High PD-1 Expression Research Articles

BIOM-57. PERITUMORAL EDEMA AS A PREDICTOR OF PD-1 EXPRESSION IN BRAIN METASTASES

Abstract Brain metastases represent a significant problem in oncology with associated high morbidity and mortality. They have been associated with increased peritumoral edema which can worsen neurological symptoms and patient outcomes. Peritumoral cerebral edema may be an important metric of interest as some MRI studies have suggested an association between molecular factors and peritumoral edema. We hypothesized that an association exists between PD-1 expression in brain mets from NSCLC and the radiographic peritumoral edema to enhancing tumor ratio (ETR). A retrospective analysis of tumor characteristics was conducted to identify patients with brain metastases from non-small cell lung cancer. Patients were stratified into three groups based on their tumor PD-1 expression levels: 0%, 1-49%, and 50% or higher. Tumor and edema volumes were measured in these patients using MRIs at the time of diagnosis and converted into edema to tumor ratios. Radiographic analysis has been completed for 19 patients with 9 having no PD-1 expression, 5 having low PD-1 expression, and 5 having high PD-1 expression. The no expression group had an average ETR of 3.02 while the low expression group had an average of 15.85 and the high expression group had an average of 9.12. These initial results show increased peritumoral edema in tumors with PD-1 expression when compared to tumors with no PD-1 expression, with a non-significant trend in favor of increased edema to tumor ratio (p = 0.055). The current project is ongoing with plans to expand patient cohorts to affirm or rule out significant ETR association by MRI.

High PD-1 and CTLA-4 expression correlates with host immune suppression in patients and a mouse model infected with Echinococcus multilocularis

BackgroundAlveolar echinococcosis (AE), a fatal disease caused by Echinococcus multilocularis, often affects the liver, with tumor-like growth. However, the mechanism by which E. multilocularis evades host immune surveillance remains unclear.MethodsWe collected liver specimens from hepatic alveolar echinococcosis (HAE) patients and established a mouse model of E. multilocularis infection. Immunofluorescence staining and flow cytometry were performed to analyze programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte associated antigen 4 (CTLA-4) expression in human samples, while flow cytometry and quantitative real-time polymerase chain reaction (PCR) were performed for similar analyses in mouse samples. Cell proliferation and protoscolex (PSC) killing assays were designed to explore how E. multilocularis induces host immunosuppression.ResultsAn inflammatory reaction band with high PD-1 and CTLA-4 expression was found in close liver tissue (CLT). The ratio of regulatory T cells (Tregs) was higher in CLT than in distant liver tissue (DLT), and Tregs in CLT tended to express higher levels of PD-1 and CTLA-4 than those in DLT from HAE patients. Echinococcus multilocularis-infected mice showed significantly elevated expression of PD-1 and CTLA-4 on splenocytes and peritoneal cells. PD-1/PD-L1 or CTLA-4 pathway blockade could relieve the immunosuppressive effects of Tregs from infected mice and enhance PSC killing by mouse splenocytes.ConclusionsE. multilocularis regulated the function of T cells via the PD-1/PD-L1- and CTLA-4-dependent pathways and subsequently evaded host immune attacks. These findings provide insights for investigating the pathogenic mechanism of AE.Graphical

High Expression of Immune Checkpoint Molecules in Different Types of Thyroid Cancer

This study aimed to investigate the expression of programmed cell death protein-1 (PD-1) and its ligand (PD-L1) immune checkpoint molecules in thyroid carcinomas and determine their association with the clinicopathological characteristics of patients. Thyroid tissue specimens from 100 patients diagnosed with primary thyroid carcinomas including papillary thyroid carcinoma (PTC), follicular thyroid carcinoma (FTC), medullary thyroid carcinoma (MTC), and anaplastic thyroid carcinoma (ATC) were collected. Sections were prepared from formalin-fixed paraffin-embedded samples, and PD-1 and PD-L1 expressions were examined using immunohistochemistry. PD-1 was detected in tumor-infiltrating lymphocytes (TILs) in 88% of the patients and tumor cells in 28% of the patients with 10% in PTC, 5% in FTC, 5% in MTC, and 8% in ATC). PD-L1 was found in tumor cells and TILs in 30% and 79% of the patients, respectively. Moreover, a significant difference was observed in PD-1 and PD-L1 expression between tumor cells and TILs across different tumor types. However, their expression in tumor cells and TILs was significantly higher in ATC compared to other tumor types. Additionally, the expression of PD-1 and PD-L1 was significantly associated with an advanced stage, higher tumor size, tumor necrosis, and mitosis. A significant positive correlation was also observed between the expression of PD-1 and PD-L1 in tumor cells and TILs. The higher expression of PD-1 and PD-L1 may contribute to tumor progression. Therefore, combinational immunotherapy by these immune checkpoint inhibitors might be a promising strategy for clinical improvement in patients with thyroid cancer, especially those with ATC.

Novel Compound Heterozygous Variants in the FAS Gene Lead to Fetal Onset of Autoimmune Lymphoproliferative Syndrome (ALPS).

FAS gene defects lead to autoimmune lymphoproliferative syndrome (ALPS), which is often inherited in an autosomal dominant and rarely in an autosomal recessive manner. We report a case of a newborn girl with novel compound heterozygous variants in FAS and reveal the underlying mechanism. Whole-exome sequencing (WES) was used to identify pathogenic variants. Multiparametric flow cytometry analysis, phosflow analysis, and FAS-induced apoptosis assays were used to explore the effects of the variants on FAS expression, apoptosis, and immunophenotype. The HEK293T cells were used to assess the impact of the variants on protein expression and FAS-induced apoptosis. The patient was born with hepatosplenomegaly, anemia, and thrombocytopenia. She also experienced COVID-19, rotavirus infection, herpes simplex virus infection, and severe pneumonia. The proportion of double-negative T cells (DNTs) was significantly elevated. Novel FAS compound heterozygous variants c.310T > A (p.C104S) and c.702_704del (p.T235del) were identified. The apoptotic ability of T cells was defective, and FAS expression on the surface of T cells was deficient. The T235del variant decreased FAS expression, and the C104S protein remained in the endoplasmic reticulum (ER) and could not translocate to the cell surface. Both mutations resulted in loss-of-function in terms of FAS-induced apoptosis in HEK293T cells. The DNTs were mainly terminally differentiated T (TEMRA) and CD45RA+HLA-DR+, with high expression of CD85j, PD-1, and CD57. The percentage of Th1, Tfh, and autoreactive B cells were significantly increased in the patient. The abnormal immunophenotyping was partially attenuated by sirolimus treatment. We identified two variants that significantly affect FAS expression or localization, leading to early disease onset of in the fetus. Abnormalities in the mTOR pathway are associated with a favorable response to sirolimus.

Circulating mucosal-associated invariant T cell alterations in adults with recent-onset and long-term oral lichen planus

BackgroundMucosal-associated invariant T (MAIT) cells play key roles in many inflammatory diseases. However, their effects on the long-term course of oral lichen planus (OLP) and recent-onset OLP remain unclear. In this study, we aimed to investigate the function of MAIT cells in the different processes of OLP and to explore the immunological background of this disease.MethodsThe frequency, phenotype, cytokine secretion, and clinical relevance of MAIT cells were investigated. MAIT cells were collected from the peripheral blood of 14 adults with recent-onset OLP (7–120 days after disease onset) and 16 adults with long-term course OLP (>2 years after diagnosis) using flow cytometry and compared with 15 healthy blood donors. Statistical analyses were performed using the GraphPad Prism software.ResultsMAIT cells from adults with recent-onset OLP exhibited an activated phenotype, as indicated by an increased frequency of CD69+ (p < 0.05) and CD38+MAIT cells (p < 0.01) and elevated production of the proinflammatory cytokine IL-17 A (p < 0.01), compared with healthy adult donors. In adults with long-term OLP, MAIT cells exhibited an activated and exhausted phenotype, characterized by high expression of CD69 (p < 0.01) and PD-1 (p < 0.001) and increased production of granzyme B released (p < 0.01). Compared with recent-onset OLP patients, long-term OLP patients showed a decreased production of CD8+, and CD4−CD8− cells, but an increase in PD-1+ production (p < 0.05).ConclusionsCirculating MAIT cells exhibited activation in OLP patients across varying disease durations. Given that PD-1 expression is elevated in adults with long-term OLP, it is reasonable to infer that circulating MAIT cells in long-term OLP may exhibit a more exhausted state than those in recent-onset OLP.

Multi-dimensional immunotyping of human NF1-associated peripheral nerve sheath tumors uncovers tumor-associated macrophages as key drivers of immune evasion in the tumor microenvironment.

Malignant peripheral nerve sheath tumors (MPNST) are aggressive soft tissue sarcomas and the leading cause of mortality in individuals with neurofibromatosis type 1 (NF1). Despite many clinical trials, outcomes for patients with MPNST have remained stagnant and most succumb to their disease; thus, novel therapeutic approaches are needed. A better understanding of the MPNST immune ecosystem will aid in the development of strategies to activate the immune system against the tumor. Herein, we profile the tumor immune microenvironment (TIME) in NF1-associated peripheral nerve sheath tumors (PNST) to discover insights on the role that tumor-infiltrating immune cells play in malignant transformation. Utilizing fresh and formalin-fixed, paraffin-embedded tissue from patients diagnosed with NF1-PNST, we dissected the TIME by using immunohistochemistry, multiparameter flow cytometry, and comparative transcriptomic studies. Immunophenotyping confirmed increased immune cells infiltration during malignant progression, with a predominance of infiltrating myeloid cells, particularly CD163+ tumor-associated macrophages (TAM). The T cells within MPNST exhibited signs of tumor activation, characterized by high PD-1 expression. Additionally, MPNST specimens demonstrated elevated levels of immunosuppressive TAM, with heightened PD-L1 expression. The proportion of CD163+ myeloid cells within the TIME correlated with poorer progression free survival. Notably, loss of H3K27 trimethylation correlated with low immune cell infiltration in MPNST. Malignant transformation of NF1-PNST is characterized by an immunosuppressive microenvironment comprising of TAM with high expression of PD-L1, which are associated with inferior outcomes. These findings suggest a clinical potential of immune modulating therapeutics that can unleash an anti-tumor immune response.

Epigenetic tuning of PD-1 expression improves exhausted T cell function and viral control.

PD-1 is a key negative regulator of CD8+ T cell activation and is highly expressed by exhausted T cells in cancer and chronic viral infection. Although PD-1 blockade can improve viral and tumor control, physiological PD-1 expression prevents immunopathology and improves memory formation. The mechanisms driving high PD-1 expression in exhaustion are not well understood and could be critical to disentangling its beneficial and detrimental effects. Here, we functionally interrogated the epigenetic regulation of PD-1 using a mouse model with deletion of an exhaustion-specific PD-1 enhancer. Enhancer deletion exclusively alters PD-1 expression in CD8+ T cells in chronic infection, creating a 'sweet spot' of intermediate expression where T cell function is optimized compared to wild-type and Pdcd1-knockout cells. This permits improved control of chronic infection without additional immunopathology. Together, these results demonstrate that tuning PD-1 via epigenetic editing can reduce CD8+ T cell dysfunction while avoiding excess immunopathology.

47 Generation and Characterization of Ex Vivo Expanded Tumor-Infiltrating Lymphocytes from Renal Cell Carcinoma Tumors for Adoptive Cell Therapy

Abstract Background Autologous therapeutic tumor-infiltrating lymphocyte (TIL) therapy is a promising strategy to enhance anti-tumor immunity. Optimization of ex vivo TIL expansion could expand current immunotherapy options. Previous attempts to generate TIL in renal cell carcinoma (RCC) have been technically challenging. We applied a second-generation manufacturing process, currently used to generate the melanoma TIL product lifileucel, in RCC. Methods Resected primary and metastatic RCC samples were processed using the Gen 2 manufacturing process comprising of pre-Rapid Expansion Protocol (pre-REP) and REP steps. We assessed REP TILs for viability and performed phenotypic and functional characterization. We correlated the tumor immune microenvironment (TIME) with successful TIL expansion. Results Eight of 11 RCC samples underwent successful REP. Three failed cases demonstrated low CD8/FoxP3 ratio and high expression of PD-1 within FoxP3 cells. Expression of exhaustion markers differed between the TIME and expanded TILs; the latter had a TIM3-high/PD-1-low phenotype but retained functional capacity comparable to lifileucel. Figure: Assessment of tumor immune microenvironment (TIME) via multiplex immunofluorescence (IF). Cases that failed REP are indicated in red. (A) Representative image at 60x magnification, fluorophore colors as indicated. (B) Density of tumor-infiltrating CD8+ and FoxP3+ cells. (C) Expression of co-inhibitory exhaustion markers as a percentage of total CD8+ cells. (D) Expression of PD-1 in FoxP3+ cells. (E) Comparison of tissue TIME (dots) with TIL product post-REP (open circles) exhaustion profiles as a percentage of total CD8+ cells. Conclusions The Gen 2 manufacturing process used for lifileucel successfully expanded functional TILs from RCC samples, enabling further study in a clinical trial. TIME features such as low CD8/FoxP3 ratio and high PD-1 expression within FoxP3 cells warrant study as potential biomarkers of successful TIL expansion.

Expression and Significance of PD-1/PD-L1 in MDS Blast Cells, T Lymphocyte Cell Subsets and Treg Cells

To investigate the expression and significance of PD-1/PD-L1 in MDS blast cells, T lymphocyte cell subsets and Treg cells. Eighty-eight MDS patients and 19 AML patients were collectd as the study subjects, and Iron deficiency anemia and healthy bone marrow donors were used as control group. The expression of PD-1/PD-L1 in MDS/AML blast cells, T lymphocyte cell subsets and Treg cells was detected by flow cytometry, and the expression level of Th1/Th2/Th17-related cytokines in peripheral serum was detected. The expression of PD-1/PD-L1 in blast cells, T lymphocyte cell subsets and Treg cells in low risk MDS group were lower than that in control group, medium and high risk MDS group and AML group（all P < 0.01）, and Th1/Th17 type cytokines were dominant. The expression of PD-1/PD-L1 in blast cells, T lymphocyte cell subsets and Treg cells of intermediate and high risk MDS group and AML group were higher than that of control group and low risk MDS group (all P < 0.01), and Th2 type and Treg type (IL-10、TGF-β) cytokines were dominant. After treatment, the differences of PD-1/PD-L1 expression were not statisticatly significant in blast cells, T lymphocyte cell subsets and Treg cells between the MDS remission group and the control group (all P >0.05). The expression of PD-1/PD-L1 in blast cells, T lymphocyte cell subsets and Treg cells in MDS non-remission group were significantly higher than that in remission group and control group (all P < 0.01). The high expression of PD-1/PD-L1, dominance of Treg (IL-10、TGF-β) and Th2-related cytokines and inhibition of effector T lymphocyte cells in patients with MDS is conducive to tumor cell proliferation and immune escape, which may promote the progression of MDS disease.

Epidermal growth factor receptor and programmed cell death-1 expression levels in peripheral T cell subsets of patients with non-small cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths, in part due to its late diagnosis. Increased epidermal growth factor receptor (EGFR) expression in cancer cells is associated with a poor prognosis, and EGFR tyrosine kinase inhibitors are widely used in cancer treatment. This study aimed to clarify the relationship between EGFR expression on T cells and cancer prognosis in patients with non-small cell lung cancer (NSCLC). Forty patients with NSCLC and 40 healthy volunteers were included in this study. Peripheral CD4+T helper (Th1, Th2, Th9, Th17, Th1Th17, follicular and peripheral Th) and cytotoxic T lymphocyte (CD8+follicular and peripheral T) subsets were identified with flow cytometry according to their chemokine receptors. EGFR expression on T lymphocytes in relation to overall survival (OS) was investigated in patients with NSCLC. The patients [mean age (min-max) = 64.03 (45-83); 20 stage I-III and 20 stage IV] had increased EGFR expression on CD3+T, CD4+Th, Th1, Th2, and Th17 cells compared to the controls (p < 0.05). High EGFR expression on CD3+T, CD4+Th, Th1, and Th2 cells was associated with poor OS. Also, PD-1 expression on lymphocytes, CD3+T, and Th cells was increased in patients with NSCLC compared to controls. The high expression of EGFR and PD-1 on Th cells and the reduced percentage of lymphocytes and Th cells, especially in stage IV patients with NSCLC, revealed that increased EGFR activity may trigger apoptosis of Th cells and promote the development of metastases, while high EGFR expression on CD3+T, CD4+Th, Th1, and Th2 cells may be an independent poor prognostic marker in NSCLC.

Generation and Characterization of Ex Vivo Expanded Tumor-infiltrating Lymphocytes From Renal Cell Carcinoma Tumors for Adoptive Cell Therapy.

Autologous therapeutic tumor-infiltrating lymphocyte (TIL) therapy is a promising strategy to enhance antitumor immunity. Optimization of ex vivo TIL expansion could expand current immunotherapy options. Previous attempts to generate TIL in renal cell carcinoma (RCC) have been technically challenging. We applied a second-generation manufacturing process, currently used to generate the melanoma TIL product lifileucel, in RCC. Resected primary and metastatic RCC samples were processed using the Gen 2 manufacturing process comprising of pre-Rapid Expansion Protocol (pre-REP) and REP steps. We assessed REP TILs for viability and performed phenotypic and functional characterization. We correlated the tumor immune microenvironment (TIME) with successful TIL expansion. Eight of 11 RCC samples underwent successful REP. Three failed cases demonstrated low CD8/FoxP3 ratio and high expression of PD-1 within FoxP3 cells. Expression of exhaustion markers differed between the TIME and expanded TILs; the latter had a TIM3-high/PD-1-low phenotype but retained functional capacity comparable to lifileucel. The Gen 2 manufacturing process used for lifileucel successfully expanded functional TILs from RCC samples, enabling further study in a clinical trial. TIME features such as low CD8/FoxP3 ratio and high PD-1 expression within FoxP3 cells warrant study as potential biomarkers of successful TIL expansion.

Clinical Significance of SIRPα Expression on Tumor-Associated Macrophages in Patients with Lung Squamous Cell Carcinoma.

Signal-regulatory protein alpha (SIRPα) is an immune checkpoint molecule expressed on macrophages that functions to inhibit phagocytosis by binding to CD47 expressed on tumor cells. SIRPα has attracted increasing attention as a novel target for cancer immunotherapy; however, the expression and immune function of SIRPα in lung squamous cell carcinoma (LUSC) remain unclear. Therefore, this study aimed to identify the clinical importance of SIRPα expression in LUSC and to explore the factors that elevate SIRPα expression. Primary LUSC specimens surgically resected from 172 patients underwent immunohistochemical evaluation of the association of SIRPα expression on tumor-associated macrophages with clinicopathological features and clinical outcomes. Furthermore, we analyzed the association of SIRPα expression with tumor-infiltrating lymphocytes and the expression of programmed cell death ligand 1 (PD-L1). In vitro, monocytes were treated with cytokines, and SIRPα protein expression was assessed by flow cytometry. There were no differences in SIRPα expression and clinicopathological factors. High SIRPα expression was significantly associated with PD-L1-positive expression, and high CD8, PD-1, and CD163 expression. The high SIRPα expression group showed significantly shorter recurrence-free survival (RFS) and overall survival (OS). On multivariate analysis, high SIRPα expression was an independent poor prognostic factor for RFS and OS. The expression of SIRPα protein in monocytes was upregulated by treatment with IFNγ. Our analysis revealed that high SIRPα expression significantly predicts poor prognosis in patients with surgically resected LUSC.

Peripheral PD-1+NK cells could predict the 28-day mortality in sepsis patients.

Unbalanced inflammatory response is a critical feature of sepsis, a life-threatening condition with significant global health burdens. Immune dysfunction, particularly that involving different immune cells in peripheral blood, plays a crucial pathophysiological role and shows early warning signs in sepsis. The objective is to explore the relationship between sepsis and immune subpopulations in peripheral blood, and to identify patients with a higher risk of 28-day mortality based on immunological subtypes with machine-learning (ML) model. Patients were enrolled according to the sepsis-3 criteria in this retrospective observational study, along with age- and sex-matched healthy controls (HCs). Data on clinical characteristics, laboratory tests, and lymphocyte immunophenotyping were collected. XGBoost and k-means clustering as ML approaches, were employed to analyze the immune profiles and stratify septic patients based on their immunological subtypes. Cox regression survival analysis was used to identify potential biomarkers and to assess their association with 28-day mortality. The accuracy of biomarkers for mortality was determined by the area under the receiver operating characteristic (ROC) curve (AUC) analysis. The study enrolled 100 septic patients and 89 HCs, revealing distinct lymphocyte profiles between the two groups. The XGBoost model discriminated sepsis from HCs with an area under the receiver operating characteristic curve of 1.0 and 0.99 in the training and testing set, respectively. Within the model, the top three highest important contributions were the percentage of CD38+CD8+T cells, PD-1+NK cells, HLA-DR+CD8+T cells. Two clusters of peripheral immunophenotyping of septic patients by k-means clustering were conducted. Cluster 1 featured higher proportions of PD1+ NK cells, while cluster 2 featured higher proportions of naïve CD4+T cells. Furthermore, the level of PD-1+NK cells was significantly higher in the non-survivors than the survivors (15.1% vs 8.6%, P<0.01). Moreover, the levels of PD1+ NK cells combined with SOFA score showed good performance in predicting the 28-day mortality in sepsis (AUC=0.91,95%CI 0.82-0.99), which is superior to PD1+ NK cells only(AUC=0.69, sensitivity 0.74, specificity 0.64, cut-off value of 11.25%). In the multivariate Cox regression, high expression of PD1+ NK cells proportion was related to 28-day mortality (aHR=1.34, 95%CI 1.19 to 1.50; P<0.001). The study provides novel insights into the association between PD1+NK cell profiles and prognosis of sepsis. Peripheral immunophenotyping could potentially stratify the septic patients and identify those with a high risk of 28-day mortality.

Subclone from CT26 resistant to anti-PD-1 therapy associated with increased expression of genes related to glucocorticoids

BackgroundAlthough the use of anti-PD-1 antibodies has fundamentally changed traditional cancer treatment, most patients are resistant to anti-PD-1 treatment. Glucocorticoids (GCs) play an important role in tumorigenesis and tumor progression, but the role of endogenous GCs in resistance to anti-PD-1 antibody therapy remains unclear. MethodsSingle cell-derived cell lines (SCDCLs) were generated from a colorectal cancer cell line (CT26) using limiting dilution. We analyzed tumor tissues from anti-PD-1 antibody-treated and untreated mice inoculated with SCDCLs via transcriptome sequencing and flow cytometry to detect pathway activity and immune cell composition changes in the tumor microenvironment. ResultsFive SCDCLs were inoculated into wild-type BALB/c mice (all tumorigenic). Single-cell clone (SCC)-2 exhibited the slowest growth rates both in vivo and in vitro compared to other single-cell clones, and better long-term survival than SCC1 and CT26. Flow cytometry showed that SCC2 tumor-bearing mice exhibited significantly higher infiltration of T cells within the tumor tissue, and higher expression of PD-1 on these T cells than the other groups in vivo. However, the SCC2 group showed no response to anti-PD-1 therapy. Transcriptome analysis revealed that the SCC2 group exhibited increased expression of genes related to GC (Hsd11b1, Sgk3, Tgfbr2, and Il7r) compared to SCC2-anti-PD-1 treated tumors. ConclusionsGC pathway activation is related to resistance to anti-PD-1 therapy.

New approach in Understanding Colorectal Cancer Immunosuppression and Immunotherapy-Based Strategies in the Treatment of Microsatellite Stable Colorectal Cancer

Abstract Except the widely accepted use of immune Checkpoint inhibitors in the treatment of microsatellite instability-high (MSI-H) mismatch repair-deficient (MMRd) CRCs representing about 5% of all metastatic (m)CRC patients, new strategies are applied to cure MMR-proficient (MMRp) mCRC patients. Tumor microenvironment (TME) is decisive for cancer development. The determination of some immunoeffective and immunosuppressive immune cells and some cytokines, chemokines and growth factors in the TME gives Information about the use of immune Checkpoint inhibitors in MMRp CRCs. The increased level of IL-6 in the serum and increased number of IL-6+ immune cells in TME, the increased number of IL-17+ Th17 cells, and of FoxP3+ cells are used to determine the use of anti-IL-6 antibody and of anti-FoxP3 antibody for treatment. The determination of high CD8+, high PD-1 expression and little or no Th17 cells appoint better response to anti-PD-1 therapy. The used combination therapies are: combination of immunotherapy with chemotherapy, with radiation therapy, with targeted therapy, with vaccines, oncolytic viruses and bispeeifie antibodies. Classical treatment of CRC patients has included chemotherapy, radiotherapy and surgery. Recently, immunotherapy has been added as a mainstay for therapy of CRC. The main checkpoint inhibitors used in CRC immunotherapy are pembrolizumab and nivolumab (anti-PD-1), durvalumab (anti-PD-L1), ipilimumab (anti-CTLA-4), favezelimab (anti-LAG3), etc. They are applied after fluorapyrimidine, oxaliplain, and irinotecan therapy. In conclusion, we may state that the future treatment of MSS CRC is in combination therapies, i.e. conventional and immunotherapies. We consider that immune infiltrate in TME must be assessed in order to determine combination therapies.

TRICK-MCC: A proof-of-concept, investigator-initiated study of combination therapy with anti–PD-1, anti–LAG-3, and anti–TIM-3 in participants with advanced or metastatic PD-(L)1 refractory Merkel cell carcinoma.

TPS9618 Background: Merkel cell carcinoma (MCC) is an aggressive and highly immunogenic skin cancer often associated with the Merkel cell polyomavirus (MCPyV). Immune checkpoint inhibition (ICI) via PD-(L)1 blockade can promote durable responses among patients with metastatic MCC, yet &gt;50% of MCC patients experience disease progression. There is an unmet need for effective treatment options for these patients. One possible strategy to overcome primary and secondary resistance in PD-(L)1 refractory MCC is concurrent targeting of multiple immune checkpoints to facilitate reversal of chronic T cell exhaustion. Based on our preliminary data revealing high expression of PD-1, LAG-3 and TIM-3 on MCC-specific CD8 T cells, which appear to increase upon clinical disease progression in the context of treatment with PD-(L)1 blockade, we hypothesize that concurrent blockade of PD-1, LAG-3 and TIM-3 will overcome immune evasion in patients with PD-(L) refractory MCC. Methods: TRICK-MCC (Triple Immune Checkpoint Inhibition in MCC) is an investigator initiated, Phase 2 clinical trial investigating concurrent treatment with anti-PD-1 (retifanlimab), anti-LAG3 (INCAGN02385) and anti-TIM-3 (INCAGN02390) in patients with advanced/metastatic MCC that has progressed after treatment with anti-PD-(L)1. Target enrollment is 20 patients total, with 5 patients enrolled as of Jan 2024. Patients will receive anti-PD-1 q4w and anti-TIM-3 and LAG-3 q2w for up to 24 weeks (Induction Phase) followed by all drugs q6w (Maintenance Phase). Treatment will end 2 years after starting the study drugs, or upon disease progression, unacceptable toxicity, or study withdrawal. Primary objective is objective response rate (ORR). Secondary objectives are duration of response, progression free survival, overall survival, and incidence and severity of adverse events. Interim analysis will be done after 10 patients have been enrolled and followed sufficiently long to assess ORR. An observed ORR of 25%will be considered as clinically meaningful in this population with no reliable standard treatments. Serial tumor biopsies and blood samples will be obtained in all patients, unless deemed unsafe or not feasible. Planned correlative studies aim to better characterize the prevalence and significance of cancer-specific T cell exhaustion (through rigorous study of MCPyV-specific T cells), the immunologic effects of triple ICI, and alternative mechanisms of PD-(L)1 resistance beyond T cell exhaustion in PD-(L)1 resistant tumors. We hope to gain insights that are broadly applicable to solid tumor immunotherapy. Clinical trial information: NCT06056895 .

Abstract B008: High expression of the exhaustion markers PD1 and PD-L1 in non-muscle invasive bladder cancer is associated with poor outcome following Bacillus Calmette-Guérin immunotherapy

Abstract Introduction: The recommended treatment for high-risk non-muscle invasive bladder cancer (NMIBC) is intravesical instillations of Bacillus Calmette-Guérin (BCG). However, 40 % experience recurrence within 5 years despite completing BCG treatment. T cell exhaustion has been associated with poor outcome following treatment with BCG. Here we investigated if T cell exhaustion, characterized by protein expression of PD1 and PD-L1 measured in paired samples obtained before and after BCG treatment could further explain BCG response and help predict outcome in patients with NMIBC. Methods: We included 111 patients with NMIBC, from which we had 183 TURB-T samples obtained before and after BCG treatment. Using immunohistochemistry (IHC) staining of sections from tissue microarrays (TMAs) with triplicate core biopsies, we investigated the protein expression of the exhaustion markers PD1 (clone EP239) and PD-L1 (clone 22c3). Data was analyzed using digital pathology software (Visiopharm®), where tissue areas were defined as either carcinoma or stroma based on PanCK staining. Cells were scored as positive when detection of a nucleus was combined with a positive PD1 or PD-L1 stain, which was based on visually defined thresholds to differentiate between positive and unspecific staining. In total, 167 samples from 105 patients were considered suitable for further analysis (cell count &amp;gt; 200 in carcinoma and stromal areas) with 2-3 cores per sample, resulting in a total of 441 tissue cores. The average cell count was 5732 per core (defined by positive nuclei stain). Stratification of samples into high or low expression was based on a median split of the positive cell fraction. Results: When investigating the number of PD1 and PD-L1 positive cells in the tumors (carcinoma and stroma combined), we observed that PD-1 expression significantly increased with tumor stage in pre- (p=0.002) and post-BCG (p=0.006) tumor samples. PD1 expression was also increased in high-grade tumors compared to low-grade tumors in pre-BCG samples (p = 0.001). Furthermore, we observed a significant association between tumors of higher stage and high PD-L1 expression in the pre-BCG samples (p = 0.007). Patients with low expression of PD1 and PD-L1 in the pre-BCG tumor samples had a superior high-grade recurrence-free survival (HG-RFS) compared to patients with high PD1 (p=0.002) and PD-L1 (p=0.021) expression. In addition, low expression of PD-L1 in pre-BCG tumors was correlated with a better progression-free survival compared to the patients with high PD-L1 expression (p = 0.013). Conclusion: Protein expression of PD1 and PD-L1 in pre-BCG tumor samples were correlated to higher stage and grade as well as worse HGFRS, indicating that T cell exhaustion plays an important role in resistance to BCG treatment. Citation Format: Tine G. Andreasen, Trine Strandgaard, Jørgen B. Jensen, Lars Dyrskjøt. High expression of the exhaustion markers PD1 and PD-L1 in non-muscle invasive bladder cancer is associated with poor outcome following Bacillus Calmette-Guérin immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr B008.

The clinical significance of PD-1 expression in patients with bladder cancer without lymph node metastasis: a comparative study with drained lymph nodes and tumor tissues

ABSTRACT Objective In light of the increasing importance of immunotherapy in bladder cancer treatment, this study is aim to investigate the expression and clinical significance of programmed cell surface death-1 (PD-1) in bladder cancer patients without lymph node metastasis, and to compare and analyze the difference of PD-1 in draining lymph nodes and tumor tissues. Methods The expression of PD-1 on T cells and the proportion of positive PD-1 + T cells of IFN-γ and CD105a were detected by flow cytometry, and the correlation between PD-1 expression and clinical parameters was analyzed. Results The percentage of PD-1 positive cells in drainage lymph nodes was higher than that in tumor tissues (P < 0.001). PD-1 positive cells accounted for the highest proportion in CD3 + T cells. The proportion of IFN-γ-positive PD-1 + T cells in draining lymph nodes was significantly higher than that in tumor tissues (P < 0.001), while there was no significant difference in CD105a positive PD-1 + T cells between tumor tissues and draining lymph nodes. Pathological grade, tumor size and stage were positively correlated with PD-1 expression level in the lymph nodes. Conclusion The high expression of PD-1 in patients with bladder cancer without lymph node metastasis, especially in draining lymph nodes, suggests that PD-1 may play a key role in the regulation of tumor immune microenvironment. The correlation between PD-1 and clinical parameters indicates its potential prognostic value. These findings provide important clinical implications for PD-1 targeted therapy, but further prospective studies are needed to determine the application value of PD-1 in therapeutic strategies.

Characterization of innate lymphoid cell subsets infiltrating melanoma and epithelial ovarian tumors

ABSTRACT The innate lymphoid cell (ILC) family is composed of heterogeneous innate effector and helper immune cells that preferentially reside in tissues where they promote tissue homeostasis. In cancer, they have been implicated in driving both pro- and anti-tumor responses. This apparent dichotomy highlights the need to better understand differences in the ILC composition and phenotype within different tumor types that could drive seemingly opposite anti-tumor responses. Here, we characterized the frequency and phenotype of various ILC subsets in melanoma metastases and primary epithelial ovarian tumors. We observed high PD-1 expression on ILC subsets isolated from epithelial ovarian tumor samples, while ILC populations in melanoma samples express higher levels of LAG-3. In addition, we found that the frequency of cytotoxic ILCs and NKp46+ILC3 in tumors positively correlates with monocytic cells and conventional type 2 dendritic cells, revealing potentially new interconnected immune cell subsets in the tumor microenvironment. Consequently, these observations may have direct relevance to tumor microenvironment composition and how ILC subset may influence anti-tumor immunity.

Clinical and Biologic Correlates of ADORA2A Transcriptomic Expression in Cancer.

ADORA2A (adenosine A2a receptor) and ADORA2B propagate immunoregulatory signals, including restricting both innate and adaptive immunity, though recent data also suggest a tumor suppressor effect in certain settings. We evaluated the RNA expression from 514 tumors in a clinical-grade laboratory; 489 patients with advanced/metastatic disease had clinical outcome correlates. Transcript expression was standardized to internal housekeeping genes and ranked (0-100 scale) relative to 735 specimens from 35 different cancer types. Transcript abundance rank values were defined as "low/moderate" (0-74) or "high" (75-100) percentile RNA expression ranks. Overall, 20.8% of tumors had high ADORA2A (≥75 percentile RNA rank). The greatest proportion of high ADORA2A expressors was found in neuroendocrine and breast cancers and sarcomas, whereas the lowest was found in colorectal and ovarian cancers, albeit with patient-to-patient variability. In multivariable logistic regression analysis, there was a significant positive correlation between high ADORA2A RNA expression and a high expression of the immune checkpoint-related molecules PD-1 (p = 0.015), VISTA (p ≤ 0.001), CD38 (p = 0.031), and CD39 (p ≤ 0.001). In 217 immunotherapy-treated patients, high ADORA2A did not correlate significantly with progression-free (p = 0.51) or overall survival (OS) (p = 0.09) from the initiation of the checkpoint blockade. However, high versus not-high ADORA2A transcript expression correlated with longer OS from the time of advanced/metastatic disease (N = 489 patients; (HR 0.69 (95% CI 0.51-0.95) (p = 0.02)). Therefore, high ADORA2A transcript levels may be a favorable prognostic factor, unrelated to immunotherapy. Importantly, ascertaining co-expression patterns of ADORA2A with PD-1 and VISTA in individual tumors as a basis for the precision co-targeting of ADORA2A and these other checkpoint-related molecules warrants investigation in clinical trials.