High Expression Of TIM-3 Research Articles

Abstract 2762: Spatial distribution and immune cell infiltration at different sites of melanoma metastases (mets)

Abstract Purpose: The site of metastasis in advanced melanoma patients (pts) influences pt response to immunotherapy and overall prognosis. Liver mets are particularly resistant to immunotherapy and their presence often confers shorter survival and reduced response rates. This study characterised and compared the tumor immune microenvironment of melanoma mets at different anatomical sites. Methods: T-cell and myeloid cell markers were stained using multiplex IHC on FFPE samples from 136 untreated metastatic melanoma pts, from 5 anatomical sites (liver, lung, brain, subcutaneous, and lymph node (LN)). Cell densities, subpopulations and the spatial distribution of cells were compared between sites. Results: CD3+ T cell infiltration was less dense in the liver (med = 154.3 cells/mm2) and brain mets (med = 180.7 cells/mm2) v's lung (med = 434.7 cells/mm2) and LN mets (med = 504.8 cells/mm2) (p<0.05), and T-cells were further away from melanoma cells in liver v's lung mets (med distance = 83µm v's 57µm respectively; P<0.05). The liver displayed a unique T-cell profile, with a significantly lower proportion of CD3+ T cells expressing PD-1 (med = 0.7%) compared to all other sites (P<0.05) while brain mets expressed the highest proportion of PD-1+ T cells (med = 8.9%). There were higher proportions of Tim3+ T cells in liver (10.7%) and lung mets (12.9%) v's brain, subcut and LN mets (P<0.05). Specifically, in liver mets CD3+ T cells within 20um of a melanoma cell had a significantly higher expression of Tim3 (med = 18.57%) v's PD-1 (med = 2%). Brain mets had the lowest density of CD68+ macrophages (med = 33 cells/mm2) v's lung (med = 372 cells/mm2), liver (med = 290 cells/mm2) and Subcut mets (med = 154 cells/mm2). Lung mets also had the highest proportion of PD-L1+ macrophages (med = 18.73%) compared to liver (med = 5.2%) and brain mets (med = 0.65%). While the most highly expressed T cell population varied between sites, CD16+ macrophages were consistently the highest expressed macrophage subpopulation regardless of metastatic site. Conclusion: These data suggest that liver mets are less immunogenic, with fewer T cells and greater distances between CD3+ T cells and melanoma cells, and may account for the poor prognosis of melanoma patients with liver metastases and their reduced response rate to immunotherapy compared to other mets. While liver mets have reduced PD-1 expression, higher Tim3 expression in the liver may provide a therapeutic opportunity to improve outcomes for pts with liver mets by using anti-TIM3 immunotherapy. Overall, these data provide novel insights into the site-specific biology of melanoma mets, heterogeneity in the tumor immune microenvironment, and the need for site specific biomarkers to be considered when selecting treatment targets. Citation Format: Jordan W. Conway, Robert V. Rawson, James S. Wilmott, Georgina V. Long, Richard A. Scolyer, Inês Pires Da Silva. Spatial distribution and immune cell infiltration at different sites of melanoma metastases (mets) [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 2762.

The Immune Checkpoint Receptors PD-1, PD-L1, TIM-3 and LAG-3 in Lymphoma: Tumor Cell and Tumor Infiltrating Lymphocyte (TIL) Expression, Patient Prognostication, and Identification of Rational Therapeutic Targets

Introduction: Programmed cell death-1 (PD-1) based immunotherapy represents a significant advance in the treatment of hematologic malignancies, including lymphoma. Mechanisms of resistance and potential methods to overcome this treatment paradigm are a critical focus of research. Signaling through additional immune checkpoint receptors, such lymphocyte-activation gene-3 (LAG-3) and T cell immunoglobulin and mucin domain-3 (TIM-3), may lead to T-cell exhaustion and be a mechanism of immune escape for tumors. However, the expression patterns of TIM-3 and LAG-3 in lymphoma, including DLBCL and HL, are largely unknown. Furthermore, potential override of resistance to PD-1 blockade via rational co-targeting of checkpoint receptors represents a novel therapeutic strategy.Methods: Tissue sections (whole sections and tissue microarrays) of newly diagnosed patient cases of diffuse large B-cell lymphoma (DLBCL, n=123) and Hodgkin lymphoma (HL, n=30) were stained with antibodies to PD-L1, PD-1, TIM-3, and LAG-3. Immunohistochemical (IHC) expression of each marker on tumor cells and on tumor infiltrating lymphocytes (TILs) were scored. Staining patterns were correlated with additional pathologic and clinical parameters as well as patient outcome (i.e., progression free survival (PFS) and overall survival (OS)) among n=59 newly diagnosed advanced-stage DLBCL patients treated from 2005 to 2013 with rituximab-CHOP therapy. 4-year survival rates were estimated by Kaplan-Meier with differences assessed by log rank test. Univariate analysis and multivariate regression according to the Cox proportional hazards regression model were used (for International Prognostic Index (IPI) and TIM-3). In addition, the effects of in vitro checkpoint blockade were evaluated using investigational anti-PD-1, anti-TIM-3 or anti-LAG-3 compounds with tumor-primed T cells and Raji or SUDHL10 as target DLBCL cells. Cell viability was assessed using AcellaTox assay and IL-2 release quantified by ELISA.Results: TIM-3 showed strong, membranous staining (H-score >80) on tumor cells in over 1/3 of DLBCL (39%, 48/123) and the majority of HL cases (70%, 21/30). Furthermore, PD-L1 was expressed (>30% cells tumor cells positive) in essentially all HL cases (100%, 30/30) and in a subset of DLBCL cases (15%, 19/126). This is similar to our and others' previously reported data (Xing W et al. O ncotarget 2016). Both PD-1 and LAG-3 in the current study were positive on tumor cells in only a minority of DLBCL cases (8% and 7%, respectively), and rarely in HL tumor cells. Conversely, PD-1 and LAG-3 were widely expressed on the TILs found in DLBCL in 75% and 94% of cases, respectively. With a median follow-up of 44 months (range 5-85), 4-year PFS and OS rates were significantly inferior among DLBCL patients with high vs low/negative TIM-3 expression (i.e., PFS: 23% (95% CI 7% to 46%) vs 60% (95% CI 43% to 74%), respectively, P=0.008; and OS: 30% (95% CI 10% to 53%) vs 74% (95% CI 58% to 85%), respectively, P=0.006) (see Figure 1A/1B). Furthermore, when controlling for the IPI, both survival endpoints remained significant with high TIM-3 predicting increased risk of progression (PFS HR 2.60 (95% CI 1.22-5.53, P=0.013) and OS predicting increased risk of death (OS HR 3.36, 95% CI 1.52-7.42, P=0.003) on multivariate Cox regression analyses. In addition, we examined these checkpoint receptors as potential therapeutic targets. Co-culture of PD-L1 expressing Raji cells with tumor-dendritic cell primed T cells in the presence of anti-LAG-3 induced a potent dose-dependent increase in in vitro cell death with 15nM IC50 and IL-2 secretion (120pg/ml) as determined by AcellaTox & ELISA based assays (see Figure 1C).Conclusions: Altogether, IHC analyses showed strong expression of the TIM-3 immune checkpoint receptor in over 1/3 of DLBCL and in the majority of HL tumors. Furthermore, analysis of patient outcomes identified that DLBCL patients with high TIM-3 expression (i.e., H-score >80) had significantly inferior survival rates. PD-1 and LAG-3 were consistently expressed in the TILs of DLBCL and HL, but were infrequently expressed by tumor cells. Therapeutically, we identified a dose-dependent increase in Raji-targeted cell death and increased T cell activity with a novel anti-LAG-3 compound. Continued examination of mechanistic studies of these immune checkpoint receptors and associated rational treatment studies are warranted. [Display omitted] DisclosuresGhosh:Tesaro: Employment. Evens:Kite Pharma: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Seattle Genetics: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Millennium: Consultancy; AbbVie: Consultancy; Affimed: Consultancy; Merck: Consultancy; • Spectrum Pharmaceuticals: Consultancy.

High PD-1 and Tim-3 Expression Concurrent with Exhausted CD4+ and CD8+ T Cells in Bone Marrow Compared with Peripheral Blood from Patients with Multiple Myeloma

Immunosuppressive microenvironment plays a crucial role for T cell immunodeficiency in multiple myeloma. Increasing data showed that overexpression of the T cell immunosuppressive receptors, such as programmed death-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4) and T cell immunoglobulin mucin-3 (Tim-3) may be related to tumor immunosuppression and poor prognosis, and the malignant bone marrow microenvironment may contribute to such immunosupression. In this study, we compared the difference of PD-1 and Tim-3 expression with exhausted CD3+, CD4+ and CD8+ T cells between bone marrow (BM) and peripheral blood (PB) in patients with multiple myeloma (MM). BM and PB samples from 6 cases (4 males and 2 females, age range: 41-82 years old) with untreated MM were selected to this study, the frequencies of PD-1 and Tim-3 expression in CD3+, CD4+ and CD8+ T cells from BM and PB mononuclear cells were detected by Flow cytometry. We also detected the frequency of CD244 and CD57 expression in both CD4+ and CD8+ T cells, which are the immune markers for T cell exhaustion. The change of the frequency of T cell phenotype was followed up in two cases in different time point after therapy. The results showed that significant higher percentage of PD-1+CD3+T cells was found in BM compared with PB (P= 0.001), particularly in PD1+CD8+T cells was significantly higher in BM (P= 0.017), however, interesting, higher percentage of PD-1+CD4+T cells was showed in PB compared with BM (P= 0.043) (Figure 1). Similarly, the percentage of Tim-3+CD3+T cell in BM seemed high compared with PB, as well as in PD-1+Tim-3+CD3+, CD4+ and CD8+T cells. The percentage of CD57+, CD244+ in CD3+, CD4+ and CD8+ T cells was relative high in BM group in comparison with PB group, although the statistical significance was not significant. However, either PD-1+CD244+ (P= 0.006) or PD-1+CD57+(P = 0.02) in CD3+ T cells was significant higher in BM group than that in PB group. Moreover, PD-1+CD244+(P = 0.000) and PD-1+CD57+ (P = 0.048) in CD8+ T cells were higher distributed in BM than that in PB, while there was not significant different in CD4+ T cells (Figure 2). And the limit data also showed that the numbers of either PD-1+ or PD-1+Tim-3+T cells in different T cell subsets were decreases in both BM and PB when the patient achieved complete remission after therapy (Figure 3). In conclusions, we characterized the different distribution of PD-1 and Tim-3 concurrent with exhausted CD3+, CD4+ and CD8+ T cells between BM and PB in patients with MM. Higher numbers of PD-1+CD244+ or PD-1+CD57+ CD3+ T cells in BM from MM patients may contribute to mediate the BM immunosuppressive microenvironment, higher lever PD-1+CD244+ or PD-1+CD57+CD8+T cells may relate to lower T cell activation and T cell immunodeficiency. [Display omitted] DisclosuresLi:Guangdong Provincial Basic Research Program (No. 2015B020227003: Research Funding; National Natural Science Foundation of China (91642111): Research Funding; Guangzhou Science and Technology Project (No. 2015100010211): Research Funding; Guangdong Provincial Applied Science and Technology Research & Development Program (No. 2016B020237006): Research Funding.

Modulation of CXCR1 and CXCR3 expression on NK cells via Tim-3 in a murine model of primary biliary cholangitis

Tim-3, which is expressed on a variety of innate immune cells including NK cells, plays a key role in many autoimmune diseases. However, the immunomodulatory actions of Tim-3 on NK cells in primary biliary cholangitis (PBC) remain uncertain. Using a murine model of PBC we evaluated the expression of Tim-3 and its ligand Gal-9 in peripheral blood, liver, and spleen. Additionally, we studied Tim-3 regulation of chemokine receptors (CXCR1 and CXCR3) in vitro. Flow cytometric analysis indicated large numbers of infiltrating NK cells in the liver which exhibited high expression of Tim-3 and CXCR3. Moreover, we found overexpression of CXCR1 in liver tissue and liver-derived NK cells in PBC mice. We also observed lower levels of soluble Tim-3 in the serum of PBC mice. In vitro experiments with liver-derived NK cells from PBC mice indicated that CXCR3 was up-regulated by treatment with recombinant mouse TIM-3 Fc (rmTim-3 Fc) to activate the Tim-3 pathway. Furthermore, stimulating normal mouse spleen NK cells with poly I:C resulted in elevated expression of CXCR1 and interferon-γ release. Nonetheless, adding rmTim-3 Fc or rmGal-9 significantly down-regulated CXCR1 expression and IFN-γ release in NK cells activated by poly I:C, proposing a means to exploit the Tim-3 pathway to reverse responses in NK cells. In conclusion, our data demonstrate that dysregulation of Tim-3/Gal-9 is involved in modulating the local immune microenvironment in PBC mice. Our findings highlight the potential of Tim-3 pathway to modulate chemokine responses in NK cells during autoimmunity.

Correlation of Tim-3 expression with chemokine levels for predicting the prognosis of patients with glioblastoma

Glioblastoma (GBM) immunotherapy, which blocks the checkpoint inhibitor molecule T cell immunoglobulin domain and mucin domain-3 (Tim-3), has potential therapeutic applications. However, not all patients do benefit from the targeted therapy. This study aimed to explore Tim-3 expression correlated chemokine profiles and immune cell infiltration and investigate their potential as prognostic markers of glioblastoma (GBM) immunotherapy. We analyzed transcriptional data of GBM from TCGA database, to measure Tim-3 expression by R package DESeq2 analysis and observed differentially expressed genes in GBM samples with high Tim-3 expression levels. We also probed the relative gene enrichment pathways. Tim-3 expression was evident in biological processes including the recruitment of immune cells. We also identified some chemokines related to Tim-3 expression. The expression levels of CCL18, CXCL13 and CCL7 were significantly higher in GBM tissues with high Tim-3 expression than in GBM tissues with low Tim-3 expression. In addition, exploring the relationship between immune cell infiltration and Tim-3 expression suggested that Tim-3 expression was positively related to significant immune cell infiltration.

Low Distribution of TIM-3+ Cytotoxic Tumor-Infiltrating Lymphocytes Predicts Poor Outcomes in Gastrointestinal Stromal Tumors.

There are multiple tumor-infiltrating lymphocytes (TILs) and relevant immune checkpoints existing in gastrointestinal stromal tumor (GIST), which provides opportunities and rationales for developing effective immunotherapies. Recent studies have suggested that checkpoint TIM-3/Gal-9 plays a pivotal role on immune response in multiple tumors, similar to the PD-1/PD-L1, emerging as a potential therapeutic target. However, their functions in GIST are unrevealed. Hence, the expression of immune checkpoints TIM-3 and Gal-9, as well as the infiltration of CD8+ T cells and NK cells, is described in 299 cases of GIST specimens. The results showed that TIM-3 and Gal-9 are mainly expressed in TILs, rarely in tumor cells. Expression levels of TIM-3 and Gal-9 significantly differ in varying risks of GIST and exert opposite distribution trends. Indicated by prognosis analysis, high TIM-3 expression of TILs was associated with improved outcome, while low expression levels of TIM-3 in combination with low amounts of CD8+ and CD56+ TILs predict extremely poor survival. The integrated analysis of TIM-3+, CD8+, and CD56+ TILs as one biomarker is a reliable independent predictor of prognosis. In conclusion, low densities of TIM-3+ TILs are associated with poor survival, and integrated immune biomarkers lead to superior predictors of GIST prognosis.

Abstract PS10-04: Tucatinib favourably modulates the immune microenvironment and synergises with anti-PD1 therapy in a trastuzumab resistant HER2+ murine model

Abstract Background: The efficacy of PD-(L)1 inhibitors in patients with trastuzumab-resistant advanced HER2+ breast cancer is poor. Although many HER2 targeted therapies are used clinically, their effect on the tumor immune microenvironment (TME) and whether this contributes to efficacy is not understood. Tucatinib is a potent, highly selective, HER2 small molecule tyrosine kinase inhibitor with proven clinical benefit in the advanced setting. Methods: We used two immunocompetent, HER2+ murine cancer models (trastuzumab-sensitive H2N113 and trastuzumab-resistant fo5) to investigate the effects of tucatinib on tumor growth kinetics, as well as tucatinib anti-tumor efficacy in combination with trastuzumab and PD-1 checkpoint blockade. Effects of tucatinib on the tumor infiltrating lymphocytes were analysed using flow cytometry. To identify genes that were modulated by tucatinib treatment, we performed bulk RNA sequencing on fo5 tumors treated with vehicle or tucatinib. We evaluated healthy donor peripheral blood T cells treated with tucatinib for 96 hours and analysed secreted cytokine levels using cytometric bead array. Results: Treatment of CD4+ and CD8+ T cells with tucatinib together with TCR stimulation resulted significantly higher levels of IFNγ and TNFα compared with stimulated controls (p < 0.0001), suggesting tucatinib can directly modulate human T cell function. In both murine models, tucatinib significantly inhibited tumor growth in a dose-dependent manner and was observed at doses of 25 mg/kg, 50 mg/kg and 100 mg/kg (p < 0.05 between each dose). Median survival was 16 days in the vehicle group vs 50 days with 100 mg/kg of tucatinib (p < 0.0001). Ex vivo analysis of tumors by flow cytometry showed increased infiltration of NK cells (p=0.01) and, CD8+ T cells with high PD-1 (p = 0.001), TIM-3 (p = 0.009), IFNγ (p = 0.003) and Ki67 (p = 0.0003) expression in Tucatinib treated mice compared to vehicle treated mice. Concomitant with these changes there was a significant reduction in numbers of neutrophils (p = 0.0085) and MHC-II low expressing macrophage populations (p < 0.0001) following tucatininb treatment with an increase in frequency of MHC-II expressing dendritic cells (p < 0.0001) and macrophages (p < 0.0001) suggesting an increase in anti-tumor immunity. Similarly, in the fo5 model tucatinib treated tumors had significantly higher interferon-γ (IFNγ) produced by CD8+ T cells (p = 0.005). Gene expression profile analysis shows significant enrichment in pathways associated with immune activation, including antigen binding and presentation (p = 0.0002), adaptive immune responses (p = 0.0002) including IFNγ (p = 0.0002) and IFNα (p = 0.0002). In this model, tucatinib in combination with trastuzumab demonstrated significantly better anti-tumor activity (p = 0.03) and survival (p < 0.0001) compared with tucatinib alone, with 33% of mice achieving complete tumour regressions. Tucatinib in combination with PD-1 inhibition also demonstrated significantly greater anti-tumor efficacy compared to tucatinib alone (p = 0.0079) with increased survival (p = 0.05) and 50% of mice achieving complete tumor regression. Conclusions: This study suggests an anti-tumor immune response may be an important component of the efficacy of tucatinib. This is supported by in vitro data demonstrating tucatinib stimulated human peripheral T cells and in vivo data showing favorable effects on the TME mediated by tucatinib treatment. These changes were associated with improved efficacy when tucatinib was combined with PD-1 inhibition or trastuzumab in the setting of trastuzumab resistance. These findings suggest that the combination of tucatinib and PD-1 inhibition is a rational combination that warrants investigation in the clinical setting, particularly for trastuzumab resistant HER2+ breast tumors. Citation Format: Ran Li, Sneha Sant, Emmaline Brown, Franco Caramia, Ann Byrne, Kylie Clarke, Michael Neeson, Paul J Neeson, Phillip K Darcy, Scott Peterson, Sherene Loi. Tucatinib favourably modulates the immune microenvironment and synergises with anti-PD1 therapy in a trastuzumab resistant HER2+ murine model [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS10-04.

Multimarker scores of Th1 and Th2 immune cellular profiles in peripheral blood predict response and immune related toxicity with CTLA4 blockade and IFNα in melanoma

Neoadjuvant therapy with ipilimumab in combination with high dose IFNα was evaluated in patients with locally/regionally advanced melanoma in a previously reported clinical trial [NCT01608594]. In this study, peripheral immune cell profiling was performed in order to investigate the underlying mechanisms of tumor immune susceptibility and resistance. Peripheral blood mononuclear cells (PBMCs) from treated patients (N = 28) were collected at baseline and then at 6-weeks, 3-months and 12-months. High complexity (14-color) flow cytometry, designed to detect key immunological biomarkers was used to evaluate the frequencies of immune cell subsets. Statistical significance was determined using R-package employing Kruskal's test. We found that higher levels of Th1 cells at baseline (defined as CD45RA- CCR6- CXCR3+ CCR4-) correlated with the preoperative radiological response (p = 0.007) while higher Th2 cells (defined as CD45RA- CCR6- CXCR3- CCR4+) were associated with progressive disease (p = 0.009). A multimarker score consisting of higher levels of Th1 cells and CD8+ central memory T-cells was associated with pathologic complete response (pCR) (p = 0.041) at surgical resection. On the other hand, high TIM3 expression on T-cells correlated with gross viable tumor (p = 0.047). With regard to immune related toxicity, higher levels of phenotypically naive (defined as CCR7+CD45RA+) and effector memory (defined as CCR7-CD45RO+) CD8+ T-cells (p = 0.014) or lower levels of Th2 cells were associated with lower toxicity (p = 0.024). Furthermore, a multimarker score consisting of higher CD19+ and CD8+ cells was associated with lower toxicity (p = 0.0014). In conclusion, our study yielded mechanistic insights related to the immune impact of CTLA4 blockade and IFNα and potential biomarkers of immune response and toxicity.

PAX5 haploinsufficiency induced CD8+ T cells dysfunction or exhaustion by high expression of immune inhibitory-related molecules

PurposePAX5 haploinsufficiency promoting tumorigenesis is related to immune escape. But the mechanisms of PAX5 mutations inducing tumor immune escape have not been clarified. Our aim was to study how PAX5 haploinsufficiency influences effector CD8 + T cells in tumor microenvironment. MethodsWe estimated the proportions of 22 immune cell types and the expressions of immune inhibitory-related molecules based on gene expression profiles (GEPs) from children's B- acute lymphoblastic leukemia(B-ALL) with PAX5 mutations by CIBERSORT, an established algorithm. We constructed the PAX5 haplodeletion A20 cell lines, built allografted A20 tumor models and evaluated the effect of PAX5 haplodeletion on immune inhibitory-related molecules in the tumor microenvironment (TME). ResultsOur results indicated the percentages of T cells in bone marrow of children's B-ALL with PAX5 mutations were not statistically different from that in bone marrow of B-ALL without PAX5 mutations, except for T follicular helper (Tfh) cells. But a variety of up-regulated immune inhibitory-related molecules in bone marrow of children's B- ALL with PAX5 mutations were identified. By different approaches, we found that several immune inhibitory-related molecules of CD8+ T cells in TME of PAX5 haplodeletion clones such as TIM3, NR4A1 and BATF, were increased significantly compared with that of PAX5 wild type control. The IFN-ɤ of CD8+ T cells in TME of PAX5 haplodeletion tumors was decreased significantly compared with that of PAX5 wild type control. ConclusionOur study showed that PAX5 haploinsufficiency induced CD8+ T cells dysfunction or exhaustion by high expression of TIM3, NR4A1 and BATF in the CD8+ T cells of TME.

High Expression of TIM 3 and Galectin 9 on Immunohistochemistry Staining of Tumor Specimen at Diagnosis in Pediatric Patients with Ewing Sarcoma.

Significant progress has been made in the advancement of immune system modulation for cancer treatment in recent years. In particular, immune checkpoint inhibitors and chimeric antigen receptor (CAR) T-cell therapy have demonstrated remarkable clinical benefit in relapsed/refractory cancers. However, our understanding of the immuno-oncologic landscape in pediatric solid tumors remains limited and is a barrier to continued progress. We examined the immunohistochemical expression of checkpoint receptors PD-1, TIM-3, LAG-3 and their respective ligands in various pediatric cancers at diagnosis and found high expression of TIM-3/Galectin-9 in the infiltrating cells of Ewing sarcoma. Location of checkpoint receptor/ligand expressions is important, as some staining patterns were only seen along tumor borders. Finally, peripheral T cell function varied significantly among different tumors supporting a complex relationship between the tumor microenvironment and the global immune system.

Relationship between immune checkpoint proteins, tumour microenvironment characteristics, and prognosis in primary operable colorectal cancer.

The tumour microenvironment is an important factor for colorectal cancer prognosis, affecting the patient's immune response. Immune checkpoints, which regulate the immune functions of lymphocytes, may provide prognostic power. This study aimed to investigate the prognostic value of the immune checkpoints TIM‐3, LAG‐3 and PD‐1 in patients with stage I–III colorectal cancer. Immunohistochemistry was employed to detect TIM‐3, LAG‐3, PD‐1 and PD‐L1 in 773 patients with stage I–III colorectal cancer. Immune checkpoint protein expression was assessed in tumour cells using the weighted histoscore, and in immune cells within the stroma using point counting. Scores were analysed for associations with survival and clinical factors. High tumoural LAG‐3 (hazard ratio [HR] 1.45 95% confidence interval [CI] 1.00–2.09, p = 0.049) and PD‐1 (HR 1.34 95% CI 1.00–1.78, p = 0.047) associated with poor survival, whereas high TIM‐3 (HR 0.60 95% CI 0.42–0.84, p = 0.003), LAG‐3 (HR 0.58 95% CI 0.40–0.87, p = 0.006) and PD‐1 (HR 0.65 95% CI 0.49–0.86, p = 0.002) on immune cells within the stroma associated with improved survival, while PD‐L1 in the tumour (p = 0.487) or the immune cells within the stroma (p = 0.298) was not associated with survival. Furthermore, immune cell LAG‐3 was independently associated with survival (p = 0.017). Checkpoint expression scores on stromal immune cells were combined into a Combined Immune Checkpoint Stromal Score (CICSS), where CICSS 3 denoted all high, CICSS 2 denoted any two high, and CICSS 1 denoted other combinations. CICSS 3 was associated with improved patient survival (HR 0.57 95% CI 0.42–0.78, p = 0.001). The results suggest that individual and combined high expression of TIM‐3, LAG‐3, and PD‐1 on stromal immune cells are associated with better colorectal cancer prognosis, suggesting there is added value to investigating multiple immune checkpoints simultaneously.

Study on Tim3 Regulation of Multiple Myeloma Cell Proliferation via NF-κB Signal Pathways.

ObjectiveAs an important negative regulatory factor of immunological cells, Tim3 plays a regulating role in tumor immune microenvironment. The purpose of this study was to investigate the expression of Tim3 on MM cells and its effect on the proliferation and apoptosis of MM cells, as well as its potential mechanism.MethodsIn this study, the expression of Tim3 was detected on myeloma cells (CD38+CD138+ cells) of bone marrow by flow cytometry (FCM) from 167 patients with MM and 51 healthy donors as controls and making correlation analysis with related clinical indexes. In vitro, MM cell lines (RPMI-8226 and U266) were treated with Tim3 knock-down alone, bortezomib alone and combination of Tim3 knock-down and bortezomib, then cell proliferation, cell apoptosis and downstream signaling pathway were detected by CCK-8, FCM, RT-PCR and western blot.ResultsThe expression of Tim3 on myeloma cells in MM patients was significantly higher than normal control group and positively correlated with β2 microglobulin, creatine, and plasma cells of bone marrow, negatively correlated with hemoglobin and red blood cells. In vitro, we validated the high expression of Tim3 in RPMI-8226 and U266 cell lines. After Tim3 knock-down, the cell proliferation was inhibited and cell apoptosis was induced, the relative mRNA and protein expression of Tim3 and NF-κB signal pathway (PI3K, AKT, mTOR, NF-κB) were significantly decreased. Also, the cell proliferation was inhibited, cell apoptosis was increased, the relative mRNA and protein expression of NF-κB were decreased significantly in combination group than bortezomib or Tim3 knock-down group.ConclusionsThe high expression of Tim3 on MM cells is associated with progression of MM patients. Tim3 maybe regulate the proliferation of MM cells via NF-κB signal pathway. Down-regulation of Tim3 expression can inhibit proliferation and induce apoptosis of MM cells, also has an additive inhibitory effect of bortezomib on NF-κB signaling pathway, then inhibit proliferation and induce apoptosis. Therefore, Tim3 may be a potential target for the treatment of MM.

Higher T cell immunoglobulin mucin-3 (Tim-3) expression in cervical cancer is associated with a satisfactory prognosis.

BackgroundCervical cancer (CC), which has been increasing in incidence in recent years, is the fourth most common gynecological cancer in the world. Therapy targeting T cell immunoglobulin mucin-3 (Tim-3), known as the immune checkpoint, has been rapidly developing as oncotherapy for various carcinomas. However, few studies focus on Tim-3 in CC in terms of patient prognosis. This study demonstrates that higher Tim-3 mRNA levels in CC are associated with a favorable prognosis, which may due to active immune responses in CC.MethodsFirst, the clinical and RNA-sequencing (RNA-seq) data of 287 CC patients were downloaded from The Cancer Genome Atlas (TCGA) database and was subsequently analyzed. Then, based on the Tim-3 mRNA levels, the patients were divided into groups categorized by high and low expression, and the overall survival (OS) among the patients was determined. Next, the correlation between the expression of Tim-3 and clinicopathological variables was investigated. Finally, the Tim-3 function was carried out using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and a gene set enrichment analysis (GSEA) was performed.ResultsIn CC, the median OS of patients with high Tim-3 expression and low Tim-3 expression were 634 and 491 days (P=0.01), respectively. We found that the high expression of Tim-3 was closely associated with smoking history (P=0.012), total number of pregnancies (P=0.002), histological type (P<0.0001), M stage (P=0.036), TNM stage (P<0.0001), papillomavirus (P=0.001), hysterectomy type (P<0.0001) and survival status (P<0.0001). Univariate and multivariate logistic regression tests suggested that the level of Tim-3 was an independent prognostic factor for CC patients. In addition, GSEA further showed that Tim-3 expression was associated with macrophage differentiation, regulation of monocyte chemotaxis, positive regulation of substrate adhesion dependent cell spreading, negative regulation of interleukin 2 production, regulation of NF kappa-B signaling, STAT cascade, erk1 and erk2 cascade and regulation of vascular endothelial growth factor receptor signaling pathway.ConclusionsA higher expression of Tim-3 was associated with a favorable prognosis, which may due to the activation of immune responses in tumor tissues.

Abstract P5-04-28: Targeting TIGIT and PD-1 in triple negative breast cancer

Abstract Background: Triple negative breast cancer is aggressive and has poor prognosis. Tumor infiltrating lymphocytes (TILs) have a prognostic value in triple negative breast cancer. Increased TILs are associated with better disease-free survival and overall survival in triple negative breast cancer (TNBC). Newly approved immunotherapy drug for metastatic triple negative breast cancer patients, Atezolizumab, has shown 10 months improvement in overall survival in for PD-L1 positive group in combination with nab-paclitaxel compared to placebo and paclitaxel. Beyond PD-1 inhibitors, there is a need to improve immunotherapy options in TNBC. TIGIT gene expression in triple negative breast cancer is associated with immune function is linked with improved relapse free survival and overall survival. CD155, a high affinity receptor for TIGIT, is expressed on tumor cells including lung and melanoma. We aim to evaluate the role of TIGIT in regulating immune response to TNBC, TIGIT cooperation with PD-1 in impeding T cell responses to TNBC, and expression of TIGIT ligands on TNBC. Methods: Fresh TNBC tumor samples were obtained from 10 untreated patients and were used for single cell suspension and multi parameter flow cytometry. Results: CD8+ and CD4+ TILs in TNBC exhibited higher PD-1 and TIGIT, yet lower CD226 expression as compared to circulating T cells. LAG-3 and TIM3 were expressed at low frequency in CD 8+ and CD 4+ cells. TNBC Tregs display high expression of TIGIT, PD-1, TIM3 but low CD226 expression. TIGIT interacts with CD155 and CD112 with high affinity and both were found to be highly expressed by TNBC tumor cells and APCs. PD-1−TIGIT+ CD8+ TILs are present at high frequency and appear to represent an activated T cell subset. PD-1+Tim-3+TIGIT+ produced less cytokines including interferon gamma, IL-2, and TNF than other CD8+ T cell subsets, representing a dysfunctional T cell subset. Conclusions: TIGIT is highly upregulated by TNBC CD8+ and CD4+ TILs while the TIGIT ligands are highly expressed by tumor cells and APCs in TNBC. In sharp contrast with metastatic melanoma, high frequency PD-1−TIGIT+CD8+ TILs are present in TNBC. Additional studies will investigate the role of dual PD-1/TIGIT blockade in increasing CD8+ TIL function in TNBC. Such data may support the dual PD-1/TIGIT blockade in TNBCs. Citation Format: Apurva Kumari Pandey, Joe Marc Chauvin, Adam Brufsky, Ornella Pagliano, Mignane Ka, Carmine Menna, Priscilla McAuliffe, Hassane Zarour. Targeting TIGIT and PD-1 in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-04-28.

Expression of TIM3/VISTA checkpoints and the CD68 macrophage-associated marker correlates with anti-PD1/PDL1 resistance: implications of immunogram heterogeneity

ABSTRACT Although immunotherapies have achieved remarkable salutary effects among subgroups of advanced cancers, most patients do not respond. We comprehensively evaluated biomarkers associated with the “cancer-immunity cycle” in the pan-cancer setting in order to understand the immune landscape of metastatic malignancies as well as anti-PD-1/PD-L1 inhibitor resistance mechanisms. Interrogation of 51 markers of the cancer-immunity cycle was performed in 101 patients with diverse malignancies using a clinical-grade RNA sequencing assay. Overall, the immune phenotypes demonstrated overexpression of multiple checkpoints including VISTA (15.8% of 101 patients), PD-L2 (10.9%), TIM3 (9.9%), LAG3 (8.9%), PD-L1 (6.9%) and CTLA4 (3.0%). Additionally, aberrant expression of macrophage-associated markers (e.g. CD68 and CSF1R; 11-23%), metabolic immune escape markers (e.g. ADORA2A and IDO1; 9-16%) and T-cell priming markers (e.g. CD40, GITR, ICOS and OX40; 4-31%) were observed. Most tumors (87.1%, 88/101) expressed distinct immune portfolios, with a median of six theoretically actionable biomarkers (pharmacologically tractable by Food and Drug Administration approved agents [on- or off-label] or with agents in clinical development). Overexpression of TIM-3, VISTA and CD68 were significantly associated with shorter progression-free survival (PFS) after anti-PD-1/PD-L1-based therapies (among 39 treated patients) (all P < .01). In conclusion, cancer-immunity cycle biomarker evaluation was feasible in diverse solid tumors. High expression of alternative checkpoints TIM-3 and VISTA and of the macrophage-associated markers CD68 were associated with significantly worse PFS after anti-PD-1/PD-L1-based therapies. Most patients had distinct and complex immune expression profiles suggesting the need for customized combinations of immunotherapy.

High CD3 and ICOS and low TIM-3 expression predict favourable survival in resected oesophageal squamous cell carcinoma

With the increasing oncological potential of immunotherapy, several immune checkpoint modulators are being investigated. The value of immune markers, including programmed cell death ligand-1, programmed cell death-1 (PD-1), inducible co-stimulator (ICOS), lymphocyte activation gene-3, T-cell immunoglobulin, and mucin-dominant containing-3 (TIM-3), is not well known. Using tissue microarrays of 396 patients who underwent surgery for oesophageal squamous cell carcinoma (ESCC), infiltrated T-cell subsets (CD3, CD8, and Foxp3) and checkpoint protein expression were scored. With a median follow-up of 24.8 months, CD3+ TIL subsets (50.0%) had longer median recurrence-free survival (RFS, 55.0 vs 21.4 months) and overall survival (OS, 77.7 vs 35.8 months). Patients with high ICOS expression (46.5%) had longer median RFS (53.9 vs 25.3 months) and OS (88.8 vs 36.9 months). For PD-1, RFS (hazard ratio [HR] 0.67) and OS (HR 0.66) were significantly longer in the high-expression group (45.2%). In the multivariate analysis, high TIM-3 expression (50.8%) had a significant relationship with shorter RFS (HR = 1.52) and OS (HR = 1.60). High CD3+ TIL and T-cell ICOS expression were associated with favourable prognosis, whereas high TIM-3 expression suggested a poor prognosis. Our findings may confer new insights to improve ESCC outcomes beyond the application of PD-1 blockade.

Expression of CCR7 and Tim-3 in Childhood Patients with Acute Lymphoblastic Leukemia and Their Predictive Value for Prognosis

To analyze the expression of CCR7 and Tim-3 in childhood patients with acute lymphoblastic leukemia (ALL) and their predictive value for prognosis. Eighty-six newly diagnosed ALL childhood patients from January 2007 to January 2017 treated in our hospital were selected. The expression level of CCR7 and Tim-3 in bone marrow isolated cells of ALL patients were detected by flow cytometry, all the patients were divided into the recurrence group and non-recurrence group according to the follow-up results, the differences in the expressions of CCR7, Tim-3 between the two groups were compared. The correlation between the expression of CCR7 , Tim-3 and the clinicopathologic features of ALL patients were analyzed, the predictive value of CCR7 and Tim-3 for the prognosis of newly ALL patients were evaluated by ROC curve, and the relationship between serum CCR7, Tim-3 and prognosis were analyzed. The expression levels of CCR7 and Tim-3 in recurrence group were significantly higher than those in non-recurrence group(P<0.05). The critical value of CCR7 for diagnosis of recurrence was 45.97%, the sensitivity was 66.7%, the specificity was the 84.5% and the area under ROC curve (AUC) was 0.798 (95CI 0.777-0.939). The critical value of Tim-3 for diagnosis of recurrence was 53.54%, the sensitivity was 73.3%, the specificity was the 80.3% and the AUC was 0.806 (95CI 0.792-0.947). The AUC of the combined detection of CCR7 and Tim-3 was 0.895 (95CI 0.914-0.996), sensitivity 86.6%, specificity 78.9% (P<0.05); There was no significant correlation between CCR7, Tim-3 expression and age, sex, hemoglobin concentration, number of white blood cells, bone marrow blasts, platelets, central nervous system invasion, fusion gene (P>0.05). The exogenous infiltration rate of patients with high expression of CCR7 and Tim-3 was significantly higher than those in low expression group (P<005). The high expression rate 76.9% of Tim-3 in patients with T-ALL was significantly higher than that of B-ALL patients with Tim-3 high expression rate 45.2% (P<0.05). The median OS of patients with CCR7 level ≥45.97% and <45.97% were 9.3 months and 13.6 months respectively(P=0.004), and the Tim-3≥53.54% and Tim-3<53.54% were 9.1 months and 13.6 months respectively(P=0.001). The results of Cox's multi-factor regression analysis showed that CCR7 level(HR=1.024, 95 CI 1.001-1.049) and Tim-3 level (HR=1.879, 95 CI 1.183- 2.985) were the independent risk factors that affect the OS in ALL patients(P<0.05). The expression of CCR7 and Tim-3 in bone marrow isolated cells of ALL patients shows good predictive value for prognosis, and the combination of CCR7 and Tim-3 can improve the sensitivity of the detection, the higher expression of CCR7 and Tim-3 can be used as potential indexes in prognosis evaluate.

Expression of CCR7 and Tim-3 in Childhood Patients with Acute Lymphoblastic Leukemia and Their Predictive Value for Prognosis

To analyze the expression of CCR7 and Tim-3 in childhood patients with acute lymphoblastic leukemia (ALL) and their predictive value for prognosis. Eighty-six newly diagnosed ALL childhood patients from January 2007 to January 2017 treated in our hospital were selected. The expression level of CCR7 and Tim-3 in bone marrow isolated cells of ALL patients were detected by flow cytometry, all the patients were divided into the recurrence group and non-recurrence group according to the follow-up results, the differences in the expressions of CCR7, Tim-3 between the two groups were compared. The correlation between the expression of CCR7 , Tim-3 and the clinicopathologic features of ALL patients were analyzed, the predictive value of CCR7 and Tim-3 for the prognosis of newly ALL patients were evaluated by ROC curve, and the relationship between serum CCR7, Tim-3 and prognosis were analyzed. The expression levels of CCR7 and Tim-3 in recurrence group were significantly higher than those in non-recurrence group(P<0.05). The critical value of CCR7 for diagnosis of recurrence was 45.97%, the sensitivity was 66.7%, the specificity was the 84.5% and the area under ROC curve (AUC) was 0.798 (95CI 0.777-0.939). The critical value of Tim-3 for diagnosis of recurrence was 53.54%, the sensitivity was 73.3%, the specificity was the 80.3% and the AUC was 0.806 (95CI 0.792-0.947). The AUC of the combined detection of CCR7 and Tim-3 was 0.895 (95CI 0.914-0.996), sensitivity 86.6%, specificity 78.9% (P<0.05); There was no significant correlation between CCR7, Tim-3 expression and age, sex, hemoglobin concentration, number of white blood cells, bone marrow blasts, platelets, central nervous system invasion, fusion gene (P>0.05). The exogenous infiltration rate of patients with high expression of CCR7 and Tim-3 was significantly higher than those in low expression group (P<005). The high expression rate 76.9% of Tim-3 in patients with T-ALL was significantly higher than that of B-ALL patients with Tim-3 high expression rate 45.2% (P<0.05). The median OS of patients with CCR7 level ≥45.97% and <45.97% were 9.3 months and 13.6 months respectively(P=0.004), and the Tim-3≥53.54% and Tim-3<53.54% were 9.1 months and 13.6 months respectively(P=0.001). The results of Cox's multi-factor regression analysis showed that CCR7 level(HR=1.024, 95 CI 1.001-1.049) and Tim-3 level (HR=1.879, 95 CI 1.183- 2.985) were the independent risk factors that affect the OS in ALL patients(P<0.05). The expression of CCR7 and Tim-3 in bone marrow isolated cells of ALL patients shows good predictive value for prognosis, and the combination of CCR7 and Tim-3 can improve the sensitivity of the detection, the higher expression of CCR7 and Tim-3 can be used as potential indexes in prognosis evaluate.

Response to Anti-Bcma CAR T Cell Therapy Correlates with T Cell Exhaustion and Activation Status in T Cells at Baseline in Myeloma

Response to Anti-Bcma CAR T Cell Therapy Correlates with T Cell Exhaustion and Activation Status in T Cells at Baseline in Myeloma

Costimulatory Molecules and Immune Checkpoints Are Differentially Expressed on Different Subsets of Dendritic Cells.

Dendritic cells (DCs) play a crucial role in initiating and shaping immune responses. The effects of DCs on adaptive immune responses depend partly on functional specialization of distinct DC subsets, and partly on the activation state of DCs, which is largely dictated by environmental signals. Fully activated immunostimulatory DCs express high levels of costimulatory molecules, produce pro-inflammatory cytokines, and stimulate T cell proliferation, whereas tolerogenic DCs express low levels of costimulatory molecules, produce immunomodulatory cytokines and impair T cell proliferation. Relevant to the increasing use of immune checkpoint blockade in cancer treatment, signals generated from inhibitory checkpoint molecules on DC surface may also contribute to the inhibitory properties of tolerogenic DCs. Yet, our knowledge on the expression of inhibitory molecules on human DC subsets is fragmentary. Therefore, in this study, we investigated the expression of three immune checkpoints on peripheral blood DC subsets, in basal conditions and upon exposure to pro-inflammatory and anti-inflammatory stimuli, by using a flow cytometric panel that allows a direct comparison of the activatory/inhibitory phenotype of DC-lineage and inflammatory DC subsets. We demonstrated that functionally distinct DC subsets are characterized by differential expression of activatory and inhibitory molecules, and that cDC1s in particular are endowed with a unique immune checkpoint repertoire characterized by high TIM-3 expression, scarce PD-L1 expression and lack of ILT2. Notably, this unique cDC1 repertoire was subverted in a group of patients with myelodysplastic syndromes included in the study. Applied to the characterization of DCs in the tumor microenvironment, this panel has the potential to provide valuable information to be used for investigating the role of DC subsets in cancer, guiding DC-targeting treatments, and possibly identifying predictive biomarkers for clinical response to cancer immunotherapy.