Immune Metagenes Research Articles

Analysis of lncRNA-miRNA-mRNA interactions identified a novel biomarker LINC00657 to improve prognosis prediction of papillary thyroid carcinoma

BackgroundPapillary thyroid cancer (PTC) is the most common type of thyroid cancer. Identification of novel biomarkers can potentially help explore the underlying molecular mechanisms of PTC. Long non-coding RNAs (lncRNAs) are involved in cancer development. However, understanding the role of lncRNA in PTC remains challenging. MethodsBased on the competitive endogenous RNA (ceRNA) theory, we constructed a comprehensive PTC-related lncRNA-miRNA-mRNA network using data from The Cancer Genome Atlas. To evaluate the prognostic power, we performed survival analysis for patients with PTC with low and high lncRNA expression levels, and examined the relationship between lncRNA and immune-related functions. ResultsWe identified a hub node, long intergenic non-coding RNA, LINC00657, as a novel prognostic biomarker in PTC. LINC00657 was differentially expressed between tumor and adjacent normal samples. Low LINC00657 expression levels was significantly associated with better survival outcome. Our functional analyses showed that LINC00657 was related with infiltration of CD8+ T cell and macrophage; immune check point molecules; and immune metagenes such as IgG, LCK, MHC_I/II and etc. These results suggest that LINC00657 is an immune-related biomarker with potential clinical applicability. Additionally, cancer-related signaling pathway and high frequency of gene BRAF mutation were found in PTC samples with high LINC00657 expression level, which were consistent with previous findings. ConclusionLINC00657 is an immune-related biomarker that can potentially improve prognosis prediction in PTC. Our study provided new treatment target of PTC in clinical practice and offered the novel insights in elucidating the functional role of lncRNAs.

Prediction of Prognosis and Immunotherapy Response with a Novel Natural Killer Cell Marker Genes Signature in Osteosarcoma.

Background: Natural killer (NK) cells are characterized by their antitumor efficacy without previous sensitization, which have attracted attention in tumor immunotherapy. The heterogeneity of osteosarcoma (OS) has hindered therapeutic application of NK cell-based immunotherapy. The authors aimed to construct a novel NK cell-based signature to identify certain OS patients more responsive to immunotherapy. Materials and Methods: A total of eight publicly available datasets derived from patients with OS were enrolled in this study. Single-cell RNA sequencing data obtained from the Gene Expression Omnibus (GEO) database were analyzed to screen NK cell marker genes. Least Absolute Shrinkage and Selection Operator (LASSO) Cox regression analysis was used to construct an NK cell-based prognostic signature in the TARGET-OS dataset. The differences in immune cell infiltration, immune system-related metagenes, and immunotherapy response were evaluated among risk subgroups. Furthermore, this prognostic signature was experimentally validated by reverse transcription-quantitative real-time PCR (RT-qPCR). Results: With differentially expressed NK cell marker genes screened out, a five-gene NK cell-based prognostic signature was constructed. The prognostic predictive accuracy of the signature was validated through internal clinical subgroups and external GEO datasets. Low-risk OS patients contained higher abundances of infiltrated immune cells, especially CD8 T cells and naive CD4 T cells, indicating that T cell exhaustion states were present in the high-risk OS patients. As indicated from correlation analysis, immune system-related metagenes displayed a negative correlation with risk scores, suggesting the existence of immunosuppressive microenvironment in OS. In addition, based on responses to immune checkpoint inhibitor therapy in two immunotherapy datasets, the signature helped predict the response of OS patients to anti-programmed cell death protein 1 (PD-1) or anti-programmed cell death ligand 1 (PD-L1) therapy. RT-qPCR results demonstrated the roughly consistent relationship of these five gene expressions with predicting outcomes. Conclusions: The NK cell-based signature is likely to be available for the survival prediction and the evaluation of immunotherapy response of OS patients, which may shed light on subsequent immunotherapy choices for OS patients. In addition, the authors revealed a potential link between immunosuppressive microenvironment and OS.

The DEAD-box RNA helicase, DDX60, Suppresses immunotherapy and promotes malignant progression of pancreatic cancer

Excessive proliferation, invasion, metastasis, and immune resistance in pancreatic cancer (PC) makes it one of the most lethal malignant tumors. Recently, DDX60 was found to be involved in the development of various tumors and in immunotherapy. Therefore, we aimed to investigate whether DDX60 is a new factor involved in PC immunotherapy. The DDX60 mRNA was screened using transcriptome sequencing (RNA-seq). The Cox and survival analysis of DDX60 was performed using the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) databases. In addition, clinical and immune infiltration data in the databases were analyzed and plotted using the R language. Clinical samples and in vitro experiments were used to determine the molecular evolution of DDX60 during PC progression. We found that DDX60 was upregulated in PC tissues (P value = 0.0083) and was associated with poor prognosis and short survival time of patients with PC. Results of Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and gene set variation analyses showed that viral defense, tumor, and immune-related pathways were significantly enriched in samples with high DDX60 expression. The Pearson correlation test demonstrated that DDX60 expression correlated strongly with immune checkpoint and immune system-related metagene clusters. Our results indicated that DDX60 promoted cell proliferation, migration, and invasion and was related to poor prognosis and immune resistance. Therefore, DDX60 may be a promising novel target for PC immunotherapy.

PSEN1 is associated with colon cancer development via potential influences on PD-L1 nuclear translocation and tumor-immune interactions

Presenilin 1 (PSEN1), as a catalytical core of the γ-secretase complex, plays multiple actions through mediating transmembrane domain shedding of the substrates. Unlike extensive studies performed on investigating the functions of γ-secretase substrates or the effects of γ-secretase inhibitors, our findings uncover a potential action of PSEN1 on PD-L1 alternative truncation and nuclear translocation, broadening our understanding on how the γ-secretase contributes to colon cancer development as well as suggesting a potential strategy to improve the efficacy of PD-1/PD-L1 blockade. Immunohistochemical data showed loss of PD-L1 protein expression in all the primary colon adenocarcioma (COAD) cases in the HPA collection, while PSEN1 was scored to be highly expressed, indicating their converse expression patterns (p<0.001). Meanwhile a strongly positive gene correlation was explored by TIMER2 and GEPIA (p<0.001). Up-regulated PSEN1 expression in COAD might facilitate liberating a C-terminal PD-L1 truncation via proteolytic processing. Then following an established regulatory pathway of PD-L1 nuclear translocation, we found that PSEN1 showed significant correlations with multiple components in HDAC2-mediated deacetylation, clathrin-dependent endocytosis, vimentin-associated nucleocytoplasmic shuttling and importin family-mediated nuclear import. Moreover, connections of PSEN1 to the immune response genes transactivated by nuclear PD-L1 were tested. Additionally, contributions of PSEN1 to the tumor invasiveness (p<0.05) and the tumor infiltrating cell enrichments (p<0.001) were investigated by cBioportal and the ESTIMATE algorithm. Levels of PSEN1 were negatively correlated with infiltrating CD8+ T (p<0.05) and CD4+ T helper (Th) 1 cells (p<0.001), while positively correlated with regulatory T cells (Tregs) (p<0.001) and cancer associated fibroblasts (CAFs) (p<0.001). It also displayed significant associations with diverse immune metagenes characteristic of T cell exhaustion, Tregs and CAFs, indicating possible actions in immune escape. Despite still a preliminary stage of this study, we anticipate to deciphering a novel function of PSEN1, and supporting more researchers toward the elucidations of the mechanisms linking the γ-secretase to cancers, which has yet to be fully addressed.

Identification of three immune molecular subtypes associated with immune profiles, immune checkpoints, and clinical outcome in multiple myeloma.

PurposeTo identify the immune molecular subtype for MM to help achieve individualized and precise targeted therapy.MethodsThe GDC API was used to download the TCGA‐MM profile dataset, which contains 859 samples in total, all of which were anterior to the standard treatment after diagnosis. Moreover, 282, 298, and 258 samples were stage I, stage II, and stage III separately. We used the immune gene expression profile for consistent clustering; and used the R software package ConsensusClusterPlus to sort the immune molecular subtypes. Correlation between subtypes and clinical features, immunity, and prognosis was then analyzed.ResultsA total of 859 tumor samples were separated into these three subtypes, which were not meaningfully related to age or sex but showed a remarkable association with stage. The results suggested that obvious differences in immune metagene expression and expression of 10 immune checkpoint genes appeared among the three subtypes.ConclusionThe three subtypes are distinctly different in terms of immune metagenes, immune checkpoint molecules, and clinical prognosis. The discovery of the immune microenvironment of MM could further reveal the strategy for immunotherapy in MM and provide a promising candidate prognostic tool for survival.

CBX4 Provides an Alternate Mode of Colon Cancer Development via Potential Influences on Circadian Rhythm and Immune Infiltration.

The circadian machinery is critical for the normal physiological functions and cellular processes. Circadian rhythm disruption has been associated with immune suppression which leads to higher cancer risk, suggesting a putative tumor protective role of circadian clock homeostasis. CBX4, as an epigenetic regulator, has been explored for its involvement in tumorigenesis. However, little is known about the correlation between CBX4 and circadian rhythm disruption in colon cancer as well as the potential impact on the tumor immunity. A significant upregulation of CBX4 was identified in the TCGA colon adenocarcinoma (COAD) samples when compared with the normal controls (p < 0.001). This differential expression was confirmed at the protein level using colon adenocarcinoma tissue array (p < 0.01). CBX4 was up-regulated in the recurred/progressed colon cancer cases compared with the disease-free samples (p < 0.01), suggesting CBX4 as a potential predictor for poor prognosis. With regard to nodular metastasis, CBX4 was found to be associated with early onset of metastatic diseases but not late progression. The circadian rhythm is orchestrated by the alternating activation and suppression of the CLOCK/ARNTL-driven positive loop and the PER/CRY-controlled negative loop. In COAD, CBX4 was negatively correlated with CLOCK (p < 0.001), and positively correlated with PER1 (p < 0.001), PER3 (p < 0.01), and CRY2 (p < 0.001) as well as NR1D1 (p < 0.001), a critical negative regulator of the circadian clock. These interactions consistently impacted on patient survival based on the colorectal cancer cohorts GSE17536 and GSE14333 of PrognoScan. CBX4 showed significant negative correlations with infiltrating B cells (p < 0.05) and CD4+ T cells (p < 0.01), and positive correlations with myeloid derived suppressor cells (MDSCs) (p < 0.05) and cancer associated fibroblast (CAFs) (p < 0.001), as well as a low immunoscore. Moreover, CBX4 displayed significant correlations with diverse immune metagenes. PER1 and PER3, consistent with their coordinated expression with CBX4, also had strong correlations with these gene representatives in COAD, suggesting a potential interaction of CBX4 with the circadian machinery. Our studies implicate that CBX4 may contribute to colon cancer development via potential influence on circadian rhythm and immune infiltration. These findings provide new insights into deciphering the function of CBX4, and may contribute to the development of new targeting strategies.

Identification of a Prognostic Signature for Ovarian Cancer Based on the Microenvironment Genes.

Background: Ovarian cancer is highly malignant and has a poor prognosis in the advanced stage. Studies have shown that infiltration of tumor microenvironment cells, immune cells and stromal cells has an important impact on the prognosis of cancers. However, the relationship between tumor microenvironment genes and the prognosis of ovarian cancer has not been studied.Methods: Gene expression profiles and SNP data of ovarian cancer were downloaded from the TCGA database. Cluster analysis, WGCNA analysis and univariate survival analysis were used to identify immune microenvironment genes as prognostic signatures for predicting the survival of ovarian cancer patients. External data were used to evaluate the signature. Moreover, the top five significantly correlated genes were evaluated by immunohistochemical staining of ovarian cancer tissues.Results: We systematically analyzed the relationship between ovarian cancer and immune metagenes. Immune metagenes expression were associated with prognosis. In total, we identified 10 genes related to both immunity and prognosis in ovarian cancer according to the expression of immune metagenes. These data reveal that high expression of ETV7 (OS, HR = 1.540, 95% CI 1.023–2.390, p = 0.041), GBP4 (OS, HR = 1.834, 95% CI 1.242–3.055, p = 0.004), CXCL9 (OS, HR = 1.613, 95% CI 1.080 –2.471, p = 0.021), CD3E (OS, HR = 1.590, 95% CI 1.049 –2.459, p = 0.031), and TAP1 (OS, HR = 1.766, 95% CI 1.163 –2.723, p = 0.009) are associated with better prognosis in patients with ovarian cancer.Conclusion: Our study identified 10 immune microenvironment genes related to the prognosis of ovarian cancer. The list of tumor microenvironment-related genes provides new insights into the underlying biological mechanisms driving the tumorigenesis of ovarian cancer.

Seven interferon gamma response genes serve as a prognostic risk signature that correlates with immune infiltration in lung adenocarcinoma.

Interferon-gamma (IFN-γ) plays a complex role in modulating tumor microenvironment during lung adenocarcinoma (LUAD) development. In order to define the role of IFN-γ response genes in LUAD progression, we characterized the gene expression, mutation profile, protein-protein interaction of 24 IFN-γ response genes, which exhibited significant hazard ratio in overall survival. Two subgroups of LUAD from the TCGA cohort, which showed significant difference in the survival rate, were identified based on the expression of these genes. Furthermore, LASSO penalized cox regression model was used to derive a risk signature comprising seven IFN-γ response genes, including CD74, CSF2RB, PTPN6, MT2A, NMI, LATS2, and PFKP, which can serve as an independent prognostic predictor of LUAD. The risk signature was validated in an independent LUAD cohort. The high risk group is enriched with genes regulating cell cycle and DNA replication, as well as a high level of pro-tumor immune cells. In addition, the risk score is negatively correlated with the expression of immune metagenes, but positively correlated with DNA damage repair genes. Our findings reveal that seven-gene risk signature can be a valuable prognostic predictor for LUAD, and they are crucial participants in tumor microenvironment of LUAD.

High gene expression of estrogen and progesterone receptors is associated with decreased t cell infiltration in patients with NSCLC

ObjectivesPrior studies have demonstrated that signaling via the estrogen and progesterone receptors (ER and PR) may affect prognosis in non-small cell lung cancer (NSCLC). The precise impact of hormone signaling on clinical outcomes in NSCLC, especially in the context of immune checkpoint blockade, remains unclear. Materials and MethodsWe obtained RNA-Seq data from The Cancer Genome Atlas (TCGA) to determine mRNA expression levels of ESR1 (ER-α), ESR2 (ER-β), PGR (PR), CYP19A1 (aromatase), and immune-related genes. Tumor infiltration by activated T cells was predicted based on expression of immune metagenes. ResultsHigh levels of both ESR1 and PGR were associated with significantly decreased tumor infiltration by CD4+ and CD8+ activated T cells. CYP19A1 expression was associated with decreased CD4+ but not CD8+ T cell infiltration. There were no significant differences based on ESR2. These findings persisted after stratifying patients based on sex and tumor histology. In addition, increased ESR1 was associated with high gene expression of immune checkpoint markers, while increased PGR was associated with high levels of TGF-β genes. In a multivariate logistic regression analysis, ESR1, PGR, TGFB1, and the total number of somatic variants were identified as independent factors predicting T cell infiltration. ConclusionsIncreased gene expression of ER-α and PR was associated with decreased activated T cell infiltration in patients with NSCLC. The relevance of hormone receptor status should be validated clinically, including in the context of immune checkpoint inhibitors.

Identification of novel prognosis-related genes in the endometrial cancer immune microenvironment.

The incidence of endometrial cancer is increasing each year, and treatment effects are poor for patients with advanced and specific subtypes. Exploring immune infiltration-related factors in endometrial cancer can aid in the prognosis of patients and provide new immunotherapy targets. We downloaded immune metagene and functional data of patients with different subtypes of endometrial cancer from The Cancer Genome Atlas database and selected the lymphocyte-specific kinase (LCK) metagene as a representative genetic marker of the immune microenvironment in endometrial cancer. The results showed that LCK metagene expression is related to the prognosis of patients with endometrioid endometrial adenocarcinoma subtypes and highly correlated with the PTEN and PIK3CA mutational status. A search for LCK-related modules returned seven independent genetic predictors of survival in patients with endometrial cancer. The TIMER algorithm showed that the expression of these seven genes was positively correlated with the infiltration levels of six types of immune cells. The diagnostic value of these markers was validated using real-time quantitative PCR and immunohistochemical methods. Our results identified CD74, HLA-DRB5, CD52, HLA-DPB1 and HLA-DRB1 as possible valuable genetic markers for the diagnosis and prognosis of endometrial cancer and provided a theoretical basis for immunotherapy targets for its clinical treatment.

Immunological Differences Between Immune-Rich Estrogen Receptor-Positive and Immune-Rich Triple-Negative Breast Cancers.

PURPOSEA subset of estrogen receptor–positive (ER-positive) breast cancer (BC) contains high levels of tumor-infiltrating lymphocytes (TILs), similar to triple-negative BC (TNBC). The majority of immuno-oncology trials target TNBCs because of the greater proportion of TIL-rich TNBCs. The extent to which the immune microenvironments of immune-rich ER-positive BC and TNBC differ is unknown.PATIENTS AND METHODSRNA sequencing data from The Cancer Genome Atlas (TCGA; n = 697 ER-positive BCs; n = 191 TNBCs) were used for discovery; microarray expression data from Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n = 1,186 ER-positive BCs; n = 297 TNBCs) was used for validation. Patients in the top 25th percentile of a previously published total TIL metagene score distribution were considered immune rich. We compared expression of immune cell markers, immune function metagenes, and immuno-oncology therapeutic targets among immune-rich subtypes.RESULTSRelative fractions of resting mast cells (TCGA Padj = .009; METABRIC Padj = 4.09E-15), CD8+ T cells (TCGA Padj = .015; METABRIC Padj = 0.390), and M2-like macrophages (TCGA Padj= 4.68E-05; METABRIC Padj = .435) were higher in immune-rich ER-positive BCs, but M0-like macrophages (TCGA Padj = 0.015; METABRIC Padj = .004) and M1-like macrophages (TCGA Padj = 9.39E-08; METABRIC Padj = 6.24E-11) were higher in immune-rich TNBCs. Ninety-one immune-related genes (eg, CXCL14, CSF3R, TGF-B3, LRRC32/GARP, TGFB-R2) and a transforming growth factor β (TGF-β) response metagene were significantly overexpressed in immune-rich ER-positive BCs, whereas 41 immune-related genes (eg, IFNG, PD-L1, CTLA4, MAGEA4) were overexpressed in immune-rich TNBCs in both discovery and validation data sets. TGF-β pathway member genes correlated negatively with expression of immune activation markers (IFNG, granzyme-B, perforin) and positively with M2-like macrophages (IL4, IL10, and MMP9) and regulatory T-cell (FOXP3) markers in both subtypes.CONCLUSIONDifferent immunotherapy strategies may be optimal in immune-rich ER-positive BC and TNBC. Drugs targeting the TGF-β pathway and M2-like macrophages are promising strategies in immune-rich ER-positive BCs to augment antitumor immunity.

Prognostic Effect of Comorbid Disease and Immune Gene Expression on Mortality in Kidney Cancer-A Population Based Study.

The effect of comorbidities and the immune profiles of the kidney cancer microenvironment play a major role in patients’ prognosis and survival. Using the National Health Insurance Research Database (Taiwan), we identified patients aged >20 years with a first diagnosis of kidney cancer between 2005 and 2014. Differences in demographic characteristics and comorbidities were examined using the Pearson chi-squared test or the t test. The Cox regression model was used to construct the nomogram. RNA-seq data were applied from The Cancer Genome Atlas database, and correlations between immune metagenes and clinical characteristics were determined using a linear regression model. In this nationwide cohort study, including 5090 patients with kidney cancer, predictors in our prediction models included age, sex, chronic kidney disease, dialysis requirements, renal stones, cerebrovascular disease, and metastasis tumor. In the tumor tissue profiles, significant positive correlations between immune metagenes and clinical stage or overall survival were observed among Natural Killer (NK) cells (CD56−), CD4+ T-helper 2 (Th2) cells, and activated Dendritic Cell (aDC). A negative correlation was observed between expression level of Dendritic Cell (DC) and overall survival. Patients with kidney cancer exhibit high prevalence of comorbid disease, especially in older patients. Comorbid disease types exert unique effects, and a particular comorbidity can affect cancer mortality. Moreover, the expression of immune metagenes can be utilized as potentialbiomarkers especially for further study of molecular mechanisms as well as microenvironments in kidney cancer.

Prognostic and predictive value of an immune infiltration signature in diffuse lower-grade gliomas.

BACKGROUNDLower-grade gliomas (LGGs) vary widely in terms of the patient's overall survival (OS). There is no current, valid method that could exactly predict the survival. The effects of intratumoral immune infiltration on clinical outcome have been widely reported. Thus, we aim to develop an immune infiltration signature to predict the survival of LGG patients.METHODSWe analyzed 1216 LGGs from 5 public data sets, including 2 RNA sequencing data sets and 3 microarray data sets. Least absolute shrinkage and selection operator (LASSO) Cox regression was used to select an immune infiltration signature and build a risk score. The performance of the risk score was assessed in the training set (329 patients), internal validation set (140 patients), and 4 external validation sets (405, 118, 88, and 136 patients).RESULTSAn immune infiltration signature consisting of 20 immune metagenes was used to generate a risk score. The performance of the risk score was thoroughly verified in the training and validation sets. Additionally, we found that the risk score was positively correlated with the expression levels of TGF-β and PD-L1, which were important targets of combination immunotherapy. Furthermore, a nomogram incorporating the risk score, patient's age, and tumor grade was developed to predict the OS, and it performed well in all the training and validation sets (C-index: 0.873, 0.881, 0.781, 0.765, 0.721, and 0.753).CONCLUSIONThe risk score based on the immune infiltration signature has reliable prognostic and predictive value for patients with LGGs and is a potential biomarker for the cotargeting immunotherapy.FUNDINGThis work was supported by The National Natural Science Foundation of China (grant nos. 81472370 and 81672506), the Natural Science Foundation of Beijing (grant no. J180005), the National High Technology Research and Development Program of China (863 Program, grant no. 2014AA020610), and the National Basic Research Program of China (973 Program, grant no. 2014CB542006).

Human leucocyte antigen class I in hormone receptor-positive, HER2-negative breast cancer: association with response and survival after neoadjuvant chemotherapy

BackgroundClinical application of cancer immunotherapy requires a better understanding of tumor immunogenicity and the tumor microenvironment. HLA class I molecules present antigens to CD8+ cytotoxic cells. Their loss or downregulation is frequently found in tumors resulting in reduced T cell responses and worse prognosis.MethodsWe evaluated HLA class I heavy chain expression by immunohistochemistry in 863 biopsies (GeparTrio trial). Patients received neoadjuvant chemotherapy and adjuvant endocrine treatment if tumors were hormone receptor-positive (HR+). In parallel, the expression of HLA-A was analyzed using a microarray cohort of 320 breast cancer patients from the MD Anderson Cancer Center. We evaluated its association with clinical outcome, tumor-infiltrating lymphocytes (TILs), and immune cell metagenes.ResultsIn HR+/HER2− breast cancer, HLA class I heavy chain expression was associated with increased TILs and better response to chemotherapy (7% vs. 14% pCR rate, P = 0.029), but worse disease-free survival (hazard ratio (HR) 1.6 (1.1–2.4); P = 0.024). The effect was significant in a multivariate model adjusted for clinical and pathological variables (HR 1.7 (1.1–2.6); P = 0.016) and was confirmed by analysis of HLA-A in a microarray cohort. HLA-A was correlated to most immune cell metagenes. There was no association with response or survival in triple-negative or HER2+ disease.ConclusionsThe study confirms the negative prognostic role of lymphocytes in HR+ breast cancer and points at a complex interaction between chemotherapy, endocrine treatment, and tumor immunogenicity. The results point at a subtype-specific and potentially treatment-specific role of tumor-immunological processes in breast cancer with different implications in triple-negative and hormone receptor-positive disease.

Molecular and clinical characterization of Galectin-9 in glioma through 1,027 samples.

In recent years, research on glioma immunotherapy have grown rapidly. However, the autoimmune‐like side effects that are caused by blocking immunological checkpoints hinder their clinical application in gliomas currently. Galectin‐9, a ligand for T‐cell immunoglobulin mucin 3, has shed a new light on the treatment of malignant glioma. However, the potential mechanism of Galectin‐9 is still under discussion. In this study, first, we methodically gathered 1,027 glioma patients with RNA‐seq and 986 patients with survival data to explore the role and mechanism of Galectin‐9 in gliomas. Second, we analyzed glioma samples from 50 patients in the Department of Neurosurgery, Tianjin Medical University General Hospital. Finally, we found that Galectin‐9 was strongly upregulated in glioblastoma multiforme compared with normal brain tissues and lower‐grade glioma. Patients with Galectin‐9 overexpression had a significantly shorter overall survival. Moreover, the tissue microarray data displayed that the expression of Galectin‐9 in the core of tumor is higher than that in the border and was correlated with the shorter survival in glioma patients. Galectin‐9 is more highly expressed in the mesenchymal subtype of glioblastoma multiforme than in the other subtypes. Simultaneously, Galectin‐9 was closely associated with the immune response and lymphocyte activation, especially T‐cell activation. To further determine the underlying role of Galectin‐9 in the immune response, we selected seven immune metagenes. Through cluster analysis and correlation analysis, we discovered that Galectin‐9 was highly correlated with immune checkpoint molecules and M2 tumor‐associated macrophages. In summary, Galectin‐9 serves as a potential therapeutic target to treat glioblastoma multiforme.

Immune correlates of CD73 expression in patients with urothelial carcinoma (UC).

4548 Background: Checkpoint inhibitors have improved outcomes in UC. However, response rates are low and additional mechanisms of immune evasion need to be ascertained. CD73 (encoded by NT5E) converts extracellular AMP to adenosine, which exerts an immunosuppressive effect in the tumor microenvironment by inhibiting infiltrating T and NK cells. Utilizing The Cancer Genome Atlas (TCGA) bladder cancer dataset, we evaluated correlations between NT5E expression and the immune milieu in UC. Methods: RNA-seq data from 411 primary UC tumor samples were obtained from the TCGA. Patients were split into low, intermediate, and high NT5E expression groups (≤ -1, -1 to 1 and ≥1 standard deviation from the overall mean). A tumor inflammation signature (TIS) reflecting an inflamed tumor phenotype was calculated based on the averaged expression of 18 previously validated genes (Ayers et al, 2017). NT5E expression was compared between tumors with high and low TIS scores and among the TCGA molecular subtypes. Abundance of infiltrating immune cell subsets was estimated based on expression of previously identified 782 immune metagenes and compared between NT5E expression groups (Charoentong et al, 2017). The Mann-Whitney U test assessed statistical significance, and the Bonferroni correction was used to control for false discovery rate. Results: NT5E expression was significantly higher in tumors with a high TIS score compared to those with low TIS score (P&lt;0.0001) and correlated with expression of other immune checkpoints such as PD-L1, IDO and LAG-3 (each P&lt;0.01). Patients with basal/squamous subtype had the highest NT5E expression compared to luminal or neuronal subtypes. High NT5E expression was associated with increased infiltrating NK cells, neutrophils, Tregs and decreased Type 2 T helper cells. Conclusions: High expression of NT5E in UC patients with an inflamed tumor phenotype was associated with an increase in infiltrating Tregs, and the basal/squamous subtype. Our findings highlight a potential role of CD73-adenosine pathway as a mechanism of immune evasion and a novel therapeutic target in UC. Further studies to assess the clinical impact of NT5E expression on outcomes in UC patients treated with immunotherapy are needed. AT and NA: equal contribution.

NT5E expression and the immune landscape of prostate cancer (PC): An analysis from The Cancer Genome Atlas database.

e16591 Background: Immune checkpoint inhibitors targeting PD-1/L1 and CTLA-4 pathway have shown modest activity in patients with advanced PC. Additional immunosuppressive mechanisms in the PC tumor microenvironment need to be investigated. Increased CD73 (encoded by NT5E) expression results in generation of immunosuppressive adenosine in the tumor microenvironment and has been associated with metastasis and poor survival in PC. Utilizing the TCGA dataset, we investigated the association of NT5E expression with the immune landscape of PC. Methods: RNA-seq data for 331 PC tumor samples and 51 normal adjacent tissue (NAT) samples was downloaded and log2 transformed. Patients were split into low, intermediate, and high expression groups based on NT5E expression (≤ -1, -1 to 1 and ≥1 standard deviation from the overall mean) in tumor and NAT. A tumor inflammation signature (TIS) reflecting an inflamed tumor phenotype was calculated based on the averaged tumor expression of 18 previously validated genes (Ayers et al, 2017). Abundance of infiltrating immune cell subsets was estimated based on expression of previously identified 782 immune metagenes (Charoentong et al, 2017). Immune cell abundance scores and TIS were compared between NT5E expression groups using the Mann-Whitney U test and the Bonferroni correction was used to control for false discovery rate. Results: NT5E expression in NAT was not associated with the TIS or expression of immune cell marker genes. In contrast, NT5E expression in tumor tissue correlated positively with TIS (P &lt; 0.001). Compared to tumors with low NT5E expression, those in high NT5E expression group had higher expression of central memory CD4+, effector memory CD8+, type 1 helper, NK and regulatory T (Treg) cell markers. Conclusions: In our analysis, NT5E expression correlated with markers of inflamed tumor phenotype in PC. Although NT5E expression was associated with higher CD8+and CD4+ T cells, concurrent increase in Tregs could inhibit the infiltrating lymphocytes and promote tumor growth. Our findings indicate a possible role for the adenosine pathway as a mediator of immunosuppression in PC and a potential therapeutic target. AT and EL: Equal contribution

Effector T-cell cytolytic activity modules derived from CD3+ single cells from human primary triple-negative breast cancer (TNBC) in multiple solid tumors to predict response to immune checkpoint blockade therapy (ICB).

1073 Background: The prognostic and predictive value of tumor infiltrating lymphocytes for ICB has been recognized in a variety of tumor types, including TNBC. Nonetheless, our understanding of the mechanistic aspects of T cell activation remains incomplete. We hypothesize that a specific effector phenotype of T cell cytolytic activity (ECA) is a consistent feature of epithelial tumors, possibly varying by tumor types with a range of inflammatory features. Methods: We evaluated 6,311 purified CD3+ single cells from human primary TNBC and computed sample set enrichment scores of a set of previously published immune metagenes. Following unsupervised clustering of the enrichment scores of the entire single cell population, two subgroups of cells with highest and lowest average enrichment score of T cell cytolytic activity formed a basis for detecting functional gene expression modules. Spectral decomposition and Jackstraw analysis estimated eight modules with overlapping sets of genes. Each gene expression module was then used to train a Random Forest classifier of ECA phenotype. Results: We discovered that our module-derived classifiers were prognostic not only in TNBC samples obtained from both TCGA (N = 150) and METABRIC (N = 320) datasets but also in 14 other tumor types encompassing 6,000 samples. For example, patient samples from TCGA dataset predicted to be in group ECA ‘High’ have better progression-free survival (p-value: 0.0098l; HR: 0.30) and better overall survival (p-value: 0.0066; HR: 0.17). In both breast datasets, gene within the classifier are relatively under-expressed in ER+ tumors as opposed to HER2+ and TNBC (p-value &lt; 2.2e-16). In a dataset of normal, pure DCIS and mixed DCIS (GSE26304;N = 114), the same genes were relatively under-expressed in DCIS samples relative to invasive tumors (p-value &lt; 2.2e-16). Additionally, in a pre-therapy tumor dataset of fifty-one advanced melanoma patients treated with Nivolumab, who previously either progressed on ipilimumab or were ipilimumab-naïve, our module-derived classifier was able to classify responders and non-responders with 77% accuracy (p-value = 0.02) and was associated with progression-free survival (p-value = 0.03; HR: 0.28). Conclusions: Our study highlights one important application of single-cell genomics in our understanding of immune microenvironment and potentially identify new immunotherapy targets.

Bioinformatic Profiling of Prognosis-Related Genes in Breast Cancer Immune Microenvironment

Background: The infiltration of immune and stromal cells in breast cancer microenvironment is strongly related to prognosis. Mining potential immune-related prognostic markers or therapeutic targets is significant. Methods: We had applied multiple approaches to search for the lymphocyte-specific kinase (LCK) metagene scores and LCK metagenes were used as the objects of study to screen the representative genes using weighted gene co-expression network analysis (WGCNA); besides, the differentially ex-pressed genes (DEGs) in samples with high or low LCK metagenes scores were analyzed, and the co-expressed genes markedly correlated with the genes in LCK metagenes were mined jointly. Finally, survival analyses were applied to search for the genes, and gene functions were mined through en-richment analysis. Additionally, these genes were further verified using the external dataset and paired tumor and non-tumor tissues. Findings: LCK metagene scores have high correlations with other categories of immune-related scores, clinical stage, prognosis and the mutation levels of multiple tumor suppressor genes (BRCA1, BRCA2, TP53 and PTEN), from the four kinds of breast cancer subtypes. We screened 22 genes re-lated to immunity and prognosis in breast cancer according to the expression of immune metagenes. Interpretation: Potential immune-related therapeutic targets or markers for the diagnosis and prog-nosis of breast cancer were mined. Funding: Science and Technology Commission of Shanghai Municipality (No. 17411961100). Declaration of Interest: The authors declare no conflicts of interest. Ethical Approval: The undertaken of all the samples from patients of this study was approved by the Ethics Committees of Obstetrics and Gynecology Hospital of Fudan University and consent with all the patients.

Immune profiling of pre- and post-treatment breast cancer tissues from the SWOG S0800 neoadjuvant trial

BackgroundHow the immune microenvironment changes during neoadjuvant chemotherapy of primary breast cancer is not well understood.MethodsWe analyzed pre- and post-treatment samples from 60 patients using the NanoString PanCancer IO360™ assay to measure the expression of 750 immune-related genes corresponding to 14 immune cell types and various immune functions, and assessed TIL counts and PD-L1 protein expression by immunohistochemistry. Treatment associated changes in gene expression levels were compared using t-test with Bonferroni correction. TIL count, PD-L1 protein and immune metagenes were compared using Wilcoxon test. Baseline immune markers were correlated with pathologic complete response (pCR) using estrogen receptor and treatment arm adjusted logistic regression.ResultsAt baseline, high TIL counts and high expression of chemoattractant cytokines (CCL21, CCL19) and cytotoxic T cell markers were associated with higher pCR rate. High expression of stromal genes (VEGFB, TGFB3, PDGFB, FGFR1, IGFR1), mast and myeloid inflammatory cell metagenes, stem cell related genes (CD90, WNT11, CTNNB1) and CX3CR1, and IL11RA were associated with residual disease (RD). After treatment, in cases with pCR, TIL counts and most immune genes decreased significantly. Among RD cases, TIL counts and PD-L1 expression did not change but cellular stress and hypoxia associated genes (DUSP1, EGR1), and IL6, CD36, CXCL2, CD69 and the IL8/VEGF metagene increased.ConclusionsActivated T cells in the tumor microenvironment are associated with pCR whereas stromal functions are associated with residual disease. Most immune functions decrease during neoadjuvant chemotherapy but several immunotherapy targets (PD-L1, IL6, IL8) remain expressed in RD suggesting potential therapeutic strategies.