Immune-related Markers Research Articles

Evaluation of Molecules Involved in Intestinal Changes Induced by Lawsonia intracellularis Infection

Lawsonia intracellularis (LI)is an obligate intracellular bacterium that causes proliferative enteropathy (PE) in various species including pigs, horses, rabbits, dogs and hamsters. Infection by LI results in the expansion of proliferative SOX9 positive cells and reduction of enterocytes and goblet cells. In a previous study, RNAseq analysis of laser micro-dissected ilea of infected pigs showed up-regulation of ADAMTS1 metallopeptidase which codes for surface proteins with adhesion and protease activity. The immune related marker ­unc-93 homolog (UNC93B1, necessary for toll-like receptors response) and the tetraspanin CD63, that participates in cell differentiation, were downregulated. We hypothesize that LI regulates cell proliferation and differentiation through ADAMTS1 and CD63 genes and avoids immune response through UNC93B1. In the current study, swine enteroids were used to model LI infection. We analyzed gene expression of MUC2 (marker for goblet cells), SOX9, ADAMTS1, CD63 and UNC93B1 in swine enteroids infected with LI for 3, 5, 7, 14 and 21 days. Published studies showed that expression of MUC2 and SOX9 in infected pigs decreased on day 7 and was followed by a decreased SOX9 expression at days 14 and 21. We observed no changes in the expression of MUC2 in infected enteroids. SOX9 expression in infected compared with uninfected enteroids at the same time points reached a maximum of 44% increase on day 7 followed by a decrease in expression by 68% and 33% on days 14 and 21 respectively. Both infected enteroids (fold change: 6.63) and tissue samples (fold change: 1.4) from infected pigs showed increased expression of ADAMTS1 on day 5. Expression of UNC93B1 in infected tissues increased from days 5 (19%) to 15 (43%) while no change was observed on day 5 and increased expression was observed on day 7 (fold change: 2.98) in enteroids. CD63 expression in the tissues increased from day 5 (45%) to 15 (22%) while increase in expression on day 5 (fold change: 1.23) and day 7 (fold change: 3.28) were also observed in enteroids. Overall, swine enteroids replicated most of the changes observed in pigs for MUC2, SOX9, ADAMTS1 and UNC93B1, suggesting infected swine enteroids are a viable model to study PE-associated changes. A probable explanation for the differences in response of enteroids to LI infection in MUC2 and SOX9 expression can be the permissiveness of the culture system for infection since there is no bacterial competition, passage through the gastric environment, or anti-microbial products present in the in vitro system.

The bacterial biocontrol agent Paenibacillus alvei K165 confers inherited resistance to Verticillium dahliae.

The biocontrol agent Paenibacillus alvei K165 was previously shown to protect Arabidopsis thaliana plants against Verticillium dahliae. Here we show that K165 also confers inherited immune resistance to V. dahliae. By performing a histone acetyltransferases mutant screen, ChIP assays, and transcriptomic experiments, we were able to show that histone acetylation significantly contributes to the K165 biocontrol activity and establishment of inheritable resistance to V. dahliae. K165 treatment primed the expression of immune-related marker genes and the cinnamyl alcohol dehydrogenase gene CAD3 through the function of histone acetyltransferases. Our results reveal that offspring of plants treated with K165 have primed immunity and enhanced lignification, both contributing towards the K165-mediated inherited immune resistance. Thus, our study paves the way for the use of biocontrol agents for the establishment of inheritable resistance to agronomically important pathogens.

Post-Translational Protein Deimination Signatures in Plasma and Plasma EVs of Reindeer (Rangifer tarandus).

Simple SummaryReindeer are an important wild and domesticated species of the Arctic, Northern Europe, Siberia and North America. As reindeer have developed various strategies to adapt to extreme environments, this makes them an interesting species for studies into diversity of immune and metabolic functions in the animal kingdom. Importantly, while reindeer carry natural infections caused by viruses (including coronaviruses), bacteria and parasites, they can also act as carriers for transmitting such diseases to other animals and humans, so called zoonosis. Reindeer are also affected by chronic wasting disease, a neuronal disease caused by prions, similar to scrapie in sheep, mad cows disease in cattle and Creutzfeldt-Jakob disease in humans. The current study assessed a specific protein modification called deimination/citrullination, which can change how proteins function and allow them to take on different roles in health and disease processes. Profiling of deiminated proteins in reindeer showed that many important pathways for immune defenses, prion diseases and metabolism are enriched in deiminated proteins, both in plasma, as well as in plasma extracellular vesicles. This study provides a platform for the development of novel biomarkers to assess wild life health status and factors relating to zoonotic disease.The reindeer (caribou) Rangifer tarandus is a Cervidae in the order Artiodactyla. Reindeer are sedentary and migratory populations with circumpolar distribution in the Arctic, Northern Europe, Siberia and North America. Reindeer are an important wild and domesticated species, and have developed various adaptive strategies to extreme environments. Importantly, deer have also been identified to be putative zoonotic carriers, including for parasites, prions and coronavirus. Therefore, novel insights into immune-related markers are of considerable interest. Peptidylarginine deiminases (PADs) are a phylogenetically conserved enzyme family which causes post-translational protein deimination by converting arginine into citrulline in target proteins. This affects protein function in health and disease. Extracellular vesicles (EVs) participate in cellular communication, in physiological and pathological processes, via transfer of cargo material, and their release is partly regulated by PADs. This study assessed deiminated protein and EV profile signatures in plasma from sixteen healthy wild female reindeer, collected in Iceland during screening for parasites and chronic wasting disease. Reindeer plasma EV profiles showed a poly-dispersed distribution from 30 to 400 nm and were positive for phylogenetically conserved EV-specific markers. Deiminated proteins were isolated from whole plasma and plasma EVs, identified by proteomic analysis and protein interaction networks assessed by KEGG and GO analysis. This revealed a large number of deimination-enriched pathways for immunity and metabolism, with some differences between whole plasma and EVs. While shared KEGG pathways for whole plasma and plasma EVs included complement and coagulation pathways, KEGG pathways specific for EVs were for protein digestion and absorption, platelet activation, amoebiasis, the AGE–RAGE signaling pathway in diabetic complications, ECM receptor interaction, the relaxin signaling pathway and the estrogen signaling pathway. KEGG pathways specific for whole plasma were pertussis, ferroptosis, SLE, thyroid hormone synthesis, phagosome, Staphylococcus aureus infection, vitamin digestion and absorption, and prion disease. Further differences were also found between molecular function and biological processes GO pathways when comparing functional STRING networks for deiminated proteins in EVs, compared with deiminated proteins in whole plasma. This study highlights deiminated proteins and EVs as candidate biomarkers for reindeer health and may provide information on regulation of immune pathways in physiological and pathological processes, including neurodegenerative (prion) disease and zoonosis.

Unlocking the Mechanisms of Cutaneous Adverse Drug Reactions: Activation of the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway by EGFR Inhibitors Triggers Keratinocyte Differentiation and Polarization of Epidermal Immune Responses

EGFR inhibitors used in oncology therapy modify the keratinocyte differentiation processes, impairing proper skin barrier formation and leading to cutaneous adverse drug reactions. To uncover the molecular signatures associated with cutaneous adverse drug reactions, we applied phosphoproteomic and transcriptomic assays on reconstructed human epidermis tissues exposed to a therapeutically relevant concentration of afatinib, a second-generation EGFR inhibitor. After drug exposure, we observed activation of the phosphatidylinositol 3-kinase/protein kinase B pathway associated with an increased expression of gene families involved in keratinocyte differentiation, senescence, oxidative stress, and alterations in the epidermal immune-related markers. Furthermore, our results show that afatinib may interfere with vitamin D3 metabolism, acting via CYP27A1 and CYP24A1 to regulate calcium concentration through the phosphatidylinositol 3-kinase/protein kinase B pathway. Consequently, basal layer keratinocytes switch from a pro-proliferating to a prodifferentiative program, characterized by upregulation of biomarkers associated with increased keratinization, cornification, T helper type 2 response, and decreased innate immunity. Such effects may increase skin susceptibility to cutaneous penetration of irritants and pathogens. Taken together, these findings demonstrate a molecular mechanism of EGFR inhibitor–induced cutaneous adverse drug reactions.

Prognostic value of eight immune gene signatures in pancreatic cancer patients

BackgroundPancreatic cancer is one of the most common malignant tumors of the digestive tract, and it has a poor prognosis. Traditional methods are not effective to accurately assess the prognosis of patients with pancreatic cancer. Immunotherapy is a new promising approach for the treatment of pancreatic cancer; however, some patients do not respond well to immunotherapy, which may be related to tumor microenvironment regulation. In this study, we use gene expression database to mine important immune genes and establish a prognostic prediction model for pancreatic cancer patients. We hope to provide a feasible method to evaluate the prognosis of pancreatic cancer and provide valuable targets for pancreatic cancer immunotherapy.ResultsWe used univariate COX proportional hazard regression analysis, the least absolute shrinkage and selection operator, and multivariate COX regression analysis to screen 8 genes related to prognosis from the 314 immune-related genes, and used them to construct a new clinical prediction model in the TCGA pancreatic cancer cohort. Subsequently, we evaluated the prognostic value of the model. The Kaplan–Meier cumulative curve showed that patients with low risk scores survived significantly longer than patients with high risk scores. The area under the ROC curve (AUC value) of the risk score was 0.755. The univariate COX analysis showed that the risk score was significantly related to overall survival (HR 1.406, 95% CI 1.237–1.598, P < 0.001), and multivariate analysis showed that the risk score was an independent prognostic factor (HR 1.400, 95% CI 1.287–1.522, P < 0.001). Correlation analysis found that immune genes are closely related to tumor immune microenvironment.ConclusionsBased on the TCGA-PAAD cohort, we identified immune-related markers with independent prognostic significance, validated, and analyzed their biological functions, to provide a feasible method for the prognosis of pancreatic cancer and provide potentially valuable targets for pancreatic cancer immunotherapy.

Dim Light at Night Impairs Daily Variation of Circulating Immune Cells and Renal Immune Homeostasis.

Dim light at night (dLAN) has become a pervasive part of the modern world, and growing evidence shows its association with increased health risks. Though this link is attributed to a disturbed circadian clock, the underlying mechanisms that can explain how circadian disruption from dLAN causes negative health effects remain unclear. Here, we exposed rats to a light–dark cycle (12:12 h) with low-intensity light at night (~2 lx) for 2 and 5 weeks and explored the steady-state pattern of circulating immune cells and renal immune-related markers, which are well controlled by the circadian clock. After 5 weeks, dLAN impaired the daily variation in several types of white blood cells, especially monocytes and T cells. Two-week dLAN caused a reduction in blood monocytes and altered gene expression of macrophage marker Cd68 and monocyte-attracting chemokine Ccl2 in the kidney. Interestingly, dLAN decreased renal 3-nitrotyrosine levels and resulted in up-regulation of the main endogenous antioxidant pathways, indicating a disturbance in the renal redox balance and an activation of compensatory mechanisms. These effects paralleled the altered renal expression of the molecular clock components and increased plasma corticosterone levels. Together, our results show that chronic exposure to dLAN weakened the circadian control of daily variation of circulating immune cells and disturbed renal immune and redox homeostasis. Consequences of this dLAN-disturbed immune balance on the ability of the immune system to cope with other challenges should by clarified in further studies.

Effect of Obstructive Sleep Apnea on Immunity in Cases of Chronic Rhinosinusitis With Nasal Polyps.

Objectives.Chronic rhinosinusitis with nasal polyps (CRSwNP) is a more severe inflammatory form of CRS that often coexists with obstructive sleep apnea (OSA). However, little is known about the relationship between OSA and the immune profile in patients with CRSwNP. We aimed to investigate the immune profile of patients with CRSwNP according to OSA severity.Methods.This study included 63 patients with CRSwNP and nine control subjects. Protein levels of inflammatory mediators were determined using multiplex immunoassays. All patients underwent standard polysomnography.Results.In patients with eosinophilic CRSwNP (ECRSwNP), interleukin (IL)-6 and chemokine [C-X-C motif] ligand (CXCL)-1 (type 1 immune-related markers) were upregulated in cases of moderate-to-severe OSA. Additionally, IL-4, IL-13, C-C motif chemokine (CCL)-11, CCL-24 (type 2 immune-related markers), and IL-17A (a type 3 immune-related marker) were present at elevated levels in patients with moderate-to-severe OSA. Although there were no significant differences in type 1, 2, or 3 immune-related markers among patients with non-eosinophilic CRSwNP (NECRSwNP) according to the severity of OSA, transforming growth factor-beta expression was higher in those with moderate-to-severe OSA. Furthermore, in ECRSwNP with moderate-to-severe OSA, associations were detected between serum markers and some upregulated inflammatory markers.Conclusion.OSA may increase the heterogeneity of the immune profile (types 1, 2, and 3) in patients with ECRSwNP, but not in those with NECRSwNP.

Effects of Rifaximin on Luminal and Wall-Adhered Gut Commensal Microbiota in Mice.

Rifaximin is a broad-spectrum antibiotic that ameliorates symptomatology in inflammatory/functional gastrointestinal disorders. We assessed changes in gut commensal microbiota (GCM) and Toll-like receptors (TLRs) associated to rifaximin treatment in mice. Adult C57BL/6NCrl mice were treated (7/14 days) with rifaximin (50/150 mg/mouse/day, PO). Luminal and wall-adhered ceco-colonic GCM were characterized by fluorescent in situ hybridization (FISH) and microbial profiles determined by terminal restriction fragment length polymorphism (T-RFLP). Colonic expression of TLR2/3/4/5/7 and immune-related markers was assessed (RT-qPCR). Regardless the period of treatment or the dose, rifaximin did not alter total bacterial counts or bacterial biodiversity. Only a modest increase in Bacteroides spp. (150 mg/1-week treatment) was detected. In control conditions, only Clostridium spp. and Bifidobacterium spp. were found attached to the colonic epithelium. Rifaximin showed a tendency to favour their adherence after a 1-week, but not 2-week, treatment period. Minor up-regulation in TLRs expression was observed. Only the 50 mg dose for 1-week led to a significant increase (by 3-fold) in TLR-4 expression. No changes in the expression of immune-related markers were observed. Rifaximin, although its antibacterial properties, induces minor changes in luminal and wall-adhered GCM in healthy mice. Moreover, no modulation of TLRs or local immune systems was observed. These findings, in normal conditions, do not rule out a modulatory role of rifaximin in inflammatory and or dysbiotic states of the gut.

Concordance of Immune-Related Markers in Lymphocytes and Prefrontal Cortex in Schizophrenia.

Schizophrenia is a severe neuropsychiatric disorder associated with a wide array of transcriptomic and neurobiochemical changes. Genome-wide transcriptomic profiling conducted in postmortem brain have provided novel insights into the pathophysiology of this disorder, and identified biological processes including immune/inflammatory-related responses, metabolic, endocrine, and synaptic function. However, few studies have investigated whether similar changes are present in peripheral tissue. Here, we used RNA-sequencing to characterize transcriptomic profiles of lymphocytes in 18 nonpsychotic controls and 19 individuals with schizophrenia. We identified 2819 differentially expressed transcripts (Pnominal < .05) in the schizophrenia group when compared to controls. Bioinformatic analyses conducted on a subset of 293 genes (Pnominal < .01 and |log2 FC| > 0.5) highlighted immune/inflammatory responses as key biological processes in our dataset. Differentially expressed genes in lymphocytes were highly enriched in gene expression profiles associated with cortex layer 5a and immune cells. Thus, we investigated whether the changes in transcripts levels observed in lymphocytes could also be detected in the prefrontal cortex (PFC, BA10) in a second replication cohort of schizophrenia subjects. Remarkably, mRNA levels detected in the PFC and lymphocytes were in strong agreement, and measurements obtained using RNA-sequencing positively correlated with data obtained by reverse transcriptase-quantitative polymerase chain reaction analysis. Collectively, our work supports a role for immune dysfunction in the pathogenesis of schizophrenia and suggests that peripheral markers can be used as accessible surrogates to investigate putative central nervous system disruptions.

Immune profiling and immunotherapeutic targets in pancreatic cancer.

BackgroundImmunotherapeutic approaches for pancreatic ductal adenocarcinoma (PDAC) are less successful as compared to many other tumor types. In this study, comprehensive immune profiling was performed in order to identify novel, potentially actionable targets for immunotherapy.MethodsFormalin-fixed paraffin embedded (FFPE) specimens from 68 patients were evaluated for expression of 395 immune-related markers (RNA-seq), mutational burden by complete exon sequencing of 409 genes, PD-L1 expression by immunohistochemistry (IHC), pattern of tumor infiltrating lymphocytes (TILs) infiltration by CD8 IHC, and PD-L1/L2 copy number by fluorescent in situ hybridization (FISH).ResultsThe seven classes of actionable genes capturing myeloid immunosuppression, metabolic immunosuppression, alternative checkpoint blockade, CTLA-4 immune checkpoint, immune infiltrate, and programmed cell death 1 (PD-1) axis immune checkpoint, discerned 5 unique clinically relevant immunosuppression expression profiles (from most to least common): (I) combined myeloid and metabolic immunosuppression [affecting 25 of 68 patients (36.8%)], (II) multiple immunosuppressive mechanisms (29.4%), (III) PD-L1 positive (20.6%), (IV) highly inflamed PD-L1 negative (10.3%); and (V) immune desert (2.9%). The Wilcoxon rank-sum test was used to compare the PDAC cohort with a comparison cohort (n=1,416 patients) for the mean expressions of the 409 genes evaluated. Multiple genes including TIM3, VISTA, CCL2, CCR2, TGFB1, CD73, and CD39 had significantly higher mean expression versus the comparison cohort, while three genes (LAG3, GITR, CD38) had significantly lower mean expression.ConclusionsThis study demonstrates that a clinically relevant unique profile of immune markers can be identified in PDAC and be used as a roadmap for personalized immunotherapeutic decision-making strategies.

CD44, a marker of cancer stem cells, is positively correlated with PD-L1 expression and immune cells infiltration in lung adenocarcinoma

BackgroundPD-L1 inhibitors is widely applied in lung adenocarcinoma patients. Tumor cells with high PD-L1 expression could trigger immune evasion. Cancer stem cells (CSCs) can evade from immunesurveillance due to their immunomodulating effects. However, the correlation between CSC and PD-L1 and some immune-related markers is seldom reported in patients with lung adenocarcinoma. Therefore, we aimed to ascertain their association in lung adenocarcinoma patients.MethodsWe assessed CD44 expression and its association with PD-L1 in lung adenocarcinoma, using Tumor Immune Estimation Resource (TIMER), which was further validated in our patient cohort. The immune cells infiltration was depicted by CIBERSORT using GEO database. The correlation between CD44 and immune cells was also analyzed. We further evaluated the prognostic role of CD44 in patients with lung adenocarcinoma both using Kaplan–Meier plotter and validated in our patient cohort.ResultsPositive association between CD44 and PD-L1 were found in lung adenocarcinoma patients. T cells CD4 memory resting cells and mast cells resting cells varied significantly between patients with CD44 high and those with CD44 low. Furthermore, positive association could be found between CD44 expression and immune cells. Arm-level depletion of CD44 was linked with B cell, CD4+ T cell, neutrophil and dendritic cell infiltration. Patients with higher CD44 levels had worsened overall survival (OS).ConclusionsIn summary, these results demonstrate that CD44 was associated with PD-L1 and infiltration of immune cells, and was a negative prognostic factor for predicting worsened OS in lung adenocarcinoma.

Comprehensive analysis of prognostic gene signatures based on immune infiltration of ovarian cancer

BackgroundOvarian cancer (OV) is one of the most common malignant tumors of gynecology oncology. The lack of effective early diagnosis methods and treatment strategies result in a low five-year survival rate. Also, immunotherapy plays an important auxiliary role in the treatment of advanced OV patient, so it is of great significance to find out effective immune-related tumor markers for the diagnosis and treatment of OV.MethodsBased on the consensus clustering analysis of single-sample gene set enrichment analysis (ssGSEA) score transformed via The Cancer Genome Atlas (TCGA) mRNA profile, we obtained two groups with high and low levels of immune infiltration. Multiple machine learning methods were conducted to explore prognostic genes associated with immune infiltration. Simultaneously, the correlation between the expression of mark genes and immune cells components was explored.ResultsA prognostic classifier including 5 genes (CXCL11, S1PR4, TNFRSF17, FPR1 and DHRS95) was established and its robust efficacy for predicting overall survival was validated via 1129 OV samples. Some significant variations of copy number on gene loci were found between two risk groups and it showed that patients with fine chemosensitivity has lower risk score than patient with poor chemosensitivity (P = 0.013). The high and low-risk groups showed significantly different distribution (P < 0.001) of five immune cells (Monocytes, Macrophages M1, Macrophages M2, T cells CD4 menory and T cells CD8).ConclusionThe present study identified five prognostic genes associated with immune infiltration of OV, which may provide some potential clinical implications for OV treatment.

Comprehensive genomic analysis of microenvironment phenotypes in ovarian cancer.

BackgroundOvarian cancer is one of the leading causes of cancer-related death in women. The incidence of ovarian cancer is insidious, and the recurrence rate is high. The survival rate of ovarian cancer has not significantly improved over the past decade. Recently, immune checkpoint inhibitors such as those targeting CTLA-4, PD-1, or PD-L1 have been used to treat ovarian cancer. Therefore, a full analysis of the immune biomarkers associated with this malignancy is necessary.MethodsIn this study, we used data from The Cancer Genome Atlas (TCGA) database to analyze the infiltration patterns of specific immune cell types in tumor samples. Data from the Gene Expression Omnibus (GEO) database was used for external validation. According to the invasion patterns of immune cells, we divided the ovarian cancer microenvironment into two clusters: A and B. These tumor microenvironment (TME) subtypes were associated with genomic and clinicopathological characteristics. Subsequently, a random forest classification model was established. Differential genomic features, functional enrichment, and DNA methylation were analyzed between the two clusters. The characteristics of immune cell infiltration and the expression of immune-related cytokines or markers were analyzed. Somatic mutation analysis was also performed between clusters A and B. Finally, multivariate Cox analysis was used to analyze independent prognostic factors.ResultsThe ovarian cancer TME cluster A was characterized by less infiltration of immune cells and sparse distribution and low expression of immunomodulators. In contrast, cytotoxic T cells and immunosuppressive cells were significantly increased in the ovarian cancer TME cluster B. Additionally, immune-related cytokines or markers, including IFN-γ and TNF-β, were also expressed in large quantities. In total, 35 differentially methylated and expressed genes (DMEGs) were identified. Functional enrichment analyses revealed that the DMEGs in cluster B participated in important biological processes and immune-related pathways. The mutation load in cluster B was insignificantly higher than that of cluster A (p = 0.076). Multivariate Cox analysis showed that TME was an independent prognostic factor for ovarian cancer (hazard ratio: 1.33, 95% confidence interval: 1.01–1.75, p = 0.041).ConclusionThis study described and classified basic information about the immune invasion pattern of ovarian cancer and integrated biomarkers related to different immunophenotypes to reveal interactions between ovarian cancer and the immune system.

Using Machine Learning Modeling to Explore New Immune-Related Prognostic Markers in Non-Small Cell Lung Cancer.

ObjectiveTo find new immune-related prognostic markers for non-small cell lung cancer (NSCLC).MethodsWe found GSE14814 is related to NSCLC in GEO database. The non-small cell lung cancer observation (NSCLC-OBS) group was evaluated for immunity and divided into high and low groups for differential gene screening according to the score of immune evaluation. A single factor COX regression analysis was performed to select the genes related to prognosis. A prognostic model was constructed by machine learning, and test whether the model has a test efficacy for prognosis. A chip-in-chip non-small cell lung cancer chemotherapy (NSCLC-ACT) sample was used as a validation dataset for the same validation and prognostic analysis of the model. The coexpression genes of hub genes were obtained by pearson analysis and gene enrichment, function enrichment and protein interaction analysis. The tumor samples of patients with different clinical stages were detected by immunohistochemistry and the expression difference of prognostic genes in tumor tissues of patients with different stages was compared.ResultsBy screening, we found that LYN, C3, COPG2IT1, HLA.DQA1, and TNFRSF17 is closely related to prognosis. After machine learning, we constructed the immune prognosis model from these 5 genes, and the model AUC values were greater than 0.9 at three time periods of 1, 3, and 5 years; the total survival period of the low-risk group was significantly better than that of the high-risk group. The results of prognosis analysis in ACT samples were consistent with OBS groups. The coexpression genes are mainly involved B cell receptor signaling pathway and are mainly enriched in apoptotic cell clearance. Prognostic key genes are highly correlated with PDCD1, PDCD1LG2, LAG3, and CTLA4 immune checkpoints. The immunohistochemical results showed that the expression of COPG2IT1 and HLA.DQA1 in stage III increased significantly and the expression of LYN, C3, and TNFRSF17 in stage III decreased significantly compared with that of stage I. The experimental results are consistent with the previous analysis.ConclusionLYN, C3, COPG2IT1, LA.DQA1, and NFRSF17 may be new immune markers to judge the prognosis of patients with non-small cell lung cancer.

Abstract B10: High-dimensional (30-plex) imaging mass cytometry on tissue microarray identifies novel PD-L1-inclusive immunophenotypes associated with overall survival in stage III melanoma

Abstract We sought to determine the subcellular distribution of 30+ immune-related markers including PD-L1 in stage III melanoma tumor tissues (nuclear, cytoplasmic, surface) using novel imaging mass cytometry technology that enables simultaneous detection of over 30 immune and tumor markers. Metal-tagged antibodies were used to stain stage III melanoma tissues similarly to an IHC-based protocol, followed by laser ablation and mass cytometry using the Helios® and Hyperion® technology (Fluidigm). Markers for immune subsets included CD45, CD3, CD4, CD8, PD-1, Granzyme B, FoxP3, CD11b, CD11c, CD107, CD20, Vista, and CD68. Markers for tumor cell phenotyping included PD-L1 (intracellular and extracellular domain specific), E-cadherin, B-catenin, PD-L2, and keratin 8/18. Structural, inflammatory, survival, and signaling markers included Ki67, p53, p-tyrosine, Bcl-2, Cl-caspase 3, histone H3, collagen-1, actin, and arginase-1. We further validated subcellular distribution of PD-L1 in the same patient tumor cores using fluorochrome-labeled antibodies and 100X confocal microscopy to show difference in surface-only, cytoplasmic-only, and nuclear PD-L1. We then assessed the association between subcellular PD-L1 and immune populations in the tumor microenvironment, along with overall and recurrence-free survival. We wrote custom algorithms for data processing to identify novel immune subsets, ran SPADE and viSNE analyses for cross-validation, and employed Imaris Bitplane software to validate our own algorithms for immune population identification. Overall, we found that intracellular-only PD-L1 in tumor tissues has the highest correlation (Pearson r) to CD11b, FoxP3, PD-1, arginase, Ki67, PD-L2 and nuclear stain iridium, while extracellular (surface) PD-L1 in tumors has the highest correlation (Pearson r) to FoxP3, PD-1, arginase, Ki67, PD-L2 and Vista. We identified two novel immunophenotypes (histone-3+/p-tyrosinehi /Vistalo / PD-L1-ICDlo / PD-L1-ECDlo and CD11bhi / PD-L1-ICDhi / PD-L1-ECDhi) that are positively associated with overall patient survival. Of note, only phospho-Tyrhi was significantly associated with RFS. This work highlights the use of cutting-edge technology that can employ the highest multiplexing available for assessing novel interactions of 30+ tumor tissue markers for identification of novel immunophenotypes and biomarker in stage III melanoma patients. Our novel study showcases a novel methodologic approach to high-throughput analysis of retrospective profiling of the melanoma tumor immune microenvironment on a single IHC slide, followed by appropriate and rigorous cross-validation using Fluidigm’s novel CyTOF imaging technology. Our study provides a groundwork for this powerful, novel technology in translational and clinical research for biomarker identification and identification of novel combinatorial markers that may predict overall and recurrence-free survival in stage III melanoma. Citation Format: Heather G. Hambright, Anand V.R. Kornepati, Dai Ogata, Roland R.L. Bassett, Suhendan Ekmekcioglu, Elizabeth A. Grimm, Tyler J. Curiel. High-dimensional (30-plex) imaging mass cytometry on tissue microarray identifies novel PD-L1-inclusive immunophenotypes associated with overall survival in stage III melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr B10.

Role of LATS1/2 in Prognosis of Advanced Gastric Cancer and Its Relationship With the Tumor Immune Microenvironment.

Background: Gastric cancer (GC) remains a refractory cancer particularly in Eastern Asia. Large tumor suppressor kinases 1/2 (LATS1/2) are core members of the Hippo pathway. The role of LATS1/2 in the prognosis of different subtypes of advanced gastric cancer and its relationship with the tumor immune microenvironment in GC remain unknown. Exploring the role of LATS1/2 in GC might provide potential immunotherapeutic approaches for treating GC.Methods: Four hundred and ninety surgically resected primary GC samples were assessed for LATS1/2, CD8, FOXP3, and CD163. Correlations between LATS1/2 expression and immune-related markers were investigated and the prognoses of patients with different GC subtypes were analyzed.Results: CD8 and CD163 appeared to be favorable and adverse prognostic factors, respectively. LATS1/2 and FOXP3 did not predict patients' overall survival. However, in microsatellite-stable GC patients, high LATS1/2 and FOXP3 expression and low CD8 expression predicted poor prognoses. Furthermore, high LATS1/2 expression was significantly correlated with decreased CD8 and increased FOXP3. Combined analysis of LATS1/2, CD8, and FOXP3 had better prognostic accuracy than did each marker individually.Conclusions: Different biological molecules can predict the prognoses of different types of GC patients. LATS1/2, core kinases in the Hippo pathway, are closely related to CD8 and FOXP3. Further understanding the mechanisms of LATS1/2 in CD8+ T cells and FOXP3+ Treg cells provides further theoretical basis and potential targets for GC immunotherapy.

Abstract 2663: Identification of differential immune landscapes in high-grade serous ovarian tumors of high-risk patients by imaging mass cytometry

Abstract The immune environment of ovarian cancer has previously been linked to clinical outcomes. In particular, BRCA mutated tumors have been associated with the presence of intratumoral T cells, higher mutational load, and improved survival. To better assess the immune environment of such tumors, we used imaging mass cytometry (IMC), a novel imaging platform that allows for high-multiplex biomarker detection and simultaneous spatial evaluation, to generate and compare immune landscapes in advanced stage high-grade serous ovarian tumors (HGSC) with BRCA1 or 2 mutations against wild-type BRCA1 and 2. A total of 40 advanced stage HGSC tumors were collected under an IRB-approved protocol, 20 patients with known BRCA1 or 2 mutations and 20 wild-type patients. Formalin fixed paraffin embedded (FFPE) tissue sections of 10µm thickness were stained with 34 metal-tagged antibodies to detect various cell specific and immune related markers using the Fluidigm protocol. IMC data was obtained by the Fluidigm Helios CyTOF instrument utilizing the Hyperion Imaging System laser ablation module. Images from a 1mm2 area of each tissue section were processed and converted to tiff files using MCD Viewer (Fluidigm). Tumor tissue was identified by positive pan-Keratin and cytokeratin 8/18 activity. Stroma was noted by positive smooth muscle actin activity. Images were then analyzed for various immune marker activity including CD8, CD4, Granzyme B, CD20, CD68, PDL1, and FoxP3. Statistical analysis was performed using unpaired t-test. The results demonstrated that HGSCs with BRCA1 mutations had significantly higher number of intratumoral CD8+ cells (32 v. 7 cells/mm2, p=0.02) compared to BRCA2 mutations. A markedly higher number of intratumoral CD20+ cells and CD4+ cells were also observed in HGSCs with BRCA1 than BRCA2 mutations; although, the difference did not reach significance (6.7 v. 1.3 cells/mm2, p=0.09; 93 v. 28 cells/mm2, p=0.08). Compared to wildtype, BRCA1/2 mutated tumors had significantly higher number of CD20+ cells in the tumor aspect and in the whole tissue (4 v. 0.2 cells/mm2, p=0.03; 9 v. 1 cells/mm2, p=0.05). Furthermore, BRCA1/2 mutated tumors also have a higher number of stromal and total CD4+ than wildtype patients (28 v. 14 cells/mm2, p=0.07; 63 v 24 cells/mm2, p=0.7). In this cohort, we found that HGSC tumors with BRCA1 mutations had increased immune cells compared to BRCA2 mutations, including intratumoral CD8+, CD20+, and CD4+ cells. BRCA mutant tumors also had increased CD20+ and CD4+ cells compared to BRCA wildtype. These immune phenotypes suggest an increased immunogenicity in tumors with BRCA mutations, particularly BRCA1, consistent with previous studies. This may have clinical implications; but, furthermore, may suggest novel approaches to therapy. Overall, this study shows that IMC provides a powerful and comprehensive approach to evaluating the cellular phenotype and spatial interrelationships of the tumor immune environment. Citation Format: Han Cun, Sammy Ferri-Borgogno, Rita Cheng, Jae-Hoon Kim, Gwan Hee Han, Karen H. Lu, Samuel C. Mok. Identification of differential immune landscapes in high-grade serous ovarian tumors of high-risk patients by imaging mass cytometry [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 2663.

Abstract 3267: Identification of a novel biomarker response in a prospective clinical trial of immune checkpoint blockade in recurrent ovarian carcinoma

Abstract The role of immune checkpoint inhibitors (ICPIs) in ovarian cancer continues to be refined. The presence of intratumoral T cells in ovarian tumors correlates with improved survival and progression, suggesting that immune modulation, such as by ICPIs, may be beneficial. However, a better understanding of the immune effects following ICPI use, particularly in those with a long progression free survival, is needed. Thus, we used imaging mass cytometry (IMC) to compare the tumor immune landscape of patients with ovarian cancer before and after a CTLA4 immune checkpoint inhibitor treatment to determine the association between alterations in immune landscapes and progression free survival. Fine needle biopsies were obtained from 8 patients with recurrent platinum-resistant ovarian carcinoma enrolled in a phase 2 randomized trial evaluating the efficacy of Tremelimumab and Durvalumab (CTLA4 and PDL1 checkpoint inhibitors, respectively) in recurrent ovarian cancer treatment. Each patient had a pre-treatment biopsy and on-treatment biopsy after 3 cycles of CTLA4 inhibitor therapy. Progression free time (PFS) was recorded with 3 patients with a long PFS (&amp;gt;180 days) and 5 with short PFS (&amp;lt;60 days). Formalin fixed paraffin embedded (FFPE) tissue sections were stained for IMC analysis via Fluidigm protocol with 34 metal-tagged antibodies to detect various cell and immune related markers. The IMC data was obtained using the Fluidigm Helios CyTOF instrument and Hyperion Imaging System laser ablation module. A novel image informatics pipeline through Matlab was used to assess the image intensity of each marker and cell location. Using the developed image informatics pipeline, cell types and locations were analyzed for 16 samples from 8 patients. In all patients, mean CD8+ T cell densities had an increased trend in on-treatment vs pre-treatment samples (205.6 v 129.9 cells/mm2, p=0.086). An analysis of cell location found that in all patients on-treatment, the mean number of CD8+ T cells adjacent to M2 macrophages increased significantly (0.09 v. 0.17 cells, p = 0.0460) as well as the mean number of CD8+ T cells adjacent to B7H4+ tumor cells (0.035 v. 0.01 cells, p = 0.046). Furthermore, patients with a long PFS had a significantly higher number of CD8+ cells neighboring B cells on-treatment than short PFS (0.12 v. 0.27 cells, p=0.028). Using IMC and novel image informatics pipeline, we found that patients with recurrent ovarian cancer treated with Tremelimumab with a long PFS mounted a significant immune response compared to patients with a short PFS, with a higher number of CD8+ T cells neighboring B cells after treatment. For all patients, the immune landscape altered after treatment. To our knowledge, this is the first known use of IMC to demonstrate immune response in ovarian cancer following ICPI therapy. Overall, IMC and image informatics pipeline are robust tools that simultaneously analyze multiple biomarkers and spatial location of cells to better assess the tumor immune microenvironment and cellular interactions. Citation Format: Han Cun, Emily M. Hinchcliff, Ying Zhu, Sammy Ferri-Borgogno, Rita Cheng, Jared K. Burks, Stephen T. Wong, Amir A. Jazaeri, Samuel C. Mok. Identification of a novel biomarker response in a prospective clinical trial of immune checkpoint blockade in recurrent ovarian carcinoma [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 3267.

Expression Profiling of Primary and Recurrent Glioblastomas Reveals a Reduced Level of Pentraxin 3 in Recurrent Glioblastomas.

Glioblastomas (GBM) are highly infiltrative tumors and despite intensive treatment tumor recurrence is inevitable. The immune microenvironment in recurrent GBM is poorly characterized, but it is potentially influenced by therapeutic interventions with surgery, radiotherapy, and chemotherapy. The aim of this study was to obtain a deeper insight in the immune microenvironment in primary and recurrent GBM. Primary and recurrent glioblastoma samples from 18 patients were identified and expression profiling of 770 myeloid innate immune-related markers was performed. Leukemia inhibitory factor and pentraxin 3 were expressed at lower levels in recurrent tumors. Using in silico data and immunohistochemical staining, this was validated for pentraxin 3. Both high leukemia inhibitory factor and pentraxin 3 expression appeared to be associated with shorter survival in primary and recurrent GBM using in silico data. In primary GBM, gene set analysis also showed higher expression of genes involved in metabolism, extracellular matrix remodeling and complement activation, whereas genes involved in T cell activation and checkpoint signaling were expressed at higher levels in recurrent GBM. The reduced level of pentraxin 3 in recurrent glioblastomas and the gene set analysis results suggest an altered microenvironment in recurrent GBM that might be more active.

Development and Validation of a Five-immune Gene Pair Signature in Endometrial Carcinoma.

Endometrial cancer (EC) is a common gynecological malignancy worldwide. Immunity is closely related to the occurrence and prognosis of EC. At the same time, immune-related genes have great potential as prognostic markers in many types of cancer. Therefore, we attempt to develop immune-related gene markers to enhance prognosis prediction of EC. 542 samples of EC gene expression data and clinical follow-up information were downloaded from The Cancer Genome Atlas (TCGA). The samples were randomly divided into two groups, one group as a training set (N=271), and one set as a validation set. (N=271). In the training set, the gene pairs were established based on the relative expression levels of 271 immune genes, and the prognosis-related gene pairs were screened. The lasso was used to select the features, and finally, the robust biomarkers were screened. Finally, the prognostic model of the immune gene pair was established and verified by the validation data set. 10030 immune gene pair (IRGPs) were obtained, and univariate survival analysis was used to identify 1809 prognostic-related IRGPs (p<0.05). 5-IRGPs were obtained by lasso regression feature selection, and multivariate regression was used to establish 5-IRGPs signature, 5-IRGPs signature is an independent prognostic factor for EC patients, and could be risk stratified in patients with TCGA datasets, age, ethnicity, stage, and histological classification (p<0.05). The mean AUC of survival in both the training set and the validation set was greater than 0.7, indicating that 5-IRGPs signature has superior classification performance in patients with EC. In addition, 5-IRGPs have the highest average C index (0.795) compared to the prognostic characteristics of the three endometrial cancers reported in the past and Stage and Age. This study constructed a 5-IRGPs signature as a novel prognostic marker for predicting survival in patients with EC.