Immune Panel Research Articles

Abstract 1235: Presence of TLS and combined high densities of PD-L1+ macrophages & CD8+ T cells predict long-term overall survival for patients with advanced NSCLC treated with durvalumab

Abstract Introduction: Predictive biomarkers of anti‒PD-(L)1 therapies have largely focused on the tumor - T cell axis where tumor cell PD-L1 expression has demonstrated its clinical utility in predicting overall survival (OS) in patients with advanced non-small cell lung cancer (NSCLC). Although, other immune cell subsets were shown to be associated with clinical efficacy, their relative impact and combined effect in predicting improved long-term survival warrant further investigation. Using computational image analysis of multiplex immunofluorescence (mIF) and immunohistochemistry (IHC) immune marker panels, we sought to identify single and combined biomarkers of the tumor immune contexture in association with long-term OS in advanced NSCLC patients treated with Durvalumab. Methods: Pre-treatment tumor samples from advanced NSCLC patients (n = 210) enrolled in durvalumab nonrandomized phase 1/2 trial (10 mg/kg Q2W, CP1108/NCT01693562), were stained using IHC and 6-marker mIF panels to detect markers of immune cells, cell functional state and tertiary lymphoid structure (TLS) (PD-L1, CD8, PD-1, Ki67, CD68, CD20, CD1c, NKp46, CD66b, ICOS, FOXP3). Cell marker density (cells/mm2), distribution and proximity were quantified and analyzed in association with overall survival. Results: Computational image analysis of the tumor immune contexture revealed a greater immune inflamed phenotype, both innate (macrophages, dendritic cells) and adaptive (T and B cells), in NSCLC patients with long OS >2 years compared to those with short OS < 1 year (fold change > 2, p < 0.0001). Patient subgroup with high density of individual immune subsets show a median OS (mOS) of 10-20 months (p < 0.01 high vs. low subgroups) while combined markers of innate and adaptive immune cells show an improved mOS > 2 years (p < 0.001). Specifically, among the key findings, combined biomarkers of CD68+ PD-L1+ macrophages and CD8+ T cells predicts for a significant increase in OS for patients with high vs low marker density (HR = 0.21, 95%CI 0.12 - 0.39, p <10-7; mOS 39.5 months [high], 6.5 months [low]). Whereas, in patients with high density of either single biomarkers CD68+ PD-L1+ macrophage or CD8+ T cells mOS is 20.2 or 18.4 months respectively. Moreover, high density of CD20+ B cells, reflective of presence of TLS, associates with improved OS (mOS NR [high], 11 months [low], p = 0.003). In addition, TLS enriched tumors show an increased level of macrophages expressing PD-L1 in synapsis with CD8+ T cells (activated PD1+ or proliferative Ki67+ T cells). Conclusion: These findings demonstrate the importance of both tertiary lymphoid structure and high pre-existing innate-adaptive immunity in driving long-term overall survival of durvalumab-treated patients with NSCLC and highlight the need for the development of multiparametric predictive biomarkers beyond tumor-T cell axis. Citation Format: Lina Meinecke, Jorge Blando, Thomas Herz, Michael Surace, Thomas Padel, Monica Azqueta Gavaldon, Harald Hessel, Farzad Sekhavati, Megha Saraiya, Anmarie Boutrin, Karma Da Costa, Jaime Rodriguez Canales, Ashok Gupta, Carl Barrett, Zachary Aaron Cooper, Ikbel Achour. Presence of TLS and combined high densities of PD-L1+ macrophages & CD8+ T cells predict long-term overall survival for patients with advanced NSCLC treated with durvalumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1235.

Abstract 4177: Th17 inhibition with interleukin 6 blockade decouples immunotoxicity from tumor immunity

Abstract Durable remission rates with immune checkpoint inhibitor (ICI) monotherapy remain low. Combination ICI therapies could overcome immune resistance but are associated with higher rates of immune-related adverse events (irAEs). To elucidate the underlying irAE immunobiology, we profiled human intestinal tissue, enterocolitis (irEC), and tumor tissue from ICI-treated patients using NanoString PanCancer Immune Panel (NanoPCIP) and multiplex immunohistochemistry (mIHC); hypotheses generated from these translational results for potential irAE treatments were tested in parallel preclinical mouse model studies. In 23 patients identified with both normal intestinal and irEC tissue, we found genes encoding interleukin-6 (IL-6), Th17 differentiating cytokine, and neutrophil and monocyte chemotactic molecules were the highest significantly upregulated in irEC compared to patient-matched normal intestine. Using NanoPCIP cell scoring (23 patients) and mIHC (27 patients), Th17 cells were significantly higher in irEC than Th1 cells. T-cell subsets were similar between anti-CTLA-4 based regimen versus anti-PD-1monotherapy induced irEC, except Th17 memory cells were significantly higher CTLA-4i-induced irEC. We compared the degree of differential gene expression in irEC (normal intestine versus irEC) to the differential expression in tumors (baseline versus on-treatment tumors). The genes highest upregulated in irEC (including Th17 differentiating cytokines IL-6 and IL-1B) were not upregulated in responding tumors from patients receiving ICI. NanoPCIP Th17 cell score was significantly more upregulated than Th1 cells and neutrophils more upregulated than CD8 T cells in the irEC analysis, but not in the responding tumor analysis where IL-12 and CD8 cell score was significantly upregualted . Based on these discoveries, we used C57BL/6 and Balb/c mouse strains to address whether the antitumor effect of ICIs can be enhanced by IL-6 blockade while also improving irAEs. We found that IL-6 blockade + ICI was associated with improved tumor control and a higher density of CD4/CD8 effector T-cells, with reduced Th17, macrophages, and MDSCs compared to ICI alone. Furthermore, in the experimental autoimmune encephalomyelitis (EAE) mouse model, the pathogenesis of which is well documented to be Th17 mediated (Robinson et al., 2014), we demonstrated that combined IL-6 blockade and ICI enhanced tumor rejection while simultaneously mitigating EAE symptoms versus ICI alone. Based on the totality of the basic science, translational, and clinical data we hypothesize that IL-6 blockade combined with ICIs could de-couple autoimmunity from antitumor immunity. We are currently conducting a prospective phase 2 trials of combination ipilimumab (3mg/kg) + Nivolumab (1mg/kg) + Tocilizumab in patients with metastatic melanoma, EGFR mutant NSCLC, and urothelial carcinoma ( NCT04940299). Citation Format: Daniel H. Johnson, Yared Hailemichael, Salah-Eddine Bentebibel, Noha Abdel-Wahab, Sungnam Cho, Wai C. Foo, Khalida Wani, Stephanie S. Watowich, Suhendan Ekmekcioglu, Adi Diab. Th17 inhibition with interleukin 6 blockade decouples immunotoxicity from tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4177.

Abstract 3801: Comprehensive characterization of the biology of inflammatory bowel disease-associated colorectal cancer using the GeoMx® Digital Spatial Profiler (DSP)

Abstract Inflammatory bowel diseases (IBD) ulcerative colitis (UC) and Crohn's disease (CD) significantly increases the risk for development of colorectal cancer (CRC). However, the exact molecular mechanisms that underlie the transition from inflammation to cancer are not fully understood, and a systematic characterization of the immunopathogenesis of IBD-associated CRC is lacking. To address this issue, we will perform a comprehensive and spatial tissue analysis using the Cancer Transcriptome Atlas (CTA) on the NanoString GeoMx® Digital Spatial Profiler (DSP). The CTA panel is designed to profile the global immune response and all aspects of tumor microenvironment biology, including the 18-gene Tumor Inflammation Signature (TIS). For DSP analysis, a total of 20 FFPE samples (5 colon cancer patients, 5 UC patients, 5 CD patients, and 5 matched normal samples will be spatially profiled for up to 1,800 genes included on the CTA. Selection of regions of interest (ROI) will be guided by H&E staining and fluorescent markers (CD45, PanCK, Syto13), and profiling of tumor and TME regions will be achieved through segmenting by PanCK+/PanCK- followed by collection of indexed oligonucleotides and sequenced on NextSeq 550 Illumina instrument. NGS reads will be processed into digital counts, normalized and mapped back to each ROI, generating a map of transcript activity within each ROI. For all samples crypt and villus regions will be selected for a detailed spatial analysis. Subsequently, using the proprietary multiplex immunofluorescence (mIF) platform MultiOmyx™ we will characterize the protein immune cell landscape in IBD-associated CRC, using a custom immune panel applied to adjacent slides. With this study we hope to highlight genes and pathways involved in the transition from inflammation to cancer by combining large-scale spatial gene profiling by DSP CTA with a comprehensive spatial protein profiling by MultiOmyx. It is our hope that this will provide some much-needed understanding of the underlying mechanisms leading from an IBD disorder to the development of colon cancer. Citation Format: Anna Juncker-Jensen, Lakshmi Chandramohan. Comprehensive characterization of the biology of inflammatory bowel disease-associated colorectal cancer using the GeoMx® Digital Spatial Profiler (DSP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3801.

Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum

SummaryThe Omicron lineage of SARS-CoV-2, which was first described in November 2021, spread rapidly to become globally dominant and has split into a number of sublineages. BA.1 dominated the initial wave but has been replaced by BA.2 in many countries. Recent sequencing from South Africa’s Gauteng region uncovered two new sublineages, BA.4 and BA.5, which are taking over locally, driving a new wave. BA.4 and BA.5 contain identical spike sequences, and although closely related to BA.2, they contain further mutations in the receptor-binding domain of their spikes. Here, we study the neutralization of BA.4/5 using a range of vaccine and naturally immune serum and panels of monoclonal antibodies. BA.4/5 shows reduced neutralization by the serum from individuals vaccinated with triple doses of AstraZeneca or Pfizer vaccine compared with BA.1 and BA.2. Furthermore, using the serum from BA.1 vaccine breakthrough infections, there are, likewise, significant reductions in the neutralization of BA.4/5, raising the possibility of repeat Omicron infections.

IMMU-12. Exploring and modulating the tumour immune microenvironment to facilitate the selection of immunotherapies for paediatric-type diffuse high-grade glioma

Abstract Immune cells have the potential to selectively eradicate high-risk brain tumours such as paediatric-type diffuse high-grade glioma (PDHGG). We aim to characterize the tumour immune microenvironment (TIME) of intra-cranial syngeneic mouse models of diffuse hemispheric glioma, H3G34 (DHG-H3G34) and diffuse midline glioma, H3K27 (DMG-H3K27). We also demonstrate how an oncolytic reovirus (Reolysin) can “heat-up” the TIME of our syngeneic models. Orthotopic immunocompetent mouse models of DHG-H3G34 (C57BL/6, NRASG12V + shp53 + shATRX +/- H3.3G34R) and DMG-H3K27 (Nestin-Tv-a/p53fl/fl, RCAS-ACVR1R206H + RCAS-H3.1K27M) were profiled using single-cell RNA-sequencing (scRNA-seq) (10x genomics), a 22-colour custom flow cytometry immune panel and spatial transcriptomics. Differential marker expression was validated with immunohistochemistry and immunofluorescence in tissue sections. Syngeneic mouse tumours treated systemically with Reolysin were also profiled to evaluate the effects of the oncolytic virus on the TIME. Cell type predictions in scRNA-seq using singleR, ssGSEA and expression of individual marker genes suggested that the predominant immune cell types within hemispheric tumours were monocytes (11-21%) and macrophages (10-19%) with much smaller proportions of CD4+ and CD8+ T-cells (4-10%). By contrast, much smaller proportions of monocytes (2%) and macrophages (3%) were observed in the H3.1K27M pontine model. Flow cytometry, immunohistochemistry and immunofluorescence validated scRNA-seq immune profiles and characterised signalling of the PD-1/PD-L1 checkpoint pathway. Spatial transcriptomics allowed immune cell populations to be positioned within tumour sections and showed significant co-localization of CD4+ and CD8+ lymphocytes at tumour margins. Treatment of syngeneic mouse tumours with Reolysin resulted in reduced tumour volumes and altered the TIME, in particular increasing cytotoxic T-cell tumour infiltration. Our results highlight immunological heterogeneity within molecular subgroups of PDHGG and demonstrate ability of a systemically delivered oncolytic virus, Reolysin, to “heat-up” the TIME, contributing to a more immune actionable profile. Future work will help to identify optimal combinations for the next generation of immunotherapies in PDHGG.

Immune cell differences between patients in different stages of monoclonal plasma cell disorders.

8065 Background: Multiple myeloma (MM) is an incurable plasma cell malignancy. Precursor conditions include monoclonal gammopathy of unknown significance (MGUS) and smoldering multiple myeloma (SMM). Immune surveillance is paramount for keeping the malignancy in check, whereas a dysfunctional immune system is suspected to promote disease progression. Methods: We performed flow cytometric immune panel analyses including T-cell, B-cell, natural killer (NK) cell and dendritic cell (DC) subsets in MGUS, SMM, and MM patients between 2018-2021. Immune panels were analyzed on fresh peripheral blood samples drawn at diagnosis. The patient population were MGUS (n=28), SMM (n=19) and MM (n=94). Samples from MGUS and SMM patients were combined into a single group (asymptomatic) and were compared to MM patients (symptomatic). Lymphocytes were identified by CD45+ expression resolving against side-scattered light on a bivariate plot. B cell and NK cell antigen expression profiles were assessed from this lymphocyte population. T cell subsets were further determined by CD3+. The percent gated for each marker was summarized by cohort using the appropriate descriptive statistics and compared using the Mann-Whitney U exact test. Analyses were performed in SAS v9.4 (Cary, NC) using a two-sided α=0.05. Results: Compared to MGUS/SMM patients, MM patients had a significant lower proportion of naive B cells, NK cells, and cytolytic NK cells, and a significant higher proportion of cytolytic T cells at diagnosis (Table). Consistent with prior studies, we demonstrated an increase in exhaustion markers. There was a trend toward a higher population of CD8+ exhausted T cells in MM patients compared to MGUS/SMM patients. Since the follow up for this study was short, survival data is not mature. Conclusions: This study demonstrates the immune imbalance that occurs between myeloma precursor conditions and MM. There is a shift in certain cell subsets in the immune microenvironment that alters tumor immunosurveillance and tumor cell killing favoring disease progression. In data not shown, we are currently analyzing the bone marrow microenvironment and its relation to disease progression. Follow up studies could assess if therapeutic intervention could reverse the imbalance, restore homeostatic equilibrium with the goal of controlling disease long-term. [Table: see text]

MiRNAs hsa-miR-30a-5p and hsa-miR-99b-5p regulate chemokine-mediated immune response in colorectal cancer.

e15500 Background: The immune response plays a key role in the oncogenesis of colorectal cancer. The heterogeneity of the tumor environment determines the need for immunogenetic profiling to identify associations with the implementation of tumor-specific immune responses in colorectal cancer. The purpose of our study was to describe the expression profiles of miRNAs and genes involved in the regulation of the immune response in colorectal cancer. Methods: The study included 18 patients (median age 66 years) diagnosed with colorectal cancer who were treated at the National Medical Research Centre for Oncology in 2018-2019. Total RNA was isolated using TRIzol (Thermo Fisher, USA) followed by treatment with DNase 1 (ThermoFisher, USA). Sequencing of RNA samples was performed using two approaches: AmpliSeq for Illumina Immune Response Panel (Illumina, USA) and TruSeq Small RNA Library Prep Kit -Set A (Illumina, USA) according to the manufacturer's instructions on a NextSeq 550 device (llumina, USA). Results: The study revealed 168 differentially expressed genes and 46 differentially expressed miRNAs (p < 0.05). 29 miRNA-mRNA pairs were identified. It has been established that the key signaling mechanism involved in the regulation of the tumor-specific immune response is chemokine signaling pathway (hsa04062). Expression of the CXCL1 and CXCL10 genes encoding the chemokines of the same name is increased in tumor cells (logFC = 1.51, p = 0.006 and logFC = 2.67, p < 0.001, respectively). At the same time, the level of hsa-miR-30a-5p and hsa-miR-99b-5p, which negatively regulate the expression of CXCL1 and CXCL10, is decreased (logFC = -2.26, p < 0.001 and logFC = -1.57, p < 0.001). Conclusions: Activation of expression CXCL1 and CXCL10 with simultaneous loss of negative regulators hsa-miR-30a-5p and hsa-miR-99b-5p was determined in colorectal tumors by NGS-sequencing, which seems promising for the development of personalized therapy, based on the immunogenetic features of colon tumors.

Establishment of multiplex IHC staining panel and immune subpopulation quantificationsystem for cancer prognosis.

e20525 Background: Tumor microenvironment (TME) is now an important direction for cancer prognosis research. For analyzing the immune component within TME, illustrating the distribution of the immune cells in patient tissue samples by immunohistochemistry (IHC) and image quantification analysis are now been applied in many cancer types. However, most of the TME studies only used the tumor microarray to investigate the composition of immune cells in TME, in which the views of immune component are restricted. In addition, with single biomarker staining to stratify therapy strategy shows limited efficacy in treatment since the complicated interactions of the immune components in TME. Based on these shortages, to increase the efficiency and identify the scarce immune subsets, a more systemic analysis approach for immune subpopulations quantification is now imperative. Methods: In this study, we first established an immune cell multiplex IHC staining panel (Opal Polaris, Akoya Bioscience) in non-small cell lung cancer (NSCLC) to investigate the immune components in all the tumor blocks of each patient. In this panel, we applied markers of infiltrating immune cells (CD8, CD68), regulatory markers (PD-1, PD-L1,CD163), and tumor marker (pan-cytokeratin) to explore the TME landscape in NSCLC. Then, with an in-house built quantitative software: SIMPiE, we could analyze the staining signal intensity of each cell markers generated from in Form software to define each lymphocytic immune subsets by phenotyping and quantifying. In addition, SIMPiE (Spatial Image cytometry Multiplex IHC analysis by phyton and in Form based Elements) software allowed us to generate image cytometry data to further explore the spatial context to map the organization and interactions of immune and tumor cells in the TME, and we can define thresholds for the graph to distribute the plurality of the mark points into a plurality of quadrants. Finally, we performed the survival analysis according to the clinicopathological and immune parameters of the patients, and to examine the prognostic significance of each immune subtypes. Results: We have built up an automation quantitative image analysis software to efficiently quantity the signal density and spatial context of all the immune subpopulations in all the tissue sections of each patient, and we further performed the integrative survival analysis to explore the prognostic significance of infiltrating immune cells and immunoregulatory molecules within the TME in NSCLC. Finally, in our study, we found that M1 macrophage patamars showed the most significantly regulatory effect to the patient’s overall survival rate, and M2 macrophage showed less effect compared with M1 macrophage. Conclusions: By this way, we can generate image cytometry data to further explore the spatial context to map the organization of immune and tumor cells in the TME for prognosis analysis.

Immune cell molecular pharmacodynamics of lanreotide in relation to treatment response.

e16213 Background: Lanreotide is clinically effective in advanced neuroendocrine tumors (NETs). The inhibitory effect of lanreotide on tumor cells proliferation is due to binding to somatostatin receptors (SSTR1-5). It has been demonstrated that immune cells express SSTR1-5 differentially. The exact effect of somatostatin analogs (SSAs) on T cells function is not understood. Methods: In vitro and in vivo effects of lanreotide on immune cells were investigated, with regards to clinical response correlates. In vitro, SSTR1-5 expression was measured on CD4+ T helper cells, CD8+ cytotoxic T cells, and CD4+CD25+ T regulatory cells from healthy donors (HD), and lanreotide effect on key functional immune response parameters (NFkB, NFAT, ERK1/2 signaling, IFN, and IL-2 production) were studied. To assess in vivo effects of lanreotide on immune cells of NET pts, peripheral blood mononuclear cells (n = 16) obtained pre and 3 months post treatment were studied for gene and protein expression profiles in sorted T cell subsets using a NanoString immune cell panel. Results: HD T cells had high expression of SSTR2 and low/no expression of the other SSTRs. In vitro, lanreotide had no effect on the functional immune response parameters investigated. For the in vivo study, the patient cohort consisted of 8 responders (no progression by 19 months) and 8 non-responders. Clinicopathological features, see table. Pretreatment immunological competence of responders was greater than non-responders, as indicated by upregulation of TCR signaling (in CD4+) and interferon signaling (in CD8+ and T reg). Irrespective of clinical response, lanreotide had most significant effect on CD8+ T cells, down regulating WNT, TCR, and NF-kB signaling. Compared to non-responders, responders had down regulation of cytokine and chemokine signaling but upregulation of ubiquitination and proteasome degradation associated genes. Several myeloid specific genes were found to be significantly changed in the CD4 T helper population, possibly due to co isolated myeloid cells interacting with T cells during sorting. Conclusions: The in vivo immune effects of lanreotide seen reflect the relevance of parameters in tumor microenvironment such as interactions with myeloid components of the immune system not accounted for under the experimental in vitro conditions.[Table: see text]

Tumor mutation burden (TMB) and Notch1 gene alteration are associated with short disease-free survival in early laryngocarcinoma.

e18049 Background: Patients with early-stage laryngocarcinoma, even stage T1-2N0, are at substantial risk of relapse and death. Molecular alterations are thought to be associated with the immune microenvironment in the tumor and to have an important role in predicting the prognosis. In this study, we explored the distinct features of molecular alterations and immune-related gene expression and examined the relationship between molecular alterations and prognosis. Methods: A total of 42 stage T1-2N0 laryngocarcinoma patients were enrolled, no patients received chemotherapy or immunotherapy before surgery, tissue samples from radical operation were collected. Of these, 23 cases developed disease progression within 24 months after operation [shorter disease-free survival (shorter DFS)], while others 19 cases showed longer DFS over 24 months (longer DFS). Distinct features of molecular alterations and immune-related gene expression in Formalin-fixed paraffin-embedded (FFPE) samples through using DNA sequencing and an immune oncology panel RNA sequencing platform were performed. Results: A total of 293 mutations were detected within 42 patients, the median number of mutations in the short-DFS group was higher than that in the long-DFS group (9.0 vs. 5.2; P = 0.045). No correlation between mutation type and DFS was found. TMB-high (Top 25%) was associated with worse DFS (TMB-L: Not reached, TMB-H: 11.5 months, p = 0.036; HR, 2.4; 95% CI 1.05 to 5.48). Among 522 cancer-related genes, alterations in 51 genes were detected. TP53 (33/42, 79%) was the most common mutation gene in the entire cohort. Mutations of LRP1B (30.4% vs 5.3%, p = 0.039) and Notch1 (21.7% vs 0%, p = 0.030) were significantly occurred in the short-DFS group, patients with Notch1 mutation had a significantly shorter DFS period (P = 0.0012). Univariate and multivariate analysis showed that high TMB and Notch1 mutation were independent prognostic factors in DFS. Conclusions: In early-stage laryngocarcinoma, Notch1 mutation and high TMB were associated in patients with short-DFS, indicating the potential prognostic biomarkers and therapeutic molecular targets for early-stage laryngocarcinoma.

High-multiplexing quantitative CodePlex proteomic profiling of platelets in triple-negative breast cancer (TNBC) patients and healthy subjects.

e15017 Background: Platelets play significant roles in inflammation, immune regulation, and intracellular communication processes involved in tumor pathogenesis. Aberrant platelet activation has been associated with tumor progression and metastasis, however, cancer patient platelet deep profiling remains underutilized. New tools are needed for rapid high throughput quantification of potential altered platelet secretome in cancer patients. Such technology may provide novel insights into impactful cancer-associated biomarkers. Methods: We have developed a high throughput multiplexed proteomic technology to profile platelet cytokine content and releasates and performed a pilot study in healthy patients versus triple negative breast cancer (TNBC). Macrochambers of the CodePlex chip were pre-patterned with a 23-plex antibody IsoPlexis Human Adaptive Immune Panel: GM-CSF, Granzyme B, IFN-g, IL-10, IL-13, IL-15, IL-17A, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IP-10, MCP-1, MIP-1a, MIP-1b, perforin, sCD137, TGF-b1, TNF-a, TNF-b. 2% BSA/PBS was used as background control. The chips are loaded into an IsoLight automation system, releasates measured by fluorescence ELISA, and analysis performed by IsoSpeak software. In a pilot study to test feasibility, platelets were meticulously harvested from triple negative breast cancer patients (n=2) and healthy controls (n=2) and stimulated for 30 minutes (ADP or TRAP-6) to promote cytokine release. Both the supernatants and platelet lysates were then loaded on CodePlex chips and analyzed. Results: The methodology of bulk proteomic measurements has previously been validated in a variety of biofluids such as serum/plasma, cerebrospinal fluid, tracheal washes, and cell culture supernatants. Freshly isolated platelets activated by TRAP6 generally released more cytokines than those activated by ADP stimulation in both healthy controls and disease samples. Interestingly, platelet releasates in supernatants from TNBC patients demonstrated increased pro-tumor IL-8 and IL-9, and reduced antitumor IFN-g compared to healthy controls. Higher levels of IL-8 and IL-9 and minimal IFN-g were detected at similar activation concentrations, in platelet lysates from both healthy and TNBC platelets. This suggests that TNBC may alter platelet content and/or activation. Increased numbers are being performed to validate these initial results. Conclusions: From this small pilot study, multiplexing quantitative proteomic analysis is potentially useful in detecting differential cytokine profile in platelet releasates and lysate contents between TNBC patients and healthy controls. Quantifying the platelet proteomes in cancer patients using high throughput proteomics may provide valuable insights to dissect cancer-platelet crosstalk and potential pathogenesis mechanisms.

Full spectrum flow cytometry and mass cytometry: A 32-marker panel comparison.

High-dimensional single-cell data has become an important tool in unraveling the complexity of the immune system and its involvement in homeostasis and a large array of pathologies. As technological tools are developed, researchers are adopting them to answer increasingly complex biological questions. Up until recently, mass cytometry (MC) has been the main technology employed in cytometric assays requiring more than 29 markers. Recently, however, with the introduction of full spectrum flow cytometry (FSFC), it has become possible to break the fluorescence barrier and go beyond 29 fluorescent parameters. In this study, in collaboration with the Stanford Human Immune Monitoring Center (HIMC), we compared five patient samples using an established immune panel developed by the HIMC using their MC platform. Using split samples and the same antibody panel, we were able to demonstrate highly comparable results between the two technologies using multiple data analysis approaches. We report here a direct comparison of two technology platforms (MC and FSFC) using a 32-marker flow cytometric immune monitoring panel that can identify all the previously described and anticipated immune subpopulations defined by this panel.

Immunogenomic landscape analyses of immune molecule signature-based risk panel for patients with triple-negative breast cancer

Triple-negative breast cancer (TNBC) presented as high heterogeneous immunogenicity that lacks useful clinical signatures to risk-stratify immune-benefit subtypes. We hypothesized that molecular-based phenotypic characterization of TNBC tumors and their immunity may overcome these challenges. We enrolled 1,145 patients with TNBC for analysis. Through combining algorithm integration analysis and TNBC datasets, a tumor immune risk score (TIRS) panel consisting of 8 potential biomarkers was identified. The TIRS panel represented excellent effectiveness as an independent predictor. High- and low risk stratification of patients was further achieved by TIRS, and significant survival and immune-infiltration pattern differences were found in each cohort, both at the transcriptome and protein levels. Non-negative matrix factorization clustering further identified four different tumor immune microenvironment types (TIMTs), among which TIMT-II was associated with the best prognosis and immune status, whereas TIMT-IV had the opposite effect, TIMT-III was associated with highly unstable genomes, and TIMT-I displayed stem-cell-related characteristics along with high stromal scores and may have extensive enrichment of tumor-associated fibroblasts and vascular cells. In conclusion, our TIRS panel could serve as a robust prognostic signature and provide therapeutic benefits for immunotherapy. Additionally, coordinating four TIMTs may be helpful for clinical decision-making in TNBC patients.

T helper 17 axis and endometrial macrophage disruption in menstrual effluent provides potential insights into the pathogenesis of endometriosis

To identify immune cells, cytokines, and immune cell transcriptome in the menstrual effluent (ME) of women with endometriosis compared with that of healthy donors. Live immune cells were isolated from human ME samples and were analyzed by flow cytometry to identify various immune cell populations. Selected cytokines from the same patients were evaluated using multiplex cytokine analyses. The transcriptome of the immune cell population was subsequently profiled using NanoString nCounter's PanCancer Immune panel. Academic institution. Surgically confirmed endometriosis patients (n = 14) and healthy fertile donors (n = 19). None. In-depth immune cell profiling of ME obtained from women with endometriosis compared with that of healthy donors. ME analysis revealed that the number of T helper 17 (TH17) cells was significantly lower in patients with endometriosis compared with that of healthy donors; the number of macrophages was also lower (P=.06) in the former. Multiplex cytokine analysis revealed significantly lower transforming growth factor α in the ME "serum" of patients with endometriosis. Transcriptomic analysis of CD45+ cells revealed 47 differentially expressed genes, mainly associated with the TH17 axis (IL10, IL23A, and IL6), as well as genes associated with macrophage signaling/activation (CD74, CD83, CXCL16, and CCL3). We demonstrate for the first time that the levels of TH17 axis, macrophages, and transforming growth factor α were altered in the ME of women with endometriosis compared with that of healthy donors. These findings shed light on the potential immune pathways that could partly explain the pathogenesis and progression of endometriosis. Future large-scale studies on ME samples are warranted to exploit the use of these markers to study the pathogenesis of endometriosis.

Inflammatory phenotype and T‐cell mitochondrial reactive oxygen species in young adults with major depressive disorder

BackgroundMajor depressive disorder (MDD) is prevalent in young adults and projected to rise further with the COVID pandemic. Notably, MDD is associated with an increased risk of cardiovascular disease (CVD) and mortality. Although the mechanisms are unclear, evidence supports chronic systemic inflammation as a major contributor to CVD risk in older adults with MDD. However, whether an inflammatory phenotype is present in young adults with MDD prior to the onset of CVD and other comorbidities is equivocal. Recent work has implicated the sympathetic nervous system and specifically norepinephrine as a potent driver of inflammation via stimulating an increase in T‐cell mitochondrial reactive oxygen species (T‐cell mitoROS). Given previous findings of elevated plasma norepinephrine and inflammation in MDD, the purpose of this study was to examine the potential for a T‐cell mitoROS pro‐inflammatory pathway in young otherwise healthy adults with MDD. We hypothesized that young adults with MDD but otherwise free of clinical disease would have augmented circulating pro‐inflammatory cytokines and T‐cell mitoROS compared to young healthy adults without MDD (HA).MethodsAll participants underwent a structured diagnostic neuropsychiatric interview to determine the presence or absence of MDD based on the DSM‐5 diagnostic criteria for MDD. Systemic pro and anti‐inflammatory cytokines and T‐cell mitoROS were measured in 14 healthy young adults with MDD of mild‐to‐moderate severity (non‐medicated; age: 23±2 years) and 11 HA (age: 22±1 years). Serum cytokines were detected using LEGENDplex Human Essential Immune Response panel and assessed by flow cytometry. Seven serum cytokines related to the norepinephrine‐T‐cell mitoROS‐inflammation pathway were measured. Immune cells were isolated from whole blood by red blood cell lysis. T‐cell mitoROS was measured by labeling with antibodies against CD3+ (total T cells) and mitoROS targeted fluorogenic dye (MitoSoxRED) and assessed by flow cytometry. All data are expressed as mean ± SE.ResultsFor pro‐inflammatory cytokines, there were no differences between MDD and HA for IL‐6 (41±18 pg/mL; 92± 66 pg/mL, P=0.66), IL‐17A (7±3 pg/mL; 17±8 pg/mL, P=0.13), TNF‐α (19±9 pg/mL; 36±18 pg/mL, P=0.20), IFNγ (120±70 pg/mL; 201±137 pg/mL, P=0.34) or IL‐2 (5±2 pg/mL; 13±6 pg/mL, P=0.25), respectively. Likewise, no differences were found in anti‐inflammatory cytokines between MDD and HA for IL‐10 (18±9 pg/mL; 78±57 pg/mL, P=0.19) or IL‐4 (33±15 pg/mL; 95±46 pg/mL, P=0.13), respectively. In contrast, T‐cell mitoROS was significantly elevated in MDD (34402±13353 a.u) compared to HA (6258±2942 a.u, P<0.05).ConclusionThese preliminary findings suggest T‐cell mitoROS but not circulating cytokine profiles are different between young adults with and without MDD. Thus, an elevated T‐cell mitoROS may represent an early marker of immune system alterations and inflammation in MDD.

Combined nano cancer immunotherapy based on immune status in a tumor microenvironment.

Since the effect of cancer immunotherapy is largely dependent on the status of the immune system in the tumor microenvironment (TME), choice of therapy and the development of new therapies based on the immune status in the TME would be predicted to be effective. Unfortunately, the development of delivery systems for such therapy has been slow. Here, we defined a parameter of immune status in TME showing antitumor effects and demonstrated the cancer immunotherapy with an adjuvant loaded lipid nanoparticle (LNP), which was taken advantage the parameter. An analysis was carried out to determine the relationship between antitumor effects and gene expression (22 target genes) in tumors (MC38 and E.G7-OVA) that respond to the programmed cell death 1 (PD-1) antibody and non-responding tumors (B16-F10 and 4T1). The immune status showing an effective antitumor effect, which consisted of 10 genes, was then extracted. Treatment with the adjuvant loaded LNP caused a significant antitumor effect against an E.G7-OVA tumor, and the gene expression in the E.G7-OVA tumor was completely within the range of gene expression for showing an effective antitumor effect, as defined by the identified immune status panel (IS-panel-10). Although the treatment with the adjuvant loaded LNP failed to induce a sufficient antitumor effect against the 4T1 tumor, we succeeded in enhancing the antitumor effect by using a combination therapy that was adopted based on the analysis by the IS-panel-10 in the TME. The 10 genes were found to affect the prognosis in a variety of human cancers. Collectively, the findings reported herein demonstrate the potential of immune status analysis in the TME for developing cancer immunotherapies using a delivery system.

Effect of Zeolite Supplementation on Gene Expression in the Intestinal Mucosa in the Context of Immunosafety Support in Poultry

Zeolite is an effective and non-toxic silicate mineral. Its properties are widely used in industry due to its sorption and ion exchange properties. Due to its excellent chemical properties, it has also great potential in poultry production as a food additive or supplement to bedding. This is of great importance for the biosafety and hygiene of production. The study aimed to analyse the effects of simultaneous application of zeolite to feed and bedding on production parameters and expression of genes related to intestinal tightness, organism defence, and immune response. Male Ross 308 broiler chickens were used in the experiment. In the experimental group, an external factor in the form of a powdery zeolite was used for feed and pelleted bedding. On the day of slaughter, the caecal mucosa was collected for gene expression analysis. We showed no significant changes in the tissue composition of the carcasses, but zeolite had a beneficial effect on the carcass yield. The analysis of the immune gene panel showed a significant increase in the expression of the interleukins and interferons genes. We have demonstrated the effect of zeolite on the improvement of the intestinal barrier and increasing the tightness of the intestines. There were no changes in gene expression related to the host’s defence against infections; therefore, based on the obtained results, it was concluded that zeolite can be considered an immunomodulating factor of the immune system.

157MO Immune gene signatures for predicting pathological response of NSCLC patients treated with neoadjuvant chemoimmunotherapy

Neoadjuvant chemoimmunotherapy (CI) for NSCLC is a promising strategy that yields high rates of complete pathological responses (CPR) that are associated with long-term survival. We analyzed the tumor microenvironment by using gene expression profiling in patients from NADIM trial to identify biomarkers predicting CPR. RNAs extracted from whole sections of pre-treatment tumors of 14 patients enrolled in NADIM trial (NCT03081689) were sequenced using the Oncomine® Immune Response Research Assay panel. Differential-expressed genes between groups and pathway enrichment analysis were assessed using DESeq2 and Gene Set Enrichment Analysis (GSEA). CIBERSORTx was used to estimate the proportions of immune cells subtypes. Results were correlated with pathological response groups: complete (CPR, n=9) and non-complete (non-CPR, n=5) responders. Up to 25 genes were identified as differentially expressed between CPR and non-CPR patients. Among them, 17 were upregulated in CPR compared to non-CPR: involved in type II interferon signalling (FASLG, CXCL13, CXCL10, CXCL9 and IFNG), NK cell markers (NCR1, KIR2DL3, GNLY) or lymphocyte enrichment (NKG7, GZMB) depicting a pro-inflammatory phenotype in pre-treatment tumour samples of CPR patients. Similarly, GSEA analysis revealed in CPR patients an up-regulation of pathways related to TCR co-expression, lymphocyte infiltrate, type II interferon signalling and antigen processing, as well as a downregulation of pathways related to tumour markers, proliferation or PD-1 signalling. Finally, CPR patients had a higher proportion of follicular helper T cells, activated NK cells, M1 macrophages, resting dendritic cells and absolute score of immune cells than non-CPR patients, being the proportion of M1 macrophages significantly different (p=0.002). The AUC ROC for response prediction based on IFNG, GZMB expression or M1 macrophages proportion was 1.000 (p=0.0027), 0.911 (p=0.0136) and 0.9778 (p=0.004), respectively. In our study, a pro-inflammatory profile measured in the pre-treatment tissue by RNA-sequencing is associated with CPR following neoadjuvant CI in patients with stage IIIA NSCLC.

CMV Pneumonitis After Discontinuation of Prophylaxis Therapy Guided by CMV T-cell Immunity Panel

<h3>Introduction</h3> CMV T-cell immunity panels (TCIP) may reliably qualify T-cell specific CMV immunity, however, utility within lung transplant recipients (LTR) is unclear. We describe one of several CMV pneumonitides occurring in LTR after a positive CMV TCIP. <h3>Case Report</h3> A 61yo bilateral LTR (CMV donor IgG+/recipient IgG-) for COPD was admitted for worsening dyspnea 17mo post-LT. During admission, he experienced persistent leukopenia requiring multiple granulocyte colony-stimulating factor doses, MMF discontinuation, and frequent bronchoscopy for clearance. Respiratory cultures were positive for <i>Aspergillus</i> species only. Despite drug modification, his WBC and ANC ranged 1-4K cells/µL and ∼1K cells/µL, respectively. Durable CMV seroconversion to IgG+ occurred 9.5mo post-LT. No prior CMV viremia occurred and only 2 episodes on bronchoalveolar lavage (BAL) viral shedding ranging 1243-2762 copies/mL, with the second episode occurring 3 months prior to current admission. During hospitalization, a CMV TCIP showed immunity [(reference range >0.20%) CD4+ CMV IFN-ɣ cells = 3.08%; CD8+ CMV IFN-ɣ = 10.64%; CD4+ control = 1.93%; CD8+ control = 8.28%]. Subsequently, valganciclovir (VGCV) was discontinued to reduce further bone marrow suppression. One month after VGCV cessation, routine plasma CMV PCR monitoring resulted 740 copies/mL with BAL 37114 copies/mL. Lung tissue pathology and cytology were not performed, however, given the high BAL viral load and persistent graft dysfunction, CMV pneumonitis was diagnosed and treatment with IV ganciclovir (GCV) was initiated. He received 21d of IV GCV treatment with clinical and virologic improvement, and now remains on lifelong VGCV secondary prophylaxis. <h3>Summary</h3> This case advises caution when interpreting CMV TCIP alone to guide duration of CMV prophylaxis in LTR. Pairing CMV TCIP assay with assessment of patient factors (i.e. age, recipient CMV IgG seroconversion, hypogammaglobulinemia) or need for serial monitoring requires further exploration.

Alterations of Urinary Microbial Metabolites and Immune Indexes Linked With COVID-19 Infection and Prognosis

Coronavirus disease 2019 (COVID-19) has evolved into an established global pandemic. Metabolomic studies in COVID-19 patients is worth exploring for further available screening methods. In our study, we recruited a study cohort of 350 subjects comprising 248 COVID-19 patients (161 non-severe cases, 60 asymptomatic cases, and 27 severe cases) and 102 healthy controls (HCs), and herein present data with respect to their demographic features, urinary metabolome, immunological indices, and follow-up health status. We found that COVID-19 resulted in alterations of 39 urinary, mainly microbial, metabolites. Using random forest analysis, a simplified marker panel including three microbial metabolites (oxoglutaric acid, indoxyl, and phenylacetamide) was constructed (AUC=0.963, 95% CI, 0.930-0.983), which exhibited higher diagnostic performance than immune feature-based panels between COVID-19 and HC groups (P<0.0001). Meanwhile, we observed that urine metabolic markers enabled discriminating asymptomatic patients (ASY) from HCs (AUC = 0.981, 95% CI, 0.946-0.996), and predicting the incidence of high-risk sequalae in COVID-19 individuals (AUC=0.931, 95% CI, 0.877-0.966). Co-expression network analysis showed that 13 urinary microbial metabolites (e.g., oxoglutaric acid) were significantly correlated with alterations of CD4+, CD3+, and CD8+ T-cells, as well as IFN-γ, IL-2 and IL-4 levels, suggesting close interactions between microbial metabolites and host immune dysregulation in COVID-19. Taken together, our findings indicate that urinary metabolites may have promising potential for screening of COVID-19 in different application scenarios, and provide a new entry point to understand the microbial metabolites and related immune dysfunction in COVID-19.