Human Alzheimer's disease gene expression signatures and immune profile in APP mouse models: a discrete transcriptomic view of Aß plaque pathology.

BACKGROUND: Alzheimer's disease (AD) is a chronic neurodegenerative disease with pathological hallmarks including the formation of extracellular aggregates of amyloid-beta (Aß) known as plaques and intracellular tau tangles. Coincident with the formation of Aß plaques is recruitment and activation of glial cells to the plaque forming a plaque niche. In addition to histological data showing the formation of the niche, AD genetic studies have added to the growing appreciation of how dysfunctional glia pathways drive neuropathology, with emphasis on microglia pathways. Genomic approaches enable comparisons of human disease profiles between different mouse models informing on their utility to evaluate secondary changes to triggers such as Aß deposition. METHODS: In this study, we utilized two animal models of AD to examine and characterize the AD-associated pathology: the Tg2576 Swedish APP (KM670/671NL) and TgCRND8 Swedish plus Indiana APP (KM670/671NL + V717F) lines. We used laser capture microscopy (LCM) to isolate samples surrounding Thio-S positive plaques from distal non-plaque tissue. These samples were then analyzed using RNA sequencing. RESULTS: We determined age-associated transcriptomic differences between two similar yet distinct APP transgenic mouse models, known to differ in proportional amyloidogenic species and plaque deposition rates. In Tg2576, human AD gene signatures were not observed despite profiling mice out to 15 months of age. TgCRND8 mice however showed progressive and robust induction of lysomal, neuroimmune, and ITIM/ITAM-associated gene signatures overlapping with prior human AD brain transcriptomic studies. Notably, RNAseq analyses highlighted the vast majority of transcriptional changes observed in aging TgCRND8 cortical brain homogenates were in fact specifically enriched within the plaque niche samples. Data uncovered plaque-associated enrichment of microglia-related genes such as ITIM/ITAM-associated genes and pathway markers of phagocytosis. CONCLUSION: This work may help guide improved translational value of APP mouse models of AD, particularly for strategies aimed at targeting neuroimmune and neurodegenerative pathways, by demonstrating that TgCRND8 more closely recapitulates specific human AD-associated transcriptional responses.

Lung eQTLs to help reveal the molecular underpinnings of asthma.

Genome-wide association studies (GWAS) have identified loci reproducibly associated with pulmonary diseases; however, the molecular mechanism underlying these associations are largely unknown. The objectives of this study were to discover genetic variants affecting gene expression in human lung tissue, to refine susceptibility loci for asthma identified in GWAS studies, and to use the genetics of gene expression and network analyses to find key molecular drivers of asthma. We performed a genome-wide search for expression quantitative trait loci (eQTL) in 1,111 human lung samples. The lung eQTL dataset was then used to inform asthma genetic studies reported in the literature. The top ranked lung eQTLs were integrated with the GWAS on asthma reported by the GABRIEL consortium to generate a Bayesian gene expression network for discovery of novel molecular pathways underpinning asthma. We detected 17,178 cis- and 593 trans- lung eQTLs, which can be used to explore the functional consequences of loci associated with lung diseases and traits. Some strong eQTLs are also asthma susceptibility loci. For example, rs3859192 on chr17q21 is robustly associated with the mRNA levels of GSDMA (P = 3.55 × 10(-151)). The genetic-gene expression network identified the SOCS3 pathway as one of the key drivers of asthma. The eQTLs and gene networks identified in this study are powerful tools for elucidating the causal mechanisms underlying pulmonary disease. This data resource offers much-needed support to pinpoint the causal genes and characterize the molecular function of gene variants associated with lung diseases.

Serologic extracellular matrix remodeling markers are related to fibrosis stage and prognosis in a phase 2b trial of simtuzumab in patients with primary sclerosing cholangitis.

BACKGROUND: Novel noninvasive predictors of disease severity and prognosis in primary sclerosing cholangitis (PSC) are needed. This study evaluated the ability of extracellular matrix remodeling markers to diagnose fibrosis stage and predict PSC-related fibrosis progression and clinical events. METHODS: Liver histology and serum markers of collagen formation (propeptide of type III collagen [Pro-C3], propeptide of type IV collagen, propeptide of type V collagen), collagen degradation (type III collagen matrix metalloproteinase degradation product and type IV collagen matrix metalloproteinase degradation product), and fibrosis (enhanced liver fibrosis [ELF] score and its components [metalloproteinase-1, type III procollagen, hyaluronic acid]) were assessed in samples from baseline to week 96 in patients with PSC enrolled in a study evaluating simtuzumab (NCT01672853). Diagnostic performance for advanced fibrosis (Ishak stages 3-6) and cirrhosis (Ishak stages 5-6) was evaluated by logistic regression and AUROC. Prognostic performance for PSC-related clinical events and fibrosis progression was assessed by AUROC and Wilcoxon rank-sum test. RESULTS: Among 234 patients, 51% had advanced fibrosis and 11% had cirrhosis at baseline. Baseline Pro-C3 and ELF score and its components provided moderate diagnostic ability for discrimination of advanced fibrosis (AUROC 0.73-0.78) and cirrhosis (AUROC 0.73-0.81). Baseline Pro-C3, ELF score, and type III procollagen provided a moderate prognosis for PSC-related clinical events (AUROC 0.70-0.71). Among patients without cirrhosis at baseline, median changes in Pro-C3 and ELF score to week 96 were higher in those with than without progression to cirrhosis (both p < 0.001). CONCLUSIONS: Pro-C3 correlated with fibrosis stage, and Pro-C3 and ELF score provided discrimination of advanced fibrosis and cirrhosis and predicted PSC-related events and fibrosis progression. The results support the clinical utility of Pro-C3 and ELF score for staging and as prognostic markers in PSC.

Filgotinib Modulates Inflammation-Associated Peripheral Blood Protein Biomarkers in Adults with Active Rheumatoid Arthritis and Prior Inadequate Response to Methotrexate.

INTRODUCTION: Our aim was to evaluate protein biomarker changes related to the administration of filgotinib, a Janus kinase (JAK) 1 preferential inhibitor, in patients with moderately to severely active rheumatoid arthritis (RA) with inadequate response to methotrexate. METHODS: Plasma and serum samples were collected from patients enrolled in FINCH 1 (NCT02889796), a Phase 3 trial. Patients with stable backgrounds of methotrexate were randomly assigned once-daily oral filgotinib 200 or 100 mg, subcutaneous adalimumab 40 mg every 2 weeks (W), or placebo. Up to 35 biomarkers were analyzed at baseline, W4, and W12 with enzyme-linked immunosorbent assays and chemiluminescence and electrochemiluminescence assays. RESULTS: At baseline, four distinct biomarker clusters were identified. The strongest intragroup correlations were in bone-cartilage resorption/inflammation and JAK/signal transducer and activator of transcription (STAT) signaling activity. At baseline, significant positive correlations were identified for cytokines with patient-reported pain and with patient measures of fatigue. Filgotinib reduced levels of cytokines associated with inflammation and cell migration as early as W4 and through W12. Compared to adalimumab, filgotinib induced significant reductions in bone-related turnover biomarkers, N-telopeptide of type 1 collagen and C-telopeptide 1, as well as biomarkers associated with baseline disease activity. No baseline predictors of therapeutic response to filgotinib were identified. CONCLUSIONS: Filgotinib reduced peripheral protein biomarkers associated with JAK/STAT signaling, inflammatory signaling, immune cell migration, and bone resorption as soon as W4 in FINCH 1. Effects were dose-dependent and consistent with the clinical efficacy of filgotinib observed in FINCH 1. The changes in peripheral biomarkers associated with filgotinib treatment in methotrexate-experienced patients are consistent with changes observed in both methotrexate-naïve and biologic disease-modifying antirheumatic drug-experienced RA populations. These data demonstrate dose-dependent effects of preferential JAK1 inhibition by filgotinib on peripheral blood protein biomarkers in methotrexate-experienced patients with RA. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02889796.

Pharmacodynamic effects of filgotinib treatment driving clinical improvement in patients with active psoriatic arthritis enrolled in the EQUATOR trial.

OBJECTIVES: The goal of this study was to identify protein and transcriptional biomarkers and pathways associated with baseline disease state, the effect of filgotinib (FIL) treatment on these biomarkers, and to investigate the mechanism of action of FIL on clinical improvement in patients with active psoriatic arthritis (PsA). METHODS: The phase II EQUATOR (NCT03101670) trial evaluated the efficacy of FIL, a Janus kinase 1-preferential inhibitor, in patients with PsA. Peripheral protein and gene expression levels in association with clinical state at baseline and post-treatment were assessed in 121 patients using linear mixed effects models for repeated measures analyses. Mediation analysis and structural equation modelling (SEM) were performed to investigate the mechanism of action of FIL at week 4 on downstream clinical improvement at week 16. RESULTS: Baseline analyses showed that markers of inflammation were significantly associated with multiple PsA clinical metrics, except for Psoriasis Area and Severity Index (PASI), which corresponded to Th17 markers. FIL treatment resulted in sustained transcriptional inhibition of immune genes and pathways, a sustained increase in B-cell fraction and mature B-cells in circulation, and a transient effect on other cell fractions. Mediation analysis revealed that changes in B cells, systemic inflammatory cytokines and neutrophils at week 4 were associated with changes in clinical metrics at week 16. SEM suggested that FIL improved PASI through reduction of IL-23 p19 and IL-12 p40 proteins. CONCLUSIONS: Our results revealed that FIL treatment rapidly downregulates inflammatory and immune pathways associated with PsA disease activity corresponding to clinical improvement in PsA. TRIAL REGISTRATION NUMBER: NCT03101670.

MicroRNA-like off-target transcript regulation by siRNAs is species specific.

siRNAs mediate sequence-specific gene silencing in cultured mammalian cells but also silence unintended transcripts. Many siRNA off-target transcripts match the guide-strand "seed region," similar to the way microRNAs match their target sites. The extent to which this seed-matched, microRNA-like, off-target silencing affects the specificity of therapeutic siRNAs in vivo is currently unknown. Here, we compare microRNA-like off-target regulations in mouse liver in vivo with those seen in cell culture for a series of therapeutic candidate siRNAs targeting Apolipoprotein B (APOB). Each siRNA triggered regulation of consistent microRNA-like off-target transcripts in mouse livers and in cultured mouse liver tumor cells. In contrast, there was only random overlap between microRNA-like off-target transcripts from cultured human and mouse liver tumor cells. Therefore, siRNA therapeutics may trigger microRNA-like silencing of many unintended targets in vivo, and the potential toxicities caused by these off-target gene regulations cannot be accurately assessed in rodent models.

Correlation Between Early Endpoints and Overall Survival in Non-Small-Cell Lung Cancer: A Trial-Level Meta-Analysis.

Early endpoints, such as progression-free survival (PFS), are increasingly used as surrogates for overall survival (OS) to accelerate approval of novel oncology agents. Compiling trial-level data from randomized controlled trials (RCTs) could help to develop a predictive framework to ascertain correlation trends between treatment effects for early and late endpoints. Through trial-level correlation and random-effects meta-regression analysis, we assessed the relationship between hazard ratio (HR) OS and (1) HR PFS and (2) odds ratio (OR) PFS at 4 and 6 months, stratified according to the mechanism of action of the investigational product. Using multiple source databases, we compiled a data set including 81 phase II-IV RCTs (35 drugs and 156 observations) of patients with non-small-cell lung cancer. Low-to-moderate correlations were generally observed between treatment effects for early endpoints (based on PFS) and HR OS across trials of agents with different mechanisms of action. Moderate correlations were seen between treatment effects for HR PFS and HR OS across all trials, and in the programmed cell death-1/programmed cell death ligand-1 and epidermal growth factor receptor trial subsets. Although these results constitute an important step, caution is advised, as there are some limitations to our evaluation, and an additional patient-level analysis would be needed to establish true surrogacy.

Sputum RNA signature in allergic asthmatics following allergen bronchoprovocation test.

BACKGROUND: Inhaled allergen challenge is a validated disease model of allergic asthma offering useful pharmacodynamic assessment of pharmacotherapeutic effects in a limited number of subjects. OBJECTIVES: To evaluate whether an RNA signature can be identified from induced sputum following an inhaled allergen challenge, whether a RNA signature could be modulated by limited doses of inhaled fluticasone, and whether these gene expression profiles would correlate with the clinical endpoints measured in this study. METHODS: Thirteen non-smoking, allergic subjects with mild-to-moderate asthma participated in a randomised, placebo-controlled, 2-period cross-over study following a single-blind placebo run-in period. Each period consisted of three consecutive days, separated by a wash-out period of at least 3 weeks. Subjects randomly received inhaled fluticasone ((FP) MDI; 500 mcg BID×5 doses in total) or placebo. On day 2, house dust mite extract was inhaled and airway response was measured by FEV1 at predefined time points until 7 h post-allergen. Sputum was induced by NaCl 4.5%, processed and analysed at 24 h pre-allergen and 7 and 24 h post-allergen. RNA was isolated from eligible sputum cell pellets (<80% squamous of 500 cells), amplified according to NuGEN technology, and profiled on Affymetrix arrays. Gene expression changes from baseline and fluticasone treatment effects were evaluated using a mixed effects ANCOVA model at 7 and at 24 h post-allergen challenge. RESULTS: Inhaled allergen-induced statistically significant gene expression changes in sputum, which were effectively blunted by fluticasone (adjusted p<0.025). Forty-seven RNA signatures were selected from these responses for correlation analyses and further validation. This included Th2 mRNA levels for cytokines, chemokines, high-affinity IgE receptor FCER1A, histamine receptor HRH4, and enzymes and receptors in the arachidonic pathway. Individual messengers from the 47 RNA signatures correlated significantly with lung function and sputum eosinophil counts. CONCLUSION: Our RNA extraction and profiling protocols allowed reproducible assessments of inflammatory signatures in sputum including quantification of drug effects on this response in allergic asthmatics. This approach offers novel possibilities for the development of pharmacodynamic (PD) biomarkers in asthma.

Molecular signature of smoking in human lung tissues.

Cigarette smoking is the leading risk factor for lung cancer. To identify genes deregulated by smoking and to distinguish gene expression changes that are reversible and persistent following smoking cessation, we carried out genome-wide gene expression profiling on nontumor lung tissue from 853 patients with lung cancer. Gene expression levels were compared between never and current smokers, and time-dependent changes in gene expression were studied in former smokers. A total of 3,223 transcripts were differentially expressed between smoking groups in the discovery set (n = 344, P < 1.29 × 10(-6)). A substantial number of smoking-induced genes also were validated in two replication sets (n = 285 and 224), and a gene expression signature of 599 transcripts consistently segregated never from current smokers across all three sets. The expression of the majority of these genes reverted to never-smoker levels following smoking cessation, although the time course of normalization differed widely among transcripts. Moreover, some genes showed very slow or no reversibility in expression, including SERPIND1, which was found to be the most consistent gene permanently altered by smoking in the three sets. Our findings therefore indicate that smoking deregulates many genes, many of which reverse to normal following smoking cessation. However, a subset of genes remains altered even decades following smoking cessation and may account, at least in part, for the residual risk of lung cancer among former smokers. Cancer Res; 72(15); 3753-63. ©2012 AACR.

Multiplexed measurements of gene signatures in different analytes using the Nanostring nCounter Assay System.

BACKGROUND: We assessed NanoString's nCounter Analysis System for its ability to quantify gene expression of forty-eight genes in a single reaction with 100 ng of total RNA or an equivalent amount of tissue lysate. In the nCounter System, multiplexed gene expression target levels are directly detected, without enzymatic reactions, via two sequence-specific probes. The individual mRNA is captured with one mRNA target sequence-specific capture probe that is used in a post-hybridization affinity purification procedure. The second mRNA target specific-sequence and fluorescent-labeled colored coded probe is then used in the detection with the 3-component complex separated on a surface via an applied electric field followed by imaging. We evaluated reproducibility, accuracy, concordance with quantitative RT-PCR, linearity, dynamic range, and the ability of the system to assay different inputs (matched samples of total RNA from Flash Frozen (FF) and Formalin Fixed Paraffin Embedded Tissues (FFPET), and crude tissue lysates (CTL)). FINDINGS: The nCounter Analysis System provided data equivalent to that produced by Taqman(R)-based assays for genes expressed within the ranges of the calibration curves (above ~0.5 mRNA copies per human cell based on an assumption of 10 pg of total RNA per cell). System response was linear over more than two orders of magnitude with typical CVs of ~6% for concentrations above 1 fM (105 molecules per mL). Profiling the industry-standard MAQC data set yielded correlation coefficients of >0.83 for intensity values and >0.99 for measured ratios. Ninety percent of nCounter ratio measurements were within 1.27-1.33 fold changes of the Taqman(R) data (0.34-0.41 in log2 scale) for FF total RNA samples. CONCLUSION: The nCounter Analysis System generated robust data for multi-gene expression signatures across three different sample preparation conditions.

Six genes are preferentially transcribed by the circulating and sequestered forms of Plasmodium falciparum parasites that infect pregnant women.

In areas of stable malaria transmission, susceptibility to Plasmodium falciparum malaria increases during first pregnancy. Women become resistant to pregnancy malaria over successive pregnancies as they acquire antibodies against the parasite forms that sequester in the placenta, suggesting that a vaccine is feasible. Placental parasites are antigenically distinct and bind receptors, like chondroitin sulfate A (CSA), that are not commonly bound by other parasites. We used whole-genome-expression analysis to find transcripts that distinguish parasites of pregnant women from other parasites and employed a novel approach to define and adjust for cell cycle timing of parasites. Transcription of six genes was substantially higher in both placental parasites and peripheral parasites from pregnant women, and each gene encodes a protein with a putative export sequence and/or transmembrane domain. This cohort of genes includes var2csa, a member of the variant PfEMP1 gene family previously implicated in pregnancy malaria, as well as five conserved genes of unknown functions. Women in East Africa acquire antibodies over successive pregnancies against a protein encoded by one of these genes, PFD1140w, and this protein shows seroreactivity similar to that of VAR2CSA domains. These findings suggest that a suite of genes may be important for the genesis of the placental binding phenotype of P. falciparum and may provide novel targets for therapeutic intervention.