSCLC population; activity was numerically greater for patients whose tumors exceeded the 25% PD-L1 cutoff. The tolerability profile was manageable. Results are consistent with other anti-PD-1/PD-L1 therapies in metastatic, relapsed NSCLC and support further development of durvalumab.