PD-L1 Expression In Non-small Cell Lung Cancer Research Articles

Nonlinear association between PD-L1 expression levels and the risk of postoperative recurrence in non-small cell lung cancer

Accurate prediction of postoperative recurrence is important for optimizing the treatment strategies for non-small cell lung cancer (NSCLC). Previous studies identified the PD-L1 expression in NSCLC as a risk factor for postoperative recurrence. This study aimed to examine the contribution of PD-L1 expression to predicting postoperative recurrence using machine learning. The clinical data of 647 patients with NSCLC who underwent surgical resection were collected and stratified into training (80%), validation (10%), and testing (10%) datasets. Machine learning models were trained on the training data using clinical parameters including PD-L1 expression. The top-performing model was assessed on the test data using the SHAP analysis and partial dependence plots to quantify the contribution of the PD-L1 expression. Multivariate Cox proportional hazards model was used to validate the association between PD-L1 expression and postoperative recurrence. The random forest model demonstrated the highest predictive performance with the SHAP analysis, highlighting PD-L1 expression as an important feature, and the multivariate Cox analysis indicated a significant increase in the risk of postoperative recurrence with each increment in PD-L1 expression. These findings suggest that variations in PD-L1 expression may provide valuable information for clinical decision-making regarding lung cancer treatment strategies.

DRD1 suppresses cell proliferation and reduces EGFR activation and PD-L1 expression in NSCLC.

Dopamine (DA) acts in various key neurological and physiological processes as both a neurotransmitter and circulating hormone. Over the past several decades, the DA signaling network has been shown to regulate the progression of several types of solid tumors, and considerable evidence has shown it is a druggable pathway in the cancer cell context. However, the specific activity and effect of these pathway components appears to be tissue-type and cell-context-dependent. In the present study, expression and methylation of dopamine receptor D1 (DRD1) were measured using RNA sequencing (RNAseq) and reverse transcription polymerase chain reaction (RT-PCR) in non-small cell lung cancer (NSCLC) samples, and validated using publicly available datasets, including The Cancer Genome Atlas (TCGA). In vitro and in vivo functional experiments were performed for cell proliferation and tumor growth, respectively. Mechanistic analyses of the transcriptome and kinome in DRD1-modulated cells informed further experiments, which characterized the effects on the epidermal growth factor receptor (EGFR) pathway and programmed cell death 1 ligand 1 (PD-L1) proteins. Through these experiments, we identified the DRD1 gene as a negative regulator of disease progression in NSCLC. We show that DRD1, as well as other DA pathway components, are expressed in normal human lung tissue, and that loss of DRD1 expression through promoter hypermethylation is a common feature in NSCLC patients and is associated with worse survival. At the cellular level, DRD1 affects proliferation by inhibiting the activation of EGFR and mitogen-activated protein kinase 1/2 (ERK1/2). Interestingly, we also found that DRD1 regulates the expression of PD-L1 in lung cancer cells. Taken together, these results suggest that DRD1 methylation may constitute a biomarker of poor prognosis in NSCLC patients while other components of this pathway could be targeted to improve response to EGFR- and PD-L1-targeted therapies.

Revisiting the impact of clinicopathologic characteristics in PD-L1 profile in a large cohort of non-small cell lung cancer.

Immunotherapies using anti-programmed cell death ligand-1 (PD-L1) agents have recently shown remarkable outcomes in patients with non-small cell lung cancer (NSCLC). However, there was a poor correlation between PD-L1 expression and treatment response. Many researchers have focused on the clinicopathological factors associated with prognosis, but the results are conflicting. In the present study, we investigated the clinicopathological significance of PD-L1 overexpression in NSCLC cells. In total, 344 NSCLC cases with PD-L1 assays were retrospectively analyzed. PD-L1 expression was evaluated via immunohistochemical staining using antibodies against SP263 and SP142. The correlation between clinicopathological factors and PD-L1 expression was analyzed for various clinicopathological features. PD-L1 expression significantly correlated with several poor clinicopathological factors, including the solid component of adenocarcinoma, lymphatic invasion, and recurrence. Squamous cell carcinoma, older age, and male sex were also associated with PD-L1 expression. However, we could not observe correlation between PD-L1 expression and disease-free survival (DFS). A novel finding was that lower metastasis was associated with high PD-L1 expression of SP142 in tumor-infiltrating immune cells (ICs). PD-L1 expression in NSCLC is associated with adverse clinicopathological features and recurrence; therefore, it could be utilized to predict poor prognosis. Furthermore, the high PD-L1 expression of SP142 in tumor-infiltrating ICs could be a potential marker for low metastasis.

KEAP1 promotes anti-tumor immunity by inhibiting PD-L1 expression in NSCLC

Immunotherapy has become a prominent first-line cancer treatment strategy. In non-small cell lung cancer (NSCLC), the expression of PD-L1 induces an immuno-suppressive effect to protect cancer cells from immune elimination, which designates PD-L1 as an important target for immunotherapy. However, little is known about the regulation mechanism and the function of PD-L1 in lung cancer. In this study, we have discovered that KEAP1 serves as an E3 ligase to promote PD-L1 ubiquitination and degradation. We found that overexpression of KEAP1 suppressed tumor growth and promoted cytotoxic T-cell activation in vivo. These results indicate the important role of KEAP1 in anti-cancer immunity. Moreover, the combination of elevated KEAP1 expression with anti-PD-L1 immunotherapy resulted in a synergistic effect on both tumor growth and cytotoxic T-cell activation. Additionally, we found that the expressions of KEAP1 and PD-L1 were associated with NSCLC prognosis. In summary, our findings shed light on the mechanism of PD-L1 degradation and how NSCLC immune escape through KEAP1-PD-L1 signaling. Our results also suggest that KEAP1 agonist might be a potential clinical drug to boost anti-tumor immunity and improve immunotherapies in NSCLC.

Deep learning radiomics model based on PET/CT predicts PD-L1 expression in non-small cell lung cancer

PurposeProgrammed cell death protein-1 ligand (PD-L1) is an important prognostic predictor for immunotherapy of non-small cell lung cancer (NSCLC). This study aimed to develop a non-invasive deep learning and radiomics model based on positron emission tomography and computed tomography (PET/CT) to predict PD-L1 expression in NSCLC. MethodsA total of 136 patients with NSCLC between January 2021 and September 2022 were enrolled in this study. The patients were randomly divided into the training dataset and the validation dataset in a ratio of 7:3. Radiomics feature and deep learning feature were extracted from their PET/CT images. The Mann-whitney U-test, Least Absolute Shrinkage and Selection Operator algorithm and Spearman correlation analysis were used to select the top significant features. Then we developed a radiomics model, a deep learning model, and a fusion model based on the selected features. The performance of three models were compared by the area under the curve (AUC), sensitivity, specificity, accuracy, positive predictive value, and negative predictive value. ResultsOf the patients, 42 patients were PD-L1 negative and 94 patients were PD-L1 positive. A total of 2446 radiomics features and 4096 deep learning features were extracted per patient. In the training dataset, the fusion model achieved a highest AUC (0.954, 95% confident internal [CI]: 0.890–0.986) compared with the radiomics model (0.829, 95%CI: 0.738–0.898) and the deep learning model (0.935, 95%CI: 0.865–0.975). In the validation dataset, the AUC of the fusion model (0.910, 95% CI: 0.779–0.977) was also higher than that of the radiomics model (0.785, 95% CI: 0.628–0.897) and the deep learning model (0.867, 95% CI: 0.724–0.952). ConclusionThe PET/CT-based deep learning radiomics model can predict the PD-L1 expression accurately in NSCLC patients, and provides a non-invasive tool for clinicians to select positive PD-L1 patients.

Role of sex and sex hormones in PD-L1 expression in NSCLC: clinical and therapeutic implications.

Currently, immunotherapy based on PD-1/PD-L1 pathway blockade has improved survival of non-small cell lung cancer (NSCLC) patients. However, differential responses have been observed by sex, where men appear to respond better than women. Additionally, adverse effects of immunotherapy are mainly observed in women. Studies in some types of hormone-dependent cancer have revealed a role of sex hormones in anti-tumor response, tumor microenvironment and immune evasion. Estrogens mainly promote immune tolerance regulating T-cell function and modifying tumor microenvironment, while androgens attenuate anti-tumor immune responses. The precise mechanism by which sex and sex hormones may modulate immune response to tumor, modify PD-L1 expression in cancer cells and promote immune escape in NSCLC is still unclear, but current data show how sexual differences affect immune therapy response and prognosis. This review provides update information regarding anti-PD-1/PD-L immunotherapeutic efficacy in NSCLC by sex, analyzing potential roles for sex hormones on PD-L1 expression, and discussing a plausible of sex and sex hormones as predictive response factors to immunotherapy.

Comedication with corticosteroids and nonsteroidal antiphlogistics does not affect PD-L1 expression in non-small cell lung cancer.

BackgroundProgrammed death-ligand 1 (PD-L1) expression is a standard predictor in the selection of immunotherapy for locally advanced/advanced non-small cell lung cancer (NSCLC). However, comedication with corticosteroids or non-steroidal anti-inflammatory drugs (NSAIDs) may influence the effectiveness of this treatment as documented in several previous studies. Due to certain molecular linkages between PD-L1 and corticosteroids or NSAIDs, we therefore addressed the question of whether there is a relationship between PD-L1 expression in NSCLC and the use of this comedication.MethodsThis is a retrospective study using the Czech tumor registry LUng CAncer focuS (LUCAS), from which patient data were drawn. Independence of two categorical parameters was tested by Pearson’s chi-square test.ResultsIn our group of 1,148 patients, we observed no significant relationship between PD-L1 expression and the use of corticosteroids or NSAIDs.ConclusionsAccording to our data, treatment with corticosteroids or NSAIDs during biopsy does not affect the expression of PD-L1 and it is therefore not necessary to take this treatment into account in this regard.

EP11.02-002 A Comparative Study of PD-L1 Scoring: Humans versus AI

Immunotherapy with PD-1/PD-L1 inhibitors has revolutionized the treatment of non-small cell lung cancer (NSCLC). In clinical practice, PD-L1 expression by immunohistochemistry has been implemented as a companion or complementary diagnostic for anti-PD-1/PD-L1 agents. A widely adopted measure of PD-L1 expression in NSCLC is the tumor proportion score (TPS), defined as the ratio of positive membranous stained tumor cells and the total number of viable tumor cells. Several automatic image analysis algorithms for determining the TPS on the whole slide images (WSI) of NSCLC have been developed recently.

Andrographolide suppresses non-small-cell lung cancer progression through induction of autophagy and antitumor immune response

Despite recent advances in diagnosis and therapeutic strategies, treatment of non-small-cell lung cancer (NSCLC) remains unsatisfactory in terms of prognosis. Andrographolide (AD), a principal active component of Andrographis paniculata (Burm.f.) Nees, exerts anti-cancer therapeutic properties. AD has been used for centuries in China for clinical treatment of viral infections. However, the pharmacological biology of AD in NSCLC remains unknown. In this study, AD regulated autophagy and PD-L1 expression in NSCLC. Molecular dynamics simulations indicated that AD bound directly to signal transducer and activator of transcription-3 (STAT3) with high affinity. Proteomics analysis indicated that AD reduced the expression of tumour PD-L1 in NSCLC by suppressing JAK2/STAT3 signalling. AD modulated the P62-dependent selective autophagic degradation of PD-L1 by inhibiting STAT3 phosphorylation. In vivo study revealed that AD suppressed tumour growth in H1975 xenograft mice and Lewis lung carcinoma cell models, and better efficacy was obtained at higher concentrations. AD prolonged the survival time of the mice and enhanced the treatment efficacy of anti-PD-1 mAb immunotherapy by stimulating CD8+ T cell infiltration and function. This work elucidated the specific mechanism by which AD inhibited NSCLC. Treatment with the combination of AD and anti-PD-1 mAb immunotherapy could be a potential strategy for patients with NSCLC.

CTLA-4 Expression in Tumor-infiltrating Lymphocytes Is Irrelevant to PD-L1 Expression in NSCLC.

Treatments containing ipilimumab have shown a good outcome in patients with non-small cell lung cancer (NSCLC) regardless of the PD-L1 tumor proportion score (TPS). However, the association between PD-L1 TPS and the expression of CTLA-4 in tumor-infiltrating lymphocytes is unknown. Fifty-five NSCLC patients who underwent surgery in our hospital were included in this study. We measured the proportions of CTLA-4+ regulatory T cells, and CTLA-4+ CD8 T cells, and statistically analyzed their correlations with the PD-L1 TPS. Statistical correlations were found neither between the proportion of CTLA-4+ regulatory T cells to CD8 T cells and the PD-L1 TPS (p=0.2859) nor between the proportion of CTLA-4+ cells in CD8 T cells and the PD-L1 TPS (p=0.1919). The proportions of CTLA-4+ regulatory T cells to CD8 T cells and CTLA-4+ cells in CD8 T cells were irrelevant to the PD-L1 TPS in NSCLC patients.

Retraction Note to: Inter- and intraobserver agreement of the quantitative assessment of [99mTc]-labelled anti-programmed death-ligand 1 (PD-L1) SPECT/CT in non-small cell lung cancer

Checkpoint inhibition therapy using monoclonal antibodies against programmed cell death protein 1 (PD-1) or its ligand (PD-L1) is now standard management of non-small cell lung cancer (NSCLC). PD-L1 expression is a validated and approved prognostic and predictive biomarker for anti-PD-1/PD-L1 therapy. Technetium-99 m [99mTc]-labelled anti-PD-L1 single-domain antibody (NM-01) SPECT/CT quantification correlates with PD-L1 expression in NSCLC, presenting an opportunity for non-invasive assessment. The aim of this study was to determine the inter- and intraobserver agreement of the quantitative assessment of [99mTc]NM-01 SPECT/CT in NSCLC. [99mTc]NM-01 SPECT/CT studies of 21 consecutive NSCLC participants imaged for the evaluation of PD-L1 expression were analysed. Three independent observers measured maximum counts in a tumour region of interest (ROImax) of primary lung, metastatic lesions and normal tissue references of both 1 and 2 h post-injection (n = 42) anonymised studies using a manual technique. Intraclass correlation coefficients (ICC) were calculated, and Bland–Altman plot analysis was performed to determine inter- and intraobserver agreement. Intraclass correlation of primary lung tumour-to-blood pool (T:BP; ICC 0.83, 95% CI 0.73–0.90) and lymph node metastasis-to-blood pool (LN:BP; ICC 0.87, 0.81–0.92) measures of [99mTc]NM-01 uptake was good to excellent between observers. Freehand ROImax of T (ICC 0.94), LN (ICC 0.97), liver (ICC 0.97) and BP (ICC 0.90) reference tissues also demonstrated excellent interobserver agreement. ROImax scoring of healthy lung demonstrated moderate to excellent interobserver agreement (ICC 0.84) and improved when measured consistently at the level of the aortic arch (ICC 0.89). Manual ROImax re-scoring of T, LN, T:BP and LN:BP using [99mTc]NM-01 SPECT/CT following a 42-day interval was consistent with excellent intraobserver agreement (ICC range 0.95–0.97). Good to excellent inter- and intraobserver agreement of the quantitative assessment of [99mTc]NM-01 SPECT/CT in NSCLC was demonstrated in this study, including T:BP which has been shown to correlate with PD-L1 status. [99mTc]NM-01 SPECT/CT has the potential to reliably and non-invasively assess PD-L1 expression. ClinicalTrials.gov identifier no. NCT02978196. Registered 30th November 2016.

EGFR mutation status in non-small cell lung cancer receiving PD-1/PD-L1 inhibitors and its correlation with PD-L1 expression: a meta-analysis.

Meta-analysis was performed on the Web of Science, PubMed, Embase, and Cochrane databases to evaluate the effect of epidermal growth factor receptor (EGFR) mutation status on programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) immune checkpoint inhibitors, and the association between EGFR mutation status and PD-L1 expression in non-small cell lung cancer (NSCLC) patients. Pooled effect (hazard ratio/odds ratio, HR/OR) with 95% confidence interval (CI) was calculated, and the source of heterogeneity was explored by subgroup analysis and meta-regression using Stata/SE 15.0. Meta-analysis of the association between EGFR mutation status and overall survival (OS) in NSCLC with immunotherapy was calculated from four randomized controlled trials. We found that immune checkpoint inhibitors significantly prolonged OS over docetaxel overall (HR 0.71, 95% CI 0.64-0.79) and in the EGFR wild type (HR = 0.67, 95% CI = 0.60-0.75), but not in the EGFR mutant subgroup (HR = 1.11, 95% CI = 0.80-1.52). Meta-analysis of the association between EGFR mutation status and PD-L1 expression in NSCLC included 32 studies. The pooled OR and 95% CI were 0.60 (0.46-0.80), calculated by random effects model. No source of heterogeneity was found in subgroup analysis. Sensitivity analysis was carried out with a fixed model, and the influence of a single study on the pooled results showed no significant change with robust meta-analysis methods. Harbord's weighted linear regression test (P = 0.956) and Peters regression test (P = 0.489) indicated no significant publication bias. The limited benefit of single-agent PD-1/PD-L1 inhibitors in the second-line or later setting for EGFR-mutated NSCLC may be partly due to the lower expression of PD-L1.

Association Between EGFR Mutation Status and Immune Checkpoint Inhibitors and PD-L1 Expression in Non-Small-Cell Lung Cancer: A Meta-Analysis

Background: A systematic search was performed on the Web of Science, PubMed, Embase, and Cochrane databases. The meta-analysis was performed to evaluate the effect of epidermal growth factor receptor (EGFR) mutation status on programmed cell death protein 1/programmed death ligand 1 (PD-1/PD-L1) immune checkpoint inhibitors, and the association between EGFR mutation status and PD-L1 expression in non-small cell lung cancer (NSCLC) patients. Methods: Pooled effect (HR/OR) with 95% CI was calculated, the source of heterogeneity was explored by subgroup analysis and meta-regression using Stata/SE 15.0. Meta-analysis of the association between EGFR mutation status and overall survival in NSCLC with immunotherapy was calculated from four randomized controlled trials. Results: We found that immune checkpoint inhibitors significantly prolonged overall survival (OS) over docetaxel overall (HR: 0.71, 95% CI: 0.64-0.79) and in the EGFR wild-type (HR=0.67, 95% CI=0.60-0.75), but not in the EGFR-mutant subgroup (HR=1.11, 95% CI=0.80-1.52). Meta-analysis of the association between EGFR mutation status and PD-L1 expression in NSCLC included 32 studies. The pooled OR and 95% CI were 0.60 (0.46 0.80), calculated by random effects model. No source of heterogeneity was found in subgroup analysis. The sensitivity analysis was carried out with a fixed model, and the influence of a single study on the pooled results showed no significant change with robust meta-analysis methods. Harbord s weighted linear regression test (P=0.956) and Peters regression test (P=0.489) indicated no significant publication bias. Conclusions: The benefit of PD-1/PD-L1 immune checkpoint inhibitor treatment in patients with EGFR-mutated NSCLC is not obvious and may due to the lower expression of PD-L1. Funding: This study was supported by Fujian Program for Outstanding Young Researchers in University awarded by Education Department of Fujian (Grant Number 2017B019), Fujian Provincial Health Research Talents Training Programme Medical Innovation Project (Grant Number 2019- CX-33), Joint Funds for the innovation of science and Technology，Fujian province (Grant Number 2019Y9022), and Fujian Provincial Health Research Talents Training Programme Youth Research Project (Grant Number 2019-1- 58).The funding sponsors played no role in study design, data collection, data analysis, interpretation, writing of the report, and the decision to submit the paper for publication. Declaration of Interest: None to declare. Ethical Approval: This study was approved by the Institutional Review Board of Fujian Medical University (Fuzhou, China).

RETRACTED ARTICLE: Inter- and intraobserver agreement of the quantitative assessment of [99mTc]-labelled anti-programmed death-ligand 1 (PD-L1) SPECT/CT in non-small cell lung cancer

PurposeCheckpoint inhibition therapy using monoclonal antibodies against programmed cell death protein 1 (PD-1) or its ligand (PD-L1) is now standard management of non-small cell lung cancer (NSCLC). PD-L1 expression is a validated and approved prognostic and predictive biomarker for anti-PD-1/PD-L1 therapy. Technetium-99 m [99mTc]-labelled anti-PD-L1 single-domain antibody (NM-01) SPECT/CT quantification correlates with PD-L1 expression in NSCLC, presenting an opportunity for non-invasive assessment. The aim of this study was to determine the inter- and intraobserver agreement of the quantitative assessment of [99mTc]NM-01 SPECT/CT in NSCLC.Methods[99mTc]NM-01 SPECT/CT studies of 21 consecutive NSCLC participants imaged for the evaluation of PD-L1 expression were analysed. Three independent observers measured maximum counts in a tumour region of interest (ROImax) of primary lung, metastatic lesions and normal tissue references of both 1 and 2 h post-injection (n = 42) anonymised studies using a manual technique. Intraclass correlation coefficients (ICC) were calculated, and Bland–Altman plot analysis was performed to determine inter- and intraobserver agreement.ResultsIntraclass correlation of primary lung tumour-to-blood pool (T:BP; ICC 0.83, 95% CI 0.73–0.90) and lymph node metastasis-to-blood pool (LN:BP; ICC 0.87, 0.81–0.92) measures of [99mTc]NM-01 uptake was good to excellent between observers. Freehand ROImax of T (ICC 0.94), LN (ICC 0.97), liver (ICC 0.97) and BP (ICC 0.90) reference tissues also demonstrated excellent interobserver agreement. ROImax scoring of healthy lung demonstrated moderate to excellent interobserver agreement (ICC 0.84) and improved when measured consistently at the level of the aortic arch (ICC 0.89). Manual ROImax re-scoring of T, LN, T:BP and LN:BP using [99mTc]NM-01 SPECT/CT following a 42-day interval was consistent with excellent intraobserver agreement (ICC range 0.95–0.97).ConclusionGood to excellent inter- and intraobserver agreement of the quantitative assessment of [99mTc]NM-01 SPECT/CT in NSCLC was demonstrated in this study, including T:BP which has been shown to correlate with PD-L1 status. [99mTc]NM-01 SPECT/CT has the potential to reliably and non-invasively assess PD-L1 expression.Clinical trial registrationClinicalTrials.gov identifier no. NCT02978196. Registered 30th November 2016.

Abstract 4454: TUSC2 immunogene therapy enhances efficacy of immunotherapy and targeted drugs in human non-small cell lung cancer (NSCLC) in humanized mouse models

Abstract TUSC2 is a tumor suppressor gene, whose expression is reduced in almost all NSCLC. Systemic nanovesicle delivery of TUSC2 inhibits cancer cell growth through inhibition of a broad spectrum of kinases and mTOR downregulation as well as stimulation of the immune system through innate activation. We previously reported that TUSC2 downregulates PD-L1 expression in NSCLC and synergizes with anti-PD1 in inhibiting tumor growth in Kras mutant syngeneic mouse models through upregulation of NK and cytotoxic T cells. We developed an improved CD34-derived humanized mouse model (Hu-mice), with faster and higher human immune reconstitution than other available humanized mice, to evaluate immune responses in lung cancer. In this study, we tested whether TUSC2 immunogene therapy would enhance response to standard checkpoint blockade immunotherapy, chemotherapy and targeted therapies in humanized NSG mice implanted with highly metastatic Krasmt/LKB1− A549 cells. A significantly increased antitumor effect was found when TUSC2 was combined with pembrolizumab. Pembrolizumab alone reduced tumor burden as compared with an untreated control, whereas no antitumor effect was observed in non-Hu-mice implanted with A549 cells. The observed antitumor effect correlated with increased levels of CD8+ T and CD8+CD69+ active T, and decreased levels of MDSC and regulatory T cells in the combination group. A significantly higher percentages of CD56+ NK and CD56+CD69+ active NK cells were found in the TUSC2 alone and combination groups indicating TUSC2 related NK activation. Next, we tested whether TUSC2 enhances efficacy to carboplatin+pembrolizumab. The level of antitumor effect of carboplatin+pembrolizumab was similar to that of TUSC2 alone. However, when TUSC2 was combined with carboplatin+pembrolizumab, metastases regression was significantly greater than either TUSC2 alone or carboplatin+pembrolizumab treatments. Significantly fewer or no visible tumor nodules were found in dissected lungs in the TUSC2 combination as compared with other groups. Immune analysis of the triple combination in CMT167 syngeneic mice showed increased infiltration of CD3+ T, CD8+ T, NK cells and significantly less Treg cells into tumor, which was associated with significant tumor inhibition by the treatments. A higher percentage of CD3+CD44+ and CD8+CD44+ memory T cells were found in tumors after carbo+aPD1+TUSC2 treatment, as compared with either Carbo+aPD1 or control groups. The antitumor activity of Carbo+aPD1+TUSC2 was further enhanced when MEKi (Trametinib) was added. Moreover, we also combined TUSC2 with the anti-angiogenic agent, bevacizumab (anti-VEGF) to enhance efficacy in the highly angiogenic 786-O renal cell carcinoma. Synergistic antitumor activity was found with the combination, which was significantly stronger than either single agent. In conclusion, the addition of TUSC2 immunogene therapy with checkpoint blockade, chemotherapy, and targeted therapies showed enhanced antitumor efficacy. Citation Format: Ismail M. Meraz, Mourad Majidi, Meng Feng, RuPing Shao, Min Jin Ha, Elizabeth J. Shpall, Jack A. Roth. TUSC2 immunogene therapy enhances efficacy of immunotherapy and targeted drugs in human non-small cell lung cancer (NSCLC) in humanized mouse models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4454.

Relationship between SP142 PD-L1 Expression and 18F-FDG Uptake in Non-Small-Cell Lung Cancer.

Objectives Immune checkpoint blockers constitute the first-line treatment for advanced non-small-cell lung cancer (NSCLC) with ≥50% PD-L1 expression. In NSCLC, PD-L1 positivity is correlated with high 18F-fluorodeoxyglucose (18F-FDG) uptake. However, these studies only included patients undergoing surgical resection, almost all in their early stages. Moreover, differences in 18F-FDG uptake between NSCLC with high (≥50%) and low (49%) PD-L1 expression remain unknown. We aimed to investigate the association between metabolic parameter 18F-FDG uptake and PD-L1 expression status in NSCLC patients. Methods From February 2017 to June 2018, 428 consecutive NSCLC patients who underwent 18F-FDG positron emission tomography/computed tomography (PET/CT) and SP142 PD-L1 expression analysis were retrospectively assessed. The association between clinicopathological characteristics and PD-L1 expression was examined. Results The frequency of PD-L1-positive tumors was 38.1% (163/428), 28.5% (91/319), and 64.2% (61/95) for NSCLC, adenocarcinoma (ADC), and squamous cell carcinoma (SCC), respectively. Maximal standard uptake (SUVmax) was significantly higher in PD-L1-positive than in PD-L1-negative NSCLC (p < 0.0001), ADC (p < 0.0001), and SCC (p=0.006). SUVmax was significantly higher in NSCLC (p=0.001) and ADC (p=0.003) with high rather than low PD-L1 expression. The receiver operating characteristic curve yielded area under the curve values of 0.726 (95% CI, 0.679–0.774, p < 0.0001), 0.694 (95% CI, 0.634–0.755, p < 0.0001), and 0.625 (95% CI, 0.513–0.738, p=0.044) for NSCLC, ADC, and SCC, respectively. Conclusion 18F-FDG tumor uptake is strongly, positively correlated with PD-L1 expression in NSCLC and significantly differs between high and low PD-L1-expressing individuals.

Abstract B57: MUC3A induces PD-L1 and reduces tyrosine kinase inhibitor effects functions in EGFR-mutant non-small cell lung cancer

Abstract Objective: Non-small cell lung cancer (NSCLC) patients with specific EGFR mutations benefited from the emergence of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Unfortunately, almost all cases eventually recrudesced after a median of 10 months. Therapeutic antibodies blocking PD-L1 strikingly regressed NSCLC development. However, only 20% patients responded to the αPD-L1 therapy. EGFR activation was reported to induce PD-L1 expression in NSCLC. MUC3A is a transmembrane mucin with 2 EGF-like domains in the extracellular segment. This study aimed to investigate the effects of MUC3A on PD-L1 expression and EGFR-TKIs sensitivity in EFGR-mutant NSCLC. Methods: Expression levels of MUC3A and PD-L1 in a tissue microarray of 92 patients’ lung tumor samples were measured by immunohistochemistry. Data of lung cancer were downloaded from the TCGA database, and the correlation of PD-L1 expression between EGFR wild type and mutation in lung cancer patients was analyzed. H1975 and PC-9 cells were infected with lentiviral vectors to downregulate MUC3A. The expression pattern of PD-L1 in NSCLC cells was detected by qRT-PCR and flow cytometry. Cell proliferation was assessed with CCK8. Cell apoptosis was examined by flow cytometry. Immunoblotting was used to identify molecular markers of signaling pathways. A xenograft nude mouse transplantation model was established to examine the effects of MUC3A and EGFR-TKIs on tumorigenesis in vivo. The expression levels of MUC3A, EGFR and PD-L1 in harvested tumors were measured by immunohistochemistry. Results: The tissue microarray of 92 patients’ lung tumor samples indicated that high levels of MUC3A were positively correlated with poor prognosis and PD-L1 expression. In the TCGA database, the levels of PD-L1 were independent of EGFR protein levels but correlated with EGFR mutation. Our results showed that induction of PD-L1 by EGF stimulation was only detected in EGFR-mutated NSCLC cell lines, such as H1975 and PC-9, but not in H1299 and H460. Knockdown of MUC3A in H1975 and PC-9 reduced PD-L1 expression, as well as PI3K/Akt and MAPK pathways. In addition, MUC3A deficiency potentiated gefitinib and AZD-9291-induced growth inhibition in PC-9 and H1975 cells by downregulating proliferation and inducing apoptosis. More interestingly, knockdown of MUC3A decreased EGFR protein levels, but not mRNA levels. Our findings suggested that MUC3A deficiency improved EGFR-mutated NSCLC sensitivity to tyrosine kinase inhibitors via increasing EGFR protein stability and decreasing PD-L1 expression through PI3K/Akt and MAPK pathways. Mice inoculated with MUC3A deficiency tumor cells treated with AZD-9291 had the fastest tumor regression and delayed relapse. Conclusions: The transmembrane mucin MUC3A increased EGFR stability. MUC3A induced PD-L1 and reduced tyrosine kinase inhibitor effects via EGFR modulation in NSCLC, suggesting potential implications for a novel immunotherapeutic approach. Citation Format: Yan Gong, Yuan Luo, Shan Peng, Shijing Ma, Yingming Sun, Jiangbo Ren, Conghua Xie. MUC3A induces PD-L1 and reduces tyrosine kinase inhibitor effects functions in EGFR-mutant non-small cell lung cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B57.

EGFR-TKI resistance promotes immune escape in lung cancer via increased PD-L1 expression

BackgroundThe ATLANTIC trial reported that higher PD-L1 expression in tumors was involved in a higher objective response in patients with EGFR+/ALK+ non-small cell lung cancer (NSCLC), indicating the possibility of anti-PD-1/PD-L1 therapy as a third-line (or later) treatment for advanced NSCLC. Therefore, the determination of status and regulatory mechanisms of PD-L1 in EGFR mutant NSCLC before and after acquired EGFR-TKIs resistance are meaningful.MethodsThe correlation among PD-L1, c-MET, and HGF was analyzed based on TCGA datasheets and paired NSCLC specimens before and after acquired EGFR-TKI resistance. EGFR-TKI resistant NSCLC cells with three well-known mechanisms, c-MET amplification, hepatocyte growth factor (HGF), and EGFR-T790M, were investigated to determinate PD-L1 expression status and immune escape ability. PD-L1-deleted EGFR-TKIs sensitive and resistant cells were used to evaluate the immune escape ability of tumors in mice xenograft models.ResultsPositive correlations were found among PD-L1, c-MET, and HGF, based on TCGA datasheets and paired NSCLC specimens. Moreover, the above three resistant mechanisms increased PD-L1 expression and attenuated activation and cytotoxicity of lymphocytes in vitro and in vivo, and downregulation of PD-L1 partially restored the cytotoxicity of lymphocytes. Both MAPK and PI3K pathways were involved in the three types of resistance mechanism-induced PD-L1 overexpression, whereas the NF-kappa B pathway was only involved in T790M-induced PD-L1 expression.ConclusionsHGF, MET-amplification, and EGFR-T790M upregulate PD-L1 expression in NSCLC and promote the immune escape of tumor cells through different mechanisms.

P2.04-32 Comparison of Clinicopathological and Genomic Characteristics Between NSCLCs with a PD-L1 Tumor Proportion Score of ≥90% vs &lt;1%

Determinants of PD-L1 expression in non-small cell lung cancer (NSCLC) are poorly defined. To identify characteristics associated with high vs. absent PD-L1 expression, we compared clinicopathologic and genomic features of NSCLCs at the two ends of PD-L1 expression spectrum: a PD-L1 tumor proportion score (TPS) of ≥90% or a PD-L1 TPS of <1%. We retrospectively collected clinicopathologic and genomic data (via targeted NGS) from consecutive NSCLC patients who had consented to an IRB-approved correlative research study and whose tumor PD-L1 TPS was either ≥90% or <1%. Single nucleotide variations, insertions/deletions, and copy number alterations were compared using Fisher’s exact test. Tumor mutational burden (TMB) was compared using Mann-Whitney test. 421 NSCLCs with PD-L1 TPS ≥90% (N=133) or <1% (N=288) and successfully performed NGS were identified. There was no difference in age, sex, histology, or stage at diagnosis between the two groups. Patients with a PD-L1 TPS of ≥90% were more likely to be smokers (86.5% vs 76.4%; P=0.02) compared to patients with a PD-L1 TPS of <1%, and tumors in the PD-L1 TPS ≥90% group had higher TMB (10.89 vs 8.47 mutations/megabase; P=0.004) than those in the PD-L1 TPS <1% group. Tumors in the PD-L1 TPS ≥90% group were more likely to have KRAS (47.3% vs 31.3%; P=0.002), MET exon 14 (9.6% vs 2.1%; P=0.003), and TP53 mutations (71.0% vs 49.7%; P<0.001) than those in the PD-L1 TPS <1% group. Compared to the PD-L1 TPS ≥90% group, the PD-L1 TPS <1% group was more likely to have EGFR (23.6% vs 8.2%; P<0.001) and STK11 (23.4% vs 5.0%; P<0.001) mutations, as well as the absence of known oncogenic driver mutations (35.2% vs 24.8%; P=0.04). Chromosomal gain alterations (amplification or copy number gain) of the 9p24.1 locus, where the PD-L1, PD-L2, and JAK2 genes are located, were more common in the PD-L1 TPS ≥90% group than in the PD-L1 TPS <1% group (11.4% vs 2.8%, respectively; P<0.001). Chromosomal loss alterations (copy loss or deletion) of the 9p24.1 locus were more common in the PD-L1 TPS <1% than in the PD-L1 TPS ≥90% group (27.5% vs 3.8%, respectively; P<0.001). A repeated biopsy case showed acquired loss PD-L1 expression (PD-L1 TPS changed from 90% to 0%) with concomitant acquired loss of the 9p24.1 locus. High PD-L1 expression in NSCLC is associated with tobacco use, high TMB, gain of the 9p24.1 locus and mutations in KRAS, MET exon 14, and TP53. PD-L1 negativity is associated with never smoking status, low TMB, loss of the 9p24.1 locus, mutations in EGFR and STK11, and the absence of oncogenic driver mutations.

Monitoring the Response of PD-L1 Expression to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Nonsmall-Cell Lung Cancer Xenografts by Immuno-PET Imaging.

Accumulating evidence has suggested that the tumor microenvironment of nonsmall-cell lung cancer (NSCLC) may be impacted by chemotherapy, radiotherapy, or epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). PD-L1 is an important biomarker in the tumor microenvironment that can predict patient response to immunotherapies. Therefore, it is highly desirable to achieve a real-time, noninvasive assessment of PD-L1 expression, which can provide critical information for recruiting patients as well as monitoring therapeutic efficacy. We herein studied the EGFR-TKI-induced effects on PD-L1 levels in NSCLC tumor models using immuno-PET imaging with 89Zr-Df-KN035, an imaging tracer previously established by our group. A549 human NSCLC xenografts were established in BALB/c nude mice and treated with different doses of an EGFR-TKI gefitinib. PET imaging with 89Zr-Df-KN035 was performed before and after the treatment to evaluate PD-L1 expression, which was further verified by immunohistochemical staining. Our results demonstrate that 89Zr-Df-KN035 can specifically evaluate PD-L1 levels in NSCLC tumor models. Compared to the untreated control, the high dose of gefitinib inhibited tumor growth and lowered the tumor uptake of 89Zr-Df-KN035. In comparison, the low dose of gefitinib did not affect tumor growth, although the extensive tumor necrosis also led to the lower uptake of 89Zr-Df-KN035. In conclusion, our results demonstrate that immuno-PET imaging with 89Zr-Df-KN035 is a promising tool to noninvasively monitor PD-L1 expression in NSCLC treated with EGFR-TKIs and can be used to optimize treatment plans for immunotherapy.