Programmed Death-ligand 1 Expression In Tumor Cells Research Articles

Clinical Significance of Tumor and Immune Cell PD-L1 Expression in Gastric Adenocarcinoma.

The prognostic relevance of programmed cell death ligand-1 (PD-L1) protein expression in gastric cancer (GC) remains controversial. The aims of the present study were to determine the correlations between tumor cell (TC) and immune cell (IC) PD-L1 protein levels with prognosis, and to determine the correlation between PD-L1 expression and different molecular GC subtypes. TC and IC PD-L1 protein levels were measured in 286 GC patients. The patients were classified according to the Cancer Genome Atlas and Asian Cancer Research Group guidelines using immunohistochemistry and in situ hybridization. TC and IC PD-L1 protein levels were positively correlated with patient survival. TC PD-L1 expression was negatively correlated with tumor grade. TC and IC PD-L1 expression was associated with improved prognosis in Epstein-Barr virus negative (EBV-), microsatellite instability (MSI) rather than microsatellite stability (MSS) subgroup GC patients. PD-L1 protein expression in TCs and ICs can be used as a prognostic indicator for GC patients, particularly in the EBV-, MSI, and MSS subgroups.

Prognostic role of programmed death-ligand 1 expression in patients with biliary tract cancer: a meta-analysis

Previous studies investigated the prognostic role of programmed death-ligand 1 (PD-L1) expression in patients with biliary tract cancer (BTC); however, the results remained controversial. Therefore, we conducted the current meta-analysis with the aim of clarifying the association between PD-L1 expression and prognosis as well as with several important clinicopathological features of BTC. We searched PubMed, Embase, and Web of Science for relevant studies. Studies that detected PD-L1 expression in tumor cells by using immunohistochemistry (IHC) were selected. Pooled hazard ratios (HRs) and pooled odds ratios (ORs) with 95% confidence intervals (CIs) were calculated to estimate the correlations. In total, 15 independent studies with 1,776 patients were included in this meta-analysis. The pooled data demonstrated that high PD-L1 expression was associated with poor overall survival (n=15, HR=1.79, 95% CI=1.55–2.07, p<0.001). The correlation between PD-L1 expression and disease-free survival was not significant (n=6, HR=1.38, 95% CI=1.00–1.91, p=0.051). In addition, no significant correlation was observed between PD-L1 expression and clinical features in patients with BTC. Our study results showed that PD-L1 expression could play a pivotal role as an effective factor of poor prognosis in patients with BTC.

The effects of mesenchymal stem cells on the IDO, HLA-G and PD-L1 expression of breast tumor cells MDA-MB-231 and MCF-7

Aim: Mesenchymal stem cells (MSCs) are strong immunomodulatory cells and a component of the tumor microenvironment. In this study, we aimed to investigate the effects of MSCs derived from adipose tissue on the expressions of immune evasive molecules indoleamine 2,3-dioxygenase (IDO), human leukocyte antigen G (HLA-G) and programmed death-ligand 1 (PD-L1) of breast tumor cell lines MDA-MB-231 and MCF-7. Methods: For this purpose, MSCs, MDA-MB-231 and MCF-7 cells were cultured with increased doses of interferon gamma (IFN-g). In another plate, tumor cells were cultured in transwell inserts using the same IFN-g stimulation to evaluate the effect of MSCs. At the end of the culture period, the HLA-G and PD-L1 expression was detected by flow cytometry, and IDO expression by the Luminex method. Results: We found that in low-dose IFN-g stimulation (10 ng/mL), MSCs led to a significant increase in the HLA-G and PD-L1 expression of MCF-7 cells. On the contrary, at a high dose of IFN-g (50 ng/mL), their expression significantly decreased in both tumor cells. In addition, we observed that the IDO expression of MDA-MB-231 cells was significantly increased in the presence of MSCs, but MCF-7 cells were not affected. Conclusion: In conclusion, for MDA-MB-231 cells, MSCs may play a protective role because they reduce the expression of HLA-G and PD-L1 that are involved in the suppression of cytotoxic cells and exhaustion of T cells. On the other hand, MSCs may be an important source of high IDO levels, and therefore may negatively affect the antitumor immune response. However, our data should be supported by further studies.

Interleukin-15 (IL-5) in Combination with Avelumab in Relapsed/Refractory Mature T-Cell Malignancies

Background: Programmed death-ligand 1 (PD-L1) is expressed in many T-cell malignancies, including 75-100% of advanced cutaneous T-cell lymphomas, 75% of ALK-negaive anaplastic large cell lymphoma (ALCL), and 28% of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS). In 27% of patients with adult T-cell leukemia/lymphoma (ATLL) expression of PD-L1 is upregulated through 3'-UTR disruption; however, immune checkpoint inhibition has been associated with hyperprogression in three patients with indolent ATLL. In T-cell malignancies, PD-L1 expression was associated with worse outcomes. Avelumab is a fully human IgG1 anti-PD-L1 antibody which inhibits PD-1/PD-L1 interactions; unlike other approved immune checkpoint inhibitors, it also induces lysis of tumor cells in vitro via antibody-dependent cell-mediated cytotoxicity (ADCC). As ADCC is mediated by natural killer (NK) cells, agents which increase NK cell number and activity may act sinergistically with avelumab. Administration of recombinant human (rh)IL-15 by continuous intravenous infusion (CIV) into adult cancer patients has produced a 15-75-fold expansion in the number of circulating NK cells at well-tolerated doses. Preclinical syngeneic and xenograft murine lymphoid malignancy models have shown increased efficacy with combined administration of IL-15 and anti-tumor antibodies compared to either alone. We therefore hypothesized that rhIL-15 may increase the efficacy of avelumab in PD-L1 expressing T-cell malignancies, and are testing the combination in a phase I trial. Primary objective: determine the safety, toxicity profile and the maximum tolerated dose of CIV IL- 15 administration in combination with with a fixed dose of avelumab. Secondary objectives: 1) determine the efficacy of combined CIV rhIL-15 and avelumab treatment to patients with T-cell lymphomas other than ATL (overal response, duration of response, progression-free survival, event-free survival, and overall survival), 2) correlate response with level of PD-L1 expression in tumor cells (&amp;lt;50% and ≥50%) 3) better understand the in vivo biological effects of avelumab and rhIL-15 treatment by analyzing changes in peripheral blood lymphocyte subsets, quantify ADCC performed ex vivo on PBMCs obtained from the patients, and examine treatment-related changes in tumor deposits by immunohistochemical and molecular analysis of core biopsies obtained before and during treatment. Exploratory objectives: 1) assess the role of circulating tumor DNA (ctDNA) in T-cell malignancies, 2) assess the role of exosomes in T-cell malignancies, 3) identify other potential biomarkers of response, such as cytokine levels and T-cell clonality. Eligibility criteria: 1) age ≥18 years, 2) histologically proven PTCL-NOS, ALK-negative ALCL, or mycosis fungoides/Sézary syndrome that is refractory to at least one line of systemic treatment, 3) ECOG ≤1, 4) WBC count ≥ 3 ⨉ 109/L, with absolute neutrophil count ≥ 1.0 ⨉ 109/L, lymphocyte count ≥ 0.5 x 109/L, platelet count ≥ 100 ⨉ 109/L, and hemoglobin ≥ 9 gm/dL (may have been transfused), 5) no prior treatment with drugs targeting PD-1/PD-L1, or receipt of any organ transplantation including allogeneic stem cell transplantation. Study design: Open-label, single-center, non-randomized Phase 1 study with a 3+3 phase dose escalation design enrolling up to 30 patients. IL-15 will be administered by continuous intravenous infusion with a dose of 1 mcg/kg/day (DL1), 2 mcg/kg/day (DL2), 3 mcg/kg/day (DL3), or 4 mcg/kg/day (DL4) on days 1-5 of each cycle. Avelumab 10 mg/kg IV over 1 hour)will be administered on days 8 and 22 of the cycle. Treatment will continue for a maximum of 6 cycles, or until toxicity or progressive disease. Correlative studies: tumor (skin or lymph node) biopsy at baseline and before the second dose of avelumab (C1D22); lymphocyte subset testing, ADCC capacity of ex vivo NK cells, ctDNA, exosomes, cell and plasma banking (days 1 and 8 of each cycle). One patients has been treated so far at DL1. Enrollment is ongoing. Figure Disclosures Waldmann: Bioniz: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Avelumab off-label use for treatment of T-cell malignancies.

Prognostic significance of PD-L1 expression and evaluation of microsatellite instability status in patients with retroperitoneal leiomyosarcomas

Background. Leiomyosarcoma is one of the most common types of soft tissue sarcomas. Radical surgical resection with subsequent adjuvant chemotherapy remain the most effective treatment approach. Immunotherapy based on inhibition of PD-L1 (programmed death ligand 1) or its receptor PD1 (programmed death 1) is considered a promising treatment option. Level of PD-L1 expression in tumor cells and presence of microsatellite instability (МSI) could be considered prognostic and predictive markers of disease progression and effectiveness of immunotherapy.The study objective is to determine PD-L1 expression level and МSI status in patients with retroperitoneal leiomyosarcomas and evaluate their effect on overall and recurrence-free survival.Materials and methods. The study included 57 patients with retroperitoneal leiomyosarcomas who underwent surgical or combination treatment. Analysis of clinical and morphological characteristics was performed; results of surgical treatment were researched. Evaluation of PD-L1 expression and MSI status was performed using immunohistochemical and molecular genetic analysis.Results. PD-L1 expression and MSI status were evaluated in 41 patients of 57. In 10 (24 %) of 41 cases, positive PD-L1 expression was observed (expression level 3–50 %). In 1 (2.4 %) patient, the primary tumor and metastatic lesion had low MSI level (MSI-low, MSI-L). Median follow-up was 31 months. In patients with positive PD-L1 expression, higher Ki-67 proliferative index was observed compared to patients with PD-L1 negative tumors (58.8 and 47.8 % respectively; р = 0.02), as well as significantly lower median overall survival for grade II tumors (30 and 105 months; p = 0.043). In grade III leiomyosarcomas, a trend towards lower median overall survival in patients with PD-L1‑negative tumors (31.0 months) compared to patients with PD-L1 expression (61.2 months) (р = 0.11) was observed.Conclusion. Among patients with retroperitoneal leiomyosarcomas, positive expression of PD-L1 was observed in 24 % (10 / 41) of cases and MSI-low status was found in 2.4 % (1 / 41) of cases. In patients with grade 2 tumors, positive PD-L1 expression is associated with significantly lower overall survival. PD-L1 expression in patients with retroperitoneal leiomyosarcomas could be considered a prognostic marker and a potential therapeutic target.

PD-L1 Expression and Clinical Outcomes to Cabozantinib, Everolimus, and Sunitinib in Patients with Metastatic Renal Cell Carcinoma: Analysis of the Randomized Clinical Trials METEOR and CABOSUN.

Programmed death-ligand 1 (PD-L1) status by IHC is prognostic in metastatic renal cell carcinoma (mRCC), and its role as a potential predictive biomarker is under investigation. Using tumor tissue from the METEOR (NCT01865747) and CABOSUN (NCT01835158) clinical trials, we explored whether PD-L1 expression and the extent of the immune cell infiltrate can serve as prognostic and/or predictive biomarkers for cabozantinib and other targeted agents. IHC double staining for PD-L1 and CD45/CD163 (immune cell markers) was performed on tumor tissue from METEOR (n = 306) and CABOSUN (n = 110) clinical trials. Immune cell density and MET expression levels were also analyzed. Our primary aim was to correlate progression-free survival (PFS) by independent central review with PD-L1 status in patients treated with cabozantinib, everolimus (METEOR), or sunitinib (CABOSUN). Overall survival (OS) was also interrogated. Tumor cell (TC) PD-L1 expression (≥1% cutoff) was detected in 29% and 23% of tumors from patients in the METEOR and CABOSUN trials, respectively. On univariate analysis, patients with PD-L1-positive TC had poorer PFS and OS than patients with PD-L1-negative TC on both trials, independent of therapy. On multivariable analysis and when combining the two trials, the association between TC PD-L1 expression and OS was statistically significant for all patients (P = 0.034) and for patients treated with cabozantinib only (P = 0.038). Cabozantinib was associated with improved PFS (HR < 0.70) and OS (HR < 0.85) compared with everolimus and sunitinib irrespective of PD-L1 expression. Higher PD-L1 expression results in worse clinical outcomes in mRCC treated with targeted therapy. Furthermore, PD-L1 expression is not predictive of response to cabozantinib therapy.

The immune microenvironment and expression of PD-L1, PD-1, PRAME and MHC I in salivary duct carcinoma.

Salivary duct carcinoma (SDC) is an aggressive salivary malignancy that results in high mortality rates and is often resistant to chemotherapy. Anti-programmed death-1 (PD-1)/programmed death ligand-1 (PD-L1) checkpoint inhibitors have led to dramatic improvements in patients with various cancers. Other immunotherapeutic approaches, e.g. cancer vaccines, have shown promising results. Cancer testis antigens, e.g. preferentially expressed antigen in melanoma (PRAME), are regarded as promising vaccine targets because of their tumour-specific expression pattern. We analysed the immunoexpression of PD-L1, PD-1, major histocompatibility complex class I (MHC I) and PRAME in 53 SDCs. The immunoexpression levels of PD-L1 in tumour cells (TCs) and immune cells (ICs), PD-1 in ICs, PRAME in TCs and MHC I in TCs were analysed, and were correlated with outcome. PRAME expression was seen in 83% of SDCs. No PRAME staining was present in normal salivary gland tissue. With the three established diagnostic algorithms proposed for head and neck squamous cell carcinoma, the criteria being a combined positive score of ≥1, TC% ≥1%, and TC% ≥25%, 35 (66%), 17 (32%) and three cases (6%), respectively, were deemed to be positive for PD-L1. PD-1-positive ICs were seen in 35 (66%) cases. MHC I down-regulation was seen in 82% of SDCs. There was a significant correlation among PD-L1 expression in ICs, PD-1 expression in ICs, and PRAME expression in TCs. PD-L1 expression in TCs and lack of PD-1 expression in ICs were associated with decreased disease-specific survival in SDC patients. Alterations of the tumour immune microenvironment are common in SDCs, including expression of PD-1/PD-L1 and PRAME, which opens the way to potential novel immune therapies, such as cancer vaccination and PD-1/PD-L1 blockade, in these tumours.

EP1.12-08 Programmed Death-Ligand 1 and Human Leukocyte Antigen Class I Expression in Various Neuroendocrine Tumors of the Lung

Pulmonary neuroendocrine tumors (PNETs) encompass a broad spectrum of tumors including typical carcinoid (TC) and atypical carcinoid (AC) tumors, small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC) with important pathological, biological and clinical differences. Recently, FDA grants Atezolizumab regimen for use in combination with carboplatin and etoposide for the frontline treatment of patients with extensive stage SCLC. The aim of this study was to investigate Programmed death-ligand 1(PD-L1) and Human leukocyte antigen (HLA) class I expression patterns in PNETs. PD-L1 and HLA class I expression was evaluated by immunohistochemistry in a total of 37 resected lung neuroendocrine tumors using tissue microarray. Correlations between the expression of HLA class I/PD-L1 and clinicopathologic features and prognostic significance were analyzed. Of the 37 patients enrolled in this study, 32 patients (86.5%) were male and the median age was 66 years (range, 35–81 years), and 28 patients (75.6%) were current or ex-smokers. The pathologic stage (AJCC 8th) in the patients at presentation was IA(including IA1, IA2, IA3) in 11 patients (24.3%), IB in 4 patients (10.8%), IIA in 1 patients (2.7%), IIB in 4 patients (10.8%), IIIA in 10 patients (27.0%), IIIB in 4 patients (10.8%), and IVA in 1 patients (2.7%). Histologically, 6 patients (16.2%) were diagnosed TC, 5 patients (13.5%) with AC, 9 patients (24.3%) with SCLC, and 17 patients (45.9%) with LCNEC. Positive PD-L1 expression in tumor cells was 29.7% (11/37, 1% cut-off), and the numbers (proportions) of positive PD-L1 expression according to histologic subtypes were as follows; TC/AC/SCLC/LCNEC, 2(33.3%)/2(40.0%)/1(11.1%)/6(35.2%). HLA class I expression was reduced in 86.5% (32/37). The numbers (proportions) of reduced HLA class I expression according to histologic subtypes were as follows; TC/AC/SCLC/LCNEC, 5(83.3%)/5(100%)/8(88.9%)/14(82.4%). In survival analysis, (median overall survival (OS); 56 months) there was no significant difference in OS according to PD-L1 and HLA class I expression status. However, two cases with HLA(+)/PD-L1(+) showed more unfavorable survival curves and three cases with HLA(+)/PD-L1(-) showed a tendency of more favorable clinical course. Lung NETs shows frequent HLA class I reduction and variable PD-L1 expression. Although this is a preliminary study including small number of NETs, the finding that NETs with HLA(+)/PD-L1(-) showed favorable clinical course suggests immune microenvironment might be one of the important biomarker for NETs of the lung.

Programmed Death Ligand 1; An Immunotarget for Renal Cell Carcinoma.

Background:In this era of developing targeted therapies and immunotherapies as a treatment for renal cell carcinoma (RCC), Programmed death ligand 1 (PDL1) as a novel biomarker for RCC is analysed in our study. About 90% of all renal cancers are Renal Cell Carcinoma. Most cases are diagnosed incidentally. 17% of cases are advanced at the time of diagnosis. PDL1 being a trans-membrane cell surface protein is expressed on the tumor cells and is found to have a chief role to inhibit the T cell immune response. It is essential to improve the host immunity by targeting the PD1/PDL1 pathway, thereby destroying the tumor progression. Aim: The aim of this study was to evaluate the expression of PDL1 in tumor cells and adjacent normal tissue among the renal cell carcinoma patients and assess the relation between the PDL1 expression and the tumor characters. Methods:This is a retrospective study. Ethical clearance was obtained from the institution. 150 histopathologically proven RCC cases were chosen. Immunohistochemistry using a PD-L1 rabbit monoclonal antibody was performed on paraffin embedded formalin fixed tissue blocks. Q scoring was done to calculate the expression of PDL1. Statistical analysis:Chi square test was done to assess the comparison between the PDL1 expression in tumor cells and their characteristic features like histology, grade and stage. SPSS (version 20.0) was used for analysis. P value <0.05 was considered significant. It also explains the heterogenous nature of PDL1 as it expressed more in the aggressive pathologic characters like high grade. Results: Positive PD-L1 expression was seen in 44% of tumors. Significant association was observed between high WWHO ISUP grading and positive PDL1 expression (p=0.028). It was expressed in 75% of the sarcomatous type of RCC and 46.8% of clear cell RCCs. Conclusion: Our study suggests that blocking PD1/PDL1 pathway may become an effective mode of treatment in cancer immunotherapy especially for Renal Cell Carcinomas. Our findings confirmed the significant association between expression of PDL1 and the high graded tumors which proves it to be an important prognostic factor.

P2.09-19 Effect of Neoadjuvant (Radio)chemotherapy on PD-L1 Expression in Resected Non-Small Cell Lung Cancers

Programmed cell death ligand 1 (PD-L1) expression has been established as a predictive biomarker for checkpoint-inhibitor treatment in advanced non-small-cell lung carcinomas (NSCLC). Inclusion of PD1/PD-L1 inhibitors into existing neoadjuvant regimens is currently evaluated in clinical trials. However, data on the impact of neoadjuvant regimens on tumoral PD-L1 expression is rare. We aimed to assess the PD-L1 expression in NSCLC before/after neoadjuvant treatment, including its prognostic relevance. Our single-center, retrospective study cohort comprised 131 consecutive patients with NSCLC resected after neoadjuvant therapy, diagnosed 2000-2016, including 63 available pretreatment biopsies. Tumor types comprised 59 squamous cell carcinomas (SQCC), 66 adenocarcinomas (ADC) and 7 others. The pathological slides and clinical records were reevaluated assessing tumor-size according to current IASLC-recommendations by multiplying the vital tumor percentage with the macroscopic tumor bed. Biologically matched cohorts of 60 locally advanced (pN2) primary resected ADC and 54 SQCC served as controls. PD-L1 was immunohistochemically stained using the Ventana SP263 KIT and tumoral expression was assessed on whole slides by two investigators using the increments: <1%, 1-<25%, 25-<50%, ≥50%. The results were correlated with clinico-pathological parameters. PD-L1 could be evaluated in 112 specimens after neoadjuvant chemotherapy, including 24 following neoadjuvant radiochemotherapy: 53 cases scored <1%, 31 1-<25%, 4 25-<50% and 24 ≥50%. In pre-treatment biopsies, 50/63 (79.4%) cases scored <50%, 13/63 (20.6%) ≥50%. Eight/63 (12.7%) cases were discordant regarding the clinically relevant cut-off 50%, 4 cases (2 ADC, 2 SQCC) having a higher expression and 4 cases (2 ADC, 2 SQCC) with lower expression after neoadjuvant radiochemotherapy. PD-L1 expression was significantly higher in primary resected SQCC (n=53) compared to neoadjuvantly treated SQCC (n=52, p=0.014; n=41 neoadjuvant chemotherapy only, p=0.033), with 14 cases scoring <1%, 21 1-<25%, 4 25-49% and 14≥50%. There was no significant difference regarding PD-L1 expression for the paired ADC-groups. In neoadjuvantly treated NSCLC, there was no correlation between PD-L1 expression (increments or 50% cut-off) and ypT, ypN, UICC/AJCC-stage, histological tumor regression, tumor size or the size of the tumor bed. PD-L1 expression was not associated with overall survival nor progression free survival in any cohort. We document a discordance of 12.7% regarding the cut-off 50% between pre-treatment biopsies and resections after neoadjuvant therapy, which may be due to tumor-heterogeneity. The higher PD-L1 expression in primary resected SQCC compared to SQCC resected after treatment provides evidence that the influence of neoadjuvant therapy on PD-L1 expression might depend on the histological tumor type.

Biomarkers and clinical outcomes in nivolumab-treated patients with advanced hepatocellular carcinoma in CheckMate 040

Abstract Background Nivolumab (NIVO), a programmed death-1 (PD-1) inhibitor, is approved in several countries for sorafenib-treated patients (pts) with advanced hepatocellular carcinoma (aHCC) based on CheckMate040. We report findings on exploratory biomarker analyses. Methods In CheckMate040, pts with aHCC received NIVO regardless of programmed death ligand 1 (PD-L1) status and HCC etiology. Baseline tumor samples were analyzed by IHC for expression of PD-L1, PD-1, T-cell markers (CD3, CD4, CD8, FOXP3), and macrophages (CD68, CD163), and (in a subset of pts) by RNA sequencing for inflammatory signatures (ISs). Results were correlated with clinical outcomes: response (complete response [CR]/partial response [PR]/stable disease [SD]/progressive disease [PD]) and overall survival (OS). Associations with etiology and geographical region (non-Asia vs Asia) were assessed. Data cutoff: June 2018. Results In 184 pts with evaluable data, increased tumor cell PD-L1 expression was associated with response (CR+PR vs SD [P = 0.00009]; CR+PR vs PD [P = 0.0007]) and OS (P = 0.03), as was increased PD-1 expression (CR+PR vs SD [P = 0.05]; CR+PR vs PD [P = 0.009]; OS [P = 0.05]). Of the T-cell markers assessed, CD3 was associated with response (n = 182, CR+PR vs SD; P = 0.05). In pts positive for CD3 or CD8, there was a trend toward improved OS (P = 0.08). There was no association between CD68 and CD163 expression and clinical outcomes. For 37 pts with RNA sequencing data available, median Bristol-Myers Squibb (BMS) IS score was higher in pts with PR vs SD (P = 0.05) and was correlated with improved OS (P = 0.01). For all inflammation markers assessed, there was no association with etiology or geographical region. Conclusions In pts with aHCC, improved OS and response to NIVO may be associated with higher PD-L1, PD-1, and CD3 expression, and higher BMS IS scores, supporting the role of PD-1 inhibition in HCC treatment. Further investigation of these biomarkers is required. Originally presented at the AACR 2019.

The splicing FK506-binding protein-51 isoform plays a role in glioblastoma resistance through programmed cell death ligand-1 expression regulation

Gliomas aberrantly express programmed cell death ligand-1 (PD-L1), which has a pivotal role in immunoevasion. The splicing isoform of FKBP5, termed FKBP51s, is a PD-L1 foldase, assisting the immune checkpoint molecule in maturation and expression on the plasma membrane. The concept that PD-L1 supports tumor-intrinsic properties is increasingly emerging. The aim of the present work was to confirm the pro-tumoral effect of PD-L1 on human glioma cell survival, stemness capacity and resistance, and to address the issue of whether, by targeting its foldase either chemically or by silencing, the aggressive tumor features could be attenuated. PD-L1-depleted glioma cells have a reduced threshold for apoptosis, while PD-L1 forced expression increases resistance. Similar results were obtained with FKBP51s modulation. The ability of PD-L1 to counteract cell death was hampered by FKBP51s silencing. PD-L1 expression was particularly high in glioma cells with a cancer-stem-cell profile. Moreover, PD-L1 sustained the spheroid formation capability of glioma cells. Targeting of FKBP51s by small-interfering RNA (siRNA) or the specific inhibitor SAFit2, reduced the number of formed spheroids, along with PD-L1 expression. Finally, in an orthotopic mouse model of glioblastoma, daily treatment with SAFit2 significantly reduced tumor PD-L1 expression, and tumor growth. In treated mice, caspase-3 activation and reduced vimentin expression were observed in excised tumors. In conclusion, targeting of FKBP51s hampers PD-L1 and its pro-tumoral properties, thereby affecting the self-renewal and growth capacities of glioblastoma cells in vitro and in vivo.

Biomarkers of Targeted Therapy and Immuno-Oncology in Cancers Metastatic to the Breast.

The breast is a rare site for metastases, and their molecular characteristics have not been studied yet. Intrinsic molecular genetics, cancer characteristics, and breast tissue immune responses in diverse metastases to the breast have not been previously studied. We identified 64 patients with cancers metastatic to the breast: 51 carcinomas and 13 melanomas. Programmed death ligand 1 (PD-L1), steroid receptors, and HER2/neu expressions were evaluated using immunohistochemistry. Gene sequencing, copy number alterations, microsatellite instability, and tumor mutational burden were performed using next-generation sequencing platforms. The 3 most common primary sites for metastatic carcinomas were lung (37%), ovary (29%), and fallopian tubes/peritoneum (14%). TP53 mutations were commonly (50%) observed among the carcinoma cases, while other mutations were characteristic for the primary cancers (VHL in renal, BRCA1 in the fallopian tube, and BRAF in melanomas). High tumor mutational burden was detected in 5/14 carcinomas and 3/7 melanomas. Tumor cell PD-L1 expression was detected in 6 carcinomas, but not in any of the melanomas, whereas immune cells' expression of PD-L1 was seen in 17 carcinomas and 6 melanomas. Estrogen receptor status was positive in 13/49 carcinomas including 12 adenocarcinomas originating from the ovary and fallopian tube or peritoneum and 1 duodenal neuroendocrine carcinoma. No carcinoma was HER2/neu positive. Intrinsic genetic characteristics of the metastases to the breast followed the pattern commonly seen in primary tumors. Biomarkers of potential benefit to immune checkpoint inhibition therapy were limited to PD-L1-positive non-small cell lung cancer. No common characteristics of the heterogeneous group of tumor metastases to this organ were identified.

Inter- and intraobserver agreement of programmed death ligand 1 scoring in head and neck squamous cell carcinoma, urothelial carcinoma and breast carcinoma.

Programmed death-ligand 1 (PD-L1) expression by tumour cells (TC) is a mechanism for tumour immune escape through down-regulation of antitumour T cell responses and is a target for immunotherapy. PD-L1 status as a predictor of treatment response has led to the development of multiple biomarkers with different reference cut-offs. We assessed pathologist consistency in evaluating PD-L1 immunopositivity by examining the inter- and intraobserver agreement using various antibody clones and different cancer types. PD-L1 expression in TC and immune cells (IC) was manually scored in 27 head and neck squamous cell carcinoma (HSCC), 30 urothelial carcinoma (UC) and breast carcinoma (BC) using three commercial clones (SP263, SP142, 22C3) and one platform-independent test (E1L3N). For interobserver agreement, PD-L1 status was evaluated blindly by three pathologists. For intraobserver agreement, PD-L1 expression was re-evaluated following a wash-out period. Intraclass correlation coefficient (ICC), overall percentage agreement (OPA) and κ-values were calculated. Using clinical algorithms, the percentage of PD-L1-positive cases in HSCC, BC and UC were 15-81%, 47-67% and 7-43%, respectively. The percentage of PD-L1 positive cases relied heavily on the algorithm/cut-off values used. Almost perfect interobserver agreement was achieved using SP263 and E1L3N in HSCC, 22C3, SP142 and E1L3N in BC and 22C3 in UC. The SP142 clone in UC and HSCC showed moderate agreement and was associated with lower ICC and decreased intraobserver concordance. Excellent inter- and intraobserver agreement can be achieved using SP263, 22C3 and E1L3N, whereas PD-L1 scoring using SP142 clone is associated with a higher level of subjectivity.

Immunohistochemistry of immune checkpoint markers PD-1 and PD-L1 in prostate cancer

Recent availability of immune checkpoint inhibitors has facilitated research involving programmed cell death protein 1 (PD-1) and its ligand, programmed death-ligand 1 (PD-L1). However, the incidence and clinical implication of PD-1 and PD-L1 expression in prostate cancer remain poorly understood. The current study aimed to determine the status of PD-1/PD-L1 expression in prostate cancer specimens and its prognostic significance.We immunohistochemically stained for PD-1 and PD-L1 in our tissue microarray (TMA) consisting of radical prostatectomy specimens. The expression of PD-1/PD-L1 was designated as positive when moderate to strong staining or weak staining was seen in at least 1% or 10%, respectively, of tumor cells and/or associated immune cells. We then evaluated the relationship between the expression of each protein and clinicopathological features available for our patient cohort.PD-1 and PD-L1 were positive in 3 (1.5%) and 1 (0.5%) of 201 non-neoplastic prostate tissues, and also in 17 (7.7%) and 29 (13.2%) of 220 prostate cancers, respectively. PD-1 and PD-L1 were also expressed in tumor-infiltrating lymphocytes/macrophages in 172 (78.2%) and 33 (15.0%) cases, respectively. PD-L1 expression in tumor cells was more often seen in high pT stage (pT2: 10.8% vs pT3/4: 20.4%; P = .072; pT2/3a: 11.4% vs pT3b/4: 31.6%; P = .013) or lymph node-positive (pN0: 10.1% vs pN1: 27.3%; P = .086) cases, whereas PD-1 expression in tumor cells was not significantly associated with pT/pN stage. In addition, there were no statistically significant associations between PD-1/PD-L1 expression in tumor cells or tumor-infiltrating lymphocytes/macrophages versus patient age, preoperative prostate-specific antigen level, or Gleason score. Kaplan–Meier analysis coupled with log-rank test further revealed no significant associations between PD-1/PD-L1 expression in tumor cells (P = .619/P = .315), tumor-infiltrating lymphocytes/macrophages (P = .954/P = .155), or either or both of them (P = .964/P = .767) versus disease recurrence after radical prostatectomy.PD-1/PD-L1 expression was detected in a subset of prostate cancers. In particular, PD-L1 expression was considerably up-regulated in nonorgan-confined tumors. However, PD-1/PD-L1 expression in our TMA was found to be not very helpful in predicting tumor recurrence in prostate cancer patients who underwent radical prostatectomy.

Positive correlation between programmed death ligand-1 and p53 in triple-negative breast cancer.

PurposeTumors with high mutation load tend to have a stronger immune response in some tumors. The correlation between expression of programmed death ligand-1 (PD-L1), a biomarker of immune response in tumors, and p53, accepted as the most frequently mutated gene in many cancers, in triple-negative breast cancer (TNBC) has not been fully investigated in cancer patients.Materials and methods132 cases of TNBC and 32 cases of non-TNBC paraffin-embedded tissue sections were selected to detect the expression of PD-L1 and p53 by immunohistochemistry, and results were correlated with clinical data and survival outcomes. The staining of PD-L1 in tumor cells (TCs) and tumor-associated immune cells (TAICs) was assessed separately.ResultsStrong positive correlations were observed between expression of p53 and PD-L1 both in TCs (r=0.338, P=0.000) and TAICs (r=0.186, P=0.033). The same positive correlation was found in the expression of PD-L1 in TCs and TAICs (r=0.764, P=0.000). Like p53 (P=0.024), positive rate of PD-L1 in TCs was significantly higher in TNBC than in non-TNBC (P=0.02). PD-L1 and p53 in TCs staining were significantly associated with histological grade, tumor size and Ki67 index (P<0.05). PD-L1 in TCs staining was also associated with lymphatic metastasis status (P=0.000). However, PD-L1 in TAICs was only related to histological grade in statistically (P=0.012). Kaplan–Meier survival analysis showed that positive groups of p53, PD-L1 in TCs and TAICs had a worse overall survival and a worse progression-free survival as compared with the negative groups, but marginal significance was found only in overall survival of PD-L1 in TCs and TAICs, and progression-free survival of PD-L1 in TAICs (P=0.074, 0.097, 0.068, respectively).ConclusionOur findings suggest that positive correlation between p53 and PD-L1 in TNBC and the higher expression rates are closely correlated with some key prognostic factors and worse survival outcomes. These findings would lay the foundation for further study on the relationship of p53 and PD-L1 and the combination of mutated p53 inhibitors and PD-1/PD-L1 antibodies in TNBC.

Comparison of tumor cell numbers and 22C3 PD-L1 expression between cryobiopsy and transbronchial biopsy with endobronchial ultrasonography-guide sheath for lung cancer

BackgroundWe previously reported cryobiopsy (Cryo) with endobronchial ultrasonography-guide sheath (EBUS-GS) for peripheral pulmonary lesions (PPLs) provides significantly larger tissues than transbronchial biopsy (TBB) and provides high quantity and quality DNA for gene analysis by next generation sequencing. However, the tumor cell yields and programmed death ligand 1 (PD-L1) expression between each approach have not been compared. Here, we assessed the tumor cell numbers and PD-L1 expression for Cryo with EBUS-GS for PPLs and TBB in patients with lung cancer.MethodsSixteen patients were enrolled in this prospective study from June to November 2017 at Tokyo Women’s Medical University Hospital. The number of tumor cells from a single biopsy, total number of tumor cells, average number of tumor cells, and 22C3 PD-L1 expression (≥ 50% and ≥ 1%) were compared between Cryo and TBB.ResultsThe numbers of tumor cells from a single biopsy, total numbers of tumor cells, and average numbers of tumor cells obtained by Cryo were significantly larger than those obtained by TBB (Cryo [means ± standard errors of the means]: 1321 ± 303.7, 1981 ± 411.7, and 1406 ± 310.3; TBB: 208.8 ± 38.24, 1044 ± 189.0, and 208.8 ± 37.81; P < 0.0001, P = 0.0474, P = 0.0006, respectively). PD-L1 ≥ 50% and ≥ 1% patients for Cryo were 18.8 and 56.3%, respectively, whereas those for TBB were 12.5 and 37.5%, respectively. The sensitivity, specificity, positive predictive value, negative predictive value, concordance, and κ coefficient based on Cryo for TBB were 66.7, 100, 100, 92.9, 93.8%, and 0.7647, respectively, for PD-L1 ≥ 50%; and 44.4, 71.4, 66.7, 50, 56.3%, and 0.1515, respectively, for PD-L1 ≥ 1%.ConclusionCryo with EBUS-GS may be a useful diagnostic approach for lung cancer, with advantages over TBB for gene analysis and whole exon sequencing. Particularly, it could contribute to patients taking pembrolizumab as first-line therapy when PD-L1 was negative by evaluating TBB specimens. It could also provide ample tissue for PD-L1 expression analysis in addition to accurate diagnosis and gene analysis.

The prevalence of programmed death ligand-1 (PD-L1) expression in non-small cell lung cancer in an unselected, consecutive population.

Little is known about prevalence of PD-L1 expression in tumor cells of unselected patients with all stages of non-small cell lung cancer. The objective of this study is to assess the prevalence of PD-L1 positivity in patients with non-small cell lung cancer, to analyze the association between PD-L1 positivity and patients' clinicopathological characteristics, and to assess the use of immune-oncologic treatment in eligible patients. All non-small cell lung cancer patients diagnosed in a 10-month period in an unselected population of 1.7 million Caucasian inhabitants were evaluated with the PD-L1 IHC 22C3 pharmDx kit. A total of 819 patients were diagnosed with non-small cell lung cancer. Samples analyzable for PD-L1 expression were obtained from 97% of patients. In a multivariate analysis with cut-off at tumor proportion score ≥50%, lower stage was associated with lower prevalence of PD-L1 positivity with an odds ratio of 0.31 for stage I vs. stage IV. A significant difference in PD-L1 expression between squamous-cell carcinoma and adenocarcinoma was observed with odds ratio for adenocarcinoma 1.8. With cut-off tumor proportion score ≥1%, attenuated effects of the same direction were seen. For neither cut-off did type and location of material used for PD-L1 analysis, age, sex, smoking history, or performance status have statistically significant impact on the PD-L1 expression. Fifty four percent of the patients who were eligible for immune-oncologic treatment were actually treated in first-line with pembrolizumab monotherapy. In conclusion, 97% of the patients had material analyzable for PD-L1. If a patient in need of immuno-oncologic treatment has shifted stage, a negative or low positive PD-L1 test performed on a biopsy taken in a lower stage might not mirror the PD-L1 expression in the new metastatic lesion. Therefore, a re-biopsy should be considered.

Circulating Tumor Cell PD-L1 Expression as Biomarker for Therapeutic Efficacy of Immune Checkpoint Inhibition in NSCLC.

Over the last decade, the immune checkpoint blockade targeting the programmed death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) axis has improved progression-free and overall survival of advanced non-small cell lung cancer (NSCLC) patients. PD-L1 tumor expression, along with tumor mutational burden, is currently being explored as a predictive biomarker for responses to immune checkpoint inhibitors (ICIs). However, lung cancer patients may have insufficient tumor tissue samples and the high bleeding risk often prevents additional biopsies and, as a consequence, immunohistological evaluation of PD-L1 expression. In addition, PD-L1 shows a dynamic expression profile and can be influenced by intratumoral heterogeneity as well as the immune cell infiltrate in the tumor and its microenvironment, influencing the response rate to PD-1/PD-L1 axis ICIs. Therefore, to identify subgroups of patients with advanced NSCLC that will most likely benefit from ICI therapies, molecular characterization of PD-L1 expression in circulating tumor cells (CTCs) might be supportive. In this review, we highlight the use of CTCs as a complementary diagnostic tool for PD-L1 expression analysis in advanced NSCLC patients. In addition, we examine technical issues of PD-L1 measurement in tissue as well as in CTCs.

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

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