Comparison of the tumor immune microenvironment and checkpoint blockade biomarkers between stage III and IV non-small cell lung cancer.

BACKGROUND: Adjuvant immune checkpoint blockade (ICB) following chemoradiotherapy and adding ICB to chemotherapy have been key advances for stages III-IV non-small cell lung cancer (NSCLC) treatment. However, known biomarkers like PD-L1 are not consistently indicative of ICB response. Other markers within the tumor immune microenvironment (TIME) may better reflect ICB response and/or resistance mechanisms, but an understanding of how TIMEs differ between stage III and IV NSCLC has not been explored. METHODS: Real-world data from unresectable, stage III-IV, non-squamous, pretreatment NSCLCs (stage III n = 106, stage IV n = 285) were retrospectively analyzed. PD-L1 immunohistochemistry (IHC) was compared to CD274 gene expression. Then, differential gene expression levels, pathway enrichment, and immune infiltrate between stages were calculated from whole-transcriptome RNA-seq. Analyses were stratified by EGFR status. RESULTS: PD-L1 IHC and CD274 expression in tumor cells were highly correlated (n = 295, P < 2.2e-16, ⍴ = 0.74). CTLA4 expression was significantly increased in stage III tumors (P = 1.32e-04), while no differences were observed for other ICB-related genes. Metabolic pathway activity was significantly enriched in stage IV tumors (P = 0.004), whereas several immune-related KEGG pathways were enriched in stage III. Stage IV tumors had significantly increased macrophage infiltration (P = 0.0214), and stage III tumors had a significantly higher proportion of CD4 + T cells (P = 0.017). CD4 + T cells were also relatively more abundant in EGFR-mutant tumors vs. wild-type (P = 0.0081). CONCLUSION: Directly comparing the TIMEs of stage III and IV NSCLC, these results carry implications for further studies of ICB response in non-resectable stage III NSCLC and guide further research of prognostic biomarkers and therapeutic targets.

Integration of tumor extrinsic and intrinsic features associates with immunotherapy response in non-small cell lung cancer.

The efficacy of immune checkpoint blockade (ICB) varies greatly among metastatic non-small cell lung cancer (NSCLC) patients. Loss of heterozygosity at the HLA-I locus (HLA-LOH) has been identified as an important immune escape mechanism. However, despite HLA-I disruptions in their tumor, many patients have durable ICB responses. Here we seek to identify HLA-I-independent features associated with ICB response in NSCLC. We use single-cell profiling to identify tumor-infiltrating, clonally expanded CD4+ T cells that express a canonical cytotoxic gene program and NSCLC cells with elevated HLA-II expression. We postulate cytotoxic CD4+ T cells mediate anti-tumor activity via HLA-II on tumor cells and augment HLA-I-dependent cytotoxic CD8+ T cell interactions to drive ICB response in NSCLC. We show that integrating tumor extrinsic cytotoxic gene expression with tumor mutational burden is associated with longer time to progression in a real-world cohort of 123 NSCLC patients treated with ICB regimens, including those with HLA-LOH.

Clinical, Genomic, and Transcriptomic Data Profiling of Biliary Tract Cancer Reveals Subtype-Specific Immune Signatures.

PURPOSE: Biliary tract cancers (BTCs) are aggressive cancers that carry a poor prognosis. An enhanced understanding of the immune landscape of anatomically and molecularly defined subsets of BTC may improve patient selection for immunotherapy and inform immune-based combination treatment strategies. METHODS: We analyzed deidentified clinical, genomic, and transcriptomic data from the Tempus database to determine the mutational frequency and mutational clustering across the three major BTC subtypes (intrahepatic cholangiocarcinoma [IHC], extrahepatic cholangiocarcinoma, and gallbladder cancer). We subsequently determined the relationship between specific molecular alterations and anatomical subsets and features of the BTC immune microenvironment. RESULTS: We analyzed 454 samples of BTC, of which the most commonly detected alterations were TP53 (42.5%), CDKN2A (23.4%), ARID1A (19.6%), BAP1 (15.5%), KRAS (15%), CDKN2B (14.2%), PBRM1 (11.7%), IDH1 (11.7%), TERT (8.4%), KMT2C (10.4%) and LRP1B (8.4%), and FGFR2 fusions (8.7%). Potentially actionable molecular alterations were identified in 30.5% of BTCs including 39.1% of IHC. Integrative cluster analysis revealed four distinct molecular clusters, with cluster 4 predominately associated with FGFR2 rearrangements and BAP1 mutations in IHC. Immune-related biomarkers indicative of an inflamed tumor-immune microenvironment were elevated in gallbladder cancers and in cluster 1, which was enriched for TP53, KRAS, and ATM mutations. Multiple common driver genes, including TP53, FGFR2, IDH1, TERT, BRAF, and BAP1, were individually associated with unique BTC immune microenvironments. CONCLUSION: BTC subtypes exhibit diverse DNA alterations, RNA inflammatory signatures, and immune biomarkers. The association between specific BTC anatomical subsets, molecular alterations, and immunophenotypes highlights new opportunities for therapeutic development.

Integrated genomic profiling expands clinical options for patients with cancer.

Genomic analysis of paired tumor-normal samples and clinical data can be used to match patients to cancer therapies or clinical trials. We analyzed 500 patient samples across diverse tumor types using the Tempus xT platform by DNA-seq, RNA-seq and immunological biomarkers. The use of a tumor and germline dataset led to substantial improvements in mutation identification and a reduction in false-positive rates. RNA-seq enhanced gene fusion detection and cancer type classifications. With DNA-seq alone, 29.6% of patients matched to precision therapies supported by high levels of evidence or by well-powered studies. This proportion increased to 43.4% with the addition of RNA-seq and immunotherapy biomarker results. Combining these data with clinical criteria, 76.8% of patients were matched to at least one relevant clinical trial on the basis of biomarkers measured by the xT assay. These results indicate that extensive molecular profiling combined with clinical data identifies personalized therapies and clinical trials for a large proportion of patients with cancer and that paired tumor-normal plus transcriptome sequencing outperforms tumor-only DNA panel testing.

Clinical validation of the tempus xT next-generation targeted oncology sequencing assay.

We developed and clinically validated a hybrid capture next generation sequencing assay to detect somatic alterations and microsatellite instability in solid tumors and hematologic malignancies. This targeted oncology assay utilizes tumor-normal matched samples for highly accurate somatic alteration calling and whole transcriptome RNA sequencing for unbiased identification of gene fusion events. The assay was validated with a combination of clinical specimens and cell lines, and recorded a sensitivity of 99.1% for single nucleotide variants, 98.1% for indels, 99.9% for gene rearrangements, 98.4% for copy number variations, and 99.9% for microsatellite instability detection. This assay presents a wide array of data for clinical management and clinical trial enrollment while conserving limited tissue.

Nur77 Links Chronic Antigen Stimulation to B Cell Tolerance by Restricting the Survival of Self-Reactive B Cells in the Periphery.

Nur77 (Nr4a1) belongs to a small family of orphan nuclear receptors that are rapidly induced by BCR stimulation, yet little is known about its function in B cells. We have previously characterized a reporter of Nr4a1 transcription, Nur77-eGFP, in which GFP expression faithfully detects Ag encounter by B cells in vitro and in vivo. In this study, we report that Nur77 expression correlates with the degree of self-reactivity, counterselection, and anergy among individual B cell clones from two distinct BCR transgenic mouse models but is dispensable for all of these tolerance mechanisms. However, we identify a role for Nur77 in restraining survival of self-reactive B cells in the periphery under conditions of competition for a limited supply of the survival factor BAFF. We find that Nur77 deficiency results in the progressive accumulation of self-reactive B cells in the mature repertoire with age and is sufficient to break B cell tolerance in VH3H9 H chain transgenic mice. We thus propose that Nur77 is upregulated in self-reactive B cells in response to chronic Ag stimulation and selectively restricts the survival of these cells, gradually pruning self-reactivity from the mature repertoire to impose a novel layer of peripheral B cell tolerance.

Integrating RNA expression and visual features for immune infiltrate prediction.

Patient responses to cancer immunotherapy are shaped by their unique genomic landscape and tumor microenvironment. Clinical advances in immunotherapy are changing the treatment landscape by enhancing a patient's immune response to eliminate cancer cells. While this provides potentially beneficial treatment options for many patients, only a minority of these patients respond to immunotherapy. In this work, we examined RNA-seq data and digital pathology images from individual patient tumors to more accurately characterize the tumor-immune microenvironment. Several studies implicate an inflamed microenvironment and increased percentage of tumor infiltrating immune cells with better response to specific immunotherapies in certain cancer types. We developed NEXT (Neural-based models for integrating gene EXpression and visual Texture features) to more accurately model immune infiltration in solid tumors. To demonstrate the utility of the NEXT framework, we predicted immune infiltrates across four different cancer types and evaluated our predictions against expert pathology review. Our analyses demonstrate that integration of imaging features improves prediction of the immune infiltrate. Of note, this effect was preferentially observed for B cells and CD8 T cells. In sum, our work effectively integrates both RNA-seq and imaging data in a clinical setting and provides a more reliable and accurate prediction of the immune composition in individual patient tumors.

RNA Sequencing of the Tumor Microenvironment in Precision Cancer Immunotherapy.

RNA sequencing (RNA-seq) provides an efficient high-throughput technique to robustly characterize the tumor immune microenvironment (TME). The increasing use of RNA-seq in clinical and basic science settings provides a powerful opportunity to access novel therapeutic biomarkers in the TME. Advanced computational methods are making it possible to resolve the composition of the tumor immune infiltrate, infer the immunological phenotypes of those cells, and assess the immune receptor repertoire in RNA-seq data. These immunological characterizations have increasingly important implications for guiding immunotherapy use. Here, we highlight recent studies that demonstrate the potential utility of RNA-seq in clinical settings, review key computational methods used for characterizing the TME for precision cancer immunotherapy, and discuss important considerations in data interpretation and current technological limitations.

Myeloperoxidase aggravates pulmonary arterial hypertension by activation of vascular Rho-kinase.

Pulmonary arterial hypertension (PAH) remains a disease with limited therapeutic options and dismal prognosis. Despite its etiologic heterogeneity, the underlying unifying pathophysiology is characterized by increased vascular tone and adverse remodeling of the pulmonary circulation. Myeloperoxidase (MPO), an enzyme abundantly expressed in neutrophils, has potent vasoconstrictive and profibrotic properties, thus qualifying as a potential contributor to this disease. Here, we sought to investigate whether MPO is causally linked to the pathophysiology of PAH. Investigation of 2 independent clinical cohorts revealed that MPO plasma levels were elevated in subjects with PAH and predicted adverse outcome. Experimental analyses showed that, upon hypoxia, right ventricular pressure was less increased in Mpo-/- than in WT mice. The hypoxia-induced activation of the Rho-kinase pathway, a critical subcellular signaling pathway yielding vasoconstriction and structural vascular remodeling, was blunted in Mpo-/- mice. Mice subjected to i.v. infusion of MPO revealed activation of Rho-kinase and increased right ventricular pressure, which was prevented by coinfusion of the Rho-kinase inhibitor Y-27632. In the Sugen5416/hypoxia rat model, PAH was attenuated by the MPO inhibitor AZM198. The current data demonstrate a tight mechanistic link between MPO, the activation of Rho-kinase, and adverse pulmonary vascular function, thus pointing toward a potentially novel avenue of treatment.

Clinical validation of the Tempus xO assay.

We have developed a clinically validated NGS assay that includes tumor, germline and RNA sequencing. We apply this assay to clinical specimens and cell lines, and we demonstrate a clinical sensitivity of 98.4% and positive predictive value of 100% for the clinically actionable variants measured by the assay. We also demonstrate highly accurate copy number measurements and gene rearrangement identification.

Low CD21 expression defines a population of recent germinal center graduates primed for plasma cell differentiation.

In this study, we report that antigen-specific CD19+CD27+CD21lo (CD21lo) B cells are transiently induced 14 to 28 days after immunization, at the time germinal centers (GCs) peak. Although clonally related to memory B cells and plasmablasts, CD21lo cells form distinct clades within phylogenetic trees based on accumulated variable gene mutations, supporting exit from active GCs. CD21lo cells express a transcriptional program, suggesting that they are primed for plasma cell differentiation and are refractory to GC differentiation, although they do not spontaneously secrete antibody. In addition, CD21lo cells differentially express multiple cell surface markers and have elevated intracellular levels of Blimp-1 and T-bet protein compared with memory B cells. Together, these data support a model in which CD21lo cells are recent GC graduates that represent a distinct population from CD27+ classical memory cells, are refractory to GC reentry, and are predisposed to differentiate into long-lived plasma cells.

Myeloperoxidase Mediates Postischemic Arrhythmogenic Ventricular Remodeling.

RATIONALE: Ventricular arrhythmias remain the leading cause of death in patients suffering myocardial ischemia. Myeloperoxidase, a heme enzyme released by polymorphonuclear neutrophils, accumulates within ischemic myocardium and has been linked to adverse left ventricular remodeling. OBJECTIVE: To reveal the role of myeloperoxidase for the development of ventricular arrhythmias. METHODS AND RESULTS: In different murine models of myocardial ischemia, myeloperoxidase deficiency profoundly decreased vulnerability for ventricular tachycardia on programmed right ventricular and burst stimulation and spontaneously as assessed by ECG telemetry after isoproterenol injection. Experiments using CD11b/CD18 integrin-deficient (CD11b-/-) mice and intravenous myeloperoxidase infusion revealed that neutrophil infiltration is a prerequisite for myocardial myeloperoxidase accumulation. Ventricles from myeloperoxidase-deficient (Mpo-/-) mice showed less pronounced slowing and decreased heterogeneity of electric conduction in the peri-infarct zone than wild-type mice. Expression of the redox-sensitive gap junctional protein Cx43 (Connexin 43) was reduced in the peri-infarct area of wild-type compared with Mpo-/- mice. In isolated wild-type cardiomyocytes, Cx43 protein content decreased on myeloperoxidase/H2O2 incubation. Mapping of induced pluripotent stem cell-derived cardiomyocyte networks and in vivo investigations linked Cx43 breakdown to myeloperoxidase-dependent activation of matrix metalloproteinase 7. Moreover, Mpo-/- mice showed decreased ventricular postischemic fibrosis reflecting reduced accumulation of myofibroblasts. Ex vivo, myeloperoxidase was demonstrated to induce fibroblast-to-myofibroblast transdifferentiation by activation of p38 mitogen-activated protein kinases resulting in upregulated collagen generation. In support of our experimental findings, baseline myeloperoxidase plasma levels were independently associated with a history of ventricular arrhythmias, sudden cardiac death, or implantable cardioverter-defibrillator implantation in a cohort of 2622 stable patients with an ejection fraction >35% undergoing elective diagnostic cardiac evaluation. CONCLUSIONS: Myeloperoxidase emerges as a crucial mediator of postischemic myocardial remodeling and may evolve as a novel pharmacological target for secondary disease prevention after myocardial ischemia.

Development of nonfibrotic left ventricular hypertrophy in an ANG II-induced chronic ovine hypertension model.

Hypertension is a major risk factor for many cardiovascular diseases and leads to subsequent concomitant pathologies such as left ventricular hypertrophy (LVH). Translational approaches using large animals get more important as they allow the use of standard clinical procedures in an experimental setting. Therefore, the aim of this study was to establish a minimally invasive ovine hypertension model using chronic angiotensin II (ANG II) treatment and to characterize its effects on cardiac remodeling after 8 weeks. Sheep were implanted with osmotic minipumps filled with either vehicle control (n = 7) or ANG II (n = 9) for 8 weeks. Mean arterial blood pressure in the ANG II-treated group increased from 87.4 ± 5.3 to 111.8 ± 6.9 mmHg (P = 0.00013). Cardiovascular magnetic resonance imaging showed an increase in left ventricular mass from 112 ± 12.6 g to 131 ± 18.7 g after 7 weeks (P = 0.0017). This was confirmed by postmortem measurement of left ventricular wall thickness which was higher in ANG II-treated animals compared to the control group (18 ± 4 mm vs. 13 ± 2 mm, respectively, P = 0.002). However, ANG II-treated sheep did not reveal any signs of fibrosis or inflammatory infiltrates as defined by picrosirius red and H&E staining on myocardial full thickness paraffin sections of both atria and ventricles. Measurements of plasma high-sensitivity C-reactive protein and urinary 8-iso-prostaglandin F2α were inconspicuous in all animals. Furthermore, multielectrode surface mapping of the heart did not show any differences in epicardial conduction velocity and heterogeneity. These data demonstrate that chronic ANG II treatment using osmotic minipumps presents a reliable, minimally invasive approach to establish hypertension and nonfibrotic LVH in sheep.

Molecular Characterization of the NLRC4 Expression in Relation to Interleukin-18 Levels.

BACKGROUND: Interleukin-18 (IL-18) is a pleiotropic cytokine centrally involved in the cytokine cascade with complex immunomodulatory functions in innate and acquired immunity. Circulating IL-18 concentrations are associated with type 2 diabetes mellitus, cardiovascular events, and diverse inflammatory and autoimmune disorders. METHODS AND RESULTS: To identify causal variants affecting circulating IL-18 concentrations, we applied various omics and molecular biology approaches. By genome-wide association study, we confirmed association of IL-18 levels with a single nucleotide polymorphism in the untranslated exon 2 of the inflammasome component NLRC4 (NLR family, caspase recruitment domain-containing 4) gene on chromosome 2 (rs385076; P=2.4 × 10(-45)). Subsequent molecular analyses by gene expression analysis and reporter gene assays indicated an effect of rs385076 on NLRC4 expression and differential isoform usage by modulating binding of the transcription factor PU.1. CONCLUSIONS: Our study provides evidence for the functional causality of single nucleotide polymorphism rs385076 within the NLRC4 gene in relation to IL-18 activation.

High Affinity Antibodies against Influenza Characterize the Plasmablast Response in SLE Patients After Vaccination.

Breakdown of B cell tolerance is a cardinal feature of systemic lupus erythematosus (SLE). Increased numbers of autoreactive mature naïve B cells have been described in SLE patients and autoantibodies have been shown to arise from autoreactive and non-autoreactive precursors. How these defects, in the regulation of B cell tolerance and selection, influence germinal center (GC) reactions that are directed towards foreign antigens has yet to be investigated. Here, we examined the characteristics of post-GC foreign antigen-specific B cells from SLE patients and healthy controls by analyzing monoclonal antibodies generated from plasmablasts induced specifically by influenza vaccination. We report that many of the SLE patients had anti-influenza antibodies with higher binding affinity and neutralization capacity than those from controls. Although overall frequencies of autoreactivity in the influenza-specific plasmablasts were similar for SLE patients and controls, the variable gene repertoire of influenza-specific plasmablasts from SLE patients was altered, with increased usage of JH6 and long heavy chain CDR3 segments. We found that high affinity anti-influenza antibodies generally characterize the plasmablast responses of SLE patients with low levels of autoreactivity; however, certain exceptions were noted. The high-avidity antibody responses in SLE patients may also be correlated with cytokines that are abnormally expressed in lupus. These findings provide insights into the effects of dysregulated immunity on the quality of antibody responses following influenza vaccination and further our understanding of the underlying abnormalities of lupus.

miRNA-197 and miRNA-223 Predict Cardiovascular Death in a Cohort of Patients with Symptomatic Coronary Artery Disease.

BACKGROUND: Circulating microRNAs (miRNAs) have been described as potential diagnostic biomarkers in cardiovascular disease and in particular, coronary artery disease (CAD). Few studies were undertaken to perform analyses with regard to risk stratification of future cardiovascular events. miR-126, miR-197 and miR-223 are involved in endovascular inflammation and platelet activation and have been described as biomarkers in the diagnosis of CAD. They were identified in a prospective study in relation to future myocardial infarction. OBJECTIVES: The aim of our study was to further evaluate the prognostic value of these miRNAs in a large prospective cohort of patients with documented CAD. METHODS: Levels of miR-126, miR-197 and miR-223 were evaluated in serum samples of 873 CAD patients with respect to the endpoint cardiovascular death. miRNA quantification was performed using real time polymerase chain reaction (RT-qPCR). RESULTS: The median follow-up period was 4 years (IQR 2.78-5.04). The median age of all patients was 64 years (IQR 57-69) with 80.2% males. 38.9% of the patients presented with acute coronary syndrome (ACS), 61.1% were diagnosed with stable angina pectoris (SAP). Elevated levels of miRNA-197 and miRNA-223 reliably predicted future cardiovascular death in the overall group (miRNA-197: hazard ratio (HR) 1.77 per one standard deviation (SD) increase (95% confidence interval (CI) 1.20; 2.60), p = 0.004, C-index 0.78; miRNA-223: HR 2.23 per one SD increase (1.20; 4.14), p = 0.011, C-index 0.80). In ACS patients the prognostic power of both miRNAs was even higher (miRNA-197: HR 2.24 per one SD increase (1.25; 4.01), p = 0.006, C-index 0.89); miRA-223: HR 4.94 per one SD increase (1.42; 17.20), p = 0.012, C-index 0.89). CONCLUSION: Serum-derived circulating miRNA-197 and miRNA-223 were identified as predictors for cardiovascular death in a large patient cohort with CAD. These results reinforce the assumption that circulating miRNAs are promising biomarkers with prognostic value with respect to future cardiovascular events.

Red blood cells serve as intravascular carriers of myeloperoxidase.

Myeloperoxidase (MPO) is a heme enzyme abundantly expressed in polymorphonuclear neutrophils. MPO is enzymatically capable of catalyzing the generation of reactive oxygen species (ROS) and the consumption of nitric oxide (NO). Thus MPO has both potent microbicidal and, upon binding to the vessel wall, pro-inflammatory properties. Interestingly, MPO - a highly cationic protein - has been shown to bind to both endothelial cells and leukocyte membranes. Given the anionic surface charge of red blood cells, we investigated binding of MPO to erythrocytes. Red blood cells (RBCs) derived from patients with elevated MPO plasma levels showed significantly higher amounts of MPO by flow cytometry and ELISA than healthy controls. Heparin-induced MPO-release from patient-derived RBCs was significantly increased compared to controls. Ex vivo experiments revealed dose and time dependency for MPO-RBC binding, and immunofluorescence staining as well as confocal microscopy localized MPO-RBC interaction to the erythrocyte plasma membrane. NO-consumption by RBC-membrane fragments (erythrocyte "ghosts") increased with incrementally greater concentrations of MPO during incubation, indicating preserved catalytic MPO activity. In vivo infusion of MPO-loaded RBCs into C57BL/6J mice increased local MPO tissue concentrations in liver, spleen, lung, and heart tissue as well as within the cardiac vasculature. Further, NO-dependent relaxation of aortic rings was altered by RBC bound-MPO and systemic vascular resistance significantly increased after infusion of MPO-loaded RBCs into mice. In summary, we find that MPO binds to RBC membranes in vitro and in vivo, is transported by RBCs to remote sites in mice, and affects endothelial function as well as systemic vascular resistance. RBCs may avidly bind circulating MPO, and act as carriers of this leukocyte-derived enzyme.

Myeloperoxidase upregulates endothelin receptor type B expression.

Neutrophil recruitment and activation are principal events in inflammation. Upon activation neutrophils release myeloperoxidase (MPO), a heme enzyme, which binds to and transcytoses endothelial cells. Whereas the significance of the subendothelial deposition of MPO has evolved as a critical prerequisite for the enzyme's suppression of nitric oxide (NO⋅) bioavailability, the functional consequences of MPO binding to and interaction with endothelial and smooth muscle cells remain poorly understood. Cultured human endothelial cells (HUVECs) were exposed to MPO. Gene expression of the endothelin receptor type B (ETRB), which is critically involved not only in endothelin-1 clearance, but also in endothelin-mediated vasoconstriction, was significantly increased. Real time PCR, Western blotting and immunofluorescence confirmed up-regulation of ETRB in MPO-treated endothelial cells. Inhibition of MPO's enzymatic activity blunted the increase in ETRB protein expression. Treatment of the cells with the MAP kinase inhibitors PD98059 or SB203580 indicates that MPO activates ETRB expression via MAP kinase pathways. On human smooth muscle cells (HAoSMCs), which not only express the endothelin receptor type B (ETRB) but also express the endothelin receptor type A (ETRA), MPO also stimulated ETRB expression as opposed to ETRA expression, which remained unchanged. Functional ex vivo organ bath chamber studies with MPO-incubated rat femoral artery sections revealed increased ETRB agonist dependent constriction. Binding of MPO to endothelial and vascular smooth muscle cells increases expression of the endothelin receptor type B (ETRB) via classical MAP kinase pathways. This suggests that MPO not only affects vasomotion by reducing the bioavailability of vasodilating molecules but also by increasing responsiveness to vasoconstrictors, further advocating for MPO as a central, leukocyte-derived regulator of vascular tone.