Expression Of Inhibitory Immune Checkpoints Research Articles

Pan-cancer analysis revealing DAAM1 as a novel predictive biomarker for PD-1/PD-L1 blockade in clear cell renal cell carcinoma.

Pan-cancer analysis revealing DAAM1 as a novel predictive biomarker for PD-1/PD-L1 blockade in clear cell renal cell carcinoma.

Cuproptosis-related lncRNAs predict the prognosis and immune response in hepatocellular carcinoma.

Cuproptosis has been recently used to indicate unique biological processes triggered by Cu action as a new term. This study aimed to explore the relationship between cuproptosis-related lncRNA and hepatocellular carcinoma (HCC) with regard to immunity and prognosis. RNA sequencing and the clinical data were downloaded from the TCGA database. The cuproptosis-related genes were sorted out through literature study. The cuproptosis-related IncRNA signature was identified by Cox regression analysis and the least absolute shrinkage and selection operator analysis. The K-M survival analysis, receiver operating characteristic analysis, and C-index analysis were adopted to evaluate the prognostic prediction performance of the signature. The functional enrichment, immune infiltration and tumor mutation analysis were further analyzed. Subsequently, we predicted the differences in chemosensitivity from tumor gene expression levels for some chemotherapy drugs. The prognostic signature consisting of 5 overall survival-related CUPlncRNAs. It showed an extraordinary ability to predict the prognoses of patients with HCC. The signature can predict the abundance of immune cell infiltration, immune functions, expression of immune checkpoint inhibitors, m6A genes, which was supported by the GO biological process and KEGG analysis. And it may also have a guiding effect in the sensitivity of different chemotherapeutic drugs and tumor mutation burden. We constructed a new cuproptosis-related lncRNA signature for HCC patients. The model can be used for prognostic prediction and immune evaluation, providing a reference for immunotherapies and targeted therapies.

Multi-omic Characterization of Pancreatic Ductal Adenocarcinoma Relates CXCR4 mRNA Expression Levels to Potential Clinical Targets.

Chemokines are essential for immune cell trafficking and are considered to have a major impact on the composition of the tumor microenvironment. CX-chemokine receptor 4 (CXCR4) is associated with poor differentiation, metastasis, and prognosis in pancreatic ductal adenocarcinoma (PDAC). This study provides a comprehensive molecular portrait of PDAC according to CXCR4 mRNA expression levels. The Cancer Genome Atlas database was used to explore molecular and immunologic features associated with CXCR4 mRNA expression in PDAC. A large real-word dataset (n = 3,647) served for validation and further exploratory analyses. Single-cell RNA analyses on a publicly available dataset and in-house multiplex immunofluorescence (mIF) experiments were performed to elaborate cellular localization of CXCR4. High CXCR4 mRNA expression (CXCR4high) was associated with increased infiltration of regulatory T cells, CD8+ T cells, and macrophages, and upregulation of several immune-related genes, including immune checkpoint transcripts (e.g., TIGIT, CD274, PDCD1). Analysis of the validation cohort confirmed the CXCR4-dependent immunologic TME composition in PDAC irrespective of microsatellite instability-high/mismatch repair-deficient or tumor mutational burden. Single-cell RNA analysis and mIF revealed that CXCR4 was mainly expressed by macrophages and T-cell subsets. Clinical relevance of our finding is supported by an improved survival of CXCR4high PDAC. High intratumoral CXCR4 mRNA expression is linked to a T cell- and macrophage-rich PDAC phenotype with high expression of inhibitory immune checkpoints. Thus, our findings might serve as a rationale to investigate CXCR4 as a predictive biomarker in patients with PDAC undergoing immune checkpoint inhibition.

RGS1 and related genes as potential targets for immunotherapy in cervical cancer: computational biology and experimental validation

BackgroundEffective treatment is needed for advanced, inoperable, or chemotherapy-resistant cervical cancer patients. Immunotherapy has become a new treatment modality for cervical cancer patients, and there is an urgent need to identify additional targets for cervical cancer immunotherapy.MethodsIn this study the core gene, RGS1, which affects immune status and the FIGO stage of cervical cancer patients was identified by WGCNA analysis and differential analysis using TCGA database. 10 related genes interacting with RGS1 were identified using PPI network, and the functional and immune correlations were analyzed. Based on the expression of RGS1 and related genes, the consensus clustering method was used to divide CESC patients into two groups (group 1, high expression of RGS1; group 2, low expression of RGS1). Then, the functional enrichment analysis was used to search for the functional differences in differentially expressed genes (DEGs) between group 1 and group 2. Immune infiltration analysis was performed using ESTIMATE, CIBERSORT, and ssGSEA, and the differences in expression of immune checkpoint inhibitors (ICIs) targets were assessed between the two groups. We investigated the effect of RGS1 on the clinical relevance of CESC patients, and experimentally verified the differences in RGS1 expression between cervical cancer patient tissues and normal cervical tissues, the role of RGS1 in cell function, and the effect on tumor growth in tumor-bearing mice.ResultsWe found that RGS1 was associated with CD4, GNAI3, RGS2, GNAO1, GNAI2, RGS20, GNAZ, GNAI1, HLA-DRA and HLA-DRB1, especially CD4 and RGS2. Functional enrichment of DEGs was associated with T cell activation. Compared with group 2, group 1 had stronger immune infiltration and higher ICI target expression. RGS1 had higher expression in cervical cancer tissues than normal tissues, especially in HPV-E6 positive cancer tissues. In cervical cancer cell lines, knockdown of RGS1 can inhibited cell proliferation, migration, invasion, and tumor growth in nude mice and promoted apoptosis.ConclusionsRGS1, as an oncogenic gene of cervical cancer, affects the immune microenvironment of patients with cervical cancer and may be a target of immunotherapy.

A Novel Prognostic Signature Based on Glioma Essential Ferroptosis-Related Genes Predicts Clinical Outcomes and Indicates Treatment in Glioma.

BackgroundFerroptosis is a form of programmed cell death (PCD) that has been implicated in cancer progression, although the specific mechanism is not known. Here, we used the latest DepMap release CRISPR data to identify the essential ferroptosis-related genes (FRGs) in glioma and their role in patient outcomes.MethodsRNA-seq and clinical information on glioma cases were obtained from the Chinese Glioma Genome Atlas (CGGA) and The Cancer Genome Atlas (TCGA). FRGs were obtained from the FerrDb database. CRISPR-screened essential genes (CSEGs) in glioma cell lines were downloaded from the DepMap portal. A series of bioinformatic and machine learning approaches were combined to establish FRG signatures to predict overall survival (OS) in glioma patients. In addition, pathways analysis was used to identify the functional roles of FRGs. Somatic mutation, immune cell infiltration, and immune checkpoint gene expression were analyzed within the risk subgroups. Finally, compounds for reversing high-risk gene signatures were predicted using the GDSC and L1000 datasets.ResultsSeven FRGs (ISCU, NFS1, MTOR, EIF2S1, HSPA5, AURKA, RPL8) were included in the model and the model was found to have good prognostic value (p < 0.001) in both training and validation groups. The risk score was found to be an independent prognostic factor and the model had good efficacy. Subgroup analysis using clinical parameters demonstrated the general applicability of the model. The nomogram indicated that the model could effectively predict 12-, 36-, and 60-months OS and progression-free interval (PFI). The results showed the presence of more aggressive phenotypes (lower numbers of IDH mutations, higher numbers of EGFR and PTEN mutations, greater infiltration of immune suppressive cells, and higher expression of immune checkpoint inhibitors) in the high-risk group. The signaling pathways enriched closely related to the cell cycle and DNA damage repair. Drug predictions showed that patients with higher risk scores may benefit from treatment with RTK pathway inhibitors, including compounds that inhibit RTKs directly or indirectly by targeting downstream PI3K or MAPK pathways.ConclusionIn summary, the proposed cancer essential FRG signature predicts survival and treatment response in glioma.

Oncolytic Adenoviral Vector-Mediated Expression of an Anti-PD-L1-scFv Improves Anti-Tumoral Efficacy in a Melanoma Mouse Model.

Oncolytic virotherapy is an emerging therapeutic approach based on replication-competent viruses able to selectively infect and destroy cancer cells, inducing the release of tumor-associated antigens and thereby recruiting immune cells with a subsequent increase in antitumoral immune response. To increase the anticancer activity, we engineered a specific oncolytic adenovirus expressing a single-chain variable fragment of an antibody against PD-L1 to combine blockage of PD-1/PD-L1 interaction with the antitumoral activity of Onc.Ad5. To assess its efficacy, we infected B16.OVA cells, a murine model of melanoma, with Ad5Δ24 -anti-PD-L1-scFv and then co-cultured them with C57BL/6J naïve splenocytes. We observed that the combinatorial treatments were significantly more effective in inducing cancer cell death. Furthermore, we assessed the efficacy of intratumoral administrations of Ad5Δ24-anti-PD-L1-scFv in C57BL/6J mice engrafted with B16.OVA and compared this treatment to that of the parental Ad5Δ24 or placebo. Treatment with the scFv-expressing Onc.Ad induced a marked reduction of tumor growth concerning the parental Onc.Ad. Additionally, the evaluation of the lymphocytic population infiltrating the treated tumor reveals a favorable immune profile with an enhancement of the CD8+ population. These data suggest that Onc.Ad-mediated expression of immune checkpoint inhibitors increases oncolytic virotherapy efficacy and could be an effective and promising tool for cancer treatments, opening a new way into cancer therapy.

Integrated single-cell transcriptome analysis reveals heterogeneity of esophageal squamous cell carcinoma microenvironment

The tumor microenvironment is a highly complex ecosystem of diverse cell types, which shape cancer biology and impact the responsiveness to therapy. Here, we analyze the microenvironment of esophageal squamous cell carcinoma (ESCC) using single-cell transcriptome sequencing in 62,161 cells from blood, adjacent nonmalignant and matched tumor samples from 11 ESCC patients. We uncover heterogeneity in most cell types of the ESCC stroma, particularly in the fibroblast and immune cell compartments. We identify a tumor-specific subset of CST1+ myofibroblasts with prognostic values and potential biological significance. CST1+ myofibroblasts are also highly tumor-specific in other cancer types. Additionally, a subset of antigen-presenting fibroblasts is revealed and validated. Analyses of myeloid and T lymphoid lineages highlight the immunosuppressive nature of the ESCC microenvironment, and identify cancer-specific expression of immune checkpoint inhibitors. This work establishes a rich resource of stromal cell types of the ESCC microenvironment for further understanding of ESCC biology.

Identification of a N6-Methyladenosine (m6A)-Related lncRNA Signature for Predicting the Prognosis and Immune Landscape of Lung Squamous Cell Carcinoma.

Backgroundm6A-related lncRNAs emerged as potential targets for tumor diagnosis and treatment. This study aimed to identify m6A-regulated lncRNAs in lung squamous cell carcinoma (LUSC) patients.Materials and MethodsRNA sequencing and the clinical data of LUSC patients were downloaded from The Cancer Genome Atlas (TCGA) database. The m6A-related lncRNAs were identified by using Pearson correlation assay. Univariate and multivariate Cox regression analyses were utilized to construct a risk model. The performance of the risk model was validated using Kaplan–Meier survival analysis and receiver operating characteristics (ROC). Immune estimation of LUSC was downloaded from TIMER, and the correlations between the risk score and various immune cells infiltration were analyzed using various methods. Differences in immune functions and expression of immune checkpoint inhibitors and m6A regulators between high-risk and low-risk groups were further explored. Finally, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were utilized to explore the biological functions of AL122125.1.ResultsA total of 351 m6A-related lncRNAs were obtained from TCGA. Seven lncRNAs demonstrated prognostic values. A further multivariate Cox regression assay constructed a risk model consisting of two lncRNAs (AL122125.1 and HORMAD2-AS1). The Kaplan–Meier analysis and area under the curve indicated that this risk model could be used to predict the prognosis of LUSC patients. The m6A-related lncRNAs were immune-associated. There were significant correlations between risk score and immune cell infiltration, immune functions, and expression of immune checkpoint inhibitors. Meanwhile, there were significant differences in the expression of m6A regulators between the high- and low-risk groups. Moreover, GO and KEGG analyses revealed that the upregulated expression of AL122125.1 was tumor-related.ConclusionIn this study, we constructed an m6A-related lncRNA risk model to predict the survival of LUSC patients. This study could provide a novel insight to the prognosis and treatment of LUSC patients.

Metabolic Acidosis and Hyponatremia in a Patient With Metastatic Melanoma

Metabolic Acidosis and Hyponatremia in a Patient With Metastatic Melanoma

A Ferroptosis-Related Prognostic Signature Based on Antitumor Immunity and Tumor Protein p53 Mutation Exploration for Guiding Treatment in Patients With Head and Neck Squamous Cell Carcinoma.

Background: Due to the lack of accurate guidance of biomarkers, the treatment of head and neck squamous cell carcinoma (HNSCC) has not been ideal. Ferroptosis plays an important role in tumor suppression and treatment of patients. However, tumor protein p53 (TP53) mutation may promote tumor progression through ferroptosis. Therefore, it is particularly important to mine prognostic-related differentially expressed ferroptosis-related genes (PR-DE-FRGs) in HNSCC to construct a prognostic model for accurately guiding clinical treatment.Methods: First, the HNSCC data obtained from The Cancer Genome Atlas (TCGA) was used to identify PR-DE-FRGs for screening candidate genes to construct a prognostic model. We not only used a variety of methods to verify the accuracy of the model for predicting prognosis but also explored the role of ferroptosis in the development of HNSCC from the perspective of the immune microenvironment and mutation. Finally, we explored the correlation between the prognostic model and clinical treatment and drew a high-precision nomogram to predict the prognosis.Results: Seventeen of the 29 PR-DE-FRGs were selected to construct a prognostic model with good predictive performance. Patients in the low-risk group were found to have a greater number of CD8 + T cells, follicular helper T cells, regulatory T cells, mast cells, T-cell costimulations, and type II interferon responses. A higher tumor mutation burden (TMB) was observed in the low-risk group and was associated with a better prognosis. A higher risk score was found in the TP53 mutation group and was associated with a worse prognosis. The risk score is closely related to the expression of immune checkpoint inhibitors (ICIs)-related genes such as PD-L1 and the IC50 of six chemotherapeutic drugs. The nomogram we constructed performs well in predicting prognosis.Conclusion: Ferroptosis may participate in the progression of HNSCC through the immune microenvironment and TP53 mutation. The model we built can be used as an effective predictor of immunotherapy and chemotherapy effects and prognosis of HNSCC patients.

Immunohistochemical analysis of glassy cell carcinoma of the cervix reveals robust lymphocyte infiltrate and the expression of targetable inhibitory immune checkpoints.

To validate our previous findings of high-level EGFR expression in GCCC using an expanded cohort of specimens and to further examine the molecular and cellular features of this aggressive malignancy to identify potentially actionable therapeutic targets. The SEER database was queried to obtain the epidemiological data regarding the current national survival trends for GCCC. Immunohistochemistry (IHC) was used to examine the expression of EGFR, PD-1, and PD-L1. CiberSort analysis was used to analyze a previously published RNA-sequencing dataset obtained from a single patient diagnosed with GCCC. In comparison to squamous cell carcinomas and adenocarcinoma/adenosquamous carcinomas, GCCC was observed in younger patients (p < 0.001) and demonstrated inferior survival (p < 0.001). All (100%) of the specimens (8/8) exhibited immunoreactivity when stained for CD3ε (T-cell marker), EGFR, PD-1, and PD-L1 whereas CTLA4 expression was not detected. Analysis of RNA-sequencing data revealed that cetuximab and erlotinib altered the chemokine profile, lymphocyte abundance, and expression of inhibitory immune checkpoints in a single patient when combined with cytotoxic chemotherapy in a single patient. The data from this descriptive study suggests that immune checkpoint blockade, whether single agent or in combination, may be a suitable therapeutic option for a disease for which targeted approaches do not currently exist.

The Prognostic Value of the Lymph Node in Oesophageal Adenocarcinoma; Incorporating Clinicopathological and Immunological Profiling.

Simple SummaryOesophageal cancer rates are increasing rapidly with patients often presenting at an advanced stage. The current approach to treatment involves radiotherapy, chemotherapy, or combination chemoradiotherapy with surgery; however, only a fraction of these patients will achieve meaningful responses. Therefore, there is a need to better understand the tumour and lymph node microenvironments to inform future treatment strategies. This study measured immune markers including immune checkpoint expression in tumour and lymph node tissue in oesophageal cancer patients and patient clinical outcomes, including survival time, response to treatment, and adverse events. We report herein that nodal metastases is of equal prognostic importance to clinical tumour stage and tumour regression grade in OAC and we observed a more immunosuppressive microenvironment in the tumour compared with the lymph node.Response rates to the current gold standards of care for treating oesophageal adenocarcinoma (OAC) remain modest with 15–25% of patients achieving meaningful pathological responses, highlighting the need for novel therapeutic strategies. This study consists of immune, angiogenic, and inflammatory profiling of the tumour microenvironment (TME) and lymph node microenvironment (LNME) in OAC. The prognostic value of nodal involvement and clinicopathological features was compared using a retrospective cohort of OAC patients (n = 702). The expression of inhibitory immune checkpoints by T cells infiltrating tumour-draining lymph nodes (TDLNs) and tumour tissue post-chemo(radio)therapy at surgical resection was assessed by flow cytometry. Nodal metastases is of equal prognostic importance to clinical tumour stage and tumour regression grade (TRG) in OAC. The TME exhibited a greater immuno-suppressive phenotype than the LNME. Our data suggests that blockade of these checkpoints may have a therapeutic rationale for boosting response rates in OAC.

Abstract 1605: CERBBP mutation promotes antitumor immunity and as predictive biomarkers for immune checkpoint therapy in colorectal cancer

Abstract Background: The prognosis of colorectal cancer (CRC) remains unsatisfactory, only few CRC patients can benefit from immune checkpoint inhibitors (ICIs) treatment. Tumor immune microenvironment (TME), the Tumor mutation burden (TMB) and PD-L1 expression of ICIs have been confirmed to be promising predictive biomarkers of ICIs in pan-cancer. The roles of CREBBP gene in CRC have not been reported before, and the mutation correlation with ICIs predictive biomarkers and immune response remain unknown. Methods: Whole exome sequencing data of 526 CRC patients was obtained from the Cancer Genome Altas (TCGA). Next generation sequencing (NGS) was performed on FFPE tumor samples from 2198 Chinese CRC patients (Chinese cohort). NGS data and clinical data of 110 advanced CRC patients treated with ICIs were obtained from the MSK-IMPACT Clinical Sequencing cohort (MSKCC). The tumor-related immune cells infiltration level difference between mutant and wild-type tumors were inferred using TIMER2.0. Tumor mutation burden (TMB) examined by 387/733 gene panel (3D-Med) sequencing. PD-L1 expression was determined by immunohistochemistry (IHC) staining assay using SP263. Survival analysis was determined by Kaplan-Meier (KM) analysis. Results: In total, 8.62% (43/526) of CRC patients in TCGA harboring CREBBP mutation and 2.00% (44/2198) in Chinese cohort, the alternation frequency of CREBBP in Chinese cohorts was lower. In TCGA cohort, the level of infiltration of tumor-related immune cells, including CD8+ T cell, CD4+ T cell, T cell regulatory (Tregs) and NK cell, were all significantly increased in mutant tumors compared with wild-type tumors (Wilcoxon test, p &amp;lt; 0.05). The mutant tumors with significantly higher inferred M1 macrophages than wild-type (p=0.0029), but not in M2 macrophages. In the Chinese cohort, the TMB level was higher in mutant group compared to wild-type group (median TMB, mut vs. wt = 82.26 vs. 7.26 Mut/Mb, p &amp;lt; 0.001). There was an elevated PD-L1 expression in CREBBP mutant tumors (29.63%, n=44) compared to wild-type tumors (15.73%, n=2154, p = 0.062). The survival analysis was performed on patients in MSKCC CRC patients treated with ICIs. The overall survival (OS) of CREBBP mutation group (n=16) were significantly longer than wild-type group (n=94) (median OS, mutation vs. wild-type = 34 vs. 13 months; HR 0.23[95% CI 0.07-0.75]; P = 0.008). Conclusion: The CREBBP gene mutation was associated with higher tumor-related immune cell infiltration and TMB level. CRC patients harboring CREBBP mutation might be a survival benefit from ICIs. Citation Format: Youen Lin, Jiewei Zheng, Tingting Chen, Wenzhuan Xie, Mengli Huang. CERBBP mutation promotes antitumor immunity and as predictive biomarkers for immune checkpoint therapy in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1605.

Enhanced antitumor efficacy of a novel oncolytic vaccinia virus encoding a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT)

BackgroundOncolytic virotherapy with vaccinia virus (VV) can lead to effective anti-tumor immunity by turning “cold” tumors into “hot” tumors. However, its therapeutic potential is affected by the tumor's local immunosuppressive tumor microenvironment (TME). Therefore, it is necessary to explore the use of immune checkpoint inhibitors to arm oncolytic VVs to enhance their anti-tumor efficacy.MethodsA novel recombinant oncolytic VV, VV-α-TIGIT, which encoded a fully monoclonal antibody against T-cell immunoglobulin and ITIM domain (TIGIT) was generated by homologous recombination with a shuttle plasmid. The anti-tumor efficacy of the VV-α-TIGIT was investigated in several subcutaneous and ascites tumor models.FindingsThe functional α-TIGIT was sufficiently produced and secreted by tumor cells infected with VV-α-TIGIT, which effectively replicated in tumor cells leading to significant oncolysis. Intratumoral injection of VV-α-TIGIT improved anti-tumor efficacy in several murine subcutaneous tumor models compared to VV-Control (without α-TIGIT insertion). Intraperitoneal injection of VV-α-TIGIT achieved approximately 70% of complete tumor regression in an ascites tumor model. At the same time, treatment with VV-α-TIGIT significantly increased the recruitment and activation of T cells in TME. Moreover, the in vivo anti-tumor activity of VV-α-TIGIT was largely dependent on CD8+ T cell-mediated immunity. Finally, the tumor-bearing mice cured of VV-α-TIGIT treatment resisted rechallenge with the same tumor cells, suggesting a long-term persistence of tumor-specific immunological memory.InterpretationThe recombinant oncolytic virus VV-α-TIGIT successfully combines the advantages of oncolytic virotherapy and intratumorally expression of immune checkpoint inhibitor against TIGIT. This novel strategy can provide information on the optimal design of novel antibody-armed oncolytic viruses for cancer immunotherapy.FundingThis work was supported by the National Natural Science Foundation of China (81773255, 81472820, and 81700037), the Science and Technology Innovation Foundation of Nanjing University (14913414), and the Natural Science Foundation of Jiangsu Province of China (BK20171098).

M2 subtype tumor associated macrophages (M2-TAMs) infiltration predicts poor response rate of immune checkpoint inhibitors treatment for prostate cancer

Background Prostate cancer (PCa) is poor response to the immunotherapy for its high heterogeneity of immune microenvironment. In this study, we aim to introduce a new immune subtype for PCa involving M2 tumour associated macrophages (M2-TAMs). Methods Three hundred and sixty-two PCa patients and matched normal prostate tissues were selected from the Cancer Genome Atlas and Gene Expression Omnibus databases. Patients’ immune infiltration characters were then analyzed based on the gene expressions. The immune subtypes were identified by the method of unsupervised hierarchical clustering. Finally, the relationship between the M2-TAMs infiltration and anti-programmed death-ligand-1 (PD-L1) therapy was investigated in the IMvigor210 cohort. Results PCa expressed lower immune-related genes levels compared with the adjacent normal tissues. Based on the proved immunosuppressive mechanisms in PCa, tumour patients were classified into three independent subclasses with high infiltrated cytolytic activity (CYT), M2-TAMs and regulatory T cell (Tregs), respectively. Among these subtypes, M2-TAMs infiltration subtype showed the worst clinicopathological features and prognosis compared with the other two subtypes. The results of the IMvigor210 cohort demonstrated poor response of anti-PD-L1 therapy for patients with high M2-TAMs infiltration. Conclusion Prostate tumours involved in significant immunosuppression, and high infiltration of M2-TAMs can be applied to predict the effect of anti-PD-L1 therapy. Key Messages PCa patients can be classified into three immunotypes of high infiltrated CYT, M2-TAMS, and Tregs according to the immunosuppressive mechanisms. High M2-TAMs infiltration subtype reflected the worst clinical characters, immune infiltration, and lowest expression of immune checkpoint inhibitors among the three subclasses in PCa. High M2-TAMs infiltration predicts the low response rate of anti-PD-L1 therapy.

Immune-related microRNA signature for predicting prognosis and the immune microenvironment in hepatocellular carcinoma

AimsAs vital regulators of post-transcription gene expression, microRNAs are involved in the initiation and progression of hepatocellular carcinoma (HCC), including antitumor immune responses. We aimed to identify an immune-related microRNA signature and explore the influence of this signature on the prognosis and immunity of HCC. Materials and methodsDifferentially expressed immune-related microRNAs were identified between high- and low-immunity groups in the TCGA-HCC dataset. Then, Cox regression models were used to construct an immune-related microRNA signature. We assessed the prognostic value and clinical relevance of this signature. Furthermore, we analyzed the effect of the immune-related microRNA signature on immune cells and immune checkpoints. Key findingsWe screened 41 differentially expressed immune-related microRNAs, of which 7 microRNAs were used to construct the immune signature. Survival analysis showed that high-risk patients had a shorter survival. The immune-related microRNA signature was an independent prognostic marker and was associated with the clinical stage. To understand the functional mechanism of the seven-microRNA signature, we predicted target genes of microRNAs. Enrichment analysis indicated that these target genes participated in immune responses. Moreover, we found that the microRNA signature was related to tumor-infiltrating immune cells. In high-risk patients, there was a higher expression of inhibitory immune checkpoints. On the contrary, the expression of stimulatory immune checkpoints was lower in high-risk patients. SignificanceWe successfully identified an immune-related microRNA signature that was strongly correlated with the prognosis and immune microenvironment of HCC. The immune-related microRNAs signature might have important implications for improving the clinical outcomes of HCC patients.

Identification of a novel immune prognostic model in gastric cancer.

The tumor immune microenvironment (TIME) is now considered as an important factor during gastric cancer (GC) development. This study identified a novel immune-related risk model for predicting prognosis and assessing the immune status of GC patients. Transcriptomic data were obtained from the TCGA database. The differential expressed immune-related genes (IRGs) were identified through the ImmPort portal. Enrichment analysis was performed to explore the potential molecular mechanism of these IRGs. By the Cox regression analysis, we constructed the immune prognostic model. Then, the association between the model and the immune microenvironment was estimated. The model was validated in the GSE84433 dataset. Totally, we identified 222 differentially expressed IRGs. These IRGs were closely correlated with immune response and immune signaling pathways. Through the Cox regression analysis, we developed the immune prognostic model based on the expression of seven IRGs (CXCL3, NOX4, PROC, FAM19A4, RNASE2, IGHD2-15, CGB5). Patients were stratified into two groups according to immune-related risk scores. Survival analysis indicated that the prognosis of high-risk patients was poorer than low-risk patients. Moreover, the immune-related risk score was an independent prognostic biomarker. More importantly, we found that the infiltration level of immunosuppressive cells and the expression of inhibitory immune checkpoints were higher in high-risk patients. The immune microenvironment tended to be a suppressive status in patients with high-risk scores. This study demonstrated that our model had predictive value for prognosis and the TIME in GC. It might be a robust tool to improve personalized patient management.

Abstract 310: Immunophenotyping tumors by novel multiplex immunofluorescence and AQUA (Automated Quantitative Analyses) algorithms to guide development of immunotherapies

Abstract Purpose: Deeper understanding of immune landscape of the tumor microenvironment is critical for exploring and development of next generation immunotherapies. Multiplex fluorescence immunohistochemistry (mFIHC) combined with hypothesis driven spatial profiling algorithms (e.g., AQUA Technology) was found to provide the most powerful predictors of immunotherapies in a systematic meta-analyses of over 8000 patients treated with PD1/L1 pathway blockers (Lu et al., JAMA Oncol 2019). Study Design: To guide clinical development of next generation and/or combination immunotherapies, we built four novel mFIHC assays to simultaneously explore presence of major immune cell lineages, their spatial relationships and functional attributes. The first assay incorporates antibodies to identify major immune cells lineages (e.g., T-, B-, NK-/T- and subtypes of myeloid cells), the second assay is designed to understand T-cell proliferation (via co-expression of Ki67) or suppression (via co-expression of FoxP3), the third method is focused on quantifying the expression of immune checkpoint inhibitors (e.g., LAG3, PD1 and PD-L1), while, the final assay is designed to explore the IFNγ mediated adaptive resistance and immunosuppression (via co-expression of HLA-DR and IDO1). We will describe the successful development of these clinical grade mFIHC assays that utilize automated staining (Leica Bond RX), imaging (Vectra Polaris) and analyses (AQUA® Technology) workflows on tissue microarrays (TMAs) representing multiple regions of interest including tumor, tumor margin, tumor-associated fibrosis, various adjacent normal tissues, lymphoid structures and/or associated inflammation, and metastatic lesions collected from 100 colorectal, and pancreatic cancer patients. Results: Sensitivity, accuracy and specificity were confirmed for all mFIHC assays on known positive and negative controls. Excellent reproducibility (less than 35% CV) and precision were observed across instruments, operators and independent experiments for all markers. TMA work is underway and the latest results will be presented. Conclusion: The validated mFIHC assays helped classify unique immune phenotypes and their interactions. These assays, combined with comprehensive selection of tissues from the tumor microenvironment across multiple cancer subtypes, are expected to enable clinical development of next generation cancer therapies. Citation Format: Ju Young Kim, John Hoerter, Nathan Roscoe, John Fathman, James Santos, Lori Iaconis, Justin Santos, Minhua Qiu, Jacob Levy, Valerie Tolley, Emmanuel Pacia, Tom Carolan, Xun Li, Patrizia Barzaghi-Rinaudo, Brian Schwartz, Robbin Newlin, Karyn Colman, Andy Schumacher, Deborah Knee, Steven Bender, Margaret McLaughlin, Alexander Savchenko, Thai Tran, Shabnam Tangri, Naveen Dakappagari, Jennifer Bordeaux. Immunophenotyping tumors by novel multiplex immunofluorescence and AQUA (Automated Quantitative Analyses) algorithms to guide development of immunotherapies [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 310.

Abstract A18: Characterization of checkpoint inhibitors in the tumor microenvironment (TME) and peripheral blood in endemic Burkitt Lymphoma

Abstract Introduction: The tumor microenvironment and the interaction between immune checkpoint inhibitors (ICIs) and tumor cells are essential to understand for designing future immunotherapy strategies. In this study, we aim to characterize the tumor microenvironment (TME) in children diagnosed with endemic Burkitt lymphoma (eBL), the most common pediatric cancer in Africa. A history of chronic malaria (Plasmodium falciparum) and Epstein-Barr virus (EBV) coinfections are both risk factors in the etiology of eBL. Methods: Using multicolor flow cytometry and single-cell RNA sequencing, we analyzed the expression of immune checkpoint inhibitors in T cells from the tumor microenvironment and from peripheral blood mononuclear cells (PBMC) of eBL patients, along with age-matched controls with different malaria exposure. Additionally, CD8 T-cell infiltration was confirmed by immunohistochemistry. Results: PD-1+ CD4 T cells from eBL PBMCs were increased in number compared to healthy controls from the malaria-endemic area (p=0.03). Both CD4 and CD8 T cells from eBL PBMCs reveal higher frequency of TIGIT expressers compared to healthy controls (non-malaria endemic) (p=0.002, p=0.007 respectively). PD1+ TIGIT+ IFNG- memory CD4 T cells phenotype from eBL PBMCs was increased upon short stimulation with PMA/ionomycin, as well as “naive-like” CD95+ PD1high TIGIT+ CTLA4+ IFNG+ CD8T cells. Tumor samples from 4 eBL patient were used on single-cell analysis; we observed activated T-cells profile that highly upregulated TIGIT within the tumor microenvironment. We also noticed relatively low expression of other ICIs such as PD1, TIM3, LAG3, and CTLA4. Base on all these data, we calculated an exhaustion score that was higher than the cytotoxicity score in our eBL-derived tumor-infiltrating lymphocytes (TILs). In addition, we use immunohistochemistry staining to confirm CD8 T-cell infiltration and ICI expression in eBL tumor fine needle aspirates. Our future directions will employ TIGIT blockade inhibitor studies of CD8 T cells, in order to increase expansion and function of CD8 TILs. Our results will help support future development of ICI therapies in endemic Burkitt lymphoma. Citation Format: Priya Saikumar-Lakshmi, Cliff Oduor, Catherine Forconi, Patrick Marsh, John Michael Ong’echa, Jeffrey Bailey, Ann Moormann. Characterization of checkpoint inhibitors in the tumor microenvironment (TME) and peripheral blood in endemic Burkitt Lymphoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A18.

Beyond Cell Motility: The Expanding Roles of Chemokines and Their Receptors in Malignancy.

The anti-tumor activities of some members of the chemokine family are often overcome by the functions of many chemokines that are strongly and causatively linked with increased tumor progression. Being key leukocyte attractants, chemokines promote the presence of inflammatory pro-tumor myeloid cells and immune-suppressive cells in tumors and metastases. In parallel, chemokines elevate additional pro-cancerous processes that depend on cell motility: endothelial cell migration (angiogenesis), recruitment of mesenchymal stem cells (MSCs) and site-specific metastasis. However, the array of chemokine activities in cancer expands beyond such “typical” migration-related processes and includes chemokine-induced/mediated atypical functions that do not activate directly motility processes; these non-conventional chemokine functions provide the tumor cells with new sets of detrimental tools. Within this scope, this review article addresses the roles of chemokines and their receptors at atypical levels that are exerted on the cancer cell themselves: promoting tumor cell proliferation and survival; controlling tumor cell senescence; enriching tumors with cancer stem cells; inducing metastasis-related functions such as epithelial-to-mesenchymal transition (EMT) and elevated expression of matrix metalloproteinases (MMPs); and promoting resistance to chemotherapy and to endocrine therapy. The review also describes atypical effects of chemokines at the tumor microenvironment: their ability to up-regulate/stabilize the expression of inhibitory immune checkpoints and to reduce the efficacy of their blockade; to induce bone remodeling and elevate osteoclastogenesis/bone resorption; and to mediate tumor-stromal interactions that promote cancer progression. To illustrate this expanding array of atypical chemokine activities at the cancer setting, the review focuses on major metastasis-promoting inflammatory chemokines—including CXCL8 (IL-8), CCL2 (MCP-1), and CCL5 (RANTES)—and their receptors. In addition, non-conventional activities of CXCL12 which is a key regulator of tumor progression, and its CXCR4 receptor are described, alongside with the other CXCL12-binding receptor CXCR7 (RDC1). CXCR7, a member of the subgroup of atypical chemokine receptors (ACKRs) known also as ACKR3, opens the gate for discussion of atypical activities of additional ACKRs in cancer: ACKR1 (DARC, Duffy), ACKR2 (D6), and ACKR4 (CCRL1). The mechanisms involved in chemokine activities and the signals delivered by their receptors are described, and the clinical implications of these findings are discussed.