Co-expression Of PD-L1 Research Articles

JAK2 (V617F) positively regulates PD-L1 mRNA expression via STAT3/5 activation in MNP (PV and ET) patients

Abstract Background Escalated PD-L1 expression is reported in many cancer types initiating an immune escape mechanism. This led to the development of checkpoint inhibitors against PD-1/PD-L1. However, the mechanisms underlying escalated production of PD-L1 in many cancers is not clear yet. Methods In this study, we studied PD-L1 mRNA expression levels in concert to JAK2 (V617F) mutation in a group of 72 MPN patients (38 PV and 33 of ET). Clinical and demographical characters were noted carefully. Results Confirmed MPN patients were screened for JAK2 (V617F) mutation by tetra-primer ARMS-PCR. We next quantified JAK2 (V617F) with ASO-PCR. The patients were also screened for BCR/ABL1 fusion gene transcripts to clarify Ph negative MPN status. The mRNA expression levels of PD-L1, STAT3/5 in all MPN patients were evaluated and it was identified that PD-L1, STAT3 but not STAT5 mRNA levels were significantly high in JAK2 (V617F) patients compared to JAK2 (WT) ones. It was also identified that PD-L1, STAT3/5 mRNA expression was upregulated in MPN patients with more JAK2 (V617F) percentage/allele burden than those MPN patients with less JAK2 (V617F) percentage/allele burden. Finally, we evaluated correlation of JAK2 (V617F) percentage with PD-L1, STAT3/5 mRNA expression levels and discovered that PD-L1 and STAT3 are in strong and direct correlation with JAK2 (V617F) burden while STAT5 was found to be moderately correlated. In addition, we identified strong coexpression of PD-L1 and STAT3 and as expected STAT5 moderately coexisted with PD-L1. Conclusion In summary,this study shows that increased PD-L1 expression accompanies JAK2 (V617F) mutation. The increased expression of PD-L1 may be caused by excessive activation of STAT3/5 which are regulators of PD-L1. The study finds that PD-L1 expression is mainly mediated by JAK2 (V617F) via STAT3 and thatSTAT5 only plays a minor role.The study supports the concept of using PD-L1/STAT3/5 axes as targets for developing checkpoint inhibitors.

Enhanced B7-H4 Expression in Gliomas With Low PD-L1 Expression Identifies Cold Tumors

Abstract INTRODUCTION The expression profiles of different immune checkpoint molecules are promising for triaging personalized targeted immunotherapy. Our study was performed to determine co-expression levels of 2 major B7 immune molecules, PD-L1 and B7-H4, in gliomas where both have demonstrated to inhibit antitumor host immunity. METHODS We assessed tumor issues from primary gliomas stage II to IV (n = 505) by immunohistochemistry (IHC) for protein levels of both PD-L1 and B7-H4. Gene co-expression analysis assessing clusters based on extent of PD-L1/B7-H4 classifier genes expression were investigated in 2 transcriptome datasets (TCGA and CGGA) to validate IHC expression profiles and explore properties of the glioma immune microenvironment among specific co-expression PD-L1/B7-H4 cluster groups. RESULTS PD-L1 was detected in 61% of patients whereby 23% expressed high levels. B7-H4 was expressed in 54% whereby 20% were identified as high expression. Co-expression of PD-L1 and B7-H4 in high levels was limited to 2% cases. Comparable results were seen in RNA-sequencing datasets when PD-L1 mRNA expression level corelated negatively with B7-H4. Gene co-expression modules clustered in each grade gliomas without double-high modules (gliomas cluster with high mRNA expression of both PD-L1 and B7-H4 classifier genes) also verified restricted coexpression pattern. B7-H4 mRNA expression level had negative correlation with extent of immune cell infiltration, including tumor-infiltrating lymphocytes (TILs), and high-B7-H4 module gliomas (high B7-H4 but low PD-L1 classifier genes expression) was related to a cold tumor with less TILs. CONCLUSION The majority of gliomas express PD-L1 or B7-H4, however, co-expression of both at high levels is minimal. The high-B7-H4 module was significantly lacking in TILs, suggesting that B7-H4 might inhibit T cell trafficking into the central nervous system (CNS). This study demonstrates that PD-L1 expression alone is not fully informative in gliomas for immune targeted or active-specific immunotherapy, and PD-L1 and B7-H4 probably inhibit different aspects of the T cell functions.

PD-L1 Expression Is Associated With VEGFA and LADC Patients' Survival.

Objectives: To elucidate the relationship between VEGFA and PD-L1 expression in lung adenocarcinoma (LADC).Methods: PD-L1 and VEGFA expression were determined by immunohistochemistry with H-score on formalin-fixed paraffin-embedded resected LADC specimens of 129 cases.Results: High PD-L1 expression in 53 (41.1%) patients, high VEGFA expression in 65 (50.4%), and co-expression in 18 (14.0%) were observed. Inverse correlation between expression of PD-L1 and VEGFA was found (P = 0.002, r = −0.274). VEGFA and PD-L1 expression were not significantly associated with the clinicopathological features. High PD-L1 expression was significantly association with all patients' poor progression-free survival and overall survival in a univariate analysis, but there was no significantly association with high VEGFA expression and prognosis. Co-expression of PD-L1 and VEGFA exhibited a worst overall survival compared to negative groups (P = 0.005).Conclusions: These findings indicate that high PD-L1 expression could impact both poor overall survival and progression-free survival in patients with resected LADC. Co-expression of PD-L1 and VEGFA may be considered as an important prognostic factor for patients with resected lung adenocarcinoma.

Abstract P3-01-13: Co-expression of molecules associated with innate and adaptive immune response on single CTCs of patients with metastatic breast cancer (mBC)

Abstract Introduction The expression of CD47 on tumor cells can act as a "don't eat me signal" against phagocytosis by macrophages and dendritic cells. Moreover, PD-L1-expressing tumor cells inhibit the anti-tumor activity of cytotoxic T cells. Circulating tumor cells (CTCs) expressing these molecules could overcome elimination by the immune system. In the current study, we evaluated for the first time the co-expression of CD47 and PD-L1 on single CTCs from patients with metastatic breast cancer (mBC). Methods Triple immunofluorescence staining was performed on peripheral blood mononuclear cells (PBMC) cytospin preparations from 18 CTC-positive patients with mBC, using antibodies against cytokeratins (for CTC detection), CD47 and PD-L1. Blood samples were obtained before the initiation of first-line chemotherapy. A total of 1*106 PBMCs were analyzed per patientusing the Ariol microscopy system. The expression levels of CD47 and PD-L1 were characterized as high or low/-, after quantification by the Ariol system, using the MDA.MB.231 breast cancer cell line as positive control. Results A total of 23 CTCs (median: 1, range: 1-4) were identified. CD47-expressing CTCs were detected in 94.4% of patients and represented 91.3% of total CTCs. However, high CD47 expression was confirmed in 38.9% and 43.5% of patients and CTCs, respectively. PD-L1 expression was evident in 27.8% of patients and in 21.7% of CTCs, whereas CTCs expressing high levels of PD-L1 were identified in 16.% of patients and represented 13% of total CTCs. Co-expression of CD47 and PD-L1 (CD47+/PD-L1+) was observed in 21.7% of CTCs, whereas 69.6% of CTCs expressed CD47 only (CD47+/PD-L1-). No CTCs expressing only PD-L1 (CD47-/PD-L1+) were detected and 2 of 23 cells were negative for both markers (CD47-/PD-L1- ). Regarding the differential expression levels of CD47 and PD-L1, the phenotype CD47low/-/PD-L1low/- was the most abundant both at the patient (61.1%) and the CTC level (52.2%). CD47high/PD-L1low/- CTCswere observed in 33.3% and 34.8% of patients and CTCs, respectively whereas only 1 CD47low/-/PD-L1high CTC was detected in one patient. Interestingly, CD47high/PD-L1high CTCs were identified in only 8.7% of CTCs. Conclusions CD47 expression is identified in the great majority of CTCs in mBC and could represent a potent signal to facilitate the escape from innate immune response. PD-L1 expression on CTCs is less commonly observed and could serve for the attenuation of an adaptive anti-tumor immune response. Interestingly, CD47 and PD-L1 are co-expressed in a subset of CTCs, whereas simultaneous high expression on single CTCs is even less common. The expression of these molecules is currently further investigated in a larger cohort of patients with mBC and in patients with early disease, in order to evaluate their differential distribution that potentially reflects the equilibrium and/or escape from the immune surveillance. Citation Format: Mavroudis D, Papadaki MA, Tsoulfas PG, Troullinou K, Apostolopoulou CA, Papadaki C, Agelaki S. Co-expression of molecules associated with innate and adaptive immune response on single CTCs of patients with metastatic breast cancer (mBC) [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-01-13.

PD-1/PD-L1 expressions in medullary thyroid carcinoma: Clinicopathologic and prognostic analysis of Chinese population

IntroductionFew studies have focused on PD-L1 expression in medullary thyroid carcinoma (MTC). Expressions of PD-1 and PD-L1 and their clinicopathologic and prognostic relevance were therefore further investigated on a relatively large population of MTC patients. Materials and methodsSurgical specimens were obtained from 87 MTC patients during a median follow-up of 37.7 months. PD-1 and PD-L1 expressions on tumor and associated immune cells were studied immunohistochemically using >1% positive cells as a threshold for positivity. Their correlations with clinicopathologic and prognostic feature were analyzed. ResultsPD-1 and PD-L1 were positively stained in 22 and 19 MTC patients. Most PD-L1-positive cases (18/19) showed weak to moderate staining intensity. PD-1 and PD-L1 were co-expressed in 11 patients. PD-L1 positivity was significantly correlated with distant metastases at surgery (21.1% vs 1.5%, P = 0.007). Coexpression of PD-1 and PD-L1 in MTC was correlated with advanced pathologic TNM stage III/IV (P = 0.040) and distant metastases at surgery (P = 0.013). However, there was no other clinicopathologic and prognostic relevance regarding to PD-1, PD-L1 or their coexpression in our MTC patients. ConclusionPD-1/PD-L1 pathway was expressed in MTC patients and was significantly correlated with the distant metastases at surgery, which may shed light on PD-1/PD-L1 as a promising therapeutic target in MTC. Future better understanding of PD-1/PD-L1 expression and their relationship with immunotherapy response may provide direct evidence for management of refractory MTC.

Personalized Tumor RNA Loaded Lipid-Nanoparticles Prime the Systemic and Intratumoral Milieu for Response to Cancer Immunotherapy.

Translation of nanoparticles (NPs) into human clinical trials for patients with refractory cancers has lagged due to unknown biologic reactivities of novel NP designs. To overcome these limitations, simple well-characterized mRNA lipid-NPs have been developed as cancer immunotherapeutic vaccines. While the preponderance of RNA lipid-NPs encoding for tumor-associated antigens or neoepitopes have been designed to target lymphoid organs, they remain encumbered by the profound intratumoral and systemic immunosuppression that may stymie an activated T cell response. Herein, we show that systemic localization of untargeted tumor RNA (derived from whole transcriptome) encapsulated in lipid-NPs, with excess positive charge, primes the peripheral and intratumoral milieu for response to immunotherapy. In immunologically resistant tumor models, these RNA-NPs activate the preponderance of systemic and intratumoral myeloid cells (characterized by coexpression of PD-L1 and CD86). Addition of immune checkpoint inhibitors (ICIs) (to animals primed with RNA-NPs) augments peripheral/intratumoral PD-1+CD8+ cells and mediates synergistic antitumor efficacy in settings where ICIs alone do not confer therapeutic benefit. These synergistic effects are mediated by type I interferon released from plasmacytoid dendritic cells (pDCs). In translational studies, personalized mRNA-NPs were safe and active in a client-owned canine with a spontaneous malignant glioma. In summary, we demonstrate widespread immune activation from tumor loaded RNA-NPs concomitant with inducible PD-L1 expression that can be therapeutically exploited. While immunotherapy remains effective for only a subset of cancer patients, combination therapy with systemic immunomodulating RNA-NPs may broaden its therapeutic potency.

Indoleamine-2,3-Dioxygenase in Non-Small Cell Lung Cancer: A Targetable Mechanism of Immune Resistance Frequently Coexpressed With PD-L1.

The immune regulatory enzyme indoleamine-2,3-dioxygenase (IDO-1) suppresses T cell responses and may reduce efficacy of therapies targeting immune checkpoints such as programmed death receptor-1/programmed death ligand-1 (PD-1/PD-L1). Early phase clinical trials combining IDO-1 and PD-1/PD-L1 inhibitors have shown some promise in non-small cell lung cancers (NSCLCs). However, the coexpression of IDO-1 and PD-L1 has not been thoroughly investigated, and the potential for IDO-1 immunohistochemical expression as a therapeutic biomarker is unknown. One hundred two cases of NSCLC (51 adenocarcinomas, 9 adenosquamous carcinomas, and 42 squamous cell carcinomas) were evaluated for IDO-1 and PD-L1 expression by immunohistochemistry. IDO-1 expression was identified in 43% of NSCLC (42% of adenocarcinomas, 44% of adenosquamous carcinomas, and 43% of squamous cell carcinomas). Coexpression with PD-L1 (≥1%) was common (27% overall; 27% of adenocarcinomas, 33% of adenosquamous carcinomas, and 26% of squamous cell carcinomas). A smaller population of tumors showed isolated PD-L1 (25% overall; 16% of adenocarcinomas, 44% of adenosquamous carcinomas, and 33% of squamous cell carcinomas) or IDO-1 expression (15% overall; 14% of adenocarcinomas, 11% of adenosquamous carcinomas, and 17% of squamous cell carcinomas). In summary, IDO-1 is commonly expressed by NSCLC, and its frequent coexpression with PD-L1 may account for the increased efficacy seen with dual blockade of PD-1/PD-L1 and IDO in clinical studies. IDO-1 immunohistochemistry may be a useful biomarker for selection of patients who could benefit from dual-agent therapy and should be evaluated in prospective clinical trials using PD-1/PD-L1 and IDO inhibitors.

PD-L1 Binds to B7-1 Only In Cis on the Same Cell Surface.

Programmed death ligand 1 (PD-L1)-mediated immunosuppression regulates peripheral tolerance and is often co-opted by tumors to evade immune attack. PD-L1 binds to PD-1 but also binds to B7-1 (CD80) to regulate T-cell function. The binding interaction of PD-L1 with B7-1 and its functional role need further investigation to understand differences between PD-1 and PD-L1 tumor immunotherapy. We examined the molecular orientation of PD-L1 binding to B7-1 using cell-to-cell binding assays, ELISA, and flow cytometry. As expected, PD-L1-transfected cells bound to PD-1-transfected cells, and B7-1 cells bound to CD28 or CTLA-4-transfected cells; however, PD-L1 cells did not bind to B7-1 cells. By ELISA and flow cytometry with purified proteins, we found PD-L1 and B7-1 had a strong binding interaction only when PD-L1 was flexible. Soluble PD-1 and B7-1 competed for binding to PD-L1. Binding of native PD-L1 and B7-1 in cis on the same cell surface was demonstrated with NanoBiT proximity assays. Thus, PD-L1-B7-1 interaction can occur in cis on the same cell but not in trans between two cells, which suggests a model in which PD-L1 can bend via its 11-amino acid, flexible stalk to bind to B7-1 in cis, in a manner that can competitively block the binding of PD-L1 to PD-1 or of B7-1 to CD28. This binding orientation emphasizes the functional importance of coexpression of PD-L1 and B7-1 on the same cell. We found such coexpression on tumor-infiltrating myeloid cells. Our findings may help better utilize these pathways in cancer immunotherapy. Cancer Immunol Res; 6(8); 921-9. ©2018 AACR.

Co-expression of NGF and PD-L1 on tumor-associated immune cells in the microenvironment of Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a malignant neuroendocrine skin tumor with known viral association. The microenvironment and its interaction with the tumor via the programmed cell death protein 1 (PD-1) pathway are crucial for response to anti-PD-1/anti-PD-L1 treatments. However, not all patients respond, which is suggestive of additional mechanisms for tumor growth and/or persistence. We previously detected tropomyosin receptor kinase A (TrkA) expression on MCC tumor cells and, therefore, gained interest in the expression of its ligand nerve growth factor (NGF). Thirty-nine patients from our department were studied for immunohistochemical NGF, PD-1, and PD-L1 expression and clinico-pathological correlation. PD-L1 was expressed on the tumor cells in 42%. In 95%, PD-L1 expression was also found on CD68+ spindle cells at the tumor border, which co-expressed NGF in 71%. 66% contained PD-1+ tumor infiltrating lymphocytes. PD-1, PD-L1, and NGF expression seems to correlate with a worse outcome. The present study shows that PD-L1 and NGF are co-expressed on spindle cells in the microenvironment. The expression of NGF might be a link of the microenvironment to the TrkA-positive tumor cells. Whether this mechanism is critical for tumor growth and lack of response to anti-PD-1/L1 treatment has to be investigated in further studies.

Wide Expression and Significance of Alternative Immune Checkpoint Molecules, B7x and HHLA2, in PD-L1-Negative Human Lung Cancers.

Purpose: Immunotherapy targeting the PD-1/PD-L1 pathway has changed the treatment landscape of non-small cell lung carcinoma (NSCLC). We demonstrated that HHLA2, a newly identified immune inhibitory molecule, was widely expressed in NSCLC. We now compared the expression and function of PD-L1 with alternative immune checkpoints, B7x and HHLA2.Experimental Design: Expression was examined in tissue microarrays consisting of 392 resected NSCLC tumors. Effects of PD-L1, B7x, and HHLA2 on human T-cell proliferation and cytokine production were investigated.Results: PD-L1 expression was identified in 25% and 31% of tumors in the discovery and validation cohorts and was associated with higher stage and lymph node involvement. The multivariate analysis showed that stage, TIL status, and lymph node involvement were independently associated with PD-L1 expression. B7x was expressed in 69% and 68%, whereas HHLA2 was positive in 61% and 64% of tumors in the two sets. The coexpression of PD-L1 with B7x or HHLA2 was infrequent, 6% and 3%. The majority (78%) of PD-L1-negative cases expressed B7x, HHLA2, or both. The triple-positive group had more TIL infiltration than the triple-negative group. B7x-Ig and HHLA2-Ig inhibited TCR-mediated proliferation of CD4 and CD8 T cells more robustly than PD-L1-Ig. All three significantly suppressed cytokine productions by T cells.Conclusions: The majority of PD-L1-negative lung cancers express alternative immune checkpoints. The roles of the B7x and HHLA2 pathway in mediating immune evasion in PD-L1-negative tumors deserve to be explored to provide the rationale for an effective immunotherapy strategy in these tumors. Clin Cancer Res; 24(8); 1954-64. ©2018 AACR.

Discordance rate of PD-L1 expression between primary and metastatic lesions in urothelial carcinoma (UC).

493 Background: Immune checkpoint inhibitors (ICI) targeting PD-1 or PD-L1 are effective in select patients with advanced UC. High PD-L1 expression enriches for response to ICIs; however, the predictive value of PD-L1 expression is limited, which may be due in part to dynamic expression of PD-L1 in the tumor environment. We sought to characterize PD-L1 expression in primary UC and paired metastatic lesions to gain insight into the potential temporal discordance of tumor PD-L1 expression during the metastatic process. Methods: Immunohistochemical (IHC) staining for PD-L1 using the SP-142 antibody was performed on primary tumors and matched metastatic specimens in 83 patients with advanced UC. IHC staining was scored for the percentage of cells positive ( < 5%, ≥5%) in tumor cell (TC) and immune cell (IC) compartments. Correlation of PD-L1 expression in TCs and ICs was estimated using Spearman’s correlation coefficients (ρ). Cohen’s kappa statistics (κ) were utilized to assess the agreement in PD-L1 expression between groups. Results: High (≥5%) PD-L1 expression in primary and metastatic biopsies, respectively, was observed in 6.1% and 14.6% of TCs and in 7.8% and 11.7% of ICs. High co-expression of PD-L1 in both TC and IC compartments was infrequent in primary and metastatic lesions (3.6% and 2.6%, respectively). PD-L1 expression in TCs was positively correlated with PD-L1 expression in ICs in primary tumors (ρ = 0.47) and in metastatic lesions (ρ = 0.27). TC PD-L1 expression in primary tumors was correlated with TC PD-L1 expression in paired metastatic lesions (ρ = 0.44) but there was minimal agreement in high expression rates between primary and metastatic lesions in the TC compartment (κ = 0.147). IC PD-L1 expression in primary tumors was not correlated with IC PD-L1 expression in paired metastatic lesions (ρ = 0.05) and there was poor agreement in high expression rates between primary and metastatic lesions in the IC compartment (κ = 0.086). Conclusions: High PD-L1 IC expression is temporally discordant between primary and metastatic UC lesions. Future studies of PD-1/PD-L1 targeted therapies in patients with metastatic UC should utilize recent biopsies of metastatic lesions to define PD-L1 expression when feasible.

Association of malignant potential of BRAF mutant colorectal cancer with coexpression of PD-L1.

643 Background: PD-L1 is a member of the B7/CD28 family that regulates immune response, and also well known as targets for immune checkpoint inhibitor. On the other hand BRAF mutation (MT) is recognized as a strongly poor prognostic factor in colorectal cancer (CRC). Recently in addition to the RAS gene, a lot of new knowledge about some molecules playing an important role in the process of carcinogenesis or drug resistance, and emerging molecules as targets for new treatments have been reported. In this research, we aim to address the clinical significance of PD-L1 expression in colorectal carcinoma correlation between emerging molecules and differences through clinical stages in CRC using tissue array. Methods: Consecutive patients who underwent surgery in our hospital from June 2003 to March 2011 were enrolled in this study. Tissue array based profiling of emerging molecules was performed on archival samples using immunohistochemistry for BRAF, MLH1/MSH2/MSH6/PMS2, CDX2, HER2 and PD-L1. We analyzed PD-L1 expression and correlation with molecular profile, survival, clinicopathological findings and location of primary site. Results: A total of 1030 CRC from stage 0 to IV were analyzed. The expression rates of PD-L1 were 3.6% and increased significantly in BRAF MT, dMMR, lack of CDX-2, tumor grade3 (TG3), right sided colon of primary site (25%, 37.8%, 21.6%, 22.1%, 6.9%, respectively; P < 0.001). In the univariate analysis, lack of CDX2, BRAF MT and TG3 were identified as prognostic factors in whole population of this cohort. Furthermore the survival of BRAF MT and lack of CDX2 with co-expression of PD-L1 were extremely poor (median OS in stage IV; 2.6 months in both). In Particular the co-expression rates of PD-L1 in BRAF MT were increasing as the clinical stage progress regardless of dMMR. Conclusions: Our study comprehensively summarized the significance of PD-L1 expression associated with the recent emerging molecules and suggested that malignant potential of BRAF mutant CRC may be explained in part due to escape from immunity by co-expression of PD-L1. Immune checkpoint inhibitor could be a candidate of target of BRAF mutant CRC with PD-L1expression irrespective of MSI status.

Abstract A040: Gene and protein expression of MAGE, PD-L1, and associated immune landscape elements in non-small cell lung carcinoma (NSCLC), urothelial carcinoma (UC), squamous cell cancer of the head and neck (SCCHN), and cervical carcinoma (CC)

Abstract Purpose: Therapeutic options are limited for patients with advanced solid tumors failing conventional chemotherapy. Clinical responses have been observed with adoptively transferred tumor-specific T cells, including a T-cell receptor (TCR)-based therapy targeting the Cancer Testis Antigens MAGE-A3 and MAGE-A6 (Lu et al., J Immunother Cancer 2015;3(Suppl 2) P158). A T-cell product comprising this TCR is currently being tested in a novel phase 1 multicenter trial (NCT03139370). The programmed cell death-1/programmed cell death ligand 1 (PD-1/PD-L1) pathway may interfere with antitumor immune mechanisms. Several inhibitors of PD-1/PD-L1 signaling have shown activity and acceptable safety in multiple advanced cancer histologies. The expression of MAGE A antigens is prevalent in diverse solid tumors (Kerkar et al., J Immunother 2016;39(4):181-7). The prevalence of MAGE A3/A6 expression among MAGE A-positive tumors, and the rate and pattern of PD-L1 expression in relation to MAGE expression, are of interest as they may create a rationale for MAGE A3/A6 directed TCR therapy, alone or in conjunction with PD1/PD-L1 inhibition. Here, we describe the development and analytical validation of a two-part assay system combining immunohistochemistry (IHC) and reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for the detection of MAGE A3/A6 expression, and examine its performance in NSCLC, UC, SCCHN and CC. We also assess the coexpression of PD-L1 and other immune-related genes (e.g., HLAs) in tumor cells and/or immune cells to better understand the tumor microenvironment. Methods: Sections from approximately 200 formalin-fixed, paraffin-embedded (FFPE) tumor tissue specimens were screened by IHC for MAGE-A (mAb 6C1; recognizes MAGE A1, A2, A3, A4, A6, A10, A12) and PD-L1 (mAb E1L3N) expression. Gene expression analyses (RT-qPCR and RNA sequencing) were carried out on RNA extracted from adjacent tissue sections. Results: Analytical validation of the MAGE A3/A6 assay system successfully met predefined acceptance criteria appropriate for clinical sample testing. 75-80% of MAGE A IHC+ tumors were positive for MAGE A3/A6 mRNA. The rate of PD-L1 expression in tumor cells and/or immune cells in MAGE+ tumors was 89% for NSCLC (n=37), 41% for UC (n=32), 95% for SCCHN (n=19), and 100% for CC (n=7). Distinct tumor cell staining patterns were identified including samples in which PD-L1 and MAGE-A staining overlapped extensively, partially, or were nearly mutually exclusive. In some tumors, diffuse PD-L1+ immune cells were observed in the tumor bed; in other samples, PD-L1+ immune cells appeared to surround the tumor bed, while others showed lack of PD-L1+ immune cells. Conclusions: The results support clinical evaluation of a MAGE A3/A6 TCR T-cell product across these major histologies. This two-part MAGE A3/A6 screening assay may reduce the potential for false-positive results obtained using the 6c1 IHC assay alone , and be useful in screening subjects for anti-MAGE A3/A6 T-cell therapy. While the significance of staining patterns remains to be determined, the presence of PD-L1 positive TC and IC in MAGE A3/A6+ tumors supports the concept of checkpoint blockade in combination with T-cell therapy to treat advanced solid tumors. Citation Format: Stephanie H. Astrow, Izak Faiena, Rajul Jain, Alexandra Drakaki, Wesley S. Chang, Clark C. Fjeld, Jin Li, Adrian Bot. Gene and protein expression of MAGE, PD-L1, and associated immune landscape elements in non-small cell lung carcinoma (NSCLC), urothelial carcinoma (UC), squamous cell cancer of the head and neck (SCCHN), and cervical carcinoma (CC) [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr A040.

OA 13.06 Co-Expression of IDO1 and PD-L1 Indicates More Aggressive Features of Lung Adenocarcinoma

OA 13.06 Co-Expression of IDO1 and PD-L1 Indicates More Aggressive Features of Lung Adenocarcinoma

B7-H3 Expression in NSCLC and Its Association with B7-H4, PD-L1 and Tumor-Infiltrating Lymphocytes.

Purpose: The immune checkpoint PD-1 and its receptor B7-H1 (PD-L1) are successful therapeutic targets in cancer but less is known about other B7 family members. Here, we determined the expression level of B7-H3 protein in non-small cell lung cancer (NSCLC) and evaluated its association with tumor-infiltrating lymphocytes (TIL), PD-L1, B7-H4, and major clinicopathologic characteristics is in 3 NSCLC cohorts.Experimental design: We used multiplexed automated quantitative immunofluorescence (QIF) to assess the levels of B7-H3, PD-L1, B7-H4, and TILs in 634 NSCLC cases with validated antibodies. Associations between the marker levels, major clinicopathologic variables and survival were analyzed.Results: Expression of B7-H3 protein was found in 80.4% (510/634) of the cases. High B7-H3 protein level (top 10 percentile) was associated with poor overall survival (P < 0.05). Elevated B7-H3 was consistently associated with smoking history across the 3 cohorts, but not with sex, age, clinical stage, and histology. Coexpression of B7-H3 and PD-L1 was found in 17.6% of the cases (112/634) and with B7-H4 in 10% (63/634). B7-H4 and PD-L1 were simultaneously detected only in 1.8% of NSCLCs (12/634). The expression of B7-H3 was not associated with the levels of CD3-, CD8-, and CD20-positive TILs.Conclusions: B7-H3 protein is expressed in the majority of NSCLCs and is associated with smoking history. High levels of B7-H3 protein have a negative prognostic impact in lung carcinomas. Coexpression of B7-H3 with PD-L1 and B7-H4 is relatively low, suggesting a nonredundant biological role of these targets. Clin Cancer Res; 23(17); 5202-9. ©2017 AACR.

MiR-34a attenuates glioma cells progression and chemoresistance via targeting PD-L1.

To investigate the roles of miR-34a in progression and chemoresistance of glioma cells. Quantitative real-time PCR analysis showed that miR-34a level was lower, but PD-L1 expression level was higher in glioma tissue specimens compared with normal brain tissues and their expression levels were negatively correlated. Ectopic expression of miR-34a inhibited glioma cell proliferation, promoted cell cycle arrest in G1/S phase and cell apoptosis. Additionally, miR-34a/PD-L1 axis was again confirmed and co-expression of PD-L1 with miR-34a mimics attenuated the effects of miR-34a on cell proliferation and apoptosis in glioma cells. Importantly, PD-L1 overexpression resulted in chemoresistance in glioma cells, this effect was attenuated by miR-34a overexpression. miR-34a inhibits glioma cells progression and chemoresistance via targeting PD-L1.

PD-1 and PD-L1 co-expression predicts favorable prognosis in gastric cancer.

While the prognosis of gastric cancer (GC) remains poor, PD-1 and PD-L1/L2 are promising prognostic biomarkers. We evaluated PD-1 and PD-L1/L2 expression in tumor cells (TCs) and tumor-infiltrating immune cells (TIICs). We determined the Helicobacter pylori (Hp) and Epstein-Barr virus (EBV) infection status in a GC cohort (n=340), then analyzed the relationship between the expression of PD-1, PD-L1/L2 and GC prognosis. We found that PD-1, PD-L1, and PD-L2 mRNA levels were up-regulated in GC tissues, and were positively correlated with one another (P=0.043, P=0.008 and P=0.035). PD-1 protein expression in TIICs was observed in 22.6% of GC patients. The PD-L1 and PD-L2 positivity rates were 40.3% and 53.8% in TCs, respectively, and 60.0% and 60.9% in TIICs, respectively. PD-L1 was up-regulated in EBV-infected GC patients in both TCs (P=0.009) and TIICs (P=0.003). Hp status was not associated with PD-1 or PD-L1/PD-L2 expression. In TIICs, PD-L1 expression was independently associated with better GC prognosis (HR=0.72, 95%CI: 0.53-0.99). Co-expression of PD-1 and PD-L1, but not PD-L2, was a favorable prognostic marker that indicated a dose effect on the mortality risk of GC patients (P-value for trend=0.005). Comprehensive evaluation of PD-1 and PD-L1 in TCs and TIICs could help predict the prognosis of gastric cancers, as well as reveal patients who might benefit from targeted treatment.

Prognostic role of tumor infiltrating lymphocytes in EBV positive and EBV negative nasopharyngeal carcinoma

ObjectivesTumor infiltrating lymphocytes (TILs) correlate with both better and worse prognosis in solid tumors. As therapeutic modalities for nasopharyngeal carcinoma (NPC) are limited, immunotherapy could be a potential alternative. Up till now there is limited prognostic data on the role of TILs in NPC, so we assessed the prognostic role of TILs in Epstein-Barr-virus (EBV) positive and negative NPC. MethodsTissue of 92 NPCs was assessed for CD3, CD4, CD8, PD1 and PDL1 expression in the tumor’s micro-environment. Correlations between clinicopathological characteristics was assessed using the Pearson X2 test, Fisher’s exact test and ANOVA. Survival was analyzed with the Kaplan-Meier method and Cox regression. Differences in CD3, CD4, CD8, PD1, PDL1 counts/(co)expression between EBV positive and negative NPCs were evaluated using the Mann-Whitney U test. Two-tailed P values below 0.05 were considered statistically significant. ResultsEBV positive NPC contains significantly more CD3, CD4 and CD8 TILs than EBV negative NPC. In the whole NPC group, increased CD8 count is associated with better overall survival (OS) (HR 0.219 (95%CI 0.075–0.640)), but also in cases with PDL1 co-expression (HR 0.073 (95%CI 0.010–0.556)). In EBV positive NPC co-expression of CD8 and PDL1 showed better disease free survival (HR 0.407 (95%CI 0.195–0.850)) and OS (HR 0.170 (95%CI 0.037–0.787)). ConclusionsAlthough TILs are significantly different between EBV positive and negative NPCs, it is especially composition of the infiltrate which determines prognosis. Effects of PD1 and CD8 need more study, because these findings show much potential in using immunotherapeutic modalities in NPC treatment.

Impact of chemoradiotherapy on PD1/PDL1 expression and clinical outcomes in gastroesophageal cancers.

4031 Background: Expression of the immune modulating proteins, programmed death receptor-1 (PD1) and its ligand (PDL1), in gastrointestinal malignancies is associated with poor prognosis. PD1/PDL1 expression levels have also been identified as predictors of response to checkpoint inhibition. Minimal data is available on how expression of PD1 and PDL1 is influenced by chemoradiotherapy (CRT). In this study, we investigated the relationship between PDL1/PD1 expression, CRT, and clinical outcomes in gastroesophageal (GE) cancer. Methods: With IRB approval, we identified 28 patients with gastric cardia or GE junction tumors who underwent neoadjuvant standard CRT followed by surgical resection. Pre-CRT biopsies and post-CRT surgical specimens were analyzed using quantitative immunohistochemistry for the expression of PDL1 and PD1. Samples were categorized as trace-low (TL) or moderate-high (MH) expressors of PDL1 and PD1. The impact of these and other clinical and pathologic variables on overall survival (OS) was assessed using multivariate cox proportional hazards modeling. Co-expression of PDL1 and PD1 in matched samples was determined by regression analysis. Results: Following CRT, PDL1 and PD1 expression increased in 54% and 32% of patients, respectively. On multivariate analysis, patients with MH expression of PD1 after CRT irrespective of pre-CRT expression levels had a significant decrease in OS compared to those with TL expression (median survival 23.1 vs 74.1 months; HR,3.31; CI,1.05-10.35; p = 0.039). In patients with gastric confined tumors, an increase in PD1 expression from TL to MH after CRT was associated with significantly lower OS rates (p = 0.003). Regression analysis of PD1 to PDL1 was significant (p &lt; 0.01) both before and after CRT, with a correlation coefficient of 0.34 in pre-CRT and 0.49 in post-CRT specimens. Conclusions: Elevated expression of PD1 is associated with poor OS in patients with GE cancer. Neoadjuvant CRT upregulates both PDL1 and PD1. In gastric cancer patients, this led to significantly worse survival. These data identify potential mechanisms of resistance and suggest a role for checkpoint inhibitors in combination with CRT.

Immune microenvironment in mesothelioma: Looking beyond PD-L1.

8515 Background: Studies using immune checkpoint inhibitors in mesothelioma (MM) have shown promise. Differences in response to PD-L1 and PD-1 inhibitors (10% vs 25%) have been reported. Also, expression of PD-L1 alone appears to be a limited predictor. As the roles of the multiple check point receptors and their ligands become defined, an understanding of their expression and interplay in the mm tumour microenvironment, which could affect suitability for checkpoint inhibition therapy, has become necessary. Methods: Tissue microarrays were constructed and stained with PD-L2, LAG3 and TIM3 antibodies. Tumour infiltrating lymphocytes (TILs) were assessed in the stroma and expressed as a % of stromal area within invasive tumour. These data were combined with PD-L1 expression, CD4+ and CD8+ infiltration in the same cohort reported previously. To quantify the immunosuppressive milieu, we combined our assessment of PD-L1, PD-L2 and TIM3 expression to derive an “Immune checkpoint score (ICS)” and explored its correlation with the tumour microenvironment and clinicopathological covariates. We are also exploring its predictive value in an independent cohort of mm patients who have received anti-PD-1 treatment. Results: Amongst 329 patients evaluated, PD-L1 was positive (+) in 41.7% and PD-L2+ in 24.5%. TIM3+ lymphocytes were found in 99.4% but LAG3+ lymphocytes in only 0.2%. 28/173 (16%) of PD-L1- patients were PD-L2+ and 31/136 (22%) PD-L1 and PD-L2 negative patients had high infiltration with TIM3+ lymphocytes. High ICS was associated with non-epithelioid histology, increased TILs and poorer survival. On multivariate analysis, high TILs, non-epithelioid histology and poor physiological status remained significantly associated with poorer survival. Data on the predictive role of ICS score will also be reported. Conclusions: While co-expression of PD-L1, PD-L2 and TIM3 can occur, their expression is mutually exclusive in a large proportion of patients. The expression of PD-L2 may explain differences in responses seen between PD-1 compared to PD-L1 inhibitors. A comprehensive assessment of these multiple immunosuppressive pathways may be necessary to truly gauge the immunosuppressive environment and tailor immunotherapy for individual cases.