Peripheral blood is gaining prominence as a noninvasive alternative to tissue biopsy to develop biomarkers for glioblastoma (GBM); however, widely utilized blood-based biomarkers in clinical settings have not yet been identified due to the lack of a robust detection approach. Here, we describe the application of globin reduction in RNA sequencing of whole blood (i.e., WBGR) and perform transcriptomic analysis to identify GBM-associated transcriptomic changes. By using WBGR, we improved the detection sensitivity of informatic reads and identified differential gene expression in GBM blood. By analyzing tumor tissues, we identified transcriptomic traits of GBM blood. Further functional enrichment analyses retained the most changed genes in GBM. Subsequent validation elicited a 10-gene panel covering mRNA, long noncoding RNA, and microRNA (i.e., GBM-Dx panel) that has translational potential to aid in the early detection or clinical management of GBM. Here, we report an integrated approach, WBGR, with comprehensive analytic capacity for blood-based marker identification.

OBJECTIVE:This study was undertaken to identify blood markers of juvenile dermatomyositis (DM) disease activity (DA), which are needed to improve disease management.METHODS:The study comprised a total of 123 juvenile DM patients and 53 healthy controls. Results of laboratory tests (aldolase, creatinine kinase, lactate dehydrogenase [LDH], aspartate aminotransferase) and clinical measures of DA in patients with juvenile DM, including the Manual Muscle Testing in 8 muscles (MMT-8), Childhood Myositis Assessment Scale (CMAS), and disease activity scores (DAS) (total DAS for juvenile DM, the muscle DAS, and the skin DAS), were recorded when available. Surface phenotype of peripheral blood mononuclear cells was assessed using flow cytometry. Whole blood transcriptional profiles were studied using either RNA-sequencing or microarrays. Differential gene expression was determined using DESeq and compared by pathway and gene ontology analyses.RESULTS:Conventional memory (CD27+IgD-) B cells expressing low CXCR5 levels (CXCR5low/- CM B cells) were significantly increased in frequency and absolute numbers in 2 independent cohorts of juvenile DM patients compared with healthy controls. The frequency of CD4+ Th2 memory cells (CD45RA-CXCR5-CCR6-CXCR3-) was also increased in juvenile DM, especially in patients who were within <1 year from diagnosis. The frequency of CXCR5low/- CM B cells correlated with serum aldolase levels and with a blood interferon-stimulated gene transcriptional signature. Furthermore, both the frequency and absolute numbers of CXCR5low/- CM B cells correlated with clinical and laboratory measures of muscle DA (MMT-8, CMAS, aldolase, and LDH).CONCLUSION:These findings suggest that both CM B cells lacking the CXCR5 follicular marker and CXCR5- Th2 cells represent potential biomarkers of DA in juvenile DM and may contribute to its pathogenesis.

SYBR green qRT-PCR Primers .

Emerging evidence supports that mitochondrial dysfunction contributes to systemic lupus erythematosus (SLE) pathogenesis. Here we show that programmed mitochondrial removal, a hallmark of mammalian erythropoiesis, is defective in SLE. Specifically, we demonstrate that during human erythroid cell maturation, a hypoxia-inducible factor (HIF)-mediated metabolic switch is responsible for the activation of the ubiquitin-proteasome system (UPS), which precedes and is necessary for the autophagic removal of mitochondria. A defect in this pathway leads to accumulation of red blood cells (RBCs) carrying mitochondria (Mito(+) RBCs) in SLE patients and in correlation with disease activity. Antibody-mediated internalization of Mito(+) RBCs induces type I interferon (IFN) production through activation of cGAS in macrophages. Accordingly, SLE patients carrying both Mito(+) RBCs and opsonizing antibodies display the highest levels of blood IFN-stimulated gene (ISG) signatures, a distinctive feature of SLE.

Rationale: A human model to better understand tuberculosis immunopathogenesis and facilitate vaccine development is urgently needed. Objectives: We evaluated the feasibility, safety, and immunogenicity of live bacillus Calmette-Guerin (BCG) in a lung-oriented controlled human infection model. Methods: We recruited 106 healthy South African participants with varying degrees of tuberculosis susceptibility. Live BCG, sterile PPD, and saline were bronchoscopically instilled into separate lung segments (n = 65). A control group (n = 34) underwent a single bronchoscopy without challenge. The primary outcome was safety. Cellular and antibody immune signatures were identified in BAL before and 3 days after challenge using flow cytometry, ELISA, RNA sequencing, and mass spectrometry. Measurements and Main Results: The frequency of adverse events was low (9.4%; n = 10), similar in the challenge versus control groups (P =0.8), and all adverse events were mild and managed conservatively in an outpatient setting. The optimal PPD and BCG dose was 0.5 TU and 10(4) cfu, respectively, based on changes in BAL cellular profiles (P = 0.02) and antibody responses (P = 0.01) at incremental doses before versus after challenge. At 10(4) versus 10(3) cfu BCG, there was a significant increase in number of differentially expressed genes (367 vs. 3; P <0.001) and dysregulated proteins (64 vs. 0; P < 0.001). Immune responses were highly setting specific (in vitro vs. in vivo) and compartment specific (BAL vs. blood) and localized to the challenged lung segments. Conclusions: A lung-oriented mycobacterial controlled human infection model using live BCG and PPD is feasible and safe. These data inform the study of tuberculosis immunopathogenesis and strategies for evaluation and development of tuberculosis vaccine candidates.

Glioblastoma (GBM) is the most frequently reported primary malignant brain tumor (29.6%). The prognosis for patients who develop GBM is bleak, with average survival after diagnosis of 12-16 months. Although conventional treatment with surgery, irradiation, and temozolomide postpones tumor progression and extends patients survival, these tumors universally recur and unrelentingly result in patient death. Personalized therapies against molecular targets that drive the growth of the bulk of primary tumors have so far been unsuccessful in clinical trials, due to lack of biomarker driven approaches. Thus, there is significant unmet need to begin biomarker driven precision medicine trials for treatment of GBM. Arsenic trioxide (ATO) is an inorganic compound that induces apoptosis via multiple pathways. Arsenic trioxide (TRISENOX®) is approved by the FDA for patients with acute promyelocytic leukemia (APL). Pre-clinical studies in brain tumors suggest that ATO is synergistic with radiation therapy (RT) and may enhance effects of radiation. In an earlier Phase II clinical trial (NCT00275067) using intravenous ATO and temozolomide in combination with radiation therapy for patients with newly diagnosed malignant gliomas, a subset of patients demonstrated notable benefit (Progression free survival (avg. = 638 days) and overall survival (avg. = 967 days)). Comparing RNAseq data from preclinical models and specimen from the Phase II clinical trial, the responder group could be confidently distinguished from the non-responder cohort leading to gene signatures of differential ATO sensitivity. Applying a Relative Expression Ordering (REO) Analysis framework, we pinpointed a probability-based roster of 28 top scoring pairs (TSP) as the classifier by which to identify patients with a higher likelihood to benefit from including ATO in combination with TMZ and radiation. This method is completely independent of platform on which data is collected and can be used for analysis of individual, newly-enrolled, n = 1 patients. We are advancing a protocol using the above gene classifier as enrollment criteria for an Adaptive clinical trial testing an oral formulation of ATO for newly diagnosed IDH1 WT Primary GBM patients; the trial will test whether patients whose tumors with ATO Classifier show 6-month PFS benefit by addition of ATO to Standard-of-Care. The trial will validate and refine the comprehensive biomarker panel that could identify most likely GBM responders to ATO and TMZ treatment in combination with radiation. Supported by a grant from the Baylor Scott & White Foundation. Citation Format: Sen Peng, Jinghua Gu, Xuan Wang, Sanhita Rath, Jacob Cardenas, Nicholas Schork, George Snipes, Harshil Dhruv, Karen Fink, Michael Berens. Development of a clinical assay for predicting glioblastoma (GBM) patients most likely to respond to arsenic trioxide (ATO) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2007.

INTRODUCTION: Noting that the activated eosinophils and mast cells (MCs) that accumulate in the esophagus of eosinophilic esophagitis (EoE) patients secrete myoactive, pro-inflammatory, and cytotoxic products capable of causing the motor abnormalities and neuronal degeneration of achalasia, we proposed that an allergy-mediated, esophageal muscle-predominant form of EoE might cause achalasia. In earlier studies exploring that proposal, we found that LES muscle of achalasia patients exhibits striking MC degranulation. Although the hypercontraction and poor relaxation that characterize achalasic LES muscle have been attributed solely to its loss of inhibitory neurons, we hypothesized that abnormalities intrinsic to LES muscle cells themselves might contribute to motor disturbances in achalasia associated with MC degranulation. This study aimed to explore genetic factors that might underlie such intrinsic abnormalities. METHODS: We performed qPCR for a panel of genes known to mediate smooth muscle contraction and Ca2+ handling on LES muscle specimens obtained from 7 achalasia patients (2 type I, 4 type II, 1 type III) and 3 EGJ outflow obstruction (EGJOO) patients during Heller myotomy; control LES muscle was taken from 2 organ donors. RESULTS: Hierarchical clustering analysis revealed two discrete clusters that we call “mototype” gene patterns; one control subject grouped within each cluster (Figure 1). Mototype Cluster 1, comprised of the 2 type I and 1 type III achalasia patients as well as 2 EGJOO patients, exhibited upregulation of genes involved in smooth muscle contraction and Ca2+ handling (gene expression relative to corresponding control, Figure 2). In contrast, Mototype Cluster 2, comprised of all 4 achalasia type II patients and 1 EGJOO patient, exhibited downregulation of those same genes. Principal component analysis recapitulated this clustering of mototypes that separated achalasia types I and III from type II (Figure 3), and Rand Index calculations demonstrated that this clustering was not due to chance. CONCLUSION: We show that the genetic mototype of LES muscle can distinguish the manometric phenotypes of achalasia that are associated with MC degranulation. These findings support our proposal that there is an allergy-mediated form of achalasia, and suggest that when patients acquire this form of achalasia, their underlying genetic mototype might contribute to the resulting manometric phenotype.Figure 1.: Heat-map and hierarchical clustering with dendrogram generated from qPCR gene expression data of LES muscle for 2 control subjects, 7 achalasia patients (2 type I, 4 type II, 1 type III) and 3 EGJ outflow obstruction (EGJOO) patients reveals clustering of achalasia types I and III in mototype 1, and achalasia type II into mototype 2. Red color corresponds to high relative expression and blue color corresponds to low relative expression.Figure 2.: Heat-map and hierarchical clustering with dendrogram generated from qPCR gene expression data of LES muscle from 7 achalasia patients and 3 EGJOO patients relative to their corresponding control highlights differences between the two mototype clusters. Note the relative upregulation of the calcium handling genes PLN, RYR3, IP3R1 and the smooth muscle contraction and contractility genes RHOA, COL1A2, MYLK, and ACTA2 in mototype 2 patients and the relative downregulation of these same genes in mototype 1 patients. Red/blue colors indicate up/down-regulation with respect to corresponding control in each mototype.Figure 3.: Principal component (PC) analysis of gene expression data confirms mototype clustering of achalasia types 1 and III distinct from achalasia type II.

BACKGROUND:Eosinophils and mast cells are key effectors of allergy. When they accumulate in the esophagus, their myoactive, pro-inflammatory, and cytotoxic products potentially could cause achalasia-like motility abnormalities and neuronal degeneration. We hypothesized that there is an allergy-mediated form of achalasia. METHODS:LES muscle samples obtained during Heller myotomy from patients with achalasia or EGJ outflow obstruction (EGJOO) and from organ donor controls were immunostained for tryptase. Eosinophil and mast cell density, and mast cell degranulation were assessed. LES muscle was evaluated by qPCR for genes mediating smooth muscle Ca2+ handling and contraction. KEY RESULTS:There were 13 patients (7 men, median age 59; 10 achalasia, 3 EGJOO) and 7 controls (4 men, median age 42). Eosinophils were infrequent in LES muscle, but mast cells were plentiful. Patients and controls did not differ significantly in LES mast cell density. However, 12 of 13 patients exhibited profound LES mast cell degranulation involving perimysium and myenteric plexus nerves, while only mild degranulation was seen in 2 of 7 controls. Hierarchical clustering analysis of qPCR data revealed two "mototype" LES gene expression patterns, with all type II patients in one mototype, and type I and III patients in the other. CONCLUSIONS & INFERENCES:LES muscle of patients with achalasia or EGJOO exhibits striking mast cell degranulation, and patients with different achalasia manometric phenotypes exhibit different LES patterns of expression for genes mediating Ca2+ handling and muscle contraction. Although these findings are not definitive, they support our hypothesis that achalasia can be allergy-driven.

Tissue-resident and infiltrating immune cells are continuously exposed to molecules derived from the local cells that often come in form of secreted factors, such as cytokines. These factors are known to impact the immune cells' biology. However, very little is known about whether the tissue resident immune cells in return also affect the local environment. In this study, with the help of RNA-sequencing, we show for the first time that long-term absence of epidermal resident Langerhans cells led to significant gene expression changes in the local keratinocytes and resident dendritic epidermal T cells. Thus, immune cells might play an active role in maintaining tissue homeostasis, which should be taken in consideration at data interpretation.

BACKGROUND:Anti-IgE (omalizumab) has been used for the treatment of moderate-to-severe asthma that is not controlled by inhaled steroids. Despite its success, it does not always provide patients with significant clinical benefits.OBJECTIVE:To investigate the transcriptional variations between omalizumab responders and non-responders and to study the mechanisms of action of omalizumab.METHODS:The whole blood transcriptomes of moderate-to-severe adult asthma patients (N = 45:34 responders and 11 non-responders) were analysed over the course of omalizumab treatment. Non-asthmatic healthy controls (N = 17) were used as controls.RESULTS:Transcriptome variations between responders and non-responders were identified using the genes significant (FDR < 0.05) in at least one comparison of each patient response status and time point compared with control subjects. Using gene ontology and network analysis, eight clusters of genes were identified. Longitudinal analyses of individual clusters revealed that responders could maintain changes induced with omalizumab treatment and become more similar to the control subjects, while non-responders tend to remain more similar to their pre-treatment baseline. Further analysis of an inflammatory gene cluster revealed that genes associated with neutrophil/eosinophil activities were up-regulated in non-responders and, more importantly, omalizumab did not significantly alter their expression levels. The application of modular analysis supported our findings and further revealed variations between responders and non-responders.CONCLUSION AND CLINICAL RELEVANCE:This study provides not only transcriptional variations between omalizumab responders and non-responders, but also molecular insights for controlling asthma by omalizumab.

Circular RNAs, a family of covalently circularized RNAs with tissue-specific expression, were recently demonstrated to play important roles in mammalian biology. Regardless of extensive research to predict, quantify, and annotate circRNAs, our understanding of their functions is still in its infancy. In this study, we developed a novel computational tool: Competing Endogenous RNA for INtegrative Annotations (Cerina), to predict biological functions of circRNAs based on the competing endogenous RNA model. Pareto Frontier Analysis was employed to integrate ENCODE mRNA/miRNA data with predicted microRNA response elements to prioritize tissue-specific ceRNA interactions. Using data from several circRNA-disease databases, we demonstrated that Cerina significantly improved the functional relevance of the prioritized ceRNA interactions by several folds, in terms of precision and recall. Proof-of-concept studies on human cancers and cardiovascular diseases further showcased the efficacy of Cerina on predicting potential circRNA functions in human diseases.

Chemoresistance in cancer is linked to a subset of cancer cells termed "cancer stem cells" (CSCs), and in particular, those expressing the CD44 variant appear to represent a more aggressive disease phenotype. Herein, we demonstrate that CD44v6 represents a CSC population with increased resistance to chemotherapeutic agents, and its high expression is frequently associated with poor overall survival (OS) and disease-free survival (DFS) in patients with colorectal cancer (CRC). CD44v6+ cells showed elevated resistance to chemotherapeutic drugs and significantly high tumor initiation capacity. Inhibition of CD44v6 resulted in the attenuation of self-renewal capacity and resensitization to chemotherapeutic agents. Of note, miRNA profiling of CD44v6+ spheroid-derived CSCs identified a unique panel of miRNAs indicative of high self-renewal capacity. In particular, miR-1246 was overexpressed in CD44v6+ cells, and associated with poor OS and DFS in CRC patients. We demonstrate that CD44v6+ CSCs induced chemoresistance and enhance tumorigenicity in CRC cells, and this was in part orchestrated by a distinct panel of miRNAs with dysregulated profiles. These findings suggest that specific miRNAs could serve as therapeutic targets as well as promising prognostic biomarkers in patients with colorectal neoplasia.

Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease. It is thought that many common variant gene loci of weak effect act additively to predispose to common autoimmune diseases, while the contribution of rare variants remains unclear. Here we describe that rare coding variants in lupus-risk genes are present in most SLE patients and healthy controls. We demonstrate the functional consequences of rare and low frequency missense variants in the interacting proteins BLK and BANK1, which are present alone, or in combination, in a substantial proportion of lupus patients. The rare variants found in patients, but not those found exclusively in controls, impair suppression of IRF5 and type-I IFN in human B cell lines and increase pathogenic lymphocytes in lupus-prone mice. Thus, rare gene variants are common in SLE and likely contribute to genetic risk.

BACKGROUND:IL-10-producing regulatory B cells (Bregs) are widely ascribed immune regulatory functions. However, Breg subsets in human asthma have not been fully investigated.OBJECTIVE:We studied Breg subsets in adult allergic asthma patients by assessing two major parameters, frequency and IL-10 expression. We then investigated factors that affect these two parameters in patients.METHODS:Peripheral blood mononuclear cells (PBMCs) of adult allergic asthma patients (N = 26) and non-asthmatic controls (N = 28) were used to assess the frequency of five subsets of transitional B cells (TBs), three subsets of CD24high CD27+ B cells and B1 cells. In addition to clinical data, IL-10 expression by individual Breg subsets was assessed by flow cytometry.RESULTS:Asthma patients had decreases of CD5+ and CD1d+ CD5+ , but an increase of CD27+ TBs which was significant in patients with moderate asthma (60 < FEV1 < 80). Regardless of asthma severity, there was no significant alteration in the frequencies of 6 other Breg subsets tested. However, we found that oral corticosteroid (OCS) significantly affected the frequency of Bregs in Breg subset-specific manners. OCS decreased CD5+ and CD1d+ CD5+ TBs, but increased CD27+ TBs and CD10+ CD24high CD27+ cells. Furthermore, OCS decreased IL-10 expression by CD27+ TBs, all 3 CD24high CD27+ B cell subsets (CD5+ , CD10+ and CD1d+ ) and B1 cells. OCS-mediated inhibition of IL-10 expression was not observed in the other Breg subsets tested.CONCLUSION & CLINICAL RELEVANCE:Alterations in the frequency of Bregs and their ability to express IL-10 are Breg subset-specific. OCS treatment significantly affects the frequency as well as their ability to express IL-10 in Breg subset-specific manners.

Systemic lupus erythematosus carries an increased risk of pregnancy complications, including preeclampsia and fetal adverse outcomes. To identify the underlying molecular mechanisms, we longitudinally profiled the blood transcriptome of 92 lupus patients and 43 healthy women during pregnancy and postpartum and performed multicolor flow cytometry in a subset of them. We also profiled 25 healthy women undergoing assisted reproductive technology to monitor transcriptional changes around embryo implantation. Sustained down-regulation of multiple immune signatures, including interferon and plasma cells, was observed during healthy pregnancy. These changes appeared early after embryo implantation and were mirrored in uncomplicated lupus pregnancies. Patients with preeclampsia displayed early up-regulation of neutrophil signatures that correlated with expansion of immature neutrophils. Lupus pregnancies with fetal complications carried the highest interferon and plasma cell signatures as well as activated CD4+ T cell counts. Thus, blood immunomonitoring reveals that both healthy and uncomplicated lupus pregnancies exhibit early and sustained transcriptional modulation of lupus-related signatures, and a lack thereof associates with adverse outcomes.

Proanthocyanidins are a heterogeneous group of flavan-3-ol or flavan-3,4-diol oligomers present in various fruits and vegetables. In particular, the smaller oligomeric subset of proanthocyanidins, termed the oligomeric proanthocyanidins (OPCs) appear to have potent anti-tumorigenic properties, but the underlying mechanisms for their effectiveness remain unclear. Herein, we utilized a series of in vitro, in vivo and patient-derived organoid approaches to systematically investigate the chemoprotective role of OPCs in colorectal cancer. OPCs exerted anti-tumorigenic effects through inhibition of cellular proliferation, and induced apoptosis and cell cycle arrest. Intriguingly, OPCs suppressed spheroid derived cancer stem-like cell formation and decreased the expression of intestinal cancer stem cell markers including LGR5, CD44 and CD133. Mechanistically, RNA-sequencing results confirmed that OPCs prominently interfered with developmental and self-renewal pathways and identified several self-renewal associated oncogenes targeted by OPCs. Furthermore, OPCs inhibited Hippo pathway through downregulation of its key transcriptional regulators, YAP and TAZ. Finally, we confirmed anti-tumorigenic effects of OPCs using multiple xenograft experiments and recapitulated its protective effects using patient-derived colorectal tumor organoids. Collectively, we have comprehensively assessed anti-tumorigenic properties of OPCs and our data throws light on previously unrecognized chemopreventive mechanisms of OPCs highlighting its therapeutic potential.

Although the anticancer properties of oligomeric proanthocyanidins (OPCs) from grape seeds have been well recognized, the molecular mechanisms by which they exert anticancer effects are poorly understood. In this study, through comprehensive RNA-sequencing-based gene expression profiling in multiple colorectal cancer cell lines, we for the first time illuminate the genome-wide effects of OPCs from grape seeds in colorectal cancer. Our data revealed that OPCs affect several key cancer-associated genes. In particular, genes involved in cell cycle and DNA replication were most significantly and consistently altered by OPCs across multiple cell lines. Intriguingly, our in vivo experiments showed that OPCs were significantly more potent at decreasing xenograft tumor growth compared with the unfractionated grape seed extract (GSE) that includes the larger polymers of proanthocyanidins. These findings were further confirmed in colorectal cancer patient-derived organoids, wherein OPCs more potently inhibited the formation of organoids compared with GSE. Furthermore, we validated alteration of cell cycle and DNA replication-associated genes in cancer cell lines, mice xenografts as well as patient-derived organoids. Overall, this study provides an unbiased and comprehensive look at the mechanisms by which OPCs exert anticancer properties in colorectal cancer.

Combining anti-cancer agents in cancer therapies is becoming increasingly popular due to improved efficacy, reduced toxicity and decreased emergence of resistance. Here, we test the hypothesis that dietary agents such as oligomeric proanthocyanidins (OPCs) and curcumin cooperatively modulate cancer-associated cellular mechanisms to inhibit carcinogenesis. By a series of in vitro assays in colorectal cancer cell lines, we showed that the anti-tumorigenic properties of the OPCs-curcumin combination were superior to the effects of individual compounds. By RNA-sequencing based geneexpression profiling in six colorectal cancer cell lines, we identified the cooperative modulation of key cancer-associated pathways such as DNA replication and cell cycle pathways. Moreover, several pathways, including protein export, glutathione metabolism and porphyrin metabolism were more effectively modulated by the combination of OPCs and curcumin. We validated genes belonging to these pathways, such as HSPA5, SEC61B, G6PD, HMOX1 and PDE3B to be cooperatively modulated by the OPCs-curcumin combination. We further confirmed that the OPCs-curcumin combination more potently suppresses colorectal carcinogenesis and modulated expression of genes identified by RNA-sequencing in mice xenografts and in colorectal cancer patient-derived organoids. Overall, by delineating the cooperative mechanisms of action of OPCs and curcumin, we make a case for the clinical co-administration of curcumin and OPCs as a treatment therapy for patients with colorectal cancer.