PD-L1 Protein Expression Research Articles

Ferroptosis Characterization in Lung Adenocarcinomas Reveals Prognostic Signature With Immunotherapeutic Implication.

The iron-dependent cell death named ferroptosis has been implicated in the progression and therapeutic response of several tumors. However, potential role of ferroptosis in lung adenocarcinomas (LUAD) remained less well understood. In TCGA-LUAD cohort, unsupervised clustering was first conducted based on ferroptosis regulators extracted from FerrDb database. Comprehensive correlation analysis and comparisons were performed among ferroptosis subtypes. The ferroptosis-related prognostic (FRP) signature was identified based on filtered features and repeated LASSO and was validated in five independent cohorts. The clinical relevance between the risk score and therapeutic response was further explored by multiple algorithms. qPCR was implemented to verify gene expression. A total of 1,168 LUAD patients and 161 ferroptosis regulators were included in this study. Three ferroptosis subtypes were identified and patients in subtype B had the best prognosis among the three subtypes. Significant differences in immune microenvironment and biological function enrichment were illustrated in distinct subtypes. The Boruta algorithm was conducted on 308 common differentially expressed genes for dimensionality reduction. A total of 56 genes served as input for model construction and a six-gene signature with the highest frequencies of 881 was chosen as FRP. The prognostic significance of FRP was validated in five independent cohorts. High FRP risk score was also linked to increased tumor mutation burden, PD-L1 protein expression and number of neoantigens. Of the FRP genes, 83.3% was abnormally expressed in LUAD cell lines. In conclusion, ferroptosis plays a non-negligible role in LUAD. Exploration of the ferroptosis pattern will enhance the prognostic stratification of individual patients and move toward the purpose of personalized treatment.

Conventional Transbronchial Needle Aspiration (cTBNA) and EBUS-Guided Transbronchial Needle Aspiration (EBUS-TBNA): A Retrospective Study on the Comparison of the Two Methods for Diagnostic Adequacy in Molecular Analysis

Introduction: In recent years, there has been a growing development of molecularly targeted therapies for various types of solid tumors—in particular, in non-small-cell lung cancer (NSCLC). This has required the need for greater quantities of tissue that is able to support ancillary studies, alongside cyto-histological diagnoses for the assessment of molecular targets. Conventional TBNA (cTBNA) and EBUS-guided TBNA (EBUS-TBNA) have shown a high diagnostic yield for malignant mediastinal and/or hilar lymph node enlargement and peribronchial masses; however, few studies have compared these two procedures. We retrospectively compared TBNA patients (EBUS-TBNA and cTBNA) in order to determine the diagnostic yield and material adequacy for subsequent ancillary analyses. Materials and Methods: We retrospectively evaluated 318 patients with clinical suspicion of lung cancer or with disease recurrence. All of the patients underwent TBNA (either EBUS-TBNA or cTBNA) on enlarged mediastinal and/or hilar lymph nodes and peribronchial masses between January 2017 and June 2021 at the University Hospital of Pisa, Italy. After a definitive diagnosis, molecular analyses and an evaluation of PD-L1 expression were performed in the cases of adenocarcinoma, squamous cell carcinoma, and NSCLC, not otherwise specified (NOS). Results: EBUS-TBNA was performed in 199 patients and cTBNA was performed in 119 patients with 374 and 142 lymph nodes, respectively. The overall diagnostic yield for positive diagnoses was 59% (diagnostic rate of 61% in EBUS-TBNA, and 55% in cTBNA). Adenocarcinoma (ADC) was the most frequent diagnosis in both methods. EBUS-TBNA diagnostic adequacy was 72% for molecular analysis, while it was 55.5% for cTBNA, showing a statistical trend (p = 0.08) towards the significance of EBUS. The average percentage of neoplastic cells was also statistically different between the two methods (p = 0.05), reaching 51.19 ± 22.14 in EBUS-TBNA and 45.25 ± 22.84 in cTBNA. With regard to the PD-L1 protein expression, the percentage of positivity was similar in both procedures (86% in EBUS-TBNA, 85% in cTBNA). Conclusions: Conventional TBNA (cTBNA) and EBUS-guided TBNA (EBUS-TBNA) are minimally invasive diagnostic methods that are associated with a high diagnostic yield. However, EBUS-TBNA has an improved diagnostic adequacy for molecular analysis compared to cTBNA, and is associated with a higher average percentage of neoplastic cells.

The Frequency of DNA Mismatch Repair Deficiency Is Very Low in Surgically Resected Lung Carcinoma.

IntroductionDNA mismatch repair (MMR) deficiency leads to changes in the length of nucleotide repeat sequences of tumor DNA. In that situation, DNA replicational errors occur and accumulate during DNA replication. As a result, this mechanism frequently affects the coding regions of oncogenes and tumor suppressor genes and causes carcinogenesis. Recently, DNA MMR deficiency has been recognized as a predictive biomarker for immunotherapy. The aim of this study is to examine the frequency of DNA MMR deficiency and clinicopathological characteristics in surgically resected lung carcinoma (LC) and their correlation.MethodsA total of 1153 LCs were examined. Tissue microarrays were constructed. The status of MMR deficiency was evaluated by immunohistochemical analysis of MMR protein expression (hMLH1, hMSH2, hMSH6, and hPMS2). Microsatellite instability analysis, BRAF mutation, and MLH1 methylation analysis were performed for cases that showed MMR deficiency.ResultsOnly 2 of the 1153 cases (0.17%) showed a loss of hMLH1/hPMS2 protein expression. They also had high levels of microsatellite instability (MSI-H), had neither MLH1 promoter methylation nor BRAF mutation, and were male smokers. Histopathologically, one was a squamous cell carcinoma, and the other was combined small cell carcinoma with squamous cell carcinoma. Regarding PD-L1 protein expression, one had high expression, and the other had none.ConclusionThe frequency of MMR deficiency was very low in LC. However, our two cases were non-adenocarcinoma and differed from previous studies. Because of its very low frequency, MMR deficiency is not a practical biomarker to predict the effect of immune checkpoint inhibitors in LC.

P56.04 Pathological Features & Prognosis of NSCLC Patients With Enteric Adenocarcinomas Without Expression of TTF-1 and Napsin A

Enteric differentiation can occur in lung adenocarcinoma, and when this component exceeds 50%, the tumor is classified as pulmonary adenocarcinoma with enteric differentiation (PAED). The enteric pattern of enteric lung adenocarcinoma shares morphologic and immunohistochemical features with colorectal adenocarcinoma and it consists of glandular and/or papillary structures sometimes with a cribriform pattern, lined by tumor cells that are mostly tall-columnar with nuclear pseudostratification, luminal necrosis, and prominent nuclear debris. Poorly differentiated tumors may have a more solid pattern. These tumors show at least one immunohistologic marker of enteric differentiation (CDX-2, CK20 or MUC2). Enteric adenocarcinoma is a rare histologic type of primary lung adenocarcinoma. Detailed clinical, pathological and molecular features about these subgroup of patients is lacking, particularly if TTF-1 and Napsin A aren't expressed. NSCLC patients who were diagnosed from January 2018 until March 2021 were identified by review of the patient records. Only lung adenocarcinoma patients whose tumors didn`t express either TTF-1 or Napsin A, but were positive for either CDX-2, CK20 or MUC2 were included in the data analysis. Primary tumor of the gastrointestinal tract was excluded either by PET-CT and/or CT and endoscopy (gastroscopy, colonoscopy, and rectoscopy). A total of fourteen (eight male (57,1%) and six female (42,9%) patients were identified in the patients` records. On average, the patients were 67 years old, the median age was 68. The youngest patient was 54 and the oldest one was 83 years old. Three patients were diagnosed at local stages (I-III), eleven patients had metastatic disease (stage IV). Four of these eleven patients had a mutation of the KRAS gene, which corresponds to 37%. This percentage is higher than the average frequency of mutations in the KRAS gene in pulmonary adenocarcinomas (25%). Half of the patients had a tumors with protein expression of PD-L1 (50%). Four patients of the complete cohort underwent surgical resection of the primary tumor and five patients received radiotherapy. Every patient, who got radiotherapy, suffered from stage IV. Three patients received irradiation of brain metastasis and two of bone metastasis. A systemic therapy was administered to nine patients. Seven patients had a combined chemo-/ immuntherapy as firstline treatment (either cisplatin, pemetrexed, and pembrolizumab or carboplatin, nab-paclitaxel, and atezolizumab) and one got only a combination chemotherapy (carboplatin and nab-paclitaxel). Median disease-free survival was eight, median progression-free survival was four months. Median overall survival could not be calculated. However, six and twelve months survival rates were 35.8 and 25.0 %, respectively. This is the far largest reported series of PAED patients without any expression of TTF1- or Napsin A. It seems that this subgroup of pulmonary adenocarcinoma patients has a poorer prognosis compared to other patients with lung adenocarcinomas.

WS05.03 Treatment of Advanced and Metastatic NSCLC without Oncogenic Drivers: Balancing Cost and Efficacy

WS05.03 Treatment of Advanced and Metastatic NSCLC without Oncogenic Drivers: Balancing Cost and Efficacy

Programmed death ligand-1 protein expression difference in basal like and non-basal like triple negative breast cancer and its association with disease free survival and overall survival: A systematic review.

The study aims to summarize the literature and explore the strength of evidence for PD-L1 expression difference in basal like TNBC and non-basal like TNBC, and association of PD-L1 expression with disease free survival and overall survival in each group. A systematic search of the original research literature through November 29th, 2020, reported according to PRISMA guideline. Eligible studies investigated must have a primary outcome and at least one secondary outcome. Two reviewers independently searched, selected, and assessed quality of studies and risk of bias. Any discrepancies will be resolved by consensus or by consulting a third and fourth author. A total of 6813 articles were screened from which five articles were selected and assessed for quality of studies and risk of bias. Of 5 articles, no similar findings are found regarding the level of PD-L1 expression and its correlation with recurrence and overall survival. There is not enough substantial evidence to support the difference PD-L1 protein expression level in basal and non-basal like TNBC and its association with recurrence and overall survival. Hence, further studies are needed specifically to focus on this problem.

Expression of p53 Protein Associates with Anti-PD-L1 Treatment Response on Human-Derived Xenograft Model of GATA3/CR5/6-Negative Recurrent Nonmuscular Invasive Bladder Urothelial Carcinoma.

Background: The possible involvement of p53 signaling, FGFR3 expression, and FGFR3 mutation rates in the prediction of the NMIBC anti-PD-L1 treatment response needs to be clarified. The main aim of our study was to explore predictive value of p53 expression, FGFR3 expression, and its gene mutation status for the therapeutic success of anti-PD-L1 treatment in the patient-derived murine model of recurrent high-PD-L1(+) GATA3(−)/CR5/6(−) high-grade and low-grade NMIBC. Methods: twenty lines of patient-derived xenografts (PDXs) of relapsed high-PD-L1(+) double-negative NMIBC were developed, of which 10 lines represented high-grade tumors and the other ones—low-grade bladder cancer. Acceptors of each grade-related branch received specific anti-PD-L1 antibodies. Animals’ survival, tumor-doubling time, and remote metastasis were followed during the post-interventional period. PD-L1, GATA3, CR5/6, and p53 protein expressions in engrafted tumors were assessed by immunohistochemistry. The FGFR3 expression and FGFR3 mutations in codons 248 and 249 were detected by real-time polymerase chain reaction. Results: The expression of p53 protein is an independent factor affecting the animals’ survival time [HR = 0.036, p = 0.031] of anti-PD-L1-treated mice with low-grade high-PD-L1(+) double-negative NMIBC PDX. The FGFR3 expression and FGFR3 mutation rate have no impact on the anti-PD-L1 treatment response in the interventional groups. Conclusions: p53 expression may be considered as a prognostic factor for the anti-PD-L1 treatment efficacy of low-grade high-PD-L1-positive GATA3(−)/CR5/6(−)-relapsed noninvasive bladder cancer.

SH003 overcomes drug resistance and immune checkpoints by inhibiting JAK-STAT3 signaling in MCF7/ADR cells

BackgroundBreast cancer has the most commonly diagnosed malignancy cancer worldwide in women and has a high mortality. Various anticancer drugs to treat breast cancer have been developed and tested but have failed because of drug resistance. ObjectivesThe efficacy of SH003 against doxorubicin-resistant MCF/ADR cells has not been evaluated. In this study, we aimed to examine whether SH003 could efficiently prevent the proliferation of MCF7/ADR cells. MethodsCell viability was measured by an MTT assay. Analysis of cell cycle arrest was performed by flow cytometry. Induction of apoptosis by SH003 was measured by an annexin V-FITC/PI assay. Levels of p-STAT3, p-PD-L1, MDR and caspases were measured by western blot analysis. mRNA expression levels of MDR1, MRP1, -2, -3, -4, -5, -6, -9, BCRP and PD-L1 were measured by RT-PCR. Nuclear staining of STAT3 was measured by immunocytochemistry. The expression levels of VEGF, MMP-2 and MMP-9 were measured by ELISA. ResultsSH003 inhibited the proliferation of MCF7/ADR cells and induced their sub-G1 cell cycle arrest. SH003 also induced apoptosis, regulated apoptotic molecules, caused morphological changes and inhibited colony formation in MCF7/ADR cells. Furthermore, SH003 suppressed STAT3 transcriptional activity and, more importantly, reduced the cytokine levels of VEGF, MMP-2 and MMP-9 in MCF7/ADR cells. SH003 decreased the protein expression of PD-L1 and MDR1. Additionally, SH003 reduced the mRNA expression of PD-L1; MDR1; MRP1, -2, -3, -4, -5, -6, and -9; and BCRP. ConclusionOur results clearly demonstrate that SH003 inhibits cell growth and induces apoptosis by inhibiting STAT3 signaling. Additionally, SH003 decreased the levels of MDR1 and PD-L1 by inhibiting STAT3 signaling in MCF7/ADR breast cancer cells. These results support the possibility that SH003 could be useful as an herbal medicine to treat doxorubicin-resistant breast cancer.

PD-L1 Protein Expression and Gene Amplification Correlate with the Clinicopathological Characteristics and Prognosis of Lung Squamous Cell Carcinoma.

PurposeTo investigate PD-L1 protein expression and gene amplification in lung squamous cell carcinoma (LUSC) and analyse their correlation with the clinicopathological characteristics and prognosis of LUSC patients.Patients and MethodsTissue samples from 164 LUSC patients were collected. PD-L1 protein was detected by immunochemistry (IHC), and PD-L1 gene amplification was investigated by fluorescence in situ hybridization in LUSC patients.ResultsThe positive expression rate of PD-L1 in LUSC was 47.6% (78/164), and the amplification rate of PD-L1 was 6.7% (11/164); both rates were higher than those of paratumor tissue. Both PD-L1 positive expression and gene amplification were correlated with clinical stage and lymph node metastasis (P<0.05). PD-L1 protein expression, PD-L1 gene amplification, late stage, lymph node metastasis and distant metastasis were significantly correlated with the prognosis of patients. Among these factors, late stage, lymph node metastasis, PD-L1 protein expression and PD-L1 gene amplification were independent prognostic factors for LUSC.ConclusionPositive PD-L1 protein expression and gene amplification are involved in the malignant progression and metastasis of LUSC. Both PD-L1 protein expression and gene amplification are associated with poor prognosis.

Banxia xiexin decoction affects drug sensitivity ingastric cancer cells by regulating MGMT expression via IL‑6/JAK/STAT3‑mediated PD‑L1 activity.

Banxia xiexin decoction (BXXX) is a classic preparation used to treat gastrointestinal diseases, and also has certain therapeutic effects on gastrointestinal tumors. BXXX has been reported to regulate the expression of proteins associated with drug resistance and sensitivity in tumors, and thus, the aim of the present study was to investigate the mechanisms of BXXX drug sensitivity in gastric cancer (GC). The expression levels of programmed cell death 1 ligand 1 (PD-L1), 6-O-methylguanine-DNA methyltransferase (MGMT) and STAT3 were immunohistochemically detected in the cancer and adjacent non-cancer tissues of patients with GC, and in vitro experimentation was conducted using drug-resistant and -sensitive GC cells. The expression levels of PD-L1, MGMT and STAT3 were determined using reverse transcription-quantitative PCR. Different concentrations of BXXX drug serum were used to treat the cells and the cellular inhibition rate was assessed using a Cell Counting Kit-8 assay. Flow cytometry was used to detect apoptosis, and western blot analysis was used to detect the expression levels of IL-6, IFN-γ, JAK/STAT3 pathway proteins, PD-L1 and MGMT. The association between PD-L1 and MGMT protein expression levels was subsequently assessed via co-immunoprecipitation. Furthermore, in vivo studies were conducted following the establishment of a drug-resistant tumor-bearing mouse model, where GC tumor size was assessed under different treatment conditions, and western blot analysis was used to detect the expression of related pathway proteins. The expression levels of PD-L1, MGMT and STAT3 were significantly increased in GC tissues, GC cells and cisplatin-resistant cells. Furthermore, BXXX inhibited the proliferation of drug-resistant cells and promoted the inhibitory effects of chemotherapeutic drugs on drug-resistant cells. BXXX also inhibited the expression levels of IL-6, IFN-γ and JAK/STAT3 pathway proteins, as well as the expression levels of PD-L1 and MGMT. Colivelin, an activator of STAT3, reversed the effects of BXXX on drug-resistant GC cells, and significantly reversed the effect of BXXX on PD-L1 expression. In conclusion, BXXX was found to influence the drug sensitivity of GC cells by regulating the expression of MGMT. This process functions viaPD-L1, which was itself mediated by IL-6/JAK/STAT3 signaling.

Abstract 3190: PD-L1 protein expression in Middle Eastern breast cancer predicts favorable outcome in triple negative breast cancer

Abstract Programmed cell death ligand 1 (PD-L1) has been shown to induce potent T-cell mediated anti-tumoral immunity. The significance of PD-L1 expression on prognosis of breast cancer remains controversial and its prevalence and prognostic value in breast cancer from Middle Eastern ethnicity is lacking. A total of 1003 unselected Middle Eastern breast cancers were analyzed for PD-L1 expression using immunohistochemistry. The association with clinico-pathological markers and impact on overall survival as well as recurrence free survival was determined. PD-L1 expression was seen in 32.8% (329/1003) cases. PD-L1 positivity was significantly associated with poor prognostic indicators such as younger patients (p = 0.0499), high grade tumors (p = 0025), ER negative (p &amp;lt; 0.0001) and PR negative (p = 0.0001) status as well as high Ki-67 index (p &amp;lt; 0.0001). We also found a significant association between PD-L1 expression and deficient mismatch repair protein expression (p = 0.0009). No association was found between PD-L1 expression and clinical outcome. However, on further subgroup analysis, PD-L1 expression was found to be an independent marker for favorable prognosis in triple negative breast cancer (TNBC) (Hazard ratio = 0.27; 95% confidence interval = 0.10 - 0.63; p = 0.0018). In conclusion, we demonstrated strong association between PD-L1 and MSI in Middle Eastern BC patients and showed that PD-L1 overexpression on tumor cells was an independent favorable prognostic factor in TNBCs from this ethnicity. Overall, these findings might help in the development of more appropriate treatment strategies for BC in Middle Eastern population. Citation Format: Khawla S. Al-Kuraya, Abdul K. Siraj, Sandeep K. Parvathareddy, Saeeda Ahmed, Allianah Benito, Kaleem Iqbal. PD-L1 protein expression in Middle Eastern breast cancer predicts favorable outcome in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3190.

Abstract 3044: The unexplored immune landscape of high-risk pediatric cancers

Abstract In adult cancer, immune signatures such as the T cell-inflamed gene expression profile (GEP) have been developed to predict which patients are likely to respond to immune checkpoint inhibitors (ICIs) beyond high tumor mutation burden (TMB) and PD-L1 expression. The GEP infers T cell infiltration and activation in the tumor microenvironment (TME) from transcriptomic data. However, it is not known whether tools such as GEP are applicable in pediatric cancer, as the TME in childhood cancers is largely unexplored and response to ICIs are rare. We have undertaken an integrated analysis of the pediatric TME using RNA-sequencing (RNA-seq) and immunohistochemistry (IHC). Our goal is to identify patients with T cell-inflamed or “hot” tumors who may benefit from ICIs. Through Australia's ZERO childhood cancer precision medicine program we performed RNA-seq on 347 high-risk pediatric cancers (estimated &amp;lt;30% chance of survival) and performed IHC for CD4, CD8, CD45 and PD-L1 on 112 matching samples. Using both informatic assessments and IHC as independent measures of immune infiltration, we mapped the immune landscape of the TME across a broad range of high-risk pediatric cancers. As RNA-seq is increasingly used in the analysis of patient tumors, we investigated numerous molecular correlates of immune infiltration, tailored specifically to pediatric patients. RNA-seq was used to generate the GEP and map expression profiles of immune checkpoint genes, and deconvolution algorithms were used to extract the immune cell composition for every tumor. The correlation analysis between IHC, deconvolution of cell mixture composition and GEP were assessed, including PD-L1 protein and mRNA expression. We observed significant correlation between PD-L1 protein and mRNA expression and a weak correlation of CD8+ T cells with GEP. Deconvoluted TME estimates were most tightly correlated with the presence of T cell infiltrates (CD4 and CD8) with IHC. TMB and tumor purity estimates were derived from whole genome sequencing for each case. No correlation was observed between TMB and immune infiltration, however, tumor purity was negatively correlated with immune infiltration. Using IHC as an independent marker of a T cell-inflamed TME, we have identified a novel pediatric immune signature that includes markers of CD4 and CD8 T cells, T cell cytotoxicity, T and NK cell recruitment and activation, MHC Class II molecules and immune checkpoints. This is the first study to comprehensively analyze the pediatric TME in a cohort of this size and diversity, with matching IHC for orthogonal validation. Through the combination of RNA-seq and IHC, we have devised a novel immune signature specific to pediatrics and these techniques have identified a subset of patients that are immune “hot” and may potentially respond to ICIs. Conversely, we also highlight the potential of identifying immune “cold” patients who may need immunomodulatory combination strategies to maximize immune response. Citation Format: Chelsea Mayoh, Rachael L. Terry, Marie Wong, Loretta M. Lau, Dong Anh Khuong-Quang, Marion K. Mateos, Vanessa Tyrrell, Michelle Haber, David S. Ziegler, Mark J. Cowley, Joseph A. Trapani, Paul J. Neeson, Paul G. Ekert. The unexplored immune landscape of high-risk pediatric cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3044.

Abstract CT232: A phase II study of toripalimab plus anlotinib as maintenance therapy in extensive-stage small cell lung cancer

Abstract A phase II study of toripalimab plus anlotinib as maintenance therapy in extensive-stage small cell lung cancer Background: Maintenance therapy is a promising therapeutic approach for extensive-stage small cell lung cancer (ES-SCLC), especially in light of the IMpower 133 trial (Horn NEJM 2018). The results of the E3501, SALUTE, and CALGB 30306 trials showed that in first-line treatment of ES-SCLC, bevacizumab combined with chemotherapy improved only progression-free survival (PFS) but not overall survival (OS). However, in the ALTER 1202 study, anlotinib used as a third- or further-line treatment in patients with ES-SCLC improved PFS and OS (Cheng WCLC 2018). Toripalimab is a novel PD-1 inhibitor that, when combined with anlotinib as maintenance therapy for ES-SCLC, may improve disease control.Methods: This is a phase 2, single-arm study of toripalimab plus anlotinib, a vascular endothelial growth factor receptor inhibitor, as maintenance therapy in adult patients with ES-SCLC following their completion of platinum-based first-line chemotherapy. Eligible patients must have measurable (RECIST v1.1) ES-SCLC, an ECOG performance status of 0 or 1, and are required to have shown complete response, partial response, or stable disease as per RECIST 1.1 following 4–6 cycles of platinum-based chemotherapy. Twenty participants will be enrolled to receive toripalimab (240 mg, IV, Q3W) + anlotinib (12 mg, oral, Q3W). Patients will be treated until the disease progresses, unacceptable toxicity occurs, or for up to 2 years. Prophylactic cranial irradiation will be permitted at the investigator's discretion. The primary endpoints are PFS and OS. Secondary endpoints include safety, objective response rate, disease control rate, and time to response. Exploratory endpoints include Tumor Mutational Burden (TMB) and PD-L1 protein expression, immune gene transcript expression (PD-1, PD-L1, CTLA-4, and others) ,and correlation of these endpoints with clinical responses and drug resistance. This study is currently open and accruing patients. Clinical trial information: NCT04363255. Research Sponsor: Shanghai Junshi Biosciences Co. Citation Format: Dongqing Lv, Guixian Wu, Xiaomai Lin, Ling Lin, Weijia Pan. A phase II study of toripalimab plus anlotinib as maintenance therapy in extensive-stage small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT232.

Abstract 1560: Targeting RNA-binding protein HuR to improve anti-PD-1 immunotherapy response in breast cancer

Abstract Breast cancer has been considered as a less immune-sensitive disease. Patients with metastatic breast cancer have a particularly low objective response rate (ORR) to anti-PD-1/PD-L1 therapy (3%~24%). It will be valuable to improve the treatment efficacy of anti-PD-1 immunotherapy in breast cancer. Recent studies reported that exosomal PD-L1 derived from tumor cells mediated immune surveillance evasion by interacting with the PD-1 receptor on T cells and contributed to the low ORR in anti-PD-1/PD-L1 immunotherapy.The RNA-binding protein Hu antigen R (HuR) is a member of the embryonic lethal abnormal vision (ELAV) family that is overexpressed in a variety of cancers and promotes tumorigenesis by interacting with a subset of oncogenic mRNAs, including PD-L1 mRNA. HuR also plays an important role in the exosome secretion process by regulating RAB27B mRNA. In this study, we aim to identify HuR biological functions in the cancer-immune system and evaluate the combination strategy using a HuR inhibitor with an anti-PD-1 antibody in breast cancer. We show here the direct binding of HuR protein to PD-L1 mRNA in human breast cancer cell line MDA-MB-231 by Ribonucleoprotein immunoprecipitation (RNP-IP). We also show that PD-L1 protein expression level is decreased in HuR knockout clones and in exosomes derived from HuR knockout clones. HuR knockout downregulates exosome secretion with decreased size in MDA-MB-231 cells. KH-3 is a small molecule identified from several chemical libraries that disrupts the binding of HuR and its target mRNAs.To test the combination strategy using a HuR inhibitor and anti-PD-1 antibody in vivo, we established the EMT6 orthotopic mouse breast cancer model. A combination treatment with HuR inhibitor KH-3 and anti-PD-1 antibody inhibits tumor growth and prolongs survival in this breast cancer model. In conclusion, we preliminarily explored the role of HuR in the cancer-immune system, we showed that HuR may be a promising therapeutic target to overcome immune evasion and improve immunotherapy response in breast cancer. Citation Format: Qi Zhang, Xiaoqing Wu, Lan Lan, Lanjing Wei, Liang Xu. Targeting RNA-binding protein HuR to improve anti-PD-1 immunotherapy response in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1560.

Abstract 3192: PD-L1 is an independent prognostic marker in Middle Eastern PTC and its expression is upregulated by BRAFV600E mutation

Abstract Background: PD-L1 inhibition is a promising therapeutic target whose efficacy has been demonstrated in several cancers. This study was conducted to evaluate the prognostic significance of PD-L1 expression in a large cohort of Middle Eastern PTC patients and to explore correlation of PD-L1 and BRAFV600E mutations in PTC tumors and cell lines. Methods: Immunohistochemistry was performed to assess PD-L1 protein expression in PTC. We further conducted in vitro analysis to investigate the role of PD-L1 in regulating BRAFV600E in PTC cell lines. Results: PD-L1 over-expression was noted in 32.4% (473/1458) of cases and significantly associated with adverse clinico-pathological parameters such as tall cell variant, extra-thyroidal extension and lymph node metastasis. Importantly, PD-L1 was found to be an independent poorer prognostic marker. We also found PD-L1 to be significantly associated with BRAF mutation and patients with co-existing PD-L1 over-expression and BRAF mutation had a poor disease-free survival compared to patients with BRAF mutation alone. In vitro analysis showed high expression of PD-L1 in BRAF mutated PTC cell lines compared to BRAF wild-type cell line. Inhibition of BRAF using vemurafenib induced PD-L1 expression in BRAF mutated cell lines without affecting cell growth. Knockdown of PD-L1 in BRAF mutated cell lines significantly decreased the cell growth and induced apoptosis. Conclusion: Our data suggests that PD-L1 might represent a useful prognostic marker in Middle Eastern PTC and PD-L1 inhibition could be a potential therapeutic option for aggressive PTC cancers, such as tall cell variant, BRAF mutation positive patients that are unresponsive to standard treatment. Citation Format: Tariq Masoodi, Abdul K. Siraj, Sandeep Kumar Parvathareddy, Poyil Pratheeshkumar, Sasidharan Padmaja Divya, Rafia Begum, Roxanne Diaz, Thangavel Saravanan, Khadija Al-Obaisi, Sarah Siraj, Saud Azam, Zeeshan Qadri, Khawla S. Al-Kuraya. PD-L1 is an independent prognostic marker in Middle Eastern PTC and its expression is upregulated by BRAFV600E mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3192.

PD-L1 Expression and Gene Amplification in CD30+ Diffuse Large B-Cell Lymphoma (DLBCL): Significance of TP53 Genetic Impact and Clinicopathologic Characteristics

Introduction: Regulation of the PD-1/PD-L1 immune checkpoint has been under intense investigation in the era of immunotherapy, and PD-1 blocκade has shown promising clinical responses in several neoplasms including classical Hodgkin lymphoma (HL). Frequent amplification of the PD-L1 / PD-L2 gene locus (9p24.1) are found in most HLs, however, in other lymphoma types such as diffuse large B-cell lymphomas (DLBCL), genetic alterations of 9p24.1 locus are detected only in a small subset of cases (< 20%). Therefore, other mechanisms including transcriptional regulation of the PD-L1 gene are currently under investigation. Previous studies have shown that in another CD30+ lymphoma type, namely anaplastic large cell lymphoma of T-cell origin, PD-L1 is highly expressed despite the absence of gene amplification or other chromosomal alterations of 9p24.1 locus, and transcriptional regulation is biologically involved. However, the PD-L1 expression levels, the genetic basis of PD-L1 expression and their clinical significance in CD30+ DLBCLs are not yet studied.Methods: From a large database of 2110 DLBCLs, 78 patients with CD30+ DLBCL (44 GCB and 34 ABC-type) diagnosed and treated at the University of Texas M.D. Anderson Cancer Center (USA) and Karolinska University Hospital (Sweden) were identified. All patients were treated with R-CHOP regimen and had available clinical data. PD-L1 expression was assessed by immunohistochemistry using a monoclonal antibody and autostainer systems (Dako or Ventana). PD-L1 gene amplification and chromosomal aberrations of its gene locus were assessed by FISH. Mutations of TP53 gene were confirmed by sequencing. Statistical associations of the PD-L1 expression level (% of positive tumor cells) among various patient subgroups were based on the non-parametrical Mann-Whitney and Kruskall-Wallis tests. Survival analyses were performed using the Kaplan-Meier method (log-rank test) and Cox regression models. The DLBCL in vitro system included 3 cell lines (RC-K8, OCI-Ly3, U2932), which were treated with increasing concentrations of Nutlin 3A to stabilize and activate wild-type TP53. Western blot analysis was used to assess the protein levels of p53 and PD-L1 and quantitative RT-PCR was utilized to assess the PD-L1 mRNA level in the cell lines.Results: Using FISH, PD-L1 gene amplification, chromosomal gains and polyploidy were observed in 6 (7.7%), 3 (3.8%) and 7 (9%) cases, respectively. Expression of PD-L1 protein was found in 66 (85%) CD30+ DLBCLs and the percentage of positive tumor cells ranged from 5 to 100% (median 30%) among the positive cases. In this cohort, TP53 mutations were detected in 14 (18%) CD30+ DLBCL and were not statistically associated with PD-L1 gene amplification or expression levels. However, all PD-L1 negative tumors (n=12) were associated with a wild-type TP53 gene. High expression levels of PD-L1 correlated with ABC phenotype (p=0.04), B symptoms (p=0.03), high serum LDH (p=0.04), ECOG performance status>1 (p=0.015), and the presence of >1 extranodal sites (p=0.04) but not with Ann Arbor stage. Patients who achieved complete remission showed significantly lower PD-L1 expression level (median 20% vs. 40%, p=0.03). Given the significant survival benefit of CD30 expression in DLBCL, PD-L1 expression in this subset abolished advantage of failure-free (FFS) and overall (OS) survival after 48 months of median follow-up. The in vitro data showed that stabilization and activation of p53 in DLBCL cell lines carrying a wild-type p53 gene led to concentration-dependent increase of the PD-L1 mRNA and protein levels. As in silico analysis did not demonstrate a possible TP53 binding site on the PD-L1 gene promoter, an indirect mechanism of p53-dependent regulation of PD-L1 is suggested.Conclusions: PD-L1 is expressed in most CD30+ DLBCLs, but amplification of the PD-L1 gene locus is not a common mechanism unlike Hodgkin lymphoma. Activation of wild-type TP53 indirectly upregulates PD-L1 in DLBCL and the underlying mechanisms merit further investigation. DisclosuresSmedby:Janssen: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees.

PD-L1 Is Regulated By Interferon Gamma and Interleukin 6 through STAT1 and STAT3 Signaling in Cutaneous T-Cell Lymphoma

Background: Signaling interactions within the cutaneous T cell lymphoma (CTCL) microenvironment often create a permissive environment for CTCL growth. In response to oncogenic signals from proliferating tumor cells, the tumor microenvironment changes throughout disease progression. Studies have shown that dendritic cells and macrophages are critical tumorigenic regulators of the microenvironment in hematologic malignancies. Tumor cells escape immune surveillance by manipulating immune checkpoint receptors like PD1, and activation of the PD1 receptor by PD-L1 transduces a signal that leads to the inhibition of T-cell functions. Tumor cells, resting dendritic cells, and macrophages in the tumor microenvironment express PD-L1; and proinflammatory cytokines like IFN-γ and IL 6 upregulate PD-L1 on tumor cells. However, the mechanisms of PD-L1 upregulation in CTCL remain unknown.Methods: Immunoblots: Primary patient peripheral blood mononuclear cells (PBMCs), CTCL cell lines (Mac1 and Mac2a) and mature monocyte-derived dendritic cells (MoDCs) from healthy patients were treated with IFNγ, IL-6, a a pan JAK/STAT inhibitor (tofacitinib), and a PD-L1 inhibitor for 72 hrs. Cell lysates were collected and subjected to immunoblot analysis with antibodies against PD-L1, pSTAT1, and pSTAT3. Immunohistochemistry: Paraffin sections of patients' skin biopsies were incubated with mAbs against CD163 PD-L1, and pSTAT3 at 4°C overnight. Sections were incubated with appropriate secondary Abs for 2 hrs at room temperature. The images were taken with a Zeiss 700 confocal microscope. Flow Cytometry: Migrated skin cells from skin explant cultures taken from CTCL patients were incubated with fluorochrome-conjugated mAbs and analyzed on an LSR Fortessa (BD) flow cytometer. The gating parameters were as follows: epidermal LCs (CD1a+, CD14neg, HLA-DRbright), dermal CD1a+ DCs (CD1a+, CD14neg, HLA-DRbright, CD11b+, CD11c+), and dermal CD14+ DCs (CD1aneg, CD14+, HLA-DRbright, CD11b+, CD11c+). Gates were set for collection and analysis of at least 10,000 live events, and the data were analyzed with FlowJo 9.7.6 software (TreeStar).Results: Our idata indicate that T cells in CTCL have an exhausted phenotype, presumably due to chronic Ag stimulation by cutaneous DCs. Macrophages and dendritic cell subsets in lesional skin from mycosis fungoides patients as well as PBMCs from Sézary syndrome (SS) patients express PD-L1. To elucidate the mechanism of PD-L1 regulation, we treated PBMCs from SS patients and CTCL cell lines with IFN-γ, IL-6, a pan JAK/STAT inhibitor (tofacitinib), and a PD-L1 inhibitor (durvalumab). We found upregulation of PD-L1 protein expression by PBMCs when treated with IFN-γ and IL-6, through upregulated STAT1 and STAT3 signaling detected by Western blots. A pan JAK/STAT inhibitor abrogated the increased expression of PD-L1. Monocyte-derived dendritic cells from healthy donors exhibit upregulated PD-L1 expression by IFN-γ, but not with IL-6. Durvalumab treatment reduced pSTAT3 and PD-L1 expression. We have therefore initiated a clinical trial with a PD-L1 inhibitor (durvalumab) in patients with mycosis fungoides (MF)/Sézary syndrome (SS) and observed promising initial responses.Conclusions: PBMCs from Sezary syndrome patients and moDCs from healthy donor upregulate PD-L1 through STAT signaling initiated by proinflammatory cytokines IFN-γ and IL-6. Durvalumab also reduced PD-L1 and pSTAT3 expression in patient PBMCs. PD-L1 expression may result from the proinflammatory background of the tumor microenvironment and activation of the PD1 receptor by PD-L1 transduces a signal that leads to the inhibition of T-cell functions in CTCL. DisclosuresKadin:Allergan, Inc: Honoraria. Querfeld:Soligenix: Research Funding; Actelion: Honoraria, Research Funding; Medivir: Honoraria; Mallinckrodt: Honoraria; Kyowa: Research Funding; Elorac: Research Funding; Celgene: Honoraria, Research Funding; MiRagen: Honoraria, Research Funding; Trillium Therapeutics: Research Funding; City of Hope: Employment; Mindera: Consultancy.

Expression of Major Histocompatibility Complex (MHC) Class II, but Not MHC Class I, Predicts Outcome in Patients with Classical Hodgkin Lymphoma (cHL) Treated with Nivolumab (Programmed Death-1 [PD-1] Blockade)

Expression of Major Histocompatibility Complex (MHC) Class II, but Not MHC Class I, Predicts Outcome in Patients with Classical Hodgkin Lymphoma (cHL) Treated with Nivolumab (Programmed Death-1 [PD-1] Blockade)

Knockdown of CDK5 down-regulates PD-L1 via the ubiquitination-proteasome pathway and improves antitumor immunity in lung adenocarcinoma

Although immunotherapy (anti-PD-1/PD-L1 antibodies) has been approved for clinical treatment of lung cancer, only a small proportion of patients respond to monotherapy. Hence, understanding the regulatory mechanism of PD-L1 is particularly important to identify optimal combinations. In this study, we found that inhibition of CDK5 induced by shRNA or CDK5 inhibitor leads to reduced expression of PD-L1 protein in human lung adenocarcinoma cells, while the mRNA level is not substantially altered. The PD-L1 protein degradation is mediated by E3 ligase TRIM21 via ubiquitination-proteasome pathway. Subsequently, we studied the function of CDK5/PD-L1 axis in LUAD. In vitro, the absence of CDK5 in mouse Lewis lung cancer cell (LLC) has no effect on cell proliferation. However, the attenuation of CDK5 or combined with anti-PD-L1 greatly suppresses tumor growth in LLC implanted mouse models in vivo. Disruption of CDK5 elicits a higher level of CD3+, CD4+ and CD8+ T cells in spleens and lower PD-1 expression in CD4+ and CD8+ T cells. Our findings highlight a role for CDK5 in promoting antitumor immunity, which provide a potential therapeutic target for combined immunotherapy in LUAD.

Genetic abnormality and protein expression of C-MYC and PD-L1 in ALK-negative anaplastic large cell lymphoma

Objective: To investigate the genetic abnormality and protein expression of C-MYC and PD-L1 in the patients with ALK-negative anaplastic large cell lymphoma (ALK－ALCL), and to explore their roles in the pathogenesis of ALK－ALCL and their relationship with clinicopathological characteristics. Methods: Thirty-seven cases of ALK－ALCL diagnosed at Fujian Provincial Hospital from January 2003 to January 2017 were selected. Fluorescence in situ hybridization (FISH) was used to detect the genetic abnormality of C-MYC and PD-L1. The expression of C-MYC and PD-L1 proteins was detected by immunohistochemistry. The relationship between C-MYC and PD-L1 genes' abnormalities and protein expression was analyzed, as well as their associations with various clinicopathological parameters. Results: Among the 37 ALK－ALCL patients, 17 (45.9%) were positive for C-MYC protein, and 14 (37.8%) were positive for PD-L1 protein. There was a significant correlation between C-MYC protein and PD-L1 protein (r=0.990,P=0.014). The protein expression of C-MYC and PD-L1 (versus negative) was associated with the clinical stage of ALK－ALCL, respectively. The international prognosis index (IPI) in high-risk group was higher than that in the low-risk group (P<0.05). FISH test showed that 9 (24.3%) of the 37 cases had amplification of C-MYC gene, and no translocation of C-MYC gene was found in any of the cases. Amplification of PD-L1 gene was found in only 2 cases (5.4%). The 3-year overall survival rate of the C-MYC or PD-L1 immunohistochemistry-positive cases was significantly lower than those of the C-MYC or PD-L1 negative cases (P<0.01 and P<0.05), respectively. Conclusion: The expression of C-MYC and PD-L1 proteins are related to the clinical stage, IPI and overall survival rate of ALK－ALCL. Thus, it can be used to assess the disease's aggressiveness and to predict the prognosis of ALK－ALCL. The expression of PD-L1 in ALK－ALCL may be regulated by C-MYC, thus suggesting a possible design of combined C-MYC targeted therapy and immune checkpoint blocking for some ALK－ALCL patients.