Myeloid Signature Research Articles

Ex vivo mass cytometry analysis reveals a profound myeloid proinflammatory signature in psoriatic arthritis synovial fluid

ObjectivesA number of immune populations have been implicated in psoriatic arthritis (PsA) pathogenesis. This study used mass cytometry (CyTOF) combined with transcriptomic analysis to generate a high-dimensional dataset of matched...

Abstract 390: Identification of a novel immunosuppressive myeloid gene expression signature for clinical biomarker development

Abstract Cell-type abundance GE signatures are useful for informing drug mechanism of action and may be useful in-patient selection for cancer immunotherapies. Recently, we validated natural killer cell- and dendritic cell-type specific expression signatures using a tiered strategy that included both computational and ex vivo validation. Here, we built upon this strategy to validate expression signatures for immunosuppressive myeloid cells (IMC). IMC play a critical role in impairing anti-tumor immunity and increased levels of peripheral IMC have been associated with advanced tumor progression and poor prognosis in various cancers. However, their low abundance in normal tissues and heterogeneous surface expression render their profiling difficult. To address this, we focused on validating GE signatures for IMC abundance using mRNA expression methods which are more clinically tractable than established flow cytometry methods that require intact cells. We implemented a three-stage GE signature validation strategy. First, we generated GE data from human ex vivo differentiated IMC (LIN- CD11B+ CD33+ HLADR-). Then, we evaluated the concordance of 16 previously published myeloid signatures with cell type abundance in a series of spike-ins with varying but known quantities of ex vivo differentiated IMC in a background of undifferentiated peripheral blood mononuclear cells (PBMCs). Gene-set variation analysis (GSVA) was used to score the signatures and Spearman's rank (rS) correlation coefficient was calculated to assess the significance of correlations between GSVA scores and IMC abundance. With this method, we validated 3 of the 16 published myeloid signatures (1. MacB3/PMID:26873985 (rS = 0.87; p = 8.8e-07); 2. MacB2_3w/PMID:26873985 (rS = 0.83; p = 6.7e-06); 3. TAM/PMID:27424807 (rS = 0.80; p= 2.6e-05)). Next, we generated a ‘de novo' signature by performing differential GE analysis and identified up-regulated genes in IMC in comparison to PBMCs with log2 fold change &gt 2, p-adj &lt 0.05). We further refined this list by selecting genes with significant concordance to IMC abundance (rS &gt 0.80, p-adj &lt 0.05) and confirmed their specificity to IMC in published scRNAseq studies [PMID: 30979687; 31033233; 32302573]. This led to a 6-gene ‘de novo' signature (MRC1, APOE, C1QA, C1QB, MMP9, SPP1) associated with IMC function with highly significant concordance (rS = 0.96; p = 1.2e-11) to IMC abundance. Lastly, we showed that in whole blood baseline samples from HNSCC patients (n=53) treated with danvatirsen + durvalumab [NCT02499328], our ‘de novo' signature was significantly elevated (p=0.033) in patients with progressive disease compared to complete responders. Application to additional studies will be required to more fully determine its clinical utility. Citation Format: Srimathi S. Srinivasan, Gozde Kar, Deanna L. Russell, Peter Gathungu, Minoo Rafati, David Whitston, Grant Duclos, Ben Sidders, J Carl Barrett, Tim Slidel, Patricia McCoon. Identification of a novel immunosuppressive myeloid gene expression signature for clinical biomarker development [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 390.

Abstract 3185: Integrin signaling is critical for myeloid-mediated support of T-ALL

Abstract T-cell acute lymphoblastic leukemia (T-ALL) is a malignant transformation of immature T cells. Although T-ALL harbors multiple pro-leukemic genetic alterations, T-ALL blasts require exogenous signals to survive. We recently reported that tumor-associated myeloid cells provide critical support for T-ALL in mouse models and in pediatric patient samples, at least partially through activation of IGF1R signaling. However, IGF1 alone is not sufficient to support T-ALL survival, implicating additional mechanisms in myeloid-mediated T-ALL support. We previously found that T-ALL cells make prolonged and frequent contacts with tumor-associated myeloid cells in live tissue sections; thus, we hypothesized that cell-cell interactions are essential for myeloid-mediated support of T-ALL. Here, we confirm that cell contact is required for myeloid cells to support T-ALL cell survival in vitro. Analysis of transcriptional profiling data revealed enrichment of gene signatures associated with integrin pathways in T-ALL cells relative to healthy T cells, implicating integrin-mediated cell adhesion as a potential mechanism underlying contact-dependent T-ALL support. Blockade of integrin αL ± integrin β1 or their ligands ICAM-1 ± VCAM-1 via antibodies or pharmacologic inhibitors significantly decreased T-ALL survival and reduced activation of IGF1R in T-ALL cells co-cultured with tumor-associated myeloid cells. Consistent with these findings, inhibition of integrins or their ligands in vivo via antibody blockade or genetic manipulation resulted in a significant reduction of T-ALL burden at multiple leukemic sites and prolonged survival. Consistent with mouse models, blockade of integrin-mediated cell adhesion significantly reduced survival of primary pediatric T-ALL cells co-cultured with myeloid cells. Furthermore, analyses of published transcriptomic datasets from pediatric T-ALL patients showed that enrichment of integrin pathways correlates with elevated myeloid gene signatures and poor patient prognosis. Together, our studies demonstrate that tumor-associated myeloid cells promote survival of T-ALL cells by activating integrin signaling, implicating the associated integrins and downstream signals as targets for therapeutic intervention. Citation Format: Aram Lyu, Seo Hee Nam, Zicheng Hu, Dhivya Arasappan, Terzah M. Horton, Lauren I. Ehrlich. Integrin signaling is critical for myeloid-mediated support of T-ALL [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 3185.

Comprehensive Analysis of Myeloid Signature Genes in Head and Neck Squamous Cell Carcinoma to Predict the Prognosis and Immune Infiltration.

Myeloid cells are a major heterogeneous cell population in the tumor immune microenvironment (TIME). Imbalance of myeloid response remains a major obstacle to a favorable prognosis and successful immune therapy. Therefore, we aimed to construct a risk model to evaluate the myeloid contexture, which may facilitate the prediction of prognosis and immune infiltration in patients with head and neck squamous cell carcinoma (HNSCC). In our study, six myeloid signature genes (including CCL13, CCR7, CD276, IL1B, LYVE1 and VEGFC) analyzed from 52 differentially expressed myeloid signature genes were finally pooled to establish a prognostic risk model, termed as myeloid gene score (MGS) in a training cohort and validated in a test cohort and an independent external cohort. Furthermore, based on the MGS subgroups, we were able to effectively identify patients with a poor prognosis, aggressive clinical parameters, immune cell infiltration status and immunotherapy response. Thus, MGS may serve as an effective prognostic signature and predictive indicator for immunotherapy response in patients with HNSCC.

Innate Immune Activation Restricts Priming and Protective Efficacy of the Radiation-Attenuated Pfspz Malaria Vaccine

Baseline innate immune signatures can influence protective immunity following vaccination. Here, we used systems transcriptional analysis to assess the molecular mechanisms underlying differential immunogenicity and protective efficacy results of a clinical trial of the radiation-attenuated whole sporozoite PfSPZ Vaccine in African infants. Innate immune activation and myeloid signatures at pre-vaccination baseline correlated with protection from Plasmodium falciparum infection in placebo controls, while the same signatures predicted susceptibility to infection among infants who received the highest and most protective dose of the PfSPZ Vaccine. Machine learning identified monocytes and an antigen presentation signature as pre-vaccination features predictive of malaria infection after highest-dose PfSPZ vaccination. Consistent with these human data, innate stimulation in vivo conferred protection against malaria infection in mice while diminishing the CD8+ T cell response to radiation-attenuated sporozoites. These data establish a dichotomous role of innate stimulation for malaria protection and induction of protective immunity of whole-sporozoite malaria vaccines.

IMMU-27. SINGLE CELL RNA-SEQUENCING IDENTIFIES NOVEL BONE MARROW DERIVED MYELOID CELLS IN GLIOBLASTOMA ASSOCIATED WITH TUMOR AGGRESSION

Abstract BACKGROUND Glioma associated myeloid cells (GAMs) can be induced to adopt an immunosuppressive phenotype that can lead to inhibition of anti-tumor responses in glioblastoma (GBM). Understanding the composition and phenotypes of GAMs is essential to modulating the myeloid compartment as a therapeutic adjunct to improve anti-tumor immune response. METHODS We performed single-cell RNA-sequencing (sc-RNAseq) of 435,400 myeloid and tumor cells to identify transcriptomic and phenotypic differences in GAMs across glioma grades. We further correlated the heterogeneity of the GAM landscape with tumor cell transcriptomics to investigate interactions between GAMs and tumor cells. RESULTS sc-RNAseq revealed a diverse landscape of myeloid-lineage cells in gliomas with an increase in preponderance of bone marrow derived myeloid cells (BMDMs) with increasing tumor grade. We identified two populations of BMDMs unique to GBMs; Mac-1and Mac-2. Mac-1 demonstrates upregulation of immature myeloid gene signature and altered metabolic pathways. Mac-2 is characterized by expression of scavenger receptor MARCO. Pseudotime and RNA velocity analysis revealed the ability of Mac-1 to transition and differentiate to Mac-2 and other GAM subtypes. We further found that the presence of these two populations of BMDMs are associated with the presence of tumor cells with stem cell and mesenchymal features. Bulk RNA-sequencing data demonstrates that gene signatures of these populations are associated with worse survival in GBM. CONCLUSION We used sc-RNAseq to identify a novel population of immature BMDMs that is associated with higher glioma grades. This population exhibited altered metabolic pathways and stem-like potentials to differentiate into other GAM populations including GAMs with upregulation of immunosuppressive pathways. Our results elucidate unique interactions between BMDMs and GBM tumor cells that potentially drives GBM progression and the more aggressive mesenchymal subtype. Our discovery of these novel BMDMs have implications in new therapeutic targets in improving the efficacy of immune-based therapies in GBM.

Immunophenotypic Lineage Assessment By Multiparameter Flow Cytometry Provides More Precise MDS Prognosis

Background: Mixed phenotype acute leukemia is a rare disease characterized by an expanded blast population exhibiting multiple lineage features. It is a diagnosis of exclusion, and we and others have observed that AML-MRC or therapy-related AML cases may also exhibit similar features. We therefore sought to determine whether multi-lineage expression is present in de-novo and therapy-related MDS and whether it has an impact on clinical outcomes. Methods: We reviewed pathology, flow cytometry, cytogenetic, and molecular reports from patients seen at Memorial Sloan Kettering Cancer Center between 1996 and 2020 and identified 472 patients diagnosed with MDS using a combination of custom natural language processing tools and manual review. Cox proportional hazards modeling was performed to assess the contribution of patient characteristics, pathology, flow cytometric, cytogenetic, and molecular characteristics to overall survival (OS). Fisher's exact testing was used to assess the association of individual features. Results: We found that an abnormal myeloid lineage signature was associated with poorer OS after adjusting for age, cytogenetics, and molecular features (HR=2.3, p<0.01). In contrast, an abnormal plasma cell signature was associated with better OS (HR=0.3, p<0.01) (Figure A). Other abnormal lineages did not contribute significantly to OS although there were relatively few cases with abnormal T-cell markers (n=19). Mutations in TP53, IDH1, and EZH2 were associated with poorer OS (TP53: HR=1.5, p<0.01; IDH1: HR=2.3, p<0.01, EZH2: HR=1.9, p<0.01) and mutations in SF3B1 and IDH2 were associated with better OS (SF3B1: HR=0.35, p<0.01; IDH2: HR=0.6, p=0.05) (Figure B). TP53 mutation was enriched among patients with intermediate and poor risk cytogenetic groups (OR=11.3, p<0.01), consistent with higher risk therapy-related disease. IDH2, TET2, and ASXL1, which have been associated with clonal hematopoiesis, were enriched in the good and intermediate risk cytogenetic groups (IDH2: OR=4.3; p=0.03, TET2: OR=3.7, p<0.01; ASXL1: OR=4.3, p<0.01), although only IDH2 had a significant association with OS. Interestingly, there was a significant association of plasma cell lineage abnormalities with SRSF2 mutation (OR=3.5, p=0.05), but SRSF2 mutation was not an independent contributor to the global survival model (Figure C). Conclusion: Routine flow cytometric and molecular assessment of diagnostic bone marrows can provide added prognostic value in MDS. Identification of an abnormal myeloid lineage is associated with higher risk disease, while identification of abnormal plasma cell features is associated with lower risk disease. SRSF2 mutation is also associated with abnormal plasma cell features, but does not contribute independently to OS. Abnormal T-cell features may also provide prognostic value, although assessment in a larger cohort will be necessary. We confirm previous findings indicating that TP53 mutation is associated with higher risk disease while SF3B1 mutation is associated with better outcomes characteristic of MDS-RARS. In addition, our data suggests that IDH1 and EZH2 are associated with poorer outcomes and that IDH2 is associated with lower risk disease. Figure 1 Disclosures Tallman: Bioline rx: Membership on an entity's Board of Directors or advisory committees; Delta Fly Pharma: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Abbvie: Research Funding; Cellerant: Research Funding; Orsenix: Research Funding; ADC Therapeutics: Research Funding; BioSight: Membership on an entity's Board of Directors or advisory committees, Research Funding; Glycomimetics: Research Funding; Rafael: Research Funding; Oncolyze: Membership on an entity's Board of Directors or advisory committees; UpToDate: Patents & Royalties; Novartis: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo: Membership on an entity's Board of Directors or advisory committees; KAHR: Membership on an entity's Board of Directors or advisory committees; Rigel: Membership on an entity's Board of Directors or advisory committees. Levine:Morphosys: Consultancy; Astellas: Consultancy; Janssen: Consultancy; Lilly: Consultancy, Honoraria; Isoplexis: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Imago: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Loxo: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Qiagen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Prelude Therapeutics: Research Funding; Gilead: Honoraria; Novartis: Consultancy; Amgen: Honoraria; Roche: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding. Xiao:Stemline Therapeutics: Research Funding. Glass:Gerson Lehman Group: Consultancy.

Hepatobiliary malignancies have distinct peripheral myeloid-derived suppressor cell signatures and tumor myeloid cell profiles

Myeloid-derived suppressor cells (MDSCs) are immunosuppressive cells that are increased in patients with numerous malignancies including viral-derived hepatocellular carcinoma (HCC). Here, we report an elevation of MDSCs in the peripheral blood of patients with other hepatobiliary malignancies including non-viral HCC, neuroendocrine tumors (NET), and colorectal carcinoma with liver metastases (CRLM), but not cholangiocarcinoma (CCA). The investigation of myeloid cell infiltration in HCC, NET and intrahepatic CCA tumors further established that the frequency of antigen-presenting cells was limited compared to benign lesions, suggesting that primary and metastatic hepatobiliary cancers have distinct peripheral and tumoral myeloid signatures. Bioinformatics analysis of The Cancer Genome Atlas dataset demonstrated that a high MDSC score in HCC patients is associated with poor disease outcome. Given our observation that MDSCs are increased in non-CCA malignant liver cancers, these cells may represent suitable targets for effective immunotherapy approaches.

Engineered type 1 regulatory T cells designed for clinical use kill primary pediatric acute myeloid leukemia cells.

Type 1 regulatory (Tr1) T cells induced by enforced expression of interleukin-10 (LV-10) are being developed as a novel treatment for chemotherapy-resistant myeloid leukemias. In vivo, LV-10 cells do not cause graft-versus-host disease while mediating graft-versus-leukemia effect against adult acute myeloid leukemia (AML). Since pediatric AML (pAML) and adult AML are different on a genetic and epigenetic level, we investigate herein whether LV-10 cells also efficiently kill pAML cells. We show that the majority of primary pAML are killed by LV-10 cells, with different levels of sensitivity to killing. Transcriptionally, pAML sensitive to LV-10 killing expressed a myeloid maturation signature. Overlaying the signatures of sensitive and resistant pAML onto the public NCI TARGET pAML dataset revealed that sensitive pAML clustered with M5 monocytic pAML and pAML with MLL rearrangement. Resistant pAML clustered with myelomonocytic leukemias and those bearing the core binding factor translocations inv(16) or t(8;21)(RUNX1- RUNX1T1). Furthermore, resistant pAML upregulated the membrane glycoprotein CD200, which binds to the inhibitory receptor CD200R1 on LV-10 cells. In order to examine if CD200 expression on target cells can impair LV-10 cell function, we overexpressed CD200 in myeloid leukemia cell lines ordinarily sensitive to LV-10 killing. Indeed, LV-10 cells degranulated less and killed fewer CD200-overexpressing cells compared to controls, indicating that pAML can utilize CD200 expression for immune evasion. Altogether, the majority of pAML are killed by LV-10 cells in vitro, supporting further LV-10 cell development as an innovative cell therapy for pAML.

Longitudinal immune profiling reveals key myeloid signatures associated with COVID-19.

COVID-19 pathogenesis is associated with an exaggerated immune response. However, the specific cellular mediators and inflammatory components driving diverse clinical disease outcomes remain poorly understood. We undertook longitudinal immune profiling on both whole blood and peripheral blood mononuclear cells (PBMCs) of hospitalized patients during the peak of the COVID-19 pandemic in the UK. Here, we report key immune signatures present shortly after hospital admission that were associated with the severity of COVID-19. Immune signatures were related to shifts in neutrophil to T cell ratio, elevated serum IL-6, MCP-1 and IP-10, and most strikingly, modulation of CD14+ monocyte phenotype and function. Modified features of CD14+ monocytes included poor induction of the prostaglandin-producing enzyme, COX-2, as well as enhanced expression of the cell cycle marker Ki-67. Longitudinal analysis revealed reversion of some immune features back to the healthy median level in patients with a good eventual outcome. These findings identify previously unappreciated alterations in the innate immune compartment of COVID-19 patients and lend support to the idea that therapeutic strategies targeting release of myeloid cells from bone marrow should be considered in this disease. Moreover, they demonstrate that features of an exaggerated immune response are present early after hospital admission suggesting immune-modulating therapies would be most beneficial at early timepoints.

Tumor Infiltrating Lymphocytes Signature as a New Pan-Cancer Predictive Biomarker of Anti PD-1/PD-L1 Efficacy.

Tumor immune infiltrates are associated with tumor prognosis in many cancer types. However, their capacity to predict the efficacy of checkpoint inhibitors is poorly documented. We generate three signatures that evaluate in different ways these infiltrates: lymphoid- and myeloid-alone signatures, and a combined signature of both named the TIL (tumor-infiltrating lymphocyte) transcriptomic signature. We evaluate these signatures in The Cancer Genome Atlas Program (TCGA) Pan-Cancer cohort and four cohorts comprising patients with melanoma, lung, and head and neck cancer treated with anti-PD-1 or anti-CTLA-4 therapies. We observe using TCGA Pan-Cancer cohort that this TIL or lymphoid-alone signature accurately estimates prognosis in most cancer types and outperforms histological TIL evaluation or myeloid signature alone. Both TIL and lymphoid signatures are correlated with response rate to immunotherapy. Combining lymphoid signature or TIL with tumor mutational burden generates a score that is highly efficient in predicting response to immunotherapy. In different series of patients treated with checkpoint inhibitors for non-small cell lung cancer, head and neck cancer, and melanoma, we observed that TIL or lymphoid signature were associated with outcome. These data demonstrate that a simple TIL or lymphoid signature could be used as a Pan-Cancer prognostic and predictive biomarker to estimate patient survival under checkpoint inhibitors.

Patient ancestry significantly contributes to molecular heterogeneity of systemic lupus erythematosus.

Gene expression signatures can stratify patients with heterogeneous diseases, such as systemic lupus erythematosus (SLE), yet understanding the contributions of ancestral background to this heterogeneity is not well understood. We hypothesized that ancestry would significantly influence gene expression signatures and measured 34 gene modules in 1566 SLE patients of African ancestry (AA), European ancestry (EA), or Native American ancestry (NAA). Healthy subject ancestry-specific gene expression provided the transcriptomic background upon which the SLE patient signatures were built. Although standard therapy affected every gene signature and significantly increased myeloid cell signatures, logistic regression analysis determined that ancestral background significantly changed 23 of 34 gene signatures. Additionally, the strongest association to gene expression changes was found with autoantibodies, and this also had etiology in ancestry: the AA predisposition to have both RNP and dsDNA autoantibodies compared with EA predisposition to have only anti-dsDNA. A machine learning approach was used to determine a gene signature characteristic to distinguish AA SLE and was most influenced by genes characteristic of the perturbed B cell axis in AA SLE patients.

Myeloid signature reveals immune contexture and predicts the prognosis of hepatocellular carcinoma.

BACKGROUNDDespite an increasing appreciation of the roles that myeloid cells play in tumor progression and therapy, challenges remain in interpreting the tumor-associated myeloid response balance and its translational value. We aimed to construct a simple and reliable myeloid signature for hepatocellular carcinoma (HCC).METHODSUsing in situ immunohistochemistry, we assessed the distribution of major myeloid subtypes in both peri- and intratumoral regions of HCC. A 2-feature-based, myeloid-specific prognostic signature, named the myeloid response score (MRS), was constructed using an L1-penalized Cox regression model based on data from a training subset (n = 244), a test subset (n = 244), and an independent internal (n = 341) and 2 external (n = 94; n = 254) cohorts.RESULTSThe MRS and the MRS-based nomograms displayed remarkable discriminatory power, accuracy, and clinical usefulness for predicting recurrence and patient survival, superior to current staging algorithms. Moreover, an increase in MRS was associated with a shift in the myeloid response balance from antitumor to protumor activities, accompanied by enhanced CD8+ T cell exhaustion patterns. Additionally, we provide evidence that the MRS was associated with the efficacy of sorafenib treatment for recurrent HCC.CONCLUSIONWe identified and validated a simple myeloid signature for HCC that showed remarkable prognostic potential and may serve as a basis for the stratification of HCC immune subtypes.FUNDINGThis work was supported by the National Science and Technology Major Project of China, the National Natural Science Foundation of China, the Science and Information Technology of Guangzhou, the Fundamental Research Funds for the Central Universities, the Guangdong Basic and Applied Basic Research Foundation, and the China Postdoctoral Science Foundation.

Characterization of a Myeloid Activation Signature that Correlates with Survival in Melanoma Patients.

Understanding the cellular interactions within the tumor microenvironment (TME) of melanoma paved the way for novel therapeutic modalities, such as T cell-targeted immune checkpoint inhibitors (ICI). However, only a limited fraction of patients benefits from such therapeutic modalities, highlighting the need for novel predictive and prognostic biomarkers. As myeloid cells orchestrate the tumor-specific immune response and influence the efficacy of ICI, assessing their activation state within the TME is of clinical relevance. Here, we characterized a myeloid activation (MA) signature, comprising the three genes Cxcl11, Gbp1, and Ido1, from gene expression data of human myeloid cells stimulated with poly(I:C) or cGAMP. This MA signature positively correlated to overall survival in melanoma. In addition, increased expression of the MA signature was observed in melanoma patients responding to ICI (anti-PD-1), as compared to non-responders. Furthermore, the MA signature was validated in the murine B16F10 melanoma model where it was induced and associated with decreased tumor growth upon intratumoral administration of poly(I:C) and cGAMP. Finally, we were able to visualize co-expression of the MA signature genes in myeloid cells of human melanoma tissues using RNAscope in situ hybridization. In conclusion, the MA signature indicates the activation state of myeloid cells and represents a prognostic biomarker for the overall survival in melanoma patients.

Myeloid Response Score (MRS): A Simple Signature That Reveals the Myeloid Contexture and Predicts the Prognosis of Hepatocellular Carcinoma

Abstract Myeloid cells, including monocytes/macrophages (Mo/Mϕ), neutrophils, and dendritic cells, can mediate divergent immune responses according to different pathological conditions. In cancer, myeloid cells play important roles in disease progression and therapy, suggesting that appraising the overall and exact intratumoral myeloid response may provide valuable information that may help to predict cancer prognosis and tailor more precise therapies. In this study, we screened biomarkers that cover major myeloid subtypes in human tumors, then constructed and validated a simple prognostic signature, called Myeloid Response Score (MRS), based on a dataset from a multi-center cohort of 1,177 HCC patients. Using multiplex immunofluorescence staining, we observed a shift of predominant myeloid population from CD169+, CD169−CD11b−CD14+, and CD169−CD11b−CD163+ Mo/Mϕs in MRSlow HCC to neutrophils and CD11b+CD169− Mo/Mϕs in MRShigh tumor. Transcriptomic and flow cytometric analyses showed that MRShigh HCC exhibited patterns of CD8+ T cell exhaustion including impaired cytotoxicity and increased PD-1 expression, suggesting highly suppressive immune microenvironment. The “climb trajectory” of MRSs between HCC tumors were depicted using the TCGA dataset, and was linked to CD8+ T cell dysfunction and poorer survival of patients. Moreover, MRS-based nomograms were highly discriminative, accurate, and useful in predicting HCC prognosis and we provide additional evidence that MRS may associate with the efficacies of post-recurrence sorafenib and transcatheter arterial chemoembolization (TACE) treatments. Together, we constructed and validated a new myeloid signature with valuable immunological and clinical potential.

Expansion of Neutrophils and Classical and Nonclassical Monocytes as a Hallmark in Relapsing-Remitting Multiple Sclerosis.

Neutrophils and monocytes encompassing the classical, intermediate, and nonclassical population constitute the majority of circulating myeloid cells in humans and represent the first line of innate immune defense. As such, changes in their relative and absolute amounts serve as sensitive markers of diverse inflammatory conditions. Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system, causing demyelination and axonal loss, affecting various neuron functions and often causing irreversible neurological disability. MS disease course is individually highly heterogeneous but can be classified as progressive (PMS) or relapsing-remitting (RRMS). Each MS course type may be further characterized as active or inactive, depending on the recent disability progression and/or current relapses. Data on specific alterations of the myeloid compartment in association with MS disease course are scarce and conflicting. In the current study, we systematically immunophenotyped blood myeloid leukocytes by flow cytometry in 15 healthy and 65 MS subjects. We found a highly significant expansion of granulocytes, CD15+ neutrophils, and classical and nonclassical monocytes in inactive RRMS (RRMSi) with concomitant shrinkage of the lymphocyte compartment, which did not correlate with biochemical readouts of systemic inflammation. Each of these leukocyte populations and the combined myeloid signature accurately differentiated RRMSi from other MS forms. Additionally, nonclassical monocyte proportions were particularly elevated in RRMSi individuals receiving disease-modifying therapy (DMT), such as natalizumab. Our results suggest that flow cytometry-based myeloid cell immunophenotyping in MS may help to identify RRMSi earlier and facilitate monitoring of DMT response.

Normal myeloid progenitor cell subset-associated gene signatures for acute myeloid leukaemia subtyping with prognostic impact.

Acute myeloid leukaemia (AML) is characterised by phenotypic heterogeneity, which we hypothesise is a consequence of deregulated differentiation with transcriptional reminiscence of the normal compartment or cell-of-origin. Here, we propose a classification system based on normal myeloid progenitor cell subset-associated gene signatures (MAGS) for individual assignments of AML subtypes. We generated a MAGS classifier including the progenitor compartments CD34+/CD38- for haematopoietic stem cells (HSCs), CD34+/CD38+/CD45RA- for megakaryocyte-erythroid progenitors (MEPs), and CD34+/CD38+/CD45RA+ for granulocytic-monocytic progenitors (GMPs) using regularised multinomial regression with three discrete outcomes and an elastic net penalty. The regularisation parameters were chosen by cross-validation, and MAGS assignment accuracy was validated in an independent data set (N = 38; accuracy = 0.79) of sorted normal myeloid subpopulations. The prognostic value of MAGS assignment was studied in two clinical cohorts (TCGA: N = 171; GSE6891: N = 520) and had a significant prognostic impact. Furthermore, multivariate Cox regression analysis using the MAGS subtype, FAB subtype, cytogenetics, molecular genetics, and age as explanatory variables showed independent prognostic value. Molecular characterisation of subtypes by differential gene expression analysis, gene set enrichment analysis, and mutation patterns indicated reduced proliferation and overrepresentation of RUNX1 and IDH2 mutations in the HSC subtype; increased proliferation and overrepresentation of CEBPA mutations in the MEP subtype; and innate immune activation and overrepresentation of WT1 mutations in the GMP subtype. We present a differentiation-dependent classification system for AML subtypes with distinct pathogenetic and prognostic importance that can help identify candidates poorly responding to combination chemotherapy and potentially guide alternative treatments.

Immunogenomic signatures to predict outcome in ovarian and endometrial cancers: Potential strategies in targeting the tumor immune microenvironment to improve response to immunotherapy.

4 Background: Ovarian and MSS endometrial cancers are characterized by immunosuppressive microenvironment (TME) and low response to immunotherapy with checkpoint inhibitors (CPI). Targeting immunosuppressive factors within TMErepresents an attractive approach to enhance response to CPI. Therefore, we sought to investigate different immunogenomic signatures and immune cells within TME and correlate them with survival. Methods: We used whole transcriptome sequencing of matched tumor-normal samples from 38 uterine serous cancer and TCGA data of ovarian (n = 374) and endometrial cancers (n = 541). Immunogenomic signatures focusing on Transforming Growth Factor (TGFβ), 18-genes IFN-γ and myeloid signatures (CD47 and B7H4 expressions) and immune cell abundance were investigated. Gene expression score was calculated by averaging the normalized and log transformed individual gene read counts. The optimized score cut off was selected to best separate the survival in the pilot cohort. Then the score was tested in TCGA RNAseq datasets. Population abundance of tissue-infiltrating immune cells were estimated using MCPcounter R package from bulk transcriptome data. Results: Higher IFN-γ and lower TGF-β signatures predicted better survival for endometrial and ovarian cancers (p < 0.05). The impact of TGF-β was higher in MSI-H vs. MSS cancers (p = 0.013 vs. 0.09). High CD47 predicted poor survival in endometrial cancer. Combined IFN-γ and TGF-β signatures predicted survival in the ovarian and endometrial cohorts (p < 0.001). Combined IFN-γ and CD47 expression predicted survival in endometrial cancer (p = 0.033). Analysis of immune cell abundance revealed enrichment of monocytic lineage and neutrophils but paucity of cytotoxic T-cells, NK cells, dendritic cells and B-cells. Immune cell abundance is being correlated with survival. Conclusions: Our data support the role of immunogenomic markers in predicting survival. We are evaluating these markers in predicting response to CPI in a pilot cohort. Immunogenomic markers represent the tumor microenvironment, can potentially guide rationale combination immunotherapy.

Transcriptome reprogramming and myeloid skewing in haematopoietic stem and progenitor cells in systemic lupus erythematosus

ObjectivesHaematopoietic stem and progenitor cells (HSPCs) are multipotent cells giving rise to both myeloid and lymphoid cell lineages. We reasoned that the aberrancies of immune cells in systemic lupus erythematosus...

Critical Adjuvant Influences on Preventive Anti-Metastasis Vaccine Using a Structural Epitope Derived from Membrane Type Protease PRSS14.

We tested how adjuvants effect in a cancer vaccine model using an epitope derived from an autoactivation loop of membrane-type protease serine protease 14 (PRSS14; loop metavaccine) in mouse mammary tumor virus (MMTV)-polyoma middle tumor-antigen (PyMT) system and in 2 other orthotopic mouse systems. Earlier, we reported that loop metavaccine effectively prevented progression and metastasis regardless of adjuvant types and TH types of hosts in tail-vein injection systems. However, the loop metavaccine with Freund's complete adjuvant (CFA) reduced cancer progression and metastasis while that with alum, to our surprise, were adversely affected in 3 tumor bearing mouse models. The amounts of loop peptide specific antibodies inversely correlated with tumor burden and metastasis, meanwhile both TH1 and TH2 isotypes were present regardless of host type and adjuvant. Tumor infiltrating myeloid cells such as eosinophil, monocyte, and neutrophil were asymmetrically distributed among 2 adjuvant groups with loop metavaccine. Systemic expression profiling using the lymph nodes of the differentially immunized MMTV-PyMT mouse revealed that adjuvant types, as well as loop metavaccine can change the immune signatures. Specifically, loop metavaccine itself induces TH2 and TH17 responses but reduces TH1 and Treg responses regardless of adjuvant type, whereas CFA but not alum increased follicular TH response. Among the myeloid signatures, eosinophil was most distinct between CFA and alum. Survival analysis of breast cancer patients showed that eosinophil chemokines can be useful prognostic factors in PRSS14 positive patients. Based on these observations, we concluded that multiple immune parameters are to be considered when applying a vaccine strategy to cancer patients.