PD-L1 Gene Expression Research Articles

DNA methylation signatures provide novel diagnostic biomarkers and predict responses of immune therapy for breast cancer.

Breast cancer (BRCA) is one of the most common malignant tumors affecting women worldwide. DNA methylation modifications can influence oncogenic pathways and provide potential diagnostic and therapeutic targets for precision oncology. In this study, we used non-parametric permutation tests to identify differentially methylated positions (DMPs) between paired tumor and normal BRCA tissue samples from the Cancer Genome Atlas (TCGA) database. Then, we applied non-negative matrix factorization (NMF) to the DMPs to derive eight distinct DNA methylation signatures. Among them, signatures Hyper-S3 and Hypo-S4 signatures were associated with later tumor stages, while Hyper-S1 and Hypo-S3 exhibited higher methylation levels in earlier stages. Signature Hyper-S3 displayed an effect on overall survival. We further validated the four stage-associated signatures using an independent BRCA DNA methylation dataset from peripheral blood samples. Results demonstrated that 24 commonly hypomethylated sites in Hypo-S4 showed lower methylation in BRCA patients compared to healthy individuals, suggesting its potential as an early diagnostic biomarker. Furthermore, we found that methylation of 23 probes from four stage-related signatures exhibited predictive power for immune therapy response. Notably, methylation levels of all three probes from the Hypo-S4 and activity of the Hypo-S4 demonstrated highly positive relevance to PD-L1 gene expression, implying their significant predictive values for immunotherapy outcomes. GO and KEGG pathway enrichment analysis revealed that genes with these 23 immunotherapy-related methylation probes are mainly involved in glycan degradation or protein deglycosylation. These methylation signatures and probes may serve as novel epigenetic biomarkers for predicting tumor immunotherapy response. Our findings provide new insights into precision oncology approaches for BRCA.

Ketogenic diet enhances the anti-cancer effects of PD-L1 blockade in renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is characterized by a predominant metabolic reprogramming triggering energy production by anaerobic glycolysis at the expense of oxydative phosphorylation. Ketogenic diet (KD), which consists of high fat and low carbohydrate intake, could bring required energy substrates to healthy cells while depriving tumor cells of glucose. Our objective was to evaluate the effect of KD on renal cancer cell tumor metabolism and growth proliferation. Growth cell proliferation and mitochondrial metabolism of ACHN and Renca renal carcinoma cells were evaluated under ketone bodies (KB) exposure. In vivo studies were performed with mice (nude or Balb/c) receiving a xenograft of ACHN cells or Renca cells, respectively, and were then split into 2 feeding groups, fed either with standard diet or a 2:1 KD ad libitum. To test the effect of KD associated to immunotherapy, Balb/c mice were treated with anti-PDL1 mAb. Tumor growth was monitored. In vitro, KB exposure was associated with a significant reduction of ACHN and Renca cell proliferation and viability, while increasing mitochondrial metabolism. In mice, KD was associated with tumor growth reduction and PDL-1 gene expression up-regulation. In Balb/c mice adjuvant KD was associated to a better response to anti-PDL-1 mAb treatment. KB reduced the renal tumor cell growth proliferation and improved mitochondrial respiration and biogenesis. KD also slowed down tumor growth of ACHN and Renca in vivo. We observed that PDL-1 was significantly overexpressed in tumor in mice under KD. Response to anti-PDL-1 mAb was improved in mice under KD. Further studies are needed to confirm the therapeutic benefit of adjuvant KD combined with immunotherapy in patients with kidney cancer.

An analysis of the influencing factors of false negative autoantibodies in patients with non-small cell lung cancer.

To analyze the clinical significance of seven autoantibodies (P53, PGP9.5, SOX2, GAGE7, GBU4-5, MAGE, and CAGE) in patients with non-small cell lung cancer (NSCLC) and the factors that influence false-negative results. Seven autoantibodies were measured in the serum of 502 patients with non-small cell lung cancer (NSCLC) using ELISA, and their correlations with age, sex, smoking history, pathological type, clinical stage, and PD-L1 gene expression were analyzed. The clinicopathological data of the false-negative and positive groups for the seven autoantibodies were compared to determine the influencing factors. P53 antibody expression level was correlated with lobulation sign, PGP9.5 antibody expression level with sex and vascular convergence; SOX2 antibody expression level with pathological type, clinical stage, and enlarged lymph nodes; and MAGE antibody expression level with the pathological type (P<0.05). False-negative autoantibodies are prone to occur in lung cancer patients with ground-glass nodules, no enlarged lymph nodes, no vascular convergence, and PD-L1 gene expression <1% (P <0.05). Detection of seven autoantibodies was clinically significant in patients with NSCLC. However, poor sensitivity should be considered in clinical diagnoses to prevent missed diagnoses.

Impact of Chemotherapeutic Agents on PD-L1, CTLA-4, and VISTA Gene Expression in Cervical Cancer Cell Lines: An In Vitro Study

Impact of Chemotherapeutic Agents on PD-L1, CTLA-4, and VISTA Gene Expression in Cervical Cancer Cell Lines: An In Vitro Study

GSK0660 enhances antitumor immunotherapy by reducing PD-L1 expression

Blockade of PD-1/PD-L1 immune checkpoint is wildly used for multiple types of cancer treatment, while the low response rate for patients is still completely unknown. As nuclear hormone receptor, PPARδ (peroxisome-proliferator-activated receptor) regulates cell proliferation, inflammation, and tumor progression, while the effect of PPARδ on tumor immune escape is still unclear. Here we found that PPARδ antagonist GSK0660 significantly reduced colon cancer cell PD-L1 protein and gene expression. Luciferase analysis showed that GSK0660 decreased PD-L1 gene transcription activity. Moreover, reduced PD-L1 expression in colon cancer cells led to increased T cell activity. Further analysis showed that GSK0660 decreased PD-L1 expression in a PPARδ dependent manner. Implanted tumor model analysis showed that GSK0660 inhibited tumor immune escape and the combined PD-1 antibody with GSK0660 effectively enhanced colorectal cancer immunotherapy. These findings suggest that GSK0660 treatment could be an effective strategy for cancer immunotherapy.

Abstract 5564: The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling

Abstract Glioblastoma (GBM), the most aggressive brain cancer which is usually fatal within a year after diagnosis, poses a significant therapeutic challenge with conventional treatments providing limited success. Despite advancements in immune checkpoint blockade, PD-L1 inhibitors exhibit suboptimal efficacy in recurrent GBMs with recent clinical trials demonstrating only 8% objective responses in patients. Utilizing the R2 Genomic Analysis Visualization Platform, we identified a positive 8-18% correlation between Pyk2 and PD-L1 gene expression in human GBM specimens. We hypothesize that Pyk2 signaling is involved in the regulation of PD-L1 expression in GBM cells. In this study, primary human glioblastoma cell lines and GL261 mouse glioma cells were assessed employing CRISPR/Cas9 Pyk2 knockout (Pyk2KO) combined with pharmacological Pyk2 inhibition. Flow cytometric analysis revealed a 35% decrease in PD-L1 expression in Pyk2KO GL261 cells compared to wild-type cells. SiRNA knockdown against Pyk2 in primary human GBM cells demonstrated a similar response, with a 50% decrease in PD-L1 expression compared to MOCK control cells. Additionally, the application of the Pyk2/FAK inhibitor defactinib at a concentration of 0.2 μM reduced PD-L1 expression by 32% in both GL261 and primary human GBM cells. These findings highlight Pyk2 as a potential therapeutic target for modulating the immunosuppressive microenvironment in GBM and enhancing the effectiveness of immune checkpoint blockade therapies. This study was supported by: NIH Grant 1SC1GM122691 and PRSTRT2022. Citation Format: Tyrel Porter, Lilia Kucheryavykh. The regulation of PD-L1 in glioblastoma cells involves Pyk2 signaling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5564.

Abstract 1373: Gene expression correlation of immune checkpoint molecules Siglec-15 and PD-L1 varies widely by cancer indication

Abstract Siglec-15 is a novel target for immunotherapy in cancer and exhibits low mRNA expression in normal tissues but broad expression across cancer indications, specifically on tumor-associated macrophages and tumor cells [1]. Expression of Siglec-15 and the immune checkpoint molecule PD-L1 have previously been reported to be mutually exclusive [1][2]. However, a large-scale meta-analysis of this claim has not been undertaken across cancer indications. Here, RNA-Seq analyses were performed across single cells, cell lines, and bulk tumor samples for Siglec-15 and PD-L1 mRNA expression. Data and meta-data from TCGA, CCLE, and primary tumor samples from 62 studies were obtained through OmicSoft OncoLand and single cell analysis was performed on the BioTuring Talk2Data platform. Analyses were limited to non-small cell lung cancer (NSCLC), cholangiocarcinoma, breast, thyroid, head and neck, colon, rectal, bladder, kidney, and endometrial cancers. Expression values were binned separately into percentiles across all indications and samples in increments of 10%. These analyses revealed individual gene expression levels of Siglec-15 and PD-L1 in bulk tumor samples varied substantially by indication, while co-expression of Siglec-15 and PD-L1 varied across indications. Analysis of gene expression of Siglec-15 and PD-L1 from the TCGA database showed a positive correlation in several indications including NSCLC, head and neck, kidney, and thyroid cancers. Analyses from additional studies in primary tumor samples confirmed co-expression of Siglec-15 and PD-L1 in NSCLC and head and neck cancer. Cell lines showed a similar trend of positive correlation with varying levels of Siglec-15 and PD-L1 expression. However, when examining expression at the single cell level (not bulk tumor), in 30 single cell studies spanning &amp;gt;1.3M cells, only 731 cells in 20 studies were found to co-express Siglec-15 and PD-L1 on the same cell while each gene was expressed individually on other cells. The majority of the cells co-expressing the genes were macrophages (n=~350), followed by CD4 (n=~115) and CD8 T cells (n=~50). This study demonstrates that at the bulk tumor level, Siglec-15 and PD-L1 mRNA expression are not broadly mutually exclusive across cancer indications and instead expression of each gene varies broadly within a given indication. Additionally, their expression is frequently positively correlated, including in NSCLC, head and neck, kidney, and thyroid cancers. These results also demonstrate that Siglec-15 and PD-L1 are rarely found co-expressed on same cell. These associations enable a better understanding of the landscape of target expression in patients across a wide variety of cancer indications and can inform combination strategies with anti-Siglec-15 therapies. [1] Sun J, et al. Clin Cancer Res, 2021;27(3):680-688. [2] Wang J, et al. Nat Med, 2019;25(4):656-666. Citation Format: Krystal Watkins, Liyang Diao, Marsha Crochiere, Jan Pinkas. Gene expression correlation of immune checkpoint molecules Siglec-15 and PD-L1 varies widely by cancer indication [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1373.

Identification of an Immune signature assisted prognosis, and immunotherapy prediction for IDH wildtype glioblastoma.

IDH-wildtype glioblastoma (GBM) is the most common and malignant primary brain tumor. The purpose of this study is to establish a prognostic gene signature for IDH-wildtype GBM. RNA sequencing data of normal brain tissue and GBM patients were obtained from TCGA, CGGA, GEO and the GTEx databases. Identification of prognostic differentially expressed genes (DEGs) with | log2 fold change | > 0.5 and adjust p < 0.05 in TCGA and CGGA databases by "limma" method. By LASSO regression analysis and multivariate Cox analysis, a 3-gene prognostic signature composed of FMOD, MXRA5 and RAB36 was established. The 3-gene prognostic risk model is validated by TCGA and GSE43378 datasets. The expression of FMOD, MXRA5 and RAB36 in GBM patients was significantly higher than that in normal brain tissues in CCGA, TCGA and GSE29796 data sets. In order to further verify this result, total RNA was extracted from tumors and paracancerous tissues of 9 GBM patients. RT-PCR results showed that the expression of FMOD, MXRA5 and RAB36 in tumor tissues of most patients was higher than that in paracancerous tissues. The results of GSEA showed that the pathway enrichment of the 3-gene signature was mainly related to tumor immunity. Immune cell infiltration analyzed by ssGSEA showed that there were significant differences in macrophages between high- and low-risk groups. Immune checkpoint genes correlation analysis showed that PD-L1 gene expression is closely related to risk score. Our study identifies a prognostic-associated risk model and provides a potential effective immunotherapy target for IDH-wildtype GBM patients.

Sitravatinib combined with PD-1 blockade enhances cytotoxic T-cell infiltration by M2 to M1 tumor macrophage repolarization in esophageal adenocarcinoma.

Esophageal adenocarcinoma (EAC) is a leading cause of cancer-related mortality. Sitravatinib is a novel multi-gene tyrosine kinase inhibitor (TKI) that targets tumor-associated macrophage (TAM) receptors, VEGF, PDGF and c-Kit. Currently, sitravatinib is actively being studied in clinical trials across solid tumors and other TKIs have shown efficacy in combination with immune checkpoint inhibitors (ICI) in cancer models. In this study, we investigated the anti-tumor activity of sitravatinib alone and in combination with PD-1 blockade in an EAC rat model. Treatment response was evaluated by mortality, pre- and post-treatment MRI, gene expression, immunofluorescence and immunohistochemistry. Our results demonstrated adequate safety and significant tumor shrinkage in animals treated with sitravatinib, and more profoundly, sitravatinib and PD-1 inhibitor, AUNP-12 (P < 0.01). Suppression of TAM receptors resulted in increased gene expression of pro-inflammatory cytokines and decreased expression of anti-inflammatory cytokines, enhanced infiltration of CD8+ T cells, and M2 to M1 macrophage phenotype repolarization in the tumor microenvironment of treated animals (P < 0.01). Moreover, endpoint immunohistochemistry staining corroborated the anti-tumor activity by downregulation of Ki67 and upregulation of Caspase-3 in the treated animals. Additionally, pretreatment gene expression of TAM receptors and PD-L1 were significantly higher in major responders compared with the non-responders, in animals that received sitravatinib and AUNP-12 (P < 0.02), confirming that TAM suppression enhances the efficacy of PD-1 blockade. In conclusion, this study proposes a promising immunomodulatory strategy using a multi-gene TKI to overcome developed resistance to an ICI in EAC, establishing rationale for future clinical development.

The expression changes of PD-L1 and immune response mediators are related to the severity of primary bone tumors

The expression pattern, diagnostic value, and association of PD-L1, IFN-γ and TGF-β with bone tumor type, severity, and relapse are determined in this study. 300 human samples from patients with osteosarcoma, Ewing sarcoma, and GCT were enrolled. The PD-L1 gene and protein expression were assessed by qRT-PCR and immunohistochemistry, respectively. ELISA and flow cytometry was used to detect cytokines and CD4/CD8 T cell percentages, respectively. A considerable increase in PD-L1 level was detected in bone tumor tissues at both gene and protein levels that was considerable in osteosarcoma and Ewing sarcoma. A positive correlation was detected regarding the PD-L1 and tumor metastasis and recurrence in osteosarcoma and Ewing sarcoma. The increased IFN-γ level was detected in patients with metastatic, and recurrent osteosarcoma tumors that were in accordance with the level of TGF-β in these samples. The simultaneous elevation of IFN-γ and TGF-β was detected in Ewing sarcoma and GCT, also the CD4 + /CD8 + ratio was decreased significantly in patients with osteosarcoma compared to GCT tumors. The elevated levels of PD-L1, TGF- β, and IFN-γ were associated with bone tumor severity that can provide insights into the possible role of this axis in promoting immune system escape, suppression, and tumor invasion.

Immune-related gene-based model predicts the survival of colorectal carcinoma and reflected various biological statuses.

Bakcground: Prognosis of colorectal cancer (CRC) varies due to complex genetic-microenviromental interactions, and multiple gene-based prognostic models have been highlighted. Material and Method: In this work, the immune-related genes' expression-based model was developed and the scores of each sample were calculated. The correlation between the model and clinical information, immune infiltration, drug response and biological pathways were analyzed. Results: The high-score samples have a significantly longer survival (overall survival and progression-free survival) period than those with a low score, which was validated across seven datasets containing 1,325 samples (GSE17536 (N = 115), GSE17537 (N = 55), GSE33113 (N = 90), GSE37892 (N = 130), GSE38832 (N = 74), GSE39582 (N = 481), and TCGA (N = 380)). The score is significantly associated with clinical indicators, including age and stage, and further associated with PD-1/PD-L1 gene expression. Furthermore, high-score samples have significantly higher APC and a lower MUC5B mutation rate. The high-score samples show more immune infiltration (including CD4+ and CD8+ T cells, M1/M2 macrophages, and NK cells). Enriched pathway analyses showed that cancer-related pathways, including immune-related pathways, were significantly activated in high-score samples and that some drugs have significantly lower IC50 values than those with low score. Conclusion: The model developed based on immune-related genes is robust and reflected various statuses of CRC and may be a potential clinical indicator.

Comparison of SP142 and 22C3 PD-L1 assays in a population-based cohort of triple-negative breast cancer patients in the context of their clinically established scoring algorithms

BackgroundImmunohistochemical (IHC) PD-L1 expression is commonly employed as predictive biomarker for checkpoint inhibitors in triple-negative breast cancer (TNBC). However, IHC evaluation methods are non-uniform and further studies are needed to optimize clinical utility.MethodsWe compared the concordance, prognostic value and gene expression between PD-L1 IHC expression by SP142 immune cell (IC) score and 22C3 combined positive score (CPS; companion IHC diagnostic assays for atezolizumab and pembrolizumab, respectively) in a population-based cohort of 232 early-stage TNBC patients.ResultsThe expression rates of PD-L1 for SP142 IC ≥ 1%, 22C3 CPS ≥ 10, 22C3 CPS ≥ 1 and 22C3 IC ≥ 1% were 50.9%, 27.2%, 53.9% and 41.8%, respectively. The analytical concordance (kappa values) between SP142 IC+ and these three different 22C3 scorings were 73.7% (0.48, weak agreement), 81.5% (0.63) and 86.6% (0.73), respectively. The SP142 assay was better at identifying 22C3 positive tumors than the 22C3 assay was at detecting SP142 positive tumors. PD-L1 (CD274) gene expression (mRNA) showed a strong positive association with all two-categorical IHC scorings of the PD-L1 expression, irrespective of antibody and cut-off (Spearman Rho ranged from 0.59 to 0.62; all p-values < 0.001). PD-L1 IHC positivity and abundance of tumor infiltrating lymphocytes were of positive prognostic value in univariable regression analyses in patients treated with (neo)adjuvant chemotherapy, where it was strongest for 22C3 CPS ≥ 10 and distant relapse-free interval (HR = 0.18, p = 0.019). However, PD-L1 status was not independently prognostic when adjusting for abundance of tumor infiltrating lymphocytes in multivariable analyses.ConclusionOur findings support that the SP142 and 22C3 IHC assays, with their respective clinically applied scoring algorithms, are not analytically equivalent where they identify partially non-overlapping subpopulations of TNBC patients and cannot be substituted with one another regarding PD-L1 detection.Trial registration The Swedish Cancerome Analysis Network - Breast (SCAN-B) study, retrospectively registered 2nd Dec 2014 at ClinicalTrials.gov; ID NCT02306096.

Increased Response to Immune Checkpoint Inhibitors with Dietary Methionine Restriction in a Colorectal Cancer Model.

Dietary methionine restriction (MR), defined as a reduction of methionine intake by around 80%, has been shown to reproducibly decrease tumor growth and synergize with cancer therapies. In this study, we combined DMR with immune checkpoint inhibitors (ICIs) in a model of colon adenocarcinoma. In vitro, we observed that MR increased the expression of MHC-I and PD-L1 in both mouse and human colorectal cancer cells. We also saw an increase in the gene expression of STING, a known inducer of type I interferon signaling. Inhibition of the cGAS-STING pathway, pharmacologically or with siRNA, blunted the increase in MHC-I and PD-L1 surface and gene expression following MR. This indicated that the cGAS-STING pathway, and interferon in general, played a role in the immune response to MR. We then combined dietary MR with ICIs targeting CTLA-4 and PD-1 in an MC38 colorectal cancer tumor model developed in immunocompetent C57BL/6 mice. The combination treatment was five times more effective at reducing the tumor size than ICIs alone in male mice. We noted sex differences in the response to dietary MR, with males showing a greater response than females. Finally, we observed an increase in membrane staining for the PD-L1 protein in MC38 tumors from animals who were fed an MR diet. MHC-I was highly expressed in all tumors and showed no expression difference when comparing tumors from control and MR-treated mice. These results indicated that MR increased PD-L1 expression both in vitro and in vivo and improved the response to ICIs in mice.

Involvement of Nrf2 Activation and NF-kB Pathway Inhibition in the Antioxidant and Anti-Inflammatory Effects of Hesperetin in Activated BV-2 Microglial Cells.

Alzheimer's disease is a progressive neurodegenerative disorder leading to cognitive decline and memory loss. The incidence of this disease continues to increase due to the limited number of novel therapeutics that prevent or slow down its progression. Flavonoids have been investigated for their potential effects on cellular damage triggered by excessive reactive oxygen species (ROS) and neuroinflammatory conditions. This study investigated the effect of the flavonoid hesperetin on LPS-activated murine BV-2 microglial cells. Results show that hesperetin reduced nitric oxide levels and increased catalase, glutathione, and superoxide dismutase levels, suggesting its potential to reduce neuroinflammation and oxidative stress. Moreover, RT-PCR arrays showed that hesperetin modulated multiple genes that regulate oxidative stress. Hesperetin downregulated the mRNA expression of ERCC6, NOS2, and NCF1 and upregulated HMOX1 and GCLC. RT-PCR results showed that hesperetin-induced Nrf2 mRNA and protein expression in LPS-activated BV-2 microglial cells is involved in the transcription of several antioxidant genes, suggesting that hesperetin's antioxidant effects may be exerted via the Keap1/Nrf2 signaling pathway. Furthermore, the data demonstrated that hesperetin reduced the gene expression of PD-L1, which is upregulated as an individual ages and during chronic inflammatory processes, and inhibited the expression of genes associated with NF-kB signaling activation, which is overactivated during chronic inflammation. It was concluded from this investigation that hesperetin may have therapeutic potential to prevent or slow down the progression of neurodegenerative diseases, such as Alzheimer's disease, by reducing chronic oxidative stress and modulating neuroinflammation.

Construction and validation of a gene expression classifier to predict immunotherapy response in primary triple-negative breast cancer

BackgroundImmune checkpoint inhibitors (ICI) improve clinical outcomes in triple-negative breast cancer (TNBC) patients. However, a subset of patients does not respond to treatment. Biomarkers that show ICI predictive potential in other solid tumors, such as levels of PD-L1 and the tumor mutational burden, among others, show a modest predictive performance in patients with TNBC.MethodsWe built machine learning models based on pre-ICI treatment gene expression profiles to construct gene expression classifiers to identify primary TNBC ICI-responder patients. This study involved 188 ICI-naïve and 721 specimens treated with ICI plus chemotherapy, including TNBC tumors, HR+/HER2− breast tumors, and other solid non-breast tumors.ResultsThe 37-gene TNBC ICI predictive (TNBC-ICI) classifier performs well in predicting pathological complete response (pCR) to ICI plus chemotherapy on an independent TNBC validation cohort (AUC = 0.86). The TNBC-ICI classifier shows better performance than other molecular signatures, including PD-1 (PDCD1) and PD-L1 (CD274) gene expression (AUC = 0.67). Integrating TNBC-ICI with molecular signatures does not improve the efficiency of the classifier (AUC = 0.75). TNBC-ICI displays a modest accuracy in predicting ICI response in two different cohorts of patients with HR + /HER2- breast cancer (AUC = 0.72 to pembrolizumab and AUC = 0.75 to durvalumab). Evaluation of six cohorts of patients with non-breast solid tumors treated with ICI plus chemotherapy shows overall poor performance (median AUC = 0.67).ConclusionTNBC-ICI predicts pCR to ICI plus chemotherapy in patients with primary TNBC. The study provides a guide to implementing the TNBC-ICI classifier in clinical studies. Further validations will consolidate a novel predictive panel to improve the treatment decision-making for patients with TNBC.

Inhibition of FGFR3 upregulates MHC-I and PD-L1 via TLR3/NF-kB pathway in muscle-invasive bladder cancer.

Improving the potency of immune response is paramount among issues concerning immunotherapy of muscle-invasive bladder cancer (MIBC). On the basis of immune subtypes, we investigated possible molecular mechanisms involved in tumor immune escape in MIBC. According to the 312 immune-related genes, three MIBC immune subtypes were clustered. Cluster 2 subtype is characterized by FGFR3 mutations and has a better clinical prognosis. However, the expression levels of MHC-I and immune checkpoints genes were the lowest, indicating that this subtype is subject to immune escape and has a low response rate to immunotherapy. Bioinformatics analysis and immunofluorescence staining of clinical samples revealed that the FGFR3 is involved in the immune escape in MIBC. Besides, after FGFR3 knockout with siRNA in RT112 and UMUC14 cells, the TLR3/NF-kB pathway was significantly activated and was accompanied by upregulation of MHC-I and PD-L1 gene expression. Furthermore, the use of TLR3 agonists poly(I:C) can further improve the effect. Together, our results suggest that FGFR3 might involve in immunosuppression by inhibition of NF-kB pathway in BC. Considering that TLR3 agonists are currently approved for clinical treatment as immunoadjuvants, our study might provide more insights for improving the efficacy of immunotherapy in MIBC.

&lt;i&gt;PD-L1&lt;/i&gt; and &lt;i&gt;PD-L2&lt;/i&gt; gene expression in human glioblastoma cells resistant to chemo- and radiotherapy

Membrane molecules PD-L1 and PD-L2, ligands of T lymphocytes PD1 receptor, perform immunoregulatory functions. Their binding to the receptor leads to inhibition of proliferation, reduction of cytokine production, cytotoxic response, and apoptosis of T lymphocytes. The cells of many tumors, regardless of their histogenesis, express PD-L1 molecules, thus limiting the development of an anti-tumor immune response. Glioblastomas are highly malignant recurrent tumors of the central nervous system. The main sources of glioblastoma recurrence are resistant tumor cells initially present in gliomas with heterogeneous cellular composition, as well as resistant cells that are formed during therapy. Increasing the dose of cytostatic drugs or radiation during relapse therapy is not effective in glioblastomas. It has been shown for a number of tumors, including ovarian cancer and non-small cell lung cancer, that drugs preventing PD-L1/PD1 interaction are effective in the treatment of neoplasms resistant to chemo- and radiotherapy. Immunotherapy using drugs that inhibit the binding of PD-L molecules to their receptor is considered as a way to overcome the resistance of glioblastomas to therapy. The aim of this work was to assess the level of PD-L1 and PD-L2 gene expression in resistant glioblastoma cells lines A172, R1, T2 and T98G, which resumed proliferation after exposure to the maximum for each line, sublethal doses of cytostatic drugs (fotemustine and temozolomide), fractionated or single gamma irradiation. A172 line belongs to glioblastomas that are highly sensitive to these influences, T98G is a highly resistant cell line, while R1 and T2 lines occupy an intermediate position. In intact glioblastoma A172, R1, and T2 cells the level of PD-L1 and PD-L2 gene expression was equally high, while in T98G cells it was significantly lower. Exposure of A172 and R1 glioblastoma lines to cytostatic drugs or irradiation did not significantly change the level of PD-L1 and PD-L2 genes expression typical for intact cells. In T2 glioblastoma cells, and especially in T98G cells, a significant increase in expression of these genes was found, most pronounced for PD-L2 gene. This increase in expression may indicate an enhanced malignancy of resistant T2 and T98G cells. High expression of the genes responsible for the production of PD-L1 and PD-L2, which limit the cytotoxic response against tumor cells, is a prerequisite for the use of drugs targeted against PD-L1 and PD-L2 for the elimination of resistant cells in glioblastoma.

Abstract 4348: Validation of an integrative pan-solid tumor predictor of pembrolizumab monotherapy benefit

Abstract Background: We previously reported the development and validation of an integrative Immunotherapy Response Score (IRS) algorithm, which integrates tumor mutation burden (TMB) and quantitative gene expression of PD-L1, PD-1, ADAM12 and TOP2A, to predict PD-1 or PD-L1 (together PD-[L]1) monotherapy (mono) benefit across solid tumors from routine formalin fixed paraffin embedded samples. Herein, we evaluated IRS performance for predicting pembrolizumab (pembro) mono benefit in a second, independent validation cohort. Methods: We identified patients from an ongoing observational trial (NCT03061305) integrating molecular profiling with real world treatment data who were 1) treated with systemic pembro mono, 2) not included in the original IRS cohort, and 3) had valid molecular profiling data to generate IRS. IRS status (IRS-High [H] vs. -Low [L] by the previously validated threshold; -H with increased benefit) association with pembro real world progression free survival (rwPFS) by time-to-next therapy was determined by Cox proportional hazard modeling (adjusting for age, gender, line of systemic therapy, and PD-(L)1 mono indicated (microsatellite instable high [MSI-H], TMB-H, or approved tumor type) vs. not indicated. The predictive nature of the IRS biomarker was assessed in the subset of &amp;gt;= 2nd line patients through a case cross-over analysis (pembro rwPFS vs. immediately preceding therapy line rwPFS) as assessed by likelihood ratio test (LRT). Results: Of the 176 patients in this independent validation cohort (from 25 tumor types), 78 (44%) patients were IRS-H, while 40 (23%) were TMB-H. Pembro mono rwPFS was significantly longer in IRS-H vs. IRS-L patients (median rwPFS 18.7 vs. 6.4 months, adjusted hazard ratio [aHR] 0.46, p=0.007), while PD-(L)1 mono indicated status was not associated with pembro mono rwPFS (indicated vs. not indicated aHR 1.47, p=0.26). In the case cross-over analysis of 55 (31%) patients treated with systemic therapy prior to pembro mono, pembro rwPFS was significantly longer than preceding therapy in IRS-H patients (median 18.7 vs. 5.5 months) but not IRS-L patients (median 4.9 vs. 5.4 months); the LRT for interaction between IRS status and pembro/prior therapy was significant (p=0.046); results in the TMB-L/non-MSI-H subset (n=45) of patients was similar (LRT p=0.025). Conclusion: These results confirm the pan-solid tumor PD-(L)1 monotherapy predictive nature of the IRS biomarker. Citation Format: Benjamin J. Bulen, Nickolay A. Khazanov, Laura E. Lamb, Daniel H. Hovelson, Kat Kwiatkowski, D. Bryan Johnson, Daniel R. Rhodes, Scott A. Tomlins. Validation of an integrative pan-solid tumor predictor of pembrolizumab monotherapy benefit. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4348.

Abstract 2183: Absolute quantification of PD-L1 gene expression in single cell on tissue for accurately predicting response to ICIs

Abstract Background PD-L1 immunohistochemistry (IHC) has been traditionally used for predicting clinical responses to immune checkpoint inhibitors (ICIs). However, there are at least 4 different assays and antibodies used for PD-L1 IHC, each developed with a different ICI. Spatial transcriptomic analysis at the single cell level can be helpful for deeply characterize the complexity and heterogeneity of tumors. We set to test if in-situ hybridization-based RNA scope coupled to single cell quantification is a robust method to determine PD-L1 mRNA expression levels in each cell and furthermore efficacy of predicting response to ICIs as compared to routinely used standardized IHC procedures. Method FFPE sections from retrospectively collected surgically resected NSCLCs (adenocarcinoma and squamous cell carcinoma) tumor with progression disease or complete response from adjuvant pembrolizumab therapy were used. Target genes CD274 (PD-L1) and GZMB (Granzyme B) were assessed. For fluorescent detection, the label probe was conjugated to Opal 570 and 620 respectively. Tissues cells were counterstained with DAPI to detect the nuclei. A Digital pathology software QuPath was used for cell segmentation and dot’s detection for gene expression quantification and further classification. Results Difference in expression levels were seen between PD-L1 and GZMB across the tumor with PD-L1 staining mostly located in the tumoral area and Granzyme B in stromal area of the NSCLC tissue. This is consistent with the expected expression of PD-L1 on tumor epithelial cells and Granzyme B on T cells. This heterogeneity in the signal distribution specific to the analyzed tissue correspond to intra-tissue cells metabolism heterogeneity between cell types. This heterogeneity of expression was captured though a digital workflow capable of quantifying the number of cells expressing PD-L1 and GZMB. Among the cells expressing the markers, 96% were positive for PD-L1 for the tumor that did not respond to therapy with the threshold of positivity being set at 1 of dots, that corresponds to 1 transcript, per cell. Conclusion Our results demonstrate that PD-L1 was highly expressed in a tumor context that was not responding to PD-1 ICI inhibitor. Measurement of mRNA expression at the single cell level on tissue provides another layer of PD-L1 detection which can be exploited to predict tumor response to ICI suggesting that moving forward with this technology is a viable approach for this dynamic biomarker. Citation Format: Amandine Gerstenberg, Adele Ponzoni, Corinne Ramos, Jonathan Stauber. Absolute quantification of PD-L1 gene expression in single cell on tissue for accurately predicting response to ICIs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2183.

Abstract P4-08-03: Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC

Abstract Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC Background and Premise: Triple-negative breast cancer (TNBC) is an aggressive, metastatic disease with high mortality. The standard-of-care neoadjuvant chemotherapy (NACT) in TNBC shows an initial response but followed by an increase in tumor relapse and distant metastases. Furthermore, metastatic TNBC (mTNBC) patients often develop resistance to NACT and targeted therapies. Immunotherapy with therapeutic antibodies such as anti-programmed death ligand-1 (PD-L1) has shown efficacy in treating a subset of TNBC patients, but currently only 20% of patients qualify for that treatment. Hence, there is an unmet need for developing a precision approach against novel actionable targets along with immunotherapy to treat mTNBC. Eukaryotic initiation factor 4A1 (eIF4A1) is an integral part of the translational machinery for many oncogenic mRNAs including survivin, c-MYC, Rho kinase1, and Cyclins D1 and D3, which all require the helicase activity of eIF4A1 for their translation. Our lab previously established that the eIF4A1 is a vulnerable target in breast cancer stem-like cells (BCSCs) and bulk tumor cells. We further demonstrated a key role for eIF4A1 in cancer stemness and drug resistance. Interestingly, we also observed a reduction in levels of PD-L1 when eIF4A1 is pharmacologically inhibited or genetically ablated. Many downstream effectors of eIF4A1 such as c-MYC, STAT1 and ARF6 (involved in recycling of PD-L1 back to the plasma membrane) are indeed upstream regulators of PD-L1 gene expression and cell surface expression of PD-L1. In this study, we examine human TNBC biospecimens for total eIF4A1 level, levels of STAT1, c-MYC, ARF6 and PD-L1. This will help establish the differential operation of the eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant primary and mTNBC. Approach: Human biospecimens were obtained from CHTN NIH repositories and now we have a collection of more than 100 TNBC tumor samples as well as some lymph node, lung and brain metastases and one sample of male breast primary tumor. These specimens are analyzed for role of eIF4A1 in translation of oncogenic mRNAs that contribute to the gene expression of immune checkpoint PD-L1 and other emerging immune checkpoint markers such as VISTA, TIGIT, and LAG3. To mimic the inclusion criteria for current PD-L1 monoclonal antibody treatment, TNBC tumors are examined for PD-L1 expression via immunohistochemistry after subjecting the samples to deglycosylation for effective detection and also by immunoblotting. Results: The levels of total eIF4A1 from 16 matched, therapy naive TNBC tissue were compared to adjacent normal breast tissue from the same patient via immunoblotting. Total eIF4A1 levels were found to be significantly elevated in TNBC compared to adjacent normal tissue. The downstream effectors of eIF4A1 such as STAT1 and ARF6 were also significantly elevated indicating a robust helicase activity of eIF4A1 in tumor samples. Importantly, PD-L1 levels was found to be elevated. Additionally, the total eIF4A1 level from tumor lysates from TNBC patients that had received a variety of NACT (i.e., patients with demonstrated drug resistance) were compared against normal untreated breast tissue from volunteers who underwent reduction mammoplasty by immunoblotting. The total eIF4A1 was found to be elevated in drug-resistant TNBC specimens compared to control, therapy-naïve TNBC samples. Conclusion: The eIF4A1-STAT1-PD-L1 axis is highly active in TNBC especially in drug-resistant situations. Currently, we are evaluating emerging immune checkpoints such as VISTA, TIGIT, and LAG3. Citation Format: Andrew Boring, Dharmindra Dulal, Dayanidhi Raman. Operation of eIF4A1-PD-L1 axis in therapy-naïve and drug-resistant TNBC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-08-03.