Michael Vincent

Efficacy and safety of an interleukin 6 monoclonal antibody for the treatment of systemic lupus erythematosus: a phase II dose-ranging randomised controlled trial

Objectives This phase II trial evaluated the efficacy and safety of an interleukin (IL) 6 monoclonal antibody for systemic lupus erythematosus (SLE). Methods Patients with active disease were randomised to placebo or PF-04236921 10 mg, 50 mg or 200 mg, subcutaneously, every 8 weeks with stable background therapy. SLE Responder Index (SRI-4; primary end point) and British Isles Lupus Assessment Group-based Composite Lupus Assessment (BICLA) were assessed at week 24. Post hoc analysis identified an enriched population based upon planned univariate analyses. Results 183 patients received treatment (placebo, n=45; 10 mg, n=45; 50 mg, n=47; 200 mg, n=46). The 200 mg dose was discontinued due to safety findings and not included in the primary efficacy analysis. The SRI-4 response rates were not significant for any dose compared with placebo; however, the BICLA response rate was significant for 10 mg (p=0.026). The incidence of severe flares was significantly reduced with 10 mg (n=0) and 50 mg (n=2) combined versus placebo (n=8; p<0.01). In patients with greater baseline disease activity (enriched population), the SRI-4 (p=0.004) and BICLA (p=0.012) response rates were significantly different with 10 mg versus placebo. Four deaths (200 mg, n=3; 10 mg, n=1) occurred. The most frequently reported adverse events included headache, nausea and diarrhoea. Conclusions PF-04236921 was not significantly different from placebo for the primary efficacy end point in patients with SLE. Evidence of an effect with 10 mg was seen in a post hoc analysis. Safety was acceptable for doses up to 50 mg as the 200 mg dose was discontinued due to safety findings. Trial registration number NCT01405196; Pre-results.

QuickRNASeq lifts large-scale RNA-seq data analyses to the next level of automation and interactive visualization.

BackgroundRNA sequencing (RNA-seq), a next-generation sequencing technique for transcriptome profiling, is being increasingly used, in part driven by the decreasing cost of sequencing. Nevertheless, the analysis of the massive amounts of data generated by large-scale RNA-seq remains a challenge. Multiple algorithms pertinent to basic analyses have been developed, and there is an increasing need to automate the use of these tools so as to obtain results in an efficient and user friendly manner. Increased automation and improved visualization of the results will help make the results and findings of the analyses readily available to experimental scientists.ResultsBy combing the best open source tools developed for RNA-seq data analyses and the most advanced web 2.0 technologies, we have implemented QuickRNASeq, a pipeline for large-scale RNA-seq data analyses and visualization. The QuickRNASeq workflow consists of three main steps. In Step #1, each individual sample is processed, including mapping RNA-seq reads to a reference genome, counting the numbers of mapped reads, quality control of the aligned reads, and SNP (single nucleotide polymorphism) calling. Step #1 is computationally intensive, and can be processed in parallel. In Step #2, the results from individual samples are merged, and an integrated and interactive project report is generated. All analyses results in the report are accessible via a single HTML entry webpage. Step #3 is the data interpretation and presentation step. The rich visualization features implemented here allow end users to interactively explore the results of RNA-seq data analyses, and to gain more insights into RNA-seq datasets. In addition, we used a real world dataset to demonstrate the simplicity and efficiency of QuickRNASeq in RNA-seq data analyses and interactive visualizations. The seamless integration of automated capabilites with interactive visualizations in QuickRNASeq is not available in other published RNA-seq pipelines.ConclusionThe high degree of automation and interactivity in QuickRNASeq leads to a substantial reduction in the time and effort required prior to further downstream analyses and interpretation of the analyses findings. QuickRNASeq advances primary RNA-seq data analyses to the next level of automation, and is mature for public release and adoption.

QuickMIRSeq: a pipeline for quick and accurate quantification of both known miRNAs and isomiRs by jointly processing multiple samples from microRNA sequencing

BackgroundGenome-wide miRNA expression data can be used to study miRNA dysregulation comprehensively. Although many open-source tools for microRNA (miRNA)-seq data analyses are available, challenges remain in accurate miRNA quantification from large-scale miRNA-seq dataset. We implemented a pipeline called QuickMIRSeq for accurate quantification of known miRNAs and miRNA isoforms (isomiRs) from multiple samples simultaneously.ResultsQuickMIRSeq considers the unique nature of miRNAs and combines many important features into its implementation. First, it takes advantage of high redundancy of miRNA reads and introduces joint mapping of multiple samples to reduce computational time. Second, it incorporates the strand information in the alignment step for more accurate quantification. Third, reads potentially arising from background noise are filtered out to improve the reliability of miRNA detection. Fourth, sequences aligned to miRNAs with mismatches are remapped to a reference genome to further reduce false positives. Finally, QuickMIRSeq generates a rich set of QC metrics and publication-ready plots.ConclusionsThe rich visualization features implemented allow end users to interactively explore the results and gain more insights into miRNA-seq data analyses. The high degree of automation and interactivity in QuickMIRSeq leads to a substantial reduction in the time and effort required for miRNA-seq data analysis.

Bioinformatics Tools for RNA-seq Gene and Isoform Quantification

In recent years, RNA-seq has emerged as a powerful technology in estimation of gene or transcript expression. ‘Union-exon’ and transcript based approaches are widely used in gene quantification. The ‘Union-exon’ based approach is simple, but it does not distinguish between isoforms when multiple alternatively spliced transcripts are expressed from the same gene. Because a gene is expressed in one or more transcript isoforms, the transcript based approach is more biologically meaningful than the ‘union exon’-based approach. However, estimating the expression of individual isoform is intrinsically challenging because different isoforms of a gene usually have a high proportion of genomic overlap. Recently, a number of tools have been developed for RNA-seq isoform quantification. We review those methods and their main features to serve as guidance for users to choose suitable tools for their specific projects.

OP0185 Significant Clinical Improvement and Reduction of Severe Flares Following Administration of an IL-6 Monoclonal Antibody in Systemic Lupus Erythematosus (SLE) Subjects with High Disease Activity

Background PF-04236921 is a fully human monoclonal antibody (mAb) that binds to circulating IL-6 and neutralizes its activity. This may be beneficial in reducing the disease manifestations of active SLE. Objectives To assess the efficacy, safety, and tolerability of PF-04236921 in subjects with active SLE. Methods 183 subjects with active SLE (SLEDAI ≥6 and ≥1 BILAG 2004 A or ≥2 Bs) received 3 doses of PF-04236921 (10, 50, or 200mg) or placebo given subcutaneously every 8 wks. The primary endpoint was the proportion of SLE Responder Index 4 (SRI-4) responders at Wk 24 using a generalized linear mixed model. The BILAG-based Combined Lupus Assessment (BICLA), frequency of severe flares, and SF-36 were also evaluated. Results The majority of subjects were female (91.8%), mean age 40.4, with musculoskeletal and mucocutaneous organ system involvement. Baseline demographics were similar across groups. At Wk 24, there were more responders in the 10mg group vs placebo for the SRI (p=0.076) and BICLA (p=0.026). Improvement in the SF-36 PCS domain was also noted for the 10mg group vs placebo (p=0.092). For the 50mg group there were no significant differences vs placebo for the SRI (p=0.528) or BICLA (p=0.1). A post-hoc subgroup analysis was completed in subjects with high baseline disease activity [SLEDAI ≥10, detectable anti-dsDNA, low complement, or prednisone >7.5 mg/day (enriched population)]. In this subgroup, a significant effect size was observed for the SRI, BICLA, and SF-36 PCS domain for the 10mg group vs placebo. There was also a significant reduction in the frequency of severe SELENA-SLEDAI Flare Index (SFI) flares for the combined 10 and 50mg groups vs placebo for both the broad (p=0.004) and enriched populations (p=0.004). Adverse events (AEs), infectious AEs, and discontinuations due to AEs were comparable across groups. The rate of SAEs was highest in the placebo and 200mg groups and the rate of serious infections was highest in the 200mg group. There were 4 deaths; 3 in 200mg [cardiorespiratory arrest, urosepsis with pulmonary embolism (PE), and disseminated tuberculosis] and 1 in 10mg (suspected PE); further dosing of 200mg was subsequently terminated. Conclusions An efficacy signal was apparent in the broad population following administration of an IL-6 mAb notably with regard to severe flare reduction. Greater efficacy was observed across several key parameters for the 10mg group in the enriched population compared to the broad population. The safety profile with 10 and 50mg doses appeared acceptable for both the broad and enriched population; dosing with 200mg was terminated due to safety concerns. Disclosure of Interest J. Smolen Grant/research support from: Abbvie, Janssen, MSD, Pfizer Inc, Roche, UCB, Consultant for: Abbvie, Amgen, Astra-Zeneca, Astro, Celgene, GSK, Janssen, Eli-Lilly, Medimmune, MSD, Novartis-Sandoz, Novo-Nordisk, Pfizer Inc, Roche, Samsung, Sanofi-Aventis, UCB, S. Popa: None declared, I. Szombati: None declared, D. Wallace: None declared, M. Petri Consultant for: Pfizer Inc., P. Lipsky Consultant for: Pfizer Inc., J. Merrill Consultant for: Pfizer Inc., V. Strand Consultant for: Abbvie, Amgen, Anthera, Astra-Zeneca, Biogen Idec, Bristol-Myers Squibb, Genentech, GSK, Janssen, Merck Serono, Novartis, Pfizer Inc, Sanofi-Aventis, Takeda, UCB, P. Healey Employee of: Pfizer Inc., C. Li Employee of: Pfizer Inc., J. Christensen Employee of: Pfizer Inc., A. Diehl Employee of: Pfizer Inc., J. Beebe Employee of: Pfizer Inc., M. Vincent Employee of: Pfizer Inc., J. Wajdula Employee of: Pfizer Inc., S. Sridharan Employee of: Pfizer Inc.

Anti-MAdCAM Antibody Increases ß7+ T Cells and CCR9 Gene Expression in the Peripheral Blood of Patients With Crohn’s Disease

Abstract Objective To define pharmacodynamic biomarkers in the peripheral blood of patients with Crohn’s disease [CD] after treatment with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody. Methods In this Phase 2, randomised, double-blind, controlled study [OPERA], blood samples were analysed from patients with moderate to severe active CD who received placebo or 22.5 mg, 75 mg, or 225 mg of PF-00547659 subcutaneously at baseline and at Weeks 4 and 8, with follow-up at Week 12. Soluble MAdCAM [sMAdCAM] was measured by mass spectrometry, β7-expressing T cells by flow cytometry, and gene transcriptome by RNA sequencing. Results A slight increase in sMAdCAM was measured in the placebo group from baseline to Week 12 [6%], compared with significant decreases in all PF-00547659 groups [–87% to –98%]. A slight increase from baseline to Week 12 was observed in frequency and molecules of equivalent soluble fluorochrome for β7+ central memory T cells in the placebo group [4%], versus statistically significant increases in the active treatment groups [48% to 81%]. Similar trends were seen for β7+ effector memory T cells [placebo, 8%; PF-00547659, 84–138%] and β7+ naïve T cells [8%; 13–50%]. CCR9 gene expression had statistically significant up-regulation [p = 1.09e-06; false discovery rate < 0.1] with PF-00547659 treatment, and was associated with an increase in β7+ T cells. Conclusions Results of the OPERA study demonstrate positive pharmacology and dose-dependent changes in pharmacodynamic biomarker measurements in blood, including changes in cellular composition of lymphocytes and corresponding CCR9 gene expression changes.

Identification of drug eQTL interactions from repeat transcriptional and environmental measurements in a lupus clinical trial

Background: Cytokines are critical to human disease and are attractive therapeutic targets given their widespread influence on gene regulation and transcription. Defining the downstream regulatory mechanisms influenced by cytokines is central to defining drug and disease mechanisms. One promising strategy is to use interactions between expression quantitative trait loci (eQTLs) and cytokine levels to define target genes and mechanisms. Results: In a clinical trial for anti-IL-6 in patients with systemic lupus erythematosus we measured interferon (IFN) status, anti-IL-6 drug exposure and genome-wide gene expression at three time points (379 samples from 157 individuals). First, we show that repeat transcriptomic measurements increases the number of cis eQTLs identified compared to using a single time point by 64%. Then, after identifying 4,818 cis-eQTLs, we observed a statistically significant enrichment of in vivo eQTL interactions with IFN status (p<0.001 by permutation) and anti-IL-6 drug exposure (p<0.001). We observed 210 and 72 interactions for IFN and anti-IL-6 respectively (FDR<20%). Anti-IL-6 interactions have not yet been described while 99 of the IFN interactions are novel. Finally, we found transcription factor binding motifs interrupted by eQTL interaction SNPs, pointing to key regulatory mediators of these environmental stimuli and therefore potential therapeutic targets for autoimmune diseases. In particular, genes with IFN interactions are enriched for ISRE binding site motifs, while those with anti-IL-6 interactions are enriched for IRF4 motifs. Conclusion: This study highlights the potential to exploit clinical trial data to discover in vivo eQTL interactions with therapeutically relevant environmental variables.We hypothesize that regulatory mechanisms influenced by an environmental perturbagen may be identified with eQTL (expression quantitative trait locus) interactions, which alter the relationship between a genetic variant and transcript levels. In an anti-IL-6 clinical trial of 157 patients with systemic lupus erythematosus (SLE) we measured cell counts, interferon (IFN) signature, and drug exposure at three time points alongside genome-wide transcription. Repeated transcriptomic measurements detected 4,976 cis eQTLs, 63% more than detectable from single measurements. We identified 154, 185 and 126 nominal eQTL interactions with T cell count, IFN status and anti-IL-6 drug exposure respectively (more than expected by chance, p<0.001). IFN interactions are enriched for IRF1 motifs, and 91/126 drug-eQTL interactions are consistent with interactions using free IL-6 protein levels. This same approach can be easily used to define informative drug exposure scores, and can be applied to larger drug trials to further our understanding of the drug mechanisms.If an expression quantitative trait locus (eQTL) effect is modulated by an environmental stimulus, such as drug exposure or disease status, it can point to key regulatory mediators. In a clinical trial for anti-IL-6 in 157 patients with systemic lupus erythematosus we measured cell counts, interferon (IFN) status, drug exposure and genome-wide gene expression at three time points. First, we confirmed an increase in power using repeat transcriptomic measurements. Then, after detecting 4,976 cis eQTLs, we discovered that 154, 185 and 126 had evidence of significant eQTL interactions with T cell proportion, IFN status and anti-IL-6 drug exposure respectively. Next, we found an enrichment of transcription factor binding motifs interrupted by eQTL interaction SNPs, pointing to regulatory mediators of these environmental stimuli and therefore potential therapeutic targets for autoimmune diseases. In particular, IFN interactions are enriched for IRF1 binding site motifs, while anti-IL-6 interactions are enriched for IRF4 motifs. Finally, we used the drug-eQTL interactions to define an informative drug exposure score, reflecting a drug9s effect in an individual patient, thus highlighting the potential for utilizing drug-eQTL interactions in a pharmacogenetic framework.

Bioinformatics for RNA‐Seq Data Analysis

While RNA sequencing (RNA‐seq) has become increasingly popular for transcrip‐ tome profiling, the analysis of the massive amount of data generated by large‐scale RNA‐seq still remains a challenge. RNA‐seq data analyses typically consist of (1) accurate mapping of millions of short sequencing reads to a reference genome, including the identification of splicing events; (2) quantifying expression levels of genes, transcripts, and exons; (3) differential analysis of gene expression among different biological conditions; and (4) biological interpretation of differentially expressed genes. Despite the fact that multiple algorithms pertinent to basic analyses have been developed, there are still a variety of unresolved questions. In this chapter, we review the main tools and algorithms currently available for RNA‐seq data analyses, and our goal is to help RNA‐seq data analysts to make an informed choice of tools in practical RNA‐seq data analysis. In the meantime, RNA‐seq is evolving rapidly, and newer sequencing technologies are briefly introduced, including stranded RNA‐seq, targeted RNA‐seq, and single‐cell RNA‐seq.

The Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of a TYK2/JAK1 Inhibitor (PF-06700841) in Healthy Subjects and Patients With Plaque Psoriasis

The safety, tolerability, pharmacokinetics, and pharmacodynamics of PF‐06700841 were assessed in a randomized, double‐blind, placebo‐controlled, single‐ and multiple‐dose escalation, parallel‐group study in healthy subjects and patients with plaque psoriasis. The single ascending dose (1, 3, 10, 30, 100, or 200 mg) and multiple ascending dose (MAD; PF‐06700841; up to 175 mg once daily or 50 mg twice daily for 10 days) periods included 54 healthy participants. In addition, 30 patients with psoriasis received PF‐06700841 30 or 100 mg or placebo once daily for 28 days. Single PF‐06700841 doses were rapidly absorbed, with peak plasma concentrations ≤ 1 hour, proportional exposure up to 100 mg, and mean half‐life 3.8–7.5 hours. On day 10 of MAD, plasma concentrations peaked at ≤1.5 hours postdose (10–175 mg once daily). Elimination half‐life was 4.9–10.7 hours; steady state was reached by day 8. In psoriasis patients on day 28, peak plasma concentrations occurred at 1–2 hours. Biomarkers IP‐10 and high‐sensitivity C‐reactive protein were reduced and returned to near baseline levels after dosing. Maximal mean percent change from baseline in the Psoriasis Area and Severity Index scores for PF‐06700841 30 mg once daily and 100 mg once daily were −67.92% and −96.31%, respectively, in week 4. All adverse events were mild/moderate. PF‐06700841 was safe and well tolerated up to 200 mg once daily in healthy subjects and 100 mg once daily in patients with psoriasis, suggesting potential therapeutic utility in plaque psoriasis and other inflammatory diseases.

Discovering in vivo cytokine-eQTL interactions from a lupus clinical trial

BackgroundCytokines are critical to human disease and are attractive therapeutic targets given their widespread influence on gene regulation and transcription. Defining the downstream regulatory mechanisms influenced by cytokines is central to defining drug and disease mechanisms. One promising strategy is to use interactions between expression quantitative trait loci (eQTLs) and cytokine levels to define target genes and mechanisms.ResultsIn a clinical trial for anti-IL-6 in patients with systemic lupus erythematosus, we measure interferon (IFN) status, anti-IL-6 drug exposure, and whole blood genome-wide gene expression at three time points. We show that repeat transcriptomic measurements increases the number of cis eQTLs identified compared to using a single time point. We observe a statistically significant enrichment of in vivo eQTL interactions with IFN status and anti-IL-6 drug exposure and find many novel interactions that have not been previously described. Finally, we find transcription factor binding motifs interrupted by eQTL interaction SNPs, which point to key regulatory mediators of these environmental stimuli and therefore potential therapeutic targets for autoimmune diseases. In particular, genes with IFN interactions are enriched for ISRE binding site motifs, while those with anti-IL-6 interactions are enriched for IRF4 motifs.ConclusionsThis study highlights the potential to exploit clinical trial data to discover in vivo eQTL interactions with therapeutically relevant environmental variables.