Cell Exhaustion Markers Research Articles

Leukotriene B4 receptor 2 correlates with prognosis and immune infiltration in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common renal cancer. According to reports, leukotriene B4 receptor 2 (LTB4R2, also known as BLT2), a chemokine receptor, is upregulated in different tumors. However, the correlation between BLT2 expression and its prognostic value in ccRCC remains to be explored. This study used the The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases to evaluate the association between BLT2 expression and the clinical outcome of ccRCC. Based on TIMER2.0, the correlation between BLT2 expression in ccRCC and tumor immune characteristics was evaluated. The expression of BLT2 in ccRCC was higher than that in normal tissues. Kaplan-Meier survival analysis indicated that high BLT2 expression was significantly correlated with poor overall survival (HR = 1.75, p < 0.001) and disease-specific survival (HR = 1.60, p = 0.014) for patients with ccRCC. In addition, our findings revealed that there was no significant correlation between the M1 marker genes and the expression of BLT2 in ccRCC, while moderate correlations were observed between the BLT2 expression and the M2 marker genes. Tregs and T cell exhaustion marker genes were positively correlated with BLT2 expression in ccRCC (p < 0.001). BLT2 may serve as a novel prognostic biomarker and is related to the shaping of tumor immune microenvironment in ccRCC. The expression of BLT2 potentially contributes to the regulation of TAMs, T cell exhaustion, and Tregs activation in ccRCC, providing new approaches to promote the development of new immunotherapeutic strategies for ccRCC.

Bio-O1-04 - PD1 regulates T cell exhaustion phenotypes and disease aggressiveness in CTCL

<h3>Introduction:</h3> Cutaneous T cell lymphomas (CTCLs) are a heterogenous group of lymphomas of the skin-homing CD4+ T cell. Although the median survival for patients with stage IV disease is generally poor, there is significant heterogeneity with some stage-matched patients surviving <6 months while others survive >10 years. Currently, it is unclear why this is the case, and whether there are any biomarkers for patients with the most aggressive forms of disease. To address this knowledge gap, we sought to combine multi-omics and functional assays to a clinically annotated cohort of CTCLs to identify drivers of disease heterogeneity. <h3>Materials and methods:</h3> To explore the molecular underpinnings of disease heterogeneity in CTCL, we combined whole-genome sequencing, RNA-sequencing, immunophenotyping, and ex vivo proliferation assays on sorted malignant cells from 14 patients with leukemic CTCL. Whole genome sequencing was analyzed for single nucleotide and copy number variants. To determine ex vivo proliferation, sorted malignant cells were stained with carboxyfluorescein succinimidyl ester (CFSE), cultured with T cell receptor stimulation or cytokines and assessed via flow cytometry. <h3>Results:</h3> Unexpectedly, we observed significant heterogeneity in the ability of malignant CTCL cells to proliferate to T cell receptor and cytokine signals ex vivo. A subset of samples proliferated to more stimuli than healthy controls, another group of samples similarly to controls, and yet another group significantly less. Strikingly, unbiased DNA and RNA-seq analyses revealed a critical role for the co-inhibitory receptor and tumor suppressor PD1. PDCD1 (the gene which encodes PD1) was the most highly expressed gene in samples that failed to proliferate ex vivo compared to those that proliferate highly. The single gene significantly more frequently mutated in highly proliferating samples was PD1. Integrating genomic, transcriptomic, and functional data, we observed that most samples harbored features of T cell exhaustion, including diminished proliferation and effector cytokine production, and expression of markers of T cell exhaustion. However, samples with deletions of PD1 reversed this phenotype. These samples had higher ex vivo proliferation and effector cytokine production, and lower expression of T cell exhaustion markers. Accordingly, samples with deletions of PD1 had significantly worse prognoses in both stage IV disease and in stage II-III MF. <h3>Conclusions:</h3> By integrating genotype, transcriptome, and functional status, we have identified that a majority of CTCLs harbor features of T cell exhaustion. A subset of CTCLs which harbor PD1 deletions reverse the T cell exhaustion phenotype. Consistent with this, PD1-deleted CTCLs have significantly worse overall survival in stage-matched patients. Our data establishes T cell exhaustion status and PD1 deletions as novel drivers of heterogeneity in cutaneous T cell lymphoma.

H/ACA snoRNP Gene Family as Diagnostic and Prognostic Biomarkers for Hepatocellular Carcinoma.

BackgroundThe H/ACA small nucleolar ribonucleoprotein (snoRNP) gene family, including GAR1 ribonucleoprotein (GAR1), NHP2 ribonucleoprotein (NHP2), NOP10 ribonucleoprotein (NOP10), and dyskerin pseudouridine synthase 1 (DKC1), play important roles in ribosome biogenesis. However, the potential clinical value of the H/ACA snoRNP gene family in hepatocellular carcinoma (HCC) has not yet been reported.MethodsBioinformation databases were used to analyze the expression and roles of the H/ACA snoRNP gene family in HCC. Survival analysis, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes enrichment pathway (KEGG) analyses were performed using R software. Tumor Immune Estimation Resource (TIMER) was used to analyze the correlation between the expression of the H/ACA snoRNP gene family and immune infiltration in HCC. Finally, immunohistochemistry and Western blotting were performed to verify the protein expression of the H/ACA snoRNP gene family in HCC tissues and adjacent tissues.ResultsThe expression of the H/ACA snoRNP gene family was significantly increased in HCC samples compared to normal tissues, and the area under the curve (AUC) of GAR1, NHP2, NOP10, and DKC1 was 0.898, 0.962, 0.884, and 0.911, respectively. Increased expression of the H/ACA snoRNP gene family was associated with poor prognosis in HCC patients (Hazard Ratio, HR = 1.44 [1.02–2.04], 1.70 [1.20–2.40], 1.53 [1.09–2.17], and 1.43 [1.02–2.03], respectively; log-rank P = 0.036, 0.003, 0.014, 0.039, respectively). GO and KEGG analyses showed that co-expressed genes were primarily enriched in ribosome biogenesis. In addition, upregulated expression of H/ACA snoRNP gene family was related to the infiltration of various immune cells and multiple T cell exhaustion markers in HCC patients. Immunohistochemical analysis and Western blotting showed that the protein expression of H/ACA snoRNP gene family was higher in HCC tissues than in adjacent tissues of clinical samples.ConclusionH/ACA snoRNP gene family expression was higher in HCC tissues than in normal or adjacent tissues and was highly associated with poor prognosis of HCC patients and, therefore, has the potential to serve as diagnostic and prognostic biomarkers for HCC.

Phase II study of pembrolizumab in refractory esophageal cancer with correlates of response and survival

BackgroundImmune checkpoint inhibitors have revolutionized cancer treatment, but the benefits in refractory patients with esophageal cancer have been modest. Predictors of response as well as new targets for novel therapeutic...

Elevated mRNA Level of Y-Box Binding Protein 1 Indicates Unfavorable Prognosis Correlated with Macrophage Infiltration and T Cell Exhaustion in Luminal Breast Cancer.

PurposeThe Y-box binding protein 1 (YBX1) gene encodes the multifunctional protein YB1 that is associated with the dysregulation of numerous cancer-related genes. However, the prognostic value of YBX1 and its correlation with immune cell infiltration in breast cancer (BRCA) remain unclear.MethodsYBX1 expression data in various malignancies were obtained from Oncomine, Tumor Immune Estimation Resource (TIMER), Cancer Cell Line Encyclopedia, UALCAN and cBio Cancer Genomics Portal databases. Survival data were analyzed with Kaplan–Meier plotter. Immune cell infiltration and its association with YBX1 expression level were assessed with TIMER and LinkedOmics. YB1 expression was evaluated by immunohistochemistry and Western blotting, and changes in cancer cell viability and T cell activity following YBX1 knockdown were assessed with an immunocyte–tumor cell co-culture assay.ResultsYBX1 was downregulated in the BRCA cohort, which was closely associated with worse prognosis in the luminal A subtype (overall survival [OS]: hazard ratio [HR] 1.93, 95% confidence interval [CI] 1.22–3.05, P = 0.0042; recurrence-free survival [RFS]: HR 1.85, 95% CI 1.51–2.28, P = 3.1e-9) and luminal B subtype (OS: HR 1.08, 95% CI 0.68–1.70, P = 0.75; RFS: HR 1.29, 95% CI 1.02–1.62, P = 0.03). YBX1 expression was positively correlated with the M2 macrophage infiltration and expression of T cell exhaustion markers such as indoleamine 2,3-dioxygenase 1 (IDO1) (rs = 0.388, P = 4.93e-37) and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) (rs = 0.321, P = 2.54e-25) in luminal BRCA. Kaplan–Meier analysis revealed a correlation between YBX1 expression, M2 infiltration and survival outcome. Co-culture with macrophages or T cells enhanced the decrease in luminal BRCA cell viability induced by YBX1 knockdown.ConclusionHigh YBX1 mRNA levels predict a poor prognosis in luminal BRCA, which is correlated with M2 macrophage infiltration and T cell exhaustion in the tumor microenvironment. Combining classic therapeutics with immune checkpoint inhibitors and M1 polarization agents may be an effective treatment strategy for luminal BRCA with YBX1 overexpression.

OTEH-2. High-dimensional analysis of spatial immune cell heterogeneity in glioblastoma reveals differences between contrast-enhancing and non-contrast-enhancing tumor rims

BackgroundGlioblastoma (GBM) is the most common primary malignant brain tumor in adults. GBM remains an incurable disease, with a median survival ~20 months. Complex intercellular interactions within the tumor microenvironment and spatial heterogeneity have challenged and impeded therapeutic efficacy. The non-contrast-enhancing (by T1-weighted MRI) rim of GBM is not always safely resectable and represents a major source of recurrence. We hypothesized that differential immune infiltration is an underlying factor of spatial heterogeneity in GBM, particularly in the non-contrast-enhancing tumor rim.MethodsFive patients with newly diagnosed GBM (ages 53–84) were recruited to a device feasibility study (NCT04545177) utilizing an intraoperative high-resolution MRI-based navigation system coupled with the NICO Myriad (a non-ablative semi-automated resection tool) and a coupled automated biological Tissue Preservation System (NICO APS) to sample spatially mapped regions of tumors in a reproducible and minimally destructive manner. We obtained brain tumor tissue from: (a) tumor core, (b) contrast-enhancing tumor rim and (c) non-contrast-enhancing tumor rim. Downstream processing consisted of digestion of tumor tissue (Miltenyi human tumor digestion kit) for subsequent single-cell isolation, viability assessment and immediate staining for multiparametric flow cytometry for immune profiling.ResultsViability varied across sampled regions (median 85%, range 52–100%). With the exception of 1 sample, viability was >70% in all specimens. High-dimensional analysis with 26 marker flow cytometry revealed spatial heterogeneity in the frequency of myeloid-derived suppressor cell subsets, regulatory T cells, CD8+ T cells, as well as expression of T cell activation and exhaustion markers.ConclusionsSemi-automated, spatially mapped intraoperative sampling of GBM with high viability of specimens is feasible and reproducible with the NICO Myriad and APS devices. High-dimensional analysis of immune cells in the GBM microenvironment captured the spatial heterogeneity of GBM. Future studies will expand on these observations by analyzing more patient specimens in combination with multiple omics assays.

Abstract 1672: Overall survival in metastatic melanoma correlates with pembrolizumab exposure and T cell exhaustion markers

Abstract Background: There are few clinically validated predictive biomarkers for benefit to immune checkpoint blockade (ICB) in metastatic melanoma. Real world evidence has shown patients with higher exposure to ICB have improved best overall response (BOR) and longer survival [1]. Increased expression of T cell exhaustion markers and specific chemokine signatures have been associated with poor response to ICB in metastatic melanoma [2]. We sought to determine the relationship between pharmacokinetic (PK) and clinical outcomes of patients with metastatic melanoma treated with pembrolizumab. Translational correlative objectives included identifying expression of circulating T cell exhaustion markers and specific chemokine signatures in patients with progressive disease. Methods: Metastatic melanoma patients received weight based pembrolizumab and had serial peripheral blood draws. BRAF mutation positive patients were pre-treated with targeted therapy, otherwise pembrolizumab was first line. Plasma trough levels were assessed using the Abcam® pembrolizumab ELISA kit. BOR groups: complete response (CR), partial response (PR) and progressive disease (PD), were classified as per irRECIST. The T-cell exhaustion and chemokine markers were determined using Flow Cytometry on the BD Science Fortessa® X20. The cohort was stratified into high versus low pembrolizumab trough concentrations, split by the median. Kaplan-Meier survival analysis for progression free (PFS) and overall survival (OS) was performed based on these groups. Non-parametric correlation analysis was used to test for correlations between clinical characteristics, pembrolizumab trough concentrations and translational markers. Results: Twenty-eight patients were enrolled. Median follow up was 32.5 months. The pembrolizumab plasma trough levels were not significantly different between BOR categorical groups. Trough levels are a valid surrogate for drug exposure[3]. There were no significant correlations between pembrolizumab trough levels and expression of T-cell exhaustion and chemokine markers over time.The high pembrolizumab exposure group experienced significantly longer OS (median not reached versus 48 months, p=0.014), than the low exposure group. A similar positive exposure PFS relationship was found (median not reached versus 48 months, p=0.045). There was significantly higher frequency of co-expression of CXCR6 and TIM3 on CD4+ T cells for PD (mean 21.5% CI 14.9% - 26.06%) compared to CR (mean 12.2% CI 9.40% - 14.99%) (p=0.003) and PR (mean 9.9% CI 7.76% - 12.05%) (p=0.0001). There was also significantly higher frequency of co-expression of CXCR6 and TIM3 on CD8+ T cells for PD (mean 32.59% CI 25.76% - 39.43%) compared to PR (mean 21.9% CI 18.22% - 25.56%) (p=0.032).Conclusions: We describe a positive exposure PFS and exposure OS relationship for pembrolizumab in metastatic melanoma. The data indicate the co-expression of CXCR6 and T-cell exhaustion markers on circulating CD4+ and CD8+ T cells are potential biomarkers of disease progression on single-agent pembrolizumab.AcknowledgementsThis study was funded by a Hunter Medical Research Institute Project Grant (HMRI983).Hunter Cancer Biobank Serial Blood Collection Project Citation Format: Vishal Navani, Moira C. Graves, Giovana Celli Marchett, Hiren Mandaliya, Nikola A. Bowden, Andre Van Der Westhuizen. Overall survival in metastatic melanoma correlates with pembrolizumab exposure and T cell exhaustion markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1672.

Abstract NG12: From cholesterol regulation to tumor suppression: Pcsk9 as a novel target for cancer immunotherapy

Abstract NG12: From cholesterol regulation to tumor suppression: Pcsk9 as a novel target for cancer immunotherapy

Abstract 1527: Integrated computational and experimental analysis identifies the mitochondrial uncoupling protein 2 (Ucp2) as a key regulator of T cell anti-tumor function

Abstract T cells play a central role in cancer immunosurveillance and current cancer immunotherapies, including adoptive cell transfer (ACT), T cell receptor or chimeric antigen receptor (CAR) T cell therapies and immune checkpoint blockade. Understanding the factors regulating T cell function is hence critical for improving the success of these immunotherapies. It has been recognized that metabolism can greatly affect different aspects of T cell function, including differentiation, cytokine production, longevity and exhaustion. Here we integrate genome-scale metabolic modeling (GEM) with biological experiments to discover novel metabolic determinants of T cell function. Previously we developed the metabolic transformation algorithm (MTA), a GEM method that was successfully applied to identify driving factors and targets for different diseases. Here applying MTA to data on CAR-T cell gene expression and patient response to anti-CD19 CAR-T therapy, we predicted mitochondrial metabolite transport and specifically proton transport in mitochondrial uncoupling, as key determinants of CAR-T therapy response. Mitochondrial uncoupling is also important for the in vivo persistence of adoptively transferred tumor-infiltrating lymphocytes, as further confirmed by analyzing their gene expressions from a KRAS-targeting ACT dataset. Focusing on the mitochondrial uncoupling protein 2 (Ucp2), which is abundantly expressed in T cells, we experimentally validated that it is required for T cell longevity and anti-tumor function. Specifically, the loss of Ucp2 either via knock-out (KO) or treatment by genipin (a Ucp2 inhibitor) in mice T cells results in accelerated differentiation into “terminal effector cells”, as shown by increased levels of T cell cytotoxicity and exhaustion markers, and decreased levels of central memory and stemness markers. Adoptive transfer of Ucp2-KO Pmel-1 T cells to mice bearing B16 melanomas displayed poorer anti-tumor efficacy and worse survival than the transfer of Ucp2-wildtype T cells. We find that Ucp2 modulates oxidative stress and DNA damage by regulating the levels of mitochondrial superoxide. Reducing mitochondrial reactive oxygen species was sufficient to rescue the loss of Ucp2-mediated effector T cell differentiation, senescence, cytokine production and anti-tumor activity Ucp2-KO Pmel-1 T cells. Tumor-specific CD8+ T cells could be metabolically reprogrammed by Ucp2 overexpression, which improved T cell longevity and anti-tumor function in the Pmel-1/B16 ACT mice model. In sum, our study establishes a novel role of Ucp2 in regulating T cell longevity and anti-tumor activity by repressing increased ROS levels accompanying mitochondrial dysfunction in differentiated and exhausted cells, suggesting that manipulating Ucp2 levels in T cells can be exploited to enhance T cell-based cancer immunotherapies. Citation Format: Madhusudhanan Sukumar, Kuoyuan Cheng, Arunakumar Gangaplara, Yogin Patel, Suman K. Vodnala, Rafiqul Islam, Arash Eidizadeh, Carolyn Subramaniam, Ping Lee, Rigel Kishton, Amanda N. Henning, Michael J. Kruhlak, Zhiya Yu, Ethan M. Shevach, Toren Finkel, Eytan Ruppin, Nicholas P. Restifo. Integrated computational and experimental analysis identifies the mitochondrial uncoupling protein 2 (Ucp2) as a key regulator of T cell anti-tumor function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1527.

Abstract 505: Oral azacitidine modulates the immune microenvironment in acute myeloid leukemia (AML) patients in remission: A subanalysis from the QUAZAR AML-001 Trial

Abstract BACKGROUND The immunologic effects of maintenance therapy in patients (pts) with AML in remission are not well-characterized but of high clinical interest, as rapid recovery of bone marrow (BM) after intensive chemotherapy (IC) may help delay relapse. Post IC, immunological interactions in the BM microenvironment present several immunosuppressive mechanisms. PD-L1 is commonly overexpressed on AML blasts, which is associated with worse prognosis. Oral azacitidine (Oral-AZA [CC-486]) is a hypomethylating agent recently approved in the US for pts with AML in complete remission (CR) or CR with incomplete hematologic recovery (CRi). To better understand the effects of Oral-AZA on immune cells, checkpoint expression of PD-L1/2 on AML blasts and normal myeloid progenitors (MPs), and the kinetics of T cell recovery and activation/exhaustion (eg, PD1, TIM3) were assessed. METHODS Biomarker-evaluable pts aged ≥ 55 years with AML were randomized 1:1 to Oral-AZA 300 mg (n=56) or placebo (PBO, n=52) post IC within 4 months of achieving CR. Flow cytometry evaluations of BM aspirates were performed at screening (ie, baseline [BL]), every 3 cycles until cycle 24 and every 6 cycles thereafter to cycle 36, or as clinically indicated. Correlative analyses of baseline immune parameters with median (med) relapse-free survival (RFS) were computed using Kaplan-Meier methods. RESULTS In the biomarker-evaluable pts, PD-L1 and PD-L2 expression at BL were higher on AML blasts (med intensity 7.1 and 2.9) than normal MPs (0.7 and 1.6). Most AML blasts were PD-L2+ (79%), whereas only 1.9% were PD-L1+. When stratified by the med, higher BL CD3 T cell numbers (as a % of total BM ) were associated with favorable RFS in both Tx arms (Oral-AZA: ≥ med, 562 days[d] and &amp;lt; med, 235d [P = .0308]; PBO: ≥ med, 325d and &amp;lt; med, 155d [P = .0391]). At cycle 3, pts in the Oral-AZA arm had a 1.7-fold increase in CD3 T cells from BL (PBO, 1.1; P = .0450), suggesting Oral-AZA can promote immunologic recovery during early Tx cycles. There was an inverse correlation between T cell exhaustion marker phenotypes (PD1/TIM3+) with CD4 (r = -.5967; P &amp;lt; .0001) and CD8 (r = -.2484; P = .0095) T cell numbers. An increase in RFS was seen in the PBO arm with lower PD1/TIM3+ CD4 numbers (&amp;lt; med, 429d; ≥ med, 155d; P = .0037), with a nominal increase observed in the Oral-AZA arm (&amp;lt; med, 428d; ≥ med, 303d; P = .6764). In a subset of pts, Oral-AZA appeared to suppress CD4 T cell exhaustion (PD1/TIM3+) compared with PBO. CONCLUSIONS Pts in CR/CRi post-IC have a unique immune profile defined by high expression of PD-L1 on a subset of blasts and a high % of PD-L2+ blasts. A higher BL CD3 T cell count after IC in BM was prognostic. Additionally, Oral-AZA appears to contribute to an increase in T cells while also suppressing exhaustion, potentially promoting T cell signaling that could activate functional immune-mediated responses against residual leukemic cells. Citation Format: Daniel L. Menezes, Wendy L. See, Alberto Risueno, Jianglin Ma, Ignazia La Torre, Barry Skikne, CL Beach, Keshava Kumar, Anjan Thakurta. Oral azacitidine modulates the immune microenvironment in acute myeloid leukemia (AML) patients in remission: A subanalysis from the QUAZAR AML-001 Trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 505.

Abstract 1606: Potentiating effect of olaparib on anti-PD-1 treatment in colorectal cancer

Abstract Immune checkpoint inhibitors (ICIs) are approved for microsatellite unstable colorectal cancer (CRC), however, microsatellite stable (MSS) CRC that make up the majority of tumors in clinical practice have not seen any benefit with ICI. This is in part due to the lack of sufficient tumor infiltrating immune cells, particularly CD8+ T cells, in the milieu. Immunogenic cell death (ICD) is a particular form of cell death that elicits robust cross-priming of CD8 T cells against antigens expressed by dying cells in the absence of exogenous adjuvants. ICD-inducing drugs such as poly (ADP-ribose) polymerase inhibitors (PARPis) are poised to enhance cancer cell killing, boost tumor immunogenicity, increase in vivo immune infiltration, and thereby accelerate a tumor response to ICI treatment. Here, we tested the combinatorial therapeutic efficacy of a PARPi, olaparib (OLA), and an ICI, anti-PD-1, in in vitro and in vivo conditions. Combined treatment of OLA and anti-mouse PD-1 reduced tumor growth and increased survival in an immunocompetent, syngeneic animal model of BALB/c carrying allograft CT26 tumor that is MSS. Flow cytometry analysis revealed that the combo treatment significantly reduced percentages of anti-inflammatory macrophages and increased inflammatory monocytes. There was no significant difference in dendritic cell populations. We observed increased anti-tumor lymphocyte response via activation of CD4 and CD8 T cell populations upon combo treatment with OLA and anti-mouse PD-1. In addition, we noticed an increased association of expression of PD-L1 and its receptor PD-1 with OLA treatment in CRC cell lines and CT26 tumors. Our preliminary data suggests altered expressions of the CD8+ T cell exhaustion marker TIM-3 and its ligands galectin-9 and high mobility group protein 1 (HMGB1), an indicator of ICD. The pilot studies also revealed increased levels of STING-dependent type I interferon signaling initiator cGAS upon treatment with OLA in vivo. Mechanistically, OLA might induce DNA damage among tumor cells and trigger innate immune signaling including NF-κB activation and STING-dependent type I interferon signaling, both of which contribute to the maturation of myeloid antigen-presenting cells and subsequent activation of T cells. In conclusion, OLA potentiated the antitumor efficacy of anti-PD-1 in CT26 induced syngeneic CRC mouse model. Our findings indicate that OLA might trigger tumor immunogenicity, and enable a potential targeted-immunotherapeutic angle for exercising ICD and activating cytotoxic T lymphocytes in CT26 syngeneic CRC mouse model; however, further studies are needed to elucidate the mechanism. Despite ICD markers' identification as immuno-stimulating characteristics of dead cells, whether ICD-associated molecules can be used as a prognostic biomarker or therapeutic target, and the rational for such markers on tumor cells in eliciting antitumor immunity are still under investigation. Citation Format: Chintal Bhavsar, Peter John, Magnolia Abreu, Laura Bencosme, Radhashree Maitra, Sanjay Goel, Titto Augustine. Potentiating effect of olaparib on anti-PD-1 treatment in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1606.

PD-1 Is Not a Reliable Marker of Impending Relapse after Allogeneic Stem Cell Transplantation

INTRODUCTIONPost-transplant relapse is a major obstacle to successful allogeneic stem cell transplantation (allo-SCT) in hematologic malignancies. Failure to maintain an effective graft versus leukemia effect (GVL) is one mechanism responsible for post-transplant relapse. There is recent interest in PD-1 and other T cell exhaustion markers as therapeutic targets in cancer immunotherapy, however PD-1 inhibitors might induce graft versus host disease (GVHD) after allo-SCT. Therefore, understanding PD-1 kinetics in allo-SCT is needed before using check point inhibitors to prevent or treat relapse. Here we evaluated the relationship between leukemia relapse and the kinetics of T-cell exhaustion markers during the early post-transplant period. We further analyzed the functions of leukemia associated antigen (LAA) specific T-cells from relapsed subjects and sought for the mechanism of post-transplant relapse at a single cell level. METHODSEighty-five patients with various hematologic malignancies received ex-vivo T-cell depleted HLA matched related sibling allo-SCT at a single center between 2006 and 2015. All subjects were treated with a myeloablative conditioning regimen of cyclophosphamide, fludarabine, and total body irradiation with GVHD prophylaxis of low-dose cyclosporine for 21 days. Peripheral blood mononuclear cells (PBMCs) were collected from donors and recipients at 30, 60, and 100 days following transplant and at relapse to characterize regulatory T cells and T cell exhaustion markers by flow cytometry. Samples from six relapsed subjects with acute leukemias were evaluated for 5LAAs (WT1, PRAME, NY-ESO1, MAGEA3, AURAK) by RT-PCR and LAA specific T-cells by Elispot and flow cytometry. LAA specific T cells in one subject were further analyzed for single cell RNAseq. RESULTSAt a median follow up of 3.4 years, 35 patients had relapsed with a 5-year cumulative relapse incidence of 44% at a median time of 226 days. Post-relapse survival was poor among subjects relapsing before 180 days post allo-SCT (median post-relapse survival 126 days vs. 329 days for patients relapsing after 180 days; P=0.004). In the early post-transplant period, PD-1 expression in CD4 and CD8 T-cells was significantly and comparably elevated in both relapsed and non-relapsed cohorts as compared to donors. No association was observed between relapse incidence and pattern of PD-1 expression. In contrast, Helios+Tregs frequency was significantly higher in the relapsed cohort at day 30 (Figure 1). Among six relapsed subjects, at least one LAA was detected in PBMCs before and after relapse. LAA specific T-cells were observed in four subjects, however all samples eventually expressed new LAA different from the ones targeted by their own LAA specific T-cells suggesting immunological escape of leukemic blasts. Of note, LAA specific T-cells were predominantly enriched in the PD-1 negative fraction (Figure 2). Single cell analysis in one patient revealed a distinct cluster with the other exhaustion marker, LAG3 in PRAME specific T-cells in comparison to CMV specific T-cells. CONCLUSIONOur findings contradict the prevailing view that PD-1+ T cell exhaustion is a prominent feature of leukemic relapse after SCT. We found that PD-1 was overexpressed in T-cells during the early post-transplant period in all patients and PD-1 expression patterns did not identify patients destined to relapse. Furthermore, the leukemia antigen specific T-cells did not overexpress PD-1, instead single cell analysis revealed LAG3 overexpression in one patient at relapse. These findings do not support PD-1 is the predominate marker for leukemia specific T cell exhaustion in relapsing patients but point to other exhaustion markers or suppressor Treg cells as causes of relapse. Further studies are required to identify predictive biomarkers for check point inhibitors in hematologic malignancies and the patterns of LAA expression in residual disease following allo-SCT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Extensive Immunosuppression in the Acute Myeloid Leukemia (AML) Bone Marrow and Discovery of NK Cells As Biomarker of Superior Survival

BackgroundThe immune system has been harnessed in the treatment of AML patients by eliciting the graft-versus-leukemia response after hematopoietic stem cell transplant (HSCT). Recently, several other immunotherapy modalities such as immune checkpoint inhibitors, leukemia antigen-targeted antibodies, and adoptive cell therapy have been studied with hope for less toxic and more effective alternatives to conventional chemotherapeutic regimes. Yet, little is known on the clinical significance of the pretreatment immune microenvironment. Here, we characterized the immunologic landscape of the AML bone marrow (BM) with a novel multiplex immunohistochemistry (mIHC) method to identify prognostic immune biomarkers.MethodsDiagnostic-phase BM biopsies of AML patients (n=69) treated in the Helsinki University Hospital during 2005-2017 and non-leukemic controls (n=14) were collected. Using hematopathologic expertise, tissue microarrays (TMA) were constructed from duplicate BM spots and analyzed using mIHC combining simultaneously up to six out of 25 examined lymphoid, myeloid, and immune checkpoint marker. We complemented mIHC with machine learning-based image analysis. Immune cell subsets were compared with Mann-Whitney U test, and p-values adjusted with the Benjamini-Hochberg procedure (q-values). By combining mIHC findings with extensive diagnostic-phase demographic, peripheral blood and BM aspirate laboratory test, and treatment data, we developed a risk stratification model predicting relapse-free survival (RFS) using bootstrapped L1-penalized elastic net Cox regression. We validated results with diagnostic-phase AML BM aspirates (n=84) analyzed with flow cytometry (FC) in the centralized university hospital clinical laboratory (2005-2016).ResultsUnsupervised hierarchical clustering of integrated immune profiles separated AML patients distinctly from control subjects. In AML patients, immune cell subsets and phenotypes associated with anti-cancer immunity were decreased (e.g. lower granzyme B and CD57 expression in CD3+CD4+ and CD3+CD8+ T cells), while features of immunosuppression were accentuated (e.g. higher proportion of FOXP3+ CD4+ T cells and lower expression of class I HLA-ABC in all BM cells).Higher levels of T cell exhaustion markers PD1+ in both CD4+ and CD8+ T cells (15.7% vs. 1.7% and 13.2% vs. 2.0%, q<0.001 for both), and CTLA4+ (2.0% vs. 0.9%, q=0.02) in CD4+ T cells were noted in AML BM.Lower level of CD68+/pSTAT1+cMAF- (0.033% vs. 5.9%, q<0.001) and higher CD68+/pSTAT1-cMAF+ (24.8% vs. 1.7%, q<0.001), e.g. M1 and M2-polarized macrophages, were observed in AML vs. control BM, thus shifting the M1/M2 ratio from 3.5 in control to 0.0013 in AML BM. In addition, higher amount of CD11b+CD33+HLADR-, e.g. myeloid-derived suppressor cells (MDSC), were observed in AML BM (1.0% vs. 0.038% of all cells, q<0.001).The most predictive features from the L1-penalized regression analysis were selected with multivariate Cox regression. High patient age and low amount of CD3-CD56+ (NK) cells were significantly associated with worse RFS both in univariate and multivariate setting after adjusting for induction therapy protocol (AUC 0.84 and R2 0.45, p<0.001 log-rank test). Interestingly, the model predicted also overall survival (OS) (AUC 0.86 and R2 0.38, p<0.001). The model was categorized into even-sized low, intermediate (hazard ratio (HR) 2.5, 95%CI [1.1-5.6]) and high-risk groups (HR 8.3, 95%CI [3.7-18.8]) to predict RFS (Fig 1).In the independent validation cohort, CD45+CD2+CD3- (NK) cell count was categorized into even-sized groups based on tertiles. Low and intermediate NK cell count (<0.75% and 0.75-2.0% of all cells, respectively) predicted worse RFS (HR 3.3 p=0.005 and HR 2.2 p=0.05) and OS (HR 2.6 p=0.03 and HR 2.3 p=0.05) after adjusting for induction therapy protocol and HSCT.ConclusionTissue cytometrics of TMA slides with mIHC and automated image analysis enable fast and objective single-cell phenotyping of large patient cohorts. The AML BM is characterized by myeloid and lymphoid lineage immunosuppression. Results of immune cell analyses with both mIHC and FC suggest low NK cell count as biomarker of poor RFS and OS. Other treatment response and survival endpoints remain to be analyzed and complemented with extensive flow cytometric, cytogenetic and molecular genetic data. [Display omitted] DisclosuresPorkka:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Mustjoki:Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; BMS: Honoraria, Research Funding.

Overall survival in metastatic melanoma correlates with pembrolizumab exposure and T cell exhaustion markers.

Trial data support an absence of an exposure–survival relationship for pembrolizumab. As these relationships remain unexamined in a real‐world setting, we determined them in metastatic melanoma prospectively in an observational study. Translational objectives included identifying biomarkers of progressive disease (PD). Checkpoint blockade naïve patients receiving 2 mg/kg Q3W pembrolizumab had pharmacokinetic and clinical outcome data collected. Trough, a valid surrogate for drug exposure, was assessed using ELISA. T‐cell exhaustion and chemokine markers were determined using flow cytometry. Geometric means of exposures and biomarkers were tested against objective response groups using one‐way ANOVA. The cohort was split by the median into high versus low pembrolizumab exposure groups. Kaplan–Meier progression‐free survival (PFS) and overall survival (OS) curves were estimated for high versus low exposure, compared using the log rank test. The high pembrolizumab exposure group (n = 14) experienced substantially longer median OS (not reached vs. 48 months, p = .014), than the low exposure group (n = 14). A similar positive exposure PFS relationship was found (median not reached vs. 48 months, p = .045). The frequency of TIM‐3 expression on CD4+ T cells was significantly higher in PD (mean 27.8%) than complete response (CR) (13.38%, p = .01) and partial response (12.4%, p = .05). There was a near doubling of CXCR6 and TIM‐3 co‐expression on CD4+ T cells in PD (mean 23.3%) versus CR (mean 11.4, p = .003) and partial response (9.8%, p = .0001). We describe positive exposure‐PFS and exposure‐OS relationships for pembrolizumab in metastatic melanoma. TIM‐3, alongside co‐expression of CXCR6 and TIM‐3 on circulating CD4+ T cells are potential bio markers of treatment failure.

TACC3 is a prognostic biomarker for kidney renal clear cell carcinoma and correlates with immune cell infiltration and T cell exhaustion.

Growing evidence has demonstrated that transforming acidic coiled-coil protein 3 (TACC3), a member of the TACC family, may be involved in regulating cell mitosis, transcription, and tumorigenesis. However, the role of TACC3 in kidney renal clear cell carcinoma (KIRC) remains unknown. In this study, multiple databases were used to determine the pattern of TACC3 in KIRC. We found that high TACC3 expression was associated with poor overall survival (OS) in stage I, II, IV and grade 3 KIRC patients. Univariate and multivariate Cox regression analyses showed that TACC3 was an independent risk factor for OS among KIRC patients. Moreover, TACC3 expression correlated with immune cell infiltration levels of B cells, T cells (CD8+, CD4+, follicular helper, regulatory and gamma delta), total and resting natural killer cells, total and activated dendritic cells, and resting mast cells. Furthermore, T cell exhaustion markers, such as PD1, CTLA4, LAG3 and TIM-3 were highly expressed in TACC3 overexpressing tissues. In addition, GSEA analysis revealed that the role of TACC3 in KIRC may be closely linked to immune-associated pathways. Therefore, our study reveals that TACC3 is a prognostic biomarker for OS among KIRC patients and may be associated with immune cell infiltration and T cell exhaustion.

Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells

Non-viral platforms can be applied rapidly and cost-effectively for chimeric antigen receptor (CAR)-T cell manufacturing. In the present paper, we describe in detail a clinically relevant manufacturing process for NKG2D CAR-T cells through electroporation of CAR-encoding piggyBac transposon plasmids and in vitro expansion with K562 artificial antigen-presenting cells. With an optimized protocol, we generated the final cell therapy products with 89.2% ± 10.2% NKG2D CAR-positive cells and achieved the corresponding antigen-dependent expansion between 50,000 and 60,000 folds within 4 weeks. To facilitate repeated CAR-T cell infusions, we evaluated the practicality of cryopreservation followed by post-thaw expansion and an extended manufacturing process for up to 9 rounds of weekly K562 cell stimulation. We found that neither compromised the in vitro anti-tumor activity of NKG2D CAR-T cells. Interestingly, the expression of T cell exhaustion markers TIGIT, TIM3, and LAG3 was reduced with extended manufacturing. To enhance the safety profile of the NKG2D CAR-T cells, we incorporated a full-length CD20 transgene in tandem with the CAR construct and demonstrated that autologous NK cells could mediate efficient antibody-dependent cell-mediated cytotoxicity to remove these CAR-T cells. Collectively, our study illustrates a protocol that generates large numbers of efficacious NKG2D CAR-T cells suitable for multiple rounds of infusions.

Abstract PO075: Re-invigorating tumor infiltrating T lymphocytes against EBV positive nasopharyngeal cancer

Abstract Background/Aim: To determine if tumor-infiltrating T lymphocytes (TIL T) harvested from EBV-positive Nasopharyngeal cancer (NPC) is an effective source of cellular based immunotherapy. Methods: HLA-A02 Peripheral blood (PB; n=6; paired) and NPC tissue biopsies (n=10; undifferentiated, non-keratinizing and EBV positive) were collected from patients with written consent. Peripheral blood mononuclear cells (PBMC) were isolated from buffy coat after density gradient centrifugation of PB. Tissue biopsies were minced, dissociated with human tumour dissociation kit (Miltenyi Biotec) and passed through nylon mesh to single cell suspension. Both PBMC and dissociated tumour were used for T cell expansion by incubation with anti-CD3/CD28 microbeads (Thermo Fisher Scientific) in RPMI-1640 (Hyclone) supplemented with 10% Fetal Bovine Serum (Hyclone) and 20IU/mL recombinant IL-2 (Miltenyi Biotec). TIL T cells were isolated with pan T cell isolation kit (Miltenyi Biotec) after expansion. Functionality of both PB T and TIL T cells were assessed using flow cytometry, trans-well assay and cytotoxicity assay against C666-A2 (NPC cell line expressing HLA-A02) thereafter. Results: Successful isolation and expansion of NPC TIL T cells was achieved using 1-2 fresh NPC biopsy samples (2-3 mm); with fold increase of 210 to 867 after 28 days (1.7-4.5 × 107 cells). Comparison of primary TIL and expanded TIL T demonstrated that the former has greater percentage of CD4+ (p&amp;lt;0.006) while the latter has more CD8+ cells (p&amp;lt;0.003) cells, suggesting preferential expansion of cytotoxic T cells. Expanded TIL T cells also shown greater cytotoxicity against NPC cell line (C666-A2; E:T =1:20) than paired, expanded PB T cells (19.5% vs 4.9%, p=0.02). Furthermore, the cytotoxicity against C666-A2 was even greater (p&amp;lt;0.015) after incubating the TIL T cells with EBV peptides (LMP-1, LMP-2, EBNA-1). Primed TIL T cells also demonstrated greater lytic cell degranulation (CD107a), higher T cell activation (CD137) and IFN-γ secretion than non-primed, expanded TIL T cells. Importantly, expanded TIL T cells also demonstrated increased migration towards C666-A2 than expanded PB T cells (p=0.002). Greater anti-tumour effector function was observed with expanded TIL T despite positive expression of T inhibitory molecules (PD-1, CTLA-4 and TIM-3). Conclusion: TIL T cells can be expanded reliably from NPC biopsies after 28-day culture. Expansion protocol demonstrated preferential expression of cytotoxic T cells over CD4 T cells. The greater anti-tumour effect of expanded TIL T over paired, expanded PB T suggested greater inherent anti-tumour T cells present in NPC TIL despite positive expression of T cell exhaustion markers. This warrant further investigations to explore therapeutic potential of TIL T in NPC. Citation Format: Yitian Png, Audrey Zhi-Yi Yang, Louise Soo-Yee Tan, See-Voon Seow, Swarnalatha Lucky Asidharan, Jamie Mong, Min-Han Tan, Han ChongohOH, Chwee Ming Lim. Re-invigorating tumor infiltrating T lymphocytes against EBV positive nasopharyngeal cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO075.

Abstract PO014: PSGL-1 is an early T cell signaling regulator that drives immunometabolism and terminal differentiation in tumor-specific CD8 T cells

Abstract We previously identified that the adhesion molecule P-selectin glycoprotein ligand-1 (PSGL-1) regulates T cell function and exhaustion in responses to chronic viral infection and melanoma tumors. Subsequently, our studies have focused on investigating the mechanisms by which PSGL-1 regulates T cell activation and exhaustion and evaluating PSGL-1 as a target for cancer immunotherapy. Using the CD4cre system in combination with PSGL-1-floxed mice, we find that T cell intrinsic deletion of PSGL-1 is sufficient to promote enhanced tumor growth control of mesothelioma as well as melanoma tumors. We have found that in vivo PSGL-1 blockade recapitulates the enhanced melanoma tumor growth control observed with PSGL-1-/- CD8+ T cells and limits the development of T cell exhaustion during chronic LCMV Clone 13 viral infection. Conversely, ligation of PSGL-1 in vitro or in vivo drives enhanced upregulation T cell exhaustion markers, most notably PD-1 expression. Phosphoproteomic analysis of early T cell activation revealed the rapid upregulation of more than 30 phosphorylated proteins with significantly enhanced expression following PSGL-1 ligation during T cell activation than T cell activation alone. GSEA and western blot analysis confirmed the increased expression of several regulators of T cell signaling including pAkt1 and pErk in both activated PSGL-1-/- T cells and following PSGL-1 ligation during T cell activation. Single-cell RNA-sequencing of melanoma tumor-specific PSGL-1-/- CD8+ T cells identified differential modulation of genes associated with T cell metabolism and effector responses, including increased Mtor and Hif1a in tumors and Tcf7 in tumor-draining lymph nodes. The Seahorse glycolysis stress test identified that PSGL-1-/- T cells show increased glycolysis after 72 hours of in vitro activation at both sub-optimal and optimal levels of αCD3 stimulation, and 2-NBDG glucose uptake assays confirmed increased glucose uptake by PSGL-1-/- CD8+ T cells within two hours of stimulation. Further, 2-NBDG uptake was increased by PSGL-1-/- CD8+ T cells ex vivo from melanoma tumors. Importantly, this increased glycolytic phenotype does not come at the cost of CD8+ T cell stemness, as determined by TCF-1 staining. We hypothesize that PSGL-1 serves as a key regulator of early T cell activation and repeated signaling through PSGL-1 promotes differentiation into exhausted CD8+ T cells by sustaining TCR signaling. Conversely, we hypothesize that the absence of PSGL-1 allows for faster initiation and termination of TCR signaling, inhibiting differentiation into exhausted CD8+ T cells. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses and their metabolic profile which may drive their enhanced responses to tumors. Citation Format: Jennifer L. Hope, Dennis C. Otero, Eun-ah Bae, Christopher J Stairiker, Ashley B. Palete, Hannah A. Faso, Petrus de Jong, Garth Powis, Linda M. Bradley. PSGL-1 is an early T cell signaling regulator that drives immunometabolism and terminal differentiation in tumor-specific CD8 T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO014.

Abstract PR008: Identifying mechanisms of immune evasion in microsatellite instable endometrial cancer mouse models

Abstract Introduction: Microsatellite instability (MSI), caused by defects in DNA mismatch repair genes, including MLH1 and MSH2, occur in 30% of endometrial cancers (EC). High mutational loads associated with MSI have been associated with greater immunogenicity, and an increase in immune inhibitory factors such as PD-1 and PD-L1. While immunotherapy with anti-PD-L1 has shown promising results in MSI ECs, up to 40% did not respond, suggesting there are alternate immunosuppressive mechanisms. To improve understanding of response/resistance to immunotherapy, we evaluated immune activation and evasion using a novel immunocompetent mouse model of MSI EC. Methods: Endometrial lesions were evaluated in mice with MSH2 loss targeted to the endometrium (PRCre+MSH2flox/flox). When aged to 12-16 months, 22% develop hyperplasia and 18% develop spontaneous EC. MSI status was confirmed by PCR. Immune infiltration and function were assessed by immunohistochemistry (IHC) and Nanostring digital spatial profiling. Tumors were evaluated for tumor infiltrating lymphocytes (TIL) based on CD8a expression and found to separate into TILHIGH and TILLOW groups and further characterized to determine immune activation/resistance mechanisms using transcriptome analysis with Affymetrix Clariom D assay and validation by RT-PCR. A gene signature score approach was used to quantify immune-related expression changes for tumors from PRCre+MSH2flox/flox mice and MSI EC patient data from TCGA. Cell lines were generated from tumors and used to evaluate a syngeneic orthotopic mouse model for immunotherapy studies, where immune infiltration was characterized. Results: All endometrial tumors were MSI and included endometrioid, serous, and mixed histologies. Tumors showed varying degrees of CD8+ T cell infiltration, irrespective of histology. TILHIGH tumors had increased infiltration of dendritic cells and Type I interferon response, including genes IFI203 (fold-change, FC=18.5), IFI204 (FC=11.1) and CXCL9 (FC=5.7). There was no significant difference in type II interferon response. TILLOW tumors had low expression of IFN-associated genes. TILHIGH tumors also exhibited increased immune exhaustion markers such as TIM3 (FC=2.4) and LAG3 (FC=3.2). Tumoral PD-L1 expression was negative (&amp;lt;1% of positive cells). Similar to our mouse model, TILHIGH MSI uterine cancer patients from TCGA also showed activation of type I interferon, increased dendritic cell infiltration, and increased T cell exhaustion markers. Primary cell lines used in an orthotopic mouse model retained the immune profile of the original tumor. Conclusions: Elucidating immune activation and evasion mechanisms is critical for improving immunotherapies. The PRCre+MSH2flox/flox mouse model reflects the spectrum of immunogenicity observed in patients. A panel of primary cell lines generated from this model are an important tool that enable studies of immunotherapy efficacy and resistance mechanisms, in both immunogenic and non-immunogenic tumors. Citation Format: Brenda Melendez, Emily Hinchcliff, Nisha Gokul, Xiaoping Su, Daniel McGrail, Elizabeth Whitley, Russell Broaddus, Rosemarie Schmandt, Karen Lu, Melinda Yates. Identifying mechanisms of immune evasion in microsatellite instable endometrial cancer mouse models [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR008.

RNF183 Is a Prognostic Biomarker and Correlates With Tumor Purity, Immune Infiltrates in Uterine Corpus Endometrial Carcinoma.

RNF183, a member of the E3 ubiquitin ligase, has been shown to involve in carcinogenesis and proposed as one of the biomarkers in Uterine Corpus Endometrial Carcinoma (UCEC). However, no research focused on the role of RNF183 in UCEC. We analyzed the expression and immune infiltration of RNF183 in UCEC. TIMER, UALCAN, and GEPIA were used to analyze the gene expression of RNF183. We emplored Kaplan-Meier Plotter to examine the overall survival and progression-free survival of RNF183, and applied GeneMANIA to identify RNF183-related functional networks. LinkedOmics was helpful to identify the differential gene expression of RNF183, and to further analyze gene ontology and the genome pathways in the Kyoto Protocol. Finally, we used TIMER to investigate the immune infiltration of RNF183 in UCEC. Otherwise, we partly verified the results of bioinformatics analysis that RNF183 controlled ERα expression in ERα-positive Ishikawa cells dependent on its RING finger domain. We also found that ERα increased the stability of RNF183 through the post-translational mechanism. Together, patients with a high level of RNF183 harbor favorable overall and progression-free survival. High expression of RNF183 was associated with a low stage, endometrioid, and TP53 Non-Mutant status in endometrial cancer. The RNF183 expression was greater at higher expression and the tumor stage was greater at the lower level. On the side of immunization, high level of RNF183 in UCEC is negatively related to tumor purity, infiltrating levels of CD4 + T cells, neutrophils, and dendritic cells. Besides, the expression of RNF183 in UCEC is significantly correlated with the expression of several immune cell markers, including B cell, M1 macrophage marker, M2 Macrophage, Dendritic cell, Th1 markers, Th2 markers, Treg markers, and T cell exhaustion markers, indicating its role in regulating tumor immunity. These results suggested that RNF183 may be considered as a novel prognostic factor in endometrial cancer and an early diagnostic indicator for patients with UCEC.