Expression Levels Of PD-1 Research Articles

Follicular Helper T Cells in Pulmonary Tuberculosis: A Retrospective Study.

Follicular helper T lymphocyte (Tfh) promotes antibody production by B lymphocytes in various diseases, including Pulmonary Tuberculosis (PTB). To explore the potential role of Tfh cells and assess the expression level of PD-1, and IL-21 in PTB. 54 newly diagnosed smear-positive PTB, 27 people with latent tuberculosis (LTB) and 27 healthy controls (HC) were enrolled. The PTB group was further divided based on the range of lung field involved (focus number>=3, PTB-X3; <3, PTB-X2). After 6-month therapy, sputum smear (positive, PTB-SP; negative, PTB-SN) or imaging examinations (lesion reduction significant, PTB-os; insignificant, PTB-s) were used to evaluate the conditions of PTB patients. Blood samples were collected from PTB group at month six. CD4+CXCR5+Tfh, and its subsets, CD4+CXCR5+PD-1+Tfh and CD4+CXCR5+ICOS+Tfh in peripheral blood mononuclear cells (PBMCs) were detected. Serum IL-21 concentrations were measured. The frequencies of CD4+CXCR5+Tfh, CD4+CXCR5+ICOS+Tfh and CD4+CXCR5+PD-1+Tfh were higher in PTB group than in HC. IL-21, IL-4 and IFNγ concentrations were significantly higher in PTB group than in HC. The proportion of CD4+CXCR5+Tfh in PTB-X2 was lower than in PTB-X3 group. CD4+CXCR5+PD-1+Tfh proportion in PTB-X2 was lower than that in the PTB-X3. After treatment, CD4+CXCR5+Tfh proportion was significantly lower in the PTB-SN group. CD4+CXCR5+Tfh was lower in the PTB-os group than in the PTB-s group. However, the CD4+CXCR5+PD-1+Tfh and cytokine concentrations of IL-21 were not different. CD4+CXCR5+Tfh level might predict the sputum results, and lesion decrease rate while CD4+CXCR5+PD-1+Tfh subset and IL-21 were not associated with sputum results or lesion decrease after treatment.

GGT5 Is an Independent Prognostic Biomarker in Stomach Adenocarcinoma.

Gastric cancer is one of the cancers with the highest incidence in the world. Gamma-glutamyltransferase 5 (GGT5) is expressed in different cancers and its role in cancers remains unclear. In this study, we aimed to evaluate the value of GGT5 in stomach adenocarcinoma (STAD). In TCGA, patients with high GGT5 expression had poor overall survival (P=0.006). Based on GSE62254, high expression of GGT5 was associated with poor OS (P=0.014) and PFS (P=0.042). The same result was observed in GSE14210. We further discovered that GGT5 expression was associated with stage, grade, and T stage. Further prognostic analysis of GGT5 showed that GGT5 was associated with prognosis in both univariate analysis (P=0.032) and multivariate analysis (P=0.029). We used gene set enrichment analysis (GSEA) to explore the possible mechanism of GGT5. GSEA suggests that overexpression of GGT5 may be involved in leukocyte transendothelial migration, JAK-STAT signaling pathway, MAPK signaling pathway, and melanoma. The high-expression GGT5 group had higher concentrations of M2 macrophages, T cell regulators, and monocytes, but the contents of plasma cells and M1 macrophages were higher in the low-expression GGT5 group. The results showed that the ESTIMATEScore, ImmuneScore, and StromalScore of the high-expression GGT5 group were higher than those of the low-expression GGT5 group. PD1 and CTLA4 expression levels were higher in the high-expression GGT5 group. The high-expression GGT5 group may be more effective for immunotherapy. These results suggested that GGT5 could be a potential prognostic molecular predictor in STAD.

Exploring Immune-Related Prognostic Signatures in the Tumor Microenvironment of Colon Cancer.

Background: Colon cancer is a common malignant tumor with poor prognosis. The aim of this study is to explore the immune-related prognostic signatures and the tumor immune microenvironment of colon cancer. Methods: The mRNA expression data of TCGA-COAD from the UCSC Xena platform and the list of immune-related genes (IRGs) from the ImmPort database were used to identify immune-related differentially expressed genes (DEGs). Then, we constructed an immune-related risk score prognostic model and validated its predictive performance in the test dataset, the whole dataset, and two independent GEO datasets. In addition, we explored the differences in tumor-infiltrating immune cell types, tumor mutation burden (TMB), microsatellite status, and expression levels of immune checkpoints and their ligands between the high-risk and low-risk score groups. Moreover, the potential value of the identified immune-related signature with respect to immunotherapy was investigated based on an immunotherapeutic cohort (Imvigor210) treated with an anti-PD-L1 agent. Results: Seven immune-related DEGs were identified as prognostic signatures. The areas under the curves (AUCs) of the constructed risk score model for overall survival (OS) were calculated (training dataset: 0.780 at 3 years, 0.801 at 4 years, and 0.766 at 5 years; test dataset: 0.642 at 3 years, 0.647 at 4 years, and 0.629 at 5 years; and the whole dataset: 0.642 at 3 years, 0.647 at 4 years, and 0.629 at 5 years). In the high-risk score group of the whole dataset, patients had worse OS, higher TMN stages, advanced pathological stages, and a higher TP53 mutation rate (p < 0.05). In addition, a high level of resting NK cells or M0 macrophages, and high TMB were significantly related to poor OS (p < 0.05). Also, we observed that high-risk score patients had a high expression level of PD-L1, PD-1, and CTLA-4 (p < 0.05). The patients with high-risk scores demonstrated worse prognosis than those with low-risk scores in multiple datasets (GSE39582: p = 0.0023; GSE17536: p = 0.0008; immunotherapeutic cohort without platinum treatment: p = 0.0014; immunotherapeutic cohort with platinum treatment: p = 0.0027). Conclusion: We developed a robust immune-related prognostic signature that performed great in multiple cohorts and explored the characteristics of the tumor immune microenvironment of colon cancer patients, which may give suggestions for the prognosis and immunotherapy in the future.

Therapeutic Effect of Catgut Implantation at Acupoint in a Mouse Model of Hepatocellular Carcinoma by Suppressing Immune Escape.

Background The occurrence and development of hepatocellular carcinoma (HCC) are closely related to immune function, as is the capacity of hepatoma cells to escape. Immunosurveillance is a key mechanism. Catgut implantation at acupoint (CIAA) is a promising acupuncture improvement method that can regulate immunity and has been widely used in the clinical treatment of a variety of diseases. The aim of this study is to observe the therapeutic effect of CIAA on HCC and to investigate the potential mechanism of immune escape. Materials and Methods A total of 40 mice were randomly divided into three groups: the HCC model group (n = 15), the CIAA treatment group (n = 15), and the control group (n = 10). HCC was chemically induced in 30 mice by the combination of DEN, carbon tetrachloride, and ethanol for 150 days. Among them, 15 were selected for CIAA treatment to ascertain the therapeutic effect. The mRNA expression levels of AFP, IL-10, PD-1, and CTLA-4 in three groups were examined by using RT-PCR. AFP and AKT expressions were measured by using western blotting. PD1, CTLA-4, IL-10, CD4+, and CD8+ protein expression levels were evaluated by using IHC. The mortality rate, body weight, and psychological conditions of three groups were also compared. Results The mRNA and protein expression levels of AFP, PD-1, CTLA-4, and IL-10 were significantly downregulated in the CIAA-treated mice in comparison with HCC mice. IHC assay shows that CD4+ and CD8+ expression levels were notably upregulated after CIAA treatment. Western blotting assay shows that AKT pathway was deactivated in CIAA-treated mice. CIAA notably reduced the mortality rate and inhibited weight loss caused by HCC and improved the overall psychological condition of the mice. Conclusions Taken together, our data corroborate the effective potency of CIAA in the treatment of HCC by and inhibiting immune escape and deactivating the AKT pathway.

Regulating Histone Deacetylase Signaling Pathways of Myeloid-Derived Suppressor Cells Enhanced T Cell-Based Immunotherapy.

Immunotherapy has emerged as a promising approach to combat immunosuppressive tumor microenvironment (TME) for improved cancer treatment. FDA approval for the clinical use of programmed death receptor 1/programmed death-ligand 1 (PD-1/PD-L1) inhibitors revolutionized T cell-based immunotherapy. Although only a few cancer patients respond to this treatment due to several factors including the accumulation of immunosuppressive cells in the TME. Several immunosuppressive cells within the TME such as regulatory T cells, myeloid cells, and cancer-associated fibroblast inhibit the activation and function of T cells to promote tumor progression. The roles of epigenetic modifiers such as histone deacetylase (HDAC) in cancer have long been investigated but little is known about their impact on immune cells. Recent studies showed inhibiting HDAC expression on myeloid-derived suppressor cells (MDSCs) promoted their differentiation to less suppressive cells and reduced their immunosuppressive effect in the TME. HDAC inhibitors upregulated PD-1 or PD-L1 expression level on tumor or immune cells sensitizing tumor-bearing mice to anti-PD-1/PD-L1 antibodies. Herein we discuss how inhibiting HDAC expression on MDSCs could circumvent drawbacks to immune checkpoint inhibitors and improve cancer immunotherapy. Furthermore, we highlighted current challenges and future perspectives of HDAC inhibitors in regulating MDSCs function for effective cancer immunotherapy.

A preliminary study on Fructus Aurantii extract against hepatocarcinoma via glycolysis and PD-1/PD-L1 pathway

Fructus Aurantii (FA) is a traditional herbal medicine that has been widely used for thousands of years in China and possesses a variety of pharmacological effects. FA is full of flavonoids. Several natural bioactive compounds of FA are involved in the treatment of various types of cancers, however, the beneficial effects of FA in hepatocarcinoma and the potential mechanisms of its therapeutic effects have not been fully explored. The present study investigates the anticancer role of flavonoids extracted from FA on human hepatoblastoma cell, HepG2. Eriocitrin, neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, hesperitin, poncirin, meranzin, nobiletin and tangeretin were identified in FAE. The FAE inhibit the proliferation of HepG2 cells in a dose-dependent manner. Metabolic alteration was detected by cell metabolomics and quantitative real time polymerase chain reaction. Flavonoids decreased the level of glycolysis rate-limiting enzymes gene expression in HepG2 cells. It was also observed that the PD-1 and PD-L1 gene expression level were decreased in FAE-treated cells. Collectively, these results suggest that flavonoid extracted from FA inhibits HepG2 cell proliferation by glycolysis and its downstream of PD-1/PD-L1 pathway. These findings suggest that the possible mechanism by which FA exerts its activities in the treatment of hepatocarcinoma and lays a foundation for further experiments and the development of a rational clinical application of FA, which still needs further in vivo investigation in animals and human beings.

The Clinical, Pathological, and Prognostic Value of High PD-1 Expression and the Presence of Epstein-Barr Virus Reactivation in Patients with Laryngeal Cancer.

Simple SummaryOur immune reaction depends on some ‘immune checkpoints’, such as PD-1, PD-L1 and CTLA4, that maintain homeostasis and define new pathways in the fight against carcinogenesis. Viral infections, including EBV (Epstein-Barr Virus) are one of the risk factors for laryngeal cancer. The aim of our study was to evaluate the level of PD-1 receptor in blood, tumor and lymph node samples collected from 45 laryngeal cancer patients and 20 healthy volunteers from control group. We detected the presence of EBV molecules in cancer samples and show the relationship between tumor progression and the level of PD-1 receptor. We confirmed, that EBV infection may affect the PD-1/PD-L1 pathway and develop the laryngeal cancer. What is important, the level of PD-1 on CD4+ T cells in lymph nodes increased the risk of death, so it can be an important prognostic factor (marker) for laryngeal cancer patients’ treatment and their prognosis.Due to the development of molecular diagnostic techniques, the latest research in the diagnosis of cancer diseases, including laryngeal cancer, has been focused on the occurrence of specific types of molecular patterns, including markers expressed on cells of the immune system (e.g., PD-1, PD-L1, and CTLA-4), which may be directly or indirectly involved in the development of neoplastic diseases. Laryngeal cancer is one of the diseases that is diagnosed more often in men than in women, and many factors are involved in its development, including environmental and lifestyle factors, viral infections (e.g., HPV, HHV-1, and EBV), and disorders of the immune system. In this study, we determined the level of PD-1 receptor expression on T and B lymphocytes and their relationships based on the classification of the grade and TNM scale, in turn based on blood, tumor, and lymph node samples from patients diagnosed with laryngeal cancer. In addition, we determined the presence of EBV genetic material in the tested biological materials as well as the degree of cancer advancement and its correlation with the level of PD-1 receptor expression. The results suggested that the level of PD-1 expression on T and B lymphocytes was significantly higher in the tumor samples as compared to the lymph node samples, and their comparison with the immunophenotype results from the blood samples provided statistically significant data on changes in the incidence of individual subpopulations of T and B lymphocytes and the level of PD-1 receptor expression. The analysis of the individual parameters of the TNM scale also showed significant changes between the PD-1 expression and the tested biological material in individual subgroups of the scale. We also found that the expression of PD-1 on the CD4+ T cells from the lymph node samples caused an almost 1.5-fold increase in the risk of death. In the analyses of the presence of EBV, the highest concentration was recorded in the tumor samples, then for the lymph node samples, and followed by the blood samples. Furthermore, we showed that the presence of EBV genetic material was positively correlated with the level of PD-1 expression in the tested biological materials.

T and NK cell abundance defines two distinct subgroups of renal cell carcinoma

ABSTRACT Renal cell carcinoma (RCC) is considered as an immunogenic cancer. Because not all patients respond to current immunotherapies, we aimed to investigate the immunological heterogeneity of RCC tumors. We analyzedthe immunophenotype of the circulating, tumor, and matching adjacent healthy kidney immune cells from 52 nephrectomy patients with multi-parameter flow cytometry. Additionally, we studied the transcriptomic and mutation profiles of 20 clear cell RCC (ccRCC) tumors with bulk RNA sequencing and a customized pan-cancer gene panel. The tumor samples clustered into two distinct subgroups defined by the abundance of intratumoral CD3+ T cells (CD3high, 25/52) and NK cells (NKhigh, 27/52). CD3high tumors had an overall higher frequency of tumor infiltrating lymphocytes and PD-1 expression on the CD8+ T cells compared to NKhigh tumors. The tumor infiltrating T and NK cells had significantly elevated expression levels of LAG-3, PD-1, and HLA-DR compared to the circulating immune cells. Transcriptomic analysis revealed increased immune signaling (IFN-γ, TNF-α via NF-κB, and T cell receptor signaling) and kidney metabolism pathways in the CD3high subgroup. Genomic analysis confirmed the typical ccRCC mutation profile including VHL, PBRM1, and SETD2 mutations, and revealed PBRM1 as a uniquely mutated gene in the CD3high subgroup. Approximately half of the RCC tumors have a high infiltration of NK cells associated with a lower number of tumor infiltrating lymphocytes, lower PD-1 expression, a distinct transcriptomic and mutation profile, providing insights to the immunological heterogeneity of RCC which may impact treatment responses to immunological therapies.

Abnormal expression and clinical significance of surface receptors on natural killer cells in the peripheral blood of patients with non-small cell lung cancer.

Natural killer (NK) cells typically function as frontline lymphocytes against cancer although little is known about their engagement in non-small cell lung cancer (NSCLC). This study compared the performance and activity of NK cells and their subsets in the peripheral blood of NSCLC sufferers and healthy participants. In total, 67 healthy controls (40 males; 59.7%) and 56 patients with NSCLC (35 males; 62.5%) were included (mean age, 66.6 years). Flow cytometry identified NK cells and their subpopulations in external blood, and the total number, proportion, activity, surface activating, and inhibitory receptor expression levels were determined. Results showed that NK cell surface receptors CD107a, IFN-γ, and TNF-α activity were markedly reduced in lung cancer patients compared to healthy controls. The number and ratio of NK cells within the lymphocyte population were decreased in patients. The concentration of the inhibitory receptors TIGIT, TIM-3, CD96, PD-1, and Siglec-7 were increased in patients, whereas the expression level of the activating receptor NKP30 was decreased. Moreover, the expression levels of IFN-γ, TIGIT, CD96, PD-1, and TIM-3 were correlated with the clinical phase of NSCLC. These findings suggest that surface receptors from NK cells are likely to be involved in the evolution of NSCLC.

Abstract P017: Intra-tumoral infiltration of GZMKhigh CD8+ T effector memory cells is associated with poor clinical outcome in early-stage colo-rectal cancer

Abstract Background. A comprehensive understanding of the role of immune reactions in the progression of early-stage colorectal cancer (CRC) is currently lacking. Tumor infiltration by cytotoxic CD8+ T cells has been associated with a better prognosis in several solid tumors, including CRC, and high levels of memory CD8+ T cells prevent early metastatic invasion and are associated with better survival. However, the tumor immune infiltrate is highly heterogeneous and, although the influence of various myeloid cell populations on T cell activity has been reported, our understanding of the interplay between different cell compartments at the tumor site and their impact on the clinical outcome is still in its infancy. Methods. We sought to gain deeper mechanistic insights into the crosstalk between the TME and immune cells to elucidate their contribution to relapse and to implement patient stratification for immune-based therapeutics. Heterogeneity of immune cells within and across a novel prospective cohort of CRC patients (Stage I-III, treatment-naïve, n=60) was estimated by multiparametric flow cytometry, single-cell transcriptomics and multiplexed cytokine array while also obtaining functional information. Findings were validated on a larger independent cohort of CRC patients and a similar analysis extended to lung cancer (TCGA). Thus, we focused our attention on the crosstalk between CD8+ T cells and neutrophils, of which the contribution in inhibiting or promoting tumor progression in humans remains marginally explored and often contradictory. Experiments were conducted in a pre-clinical mouse model and the mechanistic details dissected in vitro, providing initial evidence for future therapeutic applications. Results. We defined an unique immune cell signature in which CRC tumors highly infiltrated by neutrophils contained a population of TILs with a peculiar activation and memory pattern, low levels of PD1 expression and characterized by high levels of Granzyme K (GZMKhigh CD8+ TEM cells). Remarkably, GZMKhigh CD8+ TEM cells were prospectively correlated with tumor relapse and their gene signature was found to be significantly associated with worse prognosis also in TCGA datasets. Here, we will present molecular details characterizing these interactions. Conclusions. Our study highlighted the emergence of a crosstalk between GZMKhigh CD8+ TEM and neutrophils as one of the most important hallmarks in the CRC immune-landscape, crucial to implement current stratification, develop new targets of intervention and guide next steps toward personalized therapeutics. Citation Format: Silvia Tiberti, Carlotta Catozzi, Caterina Scirgolea, Ottavio Croci, Danilo Cagnina, Stefano Campaner, Martin Shaefer, Nicola Fazio, Uberto Fumagalli-Romario, Guangwen Ren, Enrico Lugli, Luigi O. Nezi, Teresa Manzo. Intra-tumoral infiltration of GZMKhigh CD8+ T effector memory cells is associated with poor clinical outcome in early-stage colo-rectal cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P017.

Colorectal Cancer-Associated Immune Exhaustion Involves T and B Lymphocytes and Conventional NK Cells and Correlates With a Shorter Overall Survival.

Colorectal cancer (CRC) is one of the most common cancer worldwide, with a growing impact on public health and clinical management. Immunotherapy has shown promise in the treatment of advanced cancers, but needs to be improved for CRC, since only a limited fraction of patients is eligible for treatment, and most of them develop resistance due to progressive immune exhaustion. Here, we identify the transcriptional, molecular, and cellular traits of the immune exhaustion associated with CRC and determine their relationships with the patient’s clinic-pathological profile. Bioinformatic analyses of RNA-sequencing data of 594 CRCs from TCGA PanCancer collection, revealed that, in the wide range of immune exhaustion genes, those coding for PD-L1, LAG3 and T-bet were associated (Cramér’s V=0.3) with MSI/dMMR tumors and with a shorter overall survival (log-rank test: p=0.0004, p=0.0014 and p=0.0043, respectively), whereas high levels of expression of EOMES, TRAF1, PD-L1, FCRL4, BTLA and SIGLEC6 were associated with a shorter overall survival (log-rank test: p=0.0003, p=0.0188, p=0.0004, p=0.0303, p=0.0052 and p=0.0033, respectively), independently from the molecular subtype of CRC. Expression levels of PD-L1, PD-1, LAG3, EOMES, T-bet, and TIGIT were significantly correlated with each other and associated with genes coding for CD4+ and CD8+CD3+ T cell markers and NKp46+CD94+EOMES+T-bet+ cell markers, (OR >1.5, p<0.05), which identify a subset of group 1 innate lymphoid cells, namely conventional (c)NK cells. Expression of TRAF1 and BTLA co-occurred with both T cell markers, CD3γ, CD3δ, CD3ε, CD4, and B cell markers, CD19, CD20 and CD79a (OR >2, p<0.05). Expression of TGFβ1 was associated only with CD4 + and CD8+CD3ε+ T cell markers (odds ratio >2, p<0.05). Expression of PD-L2 and IDO1 was associated (OR >1.5, p<0.05) only with cNK cell markers, whereas expression of FCRL4, SIGLEC2 and SIGLEC6 was associated (OR >2.5; p<0.05) with CD19+CD20+CD79a+ B cell markers. Morphometric examination of immunostained CRC tissue sections, obtained from a validation cohort of 53 CRC patients, substantiated the biostatistical findings, showing that the highest percentage of immune exhaustion gene expressing cells were found in tumors from short-term survivors and that functional exhaustion is not confined to T lymphocytes, but also involves B cells, and cNK cells. This concept was strengthened by CYBERSORTx analysis, which revealed the expression of additional immune exhaustion genes, in particular FOXP1, SIRT1, BATF, NR4A1 and TOX, by subpopulations of T, B and NK cells. This study provides novel insight into the immune exhaustion landscape of CRC and emphasizes the need for a customized multi-targeted therapeutic approach to overcome resistance to current immunotherapy.

Comparative investigation of cell cycle and immunomodulatory genes in mucosal and cutaneous melanomas: Preliminary data suggest a potential promising clinical role for p16 and the PD-1/PD-L1 axis

Mucosal melanomas arise from the mucosal lining of various organs. Their etiology is currently unknown and there are no tissue-based methods to differentiate it from cutaneous melanomas. Furthermore, prognostic and predictive markers (e.g. for immune checkpoint inhibition) are lacking. In this study, we aimed to assess the protein expression levels of cell cycle-associated proteins and immune checkpoint markers in a cohort of mucosal melanomas in comparison to cutaneous melanomas and evaluated the effect of potential regulatory mechanisms. We performed immunohistochemistry, DNA methylation analysis and copy number profiling of 47 mucosal and 28 cutaneous melanoma samples. Protein expression of CD117, Ki67 and p16 was higher in mucosal melanomas, while BCL2, Cyclin D1, PD-1 and PD-L1 were overexpressed in cutaneous melanomas. CDKN2A deletions were the most prevalent numeric chromosomal alterations in both mucosal and cutaneous melanoma and were associated with decreased p16 expression. KIT was frequently amplified in mucosal melanomas, but not associated with CD117 expression. On the other hand, amplification of CCND1 lead to Cyclin D1 overexpression. In mucosal melanoma patients high PD-1 expression and high PD-L1 promoter methylation levels were associated with improved survival. PD-L1 expression correlated with response to immune checkpoint inhibitor therapy in the combined group of melanoma patients. Mucosal and cutaneous melanomas show different expression levels of cell cycle-associated and immunomodulatory proteins that are partially regulated by DNA methylation and copy number alterations. PD-1 expression and PD-L1 promoter methylation levels might be a prognostic marker for mucosal melanomas.

Expansion of T Follicular Helper Cells in NLC Co-Cultures Reinforces the Concept of Co-Evolution of CLL and Supportive T Helper Cell Clones

Expansion of T Follicular Helper Cells in NLC Co-Cultures Reinforces the Concept of Co-Evolution of CLL and Supportive T Helper Cell Clones

Analysis of the Tumor Microenvironment By Quantitative Immunohistochemistry Reveals Novel Predictors in Classic Hodgkin Lymphoma Treated with Anti-PD1 Therapy

Classical Hodgkin Lymphoma (cHL) is characterized histologically by the presence of neoplastic Hodgkin and Reed-Sternberg cells surrounded by mixed immune cells. Anti-PD-1 therapy is the principal treatment option for relapsed or refractory cHL. To reveal biomarkers for predicting the cHL patients' responses to immunotherapy, pathological tissues of twenty patients with cHL who have been treated with anti-PD1 therapy were collected. Tumor immune microenvironment was investigated by immunostaining and quantitative measurement of CD68 (for macrophages), CD8 (for cytotoxic T-cells), FoxP3 (for Treg), PD-1, PD-L1 and PD-L2 expression levels. The in situ hybridization for Epstein-Barr virus (EBER-ISH) were also performed. The results showed that the main immune indicators of the tumor microenvironment we had tested were independent of the histological subtypes. Patients who were positive for EBER-ISH had higher mean density of PD-L1 than those were negative for EBER-ISH. Single-factor analysis revealed the high expression of PD-L1 and positive EBER-ISH were associated with significantly increased 2-year PFS in cHL treated with anti-PD1, while CD68, CD8, FoxP3, PD-1 and PD-L2 were not. Therefore, we propose that PD-L1 and EBER-ISH may serve as novel predictors for the treatment outcomes of cHL patients with anti-PD1 therapy. DisclosuresNo relevant conflicts of interest to declare.

61 Biomarkers of favorable prognosis guides the identification of tumor reactive CD4+ and CD8+ TILs in endometrial cancer

BackgroundEndometrial cancer (EC) is the most prevalent gynecological cancer and it can be categorized into four molecular subtypes; ultramutated POLE, hypermutated MSI, CNL and CNH. To date, the prevalence, specificity and phenotype of tumor infiltrating lymphocytes (TILs), and their exact role in EC remain controversial. Thus, a thorough investigation deciphering the phenotypic landscape of EC-infiltrating T lymphocytes and their anti-tumor reactivity is still missing.MethodsIn order to study the implications of the T-cells infiltrating EC for prognosis or susceptibility to immune checkpoint inhibitors, we analyzed the phenotypic traits of CD8 and CD4 EC-resident T cells from single-cell suspensions (n=47) by multicolor flow cytometry (19 biomarkers). Correlation analyses between the phenotype of the TILs, EC molecular subgroups and the survival of the patients were performed to identify biomarkers that could predict prognosis or survival. This identification guided the isolation of specific CD8 and CD4 subpopulations based on the differential expression of the selected biomarkers to evaluate their ability to recognize tumor (figure 1).ResultsOur analysis evidenced the presence of CD8+ and CD4+ T cells with distinct levels of PD-1 expression: cells that did not express PD-1 (PD-1-), cells expressing intermediate (PD-1dim) or high (PD-1hi) levels of PD-1. We found that TIM-3+, CD39+, CXCL13+, BCL6+ or Ki67+ cells frequently co-expressed almost exclusively on the PD-1hi, but not on the PD-1- or dim CD8+ TILs. On the other hand, CD4+ TILs displayed co-expression of TIM-3, CD103, CD39, CD38, HLA-DR, CXCL13, BCL6, CXCR5 or Ki67 within the PD-1hi, but also PD-1dim cells. Importantly, our data shows that the frequency of PD-1hi or CD39+ CD8+ EC TILs, and of PD-1hi but not CD39+ CD4+ TILs was associated with improved survival. Of the 5 predominant CD8+ tumor-resident subpopulations observed (PD-1-CD39-, PD-1dimCD39-, PD-1dimCD39+, PD-1hiCD39- and PD-1hiCD39+) the PD-1hiCD39+, but not the PD-1hiCD39- lymphocytes, harbored the highest frequency of autologous tumor-reactive lymphocytes in all four EC tumors studied. However, both the CD4+ PD-1hiCD39+ and the PD-1hiCD39- contained autologous tumor-reactive lymphocytes in all four tumors screened; being the PD-1hiCD39+ cells the subpopulation containing the majority of tumor-reactive CD4+ cells.Abstract 61 Figure 1Schematic workflow of the study. A section of a primary EC tumor resection is digested and stained with specific antibodies to characterize the phenotype of CD8 and CD4 TILs by flow cytometry (upper panel). Targeted sequencing and IHC including typically altered genes in EC are performed in order to molecularly classify the patient‘s cohort into different disease subgroups. Combining the data generated by flow cytometry, targeted sequencing and IHC, specific biomarkers on CD8 and CD4 TILs are investigated for their potential role in predicting molecular subtypes or survival (middle panel). CD8 and CD4 TILs are isolated from the tumor digest based on the differential expression of PD-1 and CD39 and the specific isolated CD8 and CD4 subpopulations are cultured in vitro to expand them to large numbers. The expanded CD8 and CD4 subpopulations are subsequently screened for tumor recognition by co-culturing the isolated subpopulations with autologous tumor cell lines. Tumor recognition is assessed by measuring the up-regulation of the activation markers 4-1BB or OX40 on CD8+ or CD4+ T cells, respectively, by flow cytometry, and by measuring IFN-γ production by ELISPOT.ConclusionsOverall, our data suggest that CD39 expression on CD8+PD-1hi EC-resident T cells defines a tumor-reactive population that plays an important role in protecting patients from recurrence after surgery. However, PD-1 expression but not CD39 on CD4+ TILs, better guides the identification of lymphocyte subsets with enriched tumor-recognition potential that contribute to improved clinical benefit in EC.Ethics ApprovalThis study was approved by the ‘Comité de Ética de Investigación con Medicamentos del Hospital Universitario Vall d’Hebron’ institution’s Ethics Board; approval number PR(AG)537/2019.

MP42-05 THE EXPRESSION LEVEL OF PD-L1 IS CORRELATED WITH CLINICOPATHOLOGICAL CHARACTERISTICS OF PATIENTS WITH VHL-RELATED RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Localized: Surgical Therapy III (MP42)1 Sep 2021MP42-05 THE EXPRESSION LEVEL OF PD-L1 IS CORRELATED WITH CLINICOPATHOLOGICAL CHARACTERISTICS OF PATIENTS WITH VHL-RELATED RENAL CELL CARCINOMA Kaifang Ma, Kenan Zhang, Lin Cai, and Kan Gong Kaifang MaKaifang Ma More articles by this author , Kenan ZhangKenan Zhang More articles by this author , Lin CaiLin Cai More articles by this author , and Kan GongKan Gong More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002063.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Von Hippel-Lindau (VHL) disease is an autosomal-dominant hereditary cancer syndrome, which characterized by renal cell carcinoma and central nervous system hemangioblastoma. Currently, the expression level and clinical significance of PD-1/PD-L1 in VHL-associated renal cell carcinoma (RCC) remain unclear. This study aimed to explore the correlation of PD-L1 expression with clinicopathological features in patients with VHL-related RCC. METHODS: Patients diagnosed with VHL disease and received nephrectomy were recruited. The clinicopathological features and prognosis of the patients were evaluated. The expression of RCC-associated molecular markers was detected by immunohistochemistry. RESULTS: From Dec. 2009 to Dec. 2020, 57 patients with VHL disease were eligible and included in this study. The median age of these patients were 43 years (IQR 31.5-51), the median follow-up period were 36 months (IQR 13-68.5) and the mean size of the renal tumors were 4.19±1.96 cm (range 1.3-10.5). The study about 112 renal tumors showed that 98.2% pathological type was clear-cell RCC, 89.3% pathological stage was pT1 and 69.6% Fuhrman nuclear grade was G1 in VHL-related RCC. The positive PD-L1, TFE-3, CA-9 and CD10 expression were 24.6%, 28%, 70.6% and 43.2%, respectively. The study revealed that positive PD-L1 expression was associated with higher pathological T stage (p <0.037) and advanced Fuhrman nuclear grade (p <0.007). CONCLUSIONS: PD-L1 expression was associated with aggressive clinicopathological features in patients with VHL-related RCC. Larger prospective studies are warranted to further evaluate whether PD-L1 expression level in CCRCC is related to the effectiveness of PD-1/PD-L1 checkpoint inhibitor immunotherapy. Source of Funding: This study was funded by the National Natural Science Foundation of China (No. 81572506 and 81872081) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e772-e773 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Kaifang Ma More articles by this author Kenan Zhang More articles by this author Lin Cai More articles by this author Kan Gong More articles by this author Expand All Advertisement Loading ...

Programmed Death-Ligand 1 Tumor Proportion Score and Overall Survival From First-Line Pembrolizumab in Patients With Nonsquamous Versus Squamous NSCLC

IntroductionFor patients with NSCLC receiving immune checkpoint inhibitors, programmed death-ligand 1 (PD-L1) tumor proportion score (TPS) has been validated as a predictive biomarker for improved overall survival (OS). Nevertheless, its histology-specific predictive value in patients with advanced squamous versus nonsquamous cancers remains unclear. To evaluate the differential value of PD-L1 TPS as a predictive biomarker for OS after first-line pembrolizumab in patients with squamous versus nonsquamous NSCLC. MethodsRetrospective, observational study of patients diagnosed with having advanced NSCLC who were treated between October 2015 and April 2019 at community oncology clinics and academic medical centers in a deidentified electronic health record–derived database. Included patients were diagnosed with having advanced or metastatic NSCLC, received treatment with first-line, single-agent pembrolizumab, and had documentation of PD-L1 testing with a numeric result. Exclusion criteria included alterations in EGFR, ALK, and ROS1. The primary end point was OS from start of first-line pembrolizumab therapy by squamous or nonsquamous histology and PD-1 expression level measured by TPS (low, <50% or high, ≥50%). ResultsThe cohort of 1460 patients with NSCLC who received pembrolizumab as a first-line therapy had a mean age of 72 years. Histology was 28% squamous and 72% nonsquamous. PD-L1 expression was low in 13% and high in 87%. No meaningful differences in age, sex, or smoking history were observed by PD-L1 TPS or histology type. A generalized gamma model adjusting for sex and stage at diagnosis found that for patients with nonsquamous histology, high PD-L1 TPS was significantly associated with improved OS by a median OS difference of 8.4 months (p < 0.001). In contrast, for patients with squamous histology, there was no evidence of association between PD-L1 expression level and OS (p = 0.283). PD-L1–related incremental differences in median OS between the patients with squamous and nonsquamous tumors were significantly different (p = 0.034). ConclusionsAmong patients with NSCLC treated with first-line pembrolizumab, high PD-L1 TPS is associated with OS among patients with nonsquamous NSCLC, but not among patients with squamous NSCLC.

PD-L1 Overexpression, SWI/SNF Complex Deregulation, and Profound Transcriptomic Changes Characterize Cancer-Dependent Exhaustion of Persistently Activated CD4+ T Cells

Simple SummaryGrowing tumors induce an immune response. For proper immune response, both CD8+ and CD4+ effector T cells are required. Tumors avoid attacks from tumor-infiltrating lymphocytes (TILs) via induction of several inhibitory signals, such as PD-L1/2, which bind to the PD-1 receptor, consequently leading to T cell dysfunction, exhaustion, and apoptosis. The mechanism of T cell exhaustion has been studied mostly in CD8+ T cells, although some results suggest that CD4+ effector T cells also undergo exhaustion. In this study, we analyze global transcript profiling, PD-1 and PD-L1 expression, and chromatin status on the PD-L1 locus. We find that in exhausted CD4+ T cells, the levels of PD-L1 are increased at both the transcript and protein levels, while PD-L1 expression depends on SWI/SNF chromatin remodeling and PRC2-repressive complexes. The expression of PD-L1 in exhausted CD4+ T cells can be reversible. Growing tumors avoid recognition and destruction by the immune system. During continuous stimulation of tumor-infiltrating lymphocytes (TILs) by tumors, TILs become functionally exhausted; thus, they become unable to kill tumor cells and to produce certain cytokines and lose their ability to proliferate. This collectively results in the immune escape of cancer cells. Here, we show that breast cancer cells expressing PD-L1 can accelerate exhaustion of persistently activated human effector CD4+ T cells, manifesting in high PD-1 and PD-L1 expression level son T cell surfaces, decreased glucose metabolism genes, strong downregulation of SWI/SNF chromatin remodeling complex subunits, and p21 cell cycle inhibitor upregulation. This results in inhibition of T cell proliferation and reduction of T cell numbers. The RNAseq analysis on exhausted CD4+ T cells indicated strong overexpression of IDO1 and genes encoding pro-inflammatory cytokines and chemokines. Some interleukins were also detected in media from CD4+ T cells co-cultured with cancer cells. The PD-L1 overexpression was also observed in CD4+ T cells after co-cultivation with other cell lines overexpressing PD-L1, which suggested the existence of a general mechanism of CD4+ T cell exhaustion induced by cancer cells. The ChIP analysis on the PD-L1 promoter region indicated that the BRM recruitment in control CD4+ T cells was replaced by BRG1 and EZH2 in CD4+ T cells strongly exhausted by cancer cells. These findings suggest that epi-drugs such as EZH2 inhibitors may be used as immunomodulators in cancer treatment.

Expression of immune checkpoint PD-1 in non-small cell lung cancer is associated with tumor cell DNA-dependent protein kinase.

Immunotherapy using immune checkpoint inhibitors has demonstrated durable responses and has significantly improved survival in patients with non-small cell lung cancer (NSCLC). Moreover, immunotherapy is increasingly used in combination with cytotoxic treatments such as chemotherapy and radiotherapy. Although the combined treatments are more effective, the underling mechanisms that lead to higher antitumor activity are not fully understood. Therefore, the aim of the current retrospective study was to determine the relationship between expression of immune checkpoints [programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1)] and the enzyme DNA-dependent protein kinase (DNA-PK), which is part of a key pathway involved in the repair of cytotoxic cancer therapy induced damage. Surgically excised NSCLC tissues (n=121) were histologically examined for overall extent of inflammation (score 0-3). Expression levels of PD-1 (number of PD-1 positive cells), PD-L1 [tumor proportion score (TPS); %] and DNA-PK (proportion of DNA-PK positive tumor cells; %) were determined using immunohistochemistry. Expressions of these markers were compared in different clinicopathological subgroups and later used for nonparametric Spearman correlation analysis to determine associations. In patients with NSCLC, PD-1 was significantly expressed in males (P=0.030) and in patients with no or trivial inflammation scores (P=0.030). PD-L1 expression was also significantly higher in current smokers (P=0.025). Correlation analysis was based on the individual values of patients and revealed a significant association between one of the targets of immune checkpoint inhibitors and tumor cell DNA-PK. Tumors with higher numbers of PD-1 positive cells also demonstrated higher tumor cell DNA-PK expressions (P=0.027). The results demonstrated a significant positive correlation between the PD-1/PD-L1 axis and tumor cell DNA-PK expression in patients with NSCLC. Further studies are required to clarify the significance of this correlation and its effect on the efficacy of immunotherapy and cytotoxic cancer therapy combinations.

Comparison of two routes of administration of a cationic liposome formulation for a prophylactic DC vaccination in a murine melanoma model

Dendritic cell (DC) vaccination can be achieved via straight loading of vaccine into DCs ex vivo or administration to DCs in vivo. However, there is no certain consensus on which approach is preferable, and each strategy has its advantages and disadvantages, which affect the efficacy and safety of vaccines. It will also be more complicated when a vaccine delivery system is included. In this study, the efficacy of ex vivo pulsed DC-based vaccine compared with in vivo subcutaneous administration of a cationic liposomes (CLs) formulation containing gp100 antigen (gp100-CLs) was evaluated in a murine melanoma model. In combination with an anti-PD-1 antibody, the ex vivo approach of gp100-CLs yielded a significant (P < 0.01) increase in the number of antigen-specific tumors infiltrated lymphocytes (TILs) with a significant upregulation of IFN-γ (P < 0.0001) and PD-1 (P < 0.0001) expression level. They also dampened the function of immunosuppressive regulatory T cells (Tregs) via significant downregulation of IL-10 and TGF-β (P < 0.0001) expression level compared to in vivo approach in the tumor microenvironment (TME). Furthermore, prophylactic immunization with gp100-CLs pulsed DCs ex vivo delayed tumor growth and induced the survival benefit over in vivo immunization. Collectively, the ex vivo DC-based vaccination pulsed with gp100 encapsulated in liposomes synergizes with anti-PD-1 antibody and represents a preferable approach against melanoma.