Cells In Tumor-infiltrating Lymphocytes Research Articles

CD3D has the Potential to be a Prognostic Factor for Endometrial Carcinoma and an Indicator of Tumor Immune Microenvironment Regulation: a Study based on TCGA Data Mining

Accumulating evidence shows that tumor immune microenvironment infiltration plays a crucial role in the occurrence and development of endometrial carcinoma (EC). At present, studying the regulation mechanism of the immune microenvironment in EC remains a challenge. We calculated the amount of immune and stromal components and the proportion of tumor-infiltrating immune lymphocytes via ESTIMATE, single-sample gene enrichment analysis (ssGSEA), and CIBERSORT computational methods. Differentially expressed genes were analyzed by protein–protein interaction network construction and univariate survival analysis. CD3D expression was negatively correlated with clinicopathological features, such as age, histological type, clinical grade, and pathological stage, but positively correlated with the survival of patients with EC. Logistic regression analysis suggested that CD3D expression could be used as a biomarker to predict EC occurrence. Gene set enrichment analysis revealed that CD3D was upregulated in the immune effect pathway. Interestingly, CD3D was downregulated in the EC pathway. EC was divided into three immune cell subsets by ssGSEA. Correlation analysis between ssGSEA and CIBERSORT showed that the infiltration level of CD8 + T cells and plasma cells in tumor-infiltrating lymphocytes was lower than that in the high-immune-score group (Immunity_H). Moreover, CD3D expression in Immunity_H was low, and the prognosis was even worse. This study demonstrated that low CD3D expression is an index of poor prognosis in patients with EC. Levels of CD3D in tumor microenvironment (TME) might be a useful indicator for predicting the prognosis of patients with EC patients.

Immunophenotypic Differences in Tumor-Infiltrating Lymphocytes and Neovascularization Between Primary Cutaneous Melanoma With and Without Metastasis: An Immunohistochemical Study of 80 Cases.

The prognostic implications of the immunophenotype of the tumor-infiltrating lymphocytes (TILs) in primary cutaneous melanoma are well known. In recent years, the study of this immunophenotype has also resulted in immunotherapeutic consequences. The aims of this study were to characterize the subpopulations of TILs in primary cutaneous melanoma, in cases with and without metastasis, as well as the neovascularization associated with the primary neoplasm, and its influence on the development of metastasis. To this end, the immunophenotype of TILs and the neovascularization of 80 patients with primary cutaneous melanoma (40 each with metastatic and non-metastatic melanoma) were analyzed by immunohistochemistry for CD3, CD4, CD8, FOXP3, PD-1, CD31, and D2-40 antibodies. We found that higher frequencies of TILs with brisk pattern, and CD4+, CD8+, and CD20+ cells in TILs, and a lower frequency of CD31+ vessels were histopathological features associated with better prognosis in primary cutaneous melanoma. Our results support the notion that the immunohistochemical study of TILs and neovascularization in primary cutaneous melanoma may be helpful tools for identifying patients at increased risk of metastasis development.

Translational research of VOLTAGE-A: Efficacy predictors of preoperative chemoradiotherapy and consolidation nivolumab in patients with both microsatellite stable and microsatellite instability-high locally advanced rectal cancer.

100 Background: In VOLTAGE-A, chemoradiotherapy (CRT; 50.4Gy with capecitabine, 1,650mg/m2) followed by five cycles of consolidation nivolumab (nivo) (240mg q2 weeks) showed 30% pathological complete response [pCR; AJCC tumor regression grade (TRG) 0] and 38% major pathological responses (AJCC TRG 0-1) in patients with microsatellite stable (MSS) locally advanced primary rectal cancer (LARC). In addition, 60% pCR was observed in patients with microsatellite instability-high (MSI-H) LARC. In this study, we aimed to determine the predictive biomarkers for efficacy of sequential preoperative CRT and consolidation nivo. Methods: Serial tumor biopsies were performed at four time points:pre-CRT; post-CRT; post-3 cycles of nivo; and pre-surgery. We analyzed the immune status of the patients by flow cytometry using the collected tumor-infiltrating lymphocytes (TILs) dissociated from tumor samples. Whole exome and RNA sequencing analyses were conducted using the extracted DNA and RNA from tumor, respectively. The PD-L1 status of tumor samples was also evaluated by in vitro diagnostic immunohistochemistry staining. Results: Of the 38 MSS patients whose PD-L1 tumor proportion score (TPS) was analyzable in pre-CRT samples, the pCR rates were 67% (6/9) and 17% (5/29) in positive (≥1%) and negative PD-L1 status (p = 0.009), respectively. Among the 24 MSS patients whose samples were serially collected, the pCR rates according to CD8+ T cells/effector regulatory T cells (CD8/eTreg) ratio in TILs of pre-CRT samples ≥2.5 and < 2.5 were 78% (7/9) and 13% (2/15), respectively (p = 0.003). The CD8/eTreg ratio in TILs was consistently high in patients with pCR during the study treatments. Ki-67 and PD-1 expression by CD8+ T cells in TILs was significantly high in pre-CRT samples from patients TRG 0-1. Conversely, in patients with TRG 2-3, CTLA-4 expression by both CD4+ T cells and CD8+ T cells in TILs was significantly high after five cycles of nivo, suggesting the potential resistance mechanisms of nivo monotherapy. Of the 21 MSS patients whose consensus molecular subtype (CMS) was analyzable, those with CMS1 and CMS3 tumors achieved 100% (2/2) and 60% (4/6) TRG 0-1, respectively. In contrast, patients with CMS2 and CMS4 tumors achieved 43% (3/7) and 29% (2/7) TRG 0-1, respectively. The tumor mutational burden (TMB) of pre-CRT samples in five MSI-H patients was higher than that in the 24 MSS patients (median: 13.2 vs. 0.99 mutation/Mbp, p < 0.0001). Among MSS patients, TMB was higher in patients with TRG 0-1 than in patients with TRG 2-3 (median: 1.45 vs. 0.84 mutation/Mbp, p = 0.016). Conclusions: Positive PD-L1 TPS; high CD8/eTreg ratio; Ki-67, PD-1, and CTLA-4 expression by CD8+ T cells in TILs; CMS 1 or 3; and high TMB can be good predictors of efficacy of preoperative CRT followed by nivo. Clinical trial information: NCT02948348.

Immune-associated molecular occurrence and prognosis predictor of hepatocellular carcinoma: an integrated analysis of GEO datasets

ABSTRACT Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second most common cause of cancer-related deaths worldwide. As immune response failure is the main factor in the occurrence and poor prognosis of HCC, our study aimed to develop an immune-associated molecular occurrence and prognosis predictor (IMOPP) of HCC. To that end, we discovered a 4-gene immune-associated gene signature: C–C motif chemokine ligand 14 (CCL14), kallikrein B1 (KLKB1), vasoactive intestinal peptide receptor 1 (VIPR1), and cluster of differentiation 4 (CD4). When tested on three cohorts as an immune-associated molecular occurrence predictor (IMOP), it had high sensitivity, specificity, and area under the receiver operating characteristics curve. When tested as an immune-associated molecular prognosis predictor (IMPP), it stratified the HCC prognosis for overall survival (Kaplan–Meier analysis, log rank P = 0.0016; Cox regression, HR = 1.832, 95% CI = 1.173–2.859, P = 0.008) and disease-free survival (Kaplan-Meier analysis, log rank P = 0.0227). IMPP also significantly correlated with the clinicopathological characteristics of HCC; integrating it with clinicopathological characteristics improved the accuracy of a nomogram for overall survival prediction (C-index: 0.7097 vs. 0.6631). In HCC tumor microenviroments, the proportion of CD8+ T cells significantly differed between IMOP-stratified groups. We conclude that IMOPP can potentially predict the occurrence of HCC in high-risk populations and improve prognostic accuracy by providing new biomarkers for risk stratification. In addition, we believe that the IMOP mechanism may be related to its effect on the proportion of CD8+ T cells in tumor-infiltrating lymphocytes.

Apolipoprotein E deficiency enhances the anti-tumor immunity via repressing T cell exhaustion

Apolipoprotein E (ApoE), a component of plasma lipoproteins, also can promote tumor cell proliferation, migration and metastasis. However, the role of host ApoE in regulating anti-tumor immunity is still unknown. Here host ApoE-deficient (ApoE −/− ) mice were used to understand the role of ApoE in antitumor immunity. Tumor-infiltrating lymphocytes subsets and exhausted T cells were measured by flow cytometry in B16F10 and MC38 immunogenic tumor-bearing mice. Both B16F10 and MC38 growth were significantly repressed in ApoE −/− mice. Tumor-infiltrating T cells and pro-inflammatory M1 macrophages were significantly increased in ApoE −/− mice. Moreover, decreased PD-1+ cells representing exhausted CD8+ T cells and increased cytotoxic CD8+ T cells function and proliferation indicating as TNF- α+, IFN-γ+, GZMB+ and Ki67+ CD8+ cells in tumor-infiltrating lymphocytes of ApoE −/− mice may contribute to the antitumor function of host ApoE deficiency. Thus, inhibition of ApoE could be a good strategy for cancer immunotherapy.

Durvalumab and tremelimumab with or without stereotactic body radiation therapy in relapsed small cell lung cancer: a randomized phase II study

BackgroundImmune checkpoint blockade (ICB) targeting programmed cell death protein 1 and cytotoxic T lymphocyte-associated protein 4 has achieved modest clinical activity as salvage therapy in relapsed small cell lung cancer...

RX-5902, a novel \u03b2-catenin modulator, potentiates the efficacy of immune checkpoint inhibitors in preclinical models of triple-negative breast Cancer

BackgroundTriple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited systemic treatment options. RX-5902 is a novel anti-cancer agent that inhibits phosphorylated-p68 and thus attenuates nuclear β-catenin signaling. The purpose of this study was to evaluate the ability of β-catenin signaling blockade to enhance the efficacy of anti-CTLA-4 and anti-PD-1 immune checkpoint blockade in immunocompetent, preclinical models of TNBC.MethodsTreatment with RX-5902, anti-PD-1, anti-CTLA-4 or the combination was investigated in BALB/c mice injected with the 4 T1 TNBC cell line. Humanized BALB/c-Rag2nullIl2rγnullSIRPαNOD (hu-CB-BRGS) mice transplanted with a human immune system were implanted with MDA-MB-231 cells. Mice were randomized into treatment groups according to human hematopoietic chimerism and treated with RX-5902, anti-PD-1 or the combination. At sacrifice, bone marrow, lymph nodes, spleen and tumors were harvested for flow cytometry analysis of human immune cells.ResultsThe addition of RX-5902 to CTLA-4 or PD-1 inhibitors resulted in decreased tumor growth in the 4 T1 and human immune system and MDA-MB-231 xenograft models. Immunologic analyses demonstrated a significant increase in the number of activated T cells in tumor infiltrating lymphocytes (TILs) with RX-5902 treatment compared to vehicle (p < 0.05). In the RX-5902/nivolumab combination group, there was a significant increase in the percentage of CD4+ T cells in TILs and increased systemic granzyme B production (p < 0.01).ConclusionsConclusions: RX-5902 enhanced the efficacy of nivolumab in a humanized, preclinical model of TNBC. Several changes in immunologic profiles were noted in mice treated with RX-5902 and the combination, including an increase in activated TILs and a decrease in human myeloid populations, that are often associated with immunosuppression in a tumor microenvironment. RX-5902 also was shown to potentiate the effects of checkpoint inhibitors of CTLA4 and the PD-1 inhibitor in the 4 T-1 murine TNBC model. These findings indicate that RX-5902 may have important immunomodulatory, as well as anti-tumor activity, in TNBC when combined with a checkpoint inhibitor.

Abstract 6644: A comprehensive in vitro and in vivo system to evaluate STING agonist efficacy in cancer therapeutics

Abstract A number of stimulator of interferon genes protein (STING) agonists has recently been developed to treat cancer and infectious diseases. To investigate the binding of ligand to STING and subsequent activation of the STING pathway, we used the protein thermal shift assay, a high-throughput screening method for intermolecular interaction involving a protein of interest, to identify previously unknown compounds that bind to STING. To determine the functional consequence of ligand binding, we constructed IFN-β-Luc THP1 reporter cell line, to evaluate the pathway activation together with IFNβ secretion. In addition, we incubated human peripheral blood mononuclear cells (PBMCs) with STING agonist and measured phosphorylation of STING, phosphorylation of IRF3 and the secretion of cytokines such as IFN-β by ELISA or Luminex. To determine the in vivo therapeutic effects of STING agonists, we performed efficacy studies in syngeneic mouse models of melanoma (B16F10 cell line), colorectal cancer (CT-26 cell line) or breast cancer (4T1 cell line) with intratumoral delivery of test compounds. Test Treatment with STING agonists resulted in significant tumor growth inhibition as measured by tumor volume and significantly improved survival. Tumor-reactive IFN-γ secreting CD8+ T cells were harvested from spleens, purified by immunomagnetic sorting, and immediately used in ELISPOT assays using the mouse IFN-γ ELISPOT Kit. Tumor-infiltrating lymphocytes (TILs) have also been assessed in harvested melanoma (B16F10) tumors. We found that treatment with STING agonists resulted in increased CD69 percentage in CD8+ T cells in TILs, and decreased MHCII levels in macrophages and DCs in TILs. In summary, we have established reporter-based in vitro assays and in vivo efficacy validation platforms, which can be used for STING agonists efficacy evaluation for cancer therapy and combination IO target therapy. Citation Format: Xiaohe Shi, Feifei Fan, Ruicheng Wei, Xiangyang Zuo, Xiutao Lu, Quanhong Sun. A comprehensive in vitro and in vivo system to evaluate STING agonist efficacy in cancer therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6644.

Impact of T-cell receptor and B-cell receptor repertoire on the recurrence of early stage lung adenocarcinoma

Surgical resection is the only curative treatment for patients with early stage non-small cell lung cancer. However, approximately 33% of non-small cell lung cancer patients recur with the stage I disease, which may be attributed to a deficiency in antitumor immunity. In the present study, for early stage lung adenocarcinoma patients with early recurrence and early non-recurrence, we investigated the quantity of tumor-infiltrating T and B cells by immunohistochemistry, as well as the genes in the complementarity determining region 3 of the T-cell receptor β chain and the B-cell receptor immunoglobulin heavy chain. A decreased number of tumor-infiltrating lymphocytes cells (CD3+, CD4+, CD8+ and CD20+) was present in early recurrence patients. A significant increase in oligoclones and a reduction in T-cell receptor diversity were observed in the early recurrence group. Furthermore, there was a preference for V, J gene, and VJ gene combinations in patients with early recurrence versus non-recurrence, suggesting that this may be a new biomarker for the recurrence of early stage lung adenocarcinoma. These data indicate that T and B cell receptor repertoires influence the depth of human adaptive immune responses, and in addition to the quantity of tumor infiltrating T and B cells, may contribute to the prevention of early stage lung adenocarcinoma recurrence after surgical resection. Our study illustrates the potential value of the immune repertoire for predicting clinical efficacy and patient outcomes.

Translational research of voltage-A1: Efficacy predictors of preoperative chemoradiotherapy and subsequent nivolumab monotherapy in patients with microsatellite-stable locally advanced rectal cancer.

4073 Background: In VOLTAGE-A1, after 5 cycles of nivolumab (240 mg q2 weeks) plus radical surgery following chemoradiotherapy (CRT; 50.4 Gy with capecitabine 1,650 mg/m2), a major pathologic response is observed in 38% (AJCC tumor regression grade 0-1) of 37 patients with microsatellite-stable locally advanced primary rectal cancer. Here, biomarkers for predicting the efficacy of this treatment were investigated. Methods: Serial tumor biopsies and blood collections were performed at 4 time points; before CRT, after CRT, after 3 cycles of nivolumab, and before surgery. Tumor-infiltrating lymphocytes (TILs) and DNA/RNA were extracted from tumor samples, and peripheral blood mononuclear cells (PBMCs) were extracted from blood samples. We analyzed the immune status of the patients by flow cytometry using the collected TILs and PBMCs. Whole exome and RNA sequencing analyses were conducted using the extracted DNA and RNA, respectively. The PD-L1 status of tumor samples was also evaluated by in vitro diagnostic immunohistochemistry staining. Results: Among the 24 patients whose samples were serially collected, 11 (46%) were AJCC grade 0-1 and 13 were 2-3. Before CRT, effector regulatory T (eTreg) cells in TILs were higher in patients with AJCC grade 2-3, and both the CD8+ T cell/eTreg cell ratio in TILs and PD-L1-positive tumor cells (≥1%) were higher in patients with AJCC grade 0-1 (p = 0.047, p = 0.083, respectively). Ki67 expression by CD8+ T cells in TILs was higher before CRT in patients with AJCC grade 0-1 (p = 0.037) and increased after CRT in all patients. Patients with consensus molecular subtype (CMS) 1 and CMS3 achieved AJCC grade 0-1 at rates of 100% (2/2) and 60% (4/6), respectively. In contrast, patients with CMS2 and CMS4 achieved AJCC grade 0-1 at rates of 43% (3/7) and 29% (2/7), respectively. The tumor mutation burden of pre-CRT samples was significantly higher in patients with AJCC grade 0-1 (median 1.45/MB) than in patients with AJCC grade 2-3 (0.84/MB) (p = 0.016). Conclusions: A higher CD8+ T cell/eTreg cell ratio, PD-L1-positive, Ki67 expression by CD8+ T cells in TILs, CMS1 or 3, and higher tumor mutation burden are good predictors of the efficacy of the sequential combination of CRT and nivolumab. Further results will be reported in the meeting. Clinical trial information: NCT02948348 .

Elevated IL18 levels in Nasopharyngeal carcinoma induced PD-1 expression on NK cells in TILS leading to poor prognosis.

Characterisation of tumor-infiltrating lymphocytes (TILS) population for cancer prognostication has enabled deeper understanding of tumor immune interactions in cancer immunology. We aim to examine the significance of both the density and functional status of NK cells in a cohort of Epstein Barr Virus (EBV) associated Nasopharyngeal Cancer (NPC) patients. NK TILS of 50 NPC samples were quantified on immunohistochemistry and the density of NK TILS was correlated with clinical outcomes. Next, NK cells and a panel of cytokines of 10 newly diagnosed NPC patients were characterized in both NPC tissue and peripheral circulation. Exhausted NK cells were identified using co-expression of PD-1 and/or Tim-3. Comparison of percentage of NK cells in NPC and healthy controls was performed using student t-test for two groups; and a p value of less than 0.05 values was considered significant. NK TILS exhibited a bimodal distribution; with the NKhigh cohort demonstrating a poorer 2-year overall survival rate (p<0.035). In-vitro studies revealed a higher proportion of infiltrated NK cells in the NKhigh cohort co-expressed PD-1. Additionally, IL-18 levels in NPC tissue were significantly higher than in healthy nasopharynx; and IL-18 alone induced PD-1 expression on NK cells. Expectedly, plasma IL-18 concentration and percentage of circulating PD-1-expressing NK cells were similar among NPC patients and healthy controls. The cytotoxic function of NK TILS is mitigated by an elevated IL-18 levels within the NPC microenvironment. Hence, the functional status, and the density of NK cells in TILS should be considered when prognosticating NPC.

Abstract B41: Bis-indole derived NR4A1 antagonist induces PD-L1 degradation and enhances antitumor immunity

Abstract PD-L1 is expressed in tumor cells and interaction with PD-1 in immune cells plays an important role in evading immune surveillance, and this can be overcome by immunotherapy using antibodies against PD-L1 or PD-1. This study reports a novel approach for targeting PD-L1. Results of RNA interference, chromatin immunoprecipitation, and mutational analysis show that in MDA-MB-231 and other human breast cancer cells and 4T1 mouse mammary tumor cells the PD-L1 expression is regulated by the nuclear receptor 4A1/NR4A1/Sp1 complex bound to the proximal GC-rich region of the PD-L1 gene promoter. Treatment breast cancer cells with bis-indole derived NR4A1 antagonists including 1,1-bis(3´-indolyl)-1-(3-chloro-4-hydroxy-5-methoxyphenyl)methane (ClOCH3) decreased expression of PD-L1 mRNA promoter-dependent luciferase activity and protein. In vivo studies using a syngeneic mouse model with Balb/c mice bearing orthotopically injected 4T1 cells showed that 2.5, 7.5, and 12.5 mg/kg/d of ClOCH3 decreased tumor growth and weight and inhibited lung metastasis. The NR4A1 antagonist ClOCH3 also decreased expression of CD+/CD4+/CD25+/FoxP3 regulatory T cells in tumor-infiltrating lymphocytes and increased Teff/Treg ratios. Thus, the potent anticancer activities of NR4A1 antagonists are also accompanied by enhanced antitumor immunity in breast cancer and represent a novel class of drugs that are immunotherapy mimics. Note:This abstract was not presented at the conference. Citation Format: Keshav Karki, Gus Wright, Jin Un-Ho, Kumaravel Mohankumar, Xing Zhang, Stephen Safe. Bis-indole derived NR4A1 antagonist induces PD-L1 degradation and enhances antitumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B41.

Response to ipilimumab therapy in metastatic melanoma patients: potential relevance of CTLA-4+ tumor infiltrating lymphocytes and their in situ localization.

Immune checkpoint inhibitors, including ipilimumab (IPI), achieve a clinical benefit in a small proportion of melanoma patients highlighting the need to investigate predictive biomarkers. In this study, we characterized tumor infiltrating lymphocytes (TILs), focusing on the CTLA-4+ subset, and evaluated their possible predictive significance. We characterized TIL density, cell type, and localization in 40 melanoma lesions from 17 patients treated with IPI. Associations of TILs with IPI timing, tissue localization, and response to IPI were estimated using a linear mixed-effects modelling approach. We found that most of TIL subsets increased in situ upon IPI therapy, with particular reference to FoxP3+ cells. TILs and TIL subsets, such as CD3+, CD45RO+, CTLA-4+, CD4+, CD8+ T cells, CD20+ B cells, and NKp46+ NK cells, showed significantly different spatial distributions in the tumor microenvironment being higher at the invasive margin (IM) as compared to the tumor center (TC) (P value < 0.001 for TIL score and P value < 0.05 for all subsets). Remarkably, high TIL score and density of CD3+, CD8+ T cells, and CTLA-4+ immune cells were significantly associated with a better response to IPI (P values = 0.002, 0.023, 0.007, and 0.001, respectively, for responders vs non-responders). In conclusion, we provide a detailed analysis of CTLA-4+ TIL distribution in melanoma tissues taking into account localization, relationship with CD3+/CD8+ TILs, and changes in response to IPI treatment. We identified that CTLA-4+ TILs may represent a marker of IPI response, alone or with CD3+/CD8+ subsets, although this requires confirmation in larger studies.

Japanese Kampo Medicine Juzentaihoto Enhances Antitumor Immunity in CD1d−/− Mice Lacking NKT Cells

Although the Japanese traditional herbal medicine (Kampo), Juzentaihoto (JTT), has been reported to have antitumor effects in several tumor models, its role in tumor immunology remains controversial. In the present study, we tested whether oral administration of JTT enhances antitumor immunity in CD1d−/− mice, in which immunosuppression was partially relieved due to the lack of NKT cells. In a subcutaneous murine syngeneic CT26 colorectal tumor model, JTT had no impact on tumor growth in wild type (WT) BALB/c mice. However, the growth rate of tumors was significantly slower in CD1d−/− mice than in WT mice. Surprisingly, JTT significantly delayed tumor growth in such CD1d−/− mice. In vivo depletion of CD8+ T cells revealed that CD8+ T cells are required for JTT’s antitumor activity. Moreover, tumor-reactive cytotoxic T-lymphocytes were detected exclusively in JTT-treated mice with well-controlled tumors. JTT did not affect the number of tumor-infiltrating CD4+ regulatory T cells. On the contrary, JTT increased the degranulation marker CD107a+ CD8+ T cells and decreased Ly6G+ Ly6Clo polymorphonuclear myeloid-derived suppressor cells in tumor-infiltrating lymphocytes, most probably contributing to the suppression of tumor growth in JTT-treated mice. Nonetheless, JTT had no impact on the proportion of monocytic myeloid-derived suppressor cells. In conclusion, our results indicate that in the absence of NKT cells, JTT augments antitumor immunity by CD8+ T cells, suggesting that this Kampo medicine is a promising anticancer adjuvant when negative immune regulation is partially relieved.

PD-1 Expression and Function of T-Cell Subsets in TILs From Human Lung Cancer.

On the basis of the autologous tumor-infiltrating lymphocytes (TILs) or genetically modified TILs for adoptive cell therapy have received more attention. Programmed cell death protein 1 (PD-1) expression on the T cells exert complex response during the tumor immune response. But the composition and function of PD-1T-cell subsets in TILs from human lung cancer still limited. In blood and TILs from human lung cancer patients, we confirmed that PD-1 is expressed in higher levels in CD4T-cell subsets than in CD8T-cell subsets. To further analyze the function of PD-1T cells in TILs, we observed the cytokine production in different T-cell subsets. We found that higher interferon-γ and granzyme B production in CD4/CD8PD-1T-cell subsets in TILs than in peripheral blood mononuclear cells (PBMCs); except for PD-1Tscm, higher tumor necrosis factor-α production was observed in PD-1T-cell subsets in TILs than in PBMCs; the expression level of interleukin-17 were lower in PD-1T cells in TILs than in PBMCs; and perforin expression was significantly reduced in CD4PD-1T cells subsets in TILs compared with peripheral blood. Clarify elucidating the composition and function of PD-1T-cell subsets in TILs will have great value in clinical application for evaluating the sensitivity to PD-1 blockade and selecting the promising candidate T-cell subsets in TILs for combination immunotherapy in human lung cancer.

1242P - Clinical utility of precision immunoprofiling and monitoring of the tumour microenvironment using flow cytometry and CyTOF in patients with advanced NSCLC treated with atezolizumab: Results from a phase II study for biomarker analysis (EPOC1702)

1242P - Clinical utility of precision immunoprofiling and monitoring of the tumour microenvironment using flow cytometry and CyTOF in patients with advanced NSCLC treated with atezolizumab: Results from a phase II study for biomarker analysis (EPOC1702)

1577P - Analysis of the tumour microenvironment and tumour genotype through different stages of lung adenocarcinoma

BackgroundTreatment options for metastasized NSCLC are steadily evolving and novel therapeutic approaches are increasingly based on molecular data. Predictive biomarkers include the presence of driver mutations and PD-L1 tumor status. The contribution of the tumor microenvironment to patient outcome requires further evaluation. In this study we genetically characterized 133 lung adenocarcinomas, assessed PD-L1 status and quantified tumor-infiltrating lymphocytes (TIL) within the tumor microenvironment. Data was evaluated depending on tumor stage. MethodsTumor resections of 156 patients with lung adenocarcinomas were categorized into UICC tumor stages I-IV. Immunohistochemical analysis was performed to characterize TIL with antibodies against CD3, CD8, FoxP3 and PD-1 for T-Lymphocytes, CD19, CD20 (B cells) and CD68 (histiocytes). Total numbers of TIL and tumor cells were analyzed by counting 3 high-power fields each (40x magnification). In addition, we performed amplicon-based next-generation sequencing for 17 therapeutically relevant genes in 133 of cases were material was sufficient. ALK status was assessed by IHC. ResultsThe most common genetic alterations in our cohort of lung adenocarcinomas were TP53 mutations with 53.38% (71/133), followed by KRAS with 37.59% (50/133). In terms of targetable driver aberrations we detected frequencies of 10.53% for EGFR (14/133), 0.67% (1/133) for BRAF V600E and 3.76% (5/133) for ALK. While driver mutations were evenly distributed through tumor stages I-IV, TP53 mutations were significantly associated with tumor stages III and IV (p=0.0021). Further, we observed an unexpected correlation of driver alterations (KRAS, BRAF, ALK and EGFR) with the number of infiltrating CD19 positive B cells (p=0.0151) as well as the number of CD19 and CD20 positive B cell follicles within the tumor environment (p=0.0334). ConclusionsSupporting the concept of TP53 as a passenger mutation, we detected significantly more TP53 mutations in late stage disease. In addition, we found a pronounced B cell response in tumors carrying driver mutations. Characterization of the tumor microenvironment might become an integral part of biomarker assessment in solid tumors in the future. Legal entity responsible for the studyHematopathology Hamburg. FundingHas not received any funding. DisclosureM. Tiemann: Honoraria (self), Advisory / Consultancy, Research grant / Funding (institution), Travel / Accommodation / Expenses: Novartis; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: Boehringer Ingelheim; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: AstraZeneca; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: MSD; Honoraria (self), Advisory / Consultancy, Travel / Accommodation / Expenses: BMS. All other authors have declared no conflicts of interest.

Safety and efficacy of adoptive cell transfer using autologous tumor infiltrating lymphocytes (LN-145) for treatment of recurrent, metastatic, or persistent cervical carcinoma.

2538 Background: There is a high unmet medical need for effective treatments for patients with recurrent, metastatic, or persistent cervical cancer. Most patients are young and survival rates are poor. ORR for second line therapies is between 4 and 14% for chemotherapy and recently approved immunotherapy. Adoptive cell transfer using tumor infiltrating lymphocytes (TIL) have demonstrated durable responses in some patients with recurrent cervical cancer thus offering the potential for long-term disease control. Methods: Study C-145-04 is an ongoing, open-label, multicenter Phase 2 clinical trial evaluating the safety and efficacy of LN-145 TIL therapy in patients with advanced cervical cancer who have undergone at least one prior line of chemotherapy. Prior checkpoint inhibitor therapy is an exclusion criterion. The primary endpoint is ORR per RECIST 1.1; secondary endpoints include duration of response (DOR), disease control rate (DCR), and LN-145 safety. Tumors surgically harvested at local institutions are shipped to central GMP facilities for TIL generation in a 22-day manufacturing process. Final LN-145 TIL product is cryopreserved and shipped to sites. Patients receive one week of preconditioning lymphodepletion (cyclophosphamide, fludarabine), a single LN-145 infusion, followed by up to 6 doses of IL-2 (600,000 IU/kg). Results: As of 4 Feb 2019, 27 efficacy-c patients have received Gen 2 of LN-145, with a mean age of 47 years and 2.6 mean prior lines of therapy. Preliminary efficacy results: ORR was 44% (1 CR, 9 PR, 2 uPR), DCR was 89% at 3.5-month median study follow-up with 11/12 patients maintaining their response. Improved responses were observed in 4 patients with longer follow-up. Mean TIL cells infused was 28x109. Median IL-2 doses administered was 6.0. The adverse event profile was generally consistent with the underlying advanced disease and the profile of the lymphodepletion and IL-2 regimens. Conclusions: LN-145 results in 44% ORR in previously treated cervical cancer patients with acceptable safety and efficacy profile. LN-145 offers patients a viable therapeutic option warranting further investigation. Clinical trial information: NCT03108495.

Defective Localization With Impaired Tumor Cytotoxicity Contributes to the Immune Escape of NK Cells in Pancreatic Cancer Patients.

Tumor-infiltrating lymphocytes (TILs), found in patients with advanced pancreatic ductal adenocarcinoma (PDAC), are shown to correlate with overall survival (OS) rate. Although majority of TILs consist of CD8+/CD4+ T cells, the presence of NK cells and their role in the pathogenesis of PDAC remains elusive. We performed comprehensive analyses of TIL, PBMC, and autologous tumor cells from 80 enrolled resectable PDAC patients to comprehend the NK cell defects within PDAC. Extremely low frequencies of NK cells (<0.5%) were found within PDAC tumors, which was attributable not to the low expression of tumor chemokines, but to the lack of chemokine receptor, CXCR2. Forced expression of CXCR2 in patients' NK cells rendered them capable of trafficking into PDAC. Furthermore, NK cells exhibited impaired cell-mediated killing of autologous PDAC cells, primarily due to insufficient ligation of NKG2D and DNAM-1, and failed to proliferate within the hypoxic tumor microenvironment. Importantly, these defects could be overcome by ex-vivo stimulation of NK cells from such patients. Importantly, when the proliferative capacity of NK cells in vitro was used to stratify patients on the basis of cell expansion, patients whose NK cells proliferated <250-fold experienced significantly lower DFS and OS than those with ≥250-fold. Ex-vivo activation of NK cells restored tumor trafficking and reactivity, hence provided a therapeutic modality while their fold expansion could be a potentially significant prognostic indicator of OS and DFS in such patients.

Targeting VEGFR2 with Ramucirumab strongly impacts effector/ activated regulatory T cells and CD8+ T cells in the tumor microenvironment

BackgroundSeveral studies have established a correlation between the VEGF–VEGFR2 axis and an immunosuppressive microenvironment; this immunosuppression can be overcome by anti-angiogenic reagents, such as ramucirumab (RAM). However, little is known about the immunological impact of anti-angiogenic reagents within the tumor microenvironment in human clinical samples. This study aimed at investigating the effects of RAM on the tumor microenvironmental immune status in human cancers.MethodsWe prospectively enrolled 20 patients with advanced gastric cancer (GC) who received RAM-containing chemotherapy. We obtained paired samples from peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating lymphocytes (TILs) in primary tumors both pre- and post-RAM therapy to assess immune profiles by immunohistochemistry and flow cytometry.ResultsWithin the tumor microenvironment, both PD-L1 expression and CD8+ T-cell infiltration increased after RAM-containing therapies. In addition, CD45RA−FOXP3highCD4+ cells (effector regulatory T cells [eTreg cells]) and PD-1 expression by CD8+ T cells were significantly reduced in TILs compared with PBMCs after RAM-containing therapies. Patients with partial response and longer progression-free survival had significantly higher pre-treatment eTreg frequencies in TILs than those with progressive disease. In in vitro analysis, VEGFR2 was highly expressed by eTreg cells. Further, VEGFA promoted VEGFR2+ eTreg cell proliferation, and this effect could be inhibited by RAM.ConclusionsThis study suggests that the frequency of eTreg cells in TILs could be a biomarker for stratifying clinical responses to RAM-containing therapies. Further, we propose that RAM may be employed as an immuno-modulator in combination with immune checkpoint blockade.