Epithelioid haemangioendothelioma-a rare cause of right pleural effusion and multiple primary nodules: Case report & review of the literature.

Pulmonary epithelioid haemangioendothelioma (PEH) is a rare vascular neoplasm. The thoracic manifestation of this disorder is identified via three major imaging patterns, namely multiple primary nodules, diffuse infiltrative pleural thickening, and multiple pulmonary reticulonodular opacities. The commonest pattern of presentation is bilateral multiple nodules. Diagnosis is based on histopathological findings and verified by positive immunochemistry staining. Patients with PEH are usually asymptomatic. We report the case of a 51-year-old female who presented to our facility with a five-month history of cough, pleural nodularity, and pleural effusion. She underwent surgical washout with right pleural biopsies that showed a malignant epithelioid tumor with features of epithelioid haemangioendothelioma (EH). A CXR after treatment did not demonstrate a residual pleural effusion.

Dynamic CD8+ T cell responses to cancer immunotherapy in human regional lymph nodes are disrupted in metastatic lymph nodes.

CD8+ T cell responses are critical for anti-tumor immunity. While extensively profiled in the tumor microenvironment, recent studies in mice identified responses in lymph nodes (LNs) as essential; however, the role of LNs in human cancer patients remains unknown. We examined CD8+ T cells in human head and neck squamous cell carcinomas, regional LNs, and blood using mass cytometry, single-cell genomics, and multiplexed ion beam imaging. We identified progenitor exhausted CD8+ T cells (Tpex) that were abundant in uninvolved LN and clonally related to terminally exhausted cells in the tumor. After anti-PD-L1 immunotherapy, Tpex in uninvolved LNs reduced in frequency but localized near dendritic cells and proliferating intermediate-exhausted CD8+ T cells (Tex-int), consistent with activation and differentiation. LN responses coincided with increased circulating Tex-int. In metastatic LNs, these response hallmarks were impaired, with immunosuppressive cellular niches. Our results identify important roles for LNs in anti-tumor immune responses in humans.

Development of a novel, highly sensitive assay for quantification of minimal residual B cells in autoimmune disease and comparison to traditional methods across B-cell-depleting agents.

Targeted B-cell depletion is a useful therapy for many diseases, including autoimmune disorders and certain cancers. We developed a sensitive blood B-cell depletion assay, MRB 1.1, compared its performance with the T-cell/B-cell/NK-cell (TBNK) assay, and assessed B-cell depletion with different therapies. The empirically defined lower limit of quantification (LLOQ) for CD19+ cells in the TBNK assay was 10 cells/µL, and 0.441 cells/µL for the MRB 1.1 assay. The TBNK LLOQ was used to compare differences between B-cell depletion in similar lupus nephritis patient populations who received rituximab (LUNAR), ocrelizumab (BELONG), or obinutuzumab (NOBILITY). After 4 weeks, 10% of patients treated with rituximab retained detectable B cells vs 1.8% with ocrelizumab and 1.7% for obinutuzumab; at 24 weeks 93% of patients who received obinutuzumab remained below LLOQ vs 63% for rituximab. More-sensitive measurements of B cells may reveal differences in potency among anti-CD20 agents, which may associate with clinical outcomes.

Mechanistic convergence of the TIGIT and PD-1 inhibitory pathways necessitates co-blockade to optimize anti-tumor CD8+ T cell responses.

Dual blockade of the PD-1 and TIGIT coinhibitory receptors on T cells shows promising early results in cancer patients. Here, we studied the mechanisms whereby PD-1 and/or TIGIT blockade modulate anti-tumor CD8+ T cells. Although PD-1 and TIGIT are thought to regulate different costimulatory receptors (CD28 and CD226), effectiveness of PD-1 or TIGIT inhibition in preclinical tumor models was reduced in the absence of CD226. CD226 expression associated with clinical benefit in patients with non-small cell lung carcinoma (NSCLC) treated with anti-PD-L1 antibody atezolizumab. CD226 and CD28 were co-expressed on NSCLC infiltrating CD8+ T cells poised for expansion. Mechanistically, PD-1 inhibited phosphorylation of both CD226 and CD28 via its ITIM-containing intracellular domain (ICD); TIGIT's ICD was dispensable, with TIGIT restricting CD226 co-stimulation by blocking interaction with their common ligand PVR (CD155). Thus, full restoration of CD226 signaling, and optimal anti-tumor CD8+ T cell responses, requires blockade of TIGIT and PD-1, providing a mechanistic rationale for combinatorial targeting in the clinic.

Ulcerative colitis is characterized by a plasmablast-skewed humoral response associated with disease activity.

B cells, which are critical for intestinal homeostasis, remain understudied in ulcerative colitis (UC). In this study, we recruited three cohorts of patients with UC (primary cohort, n = 145; validation cohort 1, n = 664; and validation cohort 2, n = 143) to comprehensively define the landscape of B cells during UC-associated intestinal inflammation. Using single-cell RNA sequencing, single-cell IgH gene sequencing and protein-level validation, we mapped the compositional, transcriptional and clonotypic landscape of mucosal and circulating B cells. We found major perturbations within the mucosal B cell compartment, including an expansion of naive B cells and IgG+ plasma cells with curtailed diversity and maturation. Furthermore, we isolated an auto-reactive plasma cell clone targeting integrin αvß6 from inflamed UC intestines. We also identified a subset of intestinal CXCL13-expressing TFH-like T peripheral helper cells that were associated with the pathogenic B cell response. Finally, across all three cohorts, we confirmed that changes in intestinal humoral immunity are reflected in circulation by the expansion of gut-homing plasmablasts that correlates with disease activity and predicts disease complications. Our data demonstrate a highly dysregulated B cell response in UC and highlight a potential role of B cells in disease pathogenesis.

Intratumoral plasma cells predict outcomes to PD-L1 blockade in non-small cell lung cancer.

Inhibitors of the programmed cell death-1 (PD-1/PD-L1) signaling axis are approved to treat non-small cell lung cancer (NSCLC) patients, based on their significant overall survival (OS) benefit. Using transcriptomic analysis of 891 NSCLC tumors from patients treated with either the PD-L1 inhibitor atezolizumab or chemotherapy from two large randomized clinical trials, we find a significant B cell association with extended OS with PD-L1 blockade, independent of CD8+ T cell signals. We then derive gene signatures corresponding to the dominant B cell subsets present in NSCLC from single-cell RNA sequencing (RNA-seq) data. Importantly, we find increased plasma cell signatures to be predictive of OS in patients treated with atezolizumab, but not chemotherapy. B and plasma cells are also associated with the presence of tertiary lymphoid structures and organized lymphoid aggregates. Our results suggest an important contribution of B and plasma cells to the efficacy of PD-L1 blockade in NSCLC.

Transcriptomic profiling of adjuvant colorectal cancer identifies three key prognostic biological processes and a disease specific role for granzyme B.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer-related deaths, with a 5% 5-year survival rate for metastatic disease, yet with limited therapeutic advancements due to insufficient understanding of and inability to accurately capture high-risk CRC patients who are most likely to recur. We aimed to improve high-risk classification by identifying biological pathways associated with outcome in adjuvant stage II/III CRC. METHODS AND FINDINGS: We included 1062 patients with stage III or high-risk stage II colon carcinoma from the prospective three-arm randomized phase 3 AVANT trial, and performed expression profiling to identify a prognostic signature. Data from validation cohort GSE39582, The Cancer Genome Atlas, and cell lines were used to further validate the prognostic biology. Our retrospective analysis of the adjuvant AVANT trial uncovered a prognostic signature capturing three biological functions-stromal, proliferative and immune-that outperformed the Consensus Molecular Subtypes (CMS) and recurrence prediction signatures like Oncotype Dx in an independent cohort. Importantly, within the immune component, high granzyme B (GZMB) expression had a significant prognostic impact while other individual T-effector genes were less or not prognostic. In addition, we found GZMB to be endogenously expressed in CMS2 tumor cells and to be prognostic in a T cell independent fashion. A limitation of our study is that these results, although robust and derived from a large dataset, still need to be clinically validated in a prospective study. CONCLUSIONS: This work furthers our understanding of the underlying biology that propagates stage II/III CRC disease progression and provides scientific rationale for future high-risk stratification and targeted treatment evaluation in biomarker defined subpopulations of resectable high-risk CRC. Our results also shed light on an alternative GZMB source with context-specific implications on the disease's unique biology.

Intratumoral CD103+ CD8+ T cells predict response to PD-L1 blockade.

BACKGROUND: CD8+ tissue-resident memory T (TRM) cells, marked by CD103 (ITGAE) expression, are thought to actively suppress cancer progression, leading to the hypothesis that their presence in tumors may predict response to immunotherapy. METHODS: Here, we test this by combining high-dimensional single-cell modalities with bulk tumor transcriptomics from 1868 patients enrolled in lung and bladder cancer clinical trials of atezolizumab (anti-programmed cell death ligand 1 (PD-L1)). RESULTS: ITGAE was identified as the most significantly upregulated gene in inflamed tumors. Tumor CD103+ CD8+ TRM cells exhibited a complex phenotype defined by the expression of checkpoint regulators, cytotoxic proteins, and increased clonal expansion. CONCLUSIONS: Our analyses indeed demonstrate that the presence of CD103+ CD8+ TRM cells, quantified by tracking intratumoral CD103 expression, can predict treatment outcome, suggesting that patients who respond to PD-1/PD-L1 blockade are those who exhibit an ongoing antitumor T-cell response.

Peripheral T cell expansion predicts tumour infiltration and clinical response.

Despite the resounding clinical success in cancer treatment of antibodies that block the interaction of PD1 with its ligand PDL11, the mechanisms involved remain unknown. A major limitation to understanding the origin and fate of T cells in tumour immunity is the lack of quantitative information on the distribution of individual clonotypes of T cells in patients with cancer. Here, by performing deep single-cell sequencing of RNA and T cell receptors in patients with different types of cancer, we survey the profiles of various populations of T cells and T cell receptors in tumours, normal adjacent tissue, and peripheral blood. We find clear evidence of clonotypic expansion of effector-like T cells not only within the tumour but also in normal adjacent tissue. Patients with gene signatures of such clonotypic expansion respond best to anti-PDL1 therapy. Notably, expanded clonotypes found in the tumour and normal adjacent tissue can also typically be detected in peripheral blood, which suggests a convenient approach to patient identification. Analyses of our data together with several external datasets suggest that intratumoural T cells, especially in responsive patients, are replenished with fresh, non-exhausted replacement cells from sites outside the tumour, suggesting continued activity of the cancer immunity cycle in these patients, the acceleration of which may be associated with clinical response.

Late-differentiated effector neoantigen-specific CD8+ T cells are enriched in peripheral blood of non-small cell lung carcinoma patients responding to atezolizumab treatment.

BACKGROUND: There is strong evidence that immunotherapy-mediated tumor rejection can be driven by tumor-specific CD8+ T cells reinvigorated to recognize neoantigens derived from tumor somatic mutations. Thus, the frequencies or characteristics of tumor-reactive, mutation-specific CD8+ T cells could be used as biomarkers of an anti-tumor response. However, such neoantigen-specific T cells are difficult to reliably identify due to their low frequency in peripheral blood and wide range of potential epitope specificities. METHODS: Peripheral blood mononuclear cells (PBMC) from 14 non-small cell lung cancer (NSCLC) patients were collected pre- and post-treatment with the anti-PD-L1 antibody atezolizumab. Using whole exome sequencing and RNA sequencing we identified tumor neoantigens that are predicted to bind to major histocompatibility complex class I (MHC-I) and utilized mass cytometry, together with cellular 'barcoding', to profile immune cells from patients with objective response to therapy (n = 8) and those with progressive disease (n = 6). In parallel, a highly-multiplexed combinatorial tetramer staining was used to screen antigen-specific CD8+ T cells in peripheral blood for 782 candidate tumor neoantigens and 71 known viral-derived control peptide epitopes across all patient samples. RESULTS: No significant treatment- or response associated phenotypic difference were measured in bulk CD8+ T cells. Multiplexed peptide-MHC multimer staining detected 20 different neoantigen-specific T cell populations, as well as T cells specific for viral control antigens. Not only were neoantigen-specific T cells more frequently detected in responding patients, their phenotypes were also almost entirely distinct. Neoantigen-specific T cells from responder patients typically showed a differentiated effector phenotype, most like Cytomegalovirus (CMV) and some types of Epstein-Barr virus (EBV)-specific CD8+ T cells. In contrast, more memory-like phenotypic profiles were observed for neoantigen-specific CD8+ T cells from patients with progressive disease. CONCLUSION: This study demonstrates that neoantigen-specific T cells can be detected in peripheral blood in non-small cell lung cancer (NSCLC) patients during anti-PD-L1 therapy. Patients with an objective response had an enrichment of neoantigen-reactive T cells and these cells showed a phenotype that differed from patients without a response. These findings suggest the ex vivo identification, characterization, and longitudinal follow-up of rare tumor-specific differentiated effector neoantigen-specific T cells may be useful in predicting response to checkpoint blockade. TRIAL REGISTRATION: POPLAR trial NCT01903993 .

Quantitative Comparison of Conventional and t-SNE-guided Gating Analyses.

Dimensionality reduction using the t-Distributed Stochastic Neighbor Embedding (t-SNE) algorithm has emerged as a popular tool for visualizing high-parameter single-cell data. While this approach has obvious potential for data visualization it remains unclear how t-SNE analysis compares to conventional manual hand-gating in stratifying and quantitating the frequency of diverse immune cell populations. We applied a comprehensive 38-parameter mass cytometry panel to human blood and compared the frequencies of 28 immune cell subsets using both conventional bivariate and t-SNE-guided manual gating. t-SNE analysis was capable of stratifying every general cellular lineage and most sub-lineages with high correlation between conventional and t-SNE-guided cell frequency calculations. However, specific immune cell subsets delineated by the manual gating of continuous variables were not fully separated in t-SNE space thus causing discrepancies in subset identification and quantification between these analytical approaches. Overall, these studies highlight the consistency between t-SNE and conventional hand-gating in stratifying general immune cell lineages while demonstrating that particular cell subsets defined by conventional manual gating may be intermingled in t-SNE space.

Visualization of Mass Cytometry Signal Background to Enable Optimal Core Panel Customization and Signal Threshold Gating.

Signal interference or overlap in mass cytometry is minimal compared to flow cytometry but must still be considered for optimal panel design and assay sensitivity. Here we describe a procedure for evaluating signal interference dynamics in the context of a 25-parameter core immunophenotyping panel. Specifically, a mass-minus-many (MMM) approach was used to assess background signals in "empty" or "blank" channels intended for further customization. Through this approach cell type-specific variability in signal background is revealed. Further panel customization can thus be performed with an understanding of cell type and channel-specific background levels to enable rational panel design and the objective delineation of gating thresholds during analysis.

Single-cell Analysis of Immunophenotype and Cytokine Production in Peripheral Whole Blood via Mass Cytometry.

Cytokines play a pivotal role in the pathogenesis of autoimmune diseases. Hence, the measurement of cytokine levels has been the focus of multiple studies in an attempt to understand the precise mechanisms that lead to the breakdown of self-tolerance and subsequent autoimmunity. Approaches thus far have been based on the study of one specific aspect of the immune system (a single or few cell types or cytokines), and do not offer a global assessment of complex autoimmune disease. While patient sera-based studies have afforded important insights into autoimmunity, they do not provide the specific cellular source of the dysregulated cytokines detected. A comprehensive single-cell approach to evaluate cytokine production in multiple immune cell subsets, within the context of "intrinsic" patient-specific plasma circulating factors, is described here. This approach enables monitoring of the patient-specific immune phenotype (surface markers) and function (cytokines), either in its native "intrinsic pathogenic" disease state, or in the presence of therapeutic agents (in vivo or ex vivo).

Mass cytometry panel optimization through the designed distribution of signal interference.

Mass cytometry is capable of measuring more than 40 distinct proteins on individual cells making it a promising technology for innovating biomarker discovery. However, in order for this potential to be fully realized, best practices in panel design need to be further defined in order to achieve consistency and reproducibility in data analysis. Of particular importance are controls that reveal, and panel design principles that mitigate the effects of signal interference or overlap. We observed a disparity between the staining profiles of two noncompeting anti- integrin ß7 mAbs and hypothesized that signal interference was responsible. A mass-minus-one (MMO) control was applied and demonstrated that signal overlap caused the perceived interclonal discrepancy in ß7 expression. Panel redesign in consideration of mass-cytometry specific interference dynamics dramatically improved concordance between both mAbs by redistributing background signals caused by overlap. These studies visualize how signal overlap can complicate mass cytometry data interpretation and demonstrate how the rational distribution of interference can greatly improve panel design and data quality. © 2016 International Society for Advancement of Cytometry.

A Randomized Controlled Trial of a Trauma-Informed Support, Skills, and Psychoeducation Intervention for Survivors of Torture and Related Trauma in Kurdistan, Northern Iraq.

Supportive counseling type interventions are frequently provided to meet the mental health needs of populations in emergency and post-conflicts contexts, but it has seldom been rigorously evaluated. Existing evaluations from low- and middle-income countries provide mixed evidence of effectiveness. While Iraqi Kurdistan experienced relative stability following the fall of Saddam Hussein's government, the population in the northern Dohuk region has continued to experience periodic violence due to conflicts with neighboring Turkey as well as more recent ISIS-associated violence. We evaluated the impact of a trauma-informed support, skills, and psychoeducation intervention provided by community mental health workers (CMHWs) on depressive symptoms and dysfunction (primary outcomes) as well as post-traumatic stress, traumatic grief, and anxiety symptoms (secondary outcomes). Between June 2009 and June 2010, 295 adults were screened; 209 (71%) met eligibility criteria (trauma exposure and a symptom severity score indicating significant distress and functional impairment, among others) and consented to participate. Of these, 159 were randomized to supportive counseling while 50 were randomized to a waitlist control condition. Comparing average symptom severity scores post-treatment among those in the intervention group with those in the waitlist control group, the supportive counseling program had statistically and clinically significant impacts on the primary outcomes of depression (Cohen's d, 0.57; P = .02) and dysfunction (Cohen's d, 0.53; P = .03) and significant but smaller impacts on anxiety. Although studies by the same research team of psychotherapeutic interventions in other parts of Kurdistan and in southern Iraq found larger effects, this study adds to the global research literature on mental health and psychosocial support and shows that a well-trained and supervised program of trauma-informed support, skills, and psychoeducation that emphasizes the therapeutic relationship can also be effective.

Removal of copper ions from aqueous solutions via adsorption on carbon nanocomposites.

The development of technologies for water purification is critical to meet the global challenges of insufficient water supply and inadequate sanitation. Among all wastewater treatments, adsorption is globally recognized as the most promising method because of its versatility and economic feasibility. Herein, the removal of copper ions (Cu(II)) from aqueous solutions through adsorption on free-standing hybrid papers comprised of a mixture between graphene and different types of carbon nanotubes (CNTs) was examined. Results indicate that the rate of adsorption and long-time capacity of the metal ions on the nanocomposites significantly exceeds that of activated carbon by a factor of 4. Moreover, the combination of graphene with CNTs endows an increase in the uptake of Cu(II) up to 50% compared to that of CNTs alone, with a maximum adsorption capacity higher than 250 mg·g(-1). The removal of Cu(II) from water is sensitive to solution pH, and the presence of oxygen functional groups on the adsorbent surface promotes higher adsorption rates and capacities than pristine materials. These hybrid nanostructures show great promise for environmental remediation efforts, wastewater treatments, and separation applications, and the results presented in this study have important implications for understanding the interactions of carbonaceous materials at environmental interfaces.

Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus.

BACKGROUND: Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described. OBJECTIVE: We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes. METHODS: Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE. RESULTS: Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity. CONCLUSION: Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE.

The Split Virus Influenza Vaccine rapidly activates immune cells through Fcγ receptors.

Seasonal influenza vaccination is one of the most common medical procedures and yet the extent to which it activates the immune system beyond inducing antibody production is not well understood. In the United States, the most prevalent formulations of the vaccine consist of degraded or "split" viral particles distributed without any adjuvants. Based on previous reports we sought to determine whether the split influenza vaccine activates innate immune receptors-specifically Toll-like receptors. High-dimensional proteomic profiling of human whole-blood using Cytometry by Time-of-Flight (CyTOF) was used to compare signaling pathway activation and cytokine production between the split influenza vaccine and a prototypical TLR response ex vivo. This analysis revealed that the split vaccine rapidly and potently activates multiple immune cell types but yields a proteomic signature quite distinct from TLR activation. Importantly, vaccine induced activity was dependent upon the presence of human sera indicating that a serum factor was necessary for vaccine-dependent immune activation. We found this serum factor to be human antibodies specific for influenza proteins and therefore immediate immune activation by the split vaccine is immune-complex dependent. These studies demonstrate that influenza virus "splitting" inactivates any potential adjuvants endogenous to influenza, such as RNA, but in previously exposed individuals can elicit a potent immune response by facilitating the rapid formation of immune complexes.

Patient and family member needs during the perioperative period.

PURPOSE: The purpose of this study was to determine the needs and experiences of patients and family members throughout the entire perioperative experience. DESIGN AND METHODS: Using a descriptive study design, a convenience sample of patients and family members were surveyed about their needs and how well those needs were met during the perioperative period. Survey questions were adapted from valid and reliable patient and family needs surveys. Rank order of patient and family needs were determined based on average item scores. FINDINGS: A total of 68 patients and 63 family members were surveyed over an 8-month period. Patient needs with the highest scores were related to pain and/or nausea management, having information about the condition after surgery, and treatment with respect and dignity. Family member needs with the highest scores were related to communication with the surgeon after the procedure, opportunities to ask questions and address concerns with hospital staff, and receiving information about the surgical procedure itself before coming to the hospital. Patients and family members perceived that their needs were met most of the time. CONCLUSIONS: Results of this survey identify the needs of patients and family members throughout the perioperative time period.

Loop nucleotides control primary and mature miRNA function in target recognition and repression.

MicroRNA (miRNA) genes produce three major RNA products; primary (pri-), precursor (pre-), and mature miRNAs. Each product includes sequences complementary to cognate targets, thus they all can in principle interact with the targets. In a recent study we showed that pri-miRNAs play a direct role in target recognition and repression in the absence of functional mature miRNAs. Here we examined the functional contribution of pri-miRNAs in target regulation when full-length functional miRNAs are present. We found that pri-let-7 loop nucleotides control the production of the 5' end of mature miRNAs and modulate the activity of the miRNA gene. This insight enabled us to modulate biogenesis of functional mature miRNAs and dissect the causal relationships between mature miRNA biogenesis and target repression. We demonstrate that both pri- and mature miRNAs can contribute to target repression and that their contributions can be distinguished by the differences between the pri- and mature miRNAs' sensitivity to bind to the first seed nucleotide. Our results demonstrate that the regulatory information encoded in the pri-/pre-miRNA loop nucleotides controls the activities of pri-miRNAs and mature let-7 by influencing pri-miRNA and target complex formation and the fidelity of mature miRNA seed generation.