Possible association between polyomaviruses and gastrointestinal complications: a narrative review.

Polyomaviruses are a group of small, double-stranded DNA viruses that are known to be associated with the development of certain human diseases, but there is evidence that these viruses might be associated with gastrointestinal (GI) cancers. Several polyomaviruses have been identified, such as JC polyomavirus (JCPyV), BK polyomavirus (BKPyV) and recently Merkel cell polyomavirus (MCPyV). Although the direct effects of polyomaviruses on transformation of human cells and cancer development are not clearly recognized, their association with certain human diseases including GI cancers has been proposed through several molecular and epidemiological studies. For example, JCPyV and BKPyV have been linked to colorectal cancer, as there is growing evidence of finding viral genomes in cancerous tissues. Nevertheless, the major role of JCPyV, BKPyV and MCPyV in colorectal cancer progression is still under extensive investigation, and further surveys is required to establish a conclusive cause-and-effect relationship. Understanding the role of these viruses in cancer development has significant implications for diagnosis, treatment, and prevention strategies. It seems that proving a causal link between polyomaviruses and GI cancers might provide a novel path for targeted therapies or design and development of specific therapeutic vaccines. In addition, performing research on the possible link can provide insights into the underlying molecular mechanisms of carcinogenesis, potentially leading to the identification of novel biomarkers. This review focuses on polyomaviruses, in particular a recently discovered polyomavirus, MCPyV, and their possible link with human gastrointestinal disorders.

Comparison of Tumor Non-specific and PD-L1 Specific Imaging by Near-Infrared Fluorescence/Cerenkov Luminescence Dual-Modality In-situ Imaging.

Background: Labeled antibodies are excellent imaging agents in oncology to non-invasively visualize cancer-related antigens expression levels. However, tumor tracer uptake (TTU) of specific antibodies in-vivo may be inferior to non-specific IgG in some cases. Objectives: To explore factors affecting labeled antibody visualization by PD-L1 specific and non-specific imaging of nude mouse tumors. Methods: TTU was observed in RKO model on Cerenkov luminescence (CL) and near-infrared fluorescence (NIRF) imaging of radionuclide 131I or NIRF dyes labeled Atezolizumab and IgG. A mixture of NIRF dyes labeled Atezolizumab and 131I-labeled IgG was injected, and TTU was observed in the RKO and HCT8 model by NIRF/CL dual-modality in-situ imaging. TTU were observed by 131I-labeled Atezolizumab and IgG in-vitro distribution. Results: Labeled IgG concentrated more in tumors than Atezolizumab. NIRF/CL imaging in 24 to 168 h showed that TTU gradually decreased over time, which decreased more slowly on CL imaging compared to NIRF imaging. The distribution data in-vitro showed that TTU of 131I-labeled IgG was higher than that of 131I-labeled Atezolizumab at any time point. Conclusion: Non-specific IgG may not be suitable as a control for Atezolizumab in comparing tumor PD-L1 expression in nude mice via labeled antibody optical imaging under certain circumstances.

Brusatol improves the efficacy of an anti-mouse-PD-1 antibody via inhibiting programmed cell death 1 ligand 1 expression in a murine head and neck squamous cell carcinoma model.

OBJECTIVE: Combing PD-1/PD-L1 immune checkpoint inhibitors with natural products has exhibited better efficacy than monotherapy. Hence, the purpose of this research was to examine the anti-cancer effects of brusatol, a natural quassinoid-terpenoid derived from Brucea javanica, when used in conjunction with an anti-mouse-PD-1 antibody in a murine head and neck squamous cell carcinoma (HNSCC) model and elucidate underlying mechanisms. DESIGN: A murine HNSCC model and an SCC-15 cell xenograft nude mouse model were established to investigate the anti-cancer effects of brusatol and anti-PD-1 antibody. Mechanistic studies were performed using immunohistochemistry. Cell proliferation, migration, colony formation, and invasion were evaluated by MTT, migration, colony formation, and transwell invasion assays. PD-L1 levels in oral squamous cell carcinoma (OSCC) cells were assessed through qRT-PCR, flow cytometry, and western blotting assays. The impact of brusatol on Jurkat T cell function was assessed by an OSCC/Jurkat co-culture assay. RESULTS: Brusatol improved tumor suppression by anti-PD-1 antibody in HNSCC mouse models. Mechanistic studies revealed brusatol inhibited tumor cell growth and angiogenesis, induced apoptosis, increased T lymphocyte infiltration, and reduced PD-L1 expression in tumors. Furthermore, in vitro assays confirmed brusatol inhibited PD-L1 expression in OSCC cells and suppressed cell migration, colony formation, and invasion. Co-culture assays indicated that brusatol's PD-L1 inhibition enhanced Jurkat T cell-mediated OSCC cell death and reversed the inhibitory effect induced by OSCC cells. CONCLUSIONS: Brusatol improves anti-PD-1 antibody efficacy by targeting PD-L1, suggesting its potential as an adjuvant in anti-PD-1 immunotherapy.

Standardized Digital Image Analysis of PD-L1 Expression in Head and Neck Squamous Cell Carcinoma Reveals Intra- and Inter-Sample Heterogeneity with Therapeutic Implications.

For practical reasons, in many studies PD-L1 expression is measured by combined positive score (CPS) from a single tumor sample. This does not reflect the heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma (HNSCC). We investigated the extent and relevance of PD-L1 expression heterogeneity in HNSCC analyzing primary tumors and recurrences (LRs), as well as metastases. Tumor tissue from 200 HNSCC patients was immunohistochemically stained for PD-L1 and analyzed using image-analysis software QuPath v3.4 with multiple specimens per patient. CPS was ≥20 in 25.6% of primary tumors. Intra-tumoral heterogeneity led to a therapeutically relevant underestimation of PD-L1 expression in 28.7% of patients, when only one specimen per patient was analyzed. Inter-tumoral differences in PD-L1 expression between primary tumors and lymph node metastasis (LNM) or LR occurred in 44.4% and 61.5% (CPS) and in 40.6% and 50% of cases (TPS). Overall survival was increased in patients with CPS ≥ 1 vs. CPS < 1 in primary tumors and LNM (hazard ratio: 0.46 and 0.35; p < 0.005); CPS in LR was not prognostic. Our analysis shows clinically relevant intra- and inter-sample heterogeneity of PD-L1 expression in HNSCC. To account for heterogeneity and improve patient selection for immunotherapy, multiple sample analyses should be performed, particularly in patients with CPS/TPS < 1.

PD-1/PD-L1 pathway: Current research in breast cancer.

INTRODUCTION: Immunotherapy has shown encouraging outcomes in breast cancer (BC) treatment in recent years. The programmed cell death ligand 1 (PD-L1) transmembrane protein is suggested to function as a co-inhibitory factor in the immune response, where it collaborates with programmed cell death protein 1 (PD-1) to stimulate apoptosis, suppress cytokine release from PD-1 positive cells, and limit the growth of PD-1 positive cells. Furthermore, in many malignancies, PD-L1 reduces the immune system's response to neoplastic cells. These observations suggest that the PD-1/PD-L1 axis plays a vital role in cancer therapy and the regulation of cancer immune escape mechanisms. This review aimed to provide an overview of the functions of PD-1 and PD-L1 in BC cancer therapy. METHODS: This research design is a literature review. The style is a traditional review on topics or variables relating to the PD-1/PD-L1 pathway. A literature search was carried out using three online databases. RESULTS: The search using the keywords yielded a total of 248 studies. Each result was filtered again according to the inclusion and exclusion criteria, resulting in a final total of 4 studies to be included in the literature review. CONCLUSIONS: The combination of PD-1/PD-L1 is essential for many malignancies. According to the evidence presented, this combination presents both an opportunity and a challenge in cancer treatment. Since many solid cancers, especially BC, express high levels of PD-1/PD-L1, cancer treatment mainly involves targeted therapies.

Photodynamic therapy upregulates expression of HIF-1α and PD-L1 in related pathways and its clinical relevance in non-small-cell lung cancer.

BACKGROUND: Photodynamic therapy (PDT) is a promising interventional treatment approach that contributes to antitumor immunity. It has been reported that PDT can enhance the effectiveness of immune checkpoint inhibitors (ICIs), but its mechanism is yet unclear. Herein, we implemented bioinformatics analysis to detect common pathways and potential biomarkers in non-small cell lung cancer (NSCLC), PDT, and NSCLC immunotherapy to investigate potential links between PDT, immunotherapy and NSCLC, and their clinical impact. METHODS: Differentially expressed genes in NSCLC- and NSCLC immunotherapy-related data in the GEO database were intersected with PDT-related genes in the GeneCards database to obtain candidate genes and shared pathways. Enrichment analysis and protein-protein interaction were established to identify key genes in functionally enriched pathways. The expression profiles and the prognostic significance of key genes were depicted. RESULTS: Bioinformatics analysis showed that HIF-1α was screened as a prognostic gene in hypoxia, HIF-1, and PD-L1-related signaling pathways, which was associated with clinical response in NSCLC patients after PDT and immunotherapy. In vivo experiments showed that PDT could inhibit tumor growth and upregulate HIF-1α and PD-L1 expressions in NSCLC tissues with a positive correlation, which might influence the blocking activity of ICIs on the HIF-1, and PD-L1-related signaling pathways. CONCLUSIONS: PDT might improve the clinical response of ICIs by upregulating tumor HIF-1α and PD-L1 expressions in NSCLC.

Difference between Keratinized- and Non-Keratinized-Originating Epithelium in the Process of Immune Escape of Oral Squamous Cell Carcinoma.

Immune checkpoint inhibitors (ICIs), including anti-programmed cell death 1 ligand 1 (PD-L1) antibodies, are significantly changing treatment strategies for human malignant diseases, including oral cancer. Cancer cells usually escape from the immune system and acquire proliferative capacity and invasive/metastatic potential. We have focused on the two immune checkpoints, PD-1/PD-L1 and CD47/SIRPα, in the tumor microenvironment of oral squamous cell carcinoma (OSCC), performed a retrospective analysis of the expression of seven immune-related factors (PD-L1, PD-1, CD4, CD8, CD47, CD56 and CD11c), and examined their correlation with clinicopathological status. As a result, there were no significant findings relating to seven immune-related factors and several clinicopathological statuses. However, the immune checkpoint-related factors (PD-1, PD-L1, CD47) were highly expressed in non-keratinized epithelium-originated tumors when compared to those in keratinized epithelium-originated tumors. It is of interest that immunoediting via immune checkpoint-related factors was facilitated in non-keratinized sites. Several researchers reported that the keratinization of oral mucosal epithelia affected the immune response, but our present finding is the first study to show a difference in tumor immunity in the originating epithelium of OSCC, keratinized or non-keratinized. Tumor immunity, an immune escape status of OSCC, might be different in the originating epithelium, keratinized or non-keratinized.

Effectiveness of PD1/PD-L1 combined with anti-angiogenic drugs in patients with advanced nonsmall cell lung cancer: A systematic review and meta-analysis.

Background: Protein-1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) therapy have become an important treatment approach for patients with advanced nonsmall cell lung cancer (NSCLC), but primary or secondary resistance remains a challenge for some patients. PD-1/PD-L1 combined with anti-angiogenic drugs (AAs) in NSCLC patients have potential synergistic effects, and the survival benefit may vary based on a treatment order. To investigate the efficacy of PD-1/PD-L1 combined with AAs as the treatment for patients with advanced NSCLC. Materials and Methods: We comprehensively searched EMBASE, PubMed, Web of Science, CNKI, VIP, and Wanfang databases from January 2017 to September 2022. The Cochrane risk bias tool evaluated the quality of included randomized clinical trials. Newcastle-Ottawa-Scale score was used to evaluate the quality of retrospective studies. Publication bias was evaluated by funnel plot, Begg's test, and Egger's test. Results: Seventeen articles were finally selected, involving 5182 patients. Meta-analysis results showed that PD1/PD-L1 combined with AAs therapy significantly improved progression-free survival (PFS) (hazard ratio [HR] = 0.61, 95% confidence interval [CI]: 0.50-0.75, P < 0.00001), overall survival (OS) (HR = 0.79, 95% CI: 0.71-0.88, P < 0.00001), and objective response rate (ORR) (risk ratio = 0.88, 95% CI: 0.81-0.96, P = 0.004), with the statistically significant difference. The sensitivity analysis demonstrated the robustness of the PFS, ORR, and OS. Conclusion: The combination of PD-1/PD-L1 inhibitors with AAs in treating advanced patients has exhibited notable therapeutic advantages when contrasted with monotherapy. Specifically, the administration of PD-1/PD-L1 inhibitors in conjunction with AAs, or sequential treatment involving PD-1/PD-L1 followed by AAs, has shown enhanced therapeutic efficacy in this patient population.

Systemic immune index predicts tumor-infiltrating lymphocyte intensity and immunotherapy response in small cell lung cancer.

Background: Despite recent progresses in immune checkpoint blockade (ICB) in small-cell lung cancer (SCLC), a lack of understanding regarding the systemic tumor immune environment (STIE) and local tumor immune microenvironment (TIME) makes it difficult to accurately predict clinical outcomes and identify potential beneficiaries from ICB therapy. Methods: We enrolled 191 patients with stage I-III SCLC and comprehensively evaluated the prognostic role of STIE by several quantitative measurements, and further integrate it with a local immune score system (LISS) established by eXtreme Gradient Boosting (XGBoost) machine learning algorithm. We also test the value of STIE in beneficiary selection in our independent advanced SCLC cohort receiving programmed cell death 1 ligand 1 (PD-L1) blockade therapy. Results: Among several systemic immune markers, the STIE as assessed by prognostic nutritional index (PNI) was correlated with disease-free survival (DFS) and overall survival (OS), and remained as an independent prognostic factor for SCLC patients [hazard ratio (HR): 0.473, 95% confidence interval (CI): 0.241-0.929, P=0.030]. Higher PNI score was closely associated with inflamed SCLC molecular subtype and local tumor-infiltrating lymphocytes (TILs). We further constructed a LISS which combined top three important local immune biomarkers (CD8+ T-cell count, PD-L1 expression on CD8+ T-cell and CD4+ T-cell count) and integrated it with the PNI score. The final integrated immune risk system was an independent prognostic factor and achieved better predictive performance than Tumor Node Metastasis (TNM) stages and single immune biomarker. Furthermore, PNI-high extensive-stage SCLC patients achieved better clinical response and longer progression-free survival (PFS) (11.8 vs. 5.9 months, P=0.012) from PD-L1 blockade therapy. Conclusions: This study provides a method to investigate the prognostic value of overall immune status by combining the PNI with local immune biomarkers in SCLC. The promising clinical application of PNI in efficacy prediction and beneficiary selection for SCLC immunotherapy is also highlighted.

Human PD-L1 overexpression decreases xenogeneic human T-cell immune responses towards porcine kidneys.

Xenotransplantation offers a promising alternative to circumvent the lack of donated human organs available for transplantation. Different attempts to improve the survival of xenografts led to the generation of transgenic pigs expressing various combinations of human protective genes or knocked out for specific antigens. Currently, testing the efficiency of porcine organs carrying different genetic modifications in preventing xenogeneic immune responses completely relies on in vitro assays, humanized mouse models, or non-human primate transplantation models. However, these tests are often associated with major concerns due to reproducibility and generation of insufficient data as well as they raise ethical, logistical, and economic issues. In this study, we investigated the feasibility of specifically assessing the strength of human T-cell responses towards the kidneys of wild-type (WT) or transgenic pigs overexpressing human programmed death-1 ligand 1 (hPD-L1) during ex vivo kidney perfusion (EVKP). Human T cells were shown to adhere to the endothelium and transmigrate into WT and hPD-L1 kidneys. However, transcript levels of TNF-a and IFN-y as well as cytotoxic molecules such as granzyme B and perforin secreted by human T cells were significantly decreased in the tissue of hPD-L1 kidneys in comparison to WT kidneys. These results were confirmed via in vitro assays using renal endothelial cells (ECs) isolated from WT and hPD-L1 transgenic pigs. Both CD4+ and CD8+ T cells showed significantly lower proliferation rates after exposure to hPD-L1 porcine renal ECs in comparison to WT ECs. In addition, the secretion of pro-inflammatory cytokines was significantly reduced in cultures using hPD-L1 ECs in comparison to WT ECs. Remarkably, hPD-L1 EC survival was significantly increased in cytotoxic assays. This study demonstrates the feasibility of evaluating the human response of specific immune subsets such as human T cells towards the whole xenograft during EVKP. This may represent a robust strategy to assess the potency of different genetic modifications to prevent xenogeneic immune responses and thereby predict the risk of immune rejection of new genetically engineered xenografts.

Targeting cancer-associated fibroblast autophagy renders pancreatic cancer eradicable with immunochemotherapy by inhibiting adaptive immune resistance.

Accumulating evidence suggests that cancer-associated fibroblast (CAF) macroautophagy/autophagy is crucial in tumor development and may be a therapeutic target for pancreatic ductal adenocarcinoma (PDAC). However, the role of CAF autophagy during immune surveillance and cancer immunotherapy is unclear. The present study revealed that the inhibition of CAF autophagy suppresses in vivo tumor development in immune-deficient xenografts. This deletion compromises anti-tumor immunity and anti-tumor efficacy both in vitro and in vivo by upregulating CD274/PDL1 levels in an immune-competent mouse model. A block in CAF autophagy reduced the production of IL6 (interleukin 6), disrupting high desmoplastic TME and decreasing USP14 expression at the transcription level in pancreatic cancer cells. We further identify USP14 as the post-translational factor responsible for downregulating CD274 expression by removing K63 linked-ubiquitination at the K280 residue. Finally, chloroquine diphosphate-loaded mesenchymal stem cell (MSC)-liposomes, by accurately targeting CAFs, inhibited CAF autophagy, improving the efficacy of immunochemotherapy to combat pancreatic cancer.Abbreviation: AIR: adaptive immune resistance; ATRA: all-trans-retinoicacid; CAF: cancer-associated fibroblast; CD274/PDL1: CD274 molecule; CM: conditioned medium; CQ: chloroquine diphosphate; CyTOF: Mass cytometry; FGF2/bFGF: fibroblast growth factor 2; ICB: immune checkpoint blockade; IF: immunofluorescence; IHC: immunohistochemistry; IP: immunoprecipitation; MS: mass spectrometer; MSC: mesenchymal stem cell; PDAC: pancreatic ductal adenocarcinoma; TEM: transmission electron microscopy; TILs: tumor infiltrating lymphocytes; TME: tumor microenvironment; USP14: ubiquitin specific peptidase 14.

Pathogenicity of functionally activated PD-1+CD8+ cells and counterattacks by muscular PD-L1 through IFNγ in myositis.

Programmed-cell-death 1 (PD-1) expression is associated not only with T-cell activation but with exhaustion. Specifically, PD-1+ T cells present an exhausted phenotype in conditions of chronic antigen exposure, such as tumor microenvironments and chronic viral infection. However, the immune status regarding exhaustion of PD-1+CD8+ T cells in chronic autoimmune diseases including idiopathic inflammatory myopathies (IIMs) remains unclear. We aimed to clarify the role of PD-1+CD8+ T cells and PD-1 ligand (PD-L1) in IIMs. We showed that PD-1+ cells infiltrated into PD-L1-expressing muscles in patients with IIMs and immune checkpoint inhibitor-related myopathy. According to the peripheral blood immunophenotyping, the PD-1+CD8+ cell proportions were comparable between the active and inactive patients. Of note, PD-1+CD8+ cells in the active patients highly expressed cytolytic molecules, indicating their activation, while PD-1-CD8+ cells expressed low levels of cytolytic molecules in the active and inactive patients. A part of PD-1+CD8+ cells expressed the HMG-box transcription factor TOX highly and presented the exhausted phenotype in the active patients. Among PD-1+CD4+ T cells, PD-1highCXCR5-CD45RO+CD4+ peripheral helper T cells were increased in the active patients. PD-L1-deficient mice developed severer C-protein-induced myositis (CIM), a model of polymyositis, with abundant infiltration of PD-1+CD8+ cells expressing cytolytic molecules than wild-type mice, indicating pathogenicity of the PD-1+CD8+ cells and the protective role of PD-L1. The deficiency of IFNγ, a general PD-L1-inducer, impaired muscular PD-L1 expression and exacerbated CIM, indicating IFNγ-dependent muscular PD-L1 regulation. IFNγ-induced PD-L1 on myotubes was protective in an established muscle injury model. In conclusion, PD-1+CD8+ T cells rather than PD-1-CD8+ T cells were a pathogenic subset of IIMs. Muscular PD-L1 was regulated by IFNγ and exerted protective properties in IIMs.

Interferon-gamma and tumor necrosis factor-alpha synergistically enhance the immunosuppressive capacity of human umbilical-cord-derived mesenchymal stem cells by increasing PD-L1 expression.

BACKGROUND: The immunosuppressive capacity of mesenchymal stem cells (MSCs) is dependent on the "license" of several proinflammatory factors to express immunosuppressive factors such as programmed cell death 1 ligand 1 (PD-L1), which determines the clinical therapeutic efficacy of MSCs for inflammatory or immune diseases. In MSCs, interferon-gamma (IFN-γ) is a key inducer of PD-L1 expression, which is synergistically enhanced by tumor necrosis factor-alpha (TNF-α); however, the underlying mechanism is unclear. AIM: To reveal the mechanism of pretreated MSCs express high PD-L1 and explore the application of pretreated MSCs in ulcerative colitis. METHODS: We assessed PD-L1 expression in human umbilical-cord-derived MSCs (hUC-MSCs) induced by IFN-γ and TNF-α, alone or in combination. Additionally, we performed signal pathway inhibitor experiments as well as RNA interference experiments to elucidate the molecular mechanism by which IFN-γ alone or in combination with TNF-α induces PD-L1 expression. Moreover, we used luciferase reporter gene experiments to verify the binding sites of the transcription factors of each signal transduction pathway to the targeted gene promoters. Finally, we evaluated the immunosuppressive capacity of hUC-MSCs treated with IFN-γ and TNF-α in both an in vitro mixed lymphocyte culture assay, and in vivo in mice with dextran sulfate sodium-induced acute colitis. RESULTS: Our results suggest that IFN-γ induction alone upregulates PD-L1 expression in hUC-MSCs while TNF-α alone does not, and that the co-induction of IFN-γ and TNF-α promotes higher expression of PD-L1. IFN-γ induces hUC-MSCs to express PD-L1, in which IFN-γ activates the JAK/STAT1 signaling pathway, up-regulates the expression of the interferon regulatory factor 1 (IRF1) transcription factor, promotes the binding of IRF1 and the PD-L1 gene promoter, and finally promotes PD-L1 mRNA. Although TNF-α alone did not induce PD-L1 expression in hUC-MSCs, the addition of TNF-α significantly enhanced IFN-γ-induced JAK/STAT1/IRF1 activation. TNF-α up-regulated IFN-γ receptor expression through activation of the nuclear factor kappa-B signaling pathway, which significantly enhanced IFN-γ signaling. Finally, co-induced hUC-MSCs have a stronger inhibitory effect on lymphocyte proliferation, and significantly ameliorate weight loss, mucosal damage, inflammatory cell infiltration, and up-regulation of inflammatory factors in colitis mice. CONCLUSION: Overall, our results suggest that IFN-γ and TNF-α enhance both the immunosuppressive ability of hUC-MSCs and their efficacy in ulcerative colitis by synergistically inducing high expression of PD-L1.

Expression pattern of PD-1/PD-L1 in primary liver cancer with clinical correlation.

Immunotherapy, including ICIs, has emerged as an invaluable treatment option for advanced PLC. Nevertheless, the expression patterns of PD-L1 and PD-1 in PLC remain incompletely understood. In this study, the expression pattern and clinical correlation of PD-L1 and PD-1 were analysed in 5245 PLC patients. The positivity rates of PD-L1 and PD-1 were very low in the patient PLCs, but the positivity rates of PD-L1 and PD-1 were higher in the ICC and cHCC-ICC than in HCC. The expression of PD-L1 and PD-1 correlated with the malignant phenotypes and clinicopathological characteristics of PLC. Interestingly, PD-1 positivity might serve as an independent prognostic factor. Based on a systematic analysis of a large amount of PLC tissues, we proposed a novel classification of PD-1/PD-L1 expression in HCC and ICC. In light of this stratification, we observed a close correlation between PD-L1 levels and PD-1 expression in HCC and ICC.

On the Road towards Small-Molecule Programmed Cell Death 1 Ligand 1 Positron Emission Tomography Tracers: A Ligand-Based Drug Design Approach.

PD-1/PD-L1 immune checkpoint blockade for cancer therapy showed promising results in clinical studies. Further endeavors are required to enhance patient stratification, as, at present, only a small portion of patients with PD-L1-positive tumors (as determined by PD-L1 targeted immunohistochemistry; IHC) benefit from anti-PD-1/PD-L1 immunotherapy. This can be explained by the heterogeneity of tumor lesions and the intrinsic limitation of multiple biopsies. Consequently, non-invasive in vivo quantification of PD-L1 on tumors and metastases throughout the entire body using positron emission tomography (PET) imaging holds the potential to augment patient stratification. Within the scope of this work, six new small molecules were synthesized by following a ligand-based drug design approach supported by computational docking utilizing lead structures based on the (2-methyl-[1,1'-biphenyl]-3-yl)methanol scaffold and evaluated in vitro for potential future use as PD-L1 PET tracers. The results demonstrated binding affinities in the nanomolar to micromolar range for lead structures and newly prepared molecules, respectively. Carbon-11 labeling was successfully and selectively established and optimized with very good radiochemical conversions of up to 57%. The obtained insights into the significance of polar intermolecular interactions, along with the successful radiosyntheses, could contribute substantially to the future development of small-molecule PD-L1 PET tracers.

Analysis of Expression of Programmed Cell Death Ligand 1 (PD-L1) and BRAFV600E Mutation in Thyroid Cancer.

In thyroid cancer, it has been suggested that PD-L1 overexpression is associated with some clinicopathological factors and prognosis. The aim of this study is to characterize the expression of PD-L1, the presence of the BRAFV600E mutation, as well as cellular and humoral immunity in thyroid cancer, and to investigate the factors that predict the effectiveness of anti-PD-L1 antibody therapy. Blood samples were collected from 33 patients who were newly diagnosed with thyroid cancer after surgery or biopsy. PD-L1 expression, BRAFV600E mutation, and CD8+ expression were examined by immunohistological staining using clinical thyroid cancer specimens. With a PD-L1 staining cut-off value of 1%, 13 (39.4%) patients were classified as PD-L1 positive. Stimulation Index (SI) is an indicator of T cell activation. PD-L1 expression was significantly correlated with low SI level (p = 0.046). Moreover, BRAFV600E mutation was detected in 24 of the 33 (72.7%) patients, and was significantly associated with PD-L1 expression (p = 0.047). In addition, enhanced CD8+ expression was significantly associated with PD-L1 expression (p = 0.003). Multivariate analyses confirmed that high CRP levels (p = 0.039) were independently and significantly associated with poor progression-free survival. These findings suggest that elevated PD-L1 status can be a prognostic indicator for survival in patients with thyroid cancer when comprehensively assessed using the expression of CD8+, the presence of BRAFV600E mutation and the patient's immune status.

Evaluation of Molecular-level Changes of Programmed Cell Death Ligand-1 after Radiation Therapy in a BALB/c CT26 Colorectal Mouse Tumor Model.

The effects of radiation therapy (RT) for cancer can be systemic and partially mediated by the immune system. However, radiation alone is unlikely to transform an immunosuppressive environment into an immunostimulatory one. Therefore, an effective combination of RT and immunotherapy may provide a new, more efficient treatment approach. Here, we investigated how the expression of programmed cell death-ligand 1 (PD-L1) in the tumor microenvironment varied in different RT regimens with the same biologically effective dose. In this study, female BALB/c mice inoculated with CT26 tumor cells were irradiated with 3 different RT regimens using the same BED of 40 gray (Gy). These included ablative RT (1*15 Gy), hypo-fractionated RT (2*10 Gy), and conventional (Hyper-fractionated) RT (10*3 Gy). PD-L1 expression was analyzed with immunohistochemical staining on days 2 and 20 and when the size of tumors had reached 2 cm2 after RT. All treated groups expressed PD-L1, but the group receiving single ablative high-dose RT showed higher expression compared to the other groups. No significant differences in PD-L1 expression were observed at different times in the same group. These findings showed that different regimens of RT have different effects on the TME, so a combination of RT and immune checkpoint blockade could be clinically used in cancer patients.

PD-L1 Exon 3 Is a Hidden Switch of Its Expression and Function in Oral Cancer Cells.

The interaction between programmed cell death 1 ligand 1 (PD-L1) and programmed cell death protein 1 (PD-1) protects tumor cells from immune surveillance. PD-L1 exon 3 is a potential alternative exon and encodes an Ig variable (IgV) domain. Here, we found that a lack of exon 3 leads to the significant loss of cellular membrane locations and the dramatically reduced protein expression of PD-L1, indicating that PD-L1 exon 3 is essential for its protein expression and translocation to the cell membrane. Notably, oral cancer cells show almost no exon 3 skipping to ensure the expression of the full-length, functional PD-L1 protein. We discovered two key exonic splicing enhancers (ESEs) for exon 3 inclusion. Two efficient antisense oligonucleotides (ASOs) were identified to block these two ESEs, which can significantly trigger exon 3 skipping and decrease the production of full-length, functional PD-L1 on the surface of cancer cells. Treatment of oral cancer cells with these ASOs significantly enhanced immune cells' suppression of cancer cell proliferation. Surprisingly, these two ASOs also significantly inhibited cell growth and induced cell pyroptosis in oral cancer cells. Altogether, the results of our study demonstrate the pivotal roles of exon 3 in PD-L1 expression and provide a novel anti-PD-L1 method.

[Consistency comparison of programmed cell death 1-ligand 1 in different immuno-histochemical staining methods].

OBJECTIVE: To compare the consistency of programmed cell death 1-ligand 1 (PD-L1, clone E1L3N, 22C3, SP263) in different immunohistochemical staining methods. METHODS: The first step was to select the optimal process: The PD-L1(clone E1L3N) antibody recommended process, self-built process ①, self-built process ② and self-built process ③ were used to perform immunohistochemical staining in 5 cases of tonsil tissue. The quality of all slides was scored by expert pathologists (0-6 points). The process with the highest score was selected. The second step was to compare the consistency between the optimal procedure and the two standard procedures. Thirty-two cases of lung non-small cell carcinoma diagnosed by pathology in Peking University First Hospital in the past two years were randomly selected. The 32 cases were stained in parallel with the SP263 and 22C3 standard procedures, and all stained slides were scored by specialized pathologists for tumor proportion score (TPS). The scoring results were grouped according to < 1%, ≥1% to < 10%, ≥10% to < 50%, and ≥50%. The consistency of PD-L1 detection antibody clone E1L3N and 22C3, E1L3N and SP263 staining results was analyzed. RESULTS: Tonsil stained slides scores (0-6 points) were as follows: The recommended protocol was 5, 5, 5, 5 and 5. The self-built process ① was 5, 6, 6, 5 and 6. The self-built process ② was 4, 4, 4, 4 and 4.The self-built process ③ was 3, 3, 3, 3 and 3. The self-built process ① was the best with the highest score. The TPSs of 32 non small cell lung carcinoma (NSCLC) cases were as follows: Of self-built process ①, 6 cases were lower than 1%, 5 cases were from 1% to 10%, 10 cases were from 10% to 50%, and 11 cases were higher than 50%; of 22C3 standard procedure, 5 cases were lower than 1%, 3 cases were from 1% to 10%, 13 cases were from 10% to 50%, 11 cases were higher than 50%; of SP263 standard procedure, 7 cases were lower than 1%, 4 cases were from 1% to 10%, 11 cases were from 10% to 50%, 10 cases were higher than 50%. The results of the consistency test were as follows: The κ value for self-built process ① and 22C3 standard procedure was 0.736 (P < 0.001), the agreement was good; the κ value for self-built process ① and SP263 standard procedure was 0.914 (P < 0.001), the agreement was very good. CONCLUSION: The immunostaining using PD-L1(E1L3N) with validated self-built staining protocol ① by Ventana Benchmark GX platform can obtain high quality of slides, and the TPSs based on these slides are in good agreement with 22C3 and SP263 standard procedures.

[Thoracic SMARCA4-deficient undifferentiated tumor-pathological diagnosis and combined immune checkpoint inhibitor treatment].

We explored clinicopathological features and treatment strategies for thoracic SMARCA4-deficient undifferentiated tumor (SMARCA4-UT). Thoracic SMARCA4-UT is a new entity recently acknowledged in the 2021 edition of World Health Organization Classification of Thoracic Tumors, and doctors are relatively unfamiliar with its diagnosis, treatment, and prognosis. Taking a case of SMARCA4-UT treated in Peking University First Hospital as an example, this multi-disciplinary discussion covered several hot issues on diagnosing and treating thoracic SMARCA4-UT, including histological features, immu- nohistochemical and molecular phenotype, immune checkpoint inhibitor (ICI) therapy, and pathological assessment of neoadjuvant therapy response. The patient was an older man with a long history of smoking and was admitted due to a rapidly progressing solid tumor in the lower lobe of the right lung. Histologically, tumor cells were epithelioid, undifferentiated, diffusely positive for CD34, and partially positive for SALL4.The expression of BRG1 protein encoded by SMARCA4 gene was lost in all of tumor cells, and next-generation sequencing(NGS)confirmed SMARCA4 gene mutation (c.2196T>G, p.Y732Ter). The pathological diagnosis reached as thoracic SMARCA4-UT, and the preoperative TNM stage was T1N2M0 (ⅢA). Tumor proportion score (TPS) detected by immunohistochemistry of programmed cell death 1-ligand 1 (PD-L1, clone SP263) was 2%. Tumor mutation burden (TMB) detected by NGS of 1 021 genes was 16. 3/Mb. Microsatellite detection showed the tumor was microsatellite stable (MSS). Neo-adjuvant therapy was implemented with the combined regimen of chemotherapy and ICI. Right lower lobectomy was performed through thoracoscopy after the two weeks' neoadjuvant. The pathologic assessment of lung tumor specimens after neoadjuvant therapy revealed a complete pathological response (CPR). The post-neoadjuvant tumor TNM stage was ypT0N0M0. Then, five cycles of adjuvant therapy were completed. Until October 2022, neither tumor recurrence nor metastasis was detected, and minimal residual disease (MRD) detection was negative. At present, it is believed that if BRG1 immunohistochemical staining is negative, regardless of whether SMARCA4 gene mutation is detected, it should be classified as SMARCA4-deficient tumors. SMARCA4-deficient tumors include a variety of carcinomas and sarcomas. The essential criteria for diagnosing SMARCA4-UT includes loss of BRG1 expression, speci-fic histological morphology, and exclude other common thoracic malignant tumors with SMARCA4-deficiency, such as squamous cell carcinoma, adenocarcinoma and large cell carcinoma. SMARCA4-UT is a very aggressive malignant tumor with a poor prognosis. It has almost no targeted therapy mutations, and little response to chemotherapy, but ICI is currently the only effective drug. The successful diagnosis and treatment for this case of SMARCA4-UT should enlighten significance for various kinds of SMARCA4-deficient tumors.